CA1189856A - Semisynthetic macrolidic antibiotics, microbiological processes for their preparation and related microorganism, novel intermediate compounds for their preparation and related pharmaceutical compositions containing them - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A derivative of erythronolide A, namely (8S)-8-fluoroerythronolide A, a derivative of erythronolide B, namely (8S)-fluoroerythronolide B, or a derivative of 3-0-mycarsolyerythronolide B, namely 3-0-mycarsoyl-(8S)-fluoro-erythronolide B, which are useful as substrates in the pre-paration of the corresponding (8S)-8-fluoro derivatives of the exythromycins A, B, C and D, as well as 3-0-oleandrosyl-5-desosaminyl-(8S)-8-fluoroerythronolide A and 3-0-oleandrosyl-5-0-desosaminyl-(8S)-8-fluoroerythronolide B, all belonging to the class of the macrolide antibiotics by fermantation carried out with mutants blocked in the synthesis respec-tively of erythromycin and of oleandomycin, namely Streptomyces erythreus ATCC 31772 and Streptomyces anticio-ticus ATCC 31771. The preparation of the aforesaid substrate comprises the conversion of erythronolide A, erythronolide B
or 3-0-mycarsoyl-exythronolide B into the corresponding he?i-acetal, the reaction of the latter with a compound capable of generating eletrophilic fluorine and the opening of the resulting acetal with aqueous acid.

Description

85~;

The present invention relates to novel macrolide antibiotics, useful as antibactexial agents, and a microbio-logical process for their production starting from a novel intermediate, derived from the erythronolide A, the erythron-olide B or from the 3-O-mycarosyl-erythronolide B.

This application is a divisional application of copending application No. 393,832 filed January 8, 1982.

The present invention also relates to a ncvel micro-organism, useful for the production of the macrolide antibio-tics of the invention, as well as to the related method for the preparation thereof by mutagenesis starting from a known stock.
The present invention relates furthermore to novel intermediates derived from erythronolide A, from erythrono-lide B or from 3-O-mycarosyl-erythronolide B and to the re-lated chemical synthesis process.
As previously mentioned, the novel semisynthetic macrolide antibiotics of the present invention are useful as antibacterial agents. They in ~act show, in comparison with the erythromycins of known type, activity spectra and levels equal or broader, are less susceptible of being de~
graded in an acidic environment and thus permit better ad-sorption by the oral route; their esters or salt-esters are adsorbed in a qulcker and more complete manner giving higher and more delayed hematic levels than the free bases.
The novel ~ntibiotics of the present invention, owing to the presence of the basic groups form pharmaceutical-ly acceptable addition salts with acids both organic and in-organic. The aforesaid salts can be prepared from the free base by conventionally used methods for the preparation o~
addition salts o~ basic antibiotics.
,..",~

9~

The salts which are not water soluble are used in liquid oral suspension (they are little better). Monoester ~ 2 5~
derivatives of the novel antibiotics and pharmaceutically acceptable addition salts the~eof can be prepared by methods like those used fo~ the preparation of esters and salt-esters o~ erythromycan A.

The esters and salt-esters of erythromycin A and the methods for their preparation are known in the art.
Examples oE esters, salt-esters and salts of(8S)-8-fluoro-erythomycin A (P-80206), (8S)-8-fluoroerythromycin B (P-80203), (8S)-8-flurorerythromycin C (P-80205), (8S)-8-fluoroerythromycin D (P-80202), 3-0-oleandrosyl-5-0-deso-saminyl-(8S~ 9-fluoroerythronolide A (P-80207) and 3-0-olean-drosyl-5-0-desosaminyl- (8S)-8-fluroerythronolide B (P-80204) (wherein the abbreviations in brackets indicate the re-ferences of the Applicant), which can be prepared are acetate,p~opionate, butyrate, succinate, valerate, ethylsuccinate, propionate laurylsulphate/ stearate, lactobionate, glucohep-tonate, sulphate laurylsulphate, carbonato-derivatives and the like, among which the lactobionate and the glucoheptonate, being water solube salts, permit the administation by intravenous route. In turn, the ethylsuccinate permits the administration both by oral and by parenteral route.

