JPH0224279B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel macrolide antibiotic useful as an antibacterial agent, and further relates to erythronolide A.
(erythronolide A), erythronolide B or 3-O-mycarosyl-erythronolide B (3-
Starting from new intermediates derived from O-mycarosyl-erythronolide B), it relates to a process for their microbiological production. The present invention also relates to novel microorganisms useful for the production of the macrolide antibiotics of the invention and associated methods of producing such microorganisms by mutagenesis starting from known stocks. . The invention further relates to novel intermediates derived from erythronolide A, erythronolide B or 3-O-mycarosyl-erythronolide B, and related chemical synthesis methods. As mentioned above, the novel semi-synthetic macroloid antibiotic of the present invention is an antibacterial agent.
agents). They exhibit a spectrum and level of activity compared to virtually known types of erythromycin, and they are subject to less degradation even in acidic environments, which allows for good absorption even orally. The esters or salt-esters are absorbed more quickly and completely than the free base, giving higher and more prolonged blood levels. The novel antibiotics of the present invention form addition salts with organic or inorganic acids due to the presence of basic groups. Such salts are prepared from the free base using methods commonly used for the preparation of addition salts of basic antibiotics. Water-insoluble salts are used as liquid oral dispersions (they are less bitter). Mono-ester derivatives of the novel antibiotics and their addition salts can be prepared by methods such as those used for the preparation of erythromycin A esters and salt-esters. Erythromycin A esters and salts-esters and methods for their preparation are known in the art.
(8S)-8-fluoroerythromycin A ((8S)
-8-fluoroerythromycin A) (p-80206),
(8S)-8-fluoroerythromycin B (p-
80203), (8S)-8-fluoroerythromycin C (p-80205), (8S)-8-fluoroerythromycin D (p-80202), 3-O-oleandrosyl-5-O-desosaminyl- (8S) -8-Fluoroerythronolide B (3-O-oleandrosyl
-5-O-desosaminyl- (8S) -8-fluoro-
erythronolide B) (p-80204) (abbreviations in parentheses here are applicant's own index);
Salt - Esters or salts include acetate, propionate, butyrate, succinate, valerate, ethyl succinate, propionate/lauryl sulfate, stearate, lactobionate, glucoheptonate, sulfate, lauryl sulfate, carbonate derivatives, etc. Among them, lactobionate and glucoheptonate are water-soluble salts that can be administered intravenously. Ethyl succinate can also be administered both orally and parenterally. The novel antibiotic of the present invention is white in color and has a bitter taste.
Odorless powder, thermally stable in powder form, acidic PH
It is more stable than erythromycin A in aqueous solutions. The half-life times of the initial activity of erythromycin A and the macrolide antibiotic of the present invention in solutions of various PH values are reported in the table below. [Table] The first aspect of the present invention is a method for producing novel intermediates derived from erystronolide A, erystronolide B, and 3-O-mycarosyl-erystronolide B; Used in the microbiological production of new antibiotics. Erystronolide A is erythromycin A
It is known that it can be obtained by selectively removing cladionose sugar and desosamine sugar from (RALeMahieu et al. J. Med. Chem.,
17, 963, (1974)). Erythronolide B and 3-O-mycarosyl-erythronolide B can also be produced by micro-organisms of erythromycin by direct fermentation in suitable quantities and thus at industrially viable costs. It is also known that it is a substrate that can be obtained using microorganisms (producers) or their mutants. On the other hand, separate regular erythromycin A, these substrates have no use in other fermentations (although such fermentations have the disadvantage of lack of antibiotic properties, which also affects erythromycin itself), and Therefore, from an industrial standpoint, the use of these substrates is most desirable. The following has now been discovered: That is, erythronolide A, erythronolide B or 3-O- which can be produced by a simple and industrially possible chemical method.
Derivatives of mycarosyl-erythronolide B are useful substrates for fermentation processes, in which microorganisms obtained by mutagenesis from strains employed for the microbiological production of erythromycin A are used. This will produce the novel macroid antibiotic of the present invention, which will be described in more detail later. The chemical process for the preparation of the novel intermediates of the present invention is represented by the following scheme: When R is H, X is H or a group. and when R is OH, X represents H. and (a) a compound selected from erythronolide A, erythronolide B or 3-O-mycarosyl-erythronolide B with an anhydrous acid such as glacial acetic acid or a methanol solution of hydroxylamine hydrochloride. ) to form compound (), i.e. 8,9-anhydroerythronolide A-6,9-hemiacetal 8,9-anhydroerythronolide B-6,9-hemiacetal or 3-O-mycarosyl -8,9-anhydro-erythronolide B-6,9-hemiacetal is produced, and (b) Compound () is converted to a compound capable of generating electrophilic fluorine, preferably a fluoroxy-perfluoro-alkane (general formula CnF _2o+1 OF) and perchloryl fluoride (with CnF 2o+1 OF)
fluoride) is reacted at low humidity in the presence of an inert organic solvent to form the corresponding acetal (); (c) the compound () is reacted with an aqueous acid to form the desired compound (); It is characterized by consisting of processes. In step (b), the most commonly used fluoroxy-perfluoro-alkane closs reactant is fluoroxy-trifluoro-methane, which is commercially available. Other reactants with a positively charged fluorine atom that can be used in this reaction include fluoroxy-sulfur-pentafluoride, molecular fluorine and lead tetraacetate-hydrofluoride (lead tetraacetate-hydrofluoride).
cetate (hydrofluoric acid). Among the reaction solvents, chlorinated hydrocarbons such as trichlorofluoromethane (Freon 11), chloroform, methylene chloride, tetrahydrofuran, and mixtures thereof are preferred. Further, the reaction is preferably carried out at a low temperature, preferably within the range of -75 to -85°C, with stirring. The reaction is usually complete in about 15 minutes to 1 hour. It is also important to point out that in this process, a non-negligible amount of the desired compound (2) has already been produced and mixed with the production of the acetal (2). In the third reaction step (c), organic or mineral acids, such as acetic acid or hydrochloric acid, are used. Approximately 30℃～
A reaction temperature of about 150°C can be employed. The product () is purified and recovered by recrystallization or chromatography. Furthermore, compounds (), (), and () are new and form part of the present invention. The present invention, apart from the intermediate (), is directed to a blocked type mutant strain of Streptomyces erythrois.
erythreus) ATCC31772, and this mutant strain is mutagenized by chemical methods (i.e., chemical mutagenic agent method), ultraviolet (X-ray) irradiation, phage action, etc. can get. Streptomyces erythroid
Culture of ATCC31772 with compound () as substrate
(8S)-8-fluorinated erythromycin (scheme: P-80202, P-80203, P-
80205 and P-80206). A new class of antibiotics has been produced with R _{st of} 0.9, 1.13, 0.89,
and1.12 and R _st for erythromycin B
0.85, 1.06, 0.87and 1.04 and even have special colors when treated with their chromatic reagents: the first antibiotic has a brick red color (when hot), the second and fourth Dark purple (after cooling)
, and the third one has a dark brown color (in heat). R=CH ₃ (8S)-8-fluoroerythromycin B (P-80203) R=H(OS)-8-fluoroerythromycin D (P-80202) R=CH ₃ (8S)-8-fluoroerythromycin A (P-80206) R=H(8S)-8-fluoroerythromycin C (P-80205) The following is a further object of the present invention. It is one.
A microbiological method for the production of novel antibiotics of the erythromycin family, comprising:
is characterized in that it is used as a substrate for fermentation with Stre-ptmyces antibioticus ATCC31771, and when the substrate () is (8S)-8-fluoroerythronolide A, is an antibiotic with an R _{st of} 0.87 for oleandomycin and an R _{st of} 0.80 for erythromycin A.
o-oleandrosyl-5-o-desosaminyl-
(8S)-8-fluoroerythronolide A(3-
o-oleandro-syl-5-o-desosaminyl-
(8S)-8-fluoroerythronolide A) (Scheme:
P-80207) is produced, and the substrate () is (8S)
-8- In the case of fluoroerythronolide B,
The antibiotic 3-o- _oleandrosyl - ₅ -o-desosaminyl-(8S)-8-fluoroerythronolide B (scheme: P- 80204) is generated. 3-o-oleandrosyl-5-o desosaminyl-(8S)-8-fluoroerythronolide A (P-80207) 3-o-oleandrosyl-5-o-desosaminyl-(8S)-8-fluoroerythronolide B (P-80204) Microorganism Streptomyces erythrois
It was possible to produce the desired antibiotic by culturing ATCC 31772 and Streptomyces antibioticus ATCC 31771 as a substrate with the compound () by several fermentation methods. After fermentation is complete, various techniques can be used to isolate and purify the antibiotic. Among the various methods suitable for separation and purification, liquid
Solvent extraction methods such as liquid countercurrent extraction and gel permeation chromatography methods are preferred. In a preferred embodiment, the antibiotic produced according to the invention is recovered by separating mycelium and insoluble solids from the fermented broth by conventional means such as filtration or centrifugation. The antibiotic is then extracted from the strained or centrifuged meat juice using a batch method or a countercurrent distribution extraction method. Suitable solvents are alcohols such as methanol, ethanol etc., chlorinated hydrocarbons such as chloroform, methylene chloride etc., ethyl acetate, butyl acetate, amyl acetate, methyl isobutyl ketone and acetonitrile, with methyl isobutyl ketone being preferred. . After filtering or centrifuging the fermentation medium, thin layer chromatography or high pressure liquid phase chromatography can be used to analyze the subject antibiotic. In addition, bioautographies (bioauto-
graphy) can be used to advantage. The following examples illustrate the invention in detail without imposing any limitations. Example 1 Production of Mutant Streptomyces erythreus ATCC 31772 A suspension of spores of the Streptomyces erythreus product of erythromycin was treated with ultraviolet light (radiation maximum 250 nm) to remove approximately 99.6% of the spores. Mutant generation treatments were carried out at lethal doses (approximately 3000 ergs/cm ² ). Live spores were seeded onto plates of nutrient medium and the resulting colonies were analyzed by A. Kelner (1949) J. Bact.
