PL133485B1 - Process for preparing novel 3-/5-ribonucleotides/ of klindamycin type compounds - Google Patents

Description

The subject of the invention is a method for preparing new 3-(5'-ribonucleotide) compounds of the clindamycin type, in which the propylhyric acid radical has been replaced by a radical of another cyclic amino acid, of the general formula 1, in which Rj represents a hydrogen atom, an optionally substituted alkyl radical having in the alkyl part a total of 1-8 carbon atoms, also its isomeric forms R3 means a hydrogen atom or a CH3, -C2H5 or -CH2CH2OH group, n means an integer from 1 to 4 inclusive, Y means 7 (S) chlorine or 7(R) chlorine, optionally in the form of pharmaceutically acceptable salts thereof. The term alkyl group means a straight or branched chain alkyl group. The characteristics and preparation of clindamycin of formula 9 are described in U.S. patents. No. 3,086,912 and 3,496,163. This antibiotic is widely used in the treatment of humans and animals. A number of patents have been granted in many countries for this antibiotic and its various derivatives. Clindamycin 3-nucleotides are described in US Pat. No. 3,671,647. These compounds contain a propyl hygroic acid unit. In in vivo tests against S. aureus, the above 3-nucleotides have an activity equal to approximately 1/10 of the activity of the parent compound. The method according to the invention provides new compounds - 3-ribonucleotides of such clindamycin derivatives, in which the propylhydric acid unit has been replaced by a radical of another cyclic amino acid. Surprisingly, it turned out that these nucleotides have an in vivo antibacterial activity as high as the parent compounds. Due to these properties, the compounds whose preparation is the subject of the invention are considered candidates for use in medicine. They are also useful in the prevention and treatment of patients infected with protozoa. Clindamycin-type compounds that can be converted into 3-ribonucleotides according to the invention can be represented by the formula 10. In these compounds, the hydroxyl group in the 3rd position of the lincosamidine unit has been replaced with a nucleotide from the group including adenylic, guanylic, cytidylic or uridylic acid. In formula 10, Rj may be substituted at any position of the pyridine ring not substituted by R2 and has the meaning given above, and R2, which may occur once at any position of the pyridine ring not occupied by Rr, denotes the group -C(0 )X, wherein 133 485 r133 485 According to the method of the invention, the discussed 3-(5'-ribonucleotides) of the general formula 1, in which Rj, R3, n and Y have the given meanings, are prepared by microbiological transformation, by culturing Streptomyces rochei, NRRL 3533, in water a medium containing a source of carbon in a digestible form as nutrients. carbohydrate and a nitrogen source in the form of an available nitrogen compound or protein substance, in the presence of the compound of formula 1, and isolates the desired 3-(5'-ribonucleotide) from the medium. Carbohydrates such as glucose, unrefined sugar, sucrose, glycerin, starch, corn starch, lactose, dextrin, molasses, etc. are used as a source of carbon in the nutrient solution. Corn soak, yeast are used as a source of available nitrogen or protein substances. , autolyzed brewer's yeast enriched with milk powder, soy flour, cottonseed flour, corn flour, milk powder, animal peptone solutions, meat and bone waste, etc. In the case of the production of 3-(5'-ribonucleotide) compounds represented by formula 2 or 3, cultures of Streptomyces rochei NRRL 3533 are grown in an aqueous medium in the presence of the compound of formula 2 or 3, respectively, and the desired 3-(5'-ribonucleotide) is isolated from the medium. In the same way, 3-(5'-ribonucleotides) of the compound of formula 4, in which R is hydrogen, marked with the code number U-57930, are obtained. 3-(5'-ribonucleotides) obtained by transformation of the compound marked with the code number U-57930 are presented in formula 4, in which R stands for the 5'-cytidyl radical (formula 5), the 5'-aderryl radical (formula 6), a 5'-uridyl radical (formula 7) or a 5'-guanyl radical (formula 8). The parent compounds of formula 10 can be obtained by methods described in US Patent No. 4,278,789. Since the compounds prepared according to the invention are amphoteric substances, they can be converted into salts with acids or bases. Examples of inorganic acids that can be used to prepare salts are hydrochloric acid, sulfuric acid, phosphoric acid and the like. Examples of inorganic bases are sodium, potassium, calcium, lithium and the like. Salts of the compounds can be used for the same purposes as the parent compounds. Prepared According to the method of the invention, nucleotides can be tested and characterized by the following methods: Determination of 3-(5'-ribonucleotides). Since the 3-ribonucleotides in question do not exhibit antibacterial activity in vitro, their formation from antibacterially active parent compounds can be easily monitored by measuring the loss of antibiotic activity. To determine the amount of antibacterially active parent compound in the culture filtrate or reaction mixture, the standard Sarcina lutea ATCC 9341 test is used. To determine the presence of 3-ribonucleotides in the fermentation liquid, extract or purified material, first the phosphodiester bond is hydrolyzed using crude alkaline phosphatase or snake venom phosphodiesterase in the manner described below. The antibacterial active compound in the hydrolyzate is determined in a standard test. Enzymatic hydrolysis. io Alkaline phosphatase: Stock solutions (0.5 mg/ml, 0.54 units/mg) of alkaline phosphatase from pigeon intestines, EC 3.1.3.1 (Sigma) are prepared in Tris hydrochloride buffer, i.e. Tris-(hydroxy-methyl)- aminomethane, 0.01 m, pH 