The novel antibiotics according to the invention are whilte, bitter, odorless powders, thermostable in the powder form, more stable than erythromycin A in aqueous solu-tion at acidic pH and, thus, more stable in the gastric acidic environment. The half life times of the initial activity of erythromycin A and the macrolide antibiotics of the present invention in solution at several pH values are reported in the following Table 1.

Stab~lity in acidic environment at 25C of the novel antibiot~cs in comparison with the erythromycin A

~L8~ 6 Erythrornycin A
pH 2.0 3.0 4.0 t 1~2 ~mirlutes) 2 6 120 (8S)-8-fluoroerythomycin A (P-80206) pH 2.0 3.0 4.0 t 1/2 (hours~ 9 82.5 100 -(8S)-8-fluoroerythromycin B (P-80203) pH 2.0 3.0 4.0 t 1/2 (hours) 3 45 100 (,8S)-8-fluoroerythrornycin C (P-80205) pH 2.0 3.0 4.0 t 1/2 (hours) 1.65 41.5 100 (8S)-8-fluoroerythromycin D- (P-80202) pH 2.0 3.0 4.0 t 1~2 (hours) 0.4 10 100 3-O-oleandrosyl-5-O-desosaminyl- (8S ) - 8-f luoroerythronolide A
P (80207) pH 2.0 3.0 4.0 t 1/2 (hours) 10 100 100 3-O-oleandrosyl-5-O- desosaminyl - (8S ) - 8 -f luoroerythronolide B
~p 80204) pH 2.0 3.0 4.0 t 1/2 (hours) 25 100 100 Note: t 1~2 represents the time for reducing to one half the initial power of the antibiotic by a rnicrobiological method through dif fusion on a plate using a test stock Micro~
ccocus luteus ( Sarcina lu tea ) ATCC 9341 ~

According to a first feature oE the invention, there are prepared the novel intermedi,ates derived from erythronolide erythronolide A, erythronolide B or 3-0-mycarosylerythronolide B, which are used in the microb.iological process for the pre-paration of the novel antibiotics.

It is well known (R.A. LeMahieu et al, J. Med. Chem.
17, 963r (1974)), that the erythronolide A is a substrate which can be obtained from ervthromycin A by selective removal of the sugars cladinose and desosamine.

lQ It is also known that the erythronolide B and the 3-0-mycarosyl-erythronolide B are substrates which can be obtained by direct fermentation in relevant amounts and thus at indus-trially feasible costs, using micro-organisms producers of erythromycin and their mutants. However, as it is also well known, apart Erom the production of normal erythromycin A, these substrates do not find use in other fermentations which may give place to antibiotics devoid of the disadvantages and problems affecting the erythromycin by itself whereas from the industrial point of view their utilization appears mostly desirable.

It has been now found that the derivatives of erythron olide A, of the erythronolide B or of 3-0-mycarosyl-erythrono-lide B, which can be preparedin achemical way in a simple and industrially feasible manner, are useful substrates for fer-mentation processes which, making use of micro-organisms ob-tained by mutagenesis from the stocks adapted for the microbiological production of erythromycin A, give place to the novel macrolide antibiotics of the pre~sent invention, as it will be more specifically described - 4a 5~56 hereinafter.
The chemical process for the preparation of the noveJ intermedial;es according to the present invention is represented by the following scheme:

HOCH~ o--~CH3 CH3"~ C~ CH ~ ~(~
C2~5 1- ~QX 3 C ~ ` GX 3 CH3 2 5 O~ H3 O II~

~ CH3 o C2H
IV
wherein when R=H, X represents H or the group:

~ \r CH3 ~`_0~1 .
CH " OH
and, when R-OH, X=H, and is characterized by the steps of:
a) treatment of a compound (1), selected among erythronolide A, erythronolide B
and 3-0-mycarosyl-erythronolkie B with an anhydrous acid, such as glacial aceticacid or a rnethanolic soiution of hydroxylamine hydrochloride, to form the compound (Il), namely 8,9-anhydroerythronolide A-6,9-hemiacetal, 8,9-anhydro-.
derythronolide B-6,9-hemiacetal or 3-O-mycarosyl-8,9-anhydrr3-erythronolide B-6,9-hemiace tal;
b) reaction of the compound (Il) with a reagent capable of generatiny electrophilic fluorine, preferably selected among fluoroxy-perflunro-alkanes ~haYing the ~9~356 general formula Cn~n~lOF) and perchloryl fluoride, to form the corresponding acetal (III) in the presence of an inert organic solvent and at low temperature; c) reaction of the compound (III) with an aqueous acid whereby the desired com-pound (IV) is ormed.

With reference to the step (b), among the reagents of the class of the fluoroxy-perfluoro-alkanes,that mostly used is the fluoroxy-trifluoromet~ane, which is commerically avail-able.

Other reagents containing fluorine atoms having posi-ti~e charge which can be used in the present reaction comprise fluoroxy-sulphur-penta-fluoride, mole~ular fluorine and lead tetraacetate-hydFofluoric acid.

Among the reaction solvents there are contemplated the chlorinated hydrocarbons such as trichlorofluoromethane ~supplied under the trade mark Freon 11), chloroform and methylene chloride, tetrahydrofuran and their mixtures.

It is preferable to carry out the reaction at low temperatures, préferably in the range of between -75C and -85C, under continuous stirring.
The reaction is normally completed in a time of between about 15 minutes and one hour.

It is important to point out that the forming of acetal (III) is accompanied by theformation already in this step of not negligible amounts of the desired compound (IV).

As regards the third reaction step (c), organic or mineral aqueous acids, such as acetic or hydrochloric acid~
3~ are used. For the reaction temperatues of between about 30C
and about 150~C can be adopted. The resulting product (IV~

~9B56 ls reco~ered in purified form by means of recrystallization or chromatography.

Also the compounds (II), (III) and (IV), being novel, are part of the invention.

The invention, besides the intermediate (IV), is based on the use of ~he mutant, Streptomyces erythreus ATCC 31772, of blocked type, which is obtained through mutagenesis by chemical methods (namely by means of chemical mutagenic agents), by irraAiation with U.V. rays, or X rays, by the action of phages and the like.

The culture of the Streptomyces erythreus ATCC 31772, using the compound (IV) as the substrate leads to the production of the novel macrolide antibiotics of the class of the (8S)-8-fluorinated erythromycins (Formula II: P-80202, P-80203, P-80205 and P-80206), respectively having P~St0.9, 1.13, 0.89, and 1.12 with respect to the erythromycin B, said antibiotics haviny furthermore specific colours if treated with chromatic reagents and heated, namely thP first one having brick red colour (hot), ~he second and the fourth dark brown violet col-our (after cooling~, and the thixd dark colour (hok).