It was analyzed for its ability not to produce erythromycin according to the technique described in 57, 73. Then a mutant blocked in the synthesis of erythromycin (approximately 2
%) was analyzed for its ability to recognize compound () as a substrate and convert it into a novel compound with antibiotic activity. Example 2 8,9-anhydroerythronolide A-6,
Preparation of 9-hemiacetal () 4.185 g (0.01 mol) of erythronolide A () in 32 ml of glacial acetic acid (RALeMahieu et al, J.
Med.Chem. 17 , 953 (1974))
The solution is allowed to stand at room temperature for 2 hours. The acetic acid is then removed in vacuo at 40° C. and the oily residue is dissolved in 150 ml of chloroform. The chloroform solution was washed with a saturated solution of sodium bicarbonate, then with water until neutral, and finally with
Dry _{with Na2SO4} . After removing the solvent in vacuo, the crude product is obtained,
Chromatograph this on a silica gel column,
Purified by elution with methylene chloride-methanol (1:1). Elution with methylene chloride-methanol (98:2) yields 8,9-anhydroerythronolide A-
A fraction containing only 6,9-hemiacetal () was given. Evaporation of these combined fractions to dryness and subsequent crystallization from acetone/n-hexane gave 2.750 g of a compound () with the following properties: mp 188 - 193°C; [α] ²⁰ _D + 20. 5゜ (C=1, methanol); UV (MeOH) 210nm (ε6720) IR (KBr) 3630, 3520, 3495, 3400, 1710,
1465, 1450, 1440, 1415, 1400, 1370, 1350,
1310, 1285, 1230, 1200, 1170, 1140, 1090,
1075, 1060, 1050, 1035, 1020, 1010, 1000,
970, 950, 940, 920, 915, 905, 890, 870, 825,
810,800cm ^-1 . Analysis of C ₂₁ H ₃₆ O ₇ : Calculated value (%): C62.97; H9.06 Actual value (%): C63.12: H9.10 Example 3 (8S)-8-Fluoroerythronolide A ―
6,9;9,11 acetal () and (8S)
-8-Fluoroerythronolide A () 8,
9-Anhydroerythronolide A-6,9-
Preparation from hemiacetal () A −80 °C solution of fluoroxy-trifluoromethane in CCl ₃ F was prepared: an excess amount of CF ₃ OF (usually approximately twice) was added to CCl ₃ F (-80 °C with dry ice). CF ₃ OF
Sartosious is a cylinder containing
(weighed continuously on an electric balance). Its concentration was determined by iodometry. CF ₃ OF / CCl ₃ F solution at −80℃ or −85℃,
slowly added to a solution of 4 g (0.010 mol) of 8,9-anhydroerythronolide A-6,9-hemiacetal in CCl ₃ F/CH ₂ Cl ₂ (295 ml/370 ml) at about -80°C; Hydrogen fluoride was removed by magnetic stirring in the presence of calcium oxide (1.920 g). The progress of the reaction was monitored using high pressure liquid phase chromatography (HPLC).
Compound () was periodically monitored for disappearance of its characteristic peak. Disappearance (or minimization) of the peak of compound ()
Stirring was then continued for 5 minutes, nitrogen was bubbled through the solution to remove excess _CF3OF at -80<0>C, and the solution was warmed to room temperature. This solution
Washed with a saturated solution of NaHCO ₃ (650 ml), then water until neutral and finally dried over Na ₂ SO ₄ . Removal of the solvent gave a solid residue, which was then purified by column chromatography on silica gel (ratio 1:50) and prepared in methylene chloride/methanol (1:1). Elution with increasing concentrations of methanol in methylene chloride yields (8S)
-8-Fluoroerythronolide A-6,9;
A fraction containing only 9,11-acetal () and a fraction containing only (8S)-8-fluoroerythronolide A () were obtained. The fraction containing compound () was evaporated to dryness and then crystallized from acetone/n-hexane, yielding 3.435 g of a product with the following characteristics: mp192 - 3°C: ; [α] ²⁰ _D +64 .7゜ (C=1, methanol) UV (methanol): No absorption corresponding to ketone group IR (KBr): 3560, 3420, 1720, 1460, 1395,
1380, 1355, 1345, 1330, 1320, 1305, 1290,
1270, 1255, 1215, 1180 (broad), 1095, 1070,
1045, 1025, 1020, 990, 980, 970, 955, 940,
930, 920, 910, 905, 895, 855, 840, 830, 805
cm ^-1 Analysis calculated value (%) for C ₂₁ H ₃₅ FO ₇ : C60.27; H8.43; F4.54 Actual value (%): C60.22; H8.51; F4.69 Compound () The fractions contained were evaporated to dryness and then crystallized from acetone/n-hexane, yielding 145 mg of a product with the following characteristics: mp 239-240°C [α] ²⁰ _D -3.1° (C=1, UV (methanol): 287-8nm (ε25.3) IR (KBr): 3610, 3550, 3480 (shoulder), 3380 (shoulder),
1735, 1700, 1460, 1405, 1390, 1380, 1350,
1325, 1300, 1290, 1270, 1175, 1105, 1090,
1050, 1035, 1020, 980, 960, 940, 920, 905,
Analysis calculation value (%) for 895, 875, 860 cm ^-1 C ₂₁ H ₃₇ FO ₈ : C57.78; H8.54; F4.35 Actual value (%): C57.87; H8.63; F4.19 Example 4 (8S)-8-fluoroerythronolide B-
6,9;9,11-hemiacetal () and (8S)-8-fluoroerythronolide B ()
8,9-Anhydroerythronolide B-
Preparation from 6,9-hemiacetal () Following the procedure of Example 3, but with 3.845 g (0.010 mol) of 8,9-anhydroerythronolide B-
6,9-hemiacetal () (U.S. Patent No.
Starting from a solution containing (as described in No. 3697547) a crude product is obtained which, crystallized from acetone/n-hexane, yields 3.450 g of (8S)-8-fluoroerythro Nolide B-6,9;9,11-acetal ()
was obtained: mp191-2°C; [α] ²⁰ _D +42.6° (C = 1.0, methanol); UV (methanol): no absorption corresponding to ketone group IR (KBr): 3540, 3450, 1735, 1460, 1260,
Analysis calculation value (%) for 1170, 1060, 1035, 980, 925, 875, 450 cm ^-1 C ₂₁ H ₃₅ FO ₆ : C62.67; H8.76; F4.72 Actual value (%): C62.63 ; H8.82; F4.81 The mother liquor was chromatographed on a silica gel column (ratio 1:50) and methylene chloride-methanol (98:
Elution with 2) gave 75 mg of a second product, (8S)-8-fluoroerythronolide B () with the following characteristics: mp247-8°C; UV (methanol): 286nm ( ε
26) [α] ²⁰ _D −30° (C=1, methanol) IR (KBr): 3540, 1727, 1703, 1460, 1330,
1270, 1175, 1130, 1075, 1050, 1015, 940,
Analysis calculation value (%) for 920, 895, 860 cm ^-1 C ₂₁ H ₃₇ FO ₇ : C59.98; H8.87; F4.52 Actual value (%): C59.92; H9.00; F4.59 Example 5 (8S)-8-fluoroerythronolide B
(8S)-8-fluoroerythronolide B-6,9 of (); Production from 9,11-acetal () 4.830g (0.012 mol) of (8S)-8-fluoroerythronolide B-6, 9; A mixture of 9.11-acetal () and 3000 ml of acetic acid aqueous solution (PH3) was refluxed at 110°C for 15 minutes with stirring,
Then 220ml of acetic acid was added. After 1 hour at 110℃,
All starting materials were dissolved. Heating was continued for 30 minutes, then the solution was cooled to room temperature as quickly as possible, neutralized with NaHCO ₃ and extracted with ethyl acetate. The ethyl acetate solution was dried over sodium sulfate and evaporated to dryness in vacuo. This crude product was applied to a column of silica gel (ratio 1:
50) and eluted with methylene chloride-methanol (95:5), 1.8 g of product was obtained, which had the same chemical and physical properties as the compound () isolated in Example 4. It had Example 6 5.465 g (0.010 mol) of 3- in 32 ml of glacial acetic acid
O-mycarosyl-erythronolide B(1) (JR
Martin, Biochemistry, 5 , 2852 (1966)) is allowed to stand at room temperature for 2 hours. The acetic acid is then removed in vacuo at a temperature of 40° C. and the oily residue is dissolved in 150 ml of chloroform. The chloroform solution was washed with a saturated solution of sodium bicarbonate, then with water until neutral, and finally with
Dry _{with Na2SO4} . Removal of the solvent in vacuo gave the crude product, which was purified on a column of silica gel prepared in chloroform (ratio 1:100). Elution with increasing concentrations of methanol in chloroform yielded fractions containing only 3-O-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal (). These fractions were combined and evaporated to dryness.
