HRP960440A2 - Sugar-modified cytostatics - Google Patents

Description

The invention relates to cytostatics, which are tumor specific due to modification of carbohydrates. Suitable spacers enable serum stability and simultaneous intracellular action.

Chemotherapy in tumor diseases is usually accompanied by severe side effects, caused by the toxicity of chemotherapeutic agents to the proliferating cells of other tissues. For many years, scientists have been dealing with the problem of improving the selectivity of added active substances. One often goes through the synthesis of prodrugs, which are more or less selectively released in the target tissue, for example by changing the pH value (Tietze et al., e.g. DE-4 229 903), enzymes (e.g. glucuronidase; Jacquesy et al. , EP-511 917; Bosslet et al., EP-595 133) or antibody-enzyme conjugates (Bagshawe et al., WO 88/07378; Senter et al., US-PS 4 975 278; Bosslet et al., EP -595 133). In these cases, the problem is, among other things, the insufficient stability of the conjugate in other tissues and organs, and especially the comprehensive distribution of the active substances, which is connected to the extracellular release of the active substance in the tumor tissue.

The distinct lectin pattern on the tumor surface (Gabius; Onkologie 12 (1989) 175) opens up essential possibilities, by binding the corresponding carbohydrate building blocks to cytostatics, to target them to tumor cells. This perspective is limited by the fact that lectins with similar carbohydrate specificities (galactose, lactose, mannose, N-acetyl-glucosamine, fucose, etc.) also appear 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). Consequently, when such unmodified sugars are used, a significant increase in glycoconjugates with the content of active substances in the liver and other lectin-rich organs should be expected.

The heterocyclic amine batacycline (1) shows good antitumor activity in various intestinal cancer models (US-PS 4 757 072).

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Peptide conjugates of (1) with good in vitro activity and favorable solubility properties (US-4 180 343) are less well tolerated than batracycline in animal experiments. The fucose conjugates described in EP-501 250 accumulate very strongly in the liver.

Quinolone-a (2), 7-[(3aRS, 4RS, 7aSR)-4-amino-1,3,3a,4,7,7a-hexahydro-isoindol-2 yl]-8-chloro-1-cyclopropyl- 6-Fluoro-1,4-dihydro-4-oxo-3-quinoline-carboxylic acid, shows a distinct antibacterial activity and very good efficacy against different tumor cell lines (EP-520 240, JP-4 253 973). However, this is opposed by significant toxicological problems (eg genotoxicity, bone marrow toxicity, high acute toxicity in vivo, etc.).

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(2) (quinolone-a)

With a new way of modifying cytostatics, we found, surprisingly, a new class of conjugates. which is characterized by the following properties:

A new way of connecting carbohydrates with cytostatics (eg batacycline, quinolone) leads to glycoconjugates that are serum stable. The action does not depend on the extracellular release of the active substance. Activities in vitro against different tumor cell lines are comparable to those of the basic cytostatic. Cell-specific uptake is carbohydrate-dependent.

Area-selective modification in the carbohydrate part of the described conjugates significantly improves cell and tissue selectivity (especially tumors towards the liver).

The conjugates according to the invention are characterized by significantly improved tolerability in vivo, compared to the active substance and the corresponding peptide conjugates.

In addition, conjugates according to the invention have significantly better solubility properties compared to basic cytostatics.

The compounds according to the invention are described by the following general formula:

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

where is

K unsubstituted or site-selectively modified carbohydrate residue,

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AA1 is an amino acid residue in the D- or in the L-configuration, which in a given case carries the second grouping K-Sp-L-, in which K, Sp, L independently of the second grouping K-Sp-L- can have the meanings mentioned above, or some direct link. The amino acid residue can be connected to L both via the α-amino group, and in a given case via side chains of amino- or hydroxy-functions, and via both functions. If AA1 carries further functional groups, they can be unblocked or protected using known protecting groups. Suitable protecting groups are, for example, acetyl, allyloxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl or tert-butyl.

AA2 is an amino acid residue in the D- or in the L-configuration, which in a given case carries the second grouping K-Sp-L-, in which K, Sp, L independently of the second grouping K-Sp-L- can have the above-mentioned meanings, or some direct link. The amino acid residue can be connected to AA1 both via the α-amino group and, in a given case, via side chains of amino- or hydroxy-functions, or via both functions. If AA2 carries further functional groups, they can be unblocked or protected using known protecting groups. Suitable protecting groups are, for example, acetyl, allyloxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl or tert-butyl.

C residues of a cytostatic agent or a cytostatic derivative, which may additionally carry an amino or hydroxy group. C can be some intercalating substance, some topoisomerase inhibitor, some antimetabolite, some alkylant, some tubulin inhibitor, some tyrosine phosphokinase inhibitor, some protein kinase-C inhibitor or some active substance with some other or unknown cytostatic or cytotoxic active mechanism. C can be, for example, a nucleoside, enediyne antibiotic, quinolone or naphthyridine carboxylic acid or a cytotoxic peptide antibiotic, for example from the dolastatin class. C can be batracycycline, quinolone-a, 5-fluorouracil, cytosinarabinoside, methotrexate, etoposide, camptothecin, daunomycin, doxorubicin, taxol, vinblastine, vincristine, dinamycin, calicamycin, esperamycin, quercetin, suramin, erbstatin, cyclophosphamide, mitomycin C, melphalan, cisplatin, bleomycin, staurosporine or some other antineoplastic active substance.

The structural element -Sp-L-AA1-AA2- together, represents the spacer connecting K and C.

Compounds of formula (I) in which

K carbohydrate residue of the general formula

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where they indicate

And methyl, hydroxymethyl, carboxy, as well as esters and amides derived from them, alkoxymethyl, acyloxymethyl or carboxyalkyloxymethyl, as well as esters and amides derived from them. A can also be CH2-B, whereby B can again be some carbohydrate residue of formula (II) connected through the anomeric center.

R2, R3, R4 individually or together H, hydroxy, alkyloxy, carboxyalkyloxy, as well as esters and amides derived from them, hydroxyalkyloxy, aminoalkyloxy, acyloxy, carboxyalkylcarbonyloxy, sulfato, phosphato, halogen, or any further, in the same framework modified via anomeric center linked carbohydrate residue (II). R2 can additionally be amino or acylamino.

Two of the residues R2, R3, R4 can together denote an epoxy group.

The stereochemistry at the anomeric center of the building carbohydrate can be α or β. Stereochemistry at other centers can produce gluco-, manno-, galacto-, gulo-, ramo- or fuco-configurations.

A aryl residue, which is always ortho-, meta- or para-modified with K and L, and can additionally have 1 to 4 further substituents, which can independently or equally be H, methyl, methoxy, hydroxy, carboxy, methyloxycarbonyl, cyano, nitro, halogen, sulfonyl or sulfonamide;

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AA1 is an amino acid residue in the D- or in the L-configuration, which in a given case carries the second grouping K-Sp-L-, in which K, Sp, L independently of the second grouping K-Sp-L- can have the above meaning, or some direct link. The amino acid residue can be connected to L both through the α-amino group, and in a given case through the side chains of amino- or hydroxy functions, and also through both functions. If AA1 carries further functional groups. they can also be unblocked or protected using known protection groups. Suitable protecting groups are, for example, acetyl, allyloxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl or tert-butyl.

AA2 is an amino acid residue in the D- or in the L-configuration, which in a given case carries the second grouping K-Sp-L-, in which K, Sp, L independently of the second grouping K-Sp-L- can have the above meaning, or some direct link. The amino acid residue can be connected to AA1 both via the α-amino group and, in a given case, via side chains of amino- or hydroxy-functions, or via both functions. If AA2 carries further functional groups, they can be unblocked or protected using known protecting groups. Suitable protecting groups are, for example, benzyloxycarbonyl, allyloxycarbonyl, acetyl, fluorenylmethoxycarbonyl, t-butoxycarbonyl, allyl, benzyl, methyl or tert-butyl.

C can be, for example, a residue of a nucleoside, enediyne antibiotic or cytotoxic peptide antibiotic, for example from the dolastatin class or some quinolone or naphthiridonecarboxylic acid defined below. C can be, for example, batacycline, 5-fluorouracil, cytosinarabinoside, methotrexate, etoposide, camptothecin, daunomycin, doxorubicin, taxol. vinblastine, vincristine, dinamycin, calicamycin, esperamycin, quercetin, suramin, erbstatin, cyclophosphamide, mitomycin C, melphalan, cisplatin, bleomycin, staurosporine or some other antineoplastic active agent. The cytostatic is linked via an amino or hydroxy function to AA2.

Compounds of formula (I) in which

K carbohydrate residue of the general formula

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where they indicate

A methyl, hydroxymethyl, carboxy, and methoxycarbonylmethyl as well as CH2-B, whereby B can again be a carbohydrate residue of formula (H) connected via an anomeric center.

R2, R3, R4 individually or together H, hydroxy, C1-C3-alkyloxy, carboxy-C1-C3-alkyloxy, as well as C1-C3-alkylesters and amides derived from them, hydroxyalkyloxy, acyloxy, carboxy-(C1-C3- alkyl)-carbonyloxy, sulfato, or some further, modified in the same frame and linked via the anomeric center carbohydrate residue in position R3 or R4.

Two of the residues R2, R3, R4 can together denote an epoxy group.

The stereochemistry at the anomeric center of the building carbohydrate can be α or β. D-manno-, D-galacto-, L-gulo-, D-gluco-, L-ramo- or L-fuco-configurations can be formed by stereochemistry at other centers.

Sp aryl residue, which is always ortho- or para-modified with K and L, and in addition to hydrogen atoms, it can have a further substituent, which can be methoxy, nitro, or chlorine;

with R = chlorine or hydroxyalkylamino

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AA1 is an amino acid residue such as lysine, alanine, aspartic acid, glutamic acid, glycine, ornithine, tyrosine, valine or serine in the D- or in the L-configuration, or some direct linkage. The amino acid residue can be connected to L both through the α-amino group and, in a given case, through the side chains of the amino functions and also through both functions, and carry some further grouping K-Sp-L-, which is the same or different from the first . If AA1 carries further functional groups, then they are first deblocked.

AA2 is an amino acid residue such as alanine, lysine, glycine, serine, ornithine or diaminopropionic acid in the D- or L-configuration, or some direct linkage. The amino acid residue can be connected to AA1 both through the α-amino group, and in a given case through the side chains of amino functions, or through both functions, and in a given case carry a further K-Sp-L- group, which is the same or different from the first. If AA2 carries further functional groups, then they are first deblocked.

C may be batacycline, methotrexate, quinolone-a, etopoxide, melphalan, taxol, camptothecin, daunomycin or doxorubicin or any quinolone- or naphthiridonecarboxylic acid defined below. The cytostatic is linked via an amino or hydroxy function to AA2.

The building blocks of quinolone- and naphthyridonecarboxylic acid C used as educts can be represented by the general structure of formula (III)

T-Q (III)

in which it indicates

Q rest of the formula

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where is

Ra in a given case with halogen or with hydroxy mono- or doubly substituted alkyl with 1 to 4 carbon atoms, vinyl, in a given case with 1 or 2 fluorine atoms substituted cycloalkyl with 3 to 6 carbon atoms, bicyclo[1.1.1]pent-1- yl, 1,1-dimethyl-propargyl, 3-oxetanyl, methoxy, amino, methylamino, dimethylamino, in a given case phenyl substituted singly or doubly by halogen, amino or hydroxy, or together with Re can form a bridge described below,

Rb hydroxy, 1- to 3-carbon alkoxy, nitromethyl,

Rc hydrogen or methyl or together with Rg forms a bridge described further,

Rd hydrogen, CH3, CH2F or =CH2,

X1 hydrogen, halogen or nitro,

X2 hydrogen, halogen, amino, hydroxy, methoxy, mercapto, methyl, halomethyl or vinyl,

Y stands for N or C-Re, where

Re means hydrogen, halogen, CF0, OCH3, OCHF2, CH3, CN, CH=CH2 or C≡CH, or together with Ra can form a bridge of the structure -*O-CH2-CH-CH3, -*S-CH2-CH2- , -*S-CH2-CH-CH3, -*CH2-CH2-CH-CH3 or -*O-CH2-N-Rf where the atom marked with * is connected to a carbon atom and where Rf can be hydrogen, methyl or formyl, a

D stands for N or C-Rg, where

Rg means hydrogen, halogen, CF3, OCH3, OCHF2 or CH3 or together with Rc can form a bridge of the structure -*O-CH2-, -*NH-CH2-, -*N(CH3)-CH2-, -*N(C2H5 )-CH2-, -N(C3H5)CH2- or -*S-CH2-, where the indicated atom is connected to the carbon atom from D,

n is 1, 2 or 3, a

T stands for the rest of the formula

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in which

[image]

where

Rk means hydrogen or methyl, a

R 1 is hydrogen, C 1 -C 3 -alkyl or cyclopropyl.

Compounds of formula (III) in which

Q denotes the rest of the formula

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where

Ra means alkyl with 2 to 4 carbon atoms substituted in the given case with 1 fluorine atom, cyclopropyl substituted in the given case with 1 fluorine atom, phenyl mono- or doubly substituted with fluorine in the given case,

Rb means hydroxy, alkoxy with 1 to 2 carbon atoms,

Rc hydrogen, methyl or together with Rg forms the bridge described there,

X1 fluorine,

X2 hydrogen or amino,

Y stands for N or C-Re, where

Re means hydrogen, fluorine, chlorine, CF3, OCH3, OCHF2, or C≡CH, or together with Ra can form a bridge of the structure -*O-CH2-CH-CH3, or -*O-CH2-N-Rf where atom marked with * connected to the carbon atom at Y and where

Rf can mean methyl,

D stands for N or C-Rg, where

Rg means hydrogen, fluorine, chlorine, CF3, OCH3, or CH3 or together with Rc can form a bridge of the structure -*O-CH2-, -*NH-CH2-, -*N(CH3)-CH2-, or -*S -CH2-, where the atom marked with * is connected to the carbon atom from D,

T stands for the rest of the formula

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where

Rh stands for -N-Rk, where

Rk means hydrogen or methyl, a

R 1 means hydrogen or methyl.

Glycoconjugates with camptothecin or its derivatives are also particularly advantageous.

Furthermore, compounds of the general formula I in which K, Sp and L are hydrogen and C is camptothecin are of particular importance. These substances are new, can be converted as intermediates into further derivatives of the general formula I, and for their part already show an interesting pharmaceutical effective spectrum, especially as cytostatics.

The compounds according to the invention can be in stereoisomeric forms, for example as enantiomers or diastereomers or as their mixtures, for example a racemate. The invention relates to both pure stereoisomers and their mixtures.

Mixtures of stereoisomers can, if desired, be separated into individual stereoisomers in a known manner, for example by chromatography or crystallization.

The compounds according to the invention can also appear in the form of their salts. In general, salts with organic or inorganic bases or acids, or inner salts, are listed here.

Acids that can be adsorbed include primarily hydrohalic acids, such as hydrochloric and hydrobromic acids, primarily hydrochloric acid, furthermore phosphoric acid, nitric acid, sulfuric acid, mono- and bifunctional carboxylic acids, such as acetic 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 sulfonic acids, for example p-toluenesulfonic acid, 1,5-naphthalene-disulfonic acid or camphorsulfonic acid .

Physiologically suitable salts can also be metal or ammonium salts of the compounds according to the invention, which possess some free carboxyl group. Sodium, potassium, magnesium or calcium salts, as well as ammonium salts derived from ammonia or from organic amines, such as ethylamine, di- or triethylamine, di- or triethinolamine, dicyclohexylamine, dimethylamino-ethanol, arginine, lysine, ethylenediamine or phenethylamine.

Examples of string A:

Biological testing

Example A.1

Growth inhibition test to determine the cytotoxic properties of batracycline and quinolone glycoconjugates:

Human colon cell lines SW 480 and HT 29 (ATCC-Nr. CCL 228 and HBT-38) as well as murine melanoma cell lines B 16 F 10 were placed in Roux dishes in RPMI 1640 medium supplemented with 10% FCS. It was then trypsinized and taken up in RPMI plus 10% FCS to a cell count of 50,000 cells/ml. In a plate with 96 microwells, 100 μl of cell suspension was added per well, and it was incubated for 1 day at 37°C in an incubator under CO2. A further 100 μl of RPMI medium and 1 μl of DMSO with test substances were then added. Growth was checked after 3 days and after 6 days. In addition, 40 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoline bromide) solution with an initial concentration of 5 mg/ml H2O was added to each microwell. It was incubated for 5 hours in a CO2 incubator at 37°C. Then the medium was aspirated and 100 μl of i-propanol was added per well. After 30 minutes of shaking with 100 μl H2O, the absorbance was measured at 540 nm using a Titertek Multiskan MCC/340 (Flow).

The cytotoxic activity of the described batracillin glycoconjugates is listed in Table 1a, as the IC50 value for each SW 480 and HT 29 cell line.

IC50 values for quinolone-a-glycoconjugates on cell lines SW 480, HT 29 and B 16 F 19 are combined in Table 1b.

Table 1b

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Table 1b

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The dependence of the biological action on carbohydrates is based additionally on the ineffectiveness, in the series of examples 5, 6 and 11, of the basic comparison compounds, N-[Nα,Nε-bis-(4-hydroxyphenylaminothiocarbonyl)-lysyl]-batracillin and N-[Nα,Nε- bis-(4-hydroxyphenylamino-thiocarbonyl)-lysyl-D-alanyl]-batracillin, or N-[Nα,Nε-bis-(4-hydroxyphenylamino-thiocarbonyl)-D-lysyl]-quinolone (IC50 values >250 ).

Example A.2

Examination of the possibility of glycoconjugate cleavage in vitro

Cleavage kinetics with human blood

1 225 ml of human blood is incubated at 37°C together with 1.25 ml of PBS and 25 μl of substrate stock solution (1 mg/ml in 3% DMSO in PBS). After 1 hour and after 24 hours, samples of 1 ml were taken, mixed with 1 ml of ethanol and left to stand for 20 minutes at 4°C. After centrifugation (5 minutes at 3500 U/min), 100 μJ of the residue is taken for HPLC analysis.

Cleavage kinetics with cells

2.25 ml of PBS was incubated at 37°C together with 225 μl of cell suspension (30 mg/ml) and 25 μl of basic substrate solution (1 mg/ml in 3% DMSO in PBS). After 1 hour and after 24 hours samples were taken from 1 ml each, mixed with 1 ml of ethanol and left to stand for 20 minutes at 4°C. After centrifugation (5 minutes at 3500 U/min), 100 μl of the residue is taken for HPLC analysis.

HPLC conditions:

device: Waters device

column: Bischoff Hypersil OCS RP 18 5 μm 250x4 mm

eluent: A: 10 mM potassium phosphate buffer pH 4.5

B; 80% acetonitrile / 20% water

flow rate: 1 ml/min

wavelength: 372 nm

gradient: 0 min 10 % B

10 min 60 % B

15 min 60 % B

18 min 10 % B

20 min 60 % B

eluent for quinolone-a-conjugates:

A: 100% methanol

B: 10 mM potassium phosphate buffer pH 2.2;

10 mM heptanesulfonic acid.

Table 2a

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* The cleavage product is N-(D-alanyl)-batracillin

Table 2b

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Example A.3

Examination of distribution in organs

Athymic mice (breeding NMRI nu/nu) were used for all experiments, bred by Prof. H.H. Fiebig, Freiburg. in "Dmg Development Laboratory, Oncotest GmbH". The animals were kept in macrolon cages under laminar flow conditions. The tissue of the cell line SW 480, which had previously undergone several passages in mice, was used as tumor material.

Each of the 6- to 8-week-old mice was implanted with two tumors subcutaneously in both flanks. The animals were kept for 26 to 27 days until the time of randomization. The average tumor size was then 500 mg, corresponding to a tumor diameter of approx. 10 mm.

The pharmacokinetics itself proceeded as follows: The mice were injected with the substance being tested, and they were returned to their cages for the next 1/2 hour, or 4 hours. until the sample is taken. Sampling began with blood collection. At the same time, the mice were stunned using ether for narcosis (duration 1/2 to one minute). Then, 0-5 hours, i.e. 4 hours after the injection of the substance to be tested, the abdominal cavity was opened, and the mouse was narcosis through the cava caudalis within 1 to 2 minutes, all the blood was drained, and then he was killed by breaking his neck. This led to a central stoppage of blood flow and interruption of organ perfusion. Furthermore, individual organs were freed and removed, a process which lasted approx. 5 minutes. Immediately afterwards, the organ samples and the rest of the body were weighed and frozen in liquid nitrogen.

The substance "conjugate 1" was given with 300 mg/kg of body weight i.p., and the substance "conjugate 2" with 100 mg/kg i.v. in the tail vein. For each substance and for each time period, 5 animals were used.

The distribution results are summarized in Table 3 for conjugate 1, and in Table 4 for conjugate 2.

A. Bajdar series:

5, 10, 50, 100 and 200 μg of the substance dissolved in an ethanol-water mixture (1:1, v/v) were added to 1 g of beef liver. It was then crushed with 1 g of sea sand and 2.5 ml of cooled ethanol-water mixture (1:1, v/v), and centrifuged at 3500 U/min for 2 minutes. After removing the upper layer, the residue was again mixed with the ethanol-water mixture, centrifuged, and the upper layers were united. 100 μl was taken from each and analyzed by HPLC.

HPLC conditions:

device: Waters device

column: Bischoff Hypersil ODS RP18 5 μn 250x4 mm

eluent: A: 80% acetonitrile / 20% water

B: 10 mM potassium phosphate buffer pH 4.5

gradient: 0 min 90 % B

10 min 40 % B

15 min 40 % B

18 min 90 % B

20 min 90 % B

flow rate: 1 ml/min

wavelength 372 nm

B. Processing of organs

The processing of the organs follows analogously to procedure A, whereby all organs are treated with 2.5 ml of the ethanol-water mixture and extracted.

C. Blood processing

The extracted amount of blood was mixed with 2 ml of ethanol-water mixture (1:1, v/v), centrifuged for 2 minutes at 3500 U/min and the upper layer was decanted. 2 ml of ethanol-water mixture (1:1, v/v) was added to the residue, centrifuged, and the upper layers were united. 100 μl of the combined upper layers were always analyzed by HPLC. The HPLC conditions specified for case A were applied.

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Table 3

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Table 4

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Example A.4

Determination of acute toxicity of batracillin glycoconjugate

(one time application)

Acute toxicity of batracillin derivatives was determined in nu/nu mice.

Substances applied i.v. were injected as aqueous solutions, and substances applied i.p. as DMSO-solutions, once in concentrations of up to 400 mg of substance per kg of mouse.

The tolerated single dose was calculated according to the reduction in the weight of the animals up to 21 days after the application and according to the number of survival of the animals

The individual dose that can be tolerated for substances can be seen from Table 5.

It is for substances 3.16; 3.33; 3.9; 6.12; 6.14; 6.2; 6.81; 8.2 was above 200 mg per kg mouse. For substances 3.33; 6.12; 6.14; 6.2; 6.81 acute toxicity could not be demonstrated even with a dose of 400 mg/kg.

In contrast, lysyl-D-alanyl-batracillin (2.13) without sugar content was clearly toxic already at individual doses between 25 and 50 mg/kg mouse.

Table 5

Maximum acceptable individual doses of batracillin derivatives and quinolone derivatives

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Example A.5

Determination of acute toxicity after repeated application

The substances were administered partly i.v. and partly i.p., either daily on days 1 to 4 and 7 to 10, or on days 1, 5 and 9. The doses were 400, 200 and 100 mg/kg/day. The assessment was based on the weight loss until the 21st day and the number of surviving animals. Five animals per substance and per dose were taken for the experiments. The results are summarized in Table 6.

Table 6

Maximum acceptable doses for repeated application

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Example A.6

Hematopoietic activity of quinolone glycoconjugates compared to the main active substance:

Materials and methods

in vitro:

Bone marrow cells were washed from mouse femurs. 105 cells were incubated in McCoy's 5A-medium (0.3% agar) together with recombinant sludge GM-CSF (genezymes; parental cell colony forming) and substances (10-4 to 100 μg/ml) at 37°C and 7% CO2. 7 days later colonies (<50 cells) and clusters (17-50 cells) were counted.

in vivo:

Mice were treated subcutaneously with 1, 3, 10, 30 mg/kg compounds. At different times (3, 24, 48, 72 p. inj.) femurs were removed and bone marrow cells were isolated. 2x10 5 cells were incubated with GM-CSF as described above, and after 7 days colonies and clusters were counted.

The results:

As shown in Table 7, the investigated glycoconjugates show a reduced inhibition of the proliferation of parental bone marrow cells by a factor of 105 to 103 compared to quinolone-a.

In comparison with quinolone-a, inhibition of proliferation by compound 12.3, up to 30 mg/kg, could not be observed either in vivo. However, already with 3 mg/kg of quinolone, a strong suppression of the proliferation of parental cells was induced (Fig. 1).

Table 7

CSF-induced proliferation of murine bone marrow progenitor cells

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Example A.7

Antineoplastic activity of quinolone conjugates

The in vitro activity of quinolone glycoconjugates was determined on human tumor xenografts in the two-layer soft agar culture system according to Hamburger and Salmon (Science 197: 461-463).

Solid tumors were first implanted into athymic mice (NMRI nu/nu), surgically obtained and mechanically minced. Individual cells were then obtained by incubation in an enzyme mixture of collagenase 0.05%, DNAse 0.07% and hyaluronidase 0.1% in RPMI at 37 °C for 30 minutes. The cells were washed twice and then crushed to 200 μm and 20 μm using a sieve.

The following culture method was applied:

The bottom layer contains 0.2 ml of Iscoves' modified Dulbecco's medium with 20% fetal calf serum and 0.7% agar. 40,000 to 200,000 cells in 0.2 ml of the same medium and 0.4% agar were applied to this layer in plates with 24 wells. Cytostatic substances were added to 0.2 ml of medium.

The cultures were incubated at 37°C in a 7% CO2 atmosphere for 6 to 15 days. Then, the grown colonies were counted using an inverted microscope, and 24 hours before the evaluation, the living colonies were stained with tetrazolium chloride-based dye.

The effect of active substances is expressed in the percentage of surviving colonies compared to the number of colonies in untreated plates (T/C = number of treated colonies x 100 / untreated control number).

The substance is active when the T/C value is ≤ 30%.

In Table 8, this value is given as the IC70 value in μg/ml.

Table 8

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Example A.8

Experiments in vivo

Method:

Mice were inoculated with 5x106 B16F10 tumor cells on day zero. Animals with a transplanted tumor develop solid peritoneal tumors and are treated daily with test substances, i.e. using vehicle controls. In the control group, 50% of the animals usually die between the 14th and 20th day. The tested substances were applied in a buffer or in an organic solvent system consisting of 20% methanol and 20% dimethylsulfoxide in a 0.7% table salt solution.

Administration of the vehicle showed no effect on the survival time of the animals. Therapeutic success is shown by prolonging the survival time of treated animals. The tolerability of the compounds is analyzed comparatively on animals without tumors. The therapeutic index can be estimated from tolerability and prolongation of survival time.

Table 9: % survival

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Table 9 shows the therapeutic efficacy of the compound from Example 11 12 in mice transplanted with B16F10 tumor.

Example A.9

Inhibition of tumor growth in vivo using a mouse model

Material:

Athymic mice (NMRI nu/nu strain) were used for all in vivo experiments examining inhibition of tumor growth. The selected large cell lung carcinoma LXFL 529 was developed in mice by serial passage. The human origin of the tumor was covered by isoenzymatic and immunohisto-chemical methods.

Creation of experiments:

The tumor was implanted subcutaneously in both flanks of 6- to 8-week-old nu/nu mice. The treatment was started, depending on the doubling time, as soon as the tumors reached a diameter of 5 - 7 mm. Mice were randomly assigned to a treatment group and a control group (5 mice per group with 8-10 evaluable tumors). All individual tumors of the control group grew progressively.

Tumor size was measured in two dimensions using a sliding scale. Tumor volume, which correlates well with cell number, was then applied for all assessments. The volume is calculated according to the formula "length x width x width/2" ([a x b2]/2, a and b denote two mutually perpendicular diameters).

Relative tumor volume (RTV) values were calculated for each tumor by dividing the tumor size on day X by the tumor size on day 0 (at the time of randomization). Mean RTV values were then applied for further evaluations.

Inhibition of growth of tumor volume (tumor volume of test group / control group, T/C, in percentage) was the final measurement value Treatment:

All compounds were applied according to the plan with interruptions, on the 1st, 5th and 9th days. Furthermore, all compounds were applied intraperitoneally (i.p.) using water as a solvent.

Table 10

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a) maximum tolerated dose (MTD)

b) 19. day

The camptothecin compounds of the series of example 18 generally showed comparable or better activity in these tests.

A series of examples B:

Examples of synthesis

Example 1.1

p-aminophenyl-2-O-methyl-ß-L-fucoside

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1.1-a) p-nitrophenyl-3,4-O-isopropylidene-ß-L-fucoside:

A solution of p-nitrophenyl-ß-L-fucoside (750 mg, 2.63 mmol) in 40 ml of DMF/dioxane 1:2 was treated at 0°C with 65 mg of p-toluenesulfonic acid and at intervals of 30 minutes with 5x100 μl of 2-methoxypropene. . After 16 hours of mixing at 20°C, it thickens and is purified by vacuum chromatography (dichloromethane/methanol 99:1). After concentration, 710 mg (83%) of a white solid is obtained.

1.1.b) p-nitrophenyl-2-O-methyl-3,4-O-isopropylidene-ß-L-fucoside:

100 mg (0.307 mmol) of the compound from example 1.1.a is placed together with 96 μl of methyl iodide in 10 ml of THF and then converted in portions with 11 mg of sodium hydride (80% vol). After 3 hours of stirring at 20°C, 96 μl of methyl iodide and 11 mg of sodium hydride are added again. After a further 16 hours of stirring at 20°C, some water and 100 ml of dichloromethane are added. The mixture is diluted twice with water, the organic phase is concentrated and then the product is purified by column chromatography (petroleum ether/ethyl acetate 8:1). Yield: 78 mg (75 %).

1.1) p-aminophenyl-2-O-methyl-ß-L-fucoside:

78 mg (0.23 mmol) of p-nitrophenyl-2-O-methyl-3,4-O-isopropylidene-ß-L-fucoside was mixed for 16 hours at 20°C in 3 ml of 80% acetic acid. Then the acetic acid is removed in vac., the mixture is converted with 10 ml of methanol and after the addition of platinum dioxide, it is hydrogenated in a hydrogen atmosphere with a slight overpressure. The suspension is filtered through celite and the filtered residue is washed with methanol. After chromatographic purification (dichloromethane/methanol 97.5:2.5), 77 mg (80%) of the desired product is obtained [TC: dichloromethane/methanol 9:1, Rf= 0.42].

Example 1.2

p-aminophenyl-3-O-methyl-ß-L-fucoside

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1.2.a) p-nitrophenyl-3-O-methyl-ß-L-fucoside:

6 g (21 mmol) p-nitrophenyl-ß-L-fucoside in 300 ml abs. of methanol is converted with 7.84 g (31.5 mmol) of dibutyltin oxide and heated for 2 h with reflux. It is then purified, the residue is dried and then taken up in 300 ml of DMF. After adding 15.7 ml of methyl iodide, it is stirred for 40 hours at 70°C. The solvent was removed in vac. and take up the residue in 300 ml of dichloromethane. The suspension is filtered, the remaining solution is concentrated again and subjected to vacuum chromatography (dichloromethane/methanol 99:1). After concentration, 3815 mg (61%) of the desired product is obtained.

1.2) p-aminophenyl-3-O-methyl-ß-L-fucoside

3.81 g (12.73 mmol) of p-nitrophenyl-3-O-methyl-ß-L-fucoside is hydrogenated analogously to example 1.1. Yield: 3 g (88 %). [TC: dichloromethane/methanol 9:1, Rf=0.53].

Example 1.3

p-aminophenyl-3-O-methyl-α-L-fucoside

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Preparation analogous to example 1.2., starting with p-nitrophenyl-α-L-fucoside.

Utilization: 63% through 2 steps. [TC: dichloromethane/methanol 9:1, Rf = 0.39].

Example 1.4

p-aminophenyl-4-O-methyl-ß-L-fucoside

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1.4.a) p-nitrophenyl-2-O-benzyl-4-O-acetyl-ß-L-fucoside:

1 g (3.5 mmol) p-aminophenyl-ß-L-fucoside in 100 ml abs. THF was treated with 31 mg of p-toluenesulfonic acid and 1134 mg (7 mmol) of triethylorthoacetate. After 15 minutes of stirring at 20°C, the solvent is removed by distillation in vac. The residue is taken up in 50 ml of THF and 3 ml of DMF and converted with 4165 μl of benzyl bromide and 210 mg of sodium hydride (60% strength). After stirring for 1 h at 20°C, 10 ml of 80% acetic acid is added, it is concentrated and the residue is purified by vacuum chromatography (dichloromethane/methanol 99:1). After concentration and drying, 1236 mg (85%) of the desired product is obtained.

