PL112672B1 - Process for preparing novel derivatives of glycosamine - Google Patents

Description

Patent description published: April 30, 1982 112672 Int. 01. ^ C07H 15/12 Inventor Patent holder: Ciba-Geigy AG., Basel (Switzerland) Method for producing new glycosamine derivatives The subject of the invention is a method for producing new derivatives of glycosanins with the general formula 1, in which R4 means hydrogen, Re means hydrogen, R7 means aphthoim hydrogen, an alkyl radical of 1-5 carbon atoms, a hydroxymethyl group or a phenyl radical, R 8 - is a carboxyl group, an alkoxycarbonyl group having 1-3 carbon atoms in the alkoxy part, a benzyloxycarbonyl group, a N-alkylcarbamoyl group having 1-4 carbon atoms in the alkyl part, a N-benylcarbamoyl group or a carbamoylmethylphenylcarbamoyl group, R 9 - is a carboxyl group, an alkoxycarbonyl group having 1-3 carbon atoms in the alkoxy part, a benzyloxycarbonyl group, a carbamoyl group, a N-alkylcarbamoyl group, 1-4 carbon atoms in the alkyl part, provided that the alkyl radical R contains more than 1 carbon atom in the case where X is a carbonyl group and R2 is a methyl radical, or in the case where X is a carbonyl group, R2 is a hydrogen atom and Rs and R9 are carboxyl groups, and their salts. In the compounds discussed above, in which R2 is an alkyl radical, the acetamido group R2 connected to the oxygen atom in the 3-position of the glycosamino group is optically active, i.e. it occurs in the D-configuration. In the case where R7 is not a hydrogen atom, the amino-acetic acid R7 occurs in the L-configuration. The new compounds prepared by the process according to the invention are, depending on their substituents, neutral, acidic or basic compounds. If excess acidic groups are present, they form salts with bases, such as ammonium salts or salts with alkali metals or alkaline earth metals, for example with sodium, potassium, calcium or magnesium. If excess basic groups are present, they form acid addition salts. Acid addition salts include pharmacologically acceptable, non-toxic acid addition salts such as salts with inorganic acids, for example hydrochloric, hydrobromic, nitric, sulfuric or phosphoric acid, or with organic acids such as organic carboxylic acids, for example acetic, propionic, glycolic, succinic, maleic, hydroxymaleic, methylmaleic, fumaric, malic, tartaric, citric, benzoic, cinnamic, mandelic, salicylic, 4-aminosalicylic, 2-phenoxybenzoic, 2-acetoxybenzoic, embolic, nicotinic or isonicotinic acids, or with organic sulfonic acids, for example methanesulfanic acid, atanesulfonic acid, 2-hydroxymethylenesulfonic acid, ethaneodiphenylphoic acid, 1,2, benzenesulfonic acid, p-itoluenesulfonic acid or na- phytaenesulfonic acid and H2, and also other acid addition salts which can be used, for example, as intermediate products, for example for the purification of free compounds or for the preparation of other salts and for their characterization, such as salts with pilcrimic acid, piicrodonic acid, flavian acid, phosphoix)woylfoam, phosphooro-molybdenum, perchloric acid and Reine- cke's. The compounds produced by the method according to the invention exhibit valuable pharmacological properties, and in particular an excellent immunity-enhancing effect. This can be proven on the basis of the following set of tests 1-i5 1. Enhancement of cellular immunity in vivo: increase in late-type hypersensitivity to ovalbumin in guinea pigs. Guinea pigs of the Piribrdigihfa variety are immunized on day 0 with 10 mg of ovalbumin in complete Ereumd adjuvant by instilling 0.1 ml of the amitrogen-adjuvant mixture into both hind paws. After 4 weeks, skin reactions are induced by intradermal injection of 100 µg ovalbumin in 0.1 ml buffered saline solution and quantitatively assessed by the volume of the reaction, calculated after 24 hours from the area of erythema and the increase in skin thickness. This observed, antigen-specific increase in the reaction volume after 24 hours (late-type reaction) serves as a cellular mediator of transferred immunity. Ovalbumin is too weak an immunoglobulinogen to induce a late-type reaction alone or in an oil-in-water emulsion with incomplete Freund's adjuvant (10 parts of egg white albumin solution in 0.9% w/w mixed with 8.5 parts of Bayol F and 1.5 parts of Arlacel A), but for effective immunization it must be applied in complete adjuvant, to which the bacilli [5 mg of killed and of freeze-dried bacillus buttyóoum has 10 ml of the Bayol F/Anlacel A system]. In order to determine the immunity-enhancing effect of the tested substances, these substances can be introduced instead of the bacilli in doses of 10-100 pg to the antibacterial-olive oil mixture. The glycosamine peptides produced according to the invention are able to imitate the effect of these bacilli in the described test system and quantitatively exceed it. A marked increase in the delayed-type reactivity to ovalbumin can also be achieved by the fact that the compounds of the type in question are no longer incorporated in an amitigen-oil mixture but are administered subcutaneously in a kitchen salt solution in doses of 10 to 100 µg per experimental animal within several days after immunization (e.g. on day 0, 1, 2, & 6 and 7). Thus, it has been proven that compounds of the type discussed are capable of significantly increasing cellular immunity, both in contact with the antigen itself (adjuvant effect in the strict sense) and when administered temporarily and locally separate from the injection of the antigen (systemic potentiation of immunity). 