Then, when crystallized from acetone/n-hexane,
1.160 g of a compound ( ) with the following properties was obtained: mp85-88°C: [α] ²⁰ _D -5° (C=1, methanol); UV (MeOH): 210 nm (ε 6850); IR ( KBr): 3500, 1730, 1465, 1415, 1380,
1340, 1185, 1120, 1085, 1055, 1005, 985,
Analysis calculation value (%) for 945, 895, 810 cm ^-1 C ₂₈ H ₄₈ O ₉ : C63.61; H9.15 Actual value (%): C63.52; H9.09 Example 7 3-O-mycarosil -(8S)-8-fluoroerythronolide B-6,9;9,11-acetanol () and 3-O-mycarosyl-
(8S)-8-fluoroerythronolide B ()
Preparation of 3-O-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal () A -80 °C solution of fluoroxy-trifluoromethane in _CCl3F was prepared; of CF ₃ OF (typically about twice as much) was dissolved in CCl ₃ F (precooled to −80°C with dry ice) by slowly adding the gas through the purge tube (while
The cylinders containing CF ₃ OF were weighed continuously on an electric Sartorious scale). Its concentration was determined by iodometry. A solution of CF ₃ OF / CCl ₃ F at −80℃ or −85℃,
5.285g in _CCl3F / _CH2Cl2 (295ml/ _370ml )
(0.010 mol) of 3-O-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal () was added slowly to a solution containing 3-O-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal () and magnetically dissolved in the presence of calcium oxide (1.920 g). Hydrogen fluoride was removed by stirring. The progress of the reaction was monitored periodically by high pressure liquid phase chromatography (HPLC) for the disappearance of the peak characteristics of compound (). After disappearance (or minimization) of the peak for compound (), stirring was continued for an additional 5 minutes, nitrogen was bubbled through the solution to remove excess _CF3OF at −80 °C, and the solution was allowed to warm to room temperature. Warmed.
This solution was washed with _NaHCO3 saturated solution (650 ml), then with water until neutral and finally with
Dried _{with Na2SO4} . Removal of the solvent gave a solid residue, which was then chromatographed on silica gel prepared in chloroform (ratio 1:
100). When elution is carried out with increasing concentrations of methanol in chloroform, 3-O-mycarosyl-
(8S)-8-fluoroerythronolide B-6,
9; Fraction containing only 9,11-acetal () and 3-O-mycarosyl-(8S)-8
- A fraction containing only fluoroerythronolide B() was obtained. The fraction containing compound () was evaporated to dryness and crystallized from acetone, yielding 1.100 g of a product with the following properties: mp 175 -7°C; [α] ²⁰ _D -9.8° (C = 1 , methanol); UV (methanol): No absorption corresponding to ketone group IR (KBr): 3540, 3490, 1720, 1460, 1395,
1380, 1365, 1355, 1330, 1320, 1290, 1275,
1265, 1245, 1205, 1185, 1165, 1145, 1120,
1100, 1080, 1060, 1045, 1015, 1005, 990,
960, 940, 925, 915, 900, 880, 865, 840, 815
cm ^-1 Analysis calculated value (%) for C ₂₈ H ₄₇ FO ₉ : C61.52; H8.67; F3.47; Actual value (%): C61.46; H8.51; F3.48 Compound () When the fraction containing is evaporated to dryness and crystallized from acetone/n-hexane,
290 mg of product with the following properties were obtained: mp 214 -5°C; [α] ²⁰ _D -73.2° (C=1, methanol); UV (methanol) 287 nm (ε 25.3); IR (KBr): 3570, 3550, 3460 (shoulder), 1745, 1735,
1465, 1380, 1365, 1340, 1300, 1275, 1250,
1180, 1115, 1080, 1055, 1010, 1000 (shoulder), 985,
Analysis calculation value (%) for 945, 935, 920, 895, 855, 840, 810 cm ^-1 C ₂₈ H ₄₉ FO ₁₀ : C59.55; H8.75; F3.37; Actual value (%): C59. 68; H8.60; F3.48. Example 8 3-O-mycarosyl-(8S)-8-fluoroerythronolide B-6,9;9,11-acetal of 3-O-mycarosyl-(8S)-8-fluoroerythronolide B() Manufacture from () 6.560g (0.012mol) of 3-O-mycarosyl-
(8S)-8-fluoroerythronolide B-6,
A mixture of 9;9,11-acetal () and 3000 ml of acetic acid aqueous solution (PH3) was stirred and kept at room temperature to completely dissolve, and then 220 ml of acetic acid was added. The solution was stirred at room temperature until complete disappearance of the starting material (controls are carried out by high-pressure liquid phase chromatography), NaHCO ₃
The mixture was made neutral and extracted with ethyl acetate. The ethyl acetate solution is dried over sodium sulfate and evaporated to dryness in vacuo. This crude product was applied to a column of silica gel (ratio 1:
50) and eluted with methylene chloride-methanol (95:5) to give the product,
This is the compound isolated in Example 7 ()
It had the same chemical and physical properties as. Example 9 (8S)-8-fluoroerythronolide A
Production of (8S)-8-fluoroerythronolide A-6,9;9.11-acetal of () 5.020 g (0.012 mol) of (8S)-8-fluoroerythronolide A-6.9;;9.11-acetal ( ) and 3000 ml of acetic acid aqueous solution (PH3) was refluxed at 110°C for 15 minutes with stirring.
Then 220ml of acetic acid was added. After 1 hour at 110°C the starting material was dissolved.
Heating was continued for 30 minutes, then the solution was cooled to room temperature as quickly as possible, neutralized with NaHCO ₃ and extracted with ethyl acetate. The ethyl acetate solution was dried over sodium sulfate and evaporated to dryness in vacuo. The crude solid was then chromatographed on a column of silica gel (ratio 1:50) and prepared with methylene chloride. Elution with increasing concentrations of methanol in methylene chloride yields a fraction still containing the starting material (8S)-8-fluoroerythronolide A-6,9;9,11-acetal ();
(8S)-8-fluoroerythronolide A ()
A fraction was obtained containing only Repeated reactions of the recovered starting material and subsequent purification by chromatography yielded (8S)
Another fraction containing -8-fluoroerythronolide A was obtained. Evaporate all of the combined fractions to dryness,
The resulting solid is then recrystallized to yield 0.725 g of (8S)-8-fluoroerythronolide A().
was obtained, which had the same properties as reported in Example 3. Example 10 Production of antibiotics (8S)-8-fluoroerythromycin C (P-80206) and (8S)-8-fluoroerythromycin A (P-80205) Streptomyces erythreus ATCC31772,
The seeding culture of the mutant blocked in the synthesis of erythromycin was mixed with the following ingredients (g/):
Prepared in a medium consisting of: sucrose 30.0; molasses 8.0; soybean oil 9.0; (NH ₄ ) ₂ SO ₄ 2.0; CaCO ₃ 7.0. This culture medium was cultured for 48 hours at 33°C on a rotary shaker. Seeds were added at a level of 5% (V/V) to a 250 ml Erlenmeyer flask containing 30 ml of fermentation medium with the following composition (g/): corn dextrin 30.0; crude corn starch 40.0; soybean. Meal 30.0; soybean oil
20.0; ( _NH4 ) _{2SO4 2.0} ; and _CaCO3 6.0. This fermentation flask was cultured for 24 hours at 33°C using a rotary shaker (220 rpm, 4 cm stroke). 15 mg of finely ground (8S)-8-fluoroerythronolide B () was sterilized with ultraviolet light for 15 minutes,
Add to each flask and incubate with shaking.