1.4.b) p-nitrophenyl-2-O-benzyl-3-O-acetyl-4-O-methyl-ß-L-fucoside:

1000 mg (2.39 mmol) of p-nitrophenyl-2-O-benzyl-4-O-acetyl-ß-L-fucoside are dissolved in 60 ml of benzene. After the addition of 2988 μl of methyl iodide and 1109 mg of silver oxide, the mixture is heated for 8 hours with reverse cooling. The resulting product mixture is separated into components by vacuum chromatography (dichloromethane/methanol 99:1). 239 mg (23 %) of p-nitrophenyl-2-O-benzyl-3-O-acetyl-4-O-methyl-ß-L-fucoside is isolated along with 653 mg (63 %) of the p-nitrophenyl-2-O isomer -benzyl-3-O-methyl-4-O-acetyl-ß-L-fucoside as a white solid.

1.4) p-aminophenyl-4-O-methyl-ß-L-fucoside

224 mg (0.52 mmol) of p-nitrophenyl-2-O-benzyl-3-O-acetyl-4-O-methyl-ß-L-fucoside were dissolved in 20 ml of methanol and treated with 390 μl of 1 M sodium methylate solution. After stirring for 16 hours at 20°C, it is neutralized with 80% acetic acid, thickened and taken up in dichloromethane. The organic phase is washed with 1 M sodium bicarbonate solution and water, dried and concentrated. The residue is taken up in 20 ml of methanol and hydrogenated over palladium/activated carbon, analogously to example 1.1. After thickening, the product is taken up in water and lipophilized. 119 mg (88%) of a white amorphous solid is isolated. [TC: dichloromethane/methanol 9:1, Rf = 0.38].

Example 1.5

p-aminophenyl-3-O-n-propyl-ß-L-fucoside

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1.5.a) p-nitrophenyl-2-O-benzyl-3-O-n-propyl-4-O-acetyl-ß-L-fucoside:

Analogously to example 1.4.b, isomeric 3- and 4-propylation products are prepared from compound 1.4.a with propyl iodide, and separated by chromatography. p-nitrophenyl-2-O-benzyl-3-O-n-propyl-4-O-acetyl-ß-L-fucoside is obtained in 49% yield, with p-nitrophenyl-2-O-benzyl-3-O -acetyl-4-O-n-propyl-ß-L-fucoside in 29% yield.

1.5.b) p-aminophenyl-3-O-n-propyl-ß-L-fucoside:

Synthesis from example 1.5.a), fraction 1 analogously to example 1.4. Yield 78% [TC: dichloromethane/methanol 9:1, Rf = 0.42].

Example 1.6

p-aminophenyl-3-deoxy-ß-L-fucoside

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1.6.a) p-nitrophenyl-3,6-dideoxy-3-chloro-4-O-acetyl-ß-L-guloside:

1 g (3.5 mmol) of p-nitrophenyl-ß-L-fucoside in 100 ml of THF was converted into 100 ml of THF with 31 mg of p-toluenesulfonic acid and 1134 mg (7 mmol) of triethyl orthoacetate. After 15 minutes of stirring at 20 °C, the solvent is removed by distillation in vac. 100 ml of a saturated solution of hydrogen chloride in dichloromethane is added. After 10 minutes of reaction time, it thickens and the product is purified by vacuum chromatography (dichloromethane/methanol 99:1). 793 mg (65%) of the desired product is obtained. (TC: dichloromethane/methanol 97.5:2.5, Rf = 0.36].

1.6.b) p-nitrophenyl-3,6-dideoxy-3-chloro-ß-L-guloside

375 mg (1.08 mmol) of p-nitrophenyl-3,6-dideoxy-3-chloro-4-O-acetyl-ß-L-guloside were dissolved in 25 ml of methanol and treated with 10 drops of 1 M sodium methylate solution. After 20 minutes, it is acidified with acetic acid, thickened and distributed between 400 ml of dichloromethane and 60 ml of water. The organic phase is dried, concentrated and precipitated from a dichloromethane/ether mixture. 315 mg (96%) of the desired product is obtained.

1.6) p-aminophenyl-3-deoxy-ß-L-fucoside:

315 mg (1.04 mg) of p-nitrophenyl-3,6-dideoxy-3-chloro-ß-L-guloside are dissolved in 40 ml of methanol, converted with 200 mg of palladium on activated carbon as well as with 290 μl of triethylamine, and hydrogenated 4 days in a hydrogen atmosphere at low overpressure. The suspension is filtered, washed, concentrated and the product is purified by vacuum chromatography (dichloromethane/methanol 97.5:2.5). 160 mg (65%) of the deoxy compound is obtained. [TC: dichloromethane/methanol 95:5, Rf = 0.18].

Example 1.7

p-aminophenyl-3,4-dideoxy-ß-L-fucoside

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400 mg (1.16 mmol) of p-nitrophenyl-3,6-dideoxy-3-chloro-4-O-acetyl-ß-L-guloside (example 1.6.a) are dissolved in 55 ml of methanol, converted with 323 μl of triethylamine and hydrates in a hydrogen atmosphere at a slightly elevated pressure above palladium/activated carbon (10%). After stirring for 16 hours at 20°C, it is filtered through celite, washed, concentrated and taken up again in 100 ml of methanol. Add 1.5 ml of 1 M sodium methylate solution and stir for 16 h at room temperature. It is neutralized with acetic acid, thickened and the resulting products are separated by vacuum chromatography (dichloromethane/methanol 97.5:2.5). After concentration of the appropriate fractions and precipitation from methanol/ether, 120 mg (46 %) of the desired compound are obtained [TC: dichloromethane/methanol 95:5, Rf = 0.31] along with 77 mg (28 %) of p-aminophenyl-3-deoxy-ß -L-fucoside [TC:dichloromethane/methanol 95 5, Rf = 0.18].

Example 1.8

p-aminophenyl-3,4-epoxy-ß-L-fucoside

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80 mg (0.23 mmol) of p-nitrophenyl-3,6-dideoxy-3-chloro-4-O-acetyl-ß-L-guloside (example 1.6.a) is taken up in 10 ml of methanol and converted with 345 μl of 1 M sodium methylate solution. After 1 hour of ultrasonic treatment, it is acidified with 80% acetic acid, concentrated and chromatographed with dichloromethane/methanol 99:1. After the concentration of the relevant fractions, it is taken up in methanol and hydrogenated over palladium/activated carbon analogously to example 1.1. 46 mg (75%) of the desired compound is obtained. FAB-MS; m/e = 238 =M+1.

Example 1.9

p-aminophenyl-4-deoxy-ß-L-fucoside

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This compound was prepared analogously to the recipe given by T. Lindhorst and J. Thiem in Carbohydr Res 209 (1991) 119, starting from p-nitrophenyl-ß-L-fucoside, via p-nitrophenyl-2,3-di-O- benzoyl-4,6-dideoxy-4-iodo-ß-L-fucoside [TC: dichloromethane/methanol 90:10, Rf = 0.3].

Example 1.10

p-aminophenyl-3-O-carboxymethyl-ß-L-fucoside

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1.10.a) p-nitrophenyl-3-O-methoxycarbonylmethyl-ß-L-fucoside:

1 g (3.5 mmol) of p-nitrophenyl-ß-L-fucoside and 1.3 g (5.2 mmol) of dibutyltin oxide are heated in 50 ml of methanol for 2 hours with reflux. The solution is concentrated, the residue is taken up in 50 ml of dioxane, converted with 2 ml of bromoacetic acid methyl ester and 100 mg of tetrabutylammonium iodide and heated for 16 hours with reflux. The solvent is evaporated and the product is purified by vacuum chromatography (dichloromethane/methanol 99:1). After concentration of the appropriate fractions and precipitation from methanol/ether, 455 mg (37%) of the final compound is obtained.

1.10) p-aminophenyl-3-O-carboxymethyl-ß-L-fucoside:

282 mg (0.79 mmol) of p-nitrophenyl-3-O-methoxycarbonylmethyl-ß-L-fucoside were dissolved in 20 ml of methanol and treated with 440 μl of 2 M lithium hydroxide solution. After 2 hours of mixing at 20°C, the pH value is adjusted to 3 using the ion exchanger SC108, and filtered. 250 mg of palladium on activated carbon is added to the filtrate. It is then hydrogenated for 1.5 hours with hydrogen at low overpressure, the catalyst is separated and washed with methanol. It is thickened, absorbed in water and freeze-dried to obtain the target product (212 mg) with a yield of 86%. [TC: acetonitrile/water/glacial acetic acid 5.1:0.2, Rf=0.24].

Example 1.11

p-aminophenyl-3-O-methoxycarbonylmethyl-ß-L-fucoside

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250 mg (0.7 mmol) of p-nitrophenyl-3-O-methoxycarbonylmethyl-ß-L-fucoside (example 1.10.a) is dissolved in 20 ml of methanol and hydrogenated for 1.5 hours with hydrogen over palladium on activated carbon, with a slight positive pressure. The catalyst is separated and washed with methanol. It thickens, is taken up in water and freeze-drying yields 195 mg (85%) of the desired product. [TC: dichloromethane/methanol 9:1, Rf = 0.43; FAB-MS: m/e = 328 = M+1]

Example 1.12

p-aminophenyl-3-O-hydroxyethyl-ß-L-fucoside

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1.12.a) p-nitrophenyl-3-O-hydroxyethyl-ß-L-fucoside:

1000 mg (2.8 mmol) of p-nitrophenyl-3-methoxycarbonylmethyl-ß-L-fucoside is dissolved in a mixture of 160 ml of THF and 40 ml of water and treated with 53 mg of sodium borohydride. After 10 minutes, the solvent is evaporated and the residue is purified by vacuum chromatography (dichloromethane/methanol 95:5). After concentration of the appropriate fractions, absorption in water and freeze-drying, 362 mg (40%) of the desired product is obtained.

1.12) p-aminophenyl-3-O-hydroxyethyl-ß-L-fucoside

After hydrogenation of 362 mg of the compound from example 1.12.a), analogously to example 1.1, 270 mg (82%) of the desired product is obtained. [TC: acetonitrile/water 10:1, Rf=0.43].

Example 1.13

p-aminophenyl-2-O-carboxymethyl-ß-L-fucoside

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1.13. a) p-nitrophenyl-2-O-methoxycarbonylmethyl-ß-L-fucoside:

250 mg (0.88 mmol) of p-nitrophenyl-ß-L-fucoside are dissolved in 25 ml of abs. THF and 3 ml DMF. Add 80 mg (2.64 mmol) of 80% sodium hydride and, after stirring for 10 minutes at 20°C, 35 μl of bromoacetic acid benzyl ester. At 10-minute intervals, 35 μl of bromoacetic acid benzyl ester is added three more times. It is stirred for another 30 minutes and the reaction is quenched with methanol. After a further 10 minutes, acidify with 5 ml of 80% acetic acid. It is concentrated and distilled with dichloromethane. Purification by vacuum chromatography begins with a dichloromethane/methanol/glacial acetic acid 90:10:1 solvent system. Later, the same system is used, with the ratio increasing to 80:20:2. After the concentration of the corresponding fractions, the residues are digested with ether, and 157 mg (42%) of the desired compound is obtained from the early eluate. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.65]. The isomeric 3-O-alkylated compound (33%) is obtained from later eluates. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.54].

1.13) p-aminophenyl-2-O-carboxymethyl-ß-L-fucoside

Saponification and hydrogenation of 150 mg of p-nitrophenyl-2-O-methoxycarbonylmethyl-ß-L-fucoside according to the procedure described in example 1.10 leads with 80% yield to 109 mg of the desired product [TC: acetonitrile/water/glacial acetic acid 5: 1:0.2, Rf=0.35].

Example 1.14.a

Synthesis of regioisomers of p-nitrophenyl-ß-L-fucoside monosuccilylation products:

1100 mg (3.86 mmol) of p-nitrophenyl-ß-L-fucoside are dissolved in 50 ml of pyridine and converted with 580 (5.79 mmol) of succinic anhydride. After 16 hours of stirring at 20°C, it thickens and is distilled twice with dichloromethane. It is precipitated from a mixture of dichloromethane/ether to obtain 1 g of a mixture of substances that cannot be separated. This is taken up in a methanol/water mixture and converted with 846 mg (2.6 mmol) of cesium carbonate. The solvent is evaporated and distilled with DMF. The residue was taken up in DMF and treated with 618 μl of benzyl bromide. After 1 hour of ultrasound treatment, the cesium bromide is filtered off and the filtrate is thickened. Distribute between 500 ml of ethyl acetate and 50 ml of water. The organic phase is dried and thickened. The separation of the ingredients by vacuum chromatography was successful in the dichloromethane/methanol 99:1 solvent system. It is obtained:

Fraction 1: 87 mg (4.8 %) of p-nitrophenyl-3-O-(3-benzyloxycarbonyl-propionyl)-ß-L-fucoside [TC: dichloromethane/methanol 95:5, Rf = 0.45].

Fraction 2: 27 mg (1.5 %) of p-nitrophenyl-2-O-(3-benzyloxycarbonyl-propionyl)-ß-L-fucoside [TC: dichloromethane/methanol 95:5, Rf = 0.34].

Fraction 3: 190 mg (10.3 %) p-nitrophenyl-4-O-(3-benzyloxycarbonyl-propionyl)-ß-L-fucoside [TC: dichloromethane/methanol 95:5, Rf = 0.28].

Example 1.14

p-aminophenyl-3-O-succinyl-ß-L-fucoside

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85 mg (0.17 mmol) of fraction 1 from example 1.14.a is dissolved in 5 ml of THF and 1 ml of water. Add 20 mg of platinum oxide and hydrate for 8 hours. The catalyst is filtered off, washed with a THF/water mixture, and the filtrate is concentrated. The rest is taken up in water and lipophilized. 57 mg (94%) of the desired product is obtained. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2. Rf=0.65].

Example 1.15

p-aminophenyl-2-O-succinyl-ß-L-fucoside

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Fraction 2 from example 1.14.a is hydrogenated analogously to the procedure from example 1.14. Ex.: 16 mg (87 %). [TC: acetonitrile/water/glacial acetic acid 5:1:0 2, Rf=0.62].

Example 1.16

p-aminophenyl-4-O-succinyl-ß-L-fucoside

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Fraction 3 from example 1.14.a is hydrogenated analogously to the procedure from example 1.14. Ex.: 125 mg (88 %). [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.63].

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1.17.a) p-nitrophenyl-2-O-benzyl-3,4-O-isopropylidene-ß-L-fucoside:

377 mg (1.16 mmol) of the compound from example 1.1.a is dissolved in 30 ml abs. THF and converted sequentially with 690 μl of benzyl bromide and 52 mg of sodium hydride, and stirred at 20°C. After 4 hours and after 6 hours, another 690 μl of benzyl bromide, i.e. sodium hydride, is added again. The mixture is processed analogously to example 1.1.b. 245 mg (51%) of the desired compound is obtained.

1.17.b) p-nitrophenyl-2-O-benzyl-3,4-di-O-methyl-ß-L-fucoside:

245 mg (0.59 mmol) of p-nitrophenyl-2-O-benzyl-3,4-O-isopropylidene-ß-L-fucoside was stirred for 16 hours at 20°C in 80% acetic acid. It thickens and the residue is mixed with an ether/pentane mixture. After suction and drying, the remaining product is collected in 20 ml. abs. THF, 45 mg of 80% sodium hydride is added and after 15 minutes, 160 μl of methyl iodide is added. After stirring for 20 hours at 20°C, the reaction was quenched with methanol and glacial acetic acid, concentrated, and the residue partitioned between dichloromethane and water. The organic phase is dried, concentrated and then purified by vacuum chromatography (dichloromethane/methanol 100:1). After concentration and drying of the respective fractions, 188 mg (79%) of the desired product is obtained.

1.17) p-aminophenyl-3,4-di-O-methyl-ß-L-fucoside

180 mg (0.45 mmol) of the compound from example 1.17.b is hydrogenated in a mixture of 15 ml of methanol and 3 ml of dichloromethane after the addition of 50 mg of palladium on activated carbon for 2 days at room temperature. The catalyst is filtered off, the filtrate is concentrated and purified by vacuum chromatography (dichloromethane/methanol 97.5:2.5). 86 mg (68%) of the desired compound is obtained. [TC: dichloromethane/methanol 95:5, Rf = 0.21].

Example 1.18

p-aminophenyl-3-O-carbamoylmethyl-ß-L-fucoside

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100 mg (0.305 mmol) of the compound from example 1.11 is dissolved in 10 ml of methanol and treated with 0.5 ml of a 17% ammonium hydroxide solution. After 4 hours it thickens, absorbs into water and lipophilizes. 95 mg (quant.) of the desired compound is obtained. [TC: acetonitrile/water 10:1, Rf= 0.43].

Example 1.19

p-aminophenyl-2-O-hydroxyethyl-ß-L-fucoside

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This compound was prepared starting from 200 mg (0.56 mmol) of p-nitrophenyl-2-O-methoxycarbonylmethyl-ß-L-fucoside (example 1.13.a) analogously to examples 1.12.a and 1.12. Ex.: 76 mg (45% through two stages). [TC: dichloromethane/methanol 9:1, Rf = 0.2].

Example 1.20

p-Aminophenyl-3,6-dideoxy-3-chloro-ß-L-fucoside

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50 mg (0.165 mmol) of the compound from example 1.6.b is hydrogenated in 5 ml of methanol over palladium/activated carbon for 1 hour. The catalyst is removed by filtration, washed, thickened, absorbed in water and lipophilized. 45 mg (89%) of the desired compound is obtained. [TC: dichloromethane/methanol 9:1, Rf = 0.35].

Example 1.21

p-aminophenyl-α-L-rhamnoside

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This compound was prepared starting from 300 mg of p-nitrophenyl-α-L-rhamnoside (Sigma) analogously to example 1.1. Ex.: 96 %.

Example 1.22

p-aminophenyl-3-O-carboxymethyl-α-L-rhamnoside

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1.22a) p-nitrophenyl-3-O-methoxycarbonylmethyl-α-L-rhamnoside

481 mg (1.63 mmol) of p-nitrophenyl-α-L-rhamnoside is taken up in 30 ml of methanol and converted with 629 mg (2.45 mmol) of dibutyltin oxide. It is heated for 2 hours with reverse cooling, thickens and takes up in 30 ml of dioxane. Add 85 mg of tetrabutylammonium iodide and 950 μl of bromoacetic acid methyl ester, and heat for 16 hours with reflux. In this case, another 1 ml of bromoacetic acid methyl ester is added, and the reaction time is extended. It is concentrated and the residue is purified by vacuum chromatography. The obtained p-nitrophenyl-3-O-methoxycarbonylmethyl-α-L-rhamnoside is eluted with a mixture of dichloromethane/methanol 99:1 and after drying, 408 mg (70%) are obtained. [TC: dichloromethane/methanol 95:5, Rf = 0.36].

1.22) p-aminophenyl-3-O-carboxymethyl-α-L-rhamnoside

The synthesis is completely analogous to example 1.10, starting from p-nitrophenyl-3-O-methoxycarbonylmethyl-α-L-rhamnoside. The desired product is obtained in 80% yield. [TC; acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.26]

Example 1.23

p-aminophenyl-ß-D-galactopyranoside

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p-nitrophenyl-ß-D-galactopyranoside (3.0 g, 10 mmol) is dissolved in a mixture of methanol/water 1:1 (50 ml) and after the addition of palladium on activated carbon (10 % Pd, 200 mg) it is hydrated for 3 hours in to a hydrogen atmosphere with a small overpressure. The suspension is filtered through celite and washed with a hot mixture of methanol/water 1:1 (100 ml). By concentrating the filtrate in vacuum and recrystallization from methanol, colorless crystals are obtained (2.11g, 78%); TC [methanol]: Rf = 0.62; [α]20 = -39.5° (c = 1.0 / H2O); tal. = 166°C.

Example 1.24

p-aminophenyl-2-O-methyl-ß-D-galactopyranoside

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1.24.a) p-nitrophenyl-6-O-triphenylmethyl-ß-D-galactopyranoside:

A solution of p-nitrophenyl-ß-D-galactopyranoside (9.0 g, 30 mmol), chlorotriphenylmethane (16.7 g, 60 mmol) and N,N-dimethylaminopyridine (609 mg, 5 mmol) in absolute pyridine (100 ml) is heated for 4 hours at 60°C. After concentration in vacuum, the residue is purified by vacuum chromatography [petroleum ether/ethyl acetate 2:1 → 3:2, each time with 0.5% triethylamine]. Colorless crystals are obtained (9.23 g, 57%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.55; tal. = 82°C.

1.24.b) p-nitrophenyl-3,4-O-isopropylidene-6-O-triphenylmethyl-ß-D-galactopyranoside:

The above compound (8.7 g, 16 mmol) was treated with dimethoxypropane (400 mL) and a catalytic amount of (±)-camphor-10-sulfonic acid (400 mg, 1.7 mmol). After 1 hour at room temperature, the reaction is terminated by the addition of triethylamine (240 ml, 1.7 mmol) and concentrated in vacuo. Vacuum chromatography [petroleum ether/ethyl acetate 2:1] gave a colorless foam (6.2 g, 66%); TC (petroleum ether/ethyl acetate 1:1): Rf = 0.46; [α]20 = -42.1° (c = 0.94 / CH2Cl2).

1.24.c) p-nitrophenyl-2-O-methyl-3,4-O-isopropylidene-6-O-triphenylmethyl-ß-D-galactopyranoside:

Dissolve compound 1.24.b (5.83 g, 10 mmol) in dimethylformamide (100 ml) and treat with methyl iodide (2.5 ml, 40 mmol) and portions of an 80% suspension of sodium hydride in mineral oil (450 mg, 15 mmol). After 2 hours at room temperature, the reaction is finished by adding methanol (10 ml) dropwise and concentrated in vacuo. The residue was taken up in dichloromethane (1000 ml) and the solution was vigorously mixed with water (500 ml). The organic phase is dried over magnesium sulfate (50 g), concentrated in vacuo and purified by vacuum chromatography [petroleum ether/ethyl acetate 12:1 → 8:1]. A colorless foam is obtained (4.72 g, 79%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.72; [α]20 = -35.7° (c = 1.0 / CH3OH)

1.24. d) p-nitrophenyl 2-O-methyl-ß-D-galactopyranoside:

A solution of the above compound (4.48 g, 7.5 mmol) in dichloromethane (200 ml) was treated with 99% trifluoroacetic acid (20 ml) and stirred for 3 hours at room temperature. After concentration in vacuum, the residue is purified by vacuum chromatography [petroleum ether/ethyl acetate 5:1 → 2:1]. Colorless crystals are obtained (1.09 g, 46%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.42; tal. = 177°C.

1.24) p-aminophenyl-2-O-methyl-ß-D-galactopyranoside:

Compound 1.24.d (946 mg, 3 mmol) is dissolved in methanol (50 ml) and after the addition of water (0.5 ml) and basic Raney nickel (approx. 200 mg) it is hydrogenated for 2 hours in a hydrogen atmosphere with a slight overpressure. The suspension is filtered through celite and thoroughly washed with methanol (100 ml). Concentration of the filtrate in vacuo gave a brownish foam (579 mg, 68%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.28; [α]20 = -39.3° (c = 0.15 / CH3OH).

Example 1.25

p-aminophenyl-3-O-methyl-ß-D-galactopyranoside

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1.25.a) p-nitrophenyl-3-O-methyl-ß-D-galactopyranoside:

A solution of p-nitrophenyl-ß-D-galactopyranoside (1.5 g, 5.0 mmol) in absolute methanol (40 ml) was treated with dibutyltin oxide (1.87 g, 7.5 mmol) and heated under reflux. After 3 hours, it thickens in a vacuum and the rest is cleaned for 1 hour with the vacuum of an oil pump. It is taken up in absolute dioxane (40 ml), the resulting solution is treated with methyl iodide (1.9 ml, 30 mmol) and stirred for 16 hours at a bath temperature of 100°C. The solvent is then distilled off in a vacuum and the residue is purified by vacuum chromatography [ethyl acetate/petroleum ether 2:1 → ethyl acetate]. Colorless crystals are obtained (1.32 g, 84%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.34; tal. = 196°C; [α]20 = -53.3° (c = 1.0 / CH3OH).

1.25) p-aminophenyl-3-O-methyl-ß-D-galactopyranoside:

The above compound (946 mg, 3 mmol) was reduced as described in Example 1.24 and worked up. Brownish crystals are obtained (656 mg, 77%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.21; tal. = 196°C; [α]20 = -25.2° (c = 1.0 / CH3OH).

Example 1.26

p-aminophenyl-4-O-methyl-ß-D-galactopyranoside, acetate

[image]

1.26.a) p-nitrophenyl-3-O-benzyl-ß-D-galactopyranoside:

A solution of p-nitrophenyl-ß-D-galactopyranoside (1.5 g, 5.0 mmol) in absolute dioxane (40 ml) was treated with dibutyltin oxide (1.87 g, 7.5 mmol) and heated under reflux. After 3 hours, the resulting solution was treated with benzyl bromide (3.6 ml, 30 mmol) and stirred for a further 48 hours under reflux. Then the solvent is distilled off in a vacuum and the residue is purified by vacuum chromatography [ethyl acetate/petroleum ether 2:1 → 1:1]. Colorless crystals are obtained (1.58 g, 81%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.69; tal. = 127°C.

1.26.b) p-nitrophenyl-3-O-benzyl-4,6-O-isopropylidene-ß-D-galactopyranoside:

Compound 1.26.a (6.26 g, 16 mmol) was converted as described in example 1.24.b. After vacuum chromatography [petroleum ether/ethyl acetate 5:1 → 3:1] a colorless foam is obtained (6.54 g, 95%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.34; [α]20 = -38.9° (c = 1.0 / CH2Cl2).

1.26. c) p-nitrophenyl-2,3-di-O-benzyl-4,6-O-isopropylidene-ß-D-galactopyranoside:

Dissolve compound 1.26.b (4.31 g, 10 mmol) in dimethylformamide (100 ml) and treat with benzyl bromide (12 ml, 100 mmol) and in portions with an 80% suspension of sodium hydride in mineral oil (450 mg, 15 mmol). After 2 hours at room temperature, the reaction is terminated by the dropwise addition of methanol (10 ml) and concentrated in vacuo. The residue was taken up in dichloromethane (1000 ml) and the solution was vigorously mixed with water (500 ml). The organic phase is dried over magnesium sulfate (50 g), concentrated in vacuo and purified by vacuum chromatography (petroleum ether/ethyl acetate 20:1 → 15:1 → 10:1). A colorless oil is obtained (2.72 g, 52 %), which still impure; TC [petroleum ether/ethyl acetate 1:1]: Rf= 0.62.

1.26.d) p-nitrophenyl-2,3-di-O-benzyl-ß-D-galactopyranoside:

The above compound (2.6 g, 5 mmol) was converted as described in example 1.24.d. After concentration in vacuo and boiling the residue with diethyl ether, colorless crystals are obtained (805 mg, 33%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.23; tal. = 160°C.

1.26.e) p-nitrophenyl-2,3-di-O-benzyl-6-O-triphenylmethyl-ß-D-galactopyranoside:

Compound 1.26.d (722 mg, 1.5 mmol) was tritulated as described in Example 1.24.a. After vacuum chromatography [petroleum ether/ethyl acetate 15:1 → 10:1 → 5:1] a colorless foam is obtained (880 mg, 81%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.79; [α]20 = -25.3° (c = 0.3 / CH2Cl2).

1.26.f) p-pitrophenyl-2,3-di-O-benzyl-4-O-methyl-6-O-triphenylmethyl-ß-D-galactopyranoside:

The above compound (724 mg, 1 mmol) was methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 5:1] a colorless foam is obtained (662 mg, 90%); TC [petroleum ether/ethyl acetate 5:1]: Rf = 0.66; [α]20 = -38.7° (c = 0.2 / CH2Cl2).

1.26) p-aminophenyl-4-O-methyl-ß-D-galactopyranoside acetate:

The above compound (590 mg, 0.8 mmol) is dissolved in 90% acetic acid (50 ml) and, after the addition of palladium on activated carbon (10% Pd, 200 mg), it is hydrogenated for 16 hours in a hydrogen atmosphere with a slight overpressure. The suspension is filtered through celite and thoroughly washed with methanol (100 ml). Concentration of the filtrate in vacuo and precipitation of the residue from a diethyl ether/petroleum ether mixture gave colorless crystals (253 mg, 92%); [TC: dichloromethane/ methanol 5:1]: Rf = 0.12.

Example 1.27

p-aminophenyl-6-O-methyl-ß-D-galactopyranoside

[image]

1.27.a) Benzylation of compound 1.24.b:

Compound 1.24.b (5.84 g, 10 mmol) was benzylated as described in example 1.26.c. After vacuum chromatography [petroleum ether/ethyl acetate 15:1 → 12:1 → 5:1 → ethyl acetate, each time with 0.5% triethylamine], two product fractions are obtained:

Fraction 1: p-nitrophenyl-2-O-benzyl-3,4-O-isopropylidene-6-O-triphenylmethyl-ß-D-galactopyranoside, yellowish foam (1.71 g, 25%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.72; [α]20 = -8.1° (c = 1.0 / CH2Cl2).

Fraction 2: p-nitrophenyl-2-O-benzyl-3,4-O-isopropylidene-ß-D-galacto-pyranoside, yellowish oil (806 mg, 19%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.45; [α]20 = +2.8° (c = 1.2 / CH3OH).

1.27.b) p-nitrophenyl-2-O-benzyl-3,4-O-isopropylidene-6-O-methyl-ß-D-galactopyranoside

Fraction 2 from Example 1.27.a (777 mg, 1.8 mmol) is methylated as described in Example 1.24.C. After vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 8:1] a brownish oil is obtained (730 mg, 91%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.54; [α]20 = -11.6° (c = 1.1 / CH2Cl2).

1.27.c) p-nitrophenyl-2-O-benzyl-6-O-methyl-ß-D-galactopyranoside

The above compound (668 mg, 1.5 mmol) was converted as described in Example 1.24.d. Concentration of the filtrate in vacuo and boiling of the residue with a little diethyl ether gave, after cooling, light beige crystals (388 mg, 64%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.15; tal. = 143°C.

1.27) p-aminophenyl-6-O-methyl-ß-D-galactopyranoside

Compound 1.27.c (324 mg, 0.8 mmol) was reduced over 16 hours, as described in Example 1.24. After concentrating the filtrate in vacuo and boiling the residue with a little diethyl ether, beige crystals are obtained after cooling (184 mg. 81%); TC [ethyl acetate]: Rf = 0.05; tal. = 115°C (decomposition).

Example 1.28

p-Aminophenyl-2,3-di-O-methyl-ß-D-galactopyranoside

[image]

1.28.a) Isopropylidenation of p-nitrophenyl-ß-D-galactopyranoside:

A solution of p-nitrophenyl-ß-D-galactopyranoside (7.5 g, 25 mmol) in absolute acetone (1000 ml) was treated with anhydrous toluenesulfonic acid (500 mg). At normal pressure, acetone (250 ml) is distilled off within 30 minutes and then immediately neutralized by adding potassium carbonate (500 mg). After concentration in vacuo, the residue was mixed with diethyl ether (1000 ml). It is filtered off, the filtrate is concentrated and purified by vacuum chromatography [petroleum ether/ethyl acetate 2:1 → 1:1 → 3:2]. In doing so, two product fractions are created:

Fraction 1: p-nitrophenyl-3,4-O-isopropylidene-ß-D-galactopyranoside; colorless foam (3.74 g; 44 %); TC

[dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.59; [α]20 = -54.2° (c = 0.38 / CH3OH).

Fraction 2: p-nitrophenyl-4,6-O-isopropylidene-ß-D-galactopyranoside; colorless foam (4.3 g; 50%); TC

[dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.54; [α]20 = -81.0° (c = 0.31 / CH3OH).

1.28.b) p-nitrophenyl-2,3-di-O-methyl-4,6-O-isopropylidene-ß-D-galacto-pyranoside:

Fraction 2 from Example 1.28.a (4.1 g, 12 mmol) is methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ ethyl acetate 5:1 → 2:1] a colorless foam is obtained (2.93 g, 66%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.42; [α]20 = -52.6° (c = 0.34 / CH3OH).

1.28 c) p-nitrophenyl-2,3-di-O-methyl-ß-D-galactopyranoside

The above compound (2.77 g, 7.5 mmol) was converted as described in Example 1.24.d. After 1 hour at room temperature, it thickens in a vacuum and the rest is dried for 1 hour in an oil pump vacuum. Colorless crystals (1.11 g, 45%) were obtained by digestion with a mixture of diethyl ether/petroleum ether 1:1 (100 ml); TC [ethyl acetate]: Rf = 0.24; tal. = 156°C.

1.28) p-aminophenyl-2,3-di-O-methyl-ß-D-galactopyranoside:

Compound 1.28.c (989 mg, 3 mmol) is reduced as described in Example 1.24. A colorless oil is obtained (396 mg, 44%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.50; [α]20 = -19.4° (c = 0.16 / CH3OH).