2. Potentiation of fluid immunity in vivo: increase in the production of antibodies to bovine serum albumin (BSA) in mice. NMU mice are immunized on day 0 by intraperitoneal (i.p.) injection of 10 µg of sediment-free bovine serum albumin (BSA). After 9, 15 and 29 days serum samples are taken and tested for the content of antibodies against BSA albumin using the haemagglutination technique. In the doses used, soluble BSA albumin is subimmunogenous, i.e. it is not able to induce at all or is only able to induce very little antibody production. Additional treatment of mice with certain immune-enhancing substances before or after the antigen dose leads to an increase in the antibody titer in serum. The effect of this treatment is expressed by the achieved value of the recording, i.e. the sum of the log2 of the difference in titers from three days of bleeding. The compounds produced by the method according to the invention are able, in the case of intraperitoneal or subcutaneous (SC) application of 100-300 mg/kg/animal on five consecutive days (day 0 to 4) after immunization with BSA albumin, to significantly increase the production of antibodies against BSA. The immunity stimulating effect of the discussed compounds is, in contrast to other, bacterial immunoleptils (for example IiPS from E. coli), dependent on the antigen: injection of the new compounds only in BSA albumin-immunized mice and never in non-immunized mice 45 causes an increase in the titer of am/ty-BSA. It is interesting that subcutaneous administration of the compounds in question is as effective as intraperitoneal administration, i.e. the observed immune-enhancing effect is systemic and does not depend on the fact that a stimulant such as amitigene or that it is mixed with an antigen must be administered by the same route, as is the case with classical adjuvants. Thanks to the described tests, it was found that compounds of the discussed type are also capable of specifically increasing fluid resistance, that they improve the immunological response to the stimulus, and that their immune-enhancing effect is based on the systemic activation of the immune apparatus. 3. In vitro potentiation of fluid immunity: the substituting effect of thymus (T) cells in the antibody response of mouse spleen cells to sheep erythrocytes (SE). Lymphocytes (T-cells) derived from the thymus are in many cases essential for the induction of antibody responses. These cells interact with the predecessors of antibody-forming lymphocytes (B-cells) and help them to respond to stimulation with so-called T-dependent antigens through cell proliferation, differentiation, and antibody synthesis. Siledidon cell suspensions from virodized athymic mu/nu mice do not contain any functional T cells and are not capable of, for example, producing any anti-SE antibodies in vitro. The compounds produced by the method of the invention are surprisingly capable of functionally replacing T cells in these cultures and of enabling an antibody response to SE erythrocytes. The addition of these substances to cultures of spleen cells in the presence of SE erythrocytes leads to a significant increase in the number of antibody-forming cells within 4 days. 20 The results show that the compounds in question are capable of increasing the production of antibody in vitro and of compensating for the defect in the T-cell composition. 25 4. Selective mitogenicity for B-cells: a pro-proliferative effect on cell proliferation in B-cell cultures. Suspensions of highly enriched B-cells (B-cells) Lymph nodes of nu/nu mice with congenital lack of athymus), and possibly the purest, immature and mature T-lymphocytes [thymic cells relatively resistant to cortisone, i.e. thymic cells of Ballb/c mice that become cortisone-resistant within 48 hours of cortisone injection] are cultured in the presence of the tested substances for three days. The incorporation of Hs-thymidine in these lymphocytes within the last 16 hours of the culture period serves as a measure of the activity of cell proliferation. The compounds prepared by the method according to the invention are mitogenic for B-lymphocytes (i.e. for the predecessors of antibody-producing cells) but not for T-lymphocytes. are myelinogenic. Thus, these compounds are able to stimulate the diversification of lymphocytes participating in the fluid response (immune reaction)/ £4 3golvibrance. Although the compounds of the discussed type exert their potentiating effect on guinea pigs, for example, after a single subcutaneous dose of 0.05 mg/kg, and on mice after subcutaneous application of a 5-fold dose of 10 mg/kg, even after intraperitoneal application of a 5-fold dose of 300 mg/kg on mice, no toxic effects are observed. 55 The discussed substances therefore have an excellent therapeutic range. The compounds, produced according to the method The compounds of the invention, firstly, in a mixture with an antigen, show the ability to increase its immunogenicity and secondly, when applied systemically, to increase the immunological reactivity of the treated organism. In this case, the compounds in question are able to promote both cellular and fluid immunity and activate lymphocytes, which are responsible for the production of antibodies, so that the new compounds can be used as adjuvants in a mixture with vaccines to improve the vaccination result and also to increase the anti-infective protection against bacterial, viral or parasitic pathogens, which is provided by fluid immunity antibodies and/or cellular immunity. Finally, the compounds in question in a mixture with antigens as adjuvants in the experimental and industrial production of antiserums for therapy and diagnostics, and in the induction of immunologically activated lymphocyte populations for the mode of cell transfer. Furthermore, the new compounds can be used even without the introduction of antigen to support already subthreshold immunological reactions in humans and animals. These compounds are therefore particularly suitable for stimulating the specific defense of the organism, for example in chronic and acute infections or in selective (antigen-specific) immune deficiencies, and in