SE459861B - LINKOMYCIN-3- (5'-RIBONUCLEOTIDES) AND PROCEDURES FOR THEIR PREPARATION - Google Patents

Description

l0 15 20 25 30 35 459 861 ducks infected with P. lophurae and chickens infected with P. gallinaceum and mammals as primates e.g. monkeys infected with P. cynomolgia and humans infected with P. falciparum, P. vivax and P. malariae.

Mammalian hosts for parasitic protozoa of the type Sporazoa order Coccidia (a microparasite that causes the disease coccidiosis) can be treated by administering the compounds of the present invention. with Eimeria zurnii, E. bovis, E. and goats infected with E. parva, terade with E. debliecki, Livestock infekte- ellipsoidalis, sheep E. faurei, swine infek- E. scabra and Isospora suis, dogs and cats infected with Isospora bigemina, I. felis, E. canis, E. felina, chickens infected with E. tenella, rabbits infected with E. stiedae, E perforans and mink infected with E. mustelae can be treated.

The lincomycin and clindamycin type compounds, which can be converted to 3-ribonucleotides, were shown in Scheme 2. Instead of hydroxyl at the 3-position in the lincosamine moiety substituted a nucleotide selected from the group consisting of adenylic acid, guanylic acid, cytidylic acid and uridylic acid. The 3-ribonucleotides of the present invention can be prepared by microbiological conversion methods. The 3- (5 'ribonucleotides) are obtained by converting U-57930 shown in Scheme 3.

Specific Description ______________ The basic compounds shown in Scheme 2 can be prepared by the method shown in U.S. Patent 4,278,789. The 3- (5'-ribonucleotides) of the compounds of Scheme 2 can be prepared according to the procedure shown in U.S. Patent 3,671,647. Salts of these nucleotides can be prepared according to the procedure described in U.S. Pat. No. 3,671,647.

The formulas for the nucleotides of the present invention may follow the examples of U.S. Patent No. 4,278,789.

The formulations are prepared by substituting a nucleotide of the present invention for the active compound of these examples. Substitution can take place on an equimolar basis.

General tests and assay procedures which can be used to determine and characterize nucleotides of the invention are as follows: Samples of 3- (5'-ribonucleotides) Since 3-ribonucleotides of the present invention lack in vitro antibacterial activity, their formation from antibacterially active basic compounds easily monitored by measuring the loss of such antibiotic activity. To determine the amount of antibacterial-active base compound in culture filtrates or reaction mixtures, a standard sample of Sarcina lutea ATCC 9341 was used. To test for the presence of the 3-ribonucleotides in culture fluids, extracts | and purified materials, the phosphorus diester is first hydrolyzed by crude alkaline phosphatase or snake venom phosphodiesterase by the procedures described below. The antibacterial activity of the compound in the hydrolyzate is determined by standard tests.

Enzymatic hydrolyses Alkaline phosphatase: Stock solutions (0.5 mg / ml, 0.54 units / mg) from the alkaline phosphatase of the Duvin River, EC 3.1.3.1 (Sigma) prepared in tris- (hydroxymethyl) -aminomethane-hydro chloride buffer, 0.01 M, pH 8.0. The samples are treated diluted 1: Z with enzyme buffer mixture and incubated at 280 ° C for 18 hours. 10 15 20 25 30 35 459 861 Snake venom phosphodiesterase: Stock solutions (100 mg / ml, 0.026 units / mg) of purified snake venom phosphodiesterase EC 3.l.4.1 (Sigma) are prepared in distilled water. Incubation mixtures containing 0.2 ml of a solution (1 mg / ml) of the sample to be treated in water, 0.6 ml of a 0.01 M tris-hydrochloride buffer, pH 9.0, 0, 1 ml 0.3 M MgCl 0.1 ml of the enzyme stock solution. at 37 ° C for 18 hours. 2 and the incubation is carried out Spleen phosphodiesterase: Stock solutions of spleen phosphodiesterase EC 3.1.4.18 (Sigma) were prepared (1 mg / ml, 19.6 units / mg) in distilled water. Incubation mixtures containing 0.4 ml of a solution (0.5 mg / ml) of the sample to be treated in water, 0.5 ml of 0.02 M Tris buffer, pH 7.0 and 0.1 ml of enzyme stock solution.

The incubation takes place at 370 C for 18 hours.

Thin layer chromatographic analysis of preparations and enzymatic hydrolysates.

Preparation and purification of 3-ribonucleotides is by sample against S. lutea (see above) and using thin layer chromatography on silica gel G and methyl ethyl ketone-acetone-water (186: 52: 20 by volume) or ethyl acetate-acetone water (8: 5: 1) as solvent system. aktiva.grundföreningen Den bio; detected by bioautography on agar incubated with S. lutea.

The products of enzymatic or chemical hydrolysis of 3-nucleotides are separated by the following thin layer chromatographic systems: A: Silica gel GF plates (Analtech Inc.); water as a solvent system.

B: Silica gel GF plates; n-propyl alcohol-concentrated ammonium hydroxide-water (S5: 10: 35, by volume). 10 15 20 25 30 35 459 861 C: NM polygram cellulose 300 (Brinkman Instruments Inc.); 1-butanol-water-formic acid (77: 13: 10, by volume).

Ultraviolet absorbing material is determined by short-wave UV radiation. Bioinactive, non-UV-absorbing material is determined by a permanganate-periodate spray reagent.

Bioactive nucleotide materials are determined by bioautography on agar incubated with S. lutea.

The following examples show culture and purification methods for the preparation of the nucleotide of the compounds called U-5793OE. The structural formula of U-5793DE is shown in Scheme 3. The following procedures in this example or obvious equivalents thereof can be prepared 3-ribonucleotides of other compounds shown in Scheme 2.

The following examples illustrate the process and products of the present invention but are not intended to be limiting. All percentages refer to% by weight and all solvent mixtures refer to volume unless otherwise stated.

Example gel A. Culture method Streptomyces rochei, NRRL 3533 grows in a medium consisting of glucose 10 g / l; Difco peptone 4 g / l; Difco yeast extract 4 g / l; MgSO 4 ° 7H 2 O 0.5 g / l; KHZPO4 2.0 g / l; KZHPO4 4 g / l for 3 days at 280 C on a rotary shaker. Mycelium from this growth was used to inoculate a culture medium containing the same ingredients. The cultivation was carried out for 48 hours at 280 ° C on a rotary shaker. At the end of this 48 hour incubation period, U57930 is added to a final concentration of 50 mg / l and the culture is continued at 320 ° C. After 12 hours, another U-57930 is added to a total concentration of 150 mg / l.

After a further 12 hours, the concentration of U-57930 10 15 20 25 30 459 861 is increased to 250 mg / l. The cultivation is continued at 320 DEG C. for 24 hours after the last addition of U-57930. At this time, the culture filtrate was harvested and was found to contain a maximum of 1 mg / l U-57930. The remaining 249 mg / l are converted to bioinactive material.

S. rochei. NRRL 3533 is a known bacterium available to the public at the request of NRRL's deposit collections.

The address of the depositary is the Northern Utilization and Research Division, Agricultural Research Service, U.S.

Department of Agriculture, Peoria, Ill. USA.

B. Isolation and purification procedures Isolation of U-57930 3-ribonucleotides from the culture broth Adsorption on Amberlite XAD-2: The culture broth (ca. 12 l) containing 3 g of inactivated U-57930 was filtered at harvest 7.7. filter aids. The mycelial cake was washed with 1.2 l of water and discarded. The clear filtrate and wash water were combined and the pH was adjusted to 6.0 and passed over a column prepared from 600 ml of Amberlite XAD-2 (Rohm and Haas Co., Philadelphia, PA, USA), at a flow rate of 40 ml / minute. Those used were tested for bioactivity before and after treatment with alkali phosphatase and discarded. with 2 l of water.

The column was washed. The aqueous wash was also found to be bioinactive before and after the treatment with alkaline phosphatase and discarded. The column was then eluted with methanol-water (70:30, by volume). Fractions of 20 ml were collected at a rate of 20 ml / minute. Samples of the biological activity before (-E) and after (fE) the treatment with alkaline phosphatase showed the following. 10 15 20 25 30 35 fraction no. 5 10 11 13 20 25 40 45 50 55 60 65 70 75 80 85 90 95 100 110 120 130 140 150 160 "170 180 190 200 Fractions 12 - 80 were combined, concentrated to an aqueous solution and lyophilized. whereby the preparation ADA-34.1, 12.22 g was obtained.

OOOC) 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 15 -459 861 zon (S. lutea) + E 0 0 0 36 55 54 50 48 39 38 37 27 26 26 26 26 26 26 26 26 21 21 21 21 21 21 21 21 21 21 21 10 15 20 25 459 861 In a further series of experiments, 6 l of culture fluid containing 2 g of "inactivated" U-57930 were described as described above.

The methanolic eluate Amberlite XAD-2 column was retained as ADA-143B. This solution was not concentrated to dryness but purified by Dowex-1 chromatography as described below.