Example 1.29

p-aminophenyl-2,4-di-O-methyl-ß-D-galactopyranoside

[image]

1.29.a) Tritulation of compound 1.26.a:

Compound 1.26.a (11.7 g, 30 mmol) was tritulated as described in Example 1.24.a. After vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 7:1 → 5:1, each time with 0.5% triethylamine] two products are obtained:

Fraction 1: triphenylmethyl-2-O-(p-nitrophenyl)-3-O-benzyl-6-O-triphenylmethyl-ß-D-galactopyranoside, colorless foam (8.5 g, 32%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.68; [α]20 = +42.8° (c = 1.0/CH2Cl2). Fraction 2: p-nitrophenyl-3-O-benzyl-6-O-triphenylmethyl-ß-D-galactopyranoside, colorless foam (9.0 g, 47%); TC [petroleum ether/ethyl acetate 2:1]: Rf= 0.22; [α]20 = -22.6° (c = 1.03 / CH2Cl2).

1.29.b) p-nitrophenyl-2,4-di-O-methyl-3-O-benzyl-6-O-triphenylmethyl-ß-D-galactopyranoside:

Fraction 2 from Example 1.29.a (7.6 g, 12 mmol) is methylated as described in Example 1.24.C. After vacuum chromatography [petroleum ether/ ethyl acetate 15:1 → 10:1] a colorless foam is obtained (7.07 g, 89%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.79; [α]20 = -35.8° (c = 1.09 / CH3OH).

1.29.c) p-aminophenyl-2,4-di-O-methyl-3-O-benzyl-ß-D-galactopyranoside:

The above compound (6.0 g, 9 mmol) was hydrogenated over 48 hours as described in Example 1.26. Colorless crystals are obtained (1.39 g, 40%); TC [ethyl acetate/petroleum ether 2:1]: Rf = 0.20; tal. = 148°C.

1.29) p-aminophenyl-2,4-di-O-methyl-ß-D-galactopyranoside:

Compound 1.29.C (779 mg, 2 mmol) was hydrogenated over 5 days as described in Example 1.24. After evaporation of the combined filtrates in vacuo and boiling of the residue with diethyl ether (2 x 50 ml), light green crystals (391 mg, 65%) were obtained; TC [ethyl acetate]: Rf = 0.16; tal. = 260°C (decomposition).

Example 1.30

p-aminophenyl-2,6-di-O-methyl-ß-D-galactopyranoside

[image]

1.30.a) p-nitrophenyl-2,6-di-O-methyl-3,4-O-isopropylidene-ß-D-galacto-pyranoside:

Fraction 1 from Example 1.28.a (4.1 g, 12 mmol) is methylated as described in Example 1.24.0. After vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 8:1 → 5:1] a colorless oil is obtained (3.25 g, 73%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.65.

1.30.b) p-nitrophenyl-2,6-di-O-methyl-ß-D-galactopyranoside:

The above compound (2.77 g, 7.5 mmol) was converted as described in Example 1.24.d. After vacuum chromatography [ethyl acetate/petroleum ether 2:1], colorless crystals are obtained (1.63 g, 66%); TC [ethyl acetate]: Rf = 0.31; tal. = 222°C.

1.30) p-aminophenyl-2,6-di-O-methyl-ß-D-galactopyranoside:

Compound 1.30.b (989 mg, 3 mmol) is reduced as described in Example 1.24. A colorless oil is obtained (597 mg, 66%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.56; [α]20 = -53.1° (c = 0.49 / CH3OH).

Example 1.31

p-Aminophenyl-3,4-di-O-methyl-ß-O-galactopyranoside, acetate

[image]

1.31.a) p-nitrophenyl-2,6-di-O-benziI-3,4-O-isopropylidene-ß-D-galacto-pyranoside:

Fraction 1 from Example 1.28.a (4.1 g, 12 mmol) of Benziline was prepared as described in Example 1.26.C. After vacuum chromatography [petroleum ether/ethyl acetate 6:1] a yellowish oil is obtained (5.3 g, 85%); TC [petroleum ether/ethyl acetate 2:1]: Rf = 0.76; [α]20 = +8.8° (c = 1.2 / CH3OH).

1.31.b) p-nitrophenyl-2,6-di-O-benzyl-ß-D-galacto-pyranoside:

The above compound (4.69 g, 9 mmol) was converted as described in Example 1.24.d. After 30 minutes at room temperature, it thickens in a vacuum and the rest is dried for 1 hour with the vacuum of an oil pump. Recrystallization from ethanol gives colorless crystals (2.89 g, 67 %): TC [ethyl acetate / petroleum ether 2:1]: Rf = 0.42; tal. = 133°C; [α]20 = -64.2° (c = 1.0 / CH3OH).

1.31.c) p-nitrophenyl-2,6-di-O-benzyl-3,4-di-O-methyl-ß-D-galacto-pyranoside:

The above compound (2.4 g, 5 mmol) is methylated as described in example 1.24.c. After recrystallization from an ethanol/n-hexane mixture, colorless crystals are obtained (1.74 g, 69%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.74; tal. = 149°C.

1.31) p-aminophenyl-3,4-di-O-methyl-ß-D-galactopyranoside, acetate:

Compound 1.31.C (1.52 g, 3 mmol) was hydrogenated as described in Example 1.26. Colorless crystals are obtained (664 mg, 62%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.47; tal. = 140°C (decomposition).

Example 1.32

p-aminophenyl-3,6-di-O-methyl-ß-D-galactopyranoside

[image]

1.32.a) p-nitrophenyl-3-O-methyl-6-O-(tert-butyldimethylsilyl)-ß-D-galactopyranoside:

A solution of compound 1.25.a (1.58 g, 5 mmol) in dimethylformamide (150 ml) was treated with imidazole (1 g, 15 mmol) and tert-butyldimethylsilyl chloride (1.25 g, 8 mmol) and stirred for 24 hours at room temperature. The reaction is then stopped by the addition of water (100 ml). It is diluted with dichloromethane (1000 ml), the organic phase is washed with water (2 x 1000 ml), dried over magnesium sulfate (20 g) and concentrated in a vacuum. After vacuum chromatography [petroleum ether/ethyl acetate 15:1 → 10:1 → 5:1] a yellowish foam (826 mg, 38%) is obtained, which is still slightly impure; TC [ethyl acetate]: Rf = 0.59; [α]20 = -56.3° (c = 1.0 / CH2Cl2).

1.32.b) p-nitrophenyl-2,4-di-O-benzyl-3-O-methyl-6-O-(tert-butyldimethyl-silyl)-ß-D-galactopyranoside:

The above compound (773 mg, 1.8 mmol) was benzylated as described in Example 1.26.C. After vacuum chromatography [petroleum ether/ethyl acetate 30:1 → 5:1] a colorless foam is obtained (810 mg, 74%); TC [petroleum ether/ethyl acetate 5:1]: Rf = 0.58.

1.32.c) p-nitrophenyl-2,4-di-O-benzyl-3-O-methyl-ß-D-galactopyranoside:

Compound 1.32.b (732 mg, 1.2 mmol) was dissolved in tetrahydrofuran and treated at 0°C with a 1 M solution of tetrabutylammonium fluoride in THF (2.4 ml). It is mixed for 40 minutes at room temperature and thickened in a vacuum. After vacuum chromatography [petroleum ether/ethyl acetate 5:1 → 3:1 → 2:1] colorless crystals are obtained (512 mg. 86%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.36; tal. = 177°C.

1.32.d) p-nitrophenyl-2,4-di-O-benzyl-3,6-di-O-methyl-ß-D-galacto-pyranoside:

The above compound (446 mg, 0.9 mmol) was methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 20:1 → 10:1 → 8:1] a colorless oil is obtained (401 mg, 87%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 070; [α]20 = -56.5° (c = 0.95 / CH2Cl2).

1.32) p-aminophenyl-3,6-di-O-methyl-ß-D-galactopyranoside:

Compound 1.32.d (357 mg, 0.7 mmol) was hydrogenated as described in Example 1.24. After evaporation of the combined filtrates in vacuo and boiling of the residue with diethyl ether (20 ml), colorless crystals (207 mg, 99%) were obtained; TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.02; tal. = >280°C (decomposition).

Example 1.33

p-aminophenyl-4,6-di-O-methyl-ß-D-galactopyranoside

[image]

1.33.a) p-nitrophenyl-2,3-di-O-benzyl-4,6-di-O-methyl-ß-D-galacto-pyranoside:

Compound 1.26.d (2.4 g, 5 mmol) is methylated as described in example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 5:1 → 3:1] colorless crystals are obtained (1.89 g, 74%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.76; tal. = 100°C.

1.33) p-aminophenyl-4,6-di-O-methyl-ß-D-galactopyranoside:

Compound 1.33.a (1.53 g, 3 mmol) was hydrogenated as described in Example 1.24. Colorless crystals are obtained (890 mg, 99%); TC [methanol/ethyl acetate 1:1]: Rf = 0.71; tal. = 180°C (decomposition).

Example 1.34

p-Aminophenyl-2,3,4-tri-O-methyl-ß-D-galactopyranoside

[image]

1.34.a) p-nitrophenyl-2,3,4-tri-O-methyl-6-O-triphenylmethyl-ß-D-galacto-pyranoside:

Compound 1.24.a (1.63 g, 3 mmol) is methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 5:1 → 3:1] a colorless foam is obtained (1.24 g, 71%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.54; [α]20 = -53.6° (c = 0.3 / CH3OH).

1-34.b) p-nitrophenyl-2,3,4-tri-O-methyl-ß-D-galacto-pyranoside:

The above compound is converted as described in example 1.24.d. After vacuum chromatography [petroleum ether/ethyl acetate 3:1 → 2:1] colorless crystals are obtained (468 mg, 68%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.12; [α]20 = -68.2° (c = 0.47 / CH3OH); tal. = 104°C.

1.34) p-aminophenyl-2,3,4-tri-O-methyl-ß-D-galactopyranoside:

Compound 1.34.b (343 mg, 1 mmol) is reduced as described in example 1.24. Beige crystals are obtained (224 mg, 71%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.67; tal. = 138°C.

[image]

1.35.a) p-nitrophenyl-2,3,6-tri-O-methyl-ß-D-galactopyranoside:

Compound 1.30.b (2.63 g, 3 mmol) is selectively methylated as described in example 1.25.a. After vacuum chromatography [petroleum ether/ethyl acetate 5:1 → 2:1] a brownish oil is obtained (890 mg, 32%); TC [ethyl acetate]; Rf = 0.37; [α]20 = -63.3° (c = 0.9 / CH2Cl2).

1.35) p-aminophenyl-2,3,6-tri-O-methyl-ß-D-galactopyranoside:

The above compound (858 mg, 2.5 mmol) is reduced as described in Example 1.24. A beige foam is obtained (519 mg, 66%); TC [ethyl acetate]: Rf = 0.23; [α]20 = -34.5° (c = 0.86 / CH3OH).

Example 1.36

p-aminophenyl-2,4,6-tri-O-methyl-ß-D-galactopyranoside

[image]

1.36.a) p-nitrophenyl-2,4,6-tri-O-methyl-3O-benzyl-ß-D-galactopyranoside:

Compound 1.26.a (1.96 g, 5 mmol) is methylated as described in example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 8:1] colorless crystals are obtained (1.47 g, 68%); TC [petroleum ether/ethyl acetate 2:1]: Rf=0.46; m.p.=164°C.

1.36) p-aminophenyl-2,4,6-tri-O-methyl-ß-D-galactopyranoside:

The above compound (1.3 g, 3 mmol) is reduced as described in Example 1.26. Colorless crystals are obtained (642 mg, 68%); TC [ethyl acetate/petroleum ether 2:1]: Rf = 0.12; tal. = 147°C (decomposition).

Example 1.37

p-Aminophenyl-3,4,6-tri-O-methyl-ß-D-galactopyranoside

[image]

1.37.a) p-nitrophenyl-2-O-benzyl-ß-D-galactopyranoside:

Fraction 1 from example 1.27.a (1.17 g. 2 mmol) is converted as described in example 1.24.d. After vacuum chromatography [petroleum ether/ethyl acetate 3:1 → 2:1] colorless crystals are obtained (468 mg, 68%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.12; tal. = 104°C; [α]20 = -68.2° (c = 0.47 / CH3OH).

1.37.b) p-nitrophenyl-2-O-benzyl-3,4,6-tri-O-methyl-ß-D-galactopyranoside:

The above compound (391 mg, 1 mmol) was methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 5:1] light yellow crystals are obtained (303 mg, 70%); TC [ethyl acetate]: Rf = 0.81; [α]20 = -76.5° (c = 1.1 / CH2Cl2).

1.37) p-aminophenyl-3,4,6-tri-O-methyl-ß-D-galactopyranoside:

Compound 1.37.b (260 mg, 0.6 mmol) is hydrogenated as described in Example 1.24. Beige crystals are obtained (161 mg, 86%); TC [ethyl acetate]: Rf = 0.20; tal. = 132°C.

Example 1.38

p-aminophenyl-2,3,4,6-tetra-O-methyl-ß-D-galactopyranoside

[image]

1.38.a) p-nitrophenyl-2,3,4,6-tetra-O-methyl-ß-D-galactopyranoside:

p-Nitrophenyl-ß-D-galactopyranoside (904 mg, 3 mmol) was methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 8:1 → 6:1 → 4:1 → 2:1] a colorless wax-like solid is obtained (633 mg, 59%); TC [ethyl acetate]: Rf = 0.67; [α]20 = -55.7° (c = 0.9 / CH2Cl2).

1.38) p-aminophenyl-2,3,4,6-tetra-O-methyl-ß-D-galactopyranoside:

Compound 1.33a (536 mg, 1.5 mmol) was hydrogenated as described in Example 1.24. After evaporation of the combined filtrates in vacuo and boiling of the residue with diethyl ether (20 ml), colorless crystals were obtained (412 mg, 84%); TC [ethyl acetate]: Rf = 0.42; tal. = 204°C (decomposition).

Example 1.39

p-aminophenyl-α-D-mannopyranoside

[image]

p-Nitrophenyl-α-D-mannopyranoside (3.0 g, 10 mmol) was hydrogenated as described in Example 1.23. Precipitation from a methanol/ether mixture gave colorless crystals (2.03 g, 75%); TC [methanol]: Rf = 0.69; [α]20 = +102.7° (c=1.0/H2O); m.p.=161°C.

Example 1.40

p-aminophenyl-3-O-methyl-α-D-mannopyranoside

[image]

1.40. a) p-nitrophenyl-6-O-triphenylmethyl-α-D-mannopyranoside:

p-Nitrophenyl-α-D-mannopyranoside (3.0 g, 10 mmol) was tritylated as described in Example 1.24.a. Colorless crystals are obtained (4.35 g, 80%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.52; [α]20 = +104.0° (c = 1.0 / CH3OH); tal. = 102-104°C.

1.40.b) p-nitrophenyl-3-O-methyl-6-O-triphenylmethyl-α-D-mannopyranoside:

The above compound (2.72 g, 5 mmol) was converted by addition of methyl iodide (2 ml, 30 mmol) as described in example 1.26.a. After vacuum chromatography [petroleum ether/ethyl acetate 2:1] and precipitation from an ethanol/n-hexane mixture, colorless crystals are obtained (1.83 g, 66%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.68; [α]20 = +106.4° (c = 1.0 / CH3OH); tal. = 104°C.

1.40) p-aminophenyl-3-O-methyl-α-D-mannopyranoside:

Compound 1.40.b (1.4 g, 2.5 mmol) was dissolved in methanol (50 ml) and after the addition of palladium on activated carbon (10 %, 300 mg) hydrin was added for 24 hours in a hydrogen atmosphere under a slight overpressure. The suspension is filtered through celite and the precipitate is thoroughly washed with methanol (100 ml). After concentrating the filtrate in vacuum, the residue is extracted with water (50 ml), filtered and the filtrate is lipophilized. A brownish amorphous solid is obtained (709 mg, 99%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.33; [α]20 = +92.9° (c = 1.1 / CH3OH).

Example 1.41

p-aminophenyl-2,3-di-O-methyl-α-D-mannopyranoside

[image]

1.41.a) p-nitrophenyl-4,6-O-benzylidene-α-D-mannopyranoside:

A solution of p-nitrophenyl-α-D-mannopyranoside (6.0 g, 20 mmol) in dimethylformamide (120 ml) was treated with benzaldehyde-dimethylacetal (3.2 ml, 21.4 mmol) and a 54% solution of tetrafluoroboric acid in diethyl ether (2.7 ml, 20 mmol). It is stirred for 5 hours at room temperature, then the reaction is stopped by the addition of triethylamine (2.8 ml, 20 mmol), and it is concentrated in a vacuum. After vacuum chromatography [toluene → toluene/ethanol 20:1] colorless crystals are obtained (6.48 g, 83%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.82; [α]20 = +170.7° (c = 1.0 / CH2Cl2); m.p.=116°C.

1.41.b) p-nitrophenyl-2,3-di-O-methyl-4,6-O-benzylidene-α-D-manno-pyranoside:

The above compound (3.9 g, 10 mmol) was methylated as described in Example 1.24.c. After vacuum chromatography [petroleum ether/ethyl acetate 20:1 → 7:1] and precipitation from a mixture of ethyl acetate/n-hexane, a colorless foam is obtained (3.2 g, 77%); TC [ethyl acetate/petroleum ether 2:1]: Rf = 0.67; [α]20 = +167.3° (c = 1.05/CH3OH).

1.41) p-aminophenyl-2,3-di-O-methyl-α-D-mannopyranoside:

Compound 1.41.b (1.25 g, 3 mmol) is hydrogenated as described in example 1.26. After vacuum chromatography [ethyl acetate/petroleum ether 2:1 → ethyl acetate, always with 0.5% triethylamine], a reddish-brown foam is obtained (480 mg, 53%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.31; [α]20 = +83.6° (c = 0.76 / CH3OH).

Example 1.42

p-aminophenyl-3-O-methoxycarbonylmethyl-ß-D-galactopyranoside

[image]

1.42.a) p-nitrophenyl-3-O-methoxycarbonylmethyl-ß-D-galactopyranoside:

A solution of p-nitrophenyl-ß-D-galactopyranoside (7.53 g, 25 mmol) in absolute dioxane (180 ml) was treated with dibutyltin oxide (9.3 g, 37.5 mmol) and heated under reflux. After 4 hours, the resulting solution is treated with bromoacetic acid methyl ester (8.3 ml, 90 mmol) and tetrabutylammonium iodide (9.25 g, 25 mmol), and stirred for a further 3 hours under reflux. The solvent is then distilled off under vacuum and the residue is purified by vacuum chromatography [dichloromethane/methanol 50:1 → 20:1]. Along with several side products, the compound 1.42.a is obtained in the form of colorless crystals (4.05 g, 43 %); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.54; [α]20 = -62.0° (c = 1.0 / CH3OH); tal. = 176°C.

1.42) p-aminophenyl-3-O-methoxycarbonylmethyl-ß-D-galactopyranoside:

Compound 1.42.a (3.73 g, 10 mmol) was hydrogenated as described in Example 1.24. After precipitation from the ethanol/n-hexane mixture, colorless crystals are obtained (2.98 g, 87%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.39; [α]20 = -36.3° (c = 1.07 / CH3OH); tal. = 155°C.

Example 1.43

p-aminophenyl-3-O-carboxymethyl-ß-D-galactopyranoside, sodium salt:

[image]

A solution of compound 1.42.a (3.73 g, 10 mmol) in methanol (100 ml) was treated with a solution of sodium hydroxide (400 mg, 10 mmol) in water (5 ml) and stirred for 2 hours at room temperature. After thickening in a vacuum, the residue is dried for 2 hours in an oil pump vacuum, and then converted with ethanol (100 ml). It is boiled for 5 minutes with reflux, filtered after cooling in an ice bath, and colorless crystals are obtained (3.66 g, 96%); TC [methanol]: Rf = 0.62; [α]20 = -50.0° (c = 1.0 / CH3OH); tal. = 180-185°C.

1.43) p-aminophenyl-3-O-carboxymethyl-ß-D-galactopyranoside, sodium salt:

The above compound (3.05 g, 8 mmol) was hydrogenated as described in Example 1.24. After boiling with ethanol (50 ml), colorless crystals are obtained (2.03 g, 72%); TC [methanol]: Rf = 0.70; [α]20 = -22.4° (c = 1.0 / CH3OH); tal. = 180-182°C.

Example 1.44

p-aminophenyl-3-O-carbamoylmethyl-ß-D-galactopyranoside

[image]

1.44.a) p-nitrophenyl-3-O-carbamoylmethyl-ß-D-galactopyranoside:

A solution of compound 1.42.a (373 mg, 1 mmol) in methanol (30 ml) was treated with 25% ammonia solution (10 ml) and stirred for 15 minutes at room temperature. After thickening in a vacuum, the residue is dried for 2 hours in an oil pump vacuum. and then converted with ethanol (30 ml). It is boiled for 5 minutes with reflux, filtered after cooling in an ice bath, and colorless crystals are obtained (306 mg, 85%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.14; [α]20 = -41.7° (c = 1.0 / CH3OH); tal. = 229°C (decomposition).

1.44) p-aminophenyl-3-O-carbamoylmethyl-ß-D-galactopyranoside:

The above compound (287 mg, 0.8 mmol) was hydrogenated as described in Example 1.24. After precipitation from a mixture of methanol/diethyl ether, colorless crystals are obtained (207 mg, 79%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.10; tal. = 205°C (decomposition).

Example 1.45

p-aminophenyl-3-O-(N-methyl-carbamoylmethyl)-ß-D-galactopyranoside

[image]

1.45.a) p-nitrophenyl-3-O-(N-methyl-carbamoylmethyl)-ß-D-galacto-pyranoside:

A solution of compound 1.42.a (373 mg, 1 mmol) in methanol (30 ml) was treated with a 30% aqueous methylamine solution (10 ml) and stirred for 2 hours at room temperature. After thickening in a vacuum, the residue is dried for 2 hours in the vacuum of an oil pump, and then recrystallized from ethanol. Colorless crystals are obtained (372 mg, 100%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.33; [α]20 = -36.7° (c = 1.0 / CH3OH); tal. = 205°C.

1.45) p-aminophenyl-3-O-(N-methyl-carbamoylmethyl)-ß-D-galacto-pyranoside:

Compound 1.45.a (298 mg. 0.8 mmol) is hydrogenated as described in example 1.24. After precipitation from a mixture of methanol/diethyl ether, colorless crystals are obtained (180 mg, 66%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.16; tal. = 239°C.

Example 1.46

p-aminophenyl-3-O-(N-propyl-carbamoylmethyl)-ß-D-galactopyranoside

[image]

1.46.a) p-nitrophenyl-3-O-(N-propyl-carbamoylmethyl)-ß-D-galacto-pyranoside

Compound 1.42.a (373 mg, 1 mmol) was treated with n-propylamine (823 μl, 10 mmol), as described in Example 1.45.a. After concentration in a vacuum, the residue is recrystallized from an ethanol/n-hexane mixture. Colorless crystals are obtained (340 mg, 85%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.49; [α]20 = -32.4° (c = 1.0 / CH3OH); tal. = 155°C.

1.46) p-aminophenyl-3-O-(N-propyl-carbamoylmethyl)-ß-D-galacto-pyranoside:

Compound 1.46.a (320 mg, 0.8 mmol) was hydrogenated as described in Example 1.24. After precipitation from a mixture of methanol/diethylether, colorless crystals are obtained (188 mg. 63%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.31; tal. = 154°C.

Example 1.47

p-aminophenyl-3-O-(N-butyl-carbamoylmethyl)-ß-D-galactopyranoside

[image]

1.47.a) p-nitrophenyl-3-O-(N-butyl-carbamoylmethyl)-ß-D-galacto-pyranoside:

Compound 1.42.a (373 mg, 1 mmol) was treated with n-butylamine (900 μl, 10 mmol), as described in Example 1.45.a. After concentration in vacuo, the residue is recrystallized from an ethanol/n-hexane mixture. Colorless crystals are obtained (413 mg, 100%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.51; [α]20 = -26.8° (c = 1.0 / CH3OH); tal. = 92°C.

1.47) p-aminophenyl-3-O-(N-butyl-carbamoylmethyl)-ß-D-galacto-pyranoside:

Compound 1.47.a (332 mg, 0.8 mmol) was hydrogenated as described in Example 1.24. After precipitation from a mixture of methanol/diethyl ether, colorless crystals are obtained (105 mg, 34%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.32; tal. = 135°C.

Example 1.48

p-aminophenyl-3-O-methoxycarbonylmethyl-α-D-mannopyranoside

[image]

1.48.a) p-nitrophenyl-3-O-methoxycarbonylmethyl-6-O-triphenylmethyl-α-D-mannopyranoside:

Compound 1.40.a (13.6 g, 25 mmol) was converted as described in Example 1.42.a. After vacuum chromatography [dichloromethane/methanol 10:1], colorless crystals (2.79 g, 18 %) were obtained along with several side products; TC [ethyl acetate/petroleum ether 2:1]: Rf = 0.50; tal. = 95-97°C.

1.48) p-aminophenyl-3-O-methoxycarbonylmethyl-α-D-mannopyranoside:

Compound 1.48.a (1.23 g, 2 mmol) was hydrogenated and worked up as described in Example 1.40. A brownish amorphous solid is obtained (250 mg, 36%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.45.

Example 1.49

p-aminophenyl-3-O-carboxymethyl-α-D-mannopyranoside

[image]

1.49.a) p-nitrophenyl-3-O-benzoxycarbonylmethyl-6-O-triphenylmethyl-α-D-mannopyranoside:

Compound 1.40.a (13.6 g, 25 mmol) was treated with bromoacetic acid benzyl ester (14.4 ml, 90 mmol), as described in Example 1.42.a. After vacuum chromatography [petroleum ether/ethyl acetate 20:1 → 10:1], a yellowish foam (5.0 g, 29%) was obtained along with several side products; TC [ethyl acetate/petroleum ether 2:1]: Rf = 0.66; [α]20 = +74.8° (c = 1.0 / CH2Cl2).

1.49) p-aminophenyl-3-O-carboxymethyl-α-D-mannopyranoside:

The above compound (2.08 g, 3 mmol) was hydrogenated over 36 hours as described in Example 1.40. After thickening the filtrate, the residue is recrystallized from an ethanol/n-hexane mixture. Colorless crystals (495 mg, 50%) were obtained by washing with ethyl acetate and recrystallization from an ethanol/diethyl ether mixture; TC [methanol]: Rf = 0.53; tal. = 205-207°C.

Example 1.50

p-aminophenyl-3-O-carbamoylmethyl-α-D-mannopyranoside

[image]

1.50.a) p-nitrophenyl-3-O-carbamoylmethyl-6-O-triphenylmethyl-α-D-mannopyranoside:

Compound 1.49.a (1.04 g, 1.5 mmol) was converted as described in Example 1.44a. After drying in the vacuum of the oil pump, the residue is purified by vacuum chromatography [petroleum ether/ethyl acetate 2:3]. Colorless crystals are obtained (561 mg, 62%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.67; [α]20 = +91.3° (c = 1.0 / CH2Cl2); tal. = 125-127°C.

1.50) p-aminophenyl-3-O-carbamoylmethyl-α-D-mannopyranoside:

The above compound (541 mg, 0.9 mmol) was hydrogenated as described in Example 1.40 over 48 hours. After concentration of the filtrate, the residue is thoroughly washed with methanol and colorless crystals are obtained (134 mg, 45%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.21; tal. = 126-128°C.

Example 1.51

p-aminophenyl-3-deoxy-ß-D-galactopyranoside

[image]

1.51.a) p-nitrophenyl-2,6-di-O-benzyl-4-O-acetyl-ß-D-galactopyranoside:

A solution of compound 1.31.b (2.7 g, 5.6 mmol) in dichloromethane (20 ml) was treated with triethylorthoacetate (3 ml, 16.3 mmol) and toluenesulfonic acid (20 mg). After 30 minutes at room temperature, the mixture is diluted with dichloromethane (200 ml), washed with saturated sodium hydrogen carbonate solution (50 ml), the organic phase is dried over magnesium sulfate and concentrated after vacuum filtration. The resulting colorless foam is dissolved in 80% acetic acid (15 ml). After a further 30 minutes at room temperature, the mixture is poured into a saturated solution of sodium bicarbonate (200 ml), extracted with chloroform (3 x 75 ml), the combined organic phases are washed with water (100 ml), dried over magnesium sulfate and concentrated after filtration under vacuum . Colorless crystals (2.43 g, 83%) were obtained by precipitation from an ethanol/petroleum ether mixture; TC [ethyl acetate/petroleum ether 2:1]: Rf = 0.64; [α]20 = -62.1° (c = 1.0 / CH2Cl2); tal. = 83°C.

1.51. b) p-nitrophenyl-2,6-di-O-benzyl-3-O-trifluoromethanesulfonyl-4-O-acetyl-ß-D-galactopyranoside:

A solution of compound 1.51.a (2.3 g, 4.4 mmol) in a mixture of dichloromethane (30 ml) and pyridine (3 ml) was converted under argon at -20°C with a solution of trifluoromethanesulfonic acid anhydride (2 ml, 11.8 mmol) in dichloromethane (30 ml). After 1 hour at -20 °C, the mixture is poured into a saturated solution of sodium bicarbonate (200 ml), the organic phase is separated, dried over magnesium sulfate and concentrated after filtration under vacuum. After vacuum chromatography [toluene → toluene/ethyl acetate 20:1], colorless crystals are obtained (2.39 g, 83%); TC [toluene/ethyl acetate 5:1]: Rf = 0.55; [α]20 = -61.4° (c = 1.0 / CH2Cl2); tal. = 105°C.

1.51.c) p-nitrophenyl-2,6-di-O-benzyl-3-deoxy-ß-D-galactopyranoside:

The above compound (1.31 g, 2 mmol) was dissolved in toluene (25 ml) and treated with tetrabutylammonium tetraborate (1.54 g, 6 mmol). After 2 hours at 80°C, the mixture is diluted with dichloromethane (200 ml), washed once with water (50 ml), the organic phase is dried over magnesium sulfate and concentrated after vacuum filtration. After vacuum chromatography [toluene/ethyl acetate 7:1] colorless crystals are obtained (596 mg, 64%); TC [toluene/ethyl acetate 5:1]: Rf = 0.10; [α]20 = -81.3° (c = 1.0 / CH2Cl2); tal. = 114°C.

1.51) p-aminophenyl-3-deoxy-ß-D-galactopyranoside:

Compound 1.51.C (465 mg, 1 mmol) was hydrogenated over 6 hours as described in Example 1.40. After thickening the filtrate, the residue was precipitated from the ethanol/petroleum ether mixture and colorless crystals were obtained (206 mg, 81%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.22.

Example 1.52

p-Aminophenyl-3,4-dideoxy-ß-D-galactopyranoside

[image]

1.52.a) p-nitrophenyl-2,6-di-O-benzyl-3,4-di-O-trifluoromethanesulfonyl-ß-D-galactopyranoside:

Compound 1.31.b (2.12 g, 4.4 mmol) was converted with trifluoromethanesulfonic acid anhydride (4 ml, 23.6 mmol) as described in example 1.51.b. After vacuum chromatography [toluene → toluene/ethyl acetate 50:1] a yellowish oil is obtained (2.75 g, 84%); TC [toluene/ethyl acetate 5:1]: Rf = 0.67; [α]20 = -22.5° (c = 1.0 / CH2Cl2).

1.52.b) p-nitrophenyl-2,6-di-O-benzyl-3,4-dideoxy-ß-D-galacto-pyranoside:

Compound 1.52.a (1.49 g, 2 mmol) was treated with tetrabutylammonium tetraborate (2.31 g, 9 mmol) as described in Example 1.51.C. After vacuum chromatography [toluene → toluene/ethyl acetate 50:1], colorless crystals are obtained (629 mg, 70%); TC [toluene/ethyl acetate 5:1]: Rf = 0.53; [α]20 = -79.1° (c = 1.0 / CH2Cl2); tal. = 89°C.

1.52) p-aminophenyl-3,4-dideoxy-ß-D-galactopyranoside:

Compound 1.52.b (450 mg, 1 mmol) was hydrogenated over 5 hours as described in Example 1.40. After thickening the filtrate, the residue was precipitated from the ethanol/petroleum ether mixture and colorless crystals were obtained (183 mg, 76%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.47; [α]20 = -115.1° (c = 1.0 / CH3OH); tal. = 187°C.

Example 1.53

p-aminophenyl-6-O-acetyl-ß-D-galactopyranoside

[image]

1.53.a) p-nitrophenyl-6-O-acetyl-ß-D-galactopyranoside:

A solution of p-nitrophenyl-ß-D-galactopyranoside (7.53 g, 25 mmol) in absolute acetonitrile (80 ml) was converted by dropwise addition of a freshly prepared solution of pyridine (2 ml, 25 mmol) and acetyl chloride (1.85 ml, 26 mmol). in acetonitrile (20 ml). It is mixed for 30 minutes at 0°C and then thickened in a vacuum. After vacuum chromatography [dichloromethane/methanol 50:1 → 20:1], colorless crystals are obtained (4.02 g, 47%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.50.

1.53) p-aminophenyl-6-O-acetyl-ß-D-galactopyranoside:

Compound 1.53.a (1.72 g, 5 mmol) was hydrogenated over 2 hours as described in Example 1.41. After thickening the filtrate, the residue was precipitated from the ethanol/petroleum ether mixture and colorless crystals were obtained (1.34 g, 86%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.34; [α]20 -41.0° (c = 0.56 / CH2OH); tal. = 180°C (decomposition).