8.0. The treated samples are diluted 1:2 with buffered enzyme and incubated at 28°C for 18 hours. Snake venom phosphodiesterase: Stock solutions (100 mg/ml, 0.026 units/mg) of purified snake venom phosphodiesterase are prepared. in distilled water. The incubation mixtures contain 0.2 ml of a solution (1 mg/ml) of the treated sample * in water, 0.6 ml of 0.01 M Tris hydrochloride buffer, pH 9.0, 0.1 ml of 0.3 M MgCl2 and 0 1 ml of the enzyme stock solution, incubation is carried out at 37°C for 18 hours. Spleen phosphodiesteresis: Stock solutions of spleen phosphodiesterase EC 3.1.4.18 (Simga) are prepared in distilled water (1 mg /ml, 19.6 units/mg). Incubation mixtures contain 0.4 ml (0.5 mg/ml) of sample for treatment in water, 0.5 ml of 0.02 M Tris buffer solution, pH 7.0 and 0.1 ml of stock solution enzyme. Incubation is carried out at a temperature of 35-37°C for 18 hours. Thin-layer chromatography of enzymatic preparations and hydrolysates. The production and purification of 3-ribonucleotides is monitored in the S, lutea assay (see above) and thin-layer chromatography (TLC) on silica gel G, using methylethyl ketone-acetone-water (186) as the developing system. :52:20, by volume) or ethyl acetate-acetone-water). Bioactive parent compounds are detected bioautographically on agar inoculated with S. lutea. The products of enzymatic or chemical hydrolysis of 3-nucleotides are separated in the following TLC systems: . 50 A: GF silica gel plate (Analtech Inc.); water as a developing system. B: GF silica gel plates; n-propyl alcohol-concentrated ammonium hydroxide-water (55:10:35, by volume). 55 C: NM-Polygram Cellulose 300 (Brinkman Instruments Inc.); butanol-1-water-formic acid (77:13:10, by volume). Materials that absorb ultraviolet (UV) are detected using a lamp that emits in the short-wavelength UV range. Biologically inactive and non-UV-absorbing materials are detected by spraying with the permanganate-periodate reagent. Bioactive nucleotide materials are detected bioautographically on agar inoculated with 65 S. lutea.5 Fermentation and purification methods are shown in the examples. Using the procedure of these examples, 3-ribonucleotide compounds of formula 10 can be obtained. Since the compounds prepared according to the invention are active against various Gram-positive and Gram-negative microorganisms, they can be used in various environments to inhibit the growth of such bacteria. For example, they can be used as disinfectants to inhibit S. aureus on washed and stored utensils for preparing food products infected with this bacterium. They can also be used as disinfectants for various dental and medical instruments. infected S. aureus. In addition, the compounds of the invention can be used as bacteriostatic rinse baths in clothing washing and for impregnating paper and fabrics. They are also useful in inhibiting the growth of susceptible organisms in plates and other microbiological environments. The compounds discussed are useful in the treatment of diseases caused by microorganisms of the Mycoplasma type. The most famous of them are PPLO (pleuropneumonia-like organisms), such as M. hominis, M. salivarium, M. mycoides, M. hyopnegomonia, M. hyorhinis, M. gallisepticum, M. arthriditis and other species in humans and animals, including domestic animals such as sheep, dogs, cattle, pigs and poultry (e.g. chickens, turkeys, ducks and geese) and laboratory animals (e.g. rats and mice). 3-(5-ribonucleotides) of the compound of formula 4, in which R stands for groups of formulas 5-8, can be used in the treatment of infections of the kidneys and other organs when L-forms of gram-negative and gram-positive bacteria are present, e.g. L-forms of the P. mirabilis bacteria. Moreover, the compounds produced by the method according to the invention can be used in the prevention and treatment of patients infected with protozoa. For example, when the protozoan is a malarial parasite, the treated object may be an animal, e.g. a mouse infected with Plasmodium bergheri; birds, e.g. ducks infected with P. lophurae or chickens infected with P. gallinaceum, and mammals, such as primates, e.g. monkeys infected with P. cynomologi and humans infected with P. falciparum, P. vivax and P. malariae. Administration of the compounds prepared according to the method of the invention, it is possible to treat mammals that are carriers of parasitic protozoa of the Sporazoa class of the order Coccidia (a microparasite causing the disease coccidiosis). For example, cattle infected with Eimeria zurnii, E. bovis, E. ellipsoidalia, sheep and goats with E. parva, E. faursi, pigs with E. debliecki, E. scabra and Isospora suis, dogs and cats may be subjected to such treatment from Isospora bigemina, I. felis, E. canis, E. felina, poultry from E. tenella, rabbits from E. stiedae, W. perforans; and minks with E. mustalae. Other protozoans that can be combated using the compounds produced according to the invention are intracellular parasites, e.g. species of Flasmodia, Toxoplasma and Leishma - 485 $ ¦::_ ¦¦-¦, - nia; protozoa that digest the red blood cells (RBCs) of treated patients, e.g. Entamoeba tica and some Trypanosoma; and parasitic worms that digest RBCs in disease processes, e.g. Schistosomes. The compounds prepared according to the invention and used as antibacterial agents are prepared in the form of compositions, in a form suitable for administration to humans and animals, such as tablets, capsules , pills, powders, granules, sterile parenteral solutions or suspensions or oral oil-water solutions, suspensions and emulsions, containing appropriate amounts of the active compound in the form of the free base or its pharmacologically acceptable salt. For oral administration, it can be used solid or fluid forms. To prepare solid compositions such as tablets, the basic active ingredient is mixed with conventional