Dowex-1 chromatography: The column was prepared with 300 ml of Dowex-1 (X-4) in acetate form. A methanolic solution, ADA-1435 with a pH of 8.2, was passed through the column. Starting material was collected at a rate of 2.5 ml / minute in 20 ml fractions. (Fractions 1 - 60). The column was washed with 1.5 L of water (10 ml / minute; fractions 66 - 108). The column was then eluted with 5% acetic acid (rate 10 ml / minute, fractions 109 - 310). The following mergers were made: pool 1 fractions l- 80 1000 ml (ADA-1A) "2" 81-110 600 ml (ADA-2A) "3" lll-130 450 ml (ADA-3A) "4" 131- 150 450 ml (ADA-4A) "5" 151-190 900 ml (ADA-5A) "6" 191-230 900 ml (ADA-6A) "7" 231-270 900 ml (ADA-7A) u 8 u 271-310 900 ml (ADA-8A) Samples before (-E) and after (+ E) the treatment with phosphatase showed the following: alkaline 10 15 20 25 30 35 459 861 zone (S. lutea) -E + E fusion 1 31 52 "2 36 49" 3 34 45 "4 18 40" 5 25 30 "6 27 29" 7 27 29 "8 29 31 Combined 1 and 2 were combined, concentrated to an aqueous solution and lyophilized to give the preparation ADA-2.1. , 1.48 g.

Combinations 3 and 4 were combined and treated in a similar manner to give ADA-2.2, 2.5 g.

Preparations ADA-2.1 and -2.2 gave U-57930 after treatment with alkaline phosphatase.

The preparations ADA-34.1, -2.1 and -2.2 were combined and purified by countercurrent distribution according to the procedure described below.

Countercurrent distribution: The material obtained by combining preparations% ADA-34, 1, -2.1 and -2.2, 16.20 g was dissolved in 25 ml of each phase in a solvent system containing equal volumes of 1-butanol. water (lzl). The solutions were added to the center tubes of a glass double-flow distributor (100 tubes, 25 ml / phase). The distribution was analyzed after 150 transfers of biological activity before (-E) and after (+ E) with alkaline phosphatase.

Results as follows: 10 15 20 25 30 35 459 861 lower collector 5 10 15 20 25 - 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 lower machine 50 45 40 35 30 25 20 15 10 10 zone (S .lutea-sensitive) -E + E 29 31 30 27 22 F '\ J OOOOOOCCJOOCDOOO zone (S. lutea-sensitive) 31 33 33 33 33 34 34 33,5 33 _34 34 35 36 38 39 40 41 42 43 43, 5 -E + E 0 45 groove 46 l5 47 17 47.5 17 47 18 47 17.5 48 17 48 16 48 15 50 groove 50 459 861 ll zone (S. lutea-sensitive) 10 15 20 25 30 35 upper machine -E * + E 5 tracks 49 10 tracks 48.5 15 tracks 48 20 tracks 48.5 25 tracks 49 30 30 50 35 tracks 51 40 15 52 45 16 53 50 21 S4 upper collector 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 l7,5 17 19 20 21 22 24 26 28 30 32,5 33 35 36 39 41 43 43 43 35 54 53,5 53,5 53,5 53,5 53, 5 53.5 53.5 53.5 52.5 52 52 52 52 49 47 48 46 43 35 10 15 20 25 30 35 459 861 12 The following mergers were made. Each pool was concentrated to an aqueous solution and lyophilized to give the corresponding preparation.

Merger I: lower collectors l - 50 IIÉ lower collectors 51 - 100; lower machine 50 - 30 III: lower machine 29 - 0; upper machine l - 50; upper collector 100 - The preparations thus obtained were: from pole I preparation ADA-47.1, 9.78 g from pole II preparation ADA-47.2, 0.30 g from pole III preparation ADA-47.3, 5 , 29 g.

Preparations ADA-47.2 and -47.3 were pooled and purified by DEAE-Sephadex chromatography described below.

DEAE-Sephadex chromatography: 300 g of DEAE-Sephadex (A-25) were stirred for 1 hour with water and 2 hours with 0.5 N aqueous sodium hydroxide solution. The ion exchanger was washed with water until its pH was about 7.5. The material was stirred for 2 hours with 0.5 N aqueous solution of acetic acid, washed with water to neutral pH and beaten in a column and packed under 1 kg of pressure to constant height. The column was washed with 4 l of water, 8 l of 0.1% aqueous solution of tris- (hydroxymethyl) aminomethane (THAM) and 3 l of 0.03 H THAM acetate buffer pH 8.0 (prepared by dissolving 3 , 64 g of THAM in 800 ml of water, adjusting the pH to 8.0 with glacial acetic acid and adjusting the volume to 1 l).

The starting material, preparations ADA-47.2 and 47.3, about 5.50 g was dissolved in 20 ml of 0.03 M TEAM acetate, pH 8.0 buffer and added to the upper end of the column. The column was then eluted through a downstream 0.3 M THAM acetate buffer 459 861 13 with a pH of 8.0. Fractions 1 - 190 (20 ml) were collected. Thereafter, the elution was continued by flowing upwards in the column. Fractions A, B, C, D and E (1 L each) were collected. Tests on the bioactivity before (-E) and after (+ E) the treatment with alkaline phosphatase show the following: zone (S. lutea-sensitive) 10 15 20 25 30 35 fraction no ~ E + E 0 O 6 0 0 9 0 0 12 0 0 15 0 O 18 0 0 21 0 0 24 0 O 27 0 O 30 O 0 33 .O 0 36 38 39 39 43,5 44 42 36 36 45 23,5 23 48 15 16 51 0 0 '54 O 0 '57 0 0 60 O 0 63 0 0 66 0 0 69 15 16 72 17 20 75 21 26 78 23 27 10 15 20 25 30 35 459 861 fraktio Dnr 81 84 87 90 93 96 99 102 105 108 lll 114 117 120 123 14 zøn (S. lutea-sensitive) The following mergers were made: Merger II! II n ..

II "III" IV "V u VI" VII fraction Combination I (ADA-69B) contained unchanged and was discarded.

-E + E 23 30 23 29 22 24 21 23 21 24 22 26 21 35 22 44 23 51 22.5 54 22.5 52.5 22 52 22 54.5 20.5 56 20 56 fractions 34- 38, 280 ml (ADA-69B) 75- 90, 330 ml (ADAf69C) 101-III, 180 ml (ADA-69D) 114-150, 580 ml (ADA-69E) 151-164, 100 ml (ADA-69F) 165- 186, 125 ml (ABA-ess) C, 11 (ADA-69A) U-57930 Merger II (ADA-69C) contained an unknown material which gave U-57930 when treated with alkaline phosphatase.

UV: Å max 275 nm. Coagulation III (ADA-69D) contains U-57930 cytidylate and was treated as described below. UV: λ max 270 nm.

Merger IV (ADA-69E) contains U-57930 adenylate and was treated as described later. UV: Å nmx 260 nm.

Combination V (ADA-69F) contained a mixture of U-57930 adenylate, U-57930 uridylate and U-57930 guanylate.

The solution was treated as described below.

Merger VI (ADA-69G) contains U-57930 guanylate and was treated as described above. UV: Å max 254 0Ch terrace point at 275.

Combination VII (ADA-69A) contained a mixture of U-57930 guanylate and U-57930 uridylate. The solution was treated as described later.

Isolation of substantially pure U-57930 cytidylate, U-§7930 adenylate and U-57930 guanylate from pool III, IV and VI. Removal of THAM acetate buffer by Amberlite XAD-2 chromatography: Merger III, IV and VI obtained as described above were transferred to a column containing Amberlite XAD-2. The review was discarded. The column was washed with water and eluted with methanol-water (70: 30 by volume). The fractions were analyzed by ultraviolet and by bioactivity test before and after the alkaline phosphatase treatment. Appropriate fractions were combined, concentrated to an aqueous solution and lyophilized. Details regarding the amount of Amberlite XAD-2 used for each pool, the amount of water and the amount of methanol eluate and the amount of material obtained are listed in the following table. 459 861 10 15 20 25 30 35 16 Amberlite XAD-2 water methanol isolated isolated wash eluate material beating (ml) (ml) (ml) (mg) III 50 200 300 150 IV 200 800 600 3510 VI 50 200 300 470 The material obtained from merger III was called ADA-73.1 from merger IV ADA-74.1 and from merger VI ADA-75.1.

Removal of THAM acetate buffer from pool V (ADA-69F) and pool VII (ADA-69A) by Amberlite XAD-2 chromatography: A column was prepared from 300 ml of Amberlite XAD-2. Combinations V and VII containing a mixture of U-57930 adenylate, U-57930 ruidylate and U-57930 guanylate passed through the column. Discontinued material was discarded. The column was washed with 600 ml of water. Outgoing material was discarded. The column was eluted with methanol-water (70:30). Fractions yielding bioactive material after treatment with alkaline phosphatase were combined with 300 ml, concentrated to an aqueous solution and lyophilized to give the preparation ADA-71.1, 670 mg. Preparations -71.1 were treated as described below.

Separation of U-57930 uridylate from U-57930 adenylate and U-57930 guanylate. DEAE-Sephadex chromatography. 600 ml of DEAE Se fi hadex in acetate form, prepared as described above, washed with 0.03 M THAM acetate pH 8.0 buffer and packed in a glass column (ID, 4.5 cm, height 40 cm) under hydrostatic pressure.