Example 1.54

p-aminophenyl-3,4-di-O-methoxycarbonylmethyl-ß-D-galactopyranoside

[image]

1.54.a) Acylation of compound 1.24.a:

Compound 1.24.a (1.36 g, 3 mmol) was dissolved in dimethylformamide (25 ml) and converted by addition of bromoacetic acid methyl ester (1 ml, 10.6 mmol) and portions of an 80% suspension of sodium hydride in mineral oil (300 mg, 10 mmol). After 3.5 hours at room temperature, bromoacetic acid methyl ester (250 μl, 2.65 mmol) and sodium hydride in mineral oil (75 mg, 2.5 mmol) are added again. After a further 2 hours, the reaction is finished by adding methanol (5 ml) dropwise and concentrated in vacuo. The residue was taken up in dichloromethane (250 ml) and the solution was vigorously mixed with water (100 ml). The organic phase is dried over magnesium sulfate (10 g), concentrated in vacuo and purified by vacuum chromatography [petroleum ether/ethyl acetate 10:1 → 7:1 → 5:1 → 3:1]. Three product fractions are obtained:

Fraction 1: p-nitrophenyl-2,3,4-tri-O-methoxycarbonylmethyl-ß-D-galacto-pyranoside; colorless foam (401 mg, 21%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.47; [α]20 = -51.9° (c = 0.26 / CH3OH); tal. = 104°C.

Fraction 2: not identified; colorless foam (88 mg); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.39; [α]20 = -61.5° (c = 0.26 / CH3OH).

Fraction 3: p-nitrophenyl-3,4-di-O-methoxycarbonylmethyl-ß-D-galacto-pyranoside; colorless foam (275 mg, 16%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.30; [α]20 = -38.6° (c = 0.28 / CH3OH).

1.54) p-aminophenyl-3,4-di-O-methoxycarbonylmethyl-ß-D-galacto-pyranoside:

Fraction 3 from Example 1.54.a (206 mg, 0.3 mmol) was hydrogenated for 16 hours as described in Example 1.40. After concentration of the filtrate, the residue is boiled with diethyl ether (20 ml) and gray crystals are obtained (45.6 mg, 37%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.22; tal. = 155°C (decomposition).

Example 1.55

p-aminophenyl-2,3,4-tri-O-methoxycarbonylmethyl-ß-D-galactopyranoside

[image]

Fraction 1 from Example 1.54.a (380 mg, 0.5 mmol) was hydrogenated for 16 hours as described in Example 1.40. After concentration of the filtrate, the residue is boiled with diethyl ether (20 ml) and a tan oil is obtained (46.8 mg, 19%); TC [petroleum ether/ethyl acetate 1:1]: Rf = 0.29; tal. = 106°C (decomposition).

Example 1.56

p-aminophenyl-4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranoside

[image]

p-Nitrophenyl-4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranoside (4.63 g, 10 mmol) was hydrogenated as described in Example 1.23. Colorless crystals are obtained (3.04 g, 70%); TC [methanol]: Rf = 0.55; tal. = 235-237°C (decomposition).

p-aminophenyl-4-O-(3'-sulfate-ß-D-galactopyranosyl)-ß-D-glucopyranoside, sodium salt

[image]

1.57.a) p-nitrophenyl-4-O-(3',4'-O-isopropylidene-ß-D-galactopyranosyl)-ß-D-glucopyranoside:

p-Nitrophenyl-4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranoside (23.2 g, 50 mmol) was treated with dimethoxypropane (400 ml) and a catalytic amount of (±)-camphor-10-sulfonic acid (400 mg, 1.7 mmol). After 3 days at room temperature, the reaction is finished by the addition of triethylamine (240 yl, 1.7 mmol), concentrated in a vacuum and dried for 2 hours in an oil pump vacuum. The resulting crystals are taken up in a mixture of methanol/water 10:1 (500 ml) and boiled for 6 hours with reflux. After concentration in vacuum and vacuum chromatography [dichloromethane/methanol 25:1 → 10:1, always with 0.5% triethylamine], colorless crystals are obtained (15.2 g, 60%); TC [dichloromethane/methanol 5:1]: Rf = 0.49; tal. = 253-255°C (decomposition).

1.57.b) p-nitrophenyl-2,3,6-tri-O-benzoyl-4-O-(2',6'-di-O-benzoyl-3',4'-O-isopropylidene-ß -D -galactopyranosyl)-ß -D-glucopyranoside:

To a solution of compound 1.57.a (15.1 g, 30 mmol) in pyridine (300 ml) was slowly added dropwise benzoyl chloride (50 ml, 430 mmol) at 0°C over 30 minutes. It is then stirred for a further 2 hours at room temperature, and the mixture is poured into ice water (2000 ml) while stirring. After 15 minutes, the formed crystals are filtered off and taken up in dichloromethane (1500 ml). The solution is washed with water (2 x 500 ml) and 1 M sodium hydrogen carbonate solution (2 x 500 ml), the organic phase is dried over magnesium sulfate (50 g), concentrated in vacuo and purified by recrystallization from methanol. Colorless crystals are obtained (26.7 g, 87%); TC [dichloromethane/methanol 50:1]: Rf = 0.49; [α]20 = +23.6° (c = 1.08 / CH2Cl2); tal. = 272-274°C.

1.57.c) p-nitrophenyl-2,3,6-tri-O-benzoyl-4-O-(2',6'-di-O-benzoyl-ß-D-galactopyranosyl)-ß-D-glucopyranoside:

A solution of compound 1.57.b (20.5 g, 20 mmol) in dichloromethane (400 ml) was treated with 99% trifluoroacetic acid (20 ml) and stirred at room temperature for 20 minutes. The solution is then washed with 1 M sodium bicarbonate solution (2 x 200 ml), the organic phase is dried over magnesium sulfate (10 g), concentrated in a vacuum and the residue is purified by precipitation from a dichloromethane/diethyl ether mixture. Colorless crystals are obtained (18.0 g, 91%); TC [dichloromethane/methanol 20:1]: Rf = 0.18; tal. = 234°C.

1.57.d) p-nitrophenyl-2,3,6-tri-O-benzoyl-4-O-(2',6'-di-O-benzoyl-4'-O-acetyl-ß-D-galactopyranosyl) -ß-D-glucopyranoside:

Compound 1.57.c (5.5 g, 5.6 mmol) was converted as described in Example 1.51.a. After vacuum chromatography [petroleum ether/ethyl acetate 3:1 → 1:1] colorless crystals are obtained (4.03 g, 70%); TC [dichloromethane/methanol 20:1]: Rf=0.67; tal. = 118°C.

1.57.e) p-nitrophenyl-2,3,6-tri-O-benzoyl-4-O-(2',6'-di-O-benzoyl-3'-sulfate-4'-O-acetyl-ß -D-galactopyranosyl)-ß-D-glucopyranoside, sodium salt:

A solution of compound 1.57.d (3.59 g, 3.5 mmol) in pyridine (200 ml) is converted with a sulfur trioxide - pyridine complex (4.5 g, 28 mmol), and it is first stirred for 2 hours at 60°C, and then for 16 hours at room temperature. The reaction is then terminated by adding methanol (50 ml) dropwise and concentrated in vacuo. The residue is purified by vacuum chromatography [dichloromethane/methanol 10:1]. A solid is obtained, which is taken up in a mixture of dichloromethane/methanol 1:1 (200 ml) and converted with Amberlite IR120 (Na+-form, 10 g). This mixture is stirred at room temperature, filtered, and the filtrate is concentrated in a vacuum. Colorless crystals are precipitated (3.64 g, 92%); TC [dichloromethane/methanol 2:1]: Rf = 0.87; tal. = 168°C.

1.57.f) p-nitrophenyl-4-O-(3'-sulfate-ß-D-galactopyranosyl)-ß-D-glucopyranoside, sodium salt:

Compound 1.57.e (3.4 g, 3 mmol) was dissolved in absolute methanol (150 ml), treated with sodium methylate (200 mg) and stirred for 7 hours at 60°C. After cooling to room temperature, it is neutralized with Lewatite SC108 (H+-form) and then filtered. The pH value of the filtrate is increased by dropwise addition of 1 M sodium alkali solution to pH 7-8, evaporated in a vacuum and precipitation from a mixture of methanol/diethyl ether gives light brownish crystals (1.30 g, 77%); TC [dichloromethane/methanol 2:1]: Rf = 0.55; tal. = 230°C (decomposition).

1.57) p-aminophenyl-4-O-(3'-sulfate-ß-D-galactopyranosyl)-ß-D-gluco-pyranoside, sodium salt:

The above compound (1.13 g, 2 mmol) is reduced as described in Example 1.24. After boiling with diethyl ether (50 ml) colorless crystals are obtained (983 mg, 92%); TC [dichloromethane/methanol 2:1]: Rf = 0.22; tal. = 176°C (decomposition).

Example 1.58

p-aminophenyl-4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-gluco-pyranoside

[image]

1.58 a) selective methylation of p-nitrophenyl-4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranoside:

p-Nitrophenyl-4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranoside (2.3 g, 5 mmol) was methylated as described in Example 1.25.a. After vacuum chromatography [dichloromethane/methanol 20:1 -> 10:1 → 5:1] two products are obtained:

Fraction 1: p-nitrophenyl-2-O-methyl-4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranoside; colorless crystals (264 mg, 11%); TC [dichloromethane/methanol 5:1]: Rf = 0.46; [α]20 = -73.9° (c = 1.0 / CH3OH); tal. = 228°C (decomposition).

Fraction 2: p-nitrophenyl-4-O-(3'-O-metal-ß-D-galactopyranosyl)-ß-D-gluco-pyranoside; colorless crystals (1.0 g, 42%); TC [dichloromethane/methanol 5:1]: Rf = 0.29; [α]20 = -65.3° (c = 1.1 / CH3OH); tal. = 220°C.

1.58) p-aminophenyl-4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-gluco-pyranoside:

Fraction 2 from Example 1.58 a (955 mg, 2 mmol) is reduced as described in Example 1.24. After washing with diethyl ether (50 ml), colorless crystals (894 mg, 100%) were obtained; TC [dichloromethane/methanol 5:1]: Rf = 0.08; m.p.= 129°C (decomposition).

Example 1.59

p-aminophenyl-2-O-methyl 4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D glucopyranoside

[image]

Fraction 1 from Example 1.58.a (246 mg, 0.5 mmol) is reduced as described in Example 1.24. After washing with diethyl ether (20 ml) colorless crystals are obtained (186 mg, 81%); TC [dichloromethane/methanol 5:1]: Rf = 0.13; [α]20 = -3.6° (c = 1.0 / CH3OH); tal. = 105°C.

Example 1.60

p-aminophenyl-4-O-(3',4'-di-O-methyl-ß-D-galactopyranosyl)-ß-D-gluco-pyranoside

[image]

1.60.a) p-nitrophenyl-2,3,6-tri-O-benzyl-4-O-(2',6'-di-O-benzyl-3',4'-O-isopropylidene-ß-D -galactopyranosyl)-ß-D-glucopyranoside:

Compound 1.57.a (5.0 g, 10 mmol) was treated with benzyl bromide (30 ml, 250 mmol) over 16 h, as described in Example 1.26.c. After concentration in vacuo, the residue is taken up in ethyl acetate (300 ml) and the solution is washed with water (200 ml). The organic phase is dried over magnesium sulfate (10 g), concentrated in vacuo and purified by vacuum chromatography [dichloromethane/petroleum ether 5:1 → dichloromethane]. A brownish oil is obtained (5.3 g, 56%); TC [dichloromethane/methanol 50:1]: Rf = 0.70; [α]20 = -23.2° (c = 1.08 / CH2Cl2)

1.60.b) p-nitrophenyl-2,3,6-tri-O-benzyl-4-O-(2',6'-di-O-benzyl-ß-D-galactopyranosyl)-ß-D-glucopyranoside:

The above compound (4.77 g, 5 mmol) was converted as described in Example 1.57.c. After concentration in vacuum and precipitation from a mixture of diethyl ether/petroleum ether, colorless crystals are obtained (3.94 g, 86%); TC [dichloromethane/methanol 50:1]: Rf = 0.36; tal. = 116°C.

1.60.c) p-nitrophenyl-2,3,6-tri-O-benzyl-4-O-(2',6'-di-O-benzyl-3',4'-di-O-methyl-ß -D-galactopyranosyl)-ß-D-glucopyranoside:

Compound 1.60.b (1.8 g, 2 mmol) is methylated as described in example 1.24.c. Colorless crystals (1.55 g, 82%) were obtained by precipitation from a dichloromethane/petroleum ether mixture; TC [dichloromethane/methanol 50:1]: Rf = 0.74; tal. = 161-162°C.

1-60) p-aminophenyl-4-O-(3',4'-di-O-methyl-ß-D-galactopyranosyl)-ß-D-gluco-pyranoside:

Compound 1.60.C (1.41 g, 1.5 mmol) is dissolved in methanol (50 ml) and after the addition of palladium hydroxide on charcoal (wet, 20%, 500 mg) it is hydrogenated for 6 days in a hydrogen atmosphere with a slight overpressure. The suspension is filtered through celite and the precipitate is thoroughly washed with methanol (100 ml). Concentration of the filtrate in vacuo and washing the residue with dichloromethane gave brownish crystals (425 mg, 61%); tal. = 124°C (decomposition).

Example 2.1

N-alanyl-batracillin

[image]

2.1.a) N-[N-(tert-butoxycarbonyl)-alanyl]-batracillin:

N-(tert-butoxycarbonyl)-alanine (3.3 g, 17.5 mmol) and 2-isobutoxy-1-isobutoxy-carbonyl-1,2-dihydro-quinoline (6.8 ml, 23 mmol) were dissolved in 100 ml of dichloromethane. After stirring for 20 minutes at room temperature, a solution of batracillin (4.1 g, 16.5 mmol) in absolute dimethylformamide (350 ml) was added and the mixture was stirred at room temperature for a further 48 hours. It is then concentrated in a vacuum to 50 ml, topped up with ethyl acetate up to 300 ml and immediately heated for 10 minutes until boiling. It is then left to cool to room temperature, filtered and the residue is boiled again with ethyl acetate (200 ml). Cooling to 0°C with stirring and filtering gave yellow crystals (6.18 g, 84%); TC [ethyl acetate]: Rf = 0.57; tal. = 246-247°C (decomposition).

2.1) N-alanyl-batracillin:

A solution of compound 2.1.a (10.5 g, 25 mmol) in anhydrous trifluoroacetic acid (150 ml) was stirred for 15 minutes at room temperature. After thickening in a vacuum to 30 ml, the mixture is poured into a saturated solution of sodium bicarbonate (1000 ml) with vigorous stirring. It is mixed for another 10 minutes, filtered and washed with water, a little isopropanol and diethyl ether. The product precipitates as yellow crystals (7.15 g, 89%); TC [ethyl acetate]: Rf = 0.06; tal. = 261-262°C (decomposition).

Example 2.2

N-[lysyl-alanyl]-batracillin, di-trifluoroacetate

[image]

2.2.a) N-[Nα, Nε]-di-(tert-butoxycarbonyl)-lysyl-alanyl]-batracillin:

N,N-di-(tert-butoxycarbonyl)-lysine (2.1 g, 6 mmol) and 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (2.4 ml, 8 mmol) were dissolved in 20 ml of dichloromethane. After stirring for 20 minutes at room temperature, a solution of compound 2.1 (1.6 g, 5 mmol) in dimethylformamide (40 ml) was added and the mixture was stirred for a further 16 hours at room temperature. It is then concentrated in a vacuum and the residue is purified by vacuum chromatography [petroleum ether/ethyl acetate 2:1 → 1:1 → ethyl acetate]. Yellow crystals are obtained (2.89 g, 89%); TC [ethyl acetate]: Rf = 0.52; tal. = 203-204°C.

2.2) N-[lysyl-alanyl]-batracillin, di-trifluoroacetate:

A suspension of the above compound (2.6 g, 4 mmol) in dichloromethane (25 ml) was treated with anhydrous trifluoroacetic acid (10 ml) and the resulting solution was stirred for 30 minutes at room temperature. After concentration in a vacuum, the residue crystallizes with the addition of diethyl ether (100 ml). The precipitate is filtered off and washed vigorously with diethyl ether. Yellow crystals are obtained (2.68 g, 99%); TC [ethyl acetate]: Rf = 0.05; tal. = 144-146°C (decomposition).

Example 2.3

N-[D-alanyl]-batracillin

[image]

2.3.a) N-[N-benzyloxycarbonyl-D-alanyl]-batracillin:

N-Benzyloxycarbonyl-D-alanine (3.9 g, 17.5 mmol) was converted and worked up as described in Example 2.1.a. The resulting yellow crystals (6.4 g. 80 %) are separated by filtration and the combined filtrates are purified by vacuum chromatography [petroleum ether/ethyl acetate 3:2 → 1:1] after concentration in a vacuum. A further 1.35 g (17%) is obtained; TC [ethyl acetate]: Rf= 0.45; tal. = 256°C; [α]20 = +75.1° (c = 1.0 / CH2Cl2 + 0.5 % CH3OH).

2.3) N-[D-alanyl]-batracillin:

Compound 2.3.a (11.4 g, 25 mmol) was dissolved in a 33% solution of hydrogen bromide in glacial acetic acid (100 ml). After 30 minutes at room temperature, the mixture is concentrated in a vacuum to 30 ml and then poured with vigorous stirring into a saturated solution of sodium hydrogencarbonate (1000 ml). It is mixed for another 10 minutes, filtered and washed with water, with a little isopropanol and diethyl ether. The product precipitates in yellow crystals (7.87 g, 98%); TC [ethyl acetate]: Rf = 0.06; tal. = 267°C (decomposition).

Example 2.4

N-[Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracillin

[image]

2.4.a) N-[Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracillin:

Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysine (5.3 g, 11.3 mmol) and 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (4 ml, 14 mmol) were dissolved in in 40 ml of dichloromethane. After stirring for 20 minutes at room temperature, a solution of compound 2.3 (3.2 g, 10 mmol) in dimethylformamide (80 ml) was added and the mixture was stirred for a further 16 hours at room temperature. It is then concentrated in vacuo and the residue is suspended in dichloromethane (100 ml). The resulting suspension is supplemented with diethyl ether (300 ml). After filtering and washing the residue with diethyl ether, yellow crystals are obtained (5.65 g, 65%); TC [ethyl acetate]: Rf = 0.45; tal. = 186°C (decomposition).

2.4) N-[Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracillin:

The above compound (5.6 g, 7.3 mmol) was dissolved in dimethylformamide (50 ml). After adding piperidine (50 ml), it is stirred for 3 hours at room temperature, then concentrated in a vacuum and the residue is purified by vacuum chromatography [dichloromethane/methanol/ammonia (25 %) 15:3-0.1 → 15:5:0.1]. Yellow crystals are obtained (2.5 g, 62%); tal. = 217°C (decomposition).

Example 2.5

N-[Nα-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracillin, trifluoroacetate

[image]

2.5.a) N-[Nα-(fluorenyl-9-methoxycarbonyl)-Nε-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracillin:

Nα-(fluorenyl-9-methoxycarbonyl)-Nε-(tert-butoxycarbonyl)-lysine (5.3 g, 11.3 mmol) was converted and purified as described in Example 2.4.a. Yellow crystals are obtained (7.0 g, 80%); TC [ethyl acetate]: Rf = 0.51; tal. = 223°C.

2.5) N-[Nα-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracillin, trifluoroacetate:

Compound 2.5.a (6.17 g, 8 mmol) was converted as described in Example 2.2. After concentration in a vacuum, the residue from the dichloromethane/diethylether mixture precipitates and yellow crystals are obtained (6.08 g, 97%); TC [ethyl acetate]: Rf = 0.05; tal. = 224°C (decomposition).

Example 2.6

N-[Nε-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-batracillin, trifluoroacetate

[image]

Compound 2.4.a (6.17 g, 8 mmol) was converted as described in Example 2.2. After concentration in a vacuum, the residue from the dichloromethane/diethyl ether mixture precipitates and yellow crystals are obtained (5.97 g, 95%); TC [ethyl acetate]: Rf = 0.04; tal. = 188°C (decomposition).

Example 2.7

N-[lysyl-D-asparagyl]-batracillin, di-trifluoroacetate

[image]

2.7.a) N-[N-(fluorenyl-9-methoxycarbonyl)-D-asparagyl-(ß-tert-butyl ester)]-batracillin:

N-(fluorenyl-9-methoxycarbonyl)-D-asparagyl-(ß-tert-butyl ester) (7.2 g, 17.5 mmol) was converted as described in Example 2.1. After concentration in vacuo, the residue is taken up in dichloromethane (1000 ml) and washed with 1 M hydrochloric acid solution (2 x 200 ml) and 1 M sodium hydrogen carbonate solution (1 x 200 ml). After drying over magnesium sulfate (20 g), filtering, concentrating to 100 ml and adding petroleum ether, compound 2.7.a precipitates in the form of yellow crystals (9.7 g, 86%); TC [ethyl acetate]: Rf = 0.71; tal. = 195°C.

2.7.b) N-[D-asparagyl-(ß-tert-butylester)]-batracillin:

The above compound (6.4 g, 10 mmol) was dissolved in dichloromethane (100 ml). After the addition of morpholine (50 ml), it is stirred for 5 hours at room temperature, then concentrated in a vacuum and the residue is purified by vacuum chromatography [ethyl acetate/petroleum ether 4:1 → ethyl acetate → ethyl acetate/ethanol 10:1]. Yellow crystals are obtained (3.44 g, 82%); TC [ethyl acetate]; Rf = 0.21; tal. = 209°C (decomposition).

2.7.c) N-[Nα,Nε-di-(tert-butoxycarbonyl)-lysyl-D-asparagyl-(ß-tert-butylester)]-batracillin:

Compound 2.7 b (2.1 g, 5 mmol) was converted as described in example 2.2.a. Yellow crystals are obtained (1.71 g, 46%); TC [ethyl acetate]: Rf = 0.61; tal. = 142°C.

2.7) N-(lysyl-D-asparagyl]-batracillin, di-trifluoroacetate:

Compound 2.7.C (1.65 g, 2.2 mmol) was converted and purified as described in Example 2.2. Yellow crystals are obtained (1.5 g. 95%); TC [methanol/acetic acid 10:1]: Rf = 0.29; tal. = 154-155°C (decomposition).

Example 2.8

N-[lysyl-D-glutamyl]-batracillin, dihydrobromide

[image]

2.8.a) N-[N-(tert-butoxycarbonyl)-D-glutamyl-(p-benzyl ester)]-batracillin:

N-(tert-butoxycarbonyl)-D-glutamyl-(ß-benzyl ester) (5.9 g, 17.5 mmol) was converted as described in Example 2.1.a. After concentration in vacuum, the residue is purified by vacuum chromatography [petroleum ether/ethyl acetate 1:1] to yellow crystals (9.45 g 95%); TC [ethyl acetate]: Rf = 0.61; [α]20 = +53.1° (c = 1.0 / CH2Cl2); tal. = 159°C.

2.8.b) N-[D-glutamyl-(p-benzyl ester)]-batracillin:

Compound 2.8.a (9.1 g, 10 mmol) was dissolved in formic acid and stirred for 6 hours at room temperature. After concentration in vacuo, the residue was taken up in methanol (100 ml) and the pH value of the solution was raised to pH 8 by careful addition of a 25% ammonium hydroxide solution. After re-concentration in vacuum and vacuum chromatography [ethyl acetate/ethanol 10:1] a yellow oil is obtained (4.2 g, 56%); TC [ethyl acetate] Rf = 0.06.

2.8.c) N-[Nα,Nε-di-(tert-butoxycarbonyl)-lysyl-D-glutamyl-(ß-benzyl ester)]-batracillin:

The above compound (3.75 g, 8 mmol) was converted and purified as described in Example 2.2.a. A yellow amorphous solid is obtained (2.26 g, 35%); TC [ethyl acetate]: Rf = 0.40; [α]20 = +32.1° (c = 1.2 / CH2Cl2).

2.8) N-[lysyl-D-glutamyl]-batracillin, dihydrobromide:

Compound 2.8.C (2.0 g, 2.5 mmol) was dissolved in a 33% solution of hydrogen bromide in glacial acetic acid (50 ml). After 1 hour at room temperature, the mixture is concentrated in a vacuum and the residue is thoroughly washed with diethyl ether. The product precipitates in yellow-red crystals (1.63 g, 98%); tal. = 207-209°C (decomposition).

Example 2.9

N-[lysyl-glycyl]-batracillin, di-trifluoroacetate

[image]

2.9.a) N-[N-(tert-butoxycarbonyl)-glycyl]-batracillin:

N-(tert-butoxycarbonyl)-glycyl (3.07 g, 17.5 mmol) was converted as described in Example 2.1.a. After 3 days at room temperature, it is concentrated in a vacuum and the residue is taken up in ethanol (200 ml). After 30 minutes of stirring with reflux, and filtration after cooling, the desired compound precipitates in the form of yellow crystals (4.73 g, 66%); TC [ethyl acetate]: Rf = 0.44; tal. = 279°C (decomposition).

2.9.b) N-glycyl-batracillin, hydrochloride:

The above compound (4.1g, 10 mmol) was dissolved in dichloromethane (1200 ml) while heating in an ultrasonic bath. After the addition of hydrogen chloride in diethyl ether (100 ml), it is stirred for 30 minutes at room temperature, concentrated in a vacuum and the residue converted with ethanol (200 ml). After 10 minutes of stirring with reflux, and filtering after cooling, the product precipitates in the form of yellow crystals (3.21 g, 94%); tal. = 297-299°C (decomposition).

2.9.c) N-[Nα,Nε-di-(tert-butoxycarbonyl)-lysyl-glycyl]-batracillin:

N,N-di-(tert-butoxycarbonyl)-lysine (2.1 g, 6 mmol) and 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-quinoline (2.4 ml, 8 mmol) were dissolved in 20 ml of dichloromethane. After stirring for 20 minutes at room temperature, a solution of compound 2.9.b (1.71 g, 5 mmol), ethyldiisopropylamine (0.86 ml, 5 mmol) and dimethylformamide (40 ml) was added, and the mixture was stirred for a further 16 hours at room temperature. It is then concentrated in a vacuum and purified by vacuum chromatography [ethyl acetate/petroleum ether 1:1 → ethyl acetate]. Yellow crystals are obtained (1.69 g, 53%); TC [ethyl acetate]; Rf = 0.31; tal. = 211°C (decomposition).

2.9) N-[lysyl-glycyl]-batracillin, di-trifluoroacetate:

Compound 2.9.C (1.4 g, 2.2 mmol) was converted and purified as described in Example 2.2. Yellow crystals are obtained (1.33 g, 91%); tal. = 153°C (decomposition).

Example 2.10

N-[lysyl-seryl]-batracillin, di-trifluoroacetate

[image]

2.10.a) N-[N-(tert-butoxycarbonyl)-seryl]-batracillin:

N-(tert-butoxycarbonyl)-serine (3.6 g, 17.5 mmol) was converted as described in Example 2.1.a. After 48 hours, it is concentrated in a vacuum to 100 ml and converted with 2 l of dichloromethane. The resulting solution is washed with water (1 x 500 ml), 0.5 M hydrochloric acid solution (2 x 250 ml) and saturated sodium bicarbonate solution (1 x 250 ml). Drying the residue over magnesium sulfate (50 g), removing the solvent by vacuum distillation, and vacuum chromatography [petroleum ether/ethyl acetate 1:1] gives compound 2.10.a (4.6 g, 64 %) in the form of yellow crystals; TC [ethyl acetate/acetic acid 100:1]: Rf = 0.38; tal. = 219°C (decomposition); [α]20 = -61.0° (c = 0.5 / CH2Cl2 + 0.5 % CH3OH).

2.10.b) N-seryl-batracillin, hydrochloride:

A suspension of the above compound (4.6 g, 10.4 mmol) in dioxane (70 ml) was stirred with concentrated hydrochloric acid (10 ml) and then stirred vigorously for 1 hour at room temperature. It is then condensed in a vacuum and the rest is dried for 2 hours in an oil pump vacuum. After the addition of ethanol (100 ml), it is boiled for 15 minutes with reverse cooling. Cooling and suction gave orange crystals (1.97 g, 96%); TC [ethyl acetate]: Rf = 0.05; [α]20 = +51.8° (c = 1.0 / H2O); melting point > 219°C (decomposition).

2.10.c) N-[Nα,Nε]di-(tert-butoxycarbonyl)-lysyl-seryl]-batracillin:

Compound 2.10.b (1.86 g, 5 mmol) was converted as described in Example 2.9.c. After vacuum chromatography [ethyl acetate/petroleum ether 2:1 → ethyl acetate], yellow crystals are obtained (1.18 g, 36%); TC [ethyl acetate/acetic acid 100:1]: Rf = 0.24; tal. = 188°C (decomposition); [α]20 = -13.1° (c = 0.5 / CH2Cl2 + 0.5 % CH3OH).

2.10) N-[lysyl-seryl]-batracillin, di-trifluoroacetate:

Compound 2.10.C (1.0 g, 1.5 mmol) was converted and purified as described in Example 2.2. Yellow crystals are obtained (1.0 g, 96%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.10; tal. = 188-190°C (decomposition).

Example 2.11

N-[lysyl-D-seryl]-batracillin, dihydrobromide

[image]

2.11.a) N-[N-(fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-seryl]-batracillin:

N-(fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-serine (6.7 g, 17.5 mmol) was converted as described in Example 2.1.a. Concentration in vacuum and vacuum chromatography [petroleum ether/ethyl acetate 1:1] gives compound 2.11.a (5.64 g, 52 %) in the form of yellow crystals; TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.87; tal. = 225-226°C (decomposition).

2.11.b) N-[O-(tert-butyl)-D-seryl]-batracillin:

The above compound (2.89 g, 4.7 mmol) was converted as described in example 2.7.b. Yellow crystals (1.15 g, 62%) were obtained by vacuum chromatography [petroleum ether/ethyl acetate/ 3:2 → ethyl acetate]; TC [ethyl acetate]: Rf = 0.11; tal. = 197°C.

2.11.c) N-[Nα,Nε-di-(tert-butoxycarbonyl)-lysyl-O-(tert-butyl)-D-seryl]-batracillin:

The above compound (1.1 g, 2.8 mmol) was converted as described in Example 2.2.a. Yellow crystals are obtained (1.94 g, 96%); TC [ethyl acetate]: Rf = 0.59; tal. = 208°C.

2.11.d) N-(lysyl-O-(tert-butyl)-D-seryl]-batracillin, di-trifluoroacetate:

Compound 2.11.C (1.08 g, 1.5 mmol) was converted and worked up as described in Example 2.2. Yellow crystals are obtained (1.1 g, 98%); TC [methanol/acetic acid 10:1]: Rf = 0.30; tal. = 128°C.

2.11) N-[lysyl-D-seryl]-batracillin, dihydrobromide:

Compound 2.11.d (1.05 g, 1.4 mmol) was converted and purified as described in Example 2.8. Yellow-red crystals are obtained (846 mg, 96%); tal. = 247-248°C.

Example 2.12

N-[lysyl-D-threonyl]-batracillin, dihydrobromide

[image]

2.12.a) N-[N-(fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-threonyl]-batracillin:

N-(fluorenyl-9-methoxycarbonyl)-O-(tert-butyl)-D-threonyl (6.96g, 17.5 mmol) was converted as described in Example 2.1.a. Concentration in vacuum and vacuum chromatography [petroleum ether/ethyl acetate 1:1] gives compound 2.12.a (7.45 g, 68 %) in the form of yellow crystals; TC [ethyl acetate]: Rf = 0.63; tal. = 225-226°C.

2.12.b) N-[O-(tert-butyl)-D-threonyl]-batracillin:

Compound 2.12a (3.8 g, 6 mmol) was converted as described in Example 2.7. b. After condensation in a vacuum, the product is obtained in the form of yellow crystals (1.9 g, 78%); TC [ethyl acetate]: Rf = 0.21; tal. = 110-111°C.

2.12.c) N-[Nα,Nε-di-(tert-butoxycarbonyl)-lysyl-O-(tert-butyl)-D-threonyl]-batracillin:

Compound 2.12.b (1.8 g, 4.5 mmol) was converted as described in example 2.2.a. Yellow crystals are obtained (2.6 g, 79%); TC [ethyl acetate]: Rf = 0.59; tal. = 212°C.

2.12.d) N-pisyl-O-(tert-butyl)-D-threonyl]-batracillin, di-trifluoroacetate:

The above compound (2.5 g, 3.4 mmol) was converted and worked up as described in Example 2.2. Yellow crystals are obtained (2.5 g, 96%); TC [methanol/acetic acid 10:1]: Rf = 0.30; tal. = 142°C (decomposition).

2.12) N-[lysyl-D-threonyl]-batracillin, dihydrobromide:

Compound 2.12.d (2.29 g, 3 mmol) was converted and worked up as described in Example 2.8. Yellow-red crystals are obtained (1.86 g, 97%); tal. = 232°C (decomposition).