congenital, but also in acquired general (i.e. non-antigen-specific) states of immune deficiencies, such as those occurring in old age, in the course of severe major diseases and above all after therapy with ionizing radiation limb hormones that stimulate immunity. The substances in question can therefore also be advantageously administered in combination with anti-infective antibiotics, chemotherapeutics or other therapeutic procedures to counteract immunological damage. Finally, the substances in question are suitable for general prophylaxis of infectious diseases in humans and animals. In J. Med. Ohm. 9 (1966) on pages 971-072 compounds of formula 1 are described, in which X o - denotes a carbonyl group, R denotes a methyl radical, R 2 denotes a hydrogen atom or a methyl radical, R 7 denotes a methyl radical and R s and H 9 denote carboxyl groups, the action of these compounds not being given. Studies Our own experiments have shown that, surprisingly, these substances are not immunologically active. In Biochem. and Biophys. Res. Com on pages 1316-(1322, among others, compounds of formula 1 are described, in which X is a carbonyl group, R and R2 are methyl radicals, R7 is a methyl radical, Rs is a carbamoyl group and R9 is a carboxyl group, and these compounds have an adjuvant effect. The new compounds of formula 1, prepared in accordance with the invention, are at least as effective as the recently reported known compounds, but are also unexpectedly much less toxic. Particularly valuable are such compounds of formula 1, in which X is a carbonyl group, R2 is a hydrogen atom and the remaining symbols have above-mentioned meaning, and their salts. Particular mention should be made of compounds of the general formula 2, in which R r O i denotes a lower alkyl or phenyl radical, R i denotes a hydrogen atom or a lower alkyl radical, R 2 denotes a hydrogen atom or a methyl radical, R 7 denotes a hydrogen atom, a lower alkyl radical or a hydroxymethyl group, R 8 denotes a R methyl group and R q denotes a carboxyl group, with the proviso that the lower alkyl radical R contains more than 1 carbon atom in the case where R 2 denotes a methyl radical, and their salts. First of all, mention should be made of compounds of the formula 2, in which R denotes a lower alkyl radical or a phenyl radical, R 1 denotes a carboxyl atom. R2 denotes a hydrogen atom or a methyl radical, R7 denotes a hydrogen atom, a methyl radical or a hydroxymethyl group, R5 denotes a carbamoyl group and R9 denotes a carboxyl group, with the proviso that the lower alkyl radical R contains more than 1 carbon atom in the case where R2 denotes a methyl radical, and salts thereof. The process for the preparation of the new compounds according to the invention consists in that in the compound of the formula III, in which R and R2 have the meanings given above, R70, R8° and R9 have the meanings given for the symbols R7, R8° and R9, and in which the hydroxyl groups are protected by easily cleaved ether or ester radicals and R5 denotes an alkylidene radical or cycloalkyldene, the oxazoline and dioxolane rings are cleaved under acidic conditions in the presence of a compound of the formula R 1 -OH, where R 1 has the meaning given above and any protecting groups present are removed. The alkylidene radical is especially the lower alkylidene radical, such as the isopropylidene radical, and the cycloalkylidene radical, such as the cyclopenityllidene or cyclohexylidene radical. This cleavage probably takes place according to the scheme shown in the drawing, in which the compounds placed in the large double clamp are hypothetical intermediate compounds which cannot be isolated. Western Europe. by means of an acidic ion exchanger, especially an ion exchanger containing sulphonic groups, such as Ambeililite HW20 (polystyrene resin with strongly acidic alpha groups) or Dowex 50 (polystyrene sulphonic acids), or by means of a strong inorganic, neutral or organic acid, such as hydrochloric acid, hydroformic acid, sulphuric acid or sulphonic acid, for example methanesulphonic acid or an optionally aromatic-substituted phenylsulphonic acid, such as toluenesulphonic acid or trifluoroacetic acid. In an aqueous medium, a free hydroxyl group is retained in the 1-position, if any, and this procedure is carried out in the presence of an alcohol of the order HO—R1, in which R1 is an optionally substituted alkyl radical, a compound is obtained containing in the -1 position the group -O—R1#. If one of the carboxyl groups Rg and/or R$ is esterified with an alcohol, especially a lower alkenyl, it can be saponified with an aqueous solution of carboxylic acid, especially at an elevated temperature. In the compounds obtained, the protecting groups on the peptide radical can be cleaved off subsequently, especially by hydrogenolysis, for example by means of catalytically activated hydrogen, or by hydrolysis. (The starting compounds used in this connection can be obtained, for example, by introducing an R2- -acetacylpeptide radical in one or more steps into the corresponding oxazoline containing a free hydroxyl group in the 3-position of the sugar residue. The compounds obtained can be converted into their salts in a known manner, for example by reacting the acidic compound obtained with alkali or alkaline earth metal hydroxides, or the basic compound obtained with acids. The above-described process routes are carried out by known methods, in the absence or preferably in the presence of solvents or solvents, and if necessary by cooling or heating, under elevated pressure and/or in a in an inert gas atmosphere, such as acetate. Taking into account all the substituents present in the molecule, if necessary, especially in the presence of easily hydrolyzable O-acyl radicals, mild reactions should be used, such as short reaction times, introduction of mild acidic and basic