Preparation ADA-71.1 (see above) was dissolved in 10 ml of 0.03 M THAM acetate, pH 8.0, and added to the upper part of the column.

The column was eluted with: 10 15 20 25 30 459 861 17 l) 0.03 M THAM acetate, pH 8-0 (fractions l ~ 79) 2) 0.12 M THAM acetate, pH 8.0 (fractions 80-395) 3) 0.25 M THAM acetate, pH 8.0 (fractions 396-750) The 20 ml fractions were collected and analyzed by UV and by testing the bioactivity before and after treatment with alkaline phosphates. Fractions 51-60 contained U-57930 adenylate, fractions 62-73 (ADA-94B) contained U-57930 uridylate, fractions 75-110 contained U-57930 guanylate.

Isolation of essentially pure U-57930 uridylate. Removal of THAM acetate buffer by Amberlite XAD-2 chromatography.

The column was prepared from 50 ml of Amberlite XAD-2. Merger ADA-94B, containing U-57930 uridylate was allowed to pass over the column at a rate of 2 ml / minute. Outgoing material was discarded. The column was washed with 200 ml of water, the washing liquid was discarded. The column was eluted with methanol-water (70:30 by volume). Fractions containing (in ultraviolet) U-57930 uridylate were combined (200 ml), concentrated to an aqueous solution and lyophilized to give ADA-95.1, 60 mg.

Characteristics of U-57930 3 '(5' cytidylate) 1. IR value The table shows IR absorption (Nujol and KBr) as follows: 10 15 20 25 30 459 861 18 bands intenband inten- freq. site type freq. site type 34l7.3 24 SH l249.0 33 SH 334l, l 19 BRD l2l4.3 21 AVG 32ll, 8 19 BRD l146.8 42 SH 3l08.6 25 BRD l089.9 15 BRD 295l, 4 BRD M l070.6 12 AVG 2926.3 BRD M l0S6, l 14 SH 2854.9 2 BRD M 992.4 39 AVG 2729.6 48 BRD M 972.2 39 AVG 2693.9 51 SH 955.8 49 SH 2535.7 65 SH 930 , 7 51 AVG l649.3 8 AVG 889.2 36 AVG l6l0.7 26 AVG 860.3 52 AVG l575.0 35 AVG 849.7 53 AVG l528,7 31 AVG 804.4 46 SH l489.2 23 AVG 788 , 9 40 AVG l462.2 9 AVG M 721.4 44 AVG M 1404.3 41 BRD 705.0 47 BRD l377.3 18 AVG M 654.9 45 SH l368.6 31 SH M 632.7 38 AVG l286, 6 34 AVG Band frequency: band frequency is given in wavelengths (cm_l) intensity: intensity in percent transmission (% T) Data type in the area next to peaks: BRD - wide; AVG - average; SHP - pointed; SH - terrace point.

Top list processed. * indicates added peaks.

M: means possible interference with mineral oil. 10 15 20 19 25 strongest gears 459 861% T frequency% T frequency l 2926.2 24 34l7.2 2 295l .3 25 3l08.5 2 2854.8 26 l6l0.6 8 l649.2 31 l528.6 9 l462, 1 31 1368,5 12 1070,5 33 l249,0 14 l056,0 34 l286,5 15 l089,8 35 l575,0 18 _ l377,2 36 889,1 19 334l, 0 38 632,6 19 3211 ,? 39 992.3 21 l214.2 39 972.1 23 1489.1 Preparations; mineral oil Max. % T: 87 @ 1848.0 æ T at 3800 (cnfln 83 Density (Cm_l / pt): 0.964 10 15 20 25 459 861 20 band inten- band inten- frequent site type frequent site type 3408.6 10 BRD l088 , 9 12 BRD 3l02,8 26 SH l07l, 5 9 AVG 2963,9 28 AVG l057, l ll SH 2930,2 27 BRD 992,4 35 AVG 2878, l 37 AVG 972,2 36 AVG 2862,7 39 SH 956 , 8 45 SH 2768, l 51 SH 928.8 48 AVG 25ll, 6 65 BRD 889.2 32 AVG l649.3 4 AVG 859.3 47 AVG 16l4.6 20 SH 851.6 47 SH l576.0 30 AVG 804 , 4 39 SH 1528,? 28 AVG 788,9 34 AVG 149l, l 21 AVG 743,6 47 SH l462,2 33 AVG 705,0 40 AVG l450,6 35 SH 654,9 39 SH 1404,3 37 AVG 634 .6 35 AVG l384.0 33 AVG 595.1 33 AVG 1360.9 42 SH 572.9 33 AVG l286.6 32 AVG 525.6 32 AVG l25l .9 30 SH 447.5 36 AVG l2l5.2 18 AVG Band frequency: band frequency in wavelengths (cm_l) Intensity: intensity in percent transmission (% T) Data type in the area next to peaks: BRD - wide; AVG - average- average; SHP - pointed; SH - terrace point Toplist processed. * indicates added peaks 459 861 21 25 strongest peaks% T frequency% T frequency l fi49.2 30 l25l, 8 l07l, 5 32 l286,5 10 3408,5 32 889,1 ll l057,0 32 525,5 12 lO88.8 33 l462, l 18 l2l5, l 33 l384,0 20 l6l4,5 33 595,0 21 l49l, 0 33 572,8 25 3lO2,7 34 788,8 27 2930, l 35 l450,5 28 2963,8 35 992,3 28 l528,6 35 634,5 30 l576,0 Preparation: KBR pellet Max% T = 100 G 403.1% T at 4000 (cm -1) = 70 Density (cm -1 / pt) = 0.964 2. UV absorption spectrum [hmax (a fl in water at pH 2.0, 279 nm ( 6.5) pH 7.0, 270 nm (9.9 pH 11.0, 271 (9.6) 3. Elemental composition Molecular formula: C26H43N5O12 SC12. Molecular Weight 715. Calculated C 43.64; H 6.01; N 9.79; O 26.88; S 4.47; Cl 4.89; P 4.33. 4. Optical rotation [25 o ujlb, +10? (c, 0.854, water) 10 15 20 459 861 22 5. Solubilities Extremely soluble in water, methanol and ethanol. Easily soluble in acetone and other ketones, ethyl acetate and other esters, chloroform, methylene chloride. Insoluble in saturated hydrocarbon solvents. 6. Antibacterial activity U-57930 3- (5'-cytidylate) is inactive in vitro. However, treatment with alkaline phosphatase or phosphodiesterase yields U-57930 which is extremely active against a number of gram-positive organisms, both in vitro and in vivo. Melting point: 205 - 2070 ° C (during decomposition).

Properties of U-57930 3 '(5'-adenylate) 1. IR values The table lists IR absorptions (Nujol and KBr) according to the following 10 15 20 25 30 459 861 23 bands intenband inten- freq. site type freq. site type 3335.3 16 BRD l245, l 24 SH 3267.8 17 BRD l213.3 17 AVG 32l0.8 17 BRD ll75.7 43 SH 2954.3 BRD M ll46.8 40 SH 2924.4 BRD M l089.9 13 AVG 2868.4 SH M 1069.6 10 AVG 2854.9 AVG M l05S, l 13 SH 2727.6 46 BRD M 991.5 35 AVG 2520.2 61 BRD 972.2 36 AVG l684.0 22 SH 957, 7 45 SH l64l, 6 14 AVG 930.7 48 AVG l600, l 28 AVG 889.2 32 AVG l576.0 31 AVG 861.3 47 AVG 1550.9 43 SH 848.7 49 AVG l509.4 53 SH 818, 8 45 AVG l463, l 15 AVG M 798,6 40 AVG 1420 ,? 35 AVG 722.4 36 AVG M l377.3 24 AVG M 708.9 40 SH l367.6 35 SH M 647.1 33 SH l332.0 36 AVG 635.6 31 AVG l299.2 34 AVG Band frequency: band frequency in speed (cm-1) Intensity: intensity in percent transmission (% T) Data type in the area next to peaks: BRD - wide; AVG - average; SHP - pointed; SH - terrace point Top list processed. * indicates added peaks M: means possible interference with mineral oil. 459 861 24 25 strongest peaks% T frequency -% T frequency 2 2924.3 22 l684.0 3 2954.2 24 l377.2 4 2854.8 24 1245.0 6 2868.3 28 l600.0 10 l069.5 31 l576,0 13 l089,8 31 635,5 13 l05S, 0 32 889,1 14 l64l, 5 33 647,0 15 l463,0 34 l299, l 16 3335,2 35 l420,6 17 3267,7 35 l367, 5 17 32l0.7 35 991.5 17 l2l3.2 Preparation: mineral oil max% T; ss G 1ss4.4% T at ssoo (cm7l) = 81 Density (cm-1 / pt): 0.964 10 15 20 25 30 459 25 band inten- band inten- freq. site type freq. site type 3375.8 7 BRD 1090.8 AVG 3223.4 10 BRD l069.6 AVG 3124.0 17 SH 1050.3 8 SH 2963.0 22 AVG 990.5 27 AVG 2929.2 22 BRD 972.2 29 AVG 2878, l 31 AVG 956.8 38 SH 2863.6 33 SH 929.8 42 AVG 2756.6 45 BRD 889.2 24 AVG 252l, 2 60 BRD 861.3 38 AVG 2188.5 75 BRD 851.6 40 SH l678.2 15 SH 818.8 36 AVG 1643.5 7 AVG 807.3 36 BRD l602.0 20 AVG 798.6 30 AVG 1576.0 23 AVG 768.7 41 BRD 1553.8 35 SH 721.4 30 AVG 15l1.4 49 SH 706.9 31 BRD 1475.7 27 AVG 648.1 26 AVG l42l.7 7 AVG 636.5 26 AVG 1384.0 32 AVG 584.5 28 SH l332.0 31 AVG 571.9 26 AVG 1301, l 28 AVG 533,3 27 SH l246, l 19 SH 522,7 25 AVG l2l5,2 12 AVG 503,4 25 AVG 1176,7 36 SH Band frequency: band frequency in carriage numbers (cm_l) Intensity: int Data type in the area next to peaks: average; SHP - pointed; SH - terrace point Top list processed. * indicates added peaks entity in percent transmission (% T) BRD - wide; AVG - genom- 861 10 15 20 25 30 35 459 861 26 25 strongest peaks frequency% T frequency 5 l069, S 9 23 l576.0 7 337s, 7 '24 889.1 7 l643.5 25 522.6 8 l090, 7 25 503.3 8 l050.2 26 647.0 10 3223.3 26 636.5 12 l2l5, l 26 571.8 15 l678, l 27 l475.6 17 3l24.0 27 990.5 19 l246.0 27 533.2 20 l602.0 28 l30l, O 22 2963.0 28 584.5 22 2929, l Preparation: KBR pellet max. 9 T = 95 @ 4os, o% T at 4000 (cm -1) = 77 Tächet (cm -1 / pt) = 0.964 2. Uvabsorption spectrum [Änmx (aü in water at pH 2.0, 258 (16.0) pH 7.0, 261 (16.5) pH 11.0, 261 (16.0) 3. Elemental composition Molecular formula C27H43N7010 SC10P Molecular weight 723.