It lasted 96 hours. Treatment of samples by thin layer chromatography and high pressure liquid phase chromatography At the end of the fermentation time, approximately 900-1000 mcg/
ml (potency expressed as erythromycin A) was centrifuged and the upper liquid was diluted with equal volumes of a 10% (w/v) aqueous solution of _ZnSO4 and 4% (w/v) of sodium hydroxide. /v) clarified by addition of aqueous solution. After centrifugation, the clear upper liquid was extracted with one third of its volume of ethyl acetate. TLC control A sample of the organic phase was spotted on a silica gel G plate, and _{CH2Cl2 -} MetOH- _H2O -concentrated _NH4OH (90:
9.5:0.5:1) for 2 hours. The spots were probed with a spray reagent consisting of methanol-anisaldehyde-concentrated acid-acetic acid (85:0.5:5:10) and the active compound was isolated from Microccus luteus (Sarcina lutea) ATCC.
This was revealed by the biological self-delineation method on plates seeded with 9341. TCL results showed the disappearance of added (8S)-8-fluoroerythronolide A () and the appearance of two active compounds. R _st values were 0.89 and 1.12 for erythromycin A, respectively, and 0.87 and 1.04 for erythromycin B, respectively. Moreover, they showed different color reactions after applying the spray reagent and heating: the slowest compound (antibiotic P-80205)
dark and color (when heated) and dark purple (after cooling) for other compounds (antibiotic P-80206). HPLC control A sample of the organic phase was evaporated to dryness, taken up in acetonitrile and injected onto a column (RP8 10 μm 25 cm; mobile phase phosphate buffer 0.01 molar PH7/acetonitrile 36:64; flow 2 ml/min; column temperature 40 °C ). 2
Two peaks were detected. Retention times were 0.68 (P-80205) and 0.87 for erythromycin A.
(P-80206). Example 11 Purification of the antibiotics (8S)-8-fluoroerythromycin C (P-80205) and (8S)-8-fluoroerythromycin A (P-80206) Following the method described in the previous example, 1.00 g of ( 8S) -8-Fluoroerythronolide A was added to several fermented meat juices, totaling 2100ml, while stirring.
(4% W/V) and then vacuum filtered.
The solid was washed with water, and the combined liquid was adjusted to pH 5.5 with acetic acid. This acidic aqueous solution was extracted three times with equal volumes of ethyl acetate. The aqueous phase was neutralized with 2N _NH4OH ,
Sterile evaporate to a volume of 1000ml with 2N _NH4OH
The pH was adjusted to 8.8 and extracted with an equal volume of methyl isobutyl ketone. The organic extracts were combined and washed twice with half the volume of 0.1M KH ₂ PO ₄ , then 1
Washed twice with water. After drying (Na ₂ SO ₄ ) and removing the solvent in vacuo,
The residue was purified by NLOleinick, J.Biol.Chem., VOl.244,
It was purified by chromatography on silica gel according to the method described in J.D. No. 3, p. 727 (1969). Fractions 90 to 174 containing only antibiotic P-80206 were combined and evaporated to dryness at 40°C. The solid residue was crystallized from absolute ethanol to yield 230 mg
(8S)-8-fluoroerythromycin A (P-80206) was obtained with the following properties: mp: 183-4°C; [α] ²⁰ _D -55° (C=1, methanol); UV ( methanol): 283nm (ε 17.9) IR (KBr): 3520, 3520 (wide), 1735, 1720,
1460, 1425, 1400, 1380, 1345, 1330, 1305,
1280, 1190, 1170, 1055, 1030, 1015, 1005,
980, 960 (shoulder), 935, 890, 870, 855, 835, 800cm
^-1 (this spectrum is shown in Figure 1). Analysis result value (%) for C ₃₇ H ₆₆ FNO ₁₃ : C59.10; H8.85; F2.52; N1.86 Actual value (%): C59.09; H8.89; F2.59; N1 .88 Fractions 280-400 containing only antibiotic P-80205 were combined and evaporated to dryness in vacuo at 40°C.
When the solid residue is crystallized from absolute ethanol,
145 mg of (8S)-8-fluoroerythromycin C (P-80205) with the following properties was obtained; mp: 217-8°C; [α] ²⁰ _D -42.35° (C = 1, methanol) UV ( methanol): 284nm (ε 23.2) IR (KBr): 3550, 3500, 3440 (shoulder), 3300 (wide),
1730, 1455, 1425, 1410, 1380, 1360, 1340,
1330, 1305, 1280, 1270, 1245, 1200 (shoulder), 1170
(wide), 1115, 1090, 1075, 1060, 1030, 1010,
1000, 980 (shoulder), 965, 955, 945, 935, 920, 905,
895, 870, 840, 830, 810 cm ^-1 (this spectrum is shown in Figure 2). Analysis calculated value (%) for C ₃₆ H ₆₄ FNO ₁₃ : C58.60; H8.74; F2.57; N1.90; Actual value (%): C58.47; H8.87; F2.60; N1 .82. Example 12 Antibiotic 3-O-oleandrosyl-5-O-
Production of desosaminyl-(8S)-8-fluoroerythronolide A (P-80207) S.antibioticus ATCC
A pre-inoculated culture of 31771, a mutant blocked in the biosynthesis of orenadomycin, was prepared in a medium consisting of the following components (grams per deionized water): soybean meal.
30.0; cellulose 15.0; yeast autolyzate 1.0; soybean _oil 30.0; _{MgSO4.7H2O1.0} ; CaCO3 _10.0 . The pH of this medium was adjusted to 7.2 before sterilization. After 24 hours at 28° C. in a rotary shaker, this culture was used to inoculate the same medium at a concentration of 2% (V/V) and further cultured under the same conditions for 16 hours. This inoculum was added to a fermentation medium with the following composition (g/):
Added to a 250 ml Erlenmeyer flask containing 30 ml at a concentration of 3% (V/V): cererose 40.0; soybean meal 20.0; corn meal
3.0; dry baker's yeast 2.0; _CaCO3 2.0. For fermentation, flasks were incubated for 32 hours at 28°C on a rotary shaker (240 rpm, 4 cm stroke). 15 mg of finely ground (8S)-8-fluoroerythronolide A () was added to each flask and the culture was continued for 64 hours with agitation. At the end of this period, the titer of the culture (expressed as erythromycin A) was 100-120 mc
g/ml. Processing of fermented broth for TLC analysis was performed using the system and conditions set forth in Example 10. TLC Control Following the technique described in Example 10, a new active compound (antibiotic P-80207) appeared and the added substrate disappeared. Its Rf is 0.80 for erythromycin A and oleandomycin.
It is 0.87. HPLC Control Following the technique described in Example 10, the new active compound (antibiotic P-80207) has a retention time of 0.69 for erythromycin A and 0.95 for oleandomycin. Example 13 Antibiotic 3-O-oleandrosyl-5-O-
Purification of Desosaminyl-(8S)-8-fluoroerythronolide A (P-80207) Total culture broth derived from 50 fermentations carried out with stirring in Erlenmeyer flasks according to previous Example 12. was treated with an equal volume of methanol. Hyflo parcel (Hyflo
Supercell) was added with stirring over 30 minutes and the mixture was filtered. The solid was washed with water/methanol (1:1) and the combined liquid was evaporated to half the starting volume under reduced pressure. The pH of this solution was adjusted to 8.2 by addition of KOH, and the solution was extracted three times with an amount corresponding to one third of the volume of methyl isobutyl ketone. The organic solvent extracts were combined and 0.1 mol Na ₂ HPO ₄
and then water. The organic solution was dried with Na ₂ SO ₄ and evaporated to dryness in vacuo. The residue was purified by column chromatography on silica gel according to the method described in Example 11. Fractions 45 to 80 containing only antibiotic P-80207 were combined and evaporated to dryness in vacuo at 40°C. The solid residue is crystallized from absolute ethanol to give 130
3-O-oleandrosyl- with the following properties of mg
5-O-desosaminyl-(8S)-8-fluoroerythronolide A (P-80207) was obtained: mp: 155-7°C: [α] ²⁰ _D -40.2° (C = 1, methanol); UV (methanol): 283nm (ε 20.3); IR (KBr): 3480 (wide), 1730, 1510, 1380,
1340 (wide), 1305, 1275, 1195, 1165, 1105,
1095, 1075, 1050, 1030, 1010, 1000, 980, 960
(Shoulder), 935, 915, 895, 875, 830 cm ^-1 (This spectrum is shown in Figure 3) Analytical calculated values for C ₃₆ H ₆₄ FNO ₁₃ (%): C58.60; H8.74; F2. 57; N1.90 Actual value (%): C58.52; H8.75; F2.63; N1.95 Example 14 Antibiotic (8S) - 8- Fluoroerythromycin D (P-80202) and (8S) - 8-Production of fluoroerythromycin B (P-80203) Streptomyces erythreus ATCC31772,
That is, a culture medium inoculated with a mutant blocked in erythromycin biosynthesis,
A medium consisting of the following ingredients (g/) was prepared: sucrose 30.0; molasses 8.0; soybean oil 9.0;
( _NH4 ) _2SO42.0 ; _{CaCO37.0 .} This culture medium was incubated at 33°C for 48 hours on a rotary shaker. This inoculum was prepared with the following composition (g/)
were added at a level of 5% (V/V) to a 250 ml Erlenmeyer flask containing 30 ml of fermentation medium with: corn dextrin 30.0; crude corn starch 40.0; soybean meal 30.0;
Soybean oil 20.0; ( _NH4 ) _{2SO4 2.0} ; _CaCO3 6.0. The fermentation flask was placed on a rotary shaker (220 rpm, 4 cm stroke) at 33°C for 24 hours. 15 mg of finely ground (8S)-8-fluoroerythronolide B (), sterilized with UV light for 15 minutes.