Formula I ~ _ 3 _ _ _ O _ ~ O

~0~ ~X
CH3 ~ OH
OR
R=CH3(8S)-8-fluoroerykhromycin B (P-80203) R=H (8S)-8-fluoroerythromycin D (P-80202) `~ ~ - O - ~ O ~ ~H3 C~ ' ~ ~ C 3 R=CH3(8S)-8-fluoroerythromycin A (P-80206) R=H (8S)-8-fluoroerythromycin C tP-80205) In a further aspectof the invention a microbiological process for the preparation of novel macrolide antibiotics of the erythromycin class is provided ~hich is characteriz~d in that a compound (IV) is used as the substrate for the fermentation with Streptomyces antibioticus ATCC 31771 which gives place respectively to the antibiotic 3-0-oleandrosyl-5-0-desosaminyl-(8S)-8-fluoroerythronolide A (Formula III: P-80207), having R ~0.87 with respect to the oleandomycin and 0.80 with respect to erythromycin A, when the substrate (IV) is (8S)-8-fluoro-erythronolide A, and to the antibiotic 3-0 oleandrosyl-5-0-desosaminyl-(8sj-8-fluoroerythrorlolide B (Formula III: P-80204), having Rst0.82 with respect to erythromycin (IV) is (8S)-8-~luoroerythronolide B~
25Formula III:

- 7a -- 8 ~ B5 C~

3-0-oleandrosyl-5-desosaminyl-(8S)-B-fluoroerythronolide A (P-80207) f ~1~, C~ll~",tHj CU j~ _ _ _, D i~1~CN, C,ll~ ~
O o~-3 3-0-oleandrosyl-5-0-desosaminyl-(SS)-8-fluoroerythronolide B (P-80204)~
The cultivation of the micro-organisms Streptomyces erythreus ATCC 31772 and Streptomyces antibioticus ATCC 31771 using the compounds (IV) as the substrate to produce the desired antibiotics can be carried out according to several fermentation methods.
After completion of the ferrnentation, various procedures can be used fnr the isolation and the purification of the antibiotics.
Among the methods suitable for the isolation and the purification the procedures of solvent extraction, both in batch form and in colums for the countercurrent liquid-liquid extraction, and the gel permeaLion chromatography are contemplated.
According to a preferred method~ the antibiotics produced according to the present invention are recovered from the culture medium by separation of the mycelium and of any undissolved solids from the fermentation broth by conventional means such as by filtration or centrifugation. The antibiotics are then extracted from the filtered or centrifugated broth using either batchwise or counter current distribution extraction techniques.
The solvent extraction may be performed using a pH range of from B to 1û and employing as the solvent an inert organic solvent. Suitable solvents include alcohols;

9 ~ 5~
such as rnethanol, etharlol and the like, cl lorinated hydrocarbon-s, such as chloroform, methylene chloride and the like, ethyl acetate, butyl acetate, amyl acetate, acetone, methylisobutylketone and acetonitrile, with methyJisobutylketone being preferred.
The final purification of the aforesaid antibiotics can be achieved hy chromatography on permeable gels.
After filtration or centrifguation of the fermentation medium, thin layer chromatogra-phy or high pressure liquid phase chromatography can be employed to analise for the subject antibiotics.
ln addition bioautography can also be used advantageously.
The following examples illustrate the invention without being an undue limitation.

-Preparation o-F the mutant Streptomyces erythreus ATCC 31772 A suspension of spores of StreptorT!yces erythreus producer of erythromycin subjected to mutagenic treatment with U.V. rays (emission maximum 25û nm) at a dose such as to kill about 99.6% of the spores (about 3000 erg/sq.cm).
The surviving spores were seeded on a plate of nutrient medium and the resultingcolonies were anali~ed with respect to their incapacity of producing erythromycin using the technique described by A. Kelner (1949) J. Bact. 57 73.
The mutants blocked in the synthesis of the erythromycin (about 2% of the surviving organisms) were then analized for their capacity of recognizing and converting the compound (IV) as a substrate into novel compounds having antibiotic activity.