Calculated C 44.81; H 5.94; N 13.55; O 22.13; S 4.42; Cl 4.84; P 4.28.

Obtained N 12.87; S 5.39; Cl 4.76; P 3.83 10 15 20 25 4. 459 861 27 Optical rotation ßi] D25, + 94 ° (c, o, ss7, water).

. Solubilities High solubility 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.

Antibacterial activity u-57930 [3- (5'-adenylate ertid however, treatment with alkaline phosphatase or phosphodiester I gives U-57930 which is extremely active against one is not active in vitro. However, large number of gram-positive organisms both in vitro and U-57930 3- (5 '- adenylate) has been shown to be active in vivo (subcutaneously, mouse) with a CD50 of 0.62 (0.48 - 0.79) mg / kg against S. pyogenes.

Melting point 203.5 DEG-205 DEG C. (during decomposition) Properties of U-57930 3 ~ (5'-uridylate). IR values The tables list IR absorptions (Nujol and KBr) as follows: 10 15 20 459 861 28 band inten band inten-freq. site type freq. site type 3330.4 19 BRD l332.9 44 BRD 3224.4 21 BRD l296.3 40 SH 2952.4 1 BRD M l25l, 9 28 BRD 2924.4 BRD M l2l5.2 19 AVG 2867.5 SH M l089, 9 13 AVG 2854.0 AVG M l07l, S 9 AVG 2733.4 49 SH M l056, l 13 SH 2695.8 53 SH 991.5 39 AVG 2532.8 67 SH 973.2 39 AVG l757.3 73 SH 957 , 7 48 SH l685,9 8 AVG 931.7 49 AVG l647,4 21 SH 890,2 34 AVG l602,0 41 AVG 858,4 50 AVG l574, l 42 AVG 8l3) 0 43 AVG 1555 ,? 43 BRD 798.6 47 AVG l462.2 12 AVG M 767.7 50 AVG l425.5 37 SH 721.4 42 AVG l378.3 22 AVG M 634.6 35 AVG l367.6 37 SH M band frequency: band frequency in wavelength (cm_l) Intensity: intensity in percent transmission (% T) Data type in the area next to peaks: BRD - wide; AVG - average; SHP - pointed; SH - terrace point The top list is unprocessed M: means possible interference with mineral oil 10 15 20 29 25 strongest peaks% frequency% frequency 0 2924.3 22 l378.2 1 2952.3 28 l25l, 8 3 2854.0 34 890.1 4 2867 .5 35 634.5 8 l685.8 37 l425.5 9 l071.5 37 l367.5 12 l462, l 39 991.5 13 l089.8 39 973.1 13 lO56.0 40 l296.2 19 3330.3 41 l602,0 19 12l5,1 42 l574,0 21 3224,3 42 721.3 21 l647,3 Preparation: mineral oil Max% _T: 86 Û 3764,5% T at 3800 (cm- 1) = ss Density 459 861 10 15 20 25 30 459 861 30 band inten- band 'inten- freq. site type freq. site type 3387.4 12 BRD l055, l 10 SH 31l4.4 25 SH 992.4 35 AVG 2962.0 26 AVG 973.2 36 AVG 293l, l 26 BRD 956.8 45 SH 2879.1 36 AVG 929.8 48 AVG 2863.6 38 SH 889.2 31 AVG 2833 ,? 43 SH 859.3 46 AVG 2509.6 64 BRD 813.0 40 AVG l685.0 6 BRD 811.1 40 SH l647.4 17 SH 798.6 43 AVG l605.9 35 SH 782.2 48 BRD l576.0 37 AVG 768.7 48 AVG 1556 ,? 39 BRD 707.9 43 AVG l463.1 31 AVG 669.3 43 SH l423.6 35 AVG 649.1 40 SH 1384.0 32 AVG 634.6 37 AVG l33l, O 43 AVG 585.4 38 SH l297.2 38 SH 567.1 34 AVG l255.8 24 AVG 523.7 35 AVG l214.3 17 AVG 447.5 39 AVG l090.8 10 AVG l070.6 7 AVG Band frequency: band frequency in wavelength (cm_1) Intensity: intensity in percent transmission (% T) Data type in the area next to peaks: BRD - wide; AVG - average; SHP - pointed; SH - terrace point Top list processed. * indicates added peaks 10 15 20 25 30 35% T 7 10 10 l2 17 17 24 25 26 26 31 31 31 459 861 25 strongest peaks frequency% T frequency 1685.0 32 1384.0 1070.5 34 567.0 1090, ? 35 l605.8 l055.0 35 1423.5 3387.3 35 992.3 1647.3 35 523.6 12l4.2 36 2879.0 1255,? 36 973.1 31l4.3 37 l576.0 2962.0 37 634.5 293l, O 38 2863.5 l463.0 38 l297, l 889.1 Preparation: KBR pellet Max% T: 101 @ 405.0% T at 4000 (cm -1: 76 rather (cm -1 / pt) = 0.964 2. UV absorption spectrum ÉÅ max (a1] In water at pH 2.0, 261 (11.5) pH 7.0, 262 (10 7) pH 11.0, 262 (11, 5) ~ 3. Elemental composition Molecular formula C Calculated C 43.57;, H 5.86; N 7.82; O 29.05; S 4.46; Cl 4, 89; P 4.33 4. Optical rotation [q] D25 + 1os ° (c, 0.94, water) 2sH42N4 ° 13 SClP Molar weight 716. 459 861 32 5. Solubilities Very soluble in water, methanol and ethanol fl Easily soluble in acetone and other ketones, ethyl acetate and other esters, chloroform and methylene chloride Insoluble in saturated hydrocarbons Z 6. Antibacterial activity U-57930 3- (5'-uridylate) is not active in vitro. gives treatment with alkaline phosphatase and phosphol10 diesterase I U-57930 which is extremely active against a number of gram-positive organisms both in vitro and in vivo 7. Melting point 15 202 - 2030 C (during decomposition) Properties of U ~ 5 793O 3- (5 '~ guanylate) 1. Tables of IR absorptions (Nujol and KBr) as follows: 10 15 20 25 459 861 33 bands intenband inten- freq. site type freky. site type 3335,3 16 BRD l250,0 36 SH 3227,2 19 BRD l2l3,3 21 AVG 2953,3 2 AVG M ll73,8 39 AVG 2925,3 l BRD M 1149 ,? 41 AVG 2868.4 6 SH M l087.9 14 SH 2855.9 4 AVG M l07l, 5 9 AVG 2737.3 51 BRD M 991.5 42 AVG 252l, 2 73 BRD 972.2 42 AVG l684.0 6 AVG 956.8 52 SH l635.8 ll AVG 929.8 51 AVG l598.2 21 AVG 890.2 36 AVG l572, l 26 AVG 860.3 51 AVG l534.5 34 AVG 800.5 46 AVG l462.2 18 AVG M 783,1 42 SHP l4l4,9 44 AVG 720,4 43 AVG l377,3 24 AVG M 707,9 45 BRD 1365 ,? 31 AVG 681.9 40 AVG 1312 ,? 44 AVG 635.6 34 AVG Band frequency: band frequency in Vâgtal (cm_l) Intensity: intensity in percent transmission (% T) Data type in the area next to peaks: BRD - wide; AVG - average; SHP - pointed; SH - terrace point Top list processed. * indicates added peaks M: means possible interference with mineral oil s, 459 861 34 25 strongest peaks% T frequency% T frequency __________________ ________________ 1 2925.2 24 l377.2 2 2953.2 26 lS72.0 4 285S, 8 3l l365, 6 6 2868,3 34 l534,5 6 l684,0 34 635,5 9 l07l, 5 36 l250,0 ll 1635 ,? 36 890,1 14 l087.8 39 ll73,7 16 3335,2 40 681,8 18 l462, l 41 ll49,6 19 3227, l 42 991,5 21 l598, l 42 972,1 21 l2l3,2 Preparat: mineral oil Max% T = 97 G 37s2.6% T at saoo (cm'l) = 97 Density (cm'1 / pt); 0.964 10 15 20 25 30 459 861 35 band inten- band inten- freq. site type freq. site type 3380.6 9 BRD ll74.7 31 AVG 3234.0 13 BRD ll47.7 33 BRD 2963.0 22 AVG 1088.9 BRD 2929.2 21 BRD l070.6 AVG 2878, l 3l AVG 991.5 33 AVG 2862.7 33 SH 972.2 34 AVG 2744.0 44 BRD 956.8 43 SH 2522.2 61 BRD 929.8 44 AVG 1683.0 BRD 889.2 28 AVG l634.8 AVG 860.4 41 AVG l598, 2 14 AVG 800.5 36 AVG l57l, 2 19 AVG 783.1 32 AVG l534.5 26 AVG 715.6 38 SH l482.4 38 AVG 705.0 38 SH l46l, 2 36 AVG 679.9 33 AVG 1448, ? 36 BRD 635.6 31 AVG l4l3.9 34 AVG 584.5 34 SH l384.0 29 AVG 571.9 32 AVG l359.9 30 AVG 523.7 31 AVG 1312 ,? 37 AVG 502.5 30 AVG l250.9 29 SH 447.5 34 AVG l2l3.3 16 AVG Band frequency: band frequency in wave numbers (cm-1 »Intensity: intensity in percent transmission (% T) Data type in the area next to peaks : BRD - wide; AVG - average; SHP - pointed; SH terrace point The top list is processed. * Indicates added peaks 10 15 20 25 30 35 459 861 36 25 strongest peaks% T fi; sequence% T frequency, 4 l683, 0 29 l384.0 6 1634,? 29 1250.8 6 1070.5 30 l359.8 9 3380.5 30 502.5 9 l088.8 31 2878.0 13 3234.0 31 1174.6 14 l598, l 31 635.5 16 1213.2 31 523.6 19 l57l, l 32 783.0 21 2929, l 32 571.8 22 2963.0 33 2862.6 26 1534.5 33 l47.6 28 889.1 Preparation: KBR pellet max 5 T = 97 @ 405.0 5 T at 4000 (cmfl) = 71 Density (cm -1 l / pt) = 0.964 2.