was added to each flask and incubation continued for 96 hours with shaking. Thin layer chromatography (TLC) of this sample
and high pressure liquid phase chromatography (HPLC)
At the end of the fermentation period, approximately 900-1000mcg/
ml of activity (titer expressed as erythromycin A) was centrifuged and the supernatant was diluted with equal volumes of a 10% (w/v) aqueous solution of ZnSO ₄ and a 4% (w/v) aqueous solution of sodium hydroxide. V) Clarified by addition of aqueous solution. After centrifugation, the upper clear liquid was extracted with one third of its volume of ethyl acetate. TLC control A sample of the organic phase is deposited on a silica gel G plate,
_{CH2Cl2 -} MetOH- _H2O -concentrated _NH4OH (90:9.5:
0.5:1) for 2 hours. The spot was mixed with methanol-anisaldehyde-concentrated sulfuric acid-acetic acid (85:
0.5:5:10) and the active compound was applied to Microccus luteus (Sarcina lutea).
It was detected by biological self-delineation method on the plate inoculated with ATCC9341. TLC results showed the disappearance of added (8S)-8-fluoroerythronolide B () and the appearance of two active compounds. R _st values are 0.9 and 1.13 for erythromycin A, respectively;
and 0.85 each for erythromycin B.
and 1.06. Moreover, they exhibit different color reactions after spray reagent application and heating: dark and colored (under hot conditions) the slowest compound (antibiotic P-80202) and dark purple (after cooling) other compounds (antibiotic P-80202) and dark purple (after cooling). -80203). HPLC Control A sample of the organic phase was evaporated to dryness, taken up in acetonitrile and injected onto the column (RP8 10μm25
cm; mobile phase phosphate buffer 0.01M PH7/acetonitrile 36:64; flow 2ml/min; column temperature 40
℃). Two peaks appeared with retention times of 0.79 for erythromycin A (P-80202) and
It was 1.06 (P-80203). Example 15 Purification of the antibiotics (8S)-8-fluoroerythromycin D (P-80202) and (8S)-8-fluoroerythromycin B (P-80203) Following the procedure described in the previous Example 14, 1.0 g of (8S)-8-Fluoroerythronolide B was added to several fermented meat juices, totaling 2100 ml, using Hyflo Supercell (4% W/V).
was added with stirring, and then filtered under vacuum. The solid was washed with water, and the combined liquid was adjusted to pH 5.5 with acetic acid. This acidic aqueous solution was extracted three times with equal volumes of ethyl acetate. Neutralize the aqueous phase with 2N _NH4OH and add 1000
Evaporated under reduced pressure to a volume of ml, adjusted to pH 8.8 with 2N NH ₄ OH, and extracted with an equal volume of methyl isobutyl ketone. The organic extracts were combined and washed twice with half volume of 0.1M KH ₂ PO ₄ and then with water. After drying (Na ₂ SO ₄ ) and removal of the solvent under vacuum, the residue was purified by N. L. Oleinick, J. Biol. Chem.
It was purified by silica gel column chromatography as described in Vol. 244, n. 3, page 727 (1969). Fractions 18 to 32 containing only antibiotic P-80203 were combined and evaporated to dryness in vacuo at 40°C. Crystallization of the solid residue from absolute ethanol gave 150 mg of (8S)-8-fluoroerythromycin B (P-80203) with the following properties: mp: 164-6°C; [α] ²⁰ _D -63 ° (C=1, methanol); UV (methanol): 285nm (ε 29.5) IR (KBr): 3480 (wide), 1735, 1465, 1435,
1385, 1375, 1330, 1305, 1280, 1170, 1115,
1090, 1075, 1055, 1035, 1020, 1000, 975,
940, 890, 835, 805 cm ^-1 (Figure 4) Analysis of C ₃₇ H ₆₆ FNO ₁₂ : Calculated value (%): C60.39; H9.04; F2.58; N1.90 Actual value (%) :C60.31;H9.09;F2.60;N1.88 The mixture was combined with fractions 55 to 105 containing only antibiotic P-80202 and evaporated to dryness in vacuum at 40°C.
Crystallization of the solid residue from absolute ethanol yielded 150 mg of (8S)-8-fluoroerythromycin D (P-80202) with the following properties: mp: 213-5°C; [α] ²⁰ _D -60 ° (C=1, methanol); UV (methanol); 285nm (ε 30.8) IR (KBr): 3600, 3520, 3300 (wide), 1730,
1460, 1420, 1385, 1370, 1355, 1345, 1330,
1310, 1275, 1190, 1160, 1120, 1100, 1060,
1040, 1030, 1010, 1000, 995, 975, 960, 935,
920, 910, 890, 875, 840, 825, 810cm ^-1 (5th
figure). Analysis for C ₃₆ H ₆₄ FNO ₁₂ : Calculated value (%): C59.87; H8.94; F2.63; N1.94 Actual value (%): C59.87; H8.85; F2.63; N1 .88 Example 16 Production of antibiotic 3-O-olenadrosyl-5-O-densamil-(8S)-8-fluoroerythronolide B (P-80204) S.antibioticus
A pre-inoculated culture of ATCC31771, a mutant blocked in the biosynthesis of orenadomycin, was prepared in a medium consisting of the following components (grams per deionized water): 30.0 soybean meal; Cererose 15.0; yeast autolysate 1.0;
Soybean oil 30.0; _{MgSO4.7H2O1.0} ; _{CaCO3 10.0} . The pH of this medium was adjusted to 7.2 before sterilization. After 24 hours at 28° C. in a rotary shaker, this culture was used to inoculate the same medium at a concentration of 2% (V/V) and further cultured under the same conditions for 16 hours. This inoculum was added to a fermentation medium with the following composition (g/):
Added to a 250 ml Erlenmeyer flask containing 30 ml at a concentration of 3% (V/V): cererose 40.0; soybean meal 20.0; corn meal
3.0; dry baker's yeast 2.0; _CaCO3 2.0. For fermentation, flasks were incubated for 32 hours at 28°C on a rotary shaker (240 rpm, 4 cm stroke). 15 mg of finely ground (8S)-8-fluoroerythronolide A () was added to each flask and the culture was continued for 64 hours with agitation. At the end of this period, the titer of the culture (expressed as erythromycin A) was 100-120 mc
g/ml. Processing of fermented broth for TLC analysis was performed using the system and conditions set forth in Example 14. HLC Control Following the technique described in Example 14, a new active compound (antibiotic P-80204) appeared and the added substrate disappeared. Its R _f is 0.82 for erythromycin B and for oleandomycin
It is 0.90. HPLC Control Following the technique described in Example 14, the new active compound (antibiotic P-80204) has a retention time of 0.62 for erythromycin B and 1.06 for oleandomycin. Example 17 Antibiotic 3-O-oleandrosyl-5-O-
Purification of densaminyl-(8S)-8-fluoroerythronolide B (P-80204) Total culture broth derived from 15 fermentations carried out with stirring in Erlenmeyer flasks according to previous Example 16 was treated with an equal volume of methanol. Hyflo Super Cell (Hyflo)
Supercell) was added with stirring over 30 minutes and the mixture was filtered. The solid was washed with water/methanol (1:1) and the combined liquid was evaporated to half the starting volume under reduced pressure. The pH of this solution was adjusted to 8.2 by addition of KOH, and the solution was extracted three times with an amount corresponding to one third of the volume of methyl isobutyl ketone. This organic solvent extract was added to 0.1 mol of Na ₂ HPO ₄
and then water. This organic solution
Dry over Na ₂ SO ₄ and evaporate to dryness in vacuo. The residue was purified by column chromatography on silica gel according to the method described in Example 15. Fractions 17 to 42 containing only antibiotic P-80204 were combined and evaporated to dryness in vacuo at 40°C. The solid residue was prepared in Sephadex LH in hexane-chloroform (1:1).