agents in low concentrations, quantitative stoichiometric ratios, selection of an appropriate catalyst, solvent, temperature and/or pressure. The process according to the invention can also be carried out in such procedures in which compounds obtained as intermediates in any process step are used as starting substances and the missing process steps are carried out, or the process is interrupted at any step, or the starting substance is prepared under reaction conditions or in the form of a reactive derivative or salt. Preferably, such starting substances are used which lead to the compounds described above as being particularly valuable by the process according to the invention. The compounds of formula I can be contained in pharmaceutical preparations. These pharmaceutical preparations are preparations for enteral administration, such as oral or rectal administration, and for parenteral administration to warm-blooded animals, which contain the pharmacologically active substance alone or in combination with a pharmacologically acceptable carrier. The dosage of this active substance depends on the type of warm-blooded animals, the age and individual condition of the patient, and on the method of administration. These pharmaceutical preparations contain about 10-90%, preferably about 20-90%, of this substance. The pharmaceutical preparations are prepared in a known manner, for example by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing methods. In addition to the above-mentioned types of administration, pharmaceutical preparations for oral use can also be obtained by combining the active substance with a single-dose tablet, optionally granulating the resulting mixture, and processing this mixture or the granules, if necessary after adding suitable excipients, to form tablets or dragee cores. Suitable Suitable additives are in particular fillers such as sugars, for example lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, additionally binders, pastes such as starch pastes using corn, wheat, rice or potato starch, gelatin, tragalcanth, methylcellulose, hyoxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if necessary, disintegrants, pastes such as the above-mentioned starches, furthermore starches carboxymethyl, cross-linked polyvinylpyrrolidone, agar, alginic acid or its salt, such as sodium auginate. Auxiliary agents are, above all, flow-regulating agents and lubricants, for example, calcium or magnesium stearate, talc, stearic acid or its salts, lubricants such as magnesium or calcium stearate and/or polyethylene glycol. The dragee cores are provided with suitable coatings, optionally resistant to gastric ulcer, whereby, among other things, concentrated sugar solutions are used, which optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions in suitable organic solvents or mixtures In order to produce coatings resistant to gastric juice, solutions of suitable cellulosic preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dyes or pigments can be incorporated into the tablet or dragee coatings, for example for identification purposes or to indicate different doses of active substance. The following examples explain the process according to the invention in more detail. The temperatures in the examples are given in degrees Celsius. Example 1: 3.77 g of 2-phenyl-4,5H-[3-O-(DH1)carboxymethyl)-5,6-O-isopropyldimethyl]-12-oxazoline in 60 ml of acetaminophen and 15 ml of ml of dimethylformamide is stirred together with 1.4 ml of triethylamine and 2.55 g of 3'-ethyl-5-phenyl-isoxazole-sulfonylmethane for 1.5 hours at 09°C, almost all of which dissolves. Then, 3.44 g of L-alanyl-diglutamic acid-amide-benzyl ester hydrochloride is added, 1.4 ml of triethylamine is added and the mixture is stirred for 24 hours at room temperature, then evaporated in a vacuum to a syrup and chromatographed on silica gel with a mixture of methylformamide and acetate (8:12). A colorless solid is obtained. syrup, It crystallizes on trituration with ether, melting at 96:99° and rotation [α]JJ = + 13° (in chloroform). The crystalline benzyl ester is hydrogenated with a 5% paliliate catalyst on a carbon support in dithiothreate at room temperature under normal pressure and, after evaporation in a vacuum, the acid is obtained in the form of a syrup. This acid is mixed in water with a W ion exchanger called Dowex^0-H+ at room temperature for 15 hours. *After filtering and freeze-drying, a colorless powder is obtained, decomposition temperature 140°. The obtained 2-benzoylamine-3-0-{D^1-CL-1- - (D^1^carbamoyl-3-(carboxypropyl)-yl-lcarbamoylethyl)-carbamoylethyl)-H2-deoxy-alpha-methyl-25-methyl-3-(carboxypropyl)-yl-l ... triethylamine is stirred in 100 ml of acetonitrile and 25 ml of dimethylformamide for 1 hour at 0°-6°C, whereupon the whole dissolves. Then 5.5 g of L-alanyl-D-glutamic acid benzyl ester hydrochloride, 85 g, and a further 2.35 ml of triethylamine are added and stirred for 48 hours at room temperature. The whole is concentrated in an oil vacuum and chromatographed on silica gel in a mixture of chloroform and ethanol (19.1 vol.). 9 g of the colourless syrup thus obtained is hydrogenated. in dioxane using 5% pallllaide catalyst on a carbon support, the catalyst is desaicated, the solution is concentrated in vacuo and hydrolyzed at room temperature in a mixture of 40 ml of tetrahydrate-rafuran and 30 ml of water with 1.5 ml of acid trifluoroiot. Then it is evaporated four times in a vacuum to dryness, dissolved in water and freeze-dried. Obtained 24enzoylamilino -3^0-{D-yl-|[l^l-/IDHl-carbamioylH3^carboxyproipyl]-lcarbamoyl ethylHDair- bamoylpropylol^^desoyloxy^a^Hglycoze it crystallizes with 0.5 mole of water, having a melting point of 114^152° and a rotationality of [a]™ = + 17° (in methane). IP r y/c l a d III. 1.21 g N-lhyde oxys'Uccymimyl, 2^16 g dicyclohexylcarboiddiimide and 1.45 ml triethylamine will dissolve in 40 ml of dimethylformamide and stirred for 24 hours at room temperature. Then it is evaporated in a vacuum, dissolved in dichloromethane and water, the precipitated dicyclohexylurea is filtered off under reduced pressure, the organic layer is shaken twice with water and the aqueous layer is shaken twice with dichloromethane. After drying and evaporating the organic layer, a solid syrup is obtained, which is chromographed on silica gel in a mixture of chloroform (ethanol/9:1). The peptide ester obtained, which crystallizes on trituration with ether, has a melting point of 167-168p and rotation \ [a] q° = —15° (in chloroform). 