UV absorption spectrum [α max (λ] in water at: pH 2.0, 256 ¶13.4; 280 (8.4) terrace point pH 7.0, 254 (14, S); 273 (9.7) terrace point pH 11.0, 259 (12.6); 266 (12.4) Terrace point ~ Elemental composition Molecular formula: C27H43N7011 SCIP Molar 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 13.32; S 4.86; C1 4.49; P 3.25. Optical rotation hd] D25, + 97 ° (c, 0.855, water) 10 15 20 25 30 35 459 861 37 5. Solubilities Highly soluble in water, methanol and ethanol. Easily soluble in acetone and other ketones, ethyl acetate and other esters, chloroform and methylene chloride. Insoluble in saturated hydrocarbons. 6. Antibacterial activity U-57930 3- (5'-guanylate) is inactive in vitro. However, treatment with alkaline phosphatase or phosphodiesterase I U-57930, which is extremely active against a number of gram-positive organisms both in vitro and in vivo. Melting point 219 - 2200 C (during decomposition).

Because the compounds of the present invention are effective against various gram-positive and gram-negative microorganisms, they can be used in various environments to prevent the growth of such microorganisms. For example, they can be used as disinfectants to prevent S. aureus on washed and stored eating utensils contaminated with this bacterium. They can also be used as disinfectants for various dental and medical equipment contaminated with S. aureus. Furthermore, the compounds of the invention can be used as bacteriostatic rinsing agents for washed clothes and for impregnated papers and fabrics and they are also useful for suppressing the growth of sensitive organisms on plates and other microbiological media.

The compounds of the present invention are also useful in the treatment of diseases caused by individuals of the genus Mycoplasma, the most commonly known being forming PPLO (pleuropneumonia-like organisms), such as M. hominis, M. salivarium, M. mycoides, M. hyopneumonia, M. hyorhinis, M. gallisepticum, M. arthriditis and other species of animals including domestic animals such as sheep, dogs, domestic pigs and poultry (eg chickens, turkeys, ducks and geese) and laboratory animals (eg rats and mice).

U-57930 3- (5'-ribonucleotides) can be used in the treatment of kidney and other infections when the L-form of gram-negative and gram-positive bacteria is present, e.g.

The L-form of P. mirabilis.

Because the compounds of the present invention are amphoteric substances, they can form salts with acids and bases using standard procedures. Examples of inorganic acids which can be used to form salts are hydrochloric acid, sulfuric acid, phosphoric acid and the like. Examples of inorganic bases are sodium, potassium, the like. calcium, lithium and salts of compounds can be used for the same purpose as the basic compounds.

The compounds of the present invention are useful as antibacterial agents in suitable compositions. These compositions are preferably prepared for administration to humans and animals in unit dosage form, such as tablets, capsules, pills, powders, granules, sterile, parenteral solutions or suspensions, and oral solutions or suspensions and oil-water emulsions containing appropriate quantities of the active compound in form of the free base or its pharmacologically acceptable salts.

For oral administration, either the solid or liquid unit dosage form can be prepared. For the preparation of the main active ingredient is mixed with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulphate, starch, lactose, acacia, methylcellulose and functionally similar materials solid compositions such as tablets, pharmaceuticals and pharmaceutical diluents . The tablets may be laminated or otherwise formulated to provide a dosage form which provides the benefit of prolonged or delayed action or a predetermined successive effect of entrapped drugs. The tablet may, for example, consist of an inner dose and an outer dose component, the latter being in the form of a cover layer over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and allows the inner component to pass undamaged through the duodenum or delays the release of the drug.

Various materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids or mixtures of polymeric acids with such materials as shellac, cetyl alcohol, cellulose acetate phthalate, styrene maleic acid copolymer and the like. Alternatively, two component systems can be used to make tablets containing two or more mutually incompatible active ingredients. Wafers are made in the same way as tablets, which differ only in the form and introduction of sucrose or other sweeteners and flavors. In its simplest embodiment, capsules as well as tablets are prepared by mixing the compound with a preparation of inert pharmaceutical diluents and fillers, after which the mixture is filled into hard gelatin capsules of suitable size. According to another embodiment, capsules can be prepared by filling hard gelatin capsules with hard polymeric acid-coated beans containing the compound of the formulas of the invention. Soft gelatin capsules can be prepared by machine encapsulation of a slurry of the compound of the invention and an acceptable vegetable oil, light liquid petroleum or other inert oils.

Liquid unit dosage forms for oral administration such as syrups, elixirs and suspensions may be prepared. A water-soluble form of the compound of the invention can be dissolved in an aqueous carrier together with sugar, aromatic flavors and preservatives to form a syrup. An elixir is prepared using hydroalcoholic (ethanol) carrier with suitable sweeteners such as sucrose together with aromatic flavors. Suspensions can be prepared from insoluble forms in a syrupy carrier using a suspending agent such as acacia, tragacanth, methylcellulose and the like.

Local liniments can be prepared by dispersing the active compound in a suitable liniment base, such as petroleum, lanolin, polyethylene glycol, mixtures thereof and the like. Preferably, the compounds are finely divided by means of a colloid mill and the use of light liquid petrolatum as a finely powdering agent before dispersion in the liniment base. Creams and lotions for local application are prepared by dispersing the compound in an oil phase before it is emulsified in the oil phase in water.

For parenteral administration, liquid unit dosage forms are prepared using utilizing compounds of the invention and a sterile vehicle, with water being preferred. The compound may, depending on the form and concentration used, be either suspended or dissolved in a carrier. In preparing solutions, a water-soluble form of the compound of the invention may be dissolved in the water for injection and filter sterilized before being filled into suitable containers or ampoules and sealed. Preferably, adjuvants such as local anesthetics, preservatives and buffering agents may be dissolved in the carrier. To improve stability, the composition can be frozen after filling into containers and the water removed in vacuo. The dry lyophilized powder can then be sealed in the container and an accompanying water container for injection is used to reconstitute the powder before use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in a carrier instead of being reconstituted. dissolved and sterilization cannot be performed by filtration.