excipients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminosilicate, calcium sulfate, starch, lactose, gum arabic, methylcellulose and functionally similar materials constituting pharmaceutical solvents or carriers. Tablets can be laminated or otherwise formed, obtaining a drug with a prolonged or delayed action or with a specific sequence of action of the active substances. For example, a tablet 30 may contain an inner and outer ingredient, the latter in the form of a coating on the former. Both components can be separated by a layer called the intestinal layer, the purpose of which is to prevent disintegration in the stomach and allow the internal component to pass intact into the duodenum or delay the secretion of the active substance. A variety of materials may be used for such enteric layers or coatings, including polymers. rical acids or mixtures of polymeric acids with materials such as shellac, cetyl alcohol, cellulose acetate phthalate, styrene-maleic acid copolymer and the like. Alternatively, a two-component system can be used to prepare tablets containing two or more active ingredients that are not you can mix them together. Wafers are prepared in the same way as tablets, which differ only in shape and content of sucrose or other sweetening and flavoring substances. 50 In the simplest version, capsules, similarly to tablets, are prepared by mixing the active compound with an inert pharmaceutical carrier and pouring the mixture into hard gelatin capsules of appropriate size. In another embodiment, capsules are prepared by filling hard gelatin capsules with polymeric acid-coated beads containing the active compound. Soft gelatin capsules are prepared by machine encapsulation of a suspension of the active compound in appropriate vegetable oil, liquid petroleum jelly or other neutral oil. Medicines can also be prepared in liquid form for oral administration, such as syrups, elixirs and potions. suspensions. Dissolve the water-soluble forms of the compound 13&47 in an aqueous vehicle, combine with sugar, aromatic flavoring agents and preservatives to obtain a syrup. The elixir is prepared using an alcoholic vehicle (ethanol) with appropriate sweetening agents, such as sucrose and aromatic fragrances. Suspensions can be prepared from insoluble forms of the compound and the vehicle and a suspending agent, such as gum arabic, tragacanth. , methyl cellulose and the like. Topical ointments may be prepared by dispersing the active compound in a suitable ointment base such as petroleum jelly, lanolin, polyethylene glycols, mixtures thereof and the like. Preferably, the compound is ground finely in a colloidal mill, first into liquid Vaseline, and then into the ointment base. Creams and lotions for topical use are prepared by distributing the compound in the oil phase and then emulsifying the oil phase in water. For parenteral use, liquid forms of the drug are prepared from the active compound and a sterile carrier, preferably water. The active compound may be suspended or dissolved in the carrier, depending on its form and concentration. When preparing solutions, soluble. the water form of the compound can be dissolved in water for injection and sterilized by filtration, and then poured into appropriate vials or ampoules and sealed. Advantageously, auxiliary substances, such as local anesthetics, preservatives and buccal agents, can be dissolved in the vehicle ¬ forums. To increase stability, the compositions can be frozen after pouring into ampoules and the water removed under reduced pressure. The dry lyophilized powder is then closed in a vial, and water for injection is added to the vial, in which the preparation is dissolved before use. ^ Enteral suspensions are prepared in essentially the same way, except that instead of dissolving, the compound is suspended in the carrier, and sterilization cannot be achieved by filtration. The compound may be sterilized by exposing it to ethylene oxide before suspending it in a sterile vehicle. To obtain a prolonged effect, an intramuscular suspension is prepared from a compound in an insoluble form, such as trimethylsilyl ether or a salt of pamic acid. It is preferable to add a surfactant or wetting agent to the composition, facilitating even distribution of the compound. For systemic treatment, unit doses are prepared containing 10, 25, 50, 100, 250 or 500 mg of the active compound, and for parenteral compositions with concentrations of 15-65% by volume. The following example illustrates the preparation of the 3-(5'-ribonucleotide) compound designated by the symbol 60 U-57930, i.e. the preparation of the compound of formula 4, in which R is a group of formula 5, 6, 7 or 8. These ribonucleotides are derivatives of adenylic, cytidylic, guanylic or uryl acid. 3-(5'-ribo-65 4 nucleotides) of the compound marked U-60970 is prepared in the same way, i.e. 4-cis-n-butyl-L-pipecolic acid amide and 7-C1 methylthiolincosamidine, prepared according to the method of United States Patent No. 4,278,789, the 3-(5'-ribonucleotides of which have the same action as ribonucleotides of the compound marked with the symbol U-57930. Unless otherwise stated, the % in the following example are % by weight and the proportions of the solvent mixtures are expressed by volume. Example A. Fermentation. Streptomyces rochei, NRRL 3533 was cultured in a medium containing 10 g/ liter of glucose, 4 g/liter of Difco peptone, 4 g/liter of Difco yeast extract, 0.5 g/liter of MgS04-7H20, 2.0 Z/liter of KH2P04, 4 g/liter of H2HP04, for 3 days at 28°C, on a rotary shaker. Mycelium from this culture was used to inoculate a fermentation medium containing the same ingredients. Fermentation was carried out for 48 hours at a temperature of 28°C on a rotary shaker. After completion of the 48 hour incubation, U-57930 was added to a final concentration of 50 mg/liter and