4.5 g of the above-mentioned ester is ionized in dioxane with the aid of 5% palladium catalyst on a charcoal support, the catalyst is filtered off and additionally extracted with ethanol. The combined filtrates are evaporated and the residue is triturated from isopropyl alcohol. The acid obtained has a melting point of 200-207°. 2.85 g of this acid is stirred in a mixture of 30 0 ml of water and 15 ml of tetrahydrofuran with 5 ml of DowexH50^H+ ion exchanger for 15 hours at room temperature, filtered through a hard filter and evaporated. under vacuum until dry. After trituration with ether, a colorless powder is obtained, which is 2-benzoylan-1-line-3-0-{[IL-l-/D-yl-,carbamoyl^3HkaiibooxypropylM^carphoanioyl ethyl]-(kairbamoylmethylmethylO}^2Hdesoloxy-a^-D-glycoze with a melting point 175-(177° (in the form of active substance)-. ,; 30 By converting the method described in Acta Chem. Scand. 18, 185 (1964), the suibstrate can be prepared as discussed below. 100 g 2-phenyl-4,545,6-0-;isopropylidene-DHglyco- furan]-zl2Joxazoline, excluding moisture and carbon dioxide, is dissolved in 1 liter of acetonitrile and, with vigorous stirring, 15.2 g of a 55% suspension of sodium hydride in mineral oil are added in portions, and stirring is continued for an hour at room temperature. Then 42 ml of ethyl chloroacetate are added dropwise at 0°C and after 1.5 hours, a further portion of 42 ml of ethyl chloroacetate is added. After 1.5 hours, a further 11.4 g of sodium hydride suspension is added, the mixture is stirred for another 45 hours and, at 0°C, a further portion of 42 ml of ethyl chloroacetate is added dropwise. After For another 2 hours the whole is left under vacuum - at the end of the procedure under oil vacuum - to the consistency of syrup. This syrup is dissolved in ethyl ether, shaken three times with water, the ether layer is dried over sodium sulphate and after evaporation 155 g of brown oil are obtained. It is dissolved in 150 ml of methanol and treated with a solution of 30 g of potassium hydroxide in 150 ml of water, extracted twice with ether and the ether layer is washed once with water. The ether is removed from the aqueous layer under vacuum and the pH is adjusted to &H. = 4.60 using 1 N hydrochloric acid. The precipitated crystalline The 2-methyl-4,5-[3-(carboxymethyl)-5,6-O-isopyrapylidene]-2-oxazoline was filtered off under reduced pressure, washed with water and dried. There were obtained 12,107 g (93% of theory) of this product, melting at 106-188°, having a rotation of α= δ-9° (c = 0.9 in chloroform) and α= -23β (c = 3 in chloroform). Example IV 6.33 g of 2-ethyl-4,5-[3-O-carboxymethyl-5,6-O-isopropylidene-glyceryl-2-oxazoline, 5.75 g of 2-ethoxy-N4carlboetoloxy-1,2-dihydrogen quinolin (KEDQ) and 9.3 ml of triethylamine are added to the solution of dibenzyl ester triflubiroacitate of Lnalanyl-D-glutamlmic acid (prepared from 8.3 g of N-tert-nbutyl- dibenzyl xycarbonyl-L-alanyl-D-glutamate with 5.1 ml of trifluoroacetic acid and 2.6 ml of dichloroethane by hydrolysis for 4 hours at a temperature of "40°) in 70 ml of dichloroethane. The mixture is reacted for 15 hours at a temperature of dilutes with chloroform, shakes the layer twice with water the water is shaken once with chloroform. After drying over sodium sulfate and evaporating the chloroform solution, 19.9 g of oil are obtained, which is purified on 400 g of silica gel (Merck's product) by elution with ether and then with the system chloroform: acetone = 17: 3. Pure 2-phenyl-4.5M[8-0-/l-L-{yl-D,3Hdiwuibenyloxycysifca;ribonylpropyl}4carbamoylmethyl-5,6-O-isopropylide eno-D-glycOfiurano]-Jf^oxazoline with temperature melting 113M1160 ik with rotationality \a]q = —47° (c = 1.57 in chloroform). 7 g of the above compound was hydrogenated with 1.8 g of palladium catalyst on carbon in a mixture of 80 ml of tetrahydrofuran and 20 ml of water until the reaction ceased, the catalyst was filtered off under reduced pressure, the filtrate was evaporated in vacuo and the residue was triturated with ether to give 4.9 g of dicarboxylic acid as a colourless powder. 4.4 g of the above dicarboxylic acid was stirred for 20 hours at 40° with 1 ml of Dowex-50IWx4 ion exchanger in a mixture of 45 ml of tetrahydrofuran and 20 ml of water. After filtration and clarification with carbon (called Darco G 60), the solution is freeze-dried, obtaining colorless, amorphous 2^benisamidOH2^deoxy-3-0- -[l-L-yylHD,3Hdikar! boxypropyl/^kaTibammolyl ethyl-ololHcarlbambiylomiethyio-DHgMcopyranose with optical rotation [a]™ = +125° (c = 0.907 in water). Example V Analogously to Example IV, 5.7 g of 2Hphenyl-4.5^3jOHcarboxymethylo^^HOHisoIpropylidene-D-glycofiuralnol-zl*-oxazoline are condensed with 4.9 g of α-amide acid y-ester hydrochloride L-seryl-D-glutaimine to the addition of 2.3 ml of triethylamline and 5.2 g of 2-toxy-methyl-ethylamine Cl,2-dihydroxy-Clhinoline in 45 ml of dichloromethane. After 18 hours at 40°, a crystallisate is formed. These crystals are added to another portion of 50 mL of dichloroethane, cooled in ice and filtered off under reduced pressure! and washed with cold dichloroethane. Analytical-pure, colorless crystals of 2-ffieinyl-^,5-[3-0-/l^-{l^Hkaiibaimoiyl-l3-III-riz.Hbuityio- xyikairboinylproipylol^kaiiitamoyl-^hyidiroxyethyl/^kairbaimoylmethyl-5,6^0-isopropyliideine-Dngli- cofuranol-^-oxazolines have a melting point of 187^188° and a rotational rate of [a]™ = +7° (c = 1.125 in methanol). '2 g of this compound is hydrolyzed at room temperature for 20 hours with a mixture of 15 ml of methylene chloride and 5 ml of trifluoroacetic acid. The whole is evaporated in an oil vacuum, the residue is triturated with ether, giving 2-benza1m,idoH2MdeiZOJksy-3-0-[! 