The compound can be sterilized by exposure to ethylene oxide before being suspended in a sterile vehicle. For delayed action, an intramuscular suspension is prepared in insoluble form, such as trimethylsilyl ether or the pamoate salt.

Preferably, a surfactant or wetting agent is included in the composition to facilitate the distribution of the compound.

The term unit dosage form is used in the description and claims to denote physically different units suitable as unit doses for humans and animals, each unit containing a predetermined amount of active material intended to provide the desired therapeutic effect in combination with necessary pharmaceutical diluents or carriers. The description of the novel unit dosage forms of the present invention is dictated by and is directly dependent on (a) the unique properties of active materials and the particular therapeutic effect desired and (b) inherent limitations on the ability to purify an active material. for therapeutic use in humans and animals which are shown in detail in the description and constitute a feature of the present invention. Examples of suitable unit dosage forms according to the present invention are tablets, capsules, pills, dragees, suppositories, powder packets, granules, wafers, capsules, teaspoonful, tablespoonful, dropwise, ampoules, containers, various multiples of the foregoing and other forms described herein. .

The active compound is combined with a suitable pharmaceutical carrier in unit form for convenient and efficient administration. According to a preferred embodiment of the present invention, the unit doses contain 10, 25, 50, 100, 250, and 500 mg amounts of a compound of the invention for systemic treatment; 5 to 65% w / v for parenteral treatment. The dose of a composition containing an active compound and one or more active ingredients is determined with reference to the usual dosages of such ingredients.

The following examples illustrate preferred embodiments in the opinion of the inventor and are not intended to be limiting.

According to the examples, 3- (5'-ribonucleotide) of U-5793OE or U-60970E is used as the active compound, but it is obvious that these are only examples of suitable compounds and other active compounds within the scope of the invention can also be used. U-60970E is 4-cis-n-butyl-L-pipecolinic acid amide of 7-C1-methylthiolinkosaminide. The preparation is shown in Example 7 of U.S. Patent No. 4,276,789.

References U-57930E or U-6097OE hereinafter refer to 3- (5'-ribonucleotide) of these compounds. The 3-ribonucleotides are those shown here.

Preparation Example 1 - Two hard gelatin capsules in two pieces for oral use, each containing 250 mg of U-57930E or U-60970E, the following types and amounts of material were prepared: U-57930E or U-6D87OE 250 g of corn starch 100 g talc 75 g magnesium stearate 25 g The materials were mixed thoroughly and encapsulated in the usual manner.

The mentioned capsule type is useful in the systemic treatment of infections in adults by oral administration of one capsule every six hours. A similar procedure was used in which capsules were prepared containing U-57930E or U-60970E in amounts of 10, 25, 50, 100 and 500 mg by inserting 10, 25, 50, 100 and 500 mg instead. g U-57930E or U-60970E instead of the 250 g used above.

Composition Example 2 Å capsules 1000 in two gelatin capsules for oral use each containing 200 mg of U-57930E or U-60970E and 250 mg of tetracycline hydrochloride were prepared from the following types and amounts of ingredients: U-57930E or U-60970E 200 g of tetracycline 250 g talc 75 g magnesium stearate 25 g The ingredients are mixed thoroughly and encapsulated in the usual way.

The foregoing capsules are useful for the systemic treatment of infections in adults by oral administration of one capsule every six hours.

Using the above procedure, capsules can be similarly prepared containing U-57930E or U-60970E and each of the following antibiotics instead of tetracycline by replacing the 250 g of tetracycline with the corresponding amount of chloramphenicol, oxytetracycline, chlorotetracycline, fumagillin, erythromycin, streptomycin, dihydronovobiooin and novobiocin. When a penicillin such as potassium penicillin G is used instead of tetracycline, 250,000 units per capsule are used.

Such combination products are useful for the systemic treatment of mixed infections in adults by oral administration of one capsule every six hours.

Composition Example 3 - Tablets 1000 tablets for oral use each containing 500 mg of U-57930E or U-60970E were prepared from the following types and amounts of materials: U-S7930E or U-609703 500 g of lactose 125 g of corn starch 65 g of magnesium stearate 25 g of light liquid petroleum 3 g Mix the ingredients thoroughly and grind. The grains are broken down by being forced through a sieve with sieve number no. 16.

The resulting granules are then compressed into tablets, each tablet containing 500 mg of U-57930E or u6o37oE.

The foregoing tables are useful for the systemic treatment of infections, including malaria infections in adults, by oral administration of 1 tablet 3 times daily.

Using the above procedure except that the amount of U-57930E or U-609703 is reduced to 250 g, tablets containing 250 mg of U-5793OE or U-6097OE may be prepared.

Composition Example 4 - Tablets 1000 oral tablets, each containing 250 mg of U-57930E or U-60970E and a total of 250 mg (83.3 mg each) of sulfadiazine, sulfamerazine and sulfamethazine were prepared from the following types and amounts of materials : 10 15 20 25 30 35 459 861 45 11-579302 or U-6o97oE 250 g sulfadiazine 83.3 g sulfamerazine 83.3 g sulfamethazine 83.3 g lactose SO g corn starch 50 g calcium stearate 25 g light liquid petrolatum 5 g The ingredients are mixed and kornas. The grains are broken down by being pressed through a sieve with the sieve numbers l6. The resulting granules are then compressed into tablets, each containing 250 mg of U-57930E or U-60970E and a total amount of 250 mg (83.3 mg each) of sulfadiazine, sulfamerazine and sulfamethazine.

The foregoing tablets are useful for the systemic treatment of infection by oral administration of 4 tablets only every 1 hour.

For the treatment of urinary tract infections, the three types of sulphate in the above preparation are suitably exchanged for 250 g of sulphamethylthiadiazole or 250 g of sulphacetamide.

Composition Example 5 - Oral Sirag 1000 ml of an aqueous suspension for oral use containing in each 5 ml 250 mg of U-57930E or U ~ 6097OE and 500 mg of total sulfa were prepared from the following types and amounts of ingredients: U-5793OE or U ~ 60970E 50 g sulfadiazine 33.3 g sulfamerazine 33.3 g sulfamethazine 33.3 g citric acid 2 g benzoic acid 1 g __.._.._.___._.._..._._.__ __ .. .________ ___ __ ____________, ____ 10 15 20 25 30 35 459 861 46 sucrose 700 g tragacanth 5 g lemon oil 2 ml deionized water, to 1000 ml Citric acid, benzoic acid, sucrose, tragacanth and lemon oil are dispersed in a sufficient amount of water to obtain 850 ml solution. U-57930E or U-60970E and the finely divided sulfa preparations are stirred in the syrup until evenly distributed. Sufficient water is added to obtain 1000 ml.

The composition thus prepared is useful for the systemic treatment of pneumonia in adults with a dose of 1 tablespoon (10 ml) 4 times daily.

Composition Example 6 - parenteral solution A sterile aqueous solution for intramuscular use containing 200 mg of U-57930E or U-60970E in ml was prepared from the following types and amounts of materials: U-57930E or U-609703 200 g of lidocaine hydrochloride 4 g of methyl paraben 2 .5 g of propyl paraben 0.17 g of water for injection up to 1000 ml The ingredients were dissolved in water and the solution was sterilized by filtration. The sterile solution is filled into containers and the containers are sealed.

Composition Example 7 - Parenteral Preparation A sterile aqueous solution for intramuscular use containing in each ml 200 mg of U-57930E or U-609703 and 400 mg of 400 mg of 400 spectral tomycin sulfate was prepared by mixing the following 459,861 in II types and amounts. -57930E or U-60970E 200 g 'spectinomycin sulphate 400 g lactose 50 g water for injection up to 1000 ml U-57930E or U-60970E, spectinomycin sulphate and lactose are dispersed in water and sterilized. The sterile mixture is aseptically filled into sterile containers in an amount of 2 ml.

Composition Example 8 ~ Local liniment 1000 g of a liniment containing 0.25% were prepared in the following types and amounts of ingredients: U-57930E or U-60970E 2.5 g zinc oxide 50 g calamine 50 g liquid petrolatum (heavy) 250 g wool fat 200 g white petrolatum to 1000 g The white apetrolatum and wool grease were melted and 100 g of liquid petrol etum was added. U-57930E or U-6097OE, zinc oxide and calamine are added to the remaining liquid petrolatum and mixed in a mixing mill until the powders are finely divided and evenly distributed. The powder mixture is stirred into the white petrolatum mixture and stirred until the liniment solidifies.

The foregoing liniment is useful for topical treatment of the skin of mammals for the treatment of infections.

The foregoing composition can be prepared by exclusively zinc oxide and calamine. If the procedure of the above was followed, similar liniments can be prepared containing U-57930E or U-60970E in amounts of 0.5, 1, 2 and 5% by replacing the above-mentioned 2.5 g U-57930E or U-60970E with 5 , 10, 20, 50 g of said compounds.