Preparation of ~,9-anhydroerythronolide A-6,9-hemiacetal (Il) A solution of 4.185 g ( 0.01 moles) of erythronolide A (1), described by R.A. LeMahieu et al in J. Med. Chem. 17, 953 (1974), in 32 mls of glacial acetic acid was maintained on standing for two hours at room temperature. The acetic acid was then removed under vacuurn at the temperature of 40C and the oily residue dissolved in 150 mls of chloroform. The chloroformic solution was washed with saturated solution of sodiurn bicarbonate and then with water upt to neutrality and lastly dried over Na25C)4.After rernovaJ of the solvent under vacuum a raw product was obtained which was purified on a silica gel column, prepared in methylene chloride-methanol (1~
The elution ~Jith methylene chloride-methanol (9S:~) gave fractions containing only - ln -8,9-anhydroerythronolide A-6,9-hemiacetal (Il).
By evaporation to dryness of these combined fractions and subsequent crystallization from acetone/n-hexane there were obtained 2.750 9 of the compound (Il) having the following characteristics:
m.p. 188-1~3C;
D ~ 20.5 (C= 1 in methanol);
UV (MeOH) 210 nm ( ~ 6720~
IR (KBI~) 3G3û, 3520, 3495, 3400, 171û, 1465, 1450, 1440, 1415, 1400, 1370, 1350, 1310, 1285, 1230, 1200, 1170, 11~I0, 1090, 1075, 10609 1050, 1035, 1020, 1010, 1000, 970, ~50, 940, 920, 915, 905, 890, f370, ~25, 810, 800 cm The analysis `For C21H3607 gave the following values:
calculated (%): C 62.97; H 9.06 found (%) : C 63.12; H 9.10 Preparation of (85)-8-fluoroerythronolide A-6,9; 9,11-acetal (III) and (85)-8-fluoroery-thnolide ~ (IV) from 8,9-anhydroerythronolide A-6,9-hemiacetal (Il) A solution of fluoroxy-trifluoromethane in Cc13F at -80C was prepared: an excess of CF3~F (usually approximately twofold) was dissolved in CCI3 (precooled at -80C on dry ice) by slow addition of the gas through a purge tube (while the cylinder containing CF30F was continously weighed on a Sartorious electric balance).
Its concentration was determined by iodometric titration.
The CF31~F/CC13 F solution at -80DC or -85C was slowly added to a solution containing 4 g (0.010 moles) of 8,9-anhydroerythronolide A-6,9-hemiacetal in Ccl3F/cH2r`l2 (295 mls/37û mls) at about -80C, magnetically stirred with calciurn oxide (1.920 9) to remove hydrogen fluoride. Progress of the reaction was periodically monitored by high pressure liquid phase chromatography (HPLC~9 with respect to the disappearance of the characteristic peak of the cornpound (Il).
After disappearance (or minimi~ation) of the peak of the compound (II) the stirring was continued for 5 rninutes and nitrogen gas was bubbled through the solution to rernove excess CF3oF at -8ûDC and the solution was allowed to warm to room ternperature.The solution was washed with a saturated.solution of NaHCO3 (650 mls) and then washr d neutral with water and finally dried over Na25O4-5~
.
Removal of the solYent afforded a solid residue which was then purified by si~ica gelcolumr) chromatorJraphy (ratio 1.50), preparated in methylene chloricie/methanol (I:l).
The elution with increasing concentrations of methanol in methylene chJoride gave fractions contain;ng only (8S)-8-fluoroerythronolide A-699; 9,11-acetal (IJI) and fractions containing only (85)-8-fluoro erythronolide A (IV). After evaporation to dryness oF the Fractions containing the compound (III) and their crystallization from acetone/n-hexane 3.435 g of product were obtained having the following characteri-st ics:
m.p. 192-3C; [D~] D ~ 64.7 (C= 1 in methanol) U.V. (methanol); no adsorption corresponding to a ketone group IR (KBr~: 3560, 3420, 1720, 1460, 13951 1380, 1355, 1345, 1330, 1320, 1305, 12909 1270, 1255, 124û, 1215, 118n (broad), 1095, 1070, 1045, 1025, 1020, 990, 980, 970, 955, 940, 930, 920, 910, 905,895, 855, 840, 830, 805 cm The analysis for C21H35FO7 gave the following values:
calculated (%): C 60.27; H 8.43; F 4.54 found (%): C 60.22; H 8.51; F 4.69 After evaporation to dryness of the fractions containing the compound (IV) and crystallization from acetone/n-hexane 145 mg were obtained with the following characteristicso m.p. 239-240C; ~1 D -3.1~ ((G1 in methanol);
UV (methanol): 287-8 nm (~ 25.3) IR (KBr): 3610, 3550, 3480 tshoulder), 3380, (shoulder), 1735, 1700, 1460, 1405, 1390, 1380, 1350, 1325, 1300, 1290" 1270, 1175, 1105, 1090, 1050, 1035, 1020, 980, 960, 940, 920, ~ns, 89~, 875, 860 crn The arlalysis for C21H37FO8 gave the following values:
claculated (%): C 57.78; H a.54; F 4.35 found (%): C 57.87; H 8.63; F 4.19 Preparation of (85)-8-fluoroerythronolide B-679; 9,11-hemiacetal (Ill) and (85)-8-fluoroerythronolide B (IV) from 8,9-anhydroerythronolide B-6,9-hemiacetal (Il).
By proceeding likewise the example 3, but starting from a solution containing 3.845 9 (0.010 rnoles) of 8,9-anhydroerythronolide B-6,9-herniacetal (Il)~ (described in the U.S.