1-L-/1-D- Hcarbamoyl-3-ikairboixypropyyl/-lka;rbamiolyl-2^hydroxyeitylol-icarbamiloimethyl-D^gfliylcopyranotze in the form of a beige-colored powder with a melting point of 100-416° and a rotation of [a]^° — = +23° (c = 0.886 in water), which crystallizes with 2 moles of water and 1 mole of trifluoroic acid. The product has an Rf value of 0.28 in the system CHOl3:CH3OH = 1:1 (thin layers of silica gel, a Merck product). Example VI. Analogously to Example IV, 5.25 g of 2-phenyao-4,5n[3,0-(carboxymethyl-5,6-0 4'zopropylidene-DHglycofuran]-^8-olkisaizoline and trifluoroacetic acid salt with α-benzyl ester of a-npropylamide acid are condensed L-alanyl-DHglutanylamine (prepared from 6.2 g of N-III-yl-propylamiiide benzyl ester of N-III-irzHbultylioxyicarbonyl^L-alanyl^DHg(1:ultaminic acid) and 4.2 g ml of trifluoroid acid in 2.5 ml of didichloroethane at this temperature for 6 hours perature 40°) in 60 ml dichloroethane in the presence of 7.75 ml of triethylamine and 4.8 g of 2-NH-carboethoxy-1-dihydrogen methyloylmethane. After 20 hours at 40°, it is diluted with 50 ml of chloroform, shaken twice with water, and the water is shaken twice with chloroform. After drying and concentrating the chloroform layer, 15 g of oil are obtained, which is purified on 200 g of silica gel (Merck product) by elution with ethyl ether and then with chloroform:acetone = -7:3. 6.4 g of a colorless, amorphous substance are obtained, having an Rf value of 0.35 in the CHO3:acetone system = 7:3. (thin layers of silica gel, a Merck product). These substances are hydrogenated with 1.8 g of 5% palladium catalyst on a carbon support in 80 ml of tetrahydrofuran and 20 ml of water until the reaction ceases, the catalyst is filtered and the filtrate is concentrated. This acid shows Rf = 0.58 in the system CHJCl3 : CH3OH = 3:1 (thin layers of silica gel, a Merck product). It is then mixed with 10 ml of Dowex 50-Wx4 ion exchanger, 50 ml of tetrahydrofuran and 25 ml of water for 15 hours at room temperature and for 1.2 hours at 40°. After filtering, clarifying, coal (called Darco G 60), repeated filtration and freeze-drying, a polymer-free, amorphous 2-benisamido-2-deoxy-3-0H[l-D1711^D^N'HnHpropyQocarbamoyl- -S^kaiixipropyl/^arbaimoiiliethyiol^kairbamolyl-methyl-D^gaiicopyranoize, with a melting point of 65^140°, a specificity of Md = +28° (€ = 1.03 in water) and a value of Rf = 0.48 in the system CHCl3: €H3OH = 1:1 (thin layers of silica gel, Merck product). ao- Example VII. 7.3 g 2-phenylJ4,3-1[3-0-carboxymethyl-5,6-0-icoproipylidene-D^glycofuraine]^A2-oxazolim, 6.5 g of L-amiminosobultyryl-D-glutamic acid γ-ester hydrochloride and 2.9 g of chloroniroscizan 15 butyl acid is dissolved in E5 ml of methylformimide and 50 ml of dichloroethane. For this purpose, a solution of 6.1 ml of triethylamine in 20 ml of dichloroetham was added dropwise at a temperature of -1115° to -40° within 30 minutes. 20 'Then it is allowed to warm up to room temperature and continue to cook for another 15 hours at room temperature. The whole mixture is diluted with 50 ml of dichloroethane, shaken twice with water, twice with 0.5 N NaOH and three times with water, and the aqueous layers are shaken twice with dichloroethane, the organic layers are dried, and after evaporation 116.5 g of oil are obtained. The oil is purified on silica gel. :(product of the company - MerCk) by elution with the system CH1OI3: 30 : Cs^OH 1= 19:1: 9.7 g of colorless, amorphous 2-phenyl-4,! -tert-butyloxycarbonylpropyl)-carboxymethylethyl]-5,6-O-isopropyllidene-D-glycofuran-ZP-oxazoline having an optical rotation of [α] = +6° (c = 1.027 in chloroform), melting point 75-89°, and Rf = 0.35 in OHCl3 : C2H5OH = 9.11 (thin layers of silica gel, product of Merck). 8.3 g of the above compound were kept for 15 hours at room temperature in a mixture of 20 ml of trifluoroacetic acid, 60 ml of methylene chloride and 2 ml of water. Then It is evaporated in vacuo and the residue is mixed with ether. The formed, differently colored powder is dissolved in 200 ml of water and clarified with 0.5 (carbon) called Dairco^G^60. After filtration and evaporation, a colorless, amorphous 24entómido^2Hdezok! sy-3- -0-i[1Hmeityl-1-/EMl-ika;rbamoylO-3Hcarboxypropy- 1o/-icarbaimoiilo eltyloj^carbamolyloimeTtyl-D^glycopyranose with a melt temperature of 110'—IIIZ1O0, with a rotation of [a]^,0 = +i310 (c — 0.88 in water) and with a value of Rf = 0.52 in the reaction: acetone: ethanol = = 1:1 (thin layers' of silica gel, a product of the Merdk company), which substance crystallizes from 0.6 mole of trifluoroacetic acid and 1.7 mole of water. 60 Prizyclade VIII. Analogously to the case of j-cladease IV and with 2-benisamido-2-deso:kisy-3-OHkanboixymethyli-^-ethyl-D-glycopyranoisidide and the trifluorocarbonate isyl acid with the IH-iriz.-buityllyl ester of Ikwaisumide L-alanyl-D-gCl'Uitarnline 65 and z 2-,ethoxy-N,(a)-betoethoxy-N,(b)-hydrogen quinoline, the corresponding glycopeptide with the rotation coefficient [α] = —f23° (c = 1.107 in methanol) and the Rf value — 0.47 in the system CH2C12:C2H5OH = 8:2 is obtained, and after hydrogenation with the help of a 5-hydroxy-palladium catalyst on a carbon support in the system etherfuran/water, 2-benzamo-2-isopropyl-3-(O-(L-1-/D-1-carboxylic acid) is obtained. bamoyl-3-carbofcsypiropyropyio/^rarbanioylethyylHcarbamoylmethyl-^-ethyl-diglyicopyranoside with a melting point of 215-217°, with rotation [a] £° = = -^22° = 0.36 in the CHOl^ system: CH3OH = neither (dark) silica gel layers, product of Merck company). 