Example 2.13

N-[lysyl-D-alanyl]-batracillin, di-trifluoroacetate

[image]

2.13.a) N-[Nα,Nε-bis-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracillin:

6 g (17.3 mmol) of Nα,Nε-bis-(tert-butoxycarbonyl)-lysine were dissolved in 75 ml of DMF and converted at 0°C with 3 g (26 mmol) of N-hydroxysuccinimide and 4.29 g (20.8 mmol) of N,N '-dicyclohexylcarbodiimide. After 3 hours, the resulting urea is filtered, 5g (15.6 mmol) of N-[D-alanyl]-batracillin (example 2.3) is added to the filtrate and stirred for 16 hours at 20°C. The rest of the urea is filtered off and discarded. The filtrate is concentrated and the residue is mixed with methanol and filtered. The filtrate is concentrated again and treated with methanol once more. It is filtered again and the filtered residues are combined. They are dissolved in a mixture of dichloromethane/methanol 10:1 and precipitated with diethyl ether. 8.22 g (81%) of the crystalline desired product is obtained.

2.13) N-[lysyl-D-alanyl]-batracillin, di-trifluoroacetate:

Preparation from 8.2 g of compound 2.13.a analogously to example 2.2. lsk.: 7.58g (89%)

Example 2.14

N-[Nα-fluorenyl-9-methoxycarbonyl)-lysyl]-batracillin, trifluoroacetate

[image]

2.14. a) N-[Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracillin:

Preparation analogous to example 2.4.a from Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysine and batracillin. Ex.: 78 %.

2.14) N-[Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracillin, trifluoroacetate:

Preparation analogous to example 2.5 from compound 2.14.a. Ex.: 90%.

Example 2.15

N-[lysyl-Nε-(fluorenyl-9-methoxycarbonyl)lysyl]-batracillin, di-trifluoroacetate

[image]

2.15.a) N-[Nα,Nε-di-(tert-butoxycarbonyl)-lysyl-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracillin:

3240 mg (4.54 mmol) of compound 2.14 was dissolved in 50 ml of dimethylformamide and converted with 2550 mg (5.45 mmol) of Nα,Nε-di-(tert-butoxycarbonyl)-lysine-p-nitrophenylester and 938 μl of ethyldiisopropylamine. It is stirred for 16 hours at 20°C, it thickens and the residue is mixed with ether. It is filtered and the filtered residue is mixed once more with a mixture of methanol/ether 1:1. Thus, after suction and drying, 3881 mg (92%) of the desired product is obtained.

2.15) N-[lysyl-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracillin, di-trifluoroacetate:

Deblocking of compound 2.15.a using an anhydrous trifluoroacetic acid/dichloromethane 1:1 mixture analogous to example 2.2. Ex.: 95 %. TC [dichloromethane/methanol/ammonia (17 %-tan) 15:6:0.6, Rf = 0.87]

Example 2.16

N-[lysyl-Nß-(fluorenyl-9-methoxycarbonyl)-α,ß-diaminopropionyl]-batracillin, di-trifluoroacetate:

[image]

This peptide conjugate was prepared through 4 steps, analogously to examples 2.14 and 2.15, from batrachitin and Nα-(tert-butoxycarbonyl)-Nß-(fluorenyl-9-methoxy-carbonyl)-diaminopropionic acid. TC [dichloromethane/methanol/glacial acetic acid 5:1:0.2, Rf = 0.15]

Example 2.17

N-[lysyl]-batracillin

[image]

"Fmoc"-cleavage analogous to example 2.4 from N-[Nε-(fluorenyl-9-methoxy-carbonyl)-lysyl]-batracillin trifluoroacetate (example 2.14). Yield: 65 %.

Example 2.18

N-[seryl-D-alanyl]-batracillin, trifluoroacetate

[image]

2.18.a) N-[N-(tert-butoxycarbonyl)-seryl-D-alanyl]-batracillin:

Preparation analogous to example 2.13.a from N-(tert-butoxycarbonyl)-serine and N-[D-alanyl]-batracillin (example 2.3). Ex.: 77 %.

2.18) N-[seryl-D-alanyl]-batracillin, trifluoroacetate:

Preparation analogous to example 2.2 from compound 2.18.a. Ex.: 98 %.

Example 2.19

N-[D-alanyl-D-alanyl]-batracillin, trifluoroacetate

[image]

Preparation through two stages analogous to example 2.18.

Example 2.20

N-[glutamyl-D-alanyl]-batracillin

[image]

Preparation through two stages analogous to example 2.18, starting from N-tert-butoxycarbonyl-glutamyl-δ-tert-butylester and N-[alanyl]-batracillin (example 2.3). After "Boc"-splitting, it thickens, takes up in water, adjusts to pH 7 using 0.1 M sodium alkali and extracts betaine.

Examples 3.1 - 3.34

General formula

[image]

Example 3.1

N-{Nε-[O-(ß-L-fucosyl)-4-hydroxy-phenylaminothiocarbonyl]-lysyl-D-alanyl}-batracillin

3.1.a) N-{Nα-(tert-butoxycarbonyl)-Nε-[O-(ß-L-fucosyl)-4-hydroxy-phenylaminothiocarbonyl]-lysyl-D-alanyl}-batracillin:

55 mg (0.22 mmol) of p-aminophenyl-ß-L-fucoside was converted into 10 ml of a 1:1 dioxane/water mixture while mixing with thiophosgene (34 μl, 0.44 mmol). After 10 minutes, it thickens in a vacuum and the rest is dried for 1 hour in a high vacuum. The obtained mustard-like oil is then combined in absolute dimethylformamide with 109 mg (0.21 mmol) of N-[Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl]batracyline (example 2.4) in the presence of 115 μl of ethyl-diisopropylamine. After double precipitation of the crude product from the methanol/isopropanol mixture, 132 mg (75%) of the desired product is obtained. TC [dichloromethane/methanol 9:1, Rf=0.15].

3.1) N-{Nε-[O-(ß-L-fucosyl)-4-hydroxy-phenylaminothiocarbonyl]-lysyl-D-alanyl}-batracillin:

127 mg (0.15 mmol) of the compound from example 3.1.a is mixed in 10 ml of dichloromethane with 6 ml of anhydrous trifluoroacetic acid for 2 hours at 0°C. It is concentrated, distilled three times with 5 ml of dichloromethane and chromatographed with dichloromethane/methanol/ammonia (17%-tan) 15:2:0.2. After drying, 80 mg (71%) of the desired product is obtained. TC [dichloromethane/methanol/ammonia (17%-ten) 15:4:0.4. Rf = 0.3].

Analogous to example 3.1, the following conjugates were prepared from the partially protected peptide conjugates in example 2.4, i.e. from the analogously prepared isomeric N-[Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl]-batracillin:

Example 3.2

N-{Nε-[O-(2-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylaminothiocarbonyl]-lysyl-D-alanyl)-batracillin

Educt: hydrocarbon from example 1.1

Vacuum chromatographic cleaning of the intermediate stage with a mixture of dichloromethane/methanol 95:5 and the final stage with a mixture of dichloromethane/methanol/ammonia (17 % tan) 15:2:0.2. Ex.: 55 %. TC [dichloromethane/methanol/ammonia (17%-tan) 5:1:0.2 Rf = 0.4].

Example 3.3

N-{Nε-[O-(2-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracillin:

Educt: carbohydrate from example 1.1

Purification of the intermediate product by precipitation from a mixture of dichloromethane/methanol 1:1 with ether and vacuum chromatography, purification of the final product with a mixture of dichloromethane/methanol/ammonia (17 %-tan) 15:2:0.2. Isk. 65%. TC [dichloromethane/methanol/ammonia (17 %-tan) 15:2:0.2 Rf = 0.21].

Example 3.4

N-{Nε-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-l isyl-D-alanyl)-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.2

Purification of the intermediate product by vacuum chromatography with a mixture of dichloromethane/methanol 97.5:2.5 and precipitation of the final product from methanol with ether; utilization: 59 %. TC [dichloromethane/methanol/ammonia (17% tan) 5:2:0.2. Rf = 0.19].

Example 3.5

N-{Nε-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracillin:

Educt: carbohydrate from example 1.2

Purification of the intermediate product by precipitation from a mixture of dichloromethane/methanol 1:1 with ether and vacuum chromatography, purification of the final product with a mixture of dichloromethane/methanol/ammonia (17 %-tan) 15:2:0.2. Ex.: 36 %. TC [dichloromethane/methanol/ammonia (17%-tan) 15:4:0.5 Rf = 0.57].

Example 3.6

N-{Nε-[O-(3-O-methyl-α-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate from example 1.3

Purification of the intermediate product by precipitation from methanol with ether and vacuum chromatography, purification of the final product with a mixture of dichloromethane/methanol/ammonia (17%-tan) 15:2:0.2. Ex.: 44 %. TC [dichloromethane/methanol/ammonia (17%-tan) 15:2:0-2 Rf = 0.15].

Example 3.7

N-{Nε-[O-(3-deoxy-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.6

Vacuum chromatographic purification of the intermediate product with a mixture of dichloromethane/methanol 95:5 and precipitation of the final product from methanol with ether. Utilization: 35%. TC [acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.42].

Example 3.8

N-{Nε-[O-(3,4-epoxy-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.8

Vacuum chromatographic cleaning of the intermediate product with a mixture of dichloromethane/methanol 95:5. Repeated precipitation of the final product from methanol with ether and then mixing with ethyl acetate. TC [acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.49].

Example 3.9

N-{Nε-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Educt: carbohydrate from example 1.10 Cleaning of the intermediate by mixing with methanol and precipitation until complete with ether. Purification of the final product by vacuum chromatography, with a mixture of dichloromethane/methanol/ammonia (17%-tan) 15:3:0.3; later in the same system with 15:6:0.6: The appropriate fractions are concentrated, taken up in water and adjusted to pH 7 with 1 M sodium hydroxide solution. Suction is applied, the filtered residue is taken up in a mixture of dimethylformamide/water 1:3 and an equivalent of 1 M sodium hydroxide solution is added. alkalis. It thickens, takes sodium salt into water and lipophilizes. Ex.: 43 %. TC [dichloromethane/methanol/ammonia (17% tan) 15:4:0.5. Rf=0.15].

Example 3.10

N-{Nε-[O-(3-O-carbamoylmethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Educt: carbohydrate from example 1.18. Purification of the intermediate by mixing with methanol and precipitation until complete with ether. Vacuum-chromatographic cleaning of the final product with a mixture of dichloromethane/methanol/ammonia (17% tan) 15:2:0.2. TC [dichloromethane/methanol/ammonia (17%-tan) 5:3:0.3 Rf = 0.38]; melting point: 190°C (decomposition).

Example 3.11

N-{Nε-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, acetate:

Educt: carbohydrate from example 1.4

Vacuum-chromatographic cleaning of the intermediate product with a mixture of dichloromethane/methanol 95:5 and the final product with dichloromethane/methanol/ammonia (17 % tan) 15:2:0.2. After thickening, the residue is converted with the equivalent of glacial acetic acid and 10 ml of water, and lipophilized. Ex.: 52 %. TC [acetonitrile/water/glacial acetic acid 5:1:0.2 Rf = 0.43]. FAB-MS: m/z = 760 = M+1.

Example 3.12

N-{Nε-[O-(α-D-glucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: p-amino-α-D-glucoside

Purification of the intermediate product and the final product by mixing with methanol and precipitation until complete with ether. Ex.: 83 %. TC [dichloromethane/methanol/ammonia (17 %-tan) 15:8:0.8 Rf = 0.48].

Example 3.13

N-{Nε-[O-(α-D-glucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracillin, trifluoroacetate:

Educt: p-amino-α-D-glucoside Preparation analogous to the isomer from example 3.12.

Example 3.14

N-{Nε-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate

3.14.a) N-{Nε-(tert-butoxycarbonyl)-Nε-[O-(3-O-methyl-ß-D-galacto-pyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl }-batracillin:

A solution of compound 1.25 (62.8 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml) was treated with thiophosgene (33.5 ml, 0.44 mmol) with stirring. After 10 minutes, it thickens in a vacuum and the rest dries for 1 hour in the vacuum of an oil pump. The obtained isothiocyanate is dissolved in absolute dimethylformamide (10 ml) and converted with compound 2.4 (109.7 mg, 0.2 mmol) and ethyldiisopropylamine (0.5 ml). It is stirred for 16 hours at room temperature, concentrated in a vacuum and the residue is purified by vacuum chromatography [dichloromethane/methanol 20:1]. Yellow crystals are obtained (108.3 mg, 62%); TC [dichloromethane/methanol 5:1]: Rf = 0.42; m.p.: 194-195°C (decomposition).

3.14) N-{Nε-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

From compound 3.14.a (105.1 mg, 0.12 mmol) the tert-butoxycarbonyl group is cleaved, as described in example 2.2. After concentration in vacuum and precipitation from a mixture of methanol/diethyl ether, yellow crystals are obtained (57.4 mg, 54%); TC [dichloromethane/methanol 5:1]: Rf = 0.16; m.p.: 188-189°C (decomposition).

Analogous to examples 3.14.a and 3.14, the following glycoconjugates were prepared from peptide conjugate 2.4 (109.7 mg, 0.2 mmol each):

Example 3.15

N-{Nε-[O-(ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.23 (59.7 mg. 0.22 mmol) by purification of the intermediate by vacuum chromatography [dichloromethane/

methanol 10:1] yellow crystals are obtained (79.5 mg. 46%); TC [methanol]: Rf = 0.74; melting point: 182°C.

Purification of the final product as in example 3.14 gives yellow crystals (77.1 mg. 44%); TC [methanol]: Rf = 0.27; m.p.: 191-192°C (decomposition).

Example 3.16

N-{Nε-[O-(3,4-di-O-methyl-ß-D-galactopyrazonyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.31 (79 mg. 0.22 mmol) Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 30:1 → 20:1] gives yellow crystals (150.7 mg, 85%); TC [dichloromethane/methanol 10:1]: Rf = 0.35; m.p.: 197-199°C (decomposition). Purification of the final product as in example 3.14 gives yellow crystals (137 mg, 76%); TC [dichloromethane/methanol 10:1]: Rf = 0.13; m.p.: 184-186°C (decomposition).

Example 3.17

N-{Nε-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.42 (75.5 mg, 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 30:1 → 25:1] yielded yellow crystals (124.1 mg, 66%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.50; melting point: 165°C. Purification of the final product as in example 3.14 gives yellow crystals (107.8 mg. 57%); TC [(dichloromethane/methanol 4:1)]: Rf = 0.53; mp: 183°C (dec).

Example 3.18

N-{Nε[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.43 (77.3 mg, 0.22 mmol)

Purification of the intermediate by precipitation from methanol/diethyl ether gives the sodium salt as yellow crystals (172 mg, 91%); TC [methanol]: Rf = 0.71; melting point: 225-228°C.

Purification of the final product as in Example 3.14 gives yellow crystals (136.5 mg. 73%); TC [methanol]: Rf = 0.12; melting point: 217-220oC (decomposition).

Example 3.19

N{Nε-[O-(3-O-carbamiolmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.44 (72.2 mg. 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 10:1] gave yellow crystals (137.7 mg, 75%); TC [dichloromethane/methanol 4:1]: Rf = 0.41; melting point: 198-201oC (decomposition).

Purification of the final product as in example 3.14 gives yellow crystals (140.2 mg, 75%); TC [dichloromethane/melanol 4:1]: Rf = 0.16; melting point: 188-190oC (decomposition).

Example 3.20

N-{Nε-[O-(3-O-(N-methyl-carbamoylmethyl)-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.45 (75.3 mg, 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol/ammonia (25%) 7:1:0.1] afforded yellow crystals (158.4 mg, 85%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.25; m.p.: 161-163°C (decomposition).

Purification of the final product as in example 3.14 yields yellow crystals (132.5 mg, 70%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.10; m.p.: 191-193°C (decomposition).

Example 3.21

N-{Nε-[O-(3-O-(N-propyl-carbamoylmethyl)-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-batracillin, trifluoroacetate:

Educt: carbohydrate 1.46 (81.5 mg, 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol/ammonia (25%) 8:1:0.1] afforded yellow crystals (153.1 mg. 80%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.33; melting point: 187°C (decomposition).

Purification of the final product as in example 3.14 yields yellow crystals (154.9 mg. 79%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.21; melting point: 179°C.

Example 3.22

N-{Nε-[O-(3-O-(N-butyl-carbamoylmethyl)-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.47 (84.6 mg. 0.22 mmol) Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 12:1] gives yellow crystals (132.7 mg, 68%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.54; melting point: 180-182°C. Purification of the final product as in example 3.14 gives yellow crystals (115.2 mg, 58%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.30; melting point: 176°C.

Example 3.23

N-{Nε-[O-(3,4-dideoxy-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.52 (52.6 mg. 0.22 mmol) Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 25:1] gives yellow crystals (127.4 mg, 77%); TC [dichloromethane/methanol/ammonia (25%) 15:3:0.2]: Rf = 0.60; melting point: 166-167°C. Purification of the final product as in example 3.14 gives yellow crystals (103.1 mg, 61%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.44; m.p.: 173-175°C (decomposition).

Example 3.24

N-{Nε-[O-(6-O-acetyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-batracillin, trifluoroacetate:

Educt: carbohydrate 1.53 (78.2 mg. 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 25:1] gave yellow crystals (88.3 mg, 49%); TC [dichloromethane/methanol 4:1]: Rf = 0.61; m.p.: 196-199°C (decomposition). Purification of the final product as in example 3.14 gives yellow crystals (89.6 mg, 49%); TC [dichloromethane/methanol 4:1]: Rf = 0.31; melting point: 186°C (decomposition).

Example 3.25

N-{Nε-[O-(α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.39 (59.7 mg. 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 10:1] gave yellow crystals (101.7 mg, 59%); TC [methanol]; Rf = 0.79; melting point: 180°C (decomposition).

Purification of the final product as in example 3.14 gives yellow crystals (103.1 mg, 59%); TC [methanol]: Rf = 0.34; m.p.: 177-178°C (decomposition).

Example 3.26

N-{Nε-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.40 (62.8 mg, 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 20:1] gave yellow crystals (56.6 mg, 32%); TC [dichloromethane/methanol 5:1]: Rf = 0.38; m.p.: 191-192°C (decomposition).

Purification of the final product as in example 3.14 gives yellow crystals (46.6 mg, 26%); TC [dichloromethane/methanol 5:1]: Rf = 0.13; m.p.: 190-191°C (decomposition).

Example 3.27

N-{Nε-[O-(2,3-di-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.41 (66 mg, 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 25:1] gave yellow crystals (77.8 mg, 44%); TC [dichloromethane/methanol 4:1]: Rf = 0.65; m.p.: 182-183°C (decomposition).

Purification of the final product as in example 3.14 gives yellow crystals (66.1 mg, 37%); TC [dichloromethane/methanol 4:1]: Rf = 0.40; melting point: 181°C.

Example 3.28

N-{Nε-[O-(3-O-methoxycarbonylmethyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}batracillin, trifluoroacetate:

Educt: carbohydrate 1.48 (75.5 mg. 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 18:1] gave yellow crystals (62.1 mg, 33%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.66; melting point: 165°C.

Purification of the final product as in example 3.14 gives yellow crystals (57.6 mg, 30%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.43; melting point: 183-184°C.

Example 3.29

N-{Nε-[O-(3-O-carboxymethyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.49 (77.3 mg, 0.22 mmol)

Purification of the intermediate product by precipitation from an ethanol/diethylether mixture gives the sodium salt in the form of yellow crystals (173.4 mg, 92%); TC [methanol]: Rf = 0.57; mp: 201-205°C (decomposition).

Purification of the final product as in example 3.14 gives yellow crystals (171.7 mg, 92%); TC [methanol]: Rf = 0.29; m.p.: 196-198°C (decomposition).

Example 3.30

N-{Nε-[O-(3-O-carbamoylmethyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate 1.50 (72.2 mg, 0.22 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 10:1] gave yellow crystals (106.6 mg, 58%); TC [dichloromethane/methanol 4:1]; Rf = 0.34; m.p.: 192-194°C (decomposition).

Purification of the final product as in example 3.14 gives yellow crystals (107.7 mg, 58%); TC [dichloromethane/methanol 4:1]: Rf = 0.13; m.p.: 186-187°C (decomposition).

Example 3.31

N-{Nε-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin

3.31.a) N-{Nα-(fluorenyl-9-methoxycarbonyl)-Nε-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl -D-alanyl)-batracillin:

A solution of compound 1.31 (79 mg, 0.22 mmol) in a 1:1 dioxane/water mixture (10 ml is converted with stirring into thiophosgene (33.5 ml, 0.44 mmol). After 10 minutes, it is concentrated in vacuo and the residue is dried for 1 hour in a vacuum of oil pump. The obtained isothiocyanate is dissolved in absolute dimethylformamide (10 ml) and converted with compound 2.5 (157 mg. 0.2 mmol) and ethyldiisopropylamine (0.5 ml). It is stirred for 16 hours at room temperature, concentrated in vacuo and taken up in dichloromethane/ methanol 1:1. The product was precipitated by addition of diethyl ether and washed with a little ice-cold methanol. Yellow crystals were obtained (191 mg, 94%); TC [dichloromethane/methanol 10:1]: Rf = 0.35; mp: 203°C (decomposition).

3.31) N-{Nε-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

From compound 3.31.a (182.2 mg, 0.18 mmol) the fluorenyl-9-methoxycarbonyl group is cleaved as described in example 2.4. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated by the addition of diethyl ether. Yellow crystals are obtained (127.1 mg, 89%); TC [methanol]: Rf = 0.46; melting point: 158°C.

Analogously to examples 3.31.a and 3.31, the following glycoconjugates were prepared from peptide conjugates 2.5 (157 mg each, 0.2 mmol):

Example 3.32

N-{Nε-[O-(3-O-(piperidyl-N)-carbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.42 (75.5 mg, 0.22 mmol)

Purification of the intermediate as in example 3.31.a gives yellow crystals (191.2 mg, 91%); TC [dichloromethane/methanol 10:1]: Rf = 0.28; melting point: 208°C (decomposition).

Purification of the final product as described in Example 3.31 afforded yellow crystals (155.8 mg, 88%); TC [methanol]: Rf = 0.47; melting point: 120°C (decomposition).

Example 3.33

N-{Nε-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Educt: carbohydrate 1.43 (77.3 mg, 0.22 mmol)

Purification of the intermediate as in example 3.31.a gives yellow crystals (192 mg, 90%); TC [ethanol/methanol 1:1]: Rf = 0.05; mp: 211-213°C (decomposition).

Purification of the final product as described in Example 3.31 afforded yellow crystals (110.6 mg, 66%); TC [methanol]: Rf = 0.33; tel.: 233-235°C (decomposition).

Example 3.34

N-{Nε-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.44 (72.2 mg, 0.22 mmol)

Purification of the intermediate as in example 3.31.a gives yellow crystals (159.2 mg, 76%); TC [dichloromethane/methanol 10:1]: Rf = 0.04; melting point: 177°C (decomposition).

Purification of the final product as described in Example 3.31 afforded yellow crystals (125.1 mg, 76%); TC [methanol]: Rf = 0.48; tel.: 106°C (decomposition).

Examples 4.1-4.12

General formula

[image]

Example 4.1

4.1.a) N-{Nα-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl}-batracillin:

140 mg (0.55 mmol) of p-aminophenyl-ß-L-fucoside is first converted into a mustard-like oil according to the recipe from example 3.1.a and then combined with 430 mg (0.55 mmol) of N-[Nε-(fluorenyl-9-methoxycarbonyl) )-lysyl-D-alanyl]-batracillin trifluoroacetate (example 2.5) in the presence of 375 μl of ethyldiisopropylamine. After precipitation of the crude product from the methanol/dichloromethane mixture, it is purified by vacuum chromatography (acetonitrile/water 10:1). After mixing the residue with ether, 358 mg (67%) of the desired product is obtained. [TC: acetonitrile/water 10:1. Rf = 0.48].

4.1) N-{Nα-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

356 mg (0.37 mmol) of the compound from example 4.1.a is dissolved in 10 ml of dimethylformamide and 5 ml of piperidine and stirred for 1 hour at 20°C. It is concentrated and the residue is chromatographed with dichloromethane/methanol/ammonia (17%) 15:6:0.6. The desired product is obtained with a yield of 46%. [TC dichloromethane/methanol/ammonia (17%) 15:4:0.5. Rf = 0.11].

Analogously to examples 4.1, the following compounds are prepared from partially protected peptide conjugates:

Example 4.2

N-{Nα-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Educt: carbohydrate from example 1.10

Chromatographic purification of the intermediate product with a mixture of dichloromethane/methanol/ammonia (17 %) 15:3:0.3; later in the same system 15:4:0.5. Cleaning of the final product in the betaine stage by mixing with water; then conversion to the sodium salt using 0.1 M sodium hydroxide solution and freeze-drying from dioxane/water. Ex.: 65%; melting point: 220°C.

Example 4.3

N-{Nα-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate from example 1.2

Purification of the intermediate by precipitation from dicormethane with ether; purification of the final product by repeated precipitation from dimethylformamide with ether.

Yield: 88% [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.28].

Example 4.4

N-{Nα-[O(4-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate from example 1.4

Purification of the intermediate product by repeated precipitation from a mixture of dichloromethane/methanol 1:1 with ether, column chromatographic purification of the final product [TC dichloromethane/methanol/ammonia (17 %) 15:8:0.8], precipitation from a mixture of dichloromethane/methanol 1:1 with ether. Yield: 74% [TC dichloromethane/methanol/ammonia (17%) 10:10:1, Rf = 0.19].

Example 4.5

N-{Nα-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin

4.5.a) N-{Nα-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl-Nε-(fluorenyl-9-methoxycarbonyl)]-lysyl -D-alanyl}-batracillin:

A solution of compound 1.31 (79 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml) was treated with thiophosgene (33.5 ml, 0.44 mmol) with stirring. After 10 minutes, it thickens in a vacuum and the rest is dried for 1 hour in the vacuum of an oil pump. The obtained isothiocyanate is dissolved in absolute dimethylformamide (10 ml) and converted with compound 2.6 (157 mg, 0.2 mmol) and ethyldiisopropylamine (0.5 ml). It is stirred for 16 hours at room temperature and then thickened in a vacuum. Repeated precipitation of the residue from a dichloromethane/methanol 1:1 mixture using diethyl ether and final washing with a little ice-cold methanol gives yellow crystals (198 mg, 98%); TC [dichloromethane/methanol 10:1]: Rf = 0.23; melting point: 175°C.

4.5) N-{Nα-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

From compound 4.5.a (172.1 mg, 0.17 mmol), the fluorenyl-9-methoxycarbonyl group is cleaved off, as described in example 2.4. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated with the addition of diethyl ether. Yellow crystals are obtained (114.5 mg, 85%); TC [dichloromethane/methanol 1:1]: Rf = 0.16; melting point: 206°C (decomposition).

Analogous to examples 4.5.a and 4.5, the following glycoconjugates are prepared from peptide conjugate 2.6 (157 mg, 0.2 mmol each):

Example 4.6

N-{Nα-[O-(ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.23 (59.7 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (185.8 mg, 94%); TC [dichloromethane/methanol 10:1]: Rf = 0.09; melting point: 182°C (decomposition).

Purification of the final product as described in Example 4.5 afforded yellow crystals (134.3 mg, 88%); TC [dichloromethane/methanol 1:1]: Rf = 0.04; melting point: 221°C (decomposition).

Example 4.7

N-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.25 (62.8 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (193.5 mg. 97%); TC [dichloromethane/methanol 10:1]: Rf = 0.27; melting point: 178°C (decomposition).

Purification of the final product by vacuum chromatography [dichloromethane/methanol 2:1 → 1:1] gives yellow crystals (130.5 mg, 84%); TC (dichloromethane/methanol 1:1): Rf = 0.09; mp: 206°C (dec).

Example 4.8

N-{Nα-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.42 (75.5 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (209.8 mg, 99%); TC [dichloromethane/methanol 10:1]: Rf = 0.32; melting point: 235°C (decomposition).

Purification of the final product as described in Example 4.5 afforded yellow crystals (164.3 mg, 99%); TC [dichloromethane/methanol 1:1]: Rf = 0.05; melting point: 217°C (decomposition).

Example 4.9

N-{Nα-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Educt: carbohydrate 1.43 (77.3 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (210.3 mg, 99%); TC [dichloromethane/methanol 10:1]: Rf = 0.02; melting point: 185°C (decomposition).

After cleaning the product as described in example 4.5, the residue is suspended in water (10 ml), and the suspension is converted by dropwise addition of 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (150.8 mg, 90%); TC [dichloromethane/methanol 1:1]: Rf = 0.04.

Example 4.10

N-{Nα-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.44 (77.2 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (152.7 mg, 73%); TC [dichloromethane/methanol 10:1]: Rf = 0.11; melting point: 229°C (decomposition).

After cleaning the product as described in example 4.5, the residue is suspended in a 1:1 dioxane/water mixture (20 ml). Lipophilization of the filtered solution gives a yellow amorphous solid (98.4 mg, 61%); TC [dichloromethane/methanol 1:1]: Rf = 0.10; [α]20 = +44.9° (c = 0.2 / H2O).

Example 4.11

N-{Nα-[O-(α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.39 (59.7 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (179.6 mg, 91%); TC [dichloromethane/methanol 10:1]: Rf = 0.07; melting point: 176°C (decomposition).

Purification of the final product as described in Example 4.5 afforded yellow crystals (137.5 mg, 90%); TC [dichloromethane/methanol 1:1]: Rf = 0.09; melting point: 213°C (decomposition).

Example 4.12

N-{Nα-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: carbohydrate 1.40 (62.8 mg, 0.22 mmol)

Intermediate cleaning as described in example 4.5.a; yellow crystals are obtained (94.2 mg, 48%); TC [dichloromethane/methanol 10:1]: Rf = 0.13; melting point: 173°C (decomposition).

Purification of the final product as described in Example 4.5 afforded yellow crystals (66.6 mg, 43 %): TC [dichloromethane/methanol 1:1]: Rf = 0.07; melting point: 215°C (decomposition).

Examples 5.1-5.23

General formula

[image]

Example 5.1

N-{Nα-[O-(3-O-carboxylmethyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[O-(3-O-methyl-ß-L-fucosyl)-4- hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

50 mg (0.16 mmol) of p-aminophenyl-3-O-carboxymethyl-ß-L-fucoside (example 1.10) was converted into 10 ml of a 1:1 dioxane/water mixture while mixing with 30 μl (0.18 mmol) of thiophosgene. After 10 minutes it thickens and dries for 1 hour in a high vacuum. The obtained mustard-like oil was then combined in absolute dimethylformamide with 109 mg (0.144 mmol) of the conjugate from example 3.4 in the presence of 82 μl of ethylenediisopropylamine. It is concentrated and purified by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:4:0.5]. The substance obtained after thickening is lipophilized from water. Ex.: 89 mg (56 %). TC (dichloromethane/methanol/ammonia (17 %) 15:6:0.6. Rf = 0.22].

Analogous to example 5.1, the following mixed substituted conjugates are prepared:

Example 5.2

N-{Nα-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4- hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educts: carbohydrate from example 1.2, conjugate from example 3.10

Purification by precipitation of the crude product from methanol using ether. Ex.: 78 %. TC [acetinitrile/water/glacial acetic acid 5:1:0.2. Rf = 0.45].

Example 5.3

N-{Nα-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educts: conjugate from example 4.3. 4-Hydroxyaniline

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]; exc.: 60%; TC [dichloromethane/methanol/ammonia (17 %) 15:2:0.2, Rf = 0.31].

Example 5.4

N-{Nα-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educts: conjugate from example 4.2,4-hydroxy-aniline

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:4:0.5]. The residue is then mixed with a methanol/ether mixture. Ex.: 55 %; TC [dichloromethane/methanol/ammonia (17%) 15:6:0.6. Rf = 0.3].

Example 5.5

N-{Nα-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educts: conjugate from the application of 4.4, 4-hydroxy-aniline

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]. The residue is then precipitated from a methanol/dichloromethane mixture using ether. Ex.: 49%; melting point: 128°C; TC [dichloromethane/methanol/ammonia (17%) 15:2:0.2. Rf = 0.26].

Example 5.6

N-{Nα-[4-hydroxyphenylamino-thiocarbonyl]-Nε-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educts: conjugate from example 3.11, 4-hydroxy-aniline

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]. Then the residue is lipophilized from a water/dioxane mixture.

Ex.: 50 %; TC [dichloromethane/methanol/ammonia (17%) 15:2:0.2. Rf = 0.29].

Example 5.7

N-{Nα-[4-hydroxyphenylamino-thiocarbonyl]-Nε-[O(3-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educts: conjugate from example 3.4, 4-hydroxy-aniline

The residue is precipitated with ether from methanol. Ex.: 76%; TC [dichloromethane/methanol/ammonia (17 %) 15:2:0.2, Rf = 0.26].

Example 5.8

N-{Nα-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[4-hydroxyethylamino-2-[O-(3-O-methyl-ß- L-fucosyl)-4-hydroxyphenylamino]triazin-6-yl]]-lysyl-D-alanyl}-batracillin:

Educts: conjugate from example 8.10, carbohydrate from example 1.2

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]. Ex.: 9 %; FAB-MS: m/z = 1165 = M+1; TC [dichloromethane/methanol/ammonia (17 %) 15:3:0.3, Rf = 0.27].