- 12 - ~8~1~35~

patent No. 3.697.547), a raw product'was ohtained which, by crystallizatiorI from acetone/n-hexane, gave 3.450 9 of (85)-B-Fluoroerythronolide B-6,9; 9,l1-acetal (111), having the following characteristics:
m.p. 191-2C; t~ D = 42.6 (C=1.0 in methanol), UV (methanol); no adsorption corresponding to a ketone group;
IR (KBr):354û, 3450, 1735, 1460, 1260, 1170, 1060, 1035, 980,925, 875, 450 cm The analysis for C21H35FO6 gave the following values:
calculated (%): C 62.67; H 8.76; F 4.72 found (%) C 62.63; H 8.82; F 4.81 When the mother liquors were purified by silica gel column chromatography (ratio1~50) with methylene chloride-methanol (98:2) as the eluant, 75 mg of a second product were obtained, (E35)-B-fluoroerythronolide B (IV) having the following characteristics:
m.p. 247-8C; lJV (methanol) 2B6 nm (~ 26) r ~, D -3û (C=1 in methanol) IR (KBr): 3540, 1727, 1703, 1460, 13:~0, 1270, 1175, 1130, 1075, 1050, 1015, 940, 920, ~95, ~60 cm The analysis for C21H37FO7 gave the following values:
calculated (%): C 59.98; H ~.87; F 4.52 found (%) : C 59.92; H 9.ûO; F ~59 Preparation of (85)-8-fluoroerythronolide B (IV) from (85)-~-fluoroerythronolide B-6,9;
9,1l-acetal (Ill) A mixture formed by 4.830 9 (0.012 moles) of (~5)-8-fluoroerythronolide B-6,9; 9,11-acetal (III) and 3000 rnls of an aqueous solution (pH) 3) of acetic acid was refluxed at 1InC for 15 minutes under stirring and then 220 mls of acetic acid were added. After one hour at 110C all the starting material was dissolved. The heating was oontinued for half a hour and the solution was then cooled as much rapidly as possible to room ternperature, made neutral with NaHCO3 and extracted with ethyl acetate.
The ethyl acetate solution after anhydrification c)ver sodium sulphate was evaporated to dryness under vacuum.
When the raw product was purified by means of silica gel column chromatography (ratio 1:5n) with methylene chloride-methanol (95:5) as the eluant, 1.8 9 of a product were obtained having chemical and physical properties equal to those of the compound (IV) isolated in the example 4.