15 2nbenzamiido^2-deisoJkisy- -3-0-! was used as the sulbstrate! Cairo! bbixymethyl-i/? ^ is obtained as discussed below. 2-phenyio^,54i3-OHkarbokisymejtyao^,6-04! zo(propy- Lidene-D^glycofuran]-D^oixazoline is dissolved in 0.1 N HOl solution in ethanol and left for 6 hours at room temperature. It is neutralized with sodium ethylate in ethanol, evaporated to dryness and dissolved in acetone. The solution is filtered through a layer of silica gel (Merok diulfite), the eluate is evaporated to dryness and the residue is extracted twice with ethyl ether at room temperature. After recrystallization from ethyl acetate, 2-benzaimido-2-deoxy-3-O-carboxymethyl-D-ethyl-D-glycopropylaminoside is obtained. 9.4 g of this ester are washed with a solution of 1.7 g of potassium hydroxide in 250 ml of ethanol and 35 ml of water for 2 hours at room temperature. The pH is adjusted to 3.5 with 1.0 hydrochloric acid and concentrated in vacuo. The residue is triturated first with ether and then three times with 20 ml of ice-water each time and filtered off under reduced pressure. Crystals of 2-benzamine are obtained in this way. doH2^deoxy-34)-ikariboxymethylethylOHD-gilylcopyranoside with a melting point of 205-2H0° and a sicreaticity of 120 45 (a]gj , = -40° lu). Example IX. 24enzaimddo «2Hdesofcsy-3-0^[D- -lncarbamoyl^^carboxypr^ tyl]Hcarbamoylmeityl4DHglycoze is obtained from ¾ 3 g of 2-phenyl^y543-0-{/lHlJkairbaimoylO'-3^isopropyl-lidene-Dig]i! cofuran]-^2-oxazolines by hydrolysis with 1.5 sun of triphylluroacetic acid in a mixture of 15 ml hydrimethoxylate and 15 ml of water at 40°C for 3 hours. The whole was concentrated in vacuo to dryness and the residue was extracted again with ether. The powder obtained as the residue was dissolved in water and treated with Darco-G-60 charcoal, filtered and lyophilized. A colorless, amorphous sulfur is obtained, melting at 11-155°, with a rotation [α] = +34° (c = 0.81 in water) and with an RF value of 0.28 in the CHGlj:CH3OH system (thin layers of acetic acid gel, product of Merck). The sulfur is prepared in the manner described below. 8.0 g of the benzyl a-amide N-III-z,N-butyloxycarboinyylglycylonD-gluitam:ine acid ester are dissolved in a mixture of 6.3 ml of trifluoroacetic acid and 7 ml of diMoroethane and reacted for 2 dhi at room temperature and for 13 hours at 45°. While cooling, 12 ml of tri-atylamine, 7.0 g of 2-ethoxy-N-carboethoxymethyl-5,2-dihydroquinoline (EEDQ), -8 ml of 2-phenyl-(1.5H[3-0- ^carboxymethyl-5.6^0-hysopropylidene^D-glycofu- in the morning]-Zl2JOxazoliiny i 20 ml of dimethylformamide. After 20 hours at 40°, the whole was concentrated in an oil vacuum and the residue was partitioned between methylene chloride and water layers. After drying and concentrating the methylene chloride layer, a solid residue was obtained, which was extracted twice with ethyl ether and recrystallized from toluene, giving 8.25 g of a product with a melting point of 1157°, an optical rotation (σ]d = +110° and (c = 1.48 in chloroform) and an RF value of 0.35 in the chloroform:ethanol system = 0:1 (thin layers of silica gel, product of Merck). The benzyl ester thus obtained was hydrogenated with 1 g of 5'/o palladium catalyst on a carbon support in 100 ml of tetrahydrofuran and 25 ml of water until the reaction stops. After filtering off the catalyst and evaporating the substances, the substances are chromatographed on 250 g of silica gel (product (from Merck) in the system CHOI3 CH^OH = 4:1. 5.8 g of colorless, amorphous 2-phenyl-4,5H[3-0-{/D-l- Hkarba.moi! lo-.3Hcarboxyiprop^l-ikaribamoylmethyl-ol^carbamoylmeltylOHS^e-O-isopropylidene-D-glycofuran]-z12-oxazoline with a value of Rf = 0.53 in the system CHCl3 : CH^OH = 3:i2 ((thin layers of silica gel, Merck product). Example L-alyanyl-D-lutamic acid diamide in the presence of 3.4 ml of triethylamine and 7.95 g of 2-ephthoxy-N-carboethoxyHl,2-dihydroquinoline (EEDQ) in a mixture of 50 ml of dichloroethane and 160 ml of dimethylformamide the entire reaction within 2 days at room temperature and for 4 hours at 40°. Then it is concentrated in an oil vacuum and the residue is extracted first twice with ether and then twice with ice-water. After drying, the product is recrystallized from chlorchloroethane, obtaining colorless crystals with a melting point of 170-1184°, with a rotation (d\™ = +3° dim) and a value of Rf = 0^4 in the system CH1Cl3 : : CH3OH = 3:1 (thin layers of silica gel, product of Merck). 6.1 g of this compound is hydrolyzed with 113.5 ml of an ion exchanger called Dowex'50 in a mixture of 60 ml of dimethyloxyethane and 60 ml of them! water in cold water for 15 hours at room temperature. After filtration, the residue is dissolved in water, clarified with carbon called Darco-G-60, filtered under reduced pressure and the filtrate is freeze-dried. Obtained isie Ibezicolored, anhydrous 2nbenziamido-2-deooxy-3-0H[Li-yl-/DHl,3^di-carbaino.ylpropyl(yl/-!car,baimoylethyl]Hcarbamium)dilomethyl-D^gilylcopyraninose, melting point 82-143°, M^0 = +04° (c = 0.98 in water) and with a value of Rf = 0.4)5 in the CHOI3 system: CH$OH = il:d (thin layers of silica/calcium gel, product of Mencik). This substance crystallizes from 1.23 moles of crystallizing water. The compounds given below, marked with the letters a.-^z, are obtained analogously. a) 3-0-{![ L-il-/Dnl-4cairbainoiilo-3Hkairiboiksyipropyl- (0^930); b) 3^)-{(Pwl/D-14catrbaimoto ikaiiibaonoylpropyl]^kainbamoylpropyl}-42-aiceltajmi- do^ndazic^y-DiglJilkoza o .s|kirecalinoiscii Md = = +46°±yl° (water, c = 0.63); c) 3-04L-1-/D-1^kainbaimoyl-3-icarboloxyipro(pyio/- ^kairbamoiioeityylJHkatfbamoiyl^ -2Hdeoxy-,/?