Further purification was carried out on a 20 column (2 x 98 cm) eluting with the same solvent. When fractions containing only the novel antibiotic P-80204 are combined and concentrated in vacuo at 40°C, 170 mg of 3-O-oleandrosyl-5-O-densaminyl-(8S)-8 with the following properties is obtained:
-Fluoroerythronolide B (P-80204) was obtained: mp: 195-6°C; [α] ²⁰ _D -48° (C = 1, methanol); UV (methanol): 285nm (ε 29) IR (KBr): 3600, 3400 (wide), 1730, 1455,
1400, 1380, 1365, 1350, 1325, 1305, 1270,
1255, 1180, 1160, 1145, 1100, 1060, 1040,
1010, 995, 975, 960, 935, 915, 890, 875,
855, 845, 830, 820 cm ^-1 (Figure 6). Analysis for C ₃₆ H ₆₄ FNO ₁₂ : Calculated value (%): C59.90; H8.94; F2.63; N1.94 Actual value (%): C59.94; H9.06; F2.66; N1 .83 Example 18 Production of antibiotics (8S)-8-fluoroerythromycin D (P-80202) and (8S)-8-fluoroerythromycin B (P-80203) Streptomyces erythreus ATCC31772,
That is, a culture medium inoculated with a mutant blocked in erythromycin biosynthesis,
A medium consisting of the following ingredients (g/) was prepared: sucrose 30.0; molasses 8.0; soybean oil 9.0;
( _NH4 ) _2SO42.0 ; _{CaCO37.0 .} This culture medium was incubated at 33°C for 48 hours on a rotary shaker. This inoculum was prepared with the following composition (g/)
crude corn starch 40.0; corn dextrin 30.0; soybean meal 30.0;
Soybean oil 20.0; ( _NH4 ) _{2SO4 2.0} ; _CaCO3 6.0. The fermentation flask was placed on a rotary shaker (220 rpm, 4 cm stroke) at 33°C for 24 hours. 15 mg of finely ground (8S)-8-fluoroerythronolide B (), sterilized with UV light for 15 minutes.
was added to each flask and incubation continued for 96 hours with shaking. Thin layer chromatography (TLC) of this sample
and high pressure liquid phase chromatography (HPLC)
At the end of the fermentation period, approximately 900-1000mcg/
ml of activity (titer expressed as erythromycin A) was centrifuged and the supernatant was diluted with equal volumes of a 10% (w/v) aqueous solution of ZnSO ₄ and a 4% (w/v) aqueous solution of sodium hydroxide. V) Clarified by addition of aqueous solution. After centrifugation, the upper clear liquid was extracted with one third of its volume of ethyl acetate. TLC control A sample of the organic phase is deposited on a silica gel G plate,
_{CH2Cl2 -} MetOH- _H2O -concentrated _NH4OH (90:9.5:
0.5:1) for 2 hours. The spot was mixed with methanol-anisaldehyde-concentrated sulfuric acid-acetic acid (85:
0.5:5:10) and the active compound was applied to microccus luteus (Sarcina luteus).
It was detected by biological self-delineation method on plates inoculated with ATCC9341. The TLC results show that the added 3-O-mycarosyl-
(8S)-8-fluoroerythronolide B ()
and the appearance of two active compounds.
R _st value is 0.9 respectively for erythromycin A
and 1.13, and 0.85 and 1.06 for erythromycin B, respectively. moreover,
They exhibit different color reactions after spray reagent application and heating; dark and colored (under hot conditions) the slowest compound (antibiotic P-80202) and dark purple (after cooling) the other compound (antibiotic P-80203). ). HPLC Control A sample of the organic phase was evaporated to dryness, taken up in acetonitrile and injected onto the column (RP8 10μm25
cm; mobile phase phosphate buffer 0.01M PH7/acetonitrile 36:64; flow 2ml/min; column temperature 40
℃). Two peaks appeared, retention times 0.79 for erythromycin A (P-80202) and
It was 1.06 (P-80203). Example 19 Purification of the antibiotics (8S)-8-fluoroerythromycin D (P-80202) and (8S)-8-fluoroerythromycin B (P-80203) Following the procedure described in Example 18 above, 1.0 g of A total of 2100 ml of several fermented meat juices containing 3-O-mycalosyl-(8S)-8-fluoroerythronolide B were added to Hyflo Supercell (Hyflo Supercell).
Supercell) (4% W/V) was added with stirring, and then filtered under vacuum. The solid was washed with water, and the combined liquid was adjusted to pH 5.5 with acetic acid. This acidic aqueous solution was extracted three times with equal volumes of ethyl acetate. water phase to 2N
of NH ₄ OH, evaporated under reduced pressure to a volume of 1000 ml, adjusted to pH 8.8 with 2N NH ₄ OH, and extracted with an equal volume of methyl isobutyl ketone. The organic extracts were combined and washed twice with half volume of 0.1M KH ₂ PO ₄ and then with water. After drying (Na ₂ SO ₄ ) and removing the solvent under vacuum, the residue was purified by NLOleinick, J.Biol.Chem,
It was purified by silica gel column chromatography as described in Vol. 244, n, 3, page 727 (1969). Fractions 23 to 38 containing only antibiotic P-80203 were included and evaporated to dryness in vacuo at 40°C. Crystallization of the solid residue from absolute ethanol gave 165 mg of (8S)-8-fluoroerythromycin B (P-80203) with the following properties: mp: 164-6°C; [α] ²⁰ _D -63 ° (C=1, methanol); UV (methanol): 285nm (ε 29.5) IR (KBr): 3480 (wide), 1735, 1465, 1435,
1385, 1375, 1330, 1305, 1280, 1170, 1115,
1090, 1075, 1055, 1035, 1020, 1000, 975,
Analysis of 940, 890, 835, 805 cm ^-1 C ₃₇ H ₆₆ FNO ₁₂ : Calculated value (%): C60.39; H9.04; F2.58; N1.90 Actual value (%): C60.31; H9.09; F2.60; N1.88 Fractions 65 to 120 containing only antibiotic P-80202 were added and dried in vacuum at 40°C. Crystallization of the solid residue from absolute ethanol yielded 125 mg of (8S)-8-fluoroerythromycin D (P-80202) with the following properties: mp: 213-5°C; [α] ²⁰ _D -60 ° (C=1, methanol); UV (methanol): 285nm (ε 30.8) IR (KBr):: 3600, 3520, 3300 (wide), 1730,
1460, 1420, 1385, 1370, 1355, 1345, 1330,
1310, 1275, 1190, 1160, 1120, 1100, 1060,
1040, 1030, 1010, 1000, 995, 975, 960, 935,
Analysis of 920, 910, 890, 875, 840, 825, 810 cm ^-1 C ₃₆ H ₆₄ FNO ₁₂ : Calculated value (%): C59.85; H8.94; F2.63; N1.94 Actual value (%) ): C59.87; H8.85; F2.63; N1.88 Example 20 Preparation of (8S)-8-fluoroerythromycin A acetate 7.520 g ( 0.010 mol) (8S)
-8- In a solution of fluoroerythromycin A,
1.23 ml (0.013 mol) of acetic anhydride was added. The mixture was stirred at 25°C for 2 hours and then poured into ice water. After 2 hours, it was extracted three times with chloroform, washed with a saturated solution of sodium bicarbonate, and then with water. The chloroform solution was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness in vacuo to yield 7.545 g of solid residue. By crystallizing this solid from ethyl ether-n-hexane, 6.325 with the following properties is obtained.