A solution of 5.465 9 (0.010 moles) of 3-0-mycarosyl-erythronolide B (I), described by J.R.Martin in biochemistry, 5, 2852 (1966), in 32 mls of glacial acetic acid wasmaintained on standing for 2 hours at room temperature.
The acetic acid was then removed under vacuum at the temperature of 40C and theoily residue was dissolved in 150 mls of chloroform.
The chloroform solution was washed with a saturated soiution of sodium bicarbonate and then with water to neutrality and lastly dried on Na2SO~- After removal of the solvent under vacuum a raw product was obtained which was purified on silica gelcolumn (ratio 1:100), prepared in chloroform.
The elution with increasing concentrations of methanol in chloroform gave fractions containing only 3-O-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal (Il).
By evaporation to dryness of these combined fractions and subsequente crystallization from acetone/n-hexane 1.16û 9 of the compound (II) were obtained having the following characteristics:
m.p. 85-88C;L~ D -5~ (C=l in methanol);
UV (MeOH): 210 nm ( ~ 6850) IR (KBr): 3500, 173û, 1465, 1415, 1380, 1340, 1185, 1120, 1085~ 1055, 1005, 985, 945, 89S, 810 cm The analysis for C28H~8Og gave the following values:
calculated (%): C 63.61; H 9,15 founcl (%): C 63.52; H 9.09 Preparation of 3-O-mycarosyl-(85)-8-fluoroerythronolide B-6,9; 9,11-acetal (III) and 3-O-mycarosyl-(8S)-fluoroerythronolide B (IV) from 3-O-mycraosyl-8,9-anhydroerythro-nolide B-6,9-hemiacetal (Il).
A solution of fluoroxy-trifluoromethane in CC13F at -80DC was prepared: an excess of CF30F (usually about tWD fold) was dissolved in CC13F (precooled at -B0C or at -85"C) was slowly added to a solution containing 5.285 9 (0.010 moles) o F 3-O-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal (II) in CC13F/CH2CI2 (295 - 14~
, .
mis/370 mls) at about-aOC; magnetieally stirred wiLh calcium oxide (1.9;~0 y) to remove the hydrogen fluoride.
The progrèss of the reaction was periodically monitorecl by higl) pressure liquid chromatography (HPLC), with respect to the disappearance of the peak characteristic of the compound (II).
After disappearance (or minimization) of the peak related to the compound (Il), the stirring was continued for further 5 minutes and nitrogen gas was bubbled through the solution tu remove the excess of CF30F at -80nC and the solution was allowed to warm to room temperature. The solution was washed with a saturated solution of NaHCO3 (65û mls) and then washed to neutrality with water and lastly dried nver Na2SO4. From the solvent removal a solid residue was obtained which was then purified by silica gel column chromatography (ratio 1:1ûû), preparated in chloroform.
The elution with increasing concentrations o~ methanol in chloroform gave fractions containing only 3-O-mycarosyl-(85)-fluoroerythronolide B-6,9; 9,11-acetal (Ill) and fractions containing only 3-O-mycarosyl-(BS)-8-fluoroerythronolide B (IV).
After evaporation to dryness cf the fractions containing the compound (III) and their crystallization from acetone 1.100 9 were obtained having the following characteri-stirhs:
m.p. 175-7~C;
L'~] 29.8(G 1 in metl-anol);
UV (methanol); no adsorption corresponding to a ketone group.
IF?c (KBr): 354û, 349û, 17207 1460, 1395, 1380, 1365, 1355, 1330, 13207 1290, 1275, 1265, 1245, 1025, 1185, 1165, 1145, 1120, 1100, 1080, 106û, 1045, 1015, 1005, 990, 960, 940, 925, 915, 90û, 880, a65, 840, 815 cm The analysis for C28H47FOg gave the following values:
ca Iculated (%): C 61.52; H 8.67; F 3.47;
found (%) : C 61.46; H 8.51; F 3.48 After evaporation to dryness of the fractions containing the compound (IV) and crystallization from acetone/n-hexane, 