Composition Example 9 - cream 1000 g of vaginal cream was prepared from the following types and amounts of ingredients: U-5793OE or U ~ 6097OE 50 g of tegacidregularl 150 g spermaceti 100 g of propylene glycol 50 g of polysorbate 80 5 g of methyl paraben 1 g of deionized water to 1000 g. , New York, NY

The tegacid and spermacetin are melted together at a temperature of 70-800 ° C. Methyl paraben is dissolved in about 500 g of water and propylene glycol, polysorbate 80 and U-57930E and U-60970E are in turn added at a temperature of 75-800 ° C. to the tegacid and the spermacetin melt with constant stirring.

The addition is continued for at least 30 minutes with continuous stirring until the temperature is lowered to 40-450 ° C. The pH of the final cream is adjusted to 3.5 by introducing 2.5 g of citric acid and 0.2 g of dibasic sodium phosphate dissolved in 50 g. g of water. Finally, a sufficient amount of water is added to give 1000 g and the preparation is stirred until homogeneous and cooled and solidifies. The foregoing composition is useful for the treatment of vaginal infections in humans.

Composition Example 10 - liniment, ophthalmic 1000 g of an ophthalmic liniment containing 0.5 U-57930E or U-60970E were prepared from the following types and amounts of ingredients: U-57930 or U-609703 5 g bacitracin 12.2 g polymyxin B sulfate (10 .000 units / mg lg light liquid petrolatum 250 g wool grease 200 g white petrolatum to 1000 g The solids were comminuted using an air atomizer and added to the light liquid petroleum. The mixture was passed through a colloid mill to obtain an even distribution of the finely divided particles.The wool fat and the white petrolatum are fused and dyed, after which the temperature is adjusted to 45 - 500 C.

The liquid petrolatum slurry was added to the liniment and stirred until the liniment solidified. Suitable liniments are packaged in ophthalmic tubes containing 3.9 g.

The foregoing liniment can be usefully applied to the eye for the treatment of local infections in humans and other animals.

Preferably the foregoing composition may contain 5 g (0.5%) of methylprednisolone for the treatment of inflammation and alternatively bacitracin and polymyxin B sulphate may be omitted. Composition Example II - Eye-Ear Drops 1000 ml of sterile eye or ear solutions containing 10 mg U-57930E or U-60970E and 5 mg methyl-prednisolone in each ml prepared from the following types and amounts of ingredients: U-57930E or U-60970E 10 g methylprednisolone phosphate sodium 5 g sodium citrate 4.5 g sodium hydrogen sulphite 1g polyethylene glycol 4000 120 g myristyl-T-picolinium chloride 0.2 g polyvinylpyrrolidone 1g deionized water to 1000 ml water and the resulting solution is sterilized by filtration. The solution is aseptically filled into sterile drip containers.

Compositions prepared in this way are useful for the local treatment of inflammations and injections in the eye and ear as well as other sensitive tissues in the animal body. Composition Example 12 - dragees 10,000 dragees are prepared from the following ingredients: types and amounts U-57930E or U-60970E 100 g neomycin sulphate 50 g polymyxin B sulphate (10,000 units / mg) 1g ethylaminobenzoate 50 g calcium stearate 150 g powdered sucrose to 5000 g 10 15 20 25 30 35 459 861 51 The powdered materials were mixed together and beaten into half-gram dragers according to the usual technique and position of compound tablets.

The dragees are kept in the mouth and allowed to dissolve slowly, whereby treatment of the mouth and throat of humans is obtained.

Composition Example 13 - Rectal suppositories 1000 suppositories, each weighing 2.5 g and containing 100 mg of U-5793OE or U-6097OE were prepared from the following types and amounts of ingredients: U-57930E or U-60970 E 100 g of polymyxin B sulfate (10,000 units / mg) 1.25 g methylprednisolone 1g ethylaminobenzoate 75g zinc oxide 62.5g propylene glycol 162.5g polyethylene glycol 4000 to 2500g U ~ 57930E or U-6097OE, polymyxin B sulfate, methylprednisolone , ethylaminobenzoate and zinc oxide are added to the propylene glycol and mixed until the powder is finely divided and evenly distributed. Polyethylene glycol 4000 is melted and the propylene glycol dispersion is added slowly with stirring. The suspension is then beaten in uncooled molds at 400 ° C.

The composition is allowed to cool and solidify and is then removed from the mold and each suppository is wrapped in foil.

Previous suppositories introduce reactants for local treatment of inflammation and infection.

Alternatively, the foregoing composition may be prepared excluding the steroid. 10 15 20 25 30 35 459 861 52 Composition example 14 - mastitis liniment 1000 g liniment for the treatment of mastitis for dairy cows was prepared from the following types and amounts of ingredients: U-5793OE or Ü-60970E 25 g methylprednisolone acetate 0.5 g light liquid petrolatum 300 g chlorobutanol , anhydrous 5 g polysorbate 80 5 g 2% aluminum monostearate-peanut oil gel 400 g white petrolatum to 1000 g U-57930E or U-60970E and methylprednisolone acetate were ground with light liquid petrolatum until the material is finely divided and evenly distributed. Chlorobutanol, polysorbate 80, peanut oil gel and white petrolatum are heated to 490 ° C and form a melt in which the liquid petrolatum dispersion is stirred. With continuous stirring, the dispersion is allowed to cool and solidify) to room temperature and is filled into disposable mastitis syringes in doses of 10 g.

Composition Example 15 - Animal feed 1000 g of food treatment were prepared from the following types and quantities of ingredients: U-57930E or U-60970E 10 g soybean meal 400 g fishmeal 400 g wheat germ oil 50 g sorghum molasses 140 g The ingredients are mixed together and beaten into pellets. The composition can be added to laboratory animals, i.e. rats, mice, guinea pigs and hamsters for prophylactic treatment 10 15 20 25 30 35 459 861 53 during transport.

For other animals such as poultry, e.g. chickens, ducks, turkeys and geese, the composition can be added to the animals' regular food in amounts calculated to give the desired dose of U-5793OE or Uf6097OE.

Composition Example 16 Following the procedure of each of the foregoing Composition Examples 1 to 15, each antibacterial compound of the present invention may be replaced with an equivalent amount of U-57930E or U-60970E listed in the examples to provide therapeutic properties.

Similarly, each of the above free base compounds may be used in the form of a pharmaceutically (or pharmacologically) acceptable salt, e.g. the hydrochloride, the sulphate, the phosphate, the sodium salt, the potassium salt, the calcium salt or the lithium salt. »V Composition Example 17 - capsules 1000 hard gelatin capsules in two pieces for oral use, each containing 200 mg of U-57930E or U-60970E and 200 mg of hydroxychloroquine sulphate, the following types and amounts of ingredients are prepared: U-5793OE or U-60970E 200 g hydroxychlorine sulphate 200 g talc 75 g magnesium stearate 25 g The ingredients are mixed thoroughly and encapsulated in the usual way. 10 15 20 25 30 35 459 861 54 The foregoing capsules are useful for preventing recurrent attacks of P. vivax in adults by oral administration of 1 capsule per week.

Composition Example 18 - Tablets 1000 oral tablets each containing 125 mg U-57930E or U-60970E and 325 mg quinine sulphate were prepared from the following types and amounts of materials: U-57930E or U-6097OE 125 g quinine sulphate 325 g lactose 50 g corn starch 50 g calcium stearate 25 g light liquid petrolatum S g The ingredients are thoroughly mixed and granulated. The granules are broken down by pressing through a sieve with a sieve number of 16. The resulting granules are pressed into tablets, each containing 125 mg of U-57930E or U-60970E and 325 mg of quinine sulphate.

These tablets are useful for the treatment of malaria by oral administration of 2 tablets every eight hours for 7 days thereafter 1 tablet 3 times daily for 7 days.

Composition Example 19 - oral syrup 1000 ml of an aqueous suspension for oral use containing in each 10 ml a 25 mg pyrimethamine, 250 mg U-5793QE or U-60970E and 500 mg sulfadiazine were prepared from the following types and amounts of ingredients: 45 g 459 861 55 U-57930E or U-60970E 25 g pyrimethamine 2.5 mg sulfadiazine 50 g citric acid 2 g benzoic acid 1 g sucrose 700 g tragacanth 5 g lemon oil 2 ml deionized water to 1000 ml Citric acid, benzoic acid, sucrose, tragacanth and the lemon oil is dispersed in a sufficient amount of water to obtain 850 ml of solution. U-57930E or U-60970E pyrimethamine and sulfadiazine are stirred in the syrup until evenly distributed. Sufficient water is added to obtain 1000 ml.

The preparation prepared in this way is useful in the prophylactic treatment of malaria in adults at a dose of 1 teaspoon (10 ml) per week.

Composition Example 20 - Parenteral Preparation Sterile water composition for intramuscular use containing 1 ml 200 mg U-57930E or U-6097OE prepared from the following types and amounts of ingredients: U-57930E or U-60970E 200 g lactose 50 g water for injection to 1000 ml U-57930E or U-60970E and the lactose was dispersed in water and sterilized. The sterile composition was aseptically filled in an amount of 2 ml into sterile containers. Composition Example 21 The procedure of each of the preceding examples was followed, substituting equivalent amounts of U-57930E or U-60970E which are shown to possess therapeutic properties for the antimalarial active compound of the present invention. Similarly, the above compounds may be used in the form of pharmaceutically (or pharmacologically) acceptable salts, e.g. the hydrochloride, sulfate, phosphate, sodium salt, potassium salt, calcium salt or lithium salt.