g (8S)-8-fluoroerythromycin A
Acetate was obtained: mp: 130-5°C; [α] ²⁰ _D -52° (C = 1, acetone); IR (KBr): 3480, 1740, 1455, 1370, 1340,
1280, 1235, 1160, 1110, 1085, 1050, 1030,
1010, 995, 975, 955, 930, 890, 870, 830, 800
cm ^-1 . Analysis for C ₃₉ H ₆₅ FNO ₁₄ : Calculated value (%): C59.00; H8.63; F2.39; N1.76 Actual value (%): C59.32; H8.75; F2.32; N1 .79 Example 21 Preparation of (8S)-8-fluoroerythromycin A propionate Using the general method of Example 20, (8S)-8-
Fluoroerythromycin A was converted to (8S)-8-fluoroerythromycin propionate by esterification with propionic anhydride. The final product had the following properties: mp: 115-20°C (ethyl ether/n-hexane); [α] ²⁰ _D -56.5° (C=1, acetone) IR (KBr): 3480, 1735, 1455, 1375, 1340,
1180 (shoulder), 1160, 1080, 1050, 1030, 995, 975,
955, 930, 890, 800 cm ^-1 . Analysis for C ₄₀ H ₇₀ FNO ₁₄ : Calculated value (%): C59.46; H8.83; F2.31; N1.70 Actual value (%): C60.13; H8.71; F2.27; N1 .75 Example 22 Preparation of (8S)-8-fluoroerythromycin A butyrate Using the general method of Example 20, (8S)-8-
Fluoroerythromycin A was converted to (8S)-8-fluoroerythromycin A butyrate by esterification with butyraldehyde. The final product had the following properties: mp: 120-5°C (ethyl ether/n-hexane); [α] ²⁰ _D -49° (C=1, acetone) IR (KBr): 3490, 1740, 1455, 1370, 1340,
1180 (shoulder), 1160, 1085, 1050, 1030, 1010, 995,
975, 955, 930, 890, 865, 830 cm ^-1 . Analysis for C ₄₁ H ₇₂ FNO ₁₄ : Calculated value (%): C59.91; H8.83; F2.31; N1.70 Actual value (%): C60.13; H8.71; F2.27; N1 .75 Example 23 Preparation of (8S)-8-fluoroerythromycin A ethyl succinate Using the general method of Example 20, (8S)-8-
Fluoroerythromycin A was converted to (8S)-8-fluoroerythromycin A ethylsuccinate by esterification with ethylsuccinyl chloride. The final product had the following properties: mp: 80-85°C (ethyl ether/n-hexane); [α] ²⁰ _D -52.7° (C=1, acetone) IR (KBr): 3480, 1735, 1450, 1370, 1345,
1190 (spalla), 1160, 1050, 1030, 1010, 995,
975, 955, 890, 800 cm ^-1 . Analysis for C ₄₃ H ₇₄ FNO ₁₆ : Calculated value (%): C58.69; H8.48; F2.16; N1.59 Actual value (%): C58.81; H8.57; F2.07; N1 .65 Example 24 Preparation of (8S)-8-fluoroerythromycin A succinate 7.520 g (0.010 mol) in 37.5 ml of anhydrous acetone
(8S)-8-fluoroerythromycin A and 1 g (0.010 mol) of succinic anhydride at 80°C.
Heat for 15 minutes, cool to room temperature and hold at temperature for 2 hours. Then follow the procedure of Example 20 and make 7.565
g of solid residue was obtained. Crystallization of this solid from ethyl ether gives 6.450 g of (8S)-8-fluoroerythromycin A succinate with the following properties: mp,: 150-5°C; [α] ²⁰ _D -52.7° (C= 1, acetone); IR (KBr): 3450, 1730, 1575, 1455, 1370,
1340, 1190 (shoulder), 1160, 1050, 990, 975, 950,
930, 883, 860, 825, 800 cm ^-1 . Analysis for C ₄₁ H ₇₀ FNO ₁₆ : Calculated value (%): C61.25; H8.77; F2.36; N1.74 Actual value (%): C61.52; H8.65; F2.32; N1 .78 Example 25 Preparation of (8S)-8-fluoroerythromycin A lactobionate A solution of 3.4 g (0.010 mol) of the delta-lactone of lactobionic acid in 20 ml of distilled water is dissolved in 7.520 g (0.010 mol) of lactobionic acid in 40 ml of acetone. molar) of (8S)-8-fluoroerythromycin A. The resulting solution is stirred until a gummy residue is obtained.
Evaporated in vacuo at 40°C. The residue was then dissolved in 50 ml of distilled water and the resulting solution was lyophilized. In this way, 10.6g of (8S)-8- with the following properties
Fluoroerythromycin A lactobionate was obtained: mp: 145-55°C; IR (KBr): 3400 (wide),
1725, 1605, 1455 (shoulder), 1370, 1340 (shoulder), 1160,
1070, 1040, 1000, 950, 885 cm ^-1 . Analysis for C ₄₉ H ₈₈ FNO ₂₅ : Calculated value (%): C53.01; H7.99; F1.71; N1.26 Actual value (%): C52.72; H7.67; F1.65; N1 .21 Example 26 Preparation of (8S)-8-fluoroerythromycin A stearate 2.85 g in 20 ml acetone-distilled water (1:1)
A solution of (0.010 mol) of stearic acid was prepared by adding 7.520 g (0.010 mol) of (8S)-8- in 40 ml of acetone.
Added to fluoroerythromycin A solution. The resulting solution was then evaporated in vacuo until a solid residue was obtained. Crystallizing this solid from acetone/n-hexane yields 10.2 g with the following properties:
(8S)-8-fluoroerythromycin A stearate was obtained: mp: 100-105°C; IR (KBr): 3470, 1730,
1455, 1375, 1340, 1150, 1105, 1050, 1030,
1010, 990, 975, 950, 930, 890, 830, 800 cm ^-1 . Analysis for C ₅₅ H ₁₀₂ FNO ₁₅ : Calculated value (%): C63.74; H9.92; F1.83; N1.35 Actual value (%): C63.37; H9.81; F1.69; N1 .27 Example 27 Preparation of (8S)-8-Fluoroerythromycin A Propionate A solution of 2.885 g (0.010 mol) sodium lauryl sulfate salt in 50 ml residual water is mixed with 8.080 g (0.010 mol) ( 8S)-8-fluoroerythromycin A propionate (Example 21
(prepared in ). Add 20 ml to the solution thus obtained while stirring magnetically.
5% aqueous acetic acid solution was added. The resulting salts were filtered off, washed several times with water and then dried under vacuum at 50°C. 10.25 g of (8S)-8-fluoroerythromycin A propionate lauryl sulfate were thus obtained. Analysis of C ₅₂ H ₉₆ FNO ₁₈ S: Calculated value (%): C58.13; H9.01; F1.77; N1.30; S2.98 Actual value (%): C57.89; H8.92; F1.72; N1.26; S2.94 Example 28 Preparation of (8S)-8-fluoroerythromycin A carbonate 7.045 g (0.080 mol) in 20 ml anhydrous benzene
Heating a solution of ethylene carbonate at 7.520
g (0.010 mol) of (8S)-8-fluoroerythromycin A, 2.760 g of potassium carbonate and 20
ml of benzene mixture over about an hour,
Stir vigorously and heat to reflux. At the end of the addition, the reaction mixture was heated to reflux for an additional 15 minutes and cooled to room temperature. Then, while stirring, 40
ml of water was added. The benzene phase was then separated, washed three times with water, dried over anhydrous Na ₂ SO ₄ and then dried at 50
Evaporated to dryness at °C. The residue was dissolved in ethyl ether and evaporated to dryness in vacuo. This operation was repeated several times and the residue obtained was then purified by chromatography on a column of silica gel. (8S) -
Fractions containing only 8-fluoroerythromycin A carbonate were combined (controls were performed by high pressure liquid phase chromatography). The combined fractions 16-22 were evaporated to dryness in vacuo and crystallized from ethyl ether to yield 0.980 g of (8S)-8-fluoroerythromycin A carbonate with the following properties: mp: 234-5°C: [ α] ²⁰ _D 44.2゜ (C=1, methanol) IR (KBr): 3500, 3450, 1795, 1745, 1455,
1380, 1365, 1345, 1325, 1295, 1280, 1235,
1160, 1090, 1070, 1040, 1015, 1000, 990,
940, 930, 900, 875, 865, 830, 815, 800 cm ^-1 . Analysis for C ₃₈ H ₆₄ FNO ₁₄ : Calculated value (%): C58.67; H8.29; F2.44; N1.80 Actual value (%): C58.41; H7.80; F2.40; N1 .71 Following the same procedure, other macrolide antibiotic salts, esters and salt-esters of the invention are prepared. The novel antibiotics of the invention as well as the related derivatives mentioned above are used in the preparation of pharmaceutical compositions, primarily for oral administration. The composition is prepared according to conventional pharmaceutical techniques using conventional excipients, vehicles,
Obtained using fillers, etc. Thus 10-1000
Tablets, pills, capsules containing mg of active ingredient,
Suspensions and solutions are possible. The daily dosage is that normally compatible with a similar antibiotic, namely erythromycin. The following examples illustrate the preparation of compositions and formulations containing (8S)-8-fluoroerythromycin A. Example 29 Capsules: 1000 Unit Dosage (8S) - 8-Fluoroerythromycin A 100 g Magnesium Stearate Preparation 3 g The materials shown above are mixed homogeneously and filled into hard gelatin capsules by conventional techniques. The content of the capsule is 103 mg. Each capsule contains 100 mg of active ingredient. Example 30 Capsules: 1000 Units Dose (8S)-8-Fluoroerythromycin A 250g Magnesium Stearate Preparation 7.5g Repeat the same technique described in the previous example. Each capsule contains 257 mg of powder mixture, corresponding to 250 mg of active ingredient. Example 31 Capsules: 1000 Units Dose (8S) - 8-Fluoroerythromycin A 500g Magnesium Stearate 15g The same technique described in the previous example is repeated. Each capsule contains 515 mg of powder mixture, corresponding to 500 mg of active ingredient. Example 32 Tablets: 1000 unit dosage (8S)-8-fluoroerythromycin A 100g corn starch 50g lactose 30g talc 8g magnesium stearate 2g hydroxypropyl methylcellulose 6g ethyl cellulose preparation 4g (8S)-8-fluoroerythromycin A,