Example 5.9

N-{Nα-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[acetyl]-lysyl-D-alanyl}-batracillin:

Educts: N-[Nε-(acetyl)-lysyl-D-alanyl]-batracillin, carbohydrate from example 1.10

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2; later in the same system 15:4:0.5]. The residue is then freeze-dried from the water. Ex.: 46%; TC [acetonitrile/water/glacial acetic acid 5:1:0.2, Rf= 0.31].

Example 5.10

N-{Nα-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[acetyl]-lysyl-D-alanyl}-batracillin:

51 mg (0.067 mmol) of the conjugate from example 4.4 was converted into 5 ml of dimethylformamide with 10 μl of acetic anhydride and stirred for 30 minutes at room temperature. It is concentrated and precipitated from methanol with ether. Ex.: 46 mg (86%); TC [acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.6].

Example 5.11

N-{Nα-[O-(ß-L-fucosyl)-hydroxyphenylamino-thiocarbonyl]-Nε-[succinyl]-lysyl-D-alanyl}-batracillin, sodium salt:

20 mg (0.027 mmol) of the conjugate from example 4.1 was converted into 2 ml of dimethylformamide with 3 mg of succinic acid and stirred for 6 hours at room temperature. It is concentrated and precipitated from methanol using ether. The residue is taken up in water and 27 μl of 0.1 M sodium alkali solution is added. Ex.: 20 mg (86 %). TC [dichloromethane/methanol/glacial acetic acid 80:20:2. Rf = 0.2].

Example 5.12

N-{Nα-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-Nε-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl) )-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Carbohydrate 1.42 (37.7 mg, 0.11 mmol) was converted with peptide conjugate 3.31 (79 mg, 0.1 mmol), as described in Example 3.31.a. After concentration in vacuum and vacuum chromatography [dichloromethane/methanol 20:1 → 10:1], yellow crystals are obtained (52.9 mg, 45%); TC [dichloromethane/methanol 10:1]: Rf = 0.14; melting point: 178°C (decomposition).

Example 5.13

N-{Nα-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3,4-di-O-methyl-ß-D -galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Carbohydrate 1.43 (38.6 mg, 0.11 mmol) was converted with peptide conjugate 3.31 (79 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated by the addition of diethyl ether. The residue is suspended in water (10 ml) and the suspension is converted with stirring by the dropwise addition of 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (87.9 mg, 74%); TC [ethanol]: Rf = 0.17; [α]20 = +56.0° (c = 0.1 / H2O).

Example 5.14

N-{Nα-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3,4-di-O-methyl-ß-D -galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Carbohydrate 1.44 (36.1 mg, 0.11 mmol) was converted with peptide conjugate 3.31 (79 mg, 0.1 mmol), as described in Example 3.31. a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product precipitates with the addition of diethyl ether. Yellow crystals are obtained (45.9 mg, 39 %). TC [ethanol]: Rf = 0.38; melting point: 219°C (decomposition).

Example 5.15

N-{Nα-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-(piperidyl-N) -carbonylmethyl-ß-D-galacto-pyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Carbohydrate 1.31 (39.5 mg, 0.11 mmol) was converted with peptide conjugate 3.32 (88.7 mg, 0.1 mmol), as described in Example 3.31.a. After concentration in vacuum and vacuum chromatography [dichloromethane/methanol 20:1 → 10:1], yellow crystals are obtained (38.8 mg, 32%); TC [dichloromethane/methanol 5:1]: Rf = 0.79; melting point: 205°C (decomposition).

Example 5.16

N-{Nα-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-(piperidyl-N)-carbonylmethyl-ß -D-galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Carbohydrate 1.43 (38.6 mg, 0.11 mmol) was converted with peptide conjugate 3.32 (79 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated by the addition of diethyl ether. The residue is suspended in water (10 ml) and the suspension is converted by the dropwise addition with stirring of a 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (90.3 mg, 71%); TC [ethanol]: Rf = 0.05; [α]20 = +39.0° (c = 0.1 / H2O).

Example 5.17

N-{Nα-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-(piperidyl-N)-carbonylmethyl-ß -D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Carbohydrate 1.44 (36.1 mg, 0.11 mmol) was converted with peptide conjugate 3.32 (88.7 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated by the addition of diethyl ether. Yellow crystals are obtained (44.8 mg, 36%); TC [ethanol]: Rf = 0.06; melting point: 223°C (decomposition).

Example 5.18

N-{Nα-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-carboxymethyl-ß-D -galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Carbohydrate 1.31 (39.5 mg, 0.11 mmol) was converted with peptide conjugate 3.33 (84.2 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1 (20 ml), the product is precipitated by the addition of diethyl ether. The residue is suspended in water (10 ml) and the suspension is converted by the dropwise addition with stirring of a 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (80.7 mg, 68%); TC [ethanol]: Rf = 0.09; [α]20 = +35.0° (c = 0.1 / H2O).

Example 5.19

N-{Nα-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)- 4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Carbohydrate 1.42 (37.7 mg, 0.11 mmol) was converted with peptide conjugate 3.33 (84.2 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1 (20 ml), the product is precipitated by the addition of diethyl ether. The residue is suspended in water (10 ml) and the suspension is converted by the dropwise addition with stirring of a 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (87.5 mg, 71%); TC [ethanol]: Rf= 0.10; [α]20 = +24.6° (c=0.11/H2O).

Example 5.20

N-{Nα-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl)-Nε-[O-(3-O-carboxymethyl-ß-D-galacto-pyranosyl) )-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, sodium salt:

Carbohydrate 1.44 (36.1 mg, 0.11 mmol) was converted with peptide conjugate 3.33 (84.2 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1 (20 ml), the product is precipitated by the addition of diethyl ether. The residue is suspended in water (10 ml) and the suspension is converted by the dropwise addition with stirring of a 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (78.6 mg, 65%); TC [ethanol]: Rf = 0.11; [α]20 = -44.0° (c = 0.13/H2O).

Example 5.21

N-{Nα-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-carbamoylmethyl-ß-D -galactopyranosyl)-4-hydroxyphenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Carbohydrate 1.31 (39.5 mg, 0.11 mmol) was converted with peptide conjugate 3.34 (81.9 mg, 0.1 mmol), as described in Example 3.31.a. After concentration in vacuum and vacuum chromatography [ethanol → ethanol/methanol 10:1] yellow crystals are obtained (17.4 mg, 15%); TC [ethanol]: Rf = 0.20; m.p.> 290°C (decomposition).

Example 5.22

N-{Nα-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)- 4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Carbohydrate 1.42 (37.7 mg, 0.11 mmol) was converted with peptide conjugate 3.34 (81.9 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated by the addition of diethyl ether. Yellow crystals are obtained (72 mg, 60%); TC [ethanol]: Rf = 0.21; melting point: 222°C (decomposition).

Example 5.23

N-{Nα-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[O-(3-O-carbamoylmethyl-ß-D-galacto-pyranosyl) )-4-hydroxy-phenylamino-thiocarbonyl}-lysyl-D-alanyl}-batracillin, sodium salt:

Carbohydrate 1.43 (38.6 mg, 0.11 mmol) was converted with peptide conjugate 3.34 (81.9 mg, 0.1 mmol), as described in Example 3.31.a. After thickening in a vacuum and dissolving in a mixture of methanol/dichloromethane 1:1, the product is precipitated by the addition of diethyl ether. The residue is suspended in water (10 ml) and the suspension is converted by the dropwise addition with stirring of a 0.05 M sodium alkali solution, until a clear solution is formed (pH<10). Lipophilization of the filtered solution gives a yellow amorphous solid (78.2 mg, 65%); TC [ethanol]: Rf = 0.07; [α]20 = +33.0° (c = 0.1 / H2O).

Examples 6.1 - 6.89

General formula

[image]

Example 6.1

N-{Nα,Nε-bis-[O-(2-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

A solution of compound 1.1 (50 mg, 0.19 mmol) in dioxane/water 1:1 (10 ml) was treated with thiophosgene (30 μl, 0.4 mmol) with stirring. After 10 minutes, it thickens in a vacuum and the rest dries for 1 hour in the vacuum of an oil pump. The obtained isothiocyanate was dissolved in absolute dimethylformamide (10 ml) and converted with compound 2.13 (61 mg, 0.09 mmol) and ethyldiisopropylamine (0.5 ml). It is stirred for 16 hours at room temperature, then concentrated in a vacuum and purified by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]. The residue is precipitated from methanol using ether. 48 mg (50%) of the final product is obtained in the form of yellow crystals.

Analogous to example 6.1, the following glycoconjugates are prepared from the peptide conjugate from example 2.13, that is, the L-alanyl isomer (example 2.2):

Example 6.2

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 100 mg (0.38 mmol) of carbohydrates from example 1.2 Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:0.5:0.05; later in the same system 15:1:0.1]; then precipitation from dichloromethane/methanol/ether. Ex.: 59 %. Melting point: 178°C (decomposition). [TC: acetonitrile/water 10:1, Rf = 0.51].

Example 6.3

N-{Nα,Nε-bis-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 115 mg (0.44 mmol) of carbohydrates from example 1.4 Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]; then precipitation from dichloromethane/methanol (1:1)/ether. Ex.: 58 %. Melting point: 176°C (decomposition). [TC: acetonitrile/water 10:1, Rf = 0.52].

Example 6.4

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracillin:

Educt: 115 mg (0.44 mmol) of carbohydrates from example 1.2

Purification by precipitation from a mixture of dichloromethane/methanol (1:1)/ether. Ex.: 93 %. Melting point: 192°C (decomposition). [TC: acetonitrile/water 10:1, Rf= 0.46].

Example 6.5

N-{Nα,Nε-bis-[O-(3-O-methyl-α-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-batracillin:

Educt: 100 mg (0.38 mmol) of carbohydrates from example 1.3

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:1:0.1]; then precipitation from dichloromethane/methanol (1:1)/ether. Melting point: 178°C (decomposition). FAB-MS: m/z = 1071 = M+1.

Example 6.6

N-{Nα,Nε-bis-[O-(3-O-n-propyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 38 mg (0.127 mmol) of carbohydrates from example 1.5

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:1:0.1]; then precipitation from dichloromethane/methanol (1:1)/ether. Ex.: 42 %. Melting point: 167-170°C (decomposition). [TC: dichloromethane/methanol/ammonia (17 %), Rf = 0.34].

Example 6.7

N-{Nα,Nε-bis-[O-(3-deoxy-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 40 mg (0.167 mmol) of carbohydrates from example 1.6

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2]; then precipitation from methanol/ether. Ex.: 81 %. [TC: acetonitrile/water 10:1, Rf = 0.46].

Example 6.8

N-{Nα,Nε-bis-[O-(3,4-dideoxy-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 41 mg (0.183 mmol) of carbohydrates from example 1.7

Purification by vacuum chromatography [dichloromethane/methanol 95:5]; freeze drying from dioxane/water. Ex.: 60 %. [TC: dichloromethane/methanol 9:1, Rf=0.22].

Example 6.9

N-{Nα,Nε-bis-[O-(3-hydroxyethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 75 mg (0.25 mmol) of carbohydrates from example 1.12

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2; later in the same system 15:4:0.5]. Precipitation from methanol/ether. Ex.: 66 %. [TC: acetonitrile/water 10:1, Rf = 0.28].

Example 6.10

N-{Nα,Nε-bis-[O-(2-hydroxyethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 50 mg (0.167 mmol) of carbohydrates from example 1.19

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:3:0.3]; precipitation from methanol/ether; freeze drying from water/dioxane. Ex.: 72 %. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.39].

Example 6.11

N-{Nα,Nε-bis-[O-(2-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, disodium salt:

Educt: 50 mg (0.16 mmol) of carbohydrates from example 1.13 Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:8:0.8; later in the same system 15:10:1]. Digest the residue with ether, then freeze-dry from water/dioxane. Conversion to the disodium salt using 2 equivalents of 0.1 M sodium hydroxide solution, then freeze-drying from water. Ex.: 49 %. [TC: acetonitrile/water/glacial acetic acid 5:1:0.5, Rf = 0.38]. MS-FAB: FAB: m/z = 1157=M-2Na++H+.

Example 6.12

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, disodium salt:

Educt: 200 mg (0.64 mmol) of carbohydrates from example 1.10

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:4:0.5; later in the same system 15:8:0.8; finally 15:10:1]. Digest the residue with ether, then freeze-dry from water/dioxane. Conversion to the disodium salt using 2 equivalents of 0.1 M sodium hydroxide solution, then freeze-drying from water. Ex.: 59 %. [TC: dichloromethane/methanol/ammonia (17%), Rf = 0.1].

Example 6.13

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 60 mg (0.19 mmol) of carbohydrates from example 1.18 Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:3:0.3]. Precipitate the residue with ether from dichloromethane/methanol (1:1), suction and freeze-dry from water/dioxane 1:1. Ex.: 36 %. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.46]. Melting point: 190°C (decomposition).

Example 6.14

N-{Nα,Nε-bis-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 100 mg (0.39 mmol) p-aminophenyl-ß-L-fucoside Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:2:0.2; later in the same system 15:4:0.5]. Precipitate the residue with ether from dimethylformamide, suction. Ex.: 48 %. Melting point: 195-198°C.

Example 6.15

N-{Nα,Nε-bis-[O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Educt: 158 mg (0.61 mmol) of carbohydrates from example 1.21

Purification by vacuum chromatography (dichloromethane/methanol/ammonia (17 %) 15:3:0.3; later in the same system 15:4:0.5). Lipophilize the residue from water/dioxane. Yield: 87 %. [TC: dichloromethane/methanol/ ammonia (17 %) 15:4:0.5 Rf = 0.25].

Example 6.16

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-batracillin, disodium salt:

Educt: 200 mg (0.64 mmol) of carbohydrates from example 1.22

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:4:0.5; later in the same system 15:8:0.8; and finally 15:10:1]. Digest the residue with ether, then freeze-dry from water/dioxane 1:1.

Analogous to example 6.1, the following glycoconjugates were prepared from different peptide conjugates of batracillin, which contain a completely unblocked lysine building block at the amino-end:

Example 6.17

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin, disodium salt:

Educts: 32 mg (0.1 mmol) of carbohydrates from example 1.10 32 mg (0.045 mmol) N-[lysyl-glycyl]-batracillin di-trifluoroacetate (example 2.9) Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:8 :0.8; later in the same system 15:15:1.5]. Precipitation with ether from dimethylformamide/methanol (1:1), then freeze drying from water/dioxane. Conversion to the disodium salt using 2 equivalents of 0.1 M sodium hydroxide solution, then freeze-drying from water. Ex.: 25 %. [TC: dichloromethane/methanol/ammonia (17 %) 15:8:0.8, Rf = 0.19]. FAB-MS: m/z =1189=M+1.

Example 6.18

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Educts: 60 mg (0.22 mmol) of carbohydrates from example 1.2 66 mg (0.1 mmol) of N-[lysyl-glycyl]-batracillin di-trifluoroacetate (example 2.9)

Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1]; ether precipitation from methanol, then freeze drying from water/dioxane. Ex.: 68 %. [TC: dichloromethane/methanol/glacial acetic acid 80:20:2, Rf = 0.62].

Example 6.19

N-{Nα,Nε-bis-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-lysyl}-batracillin:

6.19.a) N-{Nα,Nε-bis-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl}-batracillin:

Educts: 297 mg (1 mmol) p-aminophenyl-ß-L-fucoside 66 mg (0.1 mmol) N-[lysyl-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-batracillin di-trifluoroacetate (example 2.15) 206 μl ethyldiisopropylamine

Cleaning by two precipitations with ether from a mixture of methanol/dichloromethane (1:1), washing the filtered residue with ether. Ex.: 89 %. [TC: dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.31].

6.19) N-{Nα,Nε-bis-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]lysyl-lysyl}-batracillin:

515 mg (0.39 mmol) of the compound from example 6.19.a is dissolved in 5 ml of dimethylformamide and 5 ml of piperidine, and stirred for 30 minutes at 20°C. The mixture is concentrated and the residue is digested with ether. It is sucked off and the filter residue is taken up in dimethylformamide. After precipitation with ether and washing of the residue, 335 mg (78%) of the crystalline final product remains. FAB-MS: m/z = 1100 = M+1.

Example 6.20

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-diaminopropionyl}-batracillin:

6.20.a) N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysiI-Nß-(fluorenyl-9-methoxycarbonyl) -diaminopropionyl}-batracillin:

Synthesis analogous to example 6.19:

Educts: 60 mg (0.22 mmol) of carbohydrates from example 1.2 100 mg (0.11 mmol) N-[lysyl-Nß-(fluorenyl-9-methoxycarbonyl)-diaminopropionyl]-batracillin di-trifluoroacetate (example 2.16) 76 μl of ethyldiisopropylamine

Purification by two precipitations with ether from methanol, washing the filter residue with ether. Ex.: 105 mg (73 %).

6.20) N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-diaminopropionyl}-batracillin:

103 mg (0.079 mmol) of the compound from example 6.20.a is deblocked with piperidine analogously to example 6.19. Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:3:0.3; later in the same system 15:6:0.6]. Freeze drying from water/dioxane. Ex.: 21 %. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.32].

Example 6.21

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-diaminopropionyl}-batracillin, disodium salt:

This compound is prepared analogously to example 6.20, through two stages, starting with the carbohydrate from example 1.10 and the peptide conjugate from example 2.16. Chromatographic purification can be omitted, since side products can be removed by digestion with methanol and ether. It is then converted into the disodium salt, using 2 equivalents of a 0.1 M sodium alkali solution. Ex.: 66 % through two degrees. [TC: acetonitrile/water/glacial acetic acid 10:3:1.5. Rf = 0.3].

Example 6.22

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Educts: 60 mg (0.22 mmol) of carbohydrates from example 1.2 76 mg (0.11 mmol) of N-[lysyl-seryl]-batracyline di-trifluoroacetate (example 2.10)

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:2:0.2]; precipitation from dichloromethane/methanol/ether. Ex.: 26 %. [TC: acetonitrile/water 10:1, Rf = 0.39].

Example 6.23

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracillin:

Educts: 60 mg (0.22 mmol) carbohydrates from example 1.2 69 mg (0.11 mmol) N-[lysyl-D-seryl]-batracillin dihydrobromide (example 2.11)

Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:2:0.2]; precipitation from dichloromethane/methanol/ether. Ex.: 29 %. [TC: acetonitrile/water 10:1, Rf = 0.36].

Example 6.24

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracillin:

Educts: 80 mg (0.3 mmol) of carbohydrates from example 1.2 53 mg (0.14 mmol) of N-[lysyl]-batracillin (example 2.17) Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:1:0.1; later in the same system 15:2:0.2]; precipitation from methanol/ether. Ex.: 26 %. [TC: dichloromethane/methanol/ammonia (17 %) 15:2:0.2, Rf = 0.22].

Example 6.25

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracillin:

Educts: 60 mg (0.19 mmol) of carbohydrates from example 1.10 24 mg (0.063 mmol) of N-[lysyl]-batracillin (example 2.17) Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:2:0.2; later in the same system 15:4:0.5]; freeze drying from water. Ex.: 26 %. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.25].

Example 6.26

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracillin:

A solution of compound 1.31 (79 mg, 0.22 mmol) in dioxane/water 1:1 (10 ml) was treated with thiophosgene (33.5 μl, 0.44 mmol), with stirring. After 10 minutes, it thickens in a vacuum and the rest dries for 1 hour in the vacuum of an oil pump. The obtained isothiocyanate was dissolved in absolute dimethylformamide (10 ml) and converted with compound 2.17 (37.7 mg, 0.1 mmol) and ethyldiisopropylamine (0.5 ml). It is stirred for 16 hours at room temperature, then concentrated in a vacuum and purified by vacuum chromatography [dichloromethane/methanol 30:1 → 20:1 → 10:1]. Yellow crystals are obtained (56.7 mg, 53%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.72; melting point: 125°C (decomposition).

Example 6.27

N-{Nα,Nε-bis-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracillin:

Compound 1.42 (75.5 mg, 0.22 mmol) was converted as described in Example 6.26, with peptide conjugate 2.17 (37.7 mg, 0.1 mmol).

Purification by vacuum chromatography [dichloromethane/methanol 30:1 → 20:1 → 10:1] gave yellow crystals (51.1 mg, 44%); TC [ethanol]: Rf = 0.80; melting point: 176°C (decomposition).

Example 6.28

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted as described in Example 6.26, with peptide conjugate 2.17 (37.7 mg, 0.1 mmol). Purification by precipitation from a mixture of methanol/dichloromethane 1:1 with diethyl ether gave yellow crystals (53 mg, 47%); TC [methanol]: Rf = 0.75; m.p.>260°C (decomposition).

Example 6.29

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-batracillin:

Compound 1.44 (72.2 mg, 0.22 mmol) was converted as described in Example 6.26, with peptide conjugate 2.17 (37.7 mg, 0.1 mmol). Purification by precipitation from a mixture of methanol/dichloromethane 1:1 with diethyl ether gives yellow crystals (55.6 mg, 48%); TC [elanol]: Rf = 0.09; tal. = 260°C (decomposition).

Example 6.30

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Compound 1.25 (62.8 mg, 0.22 mmol) was converted as described in Example 6.26, with peptide conjugate 2.10 (69.3 mg, 0.1 mmol). After concentration in a vacuum, it is taken up in dichloromethane/methanol 1:1 (10 ml). precipitate the product by adding diethyl ether (15 ml) and wash with a little ice-cold methanol. Yellow crystals are obtained (46 mg, 41%); TC [ethyl acetate/ethanol 2:1]: Rf = 0 12; melting point: 190-191°C.

Example 6.31

N-{Nα,Nε-bis-[O-(ß-D-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (56.2 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate → ethanol] and precipitation from a mixture of methanol/dichloromethane 1:1 with diethyl ether yielded yellow crystals (73.8 mg, 70%); TC [ethanol]: Rf = 0.15; melting point: 123°C.

Example 6.32

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Compound 1.31 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate → ethyl acetate/ethanol 7:1] gives yellow crystals (43.8 mg, 38%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.31; melting point: 176°C.

Example 6.33

N-{Nα,Nε-bis-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Compound 1.42 (75.5 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. purification by vacuum chromatography [ethyl acetate → ethyl acetate/ethanol 3:1] gives yellow crystals (46.2 mg. 37 %); TC [ethyl acetate/ethanol 2:1]: Rf = 0.12; melting point: 161°C.

Example 6.34

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. Yellow crystals are obtained (39.2 mg, 31%); TC [methanol]: Rf = 0.77; m.p.: 213-215°C (decomposition).

Example 6.35

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Compound 1.44 (72.2 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. purification by vacuum chromatography [ethyl acetate/ethanol 2:1] gave yellow crystals (66.1 mg, 53%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.14; melting point: 192°C.

Example 6.36

N-{Nα,Nε-bis-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Compound 1.40 (62.8 mg, 0.22 mmol) was converted with peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26, and purified. Yellow crystals are obtained (48.9 mg, 44%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.10; melting point: 204°C.

Example 6.37

N-{Nα,Nε-bis-[O-(2,3-di-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl)-batracillin:

Compound 1.41 (66 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate → ethyl acetate/ethanol 7:1] gave yellow crystals (52 mg, 45%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.28; melting point: 164-165°C.

Example 6.38

N-{Nα,Nε-bis-[O-(4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-seryl}-batracillin:

Compound 1.56 (95.4 mg, 0.22 mmol) was converted to peptide conjugate 2.10 (69.3 mg, 0.1 mmol), as described in Example 6.26. After thickening in a vacuum, the product is thoroughly washed with hot methanol (50 ml). Yellow crystals are obtained (61.6 mg, 44%); TC [methanol]: Rf = 0.32; melting point: 222°C (decomposition).

Example 6.39

N-{Nα,Nε-bis-[O-(ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (56.2 mg, 0.22 mmol) was converted to peptide conjugate 2.11 (62.6 mg, 0.1 mmol), as described in Example 6.26. purification by vacuum chromatography [ethyl acetate → ethanol] and precipitation with diethyl ether from a methanol/dichloromethane 1:1 mixture yielded yellow crystals (50.9 mg, 48%); TC [ethanol]: Rf = 0.14; melting point: 133°C.

Example 6.40

N-{Nα,Nε-bis[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracillin:

Compound 1.31 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.11 (62.6 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate → ethyl acetate/ethanol 5:1] gives yellow crystals (53.8 mg. 47 %); TC [ethyl acetate/ethanol 2:1]: Rf = 0.38; melting point: 145-146°C.

Example 6.41

N-{Nα,Nε-bis-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracillin:

Compound 1.42 (75.5 mg, 0.22 mmol) was converted to peptide conjugate 2.11 (62.6 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate → ethyl acetate/ethanol 3:1] gives yellow crystals (52.4 mg, 42%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.12; melting point: 168°C.

Example 6.42

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted with peptide conjugate 2.11 (62.6 mg, 0.1 mmol), as described in Example 6.26, and purified. Yellow crystals are obtained (69.2 mg, 55%); TC [methanol]: Rf = 0.71; mp: 214-216°C (decomposition).

Example 6.43

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-seryl}-batracillin:

Compound 1.44 (72.2 mg, 0.22 mmol) was converted to peptide conjugate 2.11 (62.6 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol 2:1] gave yellow crystals (46.4 mg, 38%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.10; melting point: 173°C.

Example 6.44

N-{Nα,Nε-bis-[O-(ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (56.2 mg, 0.22 mmol) was converted to peptide conjugate 2.7 (72.1 mg, 0.1 mmol), as described in Example 6.26. purification by precipitation from methanol/dichloromethane 1:1 with diethyl ether gave yellow crystals (69.4 mg, 64%); TC [ethanol]: Rf = 0.14; melting point: 185-187°C.

Example 6.45

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracillin:

Compound 1.31 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.7 (72.1 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/acetic acid 200:1 → ethyl acetate/ethanol 3:1] gives yellow crystals (99.5 mg, 85%); TC [ethanol]: Rf = 0.59; melting point: 149°C (decomposition).

Example 6.46

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl)-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted to peptide conjugate 2.7 (72.1 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from methanol/dichloromethane 1:1, diethyl ether gave yellow crystals (93.4 mg, 73%); melting point: 220°C (decomposition).

Example 6.47

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracillin:

Compound 1.44 (72.2 mg, 0.22 mmol) was converted to peptide conjugate 2.7 (72.1 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol/acetic acid 400:100:2] gave yellow crystals (69.7 mg, 57%); TC [ethanol]: Rf = 0.11; melting point: 111°C.

Example 6.48

N-{Nα,Nε-bis-[O-(ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracillin, sodium salt:

Compound 6.44 (21.7 mg, 20 mmol) was suspended in water (10 mL) and the suspension was stirred with dropwise addition of 0.05 M sodium hydroxide solution until a clear solution (pH<10) was formed. Lipophilization of the filtered solution gives a yellow amorphous solid (20.6 mg, 93 %).

Example 6.49

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl)-batracillin, sodium salt :

Compound 6.45 (23.5 mg, 20 μmol) was converted and worked up as described in Example 6.48. A yellow amorphous solid is obtained (23.9 mg, 100 %).

Example 6.50

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracillin, trisodium salt:

Compound 6.46 (25.6 mg, 20 μmol) was dissolved in water (10 ml) and the solution was converted with stirring by dropwise addition of 0.05 M sodium hydroxide solution until pH 8 was reached. Lipophilization of the filtered solution gave a yellow amorphous solid ( 24 mg, 92 %).

Example 6.51

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-asparagyl}-batracillin, sodium salt:

Compound 6.47 (24.7 mg, 20 μmol) was converted and worked up as described in Example 6.48. A yellow amorphous solid is obtained (23.0 mg, 92 %).

Example 6.52

N-{Nα,Nε-bis-[O-(ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-glutamyl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (56.2 mg, 0.22 mmol) was converted to peptide conjugate 2.8 (66.8 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/acetic acid 200:1 → ethyl acetate/ethanol/acetic acid 10:1:0.1] yielded yellow crystals (39.5 mg, 36%); TC [ethanol]: Rf = 0.09; m.p.: 138-139°C (decomposition).

Example 6.53

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-glutamyl}-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted to peptide conjugate 2.8 (66.8 mg, 0.1 mmol), as described in Example 6.26, and purified. Yellow crystals are obtained (97.4 mg, 75%); melting point: 180°C (decomposition).

Example 6.54

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-glutamyl}-batracillin, trisodium salt:

Compound 6.53 (25.6 mg, 20 μmol) was converted and worked up as described in Example 6.50. A yellow amorphous solid is obtained (24.3 mg, 92%); [α]20 = +20.0° (c = 0.26 / H2O).

Example 6.55

N-{Nα,Nε-bis-[O-(ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (56.2 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. purification by vacuum chromatography [ethyl acetate → ethanol] yielded yellow crystals (62.2 mg, 60%); TC [ethanol]: Rf = 0.12; melting point: 176°C.

Example 6.56

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Compound 1.25 (62.8 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol 10:1 → 2:1] gives yellow crystals (56.2 mg, 52%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.22; tel.: 197°C.

Example 6.57

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Compound 1.31 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol 10:1 → 3:1] gives yellow crystals (26.2 mg, 23%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.39; melting point: 209°C.

Example 6.58

N-{Nα,Nε-bis-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Compound 1.42 (75.5 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol 10:1] gave yellow crystals (22 mg, 18%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.06; m.p.: 194-195°C (decomposition).

Example 6.59

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl)-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. After thickening in a vacuum, the product is thoroughly washed with methanol, dichloromethane and diethyl ether. Yellow crystals are obtained (86.8 mg, 71 %): mp; 230-232°C (decomposition).

Example 6.60

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Compound 1.44 (72.2 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. After thickening in a vacuum, the product is thoroughly washed with methanol, dichloromethane and diethyl ether. Yellow crystals are obtained (40.9 mg. 35%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.05; mp: 214-216°C (decomposition).

Example 6.61

N-{Nα,Nε-bis-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Compound 1.40 (62.8 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol 10:1 → 2:1] gives yellow crystals (72.2 mg, 66%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.18; melting point: 175°C.

Example 6.62

N-{Nα,Nε-bis-[O-(2,3-di-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-glycyl}-batracillin:

Compound 1.41 (66 mg, 0.22 mmol) was converted to peptide conjugate 2.9 (66.2 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/ethanol 10:1 → 3:1] gives yellow crystals (66.6 mg, 60%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.41; melting point: 173°C.

Example 6.63

N-{Nα,Nε-bis-[O(ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-threonyl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (56.2 mg, 0.22 mmol) was converted to peptide conjugate 2.12 (64 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate → ethanol] and precipitation of the product with diethyl ether from a mixture of methanol/dichloromethane 1:1 yielded yellow crystals (30.5 mg, 28%); TC [ethanol]: Rf = 0.10; melting point: 172°C.

Example 6.64

N-{Nα,Nε-bis-[O-(ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.23 (59.7 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 10:1 → 1:1] yielded yellow crystals (68.3 mg, 64%); TC [dichloromethane/methanol 1:1]: Rf = 0.49; mp: 222-224°C (decomposition).

Example 6.65

N-{Nα,Nε-bis-[O-(2-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.24 (62.8 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation with diethyl ether from a dichloromethane/methanol 1:1 mixture gives yellow crystals (69.6 mg, 63%); TC [ethanol]: Rf = 0.24; melting point: 208°C (decomposition).

Example 6.66

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.25 (62.8 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 15:1 → 10:1 → 5:1] gives yellow crystals (94.3 mg, 85%); TC [dichloromethane/methanol 5:1]: Rf = 0.16; melting point: 212°C (decomposition).

Example 6.67

N-{Nα,Nε-bis-[O-(4-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.26 (62.8 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 15:1 → 10:1 → 5:1] gave yellow crystals (52.6 mg, 48%); TC [dichloromethane/methanol]: Rf = 0.88; melting point: 192°C (decomposition).

Example 6.68

N-{Nα,Nε-bis-[O-(6-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.27 (62.8 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gives yellow crystals (96.7 mg. 88%); TC [dichloromethane/methanol 5:1]: Rf = 0.04; melting point: 210°C (decomposition).

Example 6.69

N-{Nα,Nε-bis-[O-(2,3-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.28 (65.9 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 20:1] gave yellow crystals (57.4 mg, 51%); TC [dichloromethane/methanol 5:1]: Rf = 0.40; melting point: 148°C (decomposition).

Example 6.70

N-{Nα,Nε-bis-[O-(2,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.29 (65.9 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 20:1 → 15:1 → 10:1] gave yellow crystals (74.2 mg, 65%); TC [dichloromethane/methanol 5:1]: Rf = 0.40; melting point: 140°C (decomposition).

Example 6.71

N-{Nα,Nε-bis-[O-(2,6-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.30 (65.9 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 20:1 → 15:1] gives yellow crystals (43.9 mg, 40%); TC [dichloromethane/methanol 5:1]: Rf = 0.43; melting point: 229°C (decomposition).

Example 6.72

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.31 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 10:1] gave yellow crystals (66.2 mg, 59%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.39; melting point: 184°C.