fermentation was continued at 32°C. After 12 hours, further amounts of U-57930 were added to a total concentration of 150 mg/liter. After another 12 hours, the concentration of U-57930 was increased to 250 mg/liter. After the last addition of U-57930, fermentation was continued at 32°C for 24 hours. After this time, the culture filtrates were collected and found to contain no more than 1 mg of U-57930 per liter. The remaining 249 mg/liter was converted into biologically inactive material. rochei NRRL 3533 is a known microorganism freely available from the NRRL collections at the Northern Utilization and Research Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, 111., USA.B. Isolation and purification. Isolation of U-57930 3-ribonucleotides from the fermentation broth. Adsorption on Amberlite filtration. The mycelium cake was washed with 1.2 liters of water and discarded. The clear filtrate and washings were combined, adjusted to pH 6.0 and passed through a column with 600 ml of Amberlite XAD-2 (Rohm Haas Co., Philadelphia, PA). at a flow rate of 40 ml/minute. The effluent from the column was tested for biological activity before and after treatment with alkaline phosphatase and discarded. The column was washed with 2 liters of water. Water washings that were also biologically inactive before and after alkaline phosphatase treatment were discarded. The column was then eluted with methanol-water (70:30 v/v). Fractions of 20 ml were collected at a rate of 20 ml/minute. Biological activity determination before (–E) and PO ( +e) treatment of alkaline phosphatase DALY "as follows: 133 485 Faction no. 5 10 11 13 20 25 40 45 50 55 60 65 70 75 75 80 85 90 95 100 110 120 130 140 150 160 170 180 190 200 Zone (S. lutea) ' -E 0 0 0 0 33 32 31 29 26 23 21 19 17 17 16 16 16 16 16 16 15 15 15 15 15 15 15 15 15 1 + E 0 0 0 36 55 54 50 43 39 38 37 27 26 26 26 26 1 26 26 26 21 21 21 21 21 21 21 21 21 21 1 15 20 25 30 35 40 Fractions 12-80 were combined, evaporated to an aqueous solution and lyophilized, obtaining 12.22 g of ADA-34.1. In another series of tests, 6 liters of fermentation broth containing 2 g of inactive U-57930 were treated as described above. The methanol eluates from the Amberlite XAD-2 column were stored as ADA-34.1. 1438. This solution was not evaporated to dryness, but was purified by chromatography on Dowex-1 resin as described below. Chromatography on Dowex-1. A column was prepared with 300 ml of Dowex-1 (X-4) in the acetate form. a methanol solution of ADA-1438 at pH 8.2 was passed through the column. The effluent was collected at a rate of 2.5 ml/minute in volume fractions of 20 ml. (Fraction 1-60). The column was washed with 1.5 liters of water (10 ml/minute, fractions 66-108). Then the column was eluted with 5% acetic acid (at a rate of 45 50 55 60 10 10 ml/minute; fractions 109-310). The fractions were combined into the following sets: set 1 fraction 1- 2 fractions 81- 3 fractions 111- 4 fractions 131- 5 fractions 151- 6 fractions 191- 7 fractions 231- 8 fractions 271- - 80 -110 -130 -150 -190 - 230 -270 -310 1000 ml (ADA-1A) 600 ml (ADA-2A) 450 ml (ADA-3A) 450 ml (ADA-4A) 900 ml (ADA-5A) 900 ml (ADA-6A) 900 ml ( ADA-7A) 900 ml (ADA-8A). Determinations before (—E) and after (+E) alkaline phosphatase treatment gave the following results: and set 1 2 3 4 5 6 7 1 8 Zone (S. lutea) -E 31 36 34 18 25 27 27 29 +E 52 49 45 40 30 29 29 31 Sets 1 and 2 were combined, evaporated to an aqueous solution and freeze-dried, obtaining 1.48 g of the ADA-2.1 preparation. Harvests 3 and 4 were also combined and treated similarly to obtain 2.5 g of ADA-2.2. After treatment with alkaline phosphatase, U-57930 was obtained from preparations ADA-2-1 and ADA-2.2. Preparations ADA-34.1, ADA-2.1 and ADA-2.2 were combined and purified by the double countercurrent separation method described below. Double countercurrent separation: 16.20 g of material obtained by combining preparations ADA-34.1, -2.1 and -2.2 , were dissolved in 25 ml of each phase of the solvent system consisting of equal volumes of butanol-1 and water (1:1). The solutions were added to the central tubes of an all-glass countercurrent dual separation apparatus (100 tubes, 25 ml/phase). The separation was determined, after 150 transfers, for biological activity before (—E) and after (+E) treatment with alkaline phosphatase. The results were as follows: Lower collector 1 5 10 15 20 25 Zone (S. lutea - sensitive) | -E 2 29 31 30 27 22 +E 3 1 31 33 33 33 3311 133 485 12 1 30 35 40 45 50 55 60 65 70 75 80 85 M 95 100 Lower machine 50 45 40 35 30 25 20 15 . 10 5 0 Upper machine 5 10 15 20 "25 30 35 ' 40 45 0 Upper collector 100 95 90 "||| -i HPK—^1 II II 2 17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Zone (S. li -E 0 trace 15 17 17 18 17.5 17 16 15 trace trace trace trace trace 30 trace 15 16 21 Zone (S. 1 -E 17.5 17 19 3 34 34 33.5 33 34 34 35 36 38 39 I 40 41 42 43 43.5 atea - sensitive) | + E '45 46 47 47, 5 47 47 48 48 48 50 50 49 48.5 48 48.5 49 50 51 52 53 54 utea - sensitive) +E 54 53.5 53.5 15- 20 25 30 35 40 45 50 60 1 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 2 20 21 22 24 26 28 30 32.5 33 35 36 39 41 43 43 43 35 3 53.5 53.5 53.5 53.5 53.5 53 ,5 52.5 52 52 52 ' 52 49 47 48 46 43 35 | The following collections were prepared. Each set was evaporated to an aqueous solution and freeze-dried to obtain appropriate preparations. Set I: lower collector 1-50; Set II: lower collector 51—100; lower machine 50—30; Set III: lower machine 29—0; upper machine 1—50; upper collector 100—30. The following preparations were obtained: From set I, preparation ADA-47.1 in an amount of 9.78 g. From set II, preparation ADA-47.2 in an amount of 0.30 g. From set III, preparation ADA-47.3 in an amount of 5.29 g. Preparations ADA-47.2 and - 47.3 was combined and purified by chromatography on DEAE-Sephadex: 300 g of DEAE-Sephadex (A-25) was stirred with water for 1 hour and with 0.5 N aqueous sodium hydroxide solution for 2 hours. The ion exchanger was washed with water until the pH was approximately 7.5. This material was then mixed for 2 hours with 0.5 N aqueous acetic acid, washed with water until neutral, poured into the column and packed under pressure of about 0.9 kg of constant height. The column was washed with 4 liters of water, 8 liters of 0, 1% aqueous solution of tris-(hydroxymethyl) aminomethane (THAM) and 3 liters of 0.03 m THAM acetate buffer, pH 8.0 (prepared by dissolving 3.64 g of THAM in 800 ml of water, adjusting with glacial acetate acid) water to pH 8.0 and make up to 1 liter). The starting material, preparations ADA-47.2 and ADA-47.3, approximately 5.50 g were dissolved in 20 ml of 0.03 M THAM acetate buffer, pH 8.0, and added to the top of the column. The column was then eluted from the top with 0.3 M THAM acetate buffer, pH 8.0. Fractions 1-190 (20 ml) were collected. At this point, the column began to elute from the bottom to the top. Fractions A, B, C, D and E were collected (133,485 13 volumes each). Determinations of biological activity before ( -E) and after ( +E) treatment with alkaline phosphotase gave the following results: ~ Fraction No. — 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 1 84 87 90 93 96 99 102 105 108 111 114 117 120 123 1 Zone (S. lutea — sensitive) 1 ~E 0 0 0 0 0 0 0 0 0 0 0 38 43.5 36 23.5 15 0 0 0 0 0 0 15 17 21 23 23 , 23 22 21 21 22 21 22 23 22.5 22, 5 22 22 20.5 20 1 | +E 0 0 0 0 0 0 0 0 0 0 0 39 44 36 23 16 . 0 ° 1 0 0 0 0 16 20 26 27 30 29 24 23 24 26 35 44 51 54 52.5 52 1 54.5 56 56 The following harvests were prepared: Harvest I fractions 34— 38, 280 ml (ADA-69B) Harvest II fractions 75—90, 330 ml (ADA-69C) Set III fractions 101—111, 180 ml (ADA-69D). Set IV, fractions 114—150, 580 ml (ADA-69E) Set V, fractions 151—164, 100 ml (ADA-69F) 15 20 35 40 45 50 55 60 14 Set VI, fractions 165—186, 125 ml (ADA-69G) ) Collection VII fraction C, liter (ADA-69A). Set I (ADA-69B) contained unchanged U-57930 and was discarded. Set II (ADA-69C) contained unknown material from which U-57930 was obtained after treatment with alkaline phosphatase. UV: it was subjected to the processing described below. UV: ^max 260 nm. Set V (ADA-69F) contained a mixture of U-57930 adenylate, U-57930 uridylate and U-57930 guanylate. The solution was processed as described below. Set VI (ADA-69G) contained U-57930 guanylate; processing. UV: This solution was treated as described below. Isolation of substantially pure U-57930 cytidylate, U-57930 adenylate and U-57930 guanylate from sets III, IV and VI, respectively, and removal of the THAM acetate buffer by chromatography on Amberlite VAD-2 was carried out as follows: Sets III, IV, and VI obtained as described above were passed through columns containing Amberlite XAD-2. Leaks were dismissed. The columns were washed with water and eluted with a mixture of methanol and water (70:30). The fractions were analyzed under UV light and the biological activity was determined before and after treatment with alkaline phosphatase. The appropriate fractions were combined, evaporated to an aqueous solution and lyophilized. Data regarding the amount of Amberlite XAD-2 used for each harvest, the amount of rinse water, the amount of methanol eluate and the amount of material obtained are summarized in the table below. Harvest III IV VI Used Amber¬lite XAD- 2 (ml) 50 200 50 Fluid water (ml) 200 800 200 Methanol eluate (ml) 300 600 300 Isolated material (mg) 150 3150 470 65 The material obtained from set III was stored as ADA-73.1; from set-IV as ADA-74.1; from set VI as ADA-75.1. Removal of THAM acetate buffer from set V (ADA-69F) and set VII (ADA-69A) by chromatography on Amberlite XAD-2: The column was prepared with 300 ml of Amberlite XAD-2. Sets V and VII containing a mixture of U-57930 adenylate, U-57930 uridylate and U-57930 guanylate were passed through the column. The impression was rejected. The column was washed with 600 ml of water. The impression was discarded. The column was washed with a mixture of methanol and water (70:30). Fractions giving biologically active material after treatment with alkaline phosphatase were combined (300 ml), evaporated to an aqueous solution and lyophilized, obtaining 670 mg of the ADA-71.1 preparation. The ADA-71.1 preparation was undergoing below the described processing. Urydylan U-57930 from Adenylan U-57930 and Guanylane U-57930 with the help of chroma-oster described, washed with 0.03 m THAM acetate buffer, pH 8.0 and packed in a glass column (internal diameter 4.5 cm, height 40 cm), under hydrostatic pressure. ADA-71.1 preparation was dissolved in 10 ml of 0.03 acetate buffer THAM with pH 8.0 was introduced onto the column from the top. The column was eluted as follows: 1) 0.03 m THAM acetate buffer pH 8.0 (fractions 1-79), 2) 0.12 m THAM acetate buffer pH 8.0 (fractions 80-395), 20 3) 0 .25 m THAM acetate buffer with pH 8.0 (fractions 396-750). Volume fractions of 20 ml were collected and analyzed under UV light and biological activity was determined before and after treatment with alkaline phosphatase. Fractions 51-60 contained U-57930 adenylate; fractions 62-73 (ADA-94. B) contained U-57930 uridylate; fractions 75-110 contained U-57930 guanylate. Isolation of essentially pure U-57930 uridylate. Removal of THAM acetate buffer by chromatography on Amberlite XAD-2. The column was prepared with 50 ml of Amberlite XAD-2. Harvesting of ADA-94B containing U-57930 uridylate was carried through the column at a rate of 2 ml/minute. The leak was dismissed. The column was washed with 200 ml of water. The charges were dismissed. The column was eluted with a mixture of methanol and water (70:30) v/v. Fractions containing (UV) U-57930 uridylate were combined (200 mL), concentrated into aqueous solution and lyophilized to obtain 60 mg of ADA-95.1. Characterization of U-57930 3-/5'-cytidylate. Infrared spectrum. Absorption bands (Nujol Band 3417.3 j 3341.1 3211.8 3108.6 2951.4 2926.3 2854.9 2729.6 1 2693.9 | 2535.7 1649.3 1 1610.7 1 1575.0 1528 .7 1489.2 1462.2 1404.3 1377.3 1368.6 1286.6 Intensity 24 19 19 25 2 1 2 48 51 65 8 26 35 31 23 9 41 18 31 34 Type bend wide wide wide wide M wide M wide M wide M inflections inflections medium medium medium medium medium medium M wide medium M inflection M medium Band 1249.0 1214.3 1146.8 1089.9 1070.6 1056.1 992.4 972.2 955.8 930 ,7 889.2 860.3 849.7 804.4 788.9 721.4 705.0 654.9 632.7 Rate 33 21 42 15 12 14 39 39 49 51 36 52 53 46 40 44 47 45 38 and KBr) Type inflections medium inflections wide medium inflections medium medium inflections medium medium medium medium inflections medium medium M wide inflections medium Band: frequency in wave number (cm-1) Intensity: in % transmittance (%T) Jtf: possible interference with mineral oil14 13349$ Absorption spectrum W UV [Xmax (i)] In water at: pH 2.0 279 nm (6.5) pH 7.0 270 nm (9.9) pH 11.0 271 (9.6) Elemental composition for the summary formula ^26iA43 Calculated '12^ H 6.01 Cl 4.89 C 43.64 S 4.47 Optical rotation (-a7 d+ 107°/C, 0.854, Water) N 9.79 P 4.33 O 26.88 5. Solubility. Highly soluble in water, methanol and ethanol. Slightly soluble in acetone and other ketones, ethyl acetate and other esters, chloroform, methylene chloride. Insoluble in saturated hydrocarbon solvents. 6. Antibacterial activity. 3-(5'-cytidylate)U-57930 is not active in vitro. However, treatment with alkaline phosphatase or phosphodiesterase I produces U-57930, which is highly active against a variety of G+ organisms, both in vitro and in vivo. 7. Melting point: 205—207°C (with decomposition) Characterization of 3-(5'-adenylate) U-57930. 25 strongest peaks %T 1 1 2 3 4 6 10 13 13 frequency 1 29S4.3 2954.2 2854.8 2868.3 1069.5 1089.8 1055.0 %T 3 22 24 24 28 31 31 32 frequency 4 1684 ,0 1377.2 1245.0 1600.0 1576.0 635.5 889.1 1. Infrared spectrum. Absorption bands (Nujol and KBr) Band: frequency in wave number (cm-1)' Intensity: in % transmittance (%T) M: possible interference from mineral oil Band 3335.3 1 3267.8 1 3210.8 2954.3 2924.4 2868.4 2854.& 2727.6 2520.2 1684.0 1641.6 1600.1 1576.0 1550.9 1509.4 | 1463.1 1420.7 1377.3 1367.6 1332.0 1299.2 Current 16 17 17 3 2 6 4 46 61 22 14 £8 81 43 53 15 35 £4 &5 36 34 Type wide wide wide wide wide M wide M bends M medium M wide M wide bends medium medium medium bends medium bends M medium medium M bends M medium medium Band 1245.1 1213.3 1175.7 1146.8 1089.9 1969.6 1055.1 991.5 972.2 957.7 930.7 889.2 861.3 848.7 818.8 798.6 722.4 708.9 647.1 635.6 Intensity 24 17 43 40 13 10 13 35 36 45 48 32 47 49 45 40 36 40 33 31 Type inflections medium inflections inflections medium medium inflections medium medium inflections medium medium medium medium medium medium medium inflections m inflections medium cut133 485 21 1 14 15 16 17 17 17 2 1641.5 1463.0 3335.2 3267.7 3210.7 1213.2 3 33 34 35 35 35 4 647.0 1299.1 1420.6 1367.5 991.5 Preparation: suspension in mineral oil Max %T: 85 at 1864.4 %T at 3800 (cm"1): 81 Density (cm-Vpt): 0.964 22 25 strongest peaks %T 1 ' 5 7 7 8 8 10 12 frequency 2 1069.5 3375.7 1643.5 1090.7 1050.2 3223.3 1215.1 %T 3 23 24 25 25 26 26 26 frequency 4 1576.0 889.1 522.6 503.3 648.0 636.5 571.8 1 Band 3375.8 3223.4 3124.0 2963.0 2929.2 2878.1 | 2863.6 | 2756.6 2521.2 ¦"i y | 2188.5 1678.2 |' 1645.5 1602.0 1576.0 1553.8 1 1511.4 1 1475.7 | 1 1421.7 | 1384.0 1322.0 1 | 1301.1 1246.1 1215.2 1176.7 | And Nate¬ zenie 1 7 1 10 1 17 22 22 1 31 33 45 60 75 16 7 20 23 35 49 27 29 32 1 31 28 19 1 12 | 36 Type wide wide inflection medium wide medium inflection wide wide wide inflection medium medium medium inflection medium overshoot average average average average inflection average inflection | Band 1090.8 1069.6 1050.3 990.5 972.2 1 956.8 1 929.8 889.2 861.3 851.6 818.8 807.3 798.6 768.7 721.4 706.9 648.1 636.5 | 584.5 | 571.9 | 533.3 522.7 503.4 1 Intensity 8 5 8 27 29 | 38 42 24 38 40 36 36 30 41 30 31 26 26 28 | 26 27 25 25 Type medium medium inflections medium medium inflections medium medium ' 1 medium inflections medium wide medium wide medium wide medium medium inflections medium 1 inflection medium medium Band: frequency freedom in wave number (cm-1) JSTatening: in % transmittance (%T)25 133 485 1 15 17 19 20 22 22 2 1678.1 3124.0 1246.0 1602.0 2963.0 3 27 27 27 28 28 4 1475.6 990.5 533, 2 1301.0 584.5 Preparation: KBr pellet Max %T: 95 at 405.0 %T at 4000 (cm-1): 77 Density (cm-Vpt): 0.964 2. UV absorption spectrum [\nax (a )] in water at: pH 2.0 258 nm (16.0) pH 7.0 261 nm (16.5) pH 11.0 261 nm (16.0) 3. Elemental composition for the summary formula C27H43N7°10SC1P- Molecular weight: 723. calculated: C 44.81 H 5.94 S 4.42 Cl 4.84 found: N 12.87 S 5.39 4. Optical rotation \a\n + 94° (0.887, water) N 13.55 P 4.28 Cl 4.76 O 22.13 P 3.83 5. Solubility Highly soluble in water, methanol and ethanol. Slightly soluble in acetone and other ketones, ethyl acetate and other esters, chloroform and methylene chloride. Insoluble in saturated hydrocarbon solvents. 6. Antibacterial activity. 3-(5'-adenylate) U-57930 is not active in vitro. However, alkaline phosphatase or phospho-diesterase I action produces U-57930, which is highly active against a variety of G+ organisms, both in vitro and in vivo. It was found that 3-(5'-adeny^lan) U-57930 is active in vivo (subcutaneously, mouse), CD50 = 0.62 (0.48-0.79) mg/kg against S.pyogonas. 7. Melting point: 203.5-205° (with decomposition). Characterization of 3(5'-uridylate) U-57930. Band: frequency in wave number (cm-1) Intensity: in % transmittance (%T) This peak list is not released M: possible interference with mineral oil 1. Infrared spectrum. Absorption bands (Nujol Band 3330.4 3224.4 2952.4 2924.4 2867.5 2854.0 2733.4 2695.8 2532.8 1757.3 1685.9 1647.4 1602.0 1574.1 1555.7 1462.2 1425.5 1378.3 1367.6 Intensity 19 21 1 0 4 3 49 53 67 73 8 21 41 42 43 12 37 22 37 Type wide wide wide M wide M medium bend M bend M bend bend medium bend average inflections * average wide average M average inflections M inflection M Band 1332.9 1296.3 1251.9 1215.2 1089.9 1071.5 1056.1 991.5 973.2 957.7 931.7 890.2 858 ,4 813.0 798.6 767.7 721.4 634.6 Intensity 44 40 28 —'TJ 19 13 9 13 39 39 48 49 34 50 43 47 50 42 35 and KBr). Type wide inflections wide medium medium medium inflections medium medium inflections medium medium medium medium medium medium medium M medium 133 485 25 25 strongest peaks *T 0 1 3 4 8 9 12 13 13 19 19 21 21 frequency 2924.3 2952.3 2854.0 2867.5 1685.8 1071.5 1462.1 1089.8 1056.0 3330.3 1215.1 3224.3 1647.3 %T 22 28 34 35 37 37 39 39 40 41 42 42 frequency 1378.2 1251, 8,890.1,634.5 | 1425.5 1367.5 991.5 973.1 1296.2 1602.0 1574.0 721.3 Preparation: suspension in mineral oil Max %T: 86 at 3764.5 %T at 3800 (cm-*): 85 Gestivity (CM-VPT): 0.964 10 2FR 25 of the strongest Pików %T 6 7 10 10 12 17 24 25 26 26 31 31 Czestotyśl 1685.0 1070.5 1090.7 1055.0 3387.3 1647.3 1214.2 1255.7 ail4.3 2962.0 2931.0 1463.0 889.1 %T 32 34 35 35 35 35 36 36 37 37 38 38 frequency 1384.0 567.0 1605.8 1423.5 992.3 ] 523 .6 2879.0 973.1 1576.0 634.5 2863.5 1297.1 15 20 Preparation: KBr pellet Max %T: 101 at 405.0 %T at 4000 (cm-1): 76 Density (cm- ypt): 0.964 Bandwidth 3387.4 3114.4 2962.0 - 1 2931.1 2879.1 2863.6 2833.7 2509.0 1685.0 1 1646.4 1605.9 1 1576.0 1 1556.7 1463 .1 | 1423.6 1384.0 1331.0 1297.2 1255.8 1214.3 1 1090.8 | 1070.6 Current 12 25. 26 26 36 3& 43 64 6 17 35 37 | 39 31 35 32 43 38 24 17 10 7 Wide type. inflections medium wide medium inflections wide inflections wide inflections inflections medium wide medium medium medium medium inflections medium medium medium medium Band I 1055.1 992.4 973.2 956.8 929.8 889.2 . 859.3 813.0 811.1 798.6 782.2 768.7 707.9 669.3 649.1 634.6 585.4 567.1 523.7 447.5 Intensity 10 35 . 36 . ¦ 45 48 31 46 40 40 43 48 48 43 43 40 37 38 34 35 39 Type inflection medium medium - inflection medium medium medium inflection medium wide medium medium inflection inflection medium inflection 1 medium 1 average average 1 Band: frequency in wavenumber ( cm-1) Intensity: in % transmittance (%T)27 133 465 2. UV absorption spectrum [A.max (a)] In water at: pH 2.0 261 nm (11.5) pH 7.0 262 nm (10.7) pH 11.0 262 nm (11.5) 3. Elemental composition for the summary formula C26H42N4013SC1P. Chain weight: 716. Calculated: O 43.57 H 5.86 N 7.82 O 29.05 S 4.46 . Cl 4.89 P 4.33 4. Optical rotation [a]g+ 105° (o, 0.94, water) 5. Solubility. Highly soluble in water, methanol and ethanol. Slabp soluble in acetone and other ketones, ethyl acetate and other esters, chloroform and methylene chloride. Insoluble in saturated hydrocarbon solvents. 6. Antibacterial activity. U-57930 3-(5'-uridylate) is not active in vitro. However, the action of alkaline phosphatase or phospho-diesterose I gives U-57930, which is highly active against various G+ organisms, both in vitro and in vivo. 7. Melting point: 202—203° (with decomposition). Characterization of 3-(5'-quanylate) U-57930. 1. Infrared spectrum. Absorption bands (Nujol Band 3335.3 3227.2 2953.3 2925.3 2868.4 2855.9 2737.3 2521.2 1684.0 1635.8 1598.2 1572.1 1534.5 1462.2 1414.9 1377.3 1365, 7 1312.7 Current 16 19 2 1 6 4 51 73 6 11 21 26 34 18 44 24 31 44 Type wide wide medium M wide M inflection M medium M wide M wide medium medium medium medium medium medium ' medium M medium medium M medium medium Band 1250.0 1213.3 1173.8 1149.7 k 1087.9 1071.5 991.5 962.2 956.8 929.8 890.2 860.3 800.5 783.1 720.4 707 .9 681.9 635.6 Intensity 36 21 39 41 14 9 42 42 52 51 36 51 46 42 43 45 40 34 and KBr) Type inflections medium medium medium inflections medium medium medium inflections medium medium medium medium sharp medium M wide average average Band: frequencies in wavenumber (cm-1) Intensity: in % transmittance (%T) M: possible interference from mineral oil £9 25 strongest peaks 133 485 30 25 strongest peaks %T 4 6 6 1 9 9 13 14 16 19 21 22 26 and 28 frequency 1683.0 1634.7 1070.5 3380.5 1088.8 3234.0 1598.1 1213.2 1571.1 2929.1 2963.0 1534.5 889.1 %T 29 29 30 30 31 31 - 31 31 32 32 33 33 frequency 1384.0 1250.8 1359.8 502.5 2878.0 1174.6" 635.5 523.6 783.0 571.8 . 2862.6 1147.6 %T 1 2 4 1 6 6 9 11 14 16 18 19 21 21 frequency 2925.2 2953.2 2855.8 2868.3 1684.0 1071.5 1635.7 1087.8 3335.2 1462.1 3227.1 1598.1 1213.2 %T 24 26 31 34 34 36 36 39 40 41 42 42 frequency 1377.2 1572.0 1365.8 1534.5 635.5 1250.0 890.1 1 1173 ,7 681.8 1149.6 991.5 972.1 Preparation: suspension in mineral oil 2o Preparation: KBr pellet Max %T: 97 at 3762.6 Max %T: 97 at 405.0 %T at 3800 (cm- 1):97 %T at 4000 (cm-1): 77 Density (cm-Vpt): 0.964 Density (cm-Vpt): 0.964 Band 3380.6 3234.0 2963.0 2929.2 2878.1 2862.7 2744.0 | 2522.2 | - 1683.0 1 1634.8 1598.2 | 1571.2 1534.5 1 1482.4 1461.2 1448.7 1413.9 1384.0 1 1359.9 1312.7 1250.9 1213.3 1 Intensity 9 13 22 21 31 33 44 61 4 6 14 19 26 38 36 36 34 29 30 37 1 29 16 | Type wide wide medium wide medium inflection wide wide wide medium medium medium medium medium medium medium wide medium medium medium medium inflection medium Band 1174.7 1147.7 1088.9 1070.6 991.5 972.2 956.8 929.8 889 , 2 860.3 ~ 800.5 783.1 715.6 705.0 679.9 635.6 584.5 571.9 523.7 502.5 447.5 Intensity 31 33 9 6 33 34 43 44 28 41 36 32 38 38 33 31 34 1 32 31 30 34 Type medium wide wide medium medium J medium inflections medium medium medium medium medium inflections medium inflections medium inflections 1 medium 1 medium medium | average 1 Band: frequency in wave number (cm-1) Intensity: in % transmittance (%T)31 133 485 32 (a)] (8.4) inflection 2. UV absorption spectrum [Xmax in water at: pH 2 .0 256 nm (13.4), 280 nm pH 7.0 254 nm (14.5), 273 nm (9.7) inflection pH 11.0 259 nm (12.6), 266 nm (12.4) ) inflection 3. Elemental composition for the summary formula: C27H43N7OnSClP. Molecular weight 739. Calculated: C 43.84 H 5.81 N 13.26 O 23.27 S 4.33 Cl 4.73 P 4.19 Found: N 13.31 S 4.86 Cl 4.49 P 3 .25 4. Optical rotation [«15+ 97° (°» °855 water 5. Solubility. Highly soluble in water, methanol and ethanol. Slightly soluble in acetone and other ketones, ethyl acetate and other esters, chloroform and methylene chloride. Insoluble in saturated hydrocarbon solvents. 6. Antibacterial activity. 3-(5/-guanylate) U-57930 is not active in vitro. However, the action of alkaline phosphotase or phospho-diesterase I gives U-57930, which is highly active against various C+ organisms, both in vitro and in vivo. 7. Melting point 219-220°C (with decomposition). Patent claims 1. Method of producing new 3-(5'-ribonucleotides) clindamycin-type compounds with the formula general number 1, in which Rx is a hydrogen atom or an alkyl radical with 1-8 carbon atoms, optionally substituted #R3 is a hydrogen atom or an alkyl group with 1-2 carbon atoms, optionally substituted hydroxyl group, n is an integer 1- 4 and Y is 7(S)-chlor or 7(R)-chlor, in the form of their isomers, optionally in the form of pharmaceutically acceptable salts, characterized in that the culture of Streptomyces rochei, NRRL 3533, is carried out in water a medium containing as nutrients a source of carbon in the form of an available carbohydrate and a source of nitrogen in the form of an available nitrogen compound or a protein substance, the culture being carried out in the presence of the compound of formula 1 and isolated from the culture medium containing a mixture of ribonucleotides of the given 3-(5) '-ribonucleotide), which is optionally converted into a salt. 2. The method according to claim 1, characterized in that in the case of the production of 3-(5)-ribonucleotides of the compound of formula 2, the culture of Streptomyces rochei, NRRL 3533, is carried out in an aqueous medium containing a carbon source in the form of digestible carbohydrate and a nitrogen source as nutrients. in the form of an assimilable nitrogen compound or protein substance in the presence of the compound of formula 2 and isolates the desired 3-(5'-ribonucleotide) from the culture medium. 3. The method according to claim 1, characterized in that in the case of the production of 3-(5,-ribonucleotides) of the compound of formula 3, the culture of Streptomyces rochei, NRRL 3533, is carried out in an aqueous medium containing a carbon source in the form of digestible carbohydrate and a nitrogen source as nutrients. in the form of an assimilable nitrogen compound or protein substance, in the presence of the compound of formula 3, and isolates the desired 3-(5'-ribonucleotide) from the culture medium. MV N I R<2 o o C-NH Formula 1 -v CH3 - Y CH CH, OH HC -Cl I N^C-NH—CH H HOl-0. Formula 2133 4S5 Cri* I "HC-Cl N^C-NH-CH H HO/!--O Formula 3 WS~CH, OH CH2CH3 I" -P-0-CH2 OH rrO I 3' 1 Formula'5 I2 ' OH OH NH2 N 5 k N"^N OH 12 l| - P- O -CH2 O i OH Formula e \ OH OH -P-0-CH2 OH Lonv Formula7 oj^ ^U3 485 OH NAf N 0 m/hAn ^ II 5' ' P-0—CH2 OH & OH OH Formula 8 CH3 f\C3H7 \jf H H ii Formula 9 CK H-C-Cl H | N-C-H hoJ-Qh U%CH3 ,(CH?} n\^. Rl ~ R2 H I r\i Pattern 10 Class Giaf. ttadom — 1537/35 85 eg;;.A4 Price PLN 100 PL PL PL PL PL

Claims (3)

1. Patent claims 1. Method for preparing new 3-(5'-ribonucleotides) compounds of the clindamycin type of the general formula 1, in which Rx is a hydrogen atom or an alkyl radical with 1-8 carbon atoms, optionally substituted #R3 is a hydrogen atom or an alkyl group with 1-2 carbon atoms optionally substituted hydroxyl group, n is an integer 1-4 and Y is 7(S)-chlor or 7(R)-chlor, in the form of their isomers, optionally in form of pharmaceutically acceptable salts, characterized in that the culture of Streptomyces rochei, NRRL 3533, is carried out in an aqueous medium containing as nutrients a carbon source in the form of an available carbohydrate and a nitrogen source in the form of an available nitrogen compound or a protein substance, with wherein the culture is carried out in the presence of the compound of formula 1 and the desired 3-(5'-ribonucleotide) is isolated from the culture medium containing a mixture of ribonucleotides, which is optionally converted into a salt. 2. The method according to claim 1, characterized in that in the case of the production of 3-(5)-ribonucleotides of the compound of formula 2, the culture of Streptomyces rochei, NRRL 3533, is carried out in an aqueous medium containing as nutrients a carbon source in the form of digestible carbohydrate and a nitrogen source in the form of an assimilable nitrogen compound or protein substance in the presence of the compound of formula 2 and isolates the desired 3-(5'-ribonucleotide) from the culture medium. 3. The method according to claim 1, characterized in that in the case of the production of 3-(5,-ribonucleotides) of the compound of formula 3, the culture of Streptomyces rochei, NRRL 3533, is carried out in an aqueous medium containing a carbon source in the form of digestible carbohydrate and a nitrogen source as nutrients. in the form of an assimilable nitrogen compound or protein substance, in the presence of the compound of formula 3, and isolates the desired 3-(5'-ribonucleotide) from the culture medium. MV N I R<2 o o C-NH Formula 1 -v CH3 - Y CH CH, OH HC -Cl I N^C-NH—CH H HOl-0. Formula 2133 4S5 Cri* I "HC-Cl N^C-NH-CH H HO/!--O Formula 3 WS~CH, OH CH2CH3 I" -P-0-CH2 OH rrO I 3' 1 Formula'5 I2 ' OH OH NH2 N 5 k N"^N OH 12 l| - P- O -CH2 O i OH Formula e \ OH OH -P-0-CH2 OH Lonv Formula7 oj^ ^U3 485 OH NAf N 0 m/hAn ^ II 5' ' P-0—CH2 OH & OH OH Formula 8 CH3 f\C3H7 \jf H H ii Formula 9 CK H-C-Cl H | N-C-H hoJ-Qh U%CH3 ,(CH?}n\^.Rl ~ R2 H I r\i Pattern 10 Class Giaf. ttadom — 1537/35 85 eg;. A4 Price PLN 100 PL PL PL PL PL