A portion of cornstarch and lactose are mixed homogeneously and granulation is carried out according to wet granulation technique using the remaining amount of cornstarch in the form of cornstarch-water as a binder. The dry granules are mixed with lubricant and compaction is carried out. You get a tablet weighing 190mg. Each tablet contains 100 mg of active substance. The tablets can be placed in a coating container and film-coated with a solution of hydroxypropylmethylcellulose and ethylcellulose. Example 33 Tablets: 1000 Unit Dosage (8S) - 8-Fluoroerythromycin A 250g Cornstarch 70g Lactose 40g Talc 16g Magnesium Stearate 4g Hydroxypropyl Methylcellulose 12g Ethylcellulose Preparation 8g The technique described in the previous example is repeated. 400mg
Each tablet weighing 250 mg of active substance. Example 34 Tablets: 1000 Unit Dosage (8S) - 8-Fluoroerythromycin 500g Cornstarch 140g Lactose 85g Talc 37g Magnesium Stearate 8g Hydroxypropyl Methylcellulose 18g Ethylcellulose Preparation 12g Repeat the same technique as the previous example. Weight 800mg
Each tablet of contains 500 mg of active substance. Example 35 Instant suspension: 60 mg suspension (8S) - 8-fluoroerythromycin A 0.6 g Sodium carboxymethylcellulose 0.010 g Methyl p-hydroxybenzoate 0.048 g Propyl p-hydroxybenzoate 0.012 g Flavoring agent 0.600 g Sucrose powder preparation Amount to make a total weight of 30 g Mix each component homogeneously and place in a 60 ml graduated becker. Before use, fill the Bezkar with water to form a 60ml suspension, which is stirred well before use. The re-formed suspension is 10
Contains mg/ml of active substance. Example 36 Instant suspension: Dosage for 60 ml of suspension (8S) - 8-fluoroerythromycin A 3 g Carboxymethyl cellulose 0.010 g Methyl p-hydroxybenzoate 0.048 g Propyl p-hydroxybenzoate 0.012 g Flavoring agent 0.600 g Sucrose preparation Amounts for a total weight of 30 g Repeat the same technique as in the previous example. The reconstituted suspension contains 50 mg/ml of active substance. Example 37 Drop: 10ml Dose (8S) - 8-Fluoroerythromycin A 0.5g Methyl p-hydroxybenzoate 0.09g Propyl p-hydroxybenzoate 0.01g Hydroxyethylcellulose 0.050g Glycerin 0.400g Sweetening and Flavoring Agents 0.100g Purified Water Preparation Amount to make 10 ml In a suitable container equipped with a mechanical stirrer, add 90% of the amount of water required for the preparation and heat to 80°C. Add parasptical to this, followed by hydroxyethyl cellulose. Add the other ingredients while stirring and fill into a 10ml small bottle. The suspension thus obtained contains 50 mg/ml of active substance. Example 38 Drop: 10ml Dose (8S)-8-Fluoroerythromycin A
2.500 g Methyl p-hydroxybenzoate 0.009 g Propyl p-hydroxybenzoate 0.001 g Hydroxyethyl cellulose 0.050 g Glycerin 0.400 g Sweetening and flavoring agents 0.100 g Purified water preparation Amount to 10 ml Repeat the same technique as the previous example. Each ml of suspension contains 250 mg of active substance. Example 39 Capsules: 1000 Units Dosage (8S) - 8-Fluoroerythromycin A Propionate Lauryl Salate (100g (8S)
-8-Equivalent to fluoroerythromycin A)
142.9g Magnesium Stearate Preparation 4.1g Repeat the same technique described in Example 29 above.
The content of each capsule is 147mg. Each capsule contains (8S)-8-fluoroerythromycin A propionate lauryl sulfate corresponding to 100 mg of (8S)-8-fluoroerythromycin A. Example 40 Capsules: 1000 Units Dosage (8S)-8-Fluoroerythromycin A Propionate Lauryl Sulfate (equivalent to 250g of (8S)-8-Fluoroerythromycin A) 357.2g Magnesium Stearate Preparation 10.8g Before Repeat the same technique as in the example. Each capsule containing 368 mg of powdered mixture contains 357.2 mg of (8S)-8-fluoroerythromycin A propionate sulfate, corresponding to 250 mg of (8S)-8-fluoroerythromycin A. Example 41 Tablets: 1000 units of dosage (8S)-8-fluoroerythromycin A stearate (equivalent to 100g of (8S)-8-fluoroerythromycin A) 137.83g Corn starch 55.00g Lactose 32.50g Talc 10.00g Stearic acid Magnesium 2.17g Hydroxypropylmethylcellulose 7.50g Enalcellulose Preparation 5.00g Repeat the same technique as in previous Example 32. Tablets having a weight of 240 mg are obtained. Each tablet is 100mg
Contains 137.83 mg of (8S)-8-fluoroerythromycin A stearate, corresponding to 137.83 mg of (8S)-8-fluoroerythromycin A. The tablets are placed in a shallow container and coated with a film of a solution of hydroxypropylmethylcellulose and ethylcellulose. The weight of the finished tablet is 250
mg. Example 42 (8S)-8-fluoroerythromycin A stearate (equivalent to 500g of (8S)-8-fluoroerythromycin A) 689.17g Corn starch 150.00g Lactose 80.00g Talc 40.83g Magnesium stearate 10.00g Hydroxypropyl methylcellulose 18.00 g Ethylcellulose Preparation 12.00 g Repeat the same technique as in previous Example 32. Each tablet weighing 1 g contains 689.17 mg (8S) corresponding to 500 mg (8S)-8-fluoroerythromycin A.
Contains 8-fluoroerythromycin A stearate. Example 43 Instant suspension: 60 ml suspension (8S)-8-fluoroerythromycin A ethyl succinate (corresponding to 0.6 g of (8S)-8-fluoroerythromycin A) 0.702 g Sodium carboxymethyl cellulose 0.010 g Methyl p-hydroxybenzoate 0.048g Propyl p-hydroxybenzoate 0.012g Flavoring agent 0.600g Sucrose Preparation Amount to make 30g Repeat the same technique as Example 35. The reconstituted suspension contains (8S)-8-fluoroerythromycin A ethyl succinate corresponding to 10 mg/ml of (8S)-8-fluoroerythromycin A. Example 44 Intravenous vial: 1000 units dose 500g
(8S)-8-fluoroerythromycin A lactobionate is dissolved in water and lyophilized after sterilization. The resulting product is divided into glass vials in a sterile environment. Each vial is
Contains 738 mg (corresponding to 500 mg of (8S)-8-fluoroerythromycin A). The macrolide antibiotics of the invention were pharmacologically tested to determine: - their ability to inhibit bacterial growth against aerobic and anaerobic Gram-positive and -negative bacterial strains, expressed as minimum inhibitory concentration (MIC); :Results in Table 2 and Table 3
Describe it in Serum concentrations for -(8S)-8-fluoroerythromycin A and erythromycin A: Results are listed in Table 4. - Inhibitory power of bacterial growth against several Gram-positive aerobics, expressed as Minimum Bacterial Inhibitory Concentration (MBC): the results are listed in Table 5. [Table] [Table] Legend: * Erythromycin resistance
** Penicillin resistance [Table] [Table] [Table] [Table] * Rat serum at 0 hours showed no antibacterial activity.
[Table] [Table] [Table]

[Brief explanation of drawings]

Figures 1-6 are IR spectra of compounds according to the invention.

Claims (1)

[Claims] 1 formula (8S)-8-Fluoroerythromycin A and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of the formula (8S)-8-Fluoroerythromycin B and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids Esters and salts of the formula (8S)-8-Fluoroerythromycin C and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of the formula (8S)-8-Fluoroerythromycin D and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of the formula 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-fluoroerythronolide A and its esters and salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of and expression 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-fluoroerythronolide B and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of A macrolide antibiotic selected from a group consisting of: 2 As an active ingredient, the formula (8S)-8-Fluoroerythromycin A and its esters, salts, -esters and salts with organic and inorganic non-toxic pharmaceutically acceptable acids of the formula (8S)-8-Fluoroerythromycin B and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids Esters and salts of the formula (8S)-8-Fluoroerythromycin C and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of the formula (8S)-8-Fluoroerythromycin D and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of the formula 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-fluoroerythronolide A and its esters and salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of and expression 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-fluoroerythronolide B and its esters, salts with organic and inorganic non-toxic pharmaceutically acceptable acids - Esters and salts of A pharmaceutical composition for antibacterial agents, characterized in that it contains a macrolide antibiotic selected from the group consisting of: together with conventional excipients, vehicles and fillers. 3. A pharmaceutical composition according to claim 2 in a form suitable for oral administration. 4. The pharmaceutical composition according to claim 2, containing a unit dose of 10 to 1000 mg of the active ingredient.