Example 6.73

N-{Nα,Nε-bis-[O-(3,6-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.32 (65.9 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 15:1 → 10:1] gives yellow crystals (57.1 mg, 50%); TC (dichloromethane/methanol 5:1): Rf = 0.42; mp: 190°C (dec).

Example 6.74

N-{Nα,Nε-bis-[O(4,6-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.33 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 20:1 → 10:1 → 5:1] gave yellow crystals (47.0 mg, 42%); TC [dichloromethane/methanol 5:1]: Rf = 0.39: mp: 169°C.

Example 6.75

N-{Nα,Nε-bis-[O-(2,3,4-tri-O-methyl-ß-O-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-batracillin:

Compound 1.34 (69 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 30:1] gave yellow crystals (83.8 mg, 72%); TC [dichloromethane/methanol 10:1]: Rf = 0.36; melting point: 165°C (decomposition).

Example 6.76

N-{Nα,Nε-bis-[O-(2,3,6-tri-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.35 (69 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gives yellow crystals (105 mg, 91%); TC [dichloromethane/methanol 5:1]: Rf = 0.48; melting point: 194°C (decomposition).

Example 6.77

N-{Nα,Nε-bis-[O-(2,4,6-tri-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Iisyl-D-alanyl)-batracillin:

Compound 1.36 (69 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 30:1 → 10:1] gave yellow crystals (67 mg, 58%); TC [dichloromethane/methanol 5:1]: Rf = 0.54; melting point: 228°C (decomposition).

Example 6.78

N-{Nα,Nε-bis-[O-(3,4,6-tri-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysiI-D-alanyl}-batracillin:

Compound 1.37 (69 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gives yellow crystals (109.3 mg, 94%); TC [dichloromethane/methanol 5:1]: Rf = 0.52; melting point: 180°C (decomposition).

Example 6.79

N-{Nα,Nε-bis-[O-(2,3,4,6-tetra-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}- batracillin:

Compound 1.38 (69 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gives yellow crystals (99.2 mg, 84%); TC [dichloromethane/methanol 10:1]: Rf = 0.73; melting point: 188°C (decomposition).

Example 6.80

N-{Nα,Nε-bis-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.42 (75.5 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gave yellow crystals (22 mg, 18%); TC [dichloromethane/methanol 5:1]: Rf = 0.33; m.p.: 194-195°C (decomposition).

Example 6.81

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin, disodium salt:

Compound 1.43 (77.3 mg, 0.22 mmol) was converted with peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26, and purified. Yellow crystals are obtained (99.7 mg, 81%); TC [methanol]: Rf = 0.80; melting point: 230°C (decomposition).

Example 6.82

N-{Nα,Nε-bis-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.44 (72.2 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 7:1] gave yellow crystals (29.4 mg, 25%); TC [dichloromethane/methanol 3:1]: Rf = 0.23; melting point: 201-202°C.

Example 6.83

N-{Nα,Nε-bis-[O-(3,4-dideoxy-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.52 (52.6 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 17:1] gave yellow crystals (38.8 mg, 37%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.40; melting point: 175°C.

Example 6.84

N-{Nα,Nε-bis-[O-(α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.39 (59.7 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 10:1 → 1:1] gave yellow crystals (52 mg, 48%); TC [dichloromethane/methanol 1:1]: Rf = 0.19; melting point: 196°C.

Example 6.85

N-{Nα,Nε-bis-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-batracillin:

Compound 1.31 (79 mg, 0.22 mmol) was converted to peptide conjugate 2.2 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 10:1] gave yellow crystals (77.6 mg, 69%); TC [dichloromethane/methanol/ammonia (25 %) 15:3:0.2]: Rf = 0.33; melting point: 186°C.

Example 6.86

N-{Nα,Nε-bis-[O(4-O-(ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.56 (95.4 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. After concentration in vacuum, the residue is thoroughly washed with hot methanol (50 ml). Yellow crystals are obtained (102.8 mg, 73%); TC [methanol]: Rf = 0.27; melting point: 225-226°C.

Example 6.87

N-{Nα,Nε-bis-[O-(4-O-(3'-sulfate-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D- alanyl}-batracillin, disodium salt:

Compound 1.57 (117.8 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gives yellow crystals (152.8 mg, 95%); melting point: 232°C (decomposition).

Example 6.88

N-{Nα,Nε-bis-[O-(4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl- D-alanyl}-batracillin:

Compound 1.58 (98.4 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation from a mixture of dichloromethane/methanol 1:1 with diethyl ether gives yellow crystals (118.2 mg, 93%); TC [dichloromethane/methanol 1:1]: Rf = 0.58; melting point: 221°C (decomposition).

Example 6.89

N-{Nα,Nε-bis-[O-(2-O-methyl-4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino -thiocarbonyl]-lysyl-D-alanyl}-batracillin:

Compound 1.59 (101.5 mg, 0.22 mmol) was converted to peptide conjugate 2.13 (67.7 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 15:1 → 10:1 → 5:1] gave yellow crystals (101.3 mg, 70%); TC [dichloromethane/methanol 2:1]: Rf = 0.58; melting point: 233°C (decomposition).

Examples 7.1-7.13

General formula

[image]

Starting from other amino acid or peptide conjugates of batracillin or from batracillin, analogously to example 4.1.a, the following glycoconjugates were prepared:

Example 7.1

N-{N-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin:

Educts: carbohydrate from example 1.2, amino acid conjugate from example 2.3

Vacuum-chromatographic purification (dichloromethane/methanol/ammonia (17 %) 15:1:0.1). Freeze drying from dioxane/water. Ex.: 42%; [TC dichloromethane/methanol/ammonia (17 %) 15:2:0.2, Rf = 0.31].

Example 7.2

N-{N-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin, sodium salt:

Educts: carbohydrate from example 1.10, amino acid conjugate from example 2.3

Vacuum-chromatographic purification (dichloromethane/methanol/ammonia (17 %) 15:3:0.3). Freeze drying from dioxane/water and then converting to sodium salt using 0.1 M sodium hydroxide solution. Ex.: 43%; [TC dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.14].

Example 7.3

N-{N-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-seryl-D-alanyl}-batracillin:

Educts: p-amino-ß-L-fucoside, amino acid conjugate from example 2.18

Vacuum-chromatographic purification (dichloromethane/methanol/ammonia (17 %) 15:3:0.3). Freeze drying from dioxane/water. Ex.: 93 %; [TC dichloromethane/methanol/ammonia (17 %) 15:3:0.3, Rf = 0.28]; FAB-MS: m/z =705=M+1.

Example 7.4

N-{N-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl-D-alanyl}-batracillin:

Educts: p-amino-ß-L-fucoside, amino acid conjugate from example 2.19

Purification by digesting with methanol and precipitation from a methanol/dichloromethane ether mixture. Eq.: 65 % [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.78].

Example 7.5

N-{N-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-glutamyl-D-alanyl}-batracillin:

Educts: p-amino-ß-L-fucoside, amino acid conjugate from example 2.20

Vacuum-chromatographic purification (dichloromethane/methanol/ammonia (17 %) 15:4:0.5, later in the same system 15:6:0.6). Freeze drying from dioxane/water Ex.: 92 %; [TC dichloromethane/methanol/ammonia (17 %) 15:8:0.8, Rf = 0.55].

Example 7.6

N-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-batracillin:

250 mg (1 mmol) of batracillin is dissolved in 50 ml of dioxane and mixed after the addition of 184 μl for 2 hours at 20°C. It is concentrated, digested with ether and filtered. The filter residue is dried for 16 hours in a high vacuum and then taken up in 30 ml of dimethylformamide. 312 mg (1 mmol) of carbohydrates from example 1.10 and 500 μl of ethyl diisopropylamine are added and treated with ultrasound for 4 hours. It is then concentrated and purified by vacuum chromatography (dichloromethane/methanol/ammonia (17%) 15:2:0.2, later in the same system 15:6:0.6). The relevant fractions are concentrated and lipophilized from a dioxane/water mixture. Ex.: 363 mg (60%); [TC dichloromethane/methanol/ammonia (17%) 15:6:0.6. Rf = 0.38].

Example 7.7

N-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-batracillin:

Preparation analogous to example 7.6 starting with batracillin and carbohydrates from example 1.2.

Vacuum-chromatographic purification (dichloromethane/methanol/ammonia (17 %) 15:1:0.1). Ex.: 58%; [TC dichloromethane/methanol/ammonia (17 %) 15:2:0.2, Rf = 0.39]. FAB-MS: m/z = 561 = M+1.

Example 7.8

N-{N-[O-(3,4-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin:

Compound 1.31 (37.5 mg, 0.11 mmol) was converted to peptide conjugate 2.3 (32 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 30:1] gave yellow crystals (49.3 mg, 75%); TC [ethyl acetate/ethanol 2:1]: Rf = 0.81; melting point: 185°C (decomposition)

Example 7.9

N-{N-[O-(2,3,4-tri-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl)-batracillin:

Compound 1.34 (34.5 mg, 0.11 mmol) was converted to peptide conjugate 2.3 (32 mg, 0.1 mmol), as described in Example 6.26. purification by vacuum chromatography [dichloromethane/methanol 40:1 → 15:1] yielded yellow crystals (54.9 mg, 81%); TC [dichloromethane/methanol 20:1]: Rf = 0.15; melting point: 190°C (decomposition).

Example 7.10

N-{N-[O-(3-O-methoxycarbonylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin:

Compound 1.42 (37.8 mg, 0.11 mmol) was converted to peptide conjugate 2.3 (32 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 20:1 → 10:1] gives yellow crystals (44.3 mg, 63%); TC [ethanol]: Rf = 0.85; melting point: 195 °C (decomposition).

Example 7.11

N-{N-[O-(3-O-carboxymethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin, sodium salt:

Compound 1.43 (38.6 mg, 0.11 mmol) was converted to peptide conjugate 2.3 (32 mg, 0.1 mmol), as described in Example 6.26. Purification by precipitation with ether from a mixture of dichloromethane/methanol 1:1 gave yellow crystals (63.8 mg, 89%); TC [ethanol]: Rf = 0.15; melting point: 217°C (decomposition).

Example 7.12

N-{N-[O-(3-O-carbamoylmethyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin:

Compound 1.44 (37.8 mg, 0.11 mmol) was converted to peptide conjugate 2.3 (32 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [dichloromethane/methanol 20:1 → 10:1-→ 5:1] gave yellow crystals (20 mg, 29%); TC [ethanol]: Rf = 0.15; melting point: 184°C (decomposition).

Example 7.13

N-{N-[O-(ß-L-fucopyranosiI)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-batracillin:

p-Aminophenyl-ß-L-fucopyranoside (28.1 mg, 0.11 mmol) was converted to peptide conjugate 2.3 (32 mg, 0.1 mmol), as described in Example 6.26. Purification by vacuum chromatography [ethyl acetate/petroleum ether 2:1 → 5:1] yielded yellow crystals (49.8 mg, 81%); TC [ethanol 20:1]: Rf = 0.50; melting point: 173°C.

Examples 8.1-8.12

General formula

[image]

Example 8.1

N-{Ne-[2-chloro-4-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}- batracillin, trifluoroacetate:

8.1. a) N-{Nα-[tert-butoxycarbonyl]-Nε-[2-chloro-4-[O-(3-O-methyl-ß-L-fucosyl)4-hydroxy-phenylamino]-triazin-6-yl ]-lysyl-D-alanyl}-batracillin:

265 mg (0.98 mmol) of p-aminophenyl-3-O-methyl-ß-L-fucoside (example 1.2) and 181 mg (0.98 mmol) of cyanuric chloride are taken up in 50 ml of a dioxane/water mixture, cooled to -5°C and after the addition of 83 mg of sodium bicarbonate, stir at that temperature. Then 538 mg (0.98 mmol) of N-[Nα-(tert-butoxycarbonyl]-lysyl-D-alanyl}-batracillin (example 2.4) dissolved in 14 ml of dimethylformamide and a further 83 mg of sodium hydrogencarbonate are added, and left to reach room temperature temperature. After stirring for 16 hours at 20°C, it thickens, and the residue is treated with water. It is suctioned off, the filter residue is dried under high vacuum and 890 mg (96%) of the desired product is obtained. TC [dichloromethane/methanol 10:1 ]: Rf = 0.26.

8.1) N-{Ne-[2-chloro-4-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl }-batracillin, trifluoroacetate:

100 mg (0.11 mmol) of the compound from example 8.1.a is mixed for 30 minutes at 0°C in a mixture of 5 ml of anhydrous trifluoroacetic acid and 5 ml of dichloromethane. It is concentrated and purified by vacuum chromatography (dichloromethane/methanol/ammonia (17%) 15:1.5:0.15). Then, 41 mg (95%) of the desired product is obtained by precipitation from the methanol/ether mixture. TC [dichloromethane/methanol/ammonia (17%) 15:2:0.2]: Rf = 0.26; FAB-MS: m/z = 829 = M+1.

Example 8.2

N-{Nα-[4-hydroxyethylamino-2-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}- batracillin, trifluoroacetate

8.2.a) N-{Nα-[tert-butoxycarbonyl]-Nε-[4-hydroxyethylamino-2-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazine- 6-yl]-lysyl-D-alanyl}-batracillin:

100 mg (0.11 mmol) of the compound from example 8.1.a is dissolved in 3 ml of dioxane. Add 60 mg of potassium carbonate and 6.5 ml of a 0.1 M solution of ethanolamine in dioxane, and mix for 18 hours at 80°C. Then it thickens and the rest is mixed with water. It is filtered and the filter residue is purified by vacuum chromatography (dichloromethane/methanol/ammonia (17%) 15:1.5:0.15). After concentration of the corresponding fractions and drying in a high vacuum, 83 mg (80%) of the final compound is obtained. TC [dichloromethane/methanol/ammonia (17%) 15:2:0.2]: Rf = 0.23.

8.2) N-{Nα-[4-hydroxyethylamino-2-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl }-batracillin, trifluoroacetate:

67 mg (0.07 mmol) of the compound from example 8.2.a is deblocked analogously to example 8.1. Purification is performed by vacuum chromatography (dichloromethane/methanol/ammonia (17 %) 15:1.5:0.15). Then, precipitation from the methanol/ether mixture gives the final product in 90% yield. FAB-MS: m/z = 854 = M+1.

Analogous to example 8.1, the following glycoconjugates are prepared:

Example 8.3

N-{Nε-2-chloro-4-[O-(2-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracillin , trifluoroacetate:

Educt: carbohydrate from example 1.1; yield: 76%, FAB-MS: m/z = 829 = M+1.

Example 8.4

N-{Ne-[2-chloro-4-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: p-Aminophenyl-ß-L-fucoside; exc.: 36%; FAB-MS: m/z = 815 = M+1. [TC dichloromethane/methanol/ammonia (17%) 15:4:0. , Rf = 0.44].

Example 8.5

N-{Ne-[2-chloro-4-[O-(3-deoxy-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.6; exc.: 56%; FAB-MS: m/z = 799 = M+1. [TC acetonitrile/water/acetic acid 5:1:0.2, Rf = 0.54].

Analogous to examples 8.1a, 8.2.a and 8.2, the following glycoconjugates are prepared from the batracycline peptide conjugate from example 2.4, that is, from the corresponding L-alanyl isomer that can be prepared:

Example 8.6

N-{Nε-[4-hydroxyethylamino-2-[O-(2-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}- batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.1; exc.: 78%; FAB-MS: m/z = 854 = M+1. [TC dichloromethane/methanol/ammonia (17%) 15:4:0.5, Rf = 0.33].

Example 8.7

N{Nε-[4-hydroxyethylamino-2-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxy phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.4; exc.: 38%; [TC dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0:4].

Example 8.8

N-{Nε-[4-hydroxyethylamino-2-[O-(3-deoxy-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.6; exc.: 77%; FAB-MS: m/z = 824 = M+1. [TC dichloromethane/methanol/ammonia (17%) 15:4:0.5, Rf = 0.37].

Example 8.9

N-{Nε-[4-hydroxyethylamino-2-[O-(ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-D-alanyl}-batracillin, trifluoroacetate:

Educt: p-Aminophenyl-ß-L-fucoside; exc.: 52%; FAB-MS: m/z = 840 = M+1. [TC dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.30].

Example 8.10

N-{Nε-[4-hydroxyethylamino-2-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.2; exc.: 88%; [TC dichloromethane/methanol/ammonia (17 %) 15:3:0.3, Rf = 0.35].

Example 8.11

N-{Nε-[4-hydroxyethylamino-2-[O-(2-O-methyl-ß-L-fucosyl)-4-hydroxy phenylamino]-triazin-6-yl]-lysyl-alanyl}-batracillin, trifluoroacetate :

Educt: carbohydrate from example 1.1; exc.: 58%; [TC dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.40].

Example 8.12

N-{Nε-[4-hydroxyethylamino-2-[O-(4-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino]-triazin-6-yl]-lysyl-alanyl}-batracillin, trifluoroacetate:

Educt: carbohydrate from example 1.4; exc.: 66%; FAB-MS: m/z=854=M+1. [TC dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.37].

Example 9.1

N-[D-alanyl]-quinolone, trifluoroacetate:

[image]

9.1.a) N-(N-(tert-butoxycarbonyl)-D-alanyl]-quinolone:

N-(tert-butoxycarbonyl)-D-alanine (3.6 g, 19.2 mmol) and 2-isobutoxy-1-isobutoxy-carbonyl-1,2-dihydro-quinoline (5.8 g, 19.2 mmol) were dissolved in 200 ml of dimethylformamide. After stirring for 8 hours at room temperature, quinolone (4 g, 9.6 mmol) and 3.3 ml of ethyldiisopropylamine were added, and the mixture was treated with ultrasound. It is concentrated, the residue is taken up in dichloromethane and precipitated with ether. After filtering, washing with ether and drying in high vacuum, 4.58 g (81%) of the final product is obtained, which is converted without further purification.

9.1) N-[D-alanyl]-quinolone-a, trifluoroacetate:

4.56 g (7.75 mmol) of the compound from example 9.1.a is dissolved in 50 ml of dichloromethane and 50 ml of anhydrous trifluoroacetic acid at 0°C, and stirred for 1 hour at that temperature. It is concentrated, distilled with dichloromethane, and precipitated from methanol with ether. 4.07 g (87 %) of the crystalline final product is obtained. [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.34].

Example 9.2

N-[alanyl]-quinolone, trifluoroacetate:

[image]

The synthesis is identical to that of the isomer from example 9.1.

Example 9.3

N-[lysyl-D-alanyl]-quinolone, di-trifluoroacetate:

[image]

9.3.a) N-[Nα,Nε-bis-(tert-butoxycarbonyl)-lysyl-D-alanyl]-quinolone:

341 mg (0 984 mmol) of Nα,Nε-bis-(tert-butoxycarbonyl)-lysine was dissolved in 10 ml of DMF and converted at 0°C with 200 mg (1.48 mmol) of N-hydroxybenzotriazole and 227 mg (1.18 mmol) of N '-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride. After 3 hours of mixing at 10°C, 432 mg (0.82 mmol) of the compound from example 9.1 was added, and the mixture was further stirred for 2 hours at 20°C. It thickens and the rest is mixed three times with 50 ml of water each. The residue is then taken up in dichloromethane and dried over sodium sulfate. Precipitation with ether gives 516 mg (78 %) of the final product. [TC acetonitrile/water 10:1, Rf = 0.55].

9.3) N [lysyl D-alanyl]-quinolone-a, di-trifluoroacetate:

Unblocking 512 mg (0.63 mmol) of the compound from example 9.3.a, analogously to example 9.1. Precipitation from ethyl acetate using ether. Ex.: 479 mg (90%); [TC acetonitrile/water/glacial acetic acid 10:3:1.5, Rf = 0.3].

Example 9.4

N-[Iisyl-lanyl]-quinolone, di-trifluoroacetate:

The synthesis proceeds identically to that of the isomer from example 9.3.

Example 9.5

N-[Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl]-quinolone-a

[image]

9.5.a) N-[Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-quinolone:

1.57 g (3.36 mmol) of Nα-(tert-butoxycarbonyl)-Nε-fluorenyl-9-methoxy-carbonyl)-lysine is dissolved in 25 ml of dimethylformamide and converted at 0°C with 600 mg (5.04 mmol) of N-hydroxysuccinimide and 820 mg (4.03 mmol) of N,N'-dicyclohexylcarbodiimide. After 3 hours, the resulting urea is filtered, 1.5 g (2.86 mmol) of the compound from example 9.1 is added to the filtrate and stirred for 16 hours at room temperature. The remaining urea is filtered off and the filtrate is purified by vacuum chromatography [dichloromethane/methanol 97.5:2.5; later in the same system 90:10]. It is then precipitated from the dichloromethane/methanol 1:1 mixture with ether. Ex.: 15 g (56 %); [TC dichloromethane/methanol 9:1, Rf = 0.47].

9.5) N-[Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl]-quinolone:

Fmoc-cleavage from example 9.5.a) with piperidine in dimethylformamide Precipitation of the crude product from dimethylformamide with ether. Ex.: 72 %; [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.43].

Example 9.6

N-[Ne-(fluorenyl-9-methoxycarbonyl)-lysyl-D-alanyl]-quinolone-a, trifluoroacetate:

[image]

Boc-cleavage from example 9.5.a) analogous to example 9.1. Precipitation of the crude product from the methanol/ether mixture. Ex.: 80 %; TC [dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.36].

Example 9.7

N-[Nα-tert-butoxycarbonyl)-lysyl-alanyl]-quinolone-a:

The synthesis proceeds identically to that of the isomer from example 9.5.

Example 9.8

N-[lysyl]-quinolone, di-trifluoroacetate:

[image]

9.8.a) N-[Nα,Nε-bis-(tert-butoxycarbonyl)-lysyl]-quinolone:

1317 mg (3.8 mmol) of Nα,Nε-bis-(tert-butoxycarbonyl)-lysine is bound with quinolone-a according to the procedure from example 9.1.a. It is purified by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:1:0.1]. 1010 mg (71%) of the final product is obtained.

9.8) N-[lysyl]-quinolone, ditrifluoroacetate:

1005 mg (1,347 mmol) of the compound from example 9.8.a is deblocked analogously to example 9.1. After precipitation with ether from a mixture of dichloromethane/methanol 1:1, 966 mg (93%) of the crystalline final product is obtained. [TC acetinitrile/water/glacial acetic acid 10:5:3, Rf = 0.33].

Example 9.9

N-[D-lysyl]-quinolone, di-trifluoroacetate:

The synthesis proceeds identically to that of the isomer from example 9.8.

Example 9.10

N-[Nα-(tert-butoxycarbonyl)-lysyl]-quinolone-a

[image]

9.10.a) N-[Nα-(tert-butoxycarbonyl)-N]-(fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone:

1350 mg (288 mmol) of Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysine is combined with quinolone-a according to the procedure from example 9.8.a. It is purified by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:1:0.1; later in the same system 15:2:0.2]. 1025 mg (82%) of the final product is obtained.

9.10) N-[Nα-(tert-butoxycarbonyl)-lysyl]-quinolone-a

Fmoc-cleavage from example 9.10.a analogously to example 9.5. Double precipitation of the crude product with ether from methanol. Ex.: 86%; [TC acetinitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.48].

Example 9.11

N-[Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone, trifluoroacetate:

[image]

Boc-cleavage from example 9.10.a analogously to example 9.1. Double precipitation of the crude product from the methanol/ether mixture. Freeze drying from a dioxane/water mixture. Ex.: 92 %; [TC dichloromethane/methanol/ammonia (17 %) 15:4:0.5, Rf = 0.44].

Examples 10.1 - 10.3

General formula

[image]

Example 10.1

N-{Nε-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone:

10.1-a) N-{Nα-(tert-butoxycarbonyl)-Nε-[O-(3-O-carboxylmethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}- quinolones:

78 mg (0.25 mmol) of p-Aminophenyl-3-O-carboxymethyl-ß-L-fucoside (Example 1.10) was converted with stirring into 15 ml of a 1:1 dioxane/water mixture with 47 μl (0.28 mmol) of thiophosgene. After 10 minutes of mixing at 20°C, it thickens and dries for 1 hour in a high vacuum. The obtained mustard oil was then combined in absolute dimethylformamide with 180 mg (0.25 mmol) of N-{Nα-(tert-butoxycarbonyl)-lysyl-D-alanyl}-quinolone (example 9.5) in the presence of 86 μl of ethyldiisopropylamine. After two precipitations of the crude product from the dichloromethane/ether mixture, and mixing with water and freeze-drying from the dioxane/water mixture, 210 mg (78%) of the final product is obtained. [TC acetonitrile/water/glacial acetic acid 5:1:0 2, Rf = 0.62].

10.1) N{Nε-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl)-quinolone:

208 mg (0.193 mmol) of the compound from example 10.1.a was mixed for 1 hour at 0°C in 10 ml of dichloromethane with 10 ml of anhydrous trifluoroacetic acid. It is concentrated, distilled with 15 ml of methanol and chromatographed with dichloromethane/methanol/ammonia (17%) 10:10:0.8. Precipitation from dimethylformamide with ether gives 52 mg (28 %) of the final product. [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.53].

Analogously to example 10.1, the following glycoconjugates are prepared from partially protected peptide conjugates from examples 9.5, 9.7, and 9.10:

Example 10.2

N-{Nε-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-quinolone:

Educt carbohydrate from example 1.2; peptide conjugate from example 9.7

Purification of the intermediate by repeated precipitation with ether from methanol. Purification of the final product by vacuum chromatography with dichloromethane/methanol/ammonia (17%) 15:4:0.5; later in the same system 15:8:0.8. Ex.: 20 %; [TC dichloromethane/methanol/ammonia (17 %) 15:8:0.8, Rf=0.15].

Example 10.3

N-{Nε-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone:

Educts: carbohydrate from example 1.2; amino acid conjugate from example 9.10

Purification of the intermediate by precipitation with ether from methanol. Purification of the final product by vacuum chromatography with dichloromethane/methanol/ammonia (17%) 15:8:0.8. Ex.: 39%; [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.33].

Examples 11.1-11.18

General formula

[image]

Example 11.1

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone:

50 mg (0.19 mmol) of p-aminophenyl-3-O-methyl-ß-L-fucoside (example 1.2) is first converted according to the prescription from example 10.1.a into mustard oil, and then combined in 5 ml of dimethylformamide with 68 mg ( 0.08 mmol) of N-[lysyl-D-alanyl]-quinolone-a di-trifluoroacetate (example 9.3) in the presence of 55 μl of ethyldiisopropylamine. It is stirred for 16 hours at room temperature, thickened and purified by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 85:15:1.5]. Then 62 mg (63%) of the final product is obtained by precipitation with ether from methanol. [TC dichloromethane/methanol/glacial acetic acid 80:20:2. Rf= 0.5] MS-MALDI: m/z = 1242 = M+1.

Analogous to example 11.1, the following glycoconjugates are prepared from the peptide conjugates in examples 9.3, 9.4, 9.8, and 9.9:

Example 11.2

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl)-quinolone-a:

Educts: 50 mg (0.19 mmol) of carbohydrates from example 1.2; 0.08 mmol of the peptide conjugate from example 9.4

Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1], and precipitation with ether from methanol. Ex.: 79 %; [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.42].

Example 11.3

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-quinolone:

Educts: 52 mg (0.166 mmol) of carbohydrates from example 1.10; 0.07 mmol of the peptide conjugate from example 9.4 Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 80:20:2], and mixing the residue with methanol. [TC acetonitrile/water/glacial acetic acid 10:3:1.5, Rf = 0.62].

Example 11.4

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-quinolone:

Educts: 44 mg (0.14 mmol) of carbohydrates from example 1.10; 0.06 mmol of peptide conjugate 9.3

Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 80:20:2], and mixing the residue with methanol. Ex.: 57%; [TC acetonitrile/water/glacial acetic acid 10:3:1.5, Rf = 0.62].

Example 11.5

N-{Nα,Nε-bis-[O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl)-quinolone:

Educts: 44 mg (0.166 mmol) of carbohydrates from example 1.21; 0.07 mmol of the peptide conjugate from example 9.4 Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 80:20:1], and mixing the residue with methanol/ether. Ex.: 90 mg (89%); [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.51] MS-ESI: m/z = 1212 = M+1.

Example 11.6

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl)-quinolone:

Educts: 70 mg (0.258 mmol) of carbohydrates from example 1.2; 011 mmol of amino acid conjugate from example 9.8 Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1], and precipitation with ether from dichloromethane/methanol 1:1. Mixing the residue with water. Ex.: 41%; [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.65].

Example 11.7

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone:

Educts: 50 mg (0.19 mmol) of carbohydrates from example 1.2; 0.08 mmol of amino acid conjugate from example 9.9 Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1], and precipitation with ether from dichloromethane/methanol 1:1. Mixing the residue with water. Ex.: 67%; [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.65] MS-FAB: m/z = 1169 = M+1.

Example 11.8

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone, disodium salt:

Educts: 50 mg (0.16 mmol) of carbohydrates from example 1.10; 0.07 mmol of the amino acid conjugate from example 9.8 After thickening the reaction mixture, taking it up in 10 ml of dimethylformamide and adding 8 ml of a 0.1 M sodium alkali solution, it is stirred for 2 hours at 20°C. After re-thickening, it is taken up in water and the pH value is adjusted to 5. It is lipophilized and digested with methanol, and then with methenol/ether. 68 mg (65%) of the final product is obtained. [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.26].

Example 11.9

N-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone, disodium salt:

Educts: 100 mg (0.32 mmol) of carbohydrates from example 1.10; 0.13 mmol of the amino acid conjugate from example 12.9

After thickening the reaction mixture, taking it up in 10 ml of dimethylformamide and adding 16 ml of 0.1 M sodium alkali solution, it is stirred for 2 hours at 20°C. After re-thickening, it is taken up in water and the pH value is adjusted to 5. It is lipophilized and digested with methanol, and then with methenol/ether. 160 mg (77%) of the final product is obtained. Melting point: 218-220°C; [TC: acetonitrile/water/glacial acetic acid 10:3:1.5, Rf = 0.69].

Example 11.10

N-{Nα,Nε-bis-[O-(3-O-methyl-α-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone:

Educts: 50 mg (0.19 mmol) of carbohydrates from example 1.3; 0.08 mmol of amino acid conjugate from example 9.9 Preparation analogous to example 11.7. Purification by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 10:10:1], and precipitation with ether from dichloromethane/methanol 1:1. Ex.: 66%; [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf=0.62].

Example 11.11

N-{Nα,Nε-bis-[O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone:

Educts: 50 mg (0.19 mmol) of carbohydrates from example 1.21; 0.08 mmol of amino acid conjugate from example 9.9 Preparation analogous to example 11.7. Ex.: 57%; [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.63].

Example 11.12

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone, sodium salt:

58 mg (0.05 mmol) of the compound from example 11.7 is suspended in water and converted into the sodium salt using one equivalent of a 0.1 M sodium hydroxide solution. After freeze-drying, 60 mg of the final compound is obtained.

Example 11.13

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone, sodium salt:

A solution of compound 1.25 (62.8 mg, 0.22 mmol) in dioxane/water 1:1 (10 mL) was stirred with thiophosgene (33.5 mL, 0.44 mmol). After 10 minutes, it thickens in a vacuum and the rest is dried for 1 hour in the vacuum of an oil pump. The obtained isothiocyanate was dissolved in absolute dimethylformamide (10 ml) and converted with compound 98 (77.4 mg, 01 mmol) and ethyldiisopropylamine (0.5 ml). It is stirred for 16 hours at room temperature, then concentrated in a vacuum and purified by vacuum chromatography [dichloromethane/methanol/ammonia (25%) 10:10:1 → methanol/ammonia (25%) 20:1]. Yellow crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.05 M sodium alkali solution, until a clear solution (pH<10) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (39.7 mg, 32%); [α]D20 = +25.8° (c = 0.26 / H2O).

Example 11.14

N-{Nα,Nε-bis-[?O-(4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-gluco-pyranosyl)-4-hydroxy-phenylamino-thiocarbonyl] -lysyl}-quinolone, sodium salt:

Compound 1.58 (98.4 mg, 0.22 mmol) was converted as described in Example 11.13 using peptide conjugate 9.8 (77.4 mg, 0.1 mmol), and purified. A yellow amorphous solid is obtained (62.5 mg, 40%); [α]D20 = +12.9° (c = 0.26 / H2O).

Example 11.15

N-{Nα,Nε-bis-[O-(2-O-methyl-4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino -thiocarbonyl]-lysyl}-quinolone, sodium salt:

Compound 1.59 (101.5 mg, 0.22 mmol) was converted as described in Example 11.13 using peptide conjugate 9.8 (77.4 mg, 0.1 mmol). Purification by precipitation from methanol/dichloromethane 1:1 using diethyl ether and boiling with ethanol gave yellow crystals which were converted to the sodium salt as described. A yellow amorphous solid is obtained (51.1 mg, 32%);

[α]D20 = +27.9 ° (c = 0.24 / H2O).

Example 11.16

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone, sodium salt:

Compound 1.25 (62.8 mg, 0.22 mmol) was converted as described in Example 11.13 using peptide conjugate 9.9 (77.4 mg, 0.1 mmol), and purified. A yellow amorphous solid is obtained (77.3 mg, 63%); [α]D20 = -23.8 ° (c = 0.63 / H2O).

Example 11.17

N-{Nα,Nε-bis-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone, sodium salt:

Compound 1.40 (62.8 mg, 0.22 mmol) was converted as described in Example 11.13 using peptide conjugate 9.9 (77.4 mg, 0.1 mmol), and purified. A yellow amorphous solid is obtained (33.6 mg, 27%); [α]D2O = +0.7 ° (c = 0.28/H2O).

Example 11.18

N-{Nα,Nε-bis-[O-(4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D- lysyl}-quinolone, sodium salt:

Compound 1.58 (98.4 mg, 0.22 mmol) was converted as described in Example 11.13 using peptide conjugate 9.9 (77.4 mg, 0.1 mmol), and purified. A yellow amorphous solid is obtained (63.0 mg. 41%); [α]D20 = -21.8o (c =0.22 / H2O).

Examples 12.1 - 12.15

General formula

[image]

Example 12.1

N-{N'-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone:

447 mg (1.66 mmol) of p-aminophenyl-3-O-methyl-ß-L-fucoside (example 1.2) is first converted as described in example 10.1.a into mustard oil, and then combined in 40 ml of dimethylformamide with 1 g (1.66 mmol) of N-[D-alanyl]-quinolone-a di-trifluoroacetate (example 9.1) in the presence of 568 μl of ethyldiisopropylamine. It is stirred for 2 hours at room temperature, thickened and purified by repeated precipitation with ether from dichloromethane/methanol 1:1. After filtering, the residue is mixed with water two more times. 876 mg (66%) of the final product is obtained. Melting point: 198°C; [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.63].

Analogous to example 12.1, the following glycoconjugates are prepared from amino acid conjugates in examples 9.1 and 9.2:

Example 12.2

N-{N'-[O-(3-O-methyl ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl}-quinolone:

Educt: 25 mg (0.092 mmol) of carbohydrates from example 1.2;

Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1], and precipitation with ether from dichloromethane/methanol 1:1 and mixing the residue with water. Ex.: 53 mg (52%); [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.65].

Example 12.3

N-{N'-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone, monosodium salt:

Educts: 523 mg (1.67 mmol) of carbohydrates from example 1.10; 840 mg (1.39 mmol) of the compound from example 12.1

After 6 hours of reaction time, it thickens and is mixed with water. Vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:8:1; later in the same system 10:1:1] is followed by freeze drying from dioxane/water. It is then taken up in water, one equivalent of 0.1 M sodium alkali solution is added, and it is lipophilized again. Ex.: 525 mg (45 %). [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.39].

Example 12.4

N-{N'-[O-(α-L-rhamnosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone:

Educt: 20 mg (0.076 mmol) of carbohydrates from example 1.21;

Purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1], and precipitation with ether from methanol. Ex.: 20 mg (34%); [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.42].

The following glycoconjugates are prepared from partially protected N-(lysyl)-quinolone-a-conjugates, or N-(lysyl-D-alanyl)-quinolone-a-conjugates:

Example 12.5

N-{Nα-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone:

12.5.a) N-{Nα-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-fluorenyl-9-methoxycarbonyl]-lysyl}-quinolone-a :

92 mg (0.34 mmol) of p-aminophenyl-3-O-methyl-ß-L-fucoside (example 1.2) is converted according to the prescription from example 10.1a first into mustard oil and then combined in 20 ml of dimethylformamide with 300 mg (0.34 mmol ) of N-[Nε-(fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-a di-trifluoroacetate (example 9.11) in the presence of 116 μl of ethyldiisopropylamine. It is stirred for 16 hours at room temperature, thickened and purified by precipitation with ether from dichloromethane. After filtering, the residue is mixed with water and lipophilized from dioxane/water. 290 mg (79%) of the final product is obtained. [TC acetonitrile/water 10:1, Rf = 0.6].

12.5) N-{Nα-(O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone:

288 mg (0.267 mmol) of the compound from example 12.5.a was dissolved in 20 ml of dichloromethane and treated with 8 ml of piperidine. After 30 minutes of stirring at 20°C, it thickens and precipitates with ether from dichloromethane. It is purified by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 10:10:2]. The residue is stirred with ether and lipophilized from water. 90 mg (39%) of the final product is obtained. [TC acetonitrile/water/ammonia (17 %) 10:10:5, Rf = 0.4]

Analogous to example 12.5, the following glycoconjugates are prepared from the conjugates in examples 9.11 and 9.6:

Example 12.6

N-{Nα-[O-(3-O-carboxymethyl-ß-L-fucosyl-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a, disodium salt:

Educts: 63 mg (0.2 mmol) of carbohydrates from example 1.10; 158 mg (0.18 mmol) of the compound from example 9.11 Purification of the intermediate by vacuum chromatography [dichloromethane/methanol/ammonia (17%) 15:4:0.5; later in the same system 10:10:1], and the final product by repeatedly mixing with methanol and washing the residue after filtering with ether. Ex.: 44 %. It is then suspended in water and the disodium salt is prepared using 2 equivalents of a 0.1 M sodium alkali solution, and the solution is lipophilized. [TC: acetonitrile/water/glacial acetic acid 10:3:1.5, Rf = 0.34].

Example 12.7

N-{Nα-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino thiocarbonyl]-lysyl-D-alanyl}-quinolone:

Educts: 147 mg (0.47 mmol) of carbohydrates from application 1.10; 448 mg (0.47 mmol) of the compound from example 9.6

Cleaning of the intermediate product by two precipitations with ether from dichloromethane/methanol 1:1; mixing the residue after filtration with water (yield: 92 %). Purification of the final product by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 10:10:2]; ether precipitation from dimethylformamide. Ex.: 59 % [TC; acetonitrile/water/glacial acetic acid 10:3:1.5, Rf = 0.4].

Example 12.8

N-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a, hydrochloride:

Educts: compound 1.25 (62.8 mg, 0.22 mmol); peptide conjugate 9.11 (180 mg, 0.2 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 10:1 → 7:1 → 2:1]. Yellow crystals were obtained (145.7 mg, 67%), TC [dichloromethane/methanol 5:1]: Rf = 0.48. Then, as described in example 4.5, the fluorenyl-9-methoxycarbonyl group is cleaved and purified. Yellow crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.1 M solution of hydrochloric acid, until a clear solution (pH>3) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (119.4 mg, 66%); [α]D20 = +33.8o (c = 0.28 / H2O).

Example 12.9

N-{Nα-[O-(3,6-di-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-a, hydrochloride:

Educts: compound 1.32 (65.9 mg, 0.22 mmol); peptide conjugate 9.11 (180 mg, 0.2 mmol)

Purification of intermediates by vacuum chromatography [dichloromethane/methanol 10:1 → 7:1 → 1:1]. Yellow crystals are obtained (115.0 mg, 52%); TC [dichloromethane/methanol 5:1]: Rf = 0.44. Then, as described in example 4.5, the fluorenyl-9-methoxycarbonyl group is cleaved and purified. Yellow crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.1 M solution of hydrochloric acid, until a clear solution (pH>3) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (94.3 mg, 51%); [α]D20 = +44.2° (c = 0.34 / H2O).

Example 12.10

N-{Nα-[O-(3-O-methyl-α-D-mannopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl)-quinolone-a, hydrochloride:

Educts: compound 1.40 (62.8 mg. 0.22 mmol); peptide conjugate 9.11 (180.0 mg, 0.2 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol 10:1 → 5:1 → 1:1]. Yellow crystals are obtained (96.7 mg, 44%); TC [dichloromethane/methanol 5:1]: Rf = 0.47. Then, as described in example 4.5, the fluorenyl-9-methoxycarbonyl group is cleaved and purified. Yellow crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.1 M solution of hydrochloric acid, until a clear solution (pH>3) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (78.2 mg, 43%); [α]D20 = -157.2o (c = 0.30 / H2O).

Example 12.11

N-{Nα-[O-(4-O(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone, hydrochloride:

Educts: compound 1.58 (98.4 mg, 0.22 mmol); peptide conjugate 9.11 (180.0 mg, 0.2 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/methanol/ammonia (25%) 20:10:1 → 10:10:1 → methanol/ammonia (25%) 20:1] Beige crystals are obtained (132.1 mg. 53%); TC [dichloromethane/methanol/ammonia (25%) 10:10:3]; Rf = 0.60. Then, as described in example 4.5, the fluorenyl-9-methoxycarbonyl group is cleaved and purified. Yellow crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.1 M solution of hydrochloric acid, until a clear solution (pH>3) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (90.0 mg, 42%); [α]D20 = +192.2° (c = 0.27 / H2O).

Starting from the unsubstituted quinolone, the following glycoconjugates are prepared according to the procedure from example 12.1:

Example 12.12

N-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-quinolone, disodium salt:

Educts: 78.5 mg (0.25 mmol) of carbohydrates from example 1.10; 70 mg (0.167 mmol) quinolone

After 6 hours of reaction time, it is thickened, taken up in dimethylformamide and mixed with 4 ml of 0.1 M sodium alkali solution. It is purified by vacuum chromatography [dichloromethane/methanol/ammonia (17 %) 15:4:0.5]. It is adjusted using a 0.1 M sodium alkali solution to pH=7, and lipophilized. Ex.: 60 mg (44 %). [TC acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.42] FAB-MS: m/z = 773 = M-2Na++3H+.

Example 12.13

N-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-quinolone:

Educts: 32 mg (0.12 mmol) of carbohydrates from example 1.2; 50 mg (0.12 mmol) quinolone

Reaction time 2 hours; purification by vacuum chromatography [dichloromethane/methanol/glacial acetic acid 90:10:1]; ether precipitation from dichloromethane/methanol 1:1. Ex.: 59 mg (51 %). [TC: acetonitrile/water 10:1, Rf = 0.43].

Example 12.14

N-{Nα-[O-(3-O-methyl ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl)-quinolone-a, hydrochloride:

86 mg (0.1 mmol) of the compound from example 12.5 was taken up in water and converted into a salt using one equivalent of 0.1 M hydrochloric acid. After freeze-drying, 88 mg of the final compound is obtained.

Example 12.15

N-{Nα-[O (3-O-methyl ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl] diamino-propionyl}-quinolone-a, hydrochloride:

Starting from Nα-(tert-butoxycarbonyl)-Nß-(fluorenyl-9-methoxycarbonyl)-L-diaminopropionic acid and quinolone, the glycoconjugate 12.15 was prepared analogously to example 12.14, through several stages. [TC acetonitrile/water/glacial acetic acid 5:1:0 2, Rf = 0.3]

Examples 13:

General formula

[image]

Example 13.1

N-{N'-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-b:

13.1.a) quinolone-b: 4-amino-7-[(3aRS, 4RS, 7aSR)-4-amino-1,3,3a,4,7,7a-hexahydro-isoindol-2-yl]-1- cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid

310 mg (1 mmol) of 5-amino-1-cyclopropyl-6,7-difluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid was converted in a mixture of 4 ml of acetonitrile and 2 ml of dimethylformamide with 170 mg (1.5 mmol) of 1,4-diazabicyclo[2.2.2]octane and 152 mg (1.1 mmol) of (3aRS, 4RS, 7aSR)-4-amino-1,3,3a,7,7a-hexahydro-isoindole, and heated 1 hour with return cooling. The mixture is evaporated in a vacuum, and the residue is mixed with approximately 20 ml of water, and the resulting precipitate is sucked off and dried in a vacuum at 100°C. Yield: 301 mg (70% of theoretical); melting point: 237-239°C (with decomposition).

13. 1b) N-[D-alanyl]-quinolone-b, trifluoroacetate:

Starting from compound 13.1 a and N-(tert-butoxycarbonyl)-D-alanine, the final compound is prepared analogously to example 9.1.

13.1) N-{N'-[O-(3-O-methyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-quinolone-b:

Starting from compound 13.1b and p-aminophenyl-3-O-methyl-ß-L-fucoside (example 1.2), the final compound was prepared analogously to example 12.1.

Examples 14;

General formula

[image]

Quinolone-c: 8-(2-amino-5-methyl-8-azabicyclo[4.3.0]non-3-en-8-yl)-1-methyl-7-fluoro-5-oxo-5H-thiazolo[ 3,2-a]quinoline-4-carboxylic acid

Example 14.1

N-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-c, hydrochloride:

14.1.a) N-[N-(fluorenyl-9-methoxycarbonyl)-lysyl]-quinolone-c, trifluoroacetate:

Educts; Nα-(tert-butoxycarbonyl)-Nβ-(fluorenyl-9-methoxycarbonyl)-lysine (1.4 g, 3.0 mmol); quinolone-c (820 mg, 1.9 mmol)

Preparation of intermediate products proceeds according to example 9.1.a. Light yellow crystals (1.37 g, 82%) are obtained by precipitation from the ethanol/diethyl ether mixture, from which compound 14.1.a is released analogously to example 9.1.b. Orange crystals are obtained (1.25 g, 74%); TC [dichloromethane/methanol/ammonia (25 %) 30:10:1]: Rf = 0.7; melting point: 180°C.

14.1) N-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-c, hydrochloride:

Analogous to example 12.5, compound 1.25 (62.8 mg, 0.22 mmol) was converted with peptide conjugate 14.1.a (178.4 mg, 0.2 mmol). Purification of the intermediate by vacuum chromatography [dichloromethane/methanol/ammonia (25 %) 30:6:1 → 30:10:1]. Light yellow crystals are obtained (97.0 mg, 44%); TC [dichloromethane/methanol/ammonia (25%) 30:10:1]: Rf = 0.23. Then, as described in example 4.5, the fluorenyl-9-methoxycarbonyl group is cleaved and purified. Yellow crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.1 M solution of hydrochloric acid, until a clear solution (pH>3) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (75.8 mg, 41%); [α]D20 = +12.5o (c = 0.27 / H2O).

Analogous to example 14.1, the following glycoconjugates are prepared from peptide conjugate 14.1.a:

Example 14.2

N-{Nα-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-c, hydrochloride:

Educts: compound 1.2 (59.5 mg, 0.22 mmol); peptide conjugate 14. 1.a (178.4 mg, 0.2 mmol)

Purification of intermediates by vacuum chromatography [dichloromethane/methanol/ammonia (25 %) 30:6:1 → 30:10:1]. Light yellow crystals are obtained (146.6 mg, 67%); TC [dichloromethane/methanol/ammonia (25%) 30:6:1] Rf = 0.48. Then, as described, the fluorenyl-9-methoxy-carbonyl group is cleaved off and converted into the hydrochloride. A yellow amorphous solid is obtained (107.7 mg, 60%); [α]D20 = +51.6° (c = 0.36 / H2O).

Example 14.3

N-{Nα-[O-(3-O-carboxymethyl-ß-L-fucosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-c, disodium salt:

Starting from compound 14.1.a, glycoconjugate 14.4 [FAB-MS: m/z = 911 = M-2Na+3H] was prepared through several steps, analogously to example 12.6.

Example 14.4

N-{Nα-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl}-quinolone-c, hydrochloride:

The conjugate is prepared analogously to the isomer from example 14.2 [FAB-MS: m/z = 867 = M+H].

Examples 15:

General formula

[image]

Quinolone-d: 4-(2-amino-8-azabicyclo[4.3.0]non-4-en-8-yl)-1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo- quinoline-3-carboxylic acid

Example 15.1

N-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-d, hydrochloride:

15.1.a) N-[Nε-(fluorenyl-9-mthoxycarbonyl)-lysyl]-quinolone-d, trifluoroacetate:

Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysine (1.4 g, 3.0 mmol) was treated with quinolone-d hydrochloride (1.28 mg, 2.8 mmol) as described in Example 9.1.a. Precipitation with diethyl ether from a mixture of dichloromethane/methanol 1.1 gives beige crystals (1.97 g, 83 %). from which compound 15.1.a is released analogously to example 9. 1.b. Beige crystals are obtained (1.7 g, 70%); TC (dichloromethane/methanol/ammonia (25 %) 28:14:1); Rf = 0.60; melting point: 215°C.

15.1) N-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-d, hydrochloride:

Analogous to example 12.5, compound 1.25 (62.8 mg, 0.22 mmol) was converted with peptide conjugate 15.1.a (173.2 mg, 0.2 mmol). Purification of the intermediate by vacuum chromatography [dichloromethane/methanol/ammonia (25 %) 28:14:1 → methanol/ammonia (25 %) 20:1]. Beige crystals are obtained (140.8 mg, 65%); TC [dichloromethane/methanol/ammonia (25%) 28:14:1]: Rf = 0.06. The fluorenyl-9-methoxycarbonyl group is then cleaved as described, and purified. Beige crystals are obtained which are suspended in water (10 ml). The suspension is converted with stirring by adding drop by drop a 0.1 M solution of hydrochloric acid, until a clear solution (pH>3) is formed. Lipophilization of the filtered solution gives a yellow amorphous solid (102.6mg, 57%); [α]D20 = -49.0° (c = 0.26 / H2O).

Analogous to example 15.1, the following glycoconjugates are prepared from peptide conjugate 15.1.a:

Example 15.2

N-{Nα-[O-(4-O-(3'-O-methyl-ß-D-galactopyranosyl)-ß-D-glucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl}-quinolone-d , hydrochloride:

Educts: compound 1.58 (98.4 mg, 0.22 mmol); peptide conjugate 15.1.a (173.2 mg, 0.2 mmol)

Purification of the intermediate by vacuum chromatography [dichloromethane/ methanol/ammonia (25 %) 20:10:1 → 10:10:1 → methanol/ammonia (25 %) 20:1]. Beige crystals are obtained (106.5 mg, 43%); TC [dichloromethane/methanol/ammonia (25%) 10:10:3]: Rf = 0.51. Then, as described, the fluorenyl-9-methoxycarbonyl group is cleaved off and converted to the hydrochloride. A yellow amorphous solid is obtained (82.0 mg, 39%); [α]D20 = +22.8° (c = 0.29 / H2O).

Examples 16: glycoconjugates with melphalan

General formula

[image]

Example 16.1

N-{N'-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-melphalan:

16.1.a) N-tert-butoxycarbonyl-D-alanyl-melphalan:

114 mg (0.6 mmol) of N-tert-butoxycarbonyl-D-alanine is dissolved in 10 ml of DMF and converted at 0°C with 138 mg of N'-(dimethylaminopropyl)-N-ethyl-carbo-diimide hydrochloride and 1-hydroxy- benzotriazole. After 10 minutes, 153 mg of melphalan is added and stirred for 16 hours at room temperature. It thickened and partitioned between dichloromethane and water. The organic phase is washed, dried over sodium sulfate, concentrated and then vacuum chromatographed with dichloromethane/methanol/ammonia (17%) 15:2:0.2 → 15:4:0.5. 134 mg (56%) of the final compound is obtained. TC [dichloromethane/methanol/ammonia (17%) 15:4:0.5]: Rf = 0.45.

16.1) N-{N'-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-alanyl}-melphalan:

Removal of the protecting group and connection with the carbohydrate is carried out as described in example 9.1, respectively 12.1. [TC: acetonitrile/water 10:1, Rf = 0.26; FAB-MS: m/z = 685 = M-H].

Example 16. 2

N-{N'-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanyl}-melphalan:

This compound can be prepared through several steps, analogous to example 16.1 [TC: acetonitrile/water 10:1. Rf = 0.2; FAB-MS: m/z = 756 = M-H].

Examples 17: glycoconjugates with doxorubicin (adriamycin)

General formula

[image]

Example 17.1

N-{N'-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanyl}-doxorubicin:

17.1.a) N-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanine:

160 mg (1 mmol) of alanyl-alanine is taken up in 20 ml of dioxane/water 1:1 and converted with 1 ml of Hunig's base. 1.2 mmol of p-Aminophenyl-3-O-methyl-ß-L-fucoside (example 1.2) is first converted according to the regulation 10.1 a into mustard oil and then added to the dipeptide solution. It is stirred for 16 hours at room temperature, then it thickens and the residue is purified by vacuum chromatography (acetonitrile/water 15:1). After concentrating the corresponding fractions, precipitate the product from methanol/ether. Ex.: 267 mg (57 %).

17.1) N-{N'-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-alanyl-alanyl}-doxorubicin:

48 mg (0.1 mmol) of the compound from example 17.1.a is dissolved in 10 ml of DMF and converted with 23.1 mg of N'-(dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and 21 mg of 1-hydroxy-benzotriazole. After 5 minutes, add 30 mg of doxorubicin and 35 μl of Hunig's base, and mix for 30 minutes at room temperature. It is concentrated and purified by vacuum chromatography (dichloromethane/methanol 88:12). Appropriate fractions are concentrated and lipophilized from dioxane/water. 20 mg (40%) of the final product is obtained. [TC: dichloromethane/methanol 10:1, Rf = 0.17: ESI: m/z = 997 = M+H].

Example 17.2

N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-alanyl}-doxorubicin:

17.2.a) Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-alanine:

580 mg (1.31 mmol) of bis-trifluoroacetate D-lysyl-alanine are bound as described in example 17.1.a in the presence of 1.3 ml of Hunig's base with 2.2 equivalents of carbohydrates from example 1.2. Vacuum-chromatographic purification is carried out with acetonitrile/water 10:1. 446 mg (41%) of the final product is obtained.

17.2) N-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-alanyl}-doxorubicin:

Binding of 59 mg of the compound from example 17.2.a with 20 mg of doxorubicin takes place as described in example 17.1. 15 mg of conjugate is obtained. [TC: dichloromethane/methanol 58:15, Rf = 0.43; FAB-MS: m/z = 1365 = M+H].

Examples 18: glycoconjugates with camptothecin

General formula

[image]

Example 18.1

20-O-{Nα,Nε-bis-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin:

18.1.a) 20-O-(alanyl)-camptothecin, trifluoroacetate:

500 mg (1.44 mmol) of camptothecin was dissolved in 20 ml of DMF and then converted with 50 mg of 4-dimethylaminopyridine and N-tert-butoxycarbonyl-alanine-N-carboxy anhydride. After 3 hours, a further 775 mg of N-tert-butoxycarbonyl-alanine-N-carboxy anhydride is added and the suspension is treated with ultrasound for 16 hours. It thickens, the raw material is taken up in 50 ml of dichloromethane and 5 ml of trifluoroacetic acid is added at 0°C. After 30 minutes of mixing, it thickens again and the product is purified by vacuum chromatography (acetonitrile/water 20:1). Appropriate fractions are pooled, concentrated and lipophilized from dioxane/water. 712 mg (93 %) of the final compound are obtained [FAB-MS: m/z = 420 = M+H].

18.1.b) 20-O-(lysyl-alanyl)-camptothecin, bis-trifluoroacetate:

The conjugate from example 18.1.a is bound according to the standard procedure with Nα,Nε-bis-(tert-butoxycarbonyl)-lysine and then deblocked. The desired product is obtained in 65% yield.

18.1) 20-O-{Nα,N'-bis-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin:

Analogously to the prescription from example 11.1, p-aminophenyl-3-O-methyl-ß-L-fucoside (example 12) is attached to the conjugate from example 18.1.b. Ex.: 40 %. [TC: acetonitrile/water 10:1, Rf = 0.44].

Example 18.2

20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin:

18.2.a) 20 O-[Nε-(fluorenyl 9-methoxycarbonyl)-lysyl-alanyl]-camptothecin, trifluoroacetate:

The conjugate from example 18.1.a is bound according to the standard procedure with Nα-(tert-butoxycarbonyl)-Nε-(fluorenyl-9-methoxycarbonyl)-lysine, and then unblocked at the α-amino function. The final compound is obtained in 24% yield. [TC acetonitrile/water 20:1. Rf = 0.15].

18.2.b) 20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-Nε-[fluorenyl-9-methoxycarbonyl]-lysyl-alanyl }-camptothecin:

The compound from example 18.1 is modified with a carbohydrate derivative from example 1.10, analogously to example 12.6 or 12.5. The crude product can be purified by digestion with water, then lipophilized from dioxane/water and included in the next step without further characterization.

18.2) 20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin:

Conjugate 18. 2.b is deblocked with piperidine in DMF. After 30 minutes, it thickens and the residue is digested twice with dichloromethane. It is then taken up in DMF and precipitated with methanol/ether. The product is filtered off with suction, washed with ether and lipophilized from dioxane/water. Ex.: 86 %. [TC: acetonitrile/water/glacial acetic acid 5:1:0 2, Rf = 0.17].

Example 18.3

20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin, sodium salt:

62 mg (0.074 mmol) of the conjugate from example 18.2 is taken up in dioxane/water and converted into the sodium salt using one equivalent of a 0.1 M sodium alkali solution. Ex.: quant. [TC: acetonitrile/water/glacial acetic acid 5:1:0.2, Rf = 0.17]

Analogous to examples 18.1 and 18.2, the following camptothecin glycoconjugates are prepared:

Example 18.4

20-O-{Nα,-Nε-bis-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-D-alanyl}-camptothecin

Example 18.6

20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin

Example 18.7

20-O-{Nα-[O-(3-O-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin

Example 18.8

20-O-{Nα-[O-(3-o-methyl-ß-D-galactopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin

Example 18.9

20-O-{Nα-[O-(3-O-carboxymethyl-ß-D-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin, hydrochloride

Compound 18.6 is converted into the hydrochloride using one equivalent of 0.01 M hydrochloric acid solution.

Example 18.10

20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin, hydrochloride

Compound 18.2 is converted into the hydrochloride using one equivalent of 0.01 M hydrochloric acid solution.

Example 18.11

20-O-{Nα-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-phenylalanyl}-camptothecin, hydrochloride

Example 18.12

20-O-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin, sodium salt

Example 18.13

20-O-{Nα,Nε-bis-[O-(3-O-carboxymethyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-valinyl}-camptothecin, sodium salt

Example 18.14

20-O-{Nα-[O-(3-O-methyl-ß-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-lysyl-alanyl}-camptothecin, hydrochloride

Claims (10)

1. Compounds of the general formula K - Sp - L - AA1 - AA2 - C (I), indicated by that K unsubstituted or site-selectively modified carbohydrate residue, Sp in the given case is substituted arylene or alkylene, [image] AA1 is an amino acid residue in the D- or L-configuration, which in a given case carries protective groups or some other grouping K-Sp-L-, in which K, Sp, L independently of the other grouping K-Sp-L- can have above meanings, or some direct connection, AA2 is an amino acid residue in the D- or in the L-configuration, which in a given case carries protective groups or another grouping K-Sp-L-, in which K, Sp,.L independently of the other grouping K-Sp-L- can have above meanings, or some direct connection, C = a cytotoxic residue or a residue of a cytostatic agent or a cytostatic derivative, which may additionally carry an amino or hydroxy group, and their isomers and salts. 2. Compounds of the general formula (I), according to claim 1, characterized in that K is a carbohydrate residue of the formula (II) [image] where they indicate A = methyl, hydroxymethyl, carboxy, as well as esters and amides derived from them, alkoxymethyl, acyloxymethyl or carboxyalkyloxymethyl, as well as esters and amides derived from them; A can also be CH2-B, where B can again be some carbohydrate residue of formula (II) connected through the anomeric center, R2, R3, R4 = individually or together are H, hydroxy, alkyloxy, carboxyalkyloxy, as well as esters and amides derived from them, hydroxyalkyloxy, aminoalkyloxy, acyloxy, carboxyalkylcarbonyloxy, sulfato, phosphato, halogen, or any other, in the same frame modified carbohydrate residue (II) connected via the anomeric center, where R2 can additionally be amino or acylamino, or two of the residues R2, R3, R4 can together denote an epoxy group, and Sp, L, AA1, AA2 and C have the meaning defined in claim 1, and their isomers and salts. 3. Compounds of formula (I), according to claim 1, characterized by the fact that they mean a Sp aryl residue, which is always ortho-, meta- or para-modified with K and L, and can additionally have 1 to 4 further substituents, which independently or equally can be H, methyl, methoxy, hydroxy, carboxy, methyloxycarbonyl, cyano, nitro, halogen, sulfonyl or sulfonamide, or Sp can be any straight-chain or branched alkylene residue, and K, L, AA1, AA2 and C have the meaning defined in claim 1, and their isomers and salts. 4. Compounds of the general formula (I) according to claim 1, characterized by the fact that C can be a nucleoside, enediyne antibiotic or cytotoxic peptide antibiotic, quinolone- or naphthiridonecarboxylic acid, or batracillin, 5-fluorouracil, cytosinarabinoside, methotrexate, etoposide, camptothecin , daunomycin, doxorubicin, taxol, vinblastine, vincristine, dinamycin, calicamycin, esperamycin, quercetin, suramin, erbstatin, cyclophosphamide, mitomycin C, melphalan, cisplatin, bleomycin, staurosporine or some other antineoplastic active substance, and K, Sp, L, AA1, AA2 have the meaning defined in claim 1, and their isomers and salts. 5. Compounds of the general formula (I) according to claim 1, characterized in that AA1 is an amino acid residue derived from lysine, alanine, aspartic acid, glutamic acid, glycine, ornithine, tyrosine, valine or serine in D- or L-configuration, which in a given case may be linked to a further grouping K-Sp-L-, or where AA1 means a bond, and K, Sp, L, AA1, AA2 and C have the meaning defined in claim 1, and their isomers and salts. 6. Compounds of general formula (I) according to claim 1, characterized in that AA2 is an amino acid residue derived from alanine. lysine, glycine, serine, ornithine or diaminopropionic acid in the D- or in the L-configuration, which in a given case may be connected to a further grouping K-Sp-L-, or where AA2 denotes a bond, and K, Sp, L, AA1 and C have the meaning defined in claim 1, and their isomers and salts. 7. Compounds of the general formula (I) according to claim 1, characterized in that C denotes the residue of the formula (III) T-Q (III) in which it indicates Q rest of the formula [image] where is Ra in the given case with halogen or with hydroxy mono- or doubly substituted alkyl with 1 to 4 carbon atoms, vinyl, in the given case with 1 or 2 fluorine atoms substituted cycloalkyl with 3 to 6 carbon atoms, bicyclo[1.1.1]pent-1- yl, 1,1-dimethyl-propargyl, 3-oxetanyl, methoxy, amino, methylamino, dimethylamino, optionally phenyl substituted singly or doubly with halogen, amino or hydroxy, or together with Re may form a bridge described therein, Rb hydroxy, 1- to 3-carbon alkoxy, nitromethyl, Rc hydrogen or methyl or together with Rg forms the bridge described there, Rd hydrogen, CH3, CH2F or =CH2, X1 hydrogen, halogen or nitro, X2 hydrogen, halogen, amino, hydroxy, methoxy, mercapto, methyl, halomethyl or vinyl, Y stands for N or C-Re where Re means hydrogen, halogen, CF3, OCH3, OCHF2, CH3, CN, CH=CH2 or C=CH, or together with Ra can form a bridge of the structure -*O-CH2-CH-CH3, -*S-CH2-CH2- , -*S-CH2-CH-CH3, -*CH2-CH2-CH-CH3 or -*O-CH2-N-Rf, where the atom marked with * is connected to a carbon atom, and R f can be hydrogen, methyl or formyl, and D stands for N or C-Rg, where Rg means hydrogen, halogen, CF3, OCH3, OCHF2 or CH3, or together with Rc can form a bridge of the structure -*O-CH2-,-*NH-CH2-,-*N(CH3)-CH2-, -*N( C2H5)-CH2-, -*N(C3H5)-CH2- or -*S-CH2-, where the indicated atom is connected to the carbon atom from D, n is 1, 2 or 3, a T stands for the rest of the formula [image] in which [image] Rk means hydrogen or methyl, a Ri means hydrogen, C1-C3-alkyl or cyclopropyl, and K, Sp, L, AA1, AA2 have the meaning defined in claim 1, and their isomers and salts. 8. Compounds of formula (I) according to claim 7, characterized in that Q denotes the rest of the formula [image] where Ra means alkyl with 2 to 4 carbon atoms substituted in a given case with 1 fluorine atom, cyclopropyl substituted in a given case with 1 fluorine atom, phenyl mono- or doubly substituted with fluorine in a given case, or together with Re can form the bridge described there, Rb means hydroxy, alkoxy with 1 to 2 carbon atoms, Rc hydrogen, methyl or together with Rg forms the bridge described there, X1 fluorine, X2 hydrogen or amino, Y stands for N or C-Re, where Re means hydrogen, fluorine, chlorine, CF3, OCH3, OCHF2, or C=CH, or together with Ra can form a bridge of the structure -*O-CH2-CH-CH3, or -*O-CH2-N-Rf, where is the atom marked with * connected to the carbon atom on Y and where Rf can mean methyl, D stands for N or C-Rg, where Rg means hydrogen, fluorine, chlorine, CF3, OCH3, or CH3 or together with Rc can form a bridge of the structure -*O-CH2-, -*NH-CH2-, -*N(CH3)-CH2-, or -*S -CH2-, where the atom marked with * is connected to the carbon atom from D, a T stands for the rest of the formula [image] where [image] Rk means hydrogen or methyl, a Ri means hydrogen or methyl, and their isomers and salts. 9. Medicines, characterized in that they contain one or more compounds according to claims 1 to 8. 10. Use of compounds of formula (I) according to claims 1 to 8, characterized in that it is for the preparation of medicines.