In vivo results against P. berghei MEDI (mg / kg) CD 2 (mg / kg) 50 compound sub. Q3 OI sub. Q OI U-57930E 0.16 1.6 16 (12-22)> 5O U-2125lF <20 - 53 (46-61) - U-24729A <1.25 - 4.7 (3.2 -6,9) - U-8284 chloroquine <5-10 l2,5 ll, 5 (8,8-15) l4 chloroquine (PO4) 2 <5 - ~ <20 - ”lMED = dose with which median survival time ( ST50) is significantly increased (p = 0.05) over ST50 for untreated control animals. 2CDšO = Median protective dose in mg / kg 95% probability.

Administration route.

Anti-malaria test (P. berghei) Test procedure. Male mice, CF-1 (18 - 20 g) were kept in cages of 10 and infected intraperitoneally with whole blood from mice infected with P. berghei 3 days before bleeding. A 0.2 ml aliquot of heparinized blood, diluted 1:10 with salt, served as inoculum. This volume contained approximately 106 parasites. 4 hours after infection, each group of 10 mice was treated either subcutaneously with 0.2 ml or orally with 0.5 ml of the desired drug concentration. The treatment continued in groups every day for 4 days. The animals were observed for 28 days and deaths were recorded. Death before the sixth day was considered traumatic.

The development of efficacy for different analogues and drug concentrations of individual analogues based on the average survival time of the animals for each level of treatment and the average protective dose for the individual analogous compounds. The calculations were performed on an IBM 370 computer. The results were based on treated groups compared with the results from untreated groups or groups treated with chloroquine.

Other protozoa within the scope of the present invention are intracellular parasites, for example species of Plasmodia, Toxoplasma and Leishmania, protozoa which have consumed the red blood cells (RBCs) in treated patients, for example Entamoeba histolytica and certain Trypanosomas and other helminths which swallow red blood cells during the procedure. . 58 459 861 Scheme 1 5 H i: 3 C113 H N H [ß HO-C-H C3H7 H I? H -NCH HH ß 10 0 Ho 5 OH HH * (1) H 3 SCH3 H OH 15 20 (2) 25 H OH 30 Å. 35 10 15 20 25 30 459 861 59 Scheme 2 where R 1 which may be single or multiple substituted in each position in the pyridine ring not already occupied by R 2 is selected from the group consisting of hydrogen, alkyl and substituted alkyl where the alkyl moiety contains 1 to 8 carbon atoms and isomeric forms thereof, cycloalkyl and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl and substituted phenyl, - (CH 2) n -OH, - (CH 2) n -NR 4 R 5 and isomeric forms thereof where n represents an integer from 1 to 8, where R 4 and R 5 represent hydrogen or alkyl having 1 to 8 carbon atoms and isomeric forms thereof, wherein R 2 which may be simply substituted at any position in the pyridine ring not already substituted with R 1 represents O 11 -cx and where X represents an amino function of a compound selected from the group 7 (R ) -hydroxy-methyl-1-thio-N, -lincosaminide, 7 (S) -hydroxy-methyl-1-thio-CL-lincosaminide, 7 (S) -halogen-methyl-1-t io-Å-~-lincosaminide, 7 (R) -halo-methyl-1-thio- & l-lincoosaminide, 7 (S) -methoxy-methyl-1-thio-OL-lincosaminide, 7-deoxy-7 (S) - ( methylthio) -methyl-1-thio-05-lincosaminide, 7-deoxy-7 (S) - (2-hydroxyethylthio) -methyl-1-thio-11-lincosaminide and 7-deoxy-7 (S) - ( 3-hydroxypropylthio) -methyl-1-thio- link, - linososamide and pharmaceutically acceptable salts thereof. 451 861 60 where R 1 and R 2, which may be present in 2, 3, 4, 5, 6, 7, 8 or 9 position in the ring are previously defined and where R 3 represents H, CH 3, C 2 H 5 and -CH 2 -CH 2 - OH, where n represents an integer from 1 to 4 and pharmaceutically acceptable salts thereof. 10 15 20 25 30 35 | H "f fl f? | Ñ": '61 Scheme_3 U-57930 U-57930 3- (5'-Cytidylase U-57930 3- (5'-adenylate) U-57930 3- (5' -uridyl fi U-57930 3- (5'-guanylate fi I coNH- ca 6 459 861 CH; a HC-C1 7 H0 5 H OR 0

Claims (12)

459 861 62 Patent claims 1. The 3- (5'-ribonucleotide) of a compound of the formula I (CH 2) n CH 0 fww C-NH 10 CH wZ OH SCH OH where R 1 may be mono- or polysubstituted in 3, 4, 5, 7 , 8 or 9 position in the ring and is selected from the group consisting of hydrogen, alkyl, and substituted alkyl wherein the alkyl moiety contains 1 to 8 carbon atoms, isomeric forms thereof, cycloalkyl and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl and substituted phenyl, - (CH 2) n -OH- - (CH 2) n -NR R more forms thereof; wherein R 3 represents H, CH 3, C 2 H 5 and -C 52 -CH 2 -OH; where n represents an integer from 1 to 4, where Y represents a group consisting of 7 (S) -halogen and 7 (R) -halogen and pharmacologically acceptable salts thereof. 30 A compound according to claim 1, characterized in that 7 (S) -halogen is 7 (S) -chloro. 3. n - G t in that it is a compound of formula 45 and isomeric forms thereof wherein n represents an integer from 1 to 8, wherein R 4 and R 5 represent hydrogen or alkyl having 1 to 8 carbon atoms and iso- The 3- (5 'ribonucleotide) of claim 1, wherein 4.; (H \ D CD = 3 \ .nå 63 Cz fl s fe É HC-Cl 5. N H C NH - CH HO O H S-CH 10 OH and pharmaceutically acceptable salts thereof. The 3- (5'-ribonucleotide) of a compound according to claim 1 having the formula C 4 H 9 β β O HC-Cl 20 || ß N C-NH-CH H H O OH 25 -CH 3 H and pharmaceutically acceptable salts thereof. The 3- (5'-ribonucride) of a compound of claim 1 having the formula N 'ff 10 15 20 25 30 459 861 64 wherein X is QIUPP selected from the group consisting of 7 (S) -halomethyl -1-thio-mf-lincosaminide and 7 (R) -halogen-methyl-1-thio-ck-lindosaminide and pharmaceutically acceptable salts thereof A 3- (5'-ribonucleotide) compound of a compound according to claim 1 having the formula C 7. - 3 '(5 "rib0 fl üklG fififi E fl) of a compound according to claim 1 having the formula 235" lozenge 211 Z O I N wherein X is defined in claim 5 and pharmaceutically acceptable salts thereof. A compound according to claim 1 of the formula Jin 01 UD CO Ch -A 65 CH 2 CH 3 ïH3 HC-Cl N CONH - CH HH I. R SCH3 OH wherein R represents NH2 / 'NI 441 \ N o O in H _.ï-O -CH2 OH 0 OH OH 459 861 66 A compound according to claim 1 of the formula CH CH 2 I 3 CH 3 I H-e-cl N coNH - ca H Ho o on scH 3 on where R represents OH OH 459 851 67 10. l0. A compound according to claim 1 of the formula CH CH CH 2 3 I 3 I-I-C-Cl N CONH "CH H HO O OR SCH 3 OH where R represents OH OH 459 861 68 11. ll. A compound according to claim 1 having the formula cH2cH3 CH I 3 Hc-cl N CONH - CH H Ho O on scH3 on wherein R represents OH Na 2 /: gQ: NN / JK _.ï-o-c fi z ou ° ~ \ OH OH 10 15 20 25 30 35 459 861 69 12. l2. A process for the preparation of a 3- (5'-ribonucleotide) of a compound according to claim 1 of the formula (wherein RL is about 3 o 'b' v YNH | c-Na ---- cH Ra Ho o oH C be mono- or double-substituted in the 3, 4, 5, 7, 8 or 9 position in the ring with a group selected from the group consisting of hydrogen, alkyl and substituted alkyl, the alkyl moiety containing 1 to 8 carbon atoms and isomeric forms thereof, cycloalkyl and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl and substituted phenyl, - (CH 2) n -OH, - (CH 2) n -NR 4 R 5 and isomeric forms thereof, where n represents an integer from 1 to 8, where R 1 and R 5 represent hydrogen or alkyl having 1 to 8 carbon atoms and isomeric forms thereof, wherein R 3 represents a group selected from the group consisting of H, CH 3, C 2 H 5 and -CH 2 -CH 2 -OH, where n represents an integer from 1 to 4, where Y represents a group selected from the group consisting of 7 (S) -halogen and 7 (R) -halogen and pharmaceutically acceptable salts thereof, characterized in that m grows Streptomyces rochei, NRRL 3533 in an aqueous medium containing nutrients in the presence of a compound of the above formula and extracts the desired 3- (5'-ribonucleotide) from the culture broth.