-eityyl-D^gLicolate:anoside with (melting point 2115-217° and sikrecailinoiaei Md = -22° (methanol; d) (e) 2-beiniza[mcyclo[H2-ideoxy-3H][iL-(D-1,3-bis-N-methylcarbamoylpropyl)carbamoylethyl]carbamoylmethyl-diglycoypyranoside with a melting point of 233°-240° and a rotational speed of −20° (methanol, c = 0.937); (e) 2-beiniza[mcyclo[H2-ideoxy-3H][iL-(D-1,3-yl)(bis-N-methylcarbamoylpropyl)carbamimoylethyl]carbamoylmethyl-diglycoypyranoside with a melting point of 125°-320° and a rotational speed of −20° (methanol, c = 0.937); = +24° (water, c — 0.93);; f) 2-benzaim! ido^2HdeoxyM3-OH0L.-yl-(D-'l,3^metoxycaiibonyylproipyl/^aribaimoiiylethyiol^aribarnoylmethyl-D^giliiikoipiraiose in the form of hydrate with a melting point of 80-90° and a siccrecity of Md = +25° and (meitano! lr c = '1.017); g) ethyl-2^benzimido^2-|deooxy-3-0-([iL^l-yiDnl,3-buis-metho-ikaorbaimioylmethyl-^-Dngiylkoipirainoiside with (melting point 127-1315° and skirecailnoisci Md° = »= ^17° (methanol, c = 1.024); i) islkrecailinoisci MD° = +21° (water, c = yl.08); bamoiylmethyl^D^glycopyranose with (melting point 114-1155° and sparkle Md = +34° (water, c = 0.83); j) 2-lbenizaimildo-3^dezoics6y-(3H0-{L-d-/D-d-ikasba- moilo-SHkainboJksyproipylo/HkaTibaimoyl ^2nmethylylcipro- pylolHlncarbaimoylmethylOHDHglycopyranose with sikre- 10 15 20 25 30 35 40 45 50 55 60 .20 total Md = 32° '(water, c = 0.78);05k) 2-aceiamide^H()^[/DHl^carbamodlo-3-carboxy^ propylZ-carbamodlomethyl^ka1^ -deoxy-D-iglycose with rotational Md = +27°± ±1° (water, c = 0.944); i) methyl-2-acetaimido-3-O-{(L-yl-/D-fl^ka;ribaimoyl-3ncarboyloxypropyl-ika^baimoylethyl]4-cartolomethylol-ideoxy-a-D-glycoipirayine iside with rotation, [a]D° = +49°±ll° (water, c = 0.913*9); 1) 2-acetamido-3-0-{[Lnln/Dnl,3-idwulka,r! baimoyl-propyl/^caa^baimoyl-eityl/Hkaribaimoyl-anetyl}^^ m) 2^ce'1aimido-3-{D-l-i[DHlHkar! bamoiyl-3-carboxypropyl/^arbamoiionietylol-kairibarnoiylpropylo}n2-dezoikisy-D^g|lyyl alcohol with sicrecality Mr. = = +46°±1° (water, c = 0.630); e) 2-acetamido-3-0-dimethylyl ester = 0.814); (p) 3-iÓ-{l[Lil-/D-tl-|karlbaimoiyl^3-ikairibok;sypropyl-/ikairibamoiilethyllolnkaribamoylmethydo}-2-kaipiynoiiloa(midone2-(deoxy^D-iglycase with accreioalinity Md = +ia°±)l° (water, c = 1.0523); 2-acetairnddoH2^deisooxy-3H0-{i[L-yl-/D-1,3-ibi3- Hmethylcaribaimoylproipyl/Jcarbaimoylproipyl]carbamoi'loathiethyl}^Iigilicoase with sicrecality Md ^ = +3yl°±yl° (water, c = i0.410); is) 2-acetate! mido-3^0-{i[Lnl-/DMlnkarbaimo! iilo^3-kair- boiksyipropy.lo/-ikairibamodiloH2Hhydirolkisejtyll]Hkarto^ moilomethyl^Hdeooxy-Dngglycosis with rotationality MD° = +H0°±(1°, (water, ic = il.053); rt) 2^ce^imddo-3-0-{i[L-l-/DHlHkai]^baimo,iyl-3-jcarboxypropyl/^carbaimoiiiolbutyl]HcarbaimoiIoimeityl}-2^deooxy-DHglylkylose with slkrecaylmosity Md =* = +.12°±1° (water, c = 0.722); u) 3-0-([L-.l-/D-l-ika'rbanioilo^3^kairboiksypropyl-lo/-karbaimoiiloet3^1oJkarbaimoliiloaiieityilo}-2-dezokisy-» 20 -2-iateairoiloaimddo-Diglycase with Md ^ = +24°±yl° (idwumetidofonmamide, c = 0.6©3; v) 2-a(cetaimiido-3-0-{[mi-ZD-d-ikaribamoiao-S^ar- bo(xipropyl/Hka;rbamoylon2Hmyethyl;pro(pylo]-kaT- baimoiylmethyl}-2ndezooxy-iD-iglylko! for o ^crecal«ness +ill0±jl° (water, c = mp,096); w) 2Hacataimiido^3-0-{[L-yl-/DnUkairibamoyl-3- boxypropyl Qo/ncarbaimodil-(3-imethylibuityl]-jka:rba moiiioimeityyl}-2-deisooxy-D-(glycose with rotation Md = +<16°±1° (water,^c = il,0i60); (water, c = (l.0yl'9); y) 2^aceitamido^3-0-{[L-H-ZD-^l-featribaimoiyl-S^kaT* mL0 Md sikrecality [a]g = +12°±1° (water, c ¦= 0.706); iz) 2-aicata'miido^3^0-{i[L-l-/D^lHkair[bamoiylmethyyl- -carifoamoiyl-34carfooyloxypropylM-.carfoainoiyl eity- -carbamoylmethyl}n2idiezo,xi-Diglycose of ealnosoic ([a]£° ,= .+il0o±l° (rwater, c = O.7i06); z) 2ntTÓ'jmety(loaceitamiido-n2Hd€isoifcsy-3-0- {[L^l- kairbaimoyl-3^kalrboiksyproipyl/-(carbaim)o,yl eityl]- Io]Hcarbam'oyl'ethyl}-a,^-Digliiko,a with a spark of M 20 = -7° (methanol, c = 0.6). alkyl with 1-18 carbon atoms or (phenyl steroid, Rj denotes an atom of hydrogen or a halyl radical with 1-3 carbon atoms, R2 denotes a hydrogen atom or an alkyl radical with 2-3 carbon atoms, R4 denotes a hydrogen atom, R6 denotes an atomic atom of water, R7 denotes a hydrogen atom, an alkyl radical with 2-5 carbon atoms vegUa, hydroxymethyl group or phenyl radical, Rg stands for carboxyl group, alikooxylcarboinyl group with 1h^3 carbon atoms 10 15 20 25 in the alkoxy part, carbamoyl group, N-alkylcarbamoyl group with 1-4 atoms carbon in the alkyl part, group N-benzylcarbamoyl group or the (carbamoylmethylylcarbamoyl group, R9 stands for a carboxyl group, aflJkolxylcarboxyl group with 1-3 carbon atoms in the alkoxy part, the [benzyl|xylcarphoonylax group, the N-alkyl carbonyl group with d-4 carbon atoms and an alkyl part, provided that the alkyl radical R contains more than 1 carbon atom and in the case where X is a carbonyl group and R2 is an immethyl radical, or and in the case where R9 denotes carboxyl groups or salts thereof, characterized in that (the compound of formula 3, above (in which R and R2 have the meaning given above, R70, Rg° and R90 have the meaning given for the symbols R7, R10 and R9, and in which the hydroxyl groups contained are protected by easily cleavable ether or ester radicals and 1R5 denotes an alkylidene or alkylidene radical, the oxyazoline and dioxolane rings are cleaved under acidic conditions in the presence of (the compound of formula R1--OH, in which R2 has the meaning given above, and any protecting groups present are cleaved off.112 672 CHOR Rfl^° M / NHXR RiC-H ^7 Re tO-NH-ChhCONH-CH (CH2)2-R9 Formula1 f D) CH? OH f? 8 H^0-R, 7 NH-CO-R Ra-C-H ^ XCO-NH-CH-CONHi5H-|CH2)2-R9 Formula Z R C°] .0 ,0 R,-CH O N-C-R 78 CONH-CH-CONH-CH-tHtRl Formula 3112 672 R5^ n o o n ril N=C-R R2-CH R° p| ^3XC0NHCH-C0NH-CH-(CH^R H+ I HOR! R,-CH r; tONHCH - C0NH-CH-(CH^2-R9° HO- tf HOR1 ,0, -ORi R2-CH HN-C-R Rz O ?: CONHCH-CONH-CH-(CH^R( I Setami 4 CH2OR6 *fto-x-R CO-NH-CH-CONH-CHfH^-Rg c.fif- Scheme i (DN-i3, izaim.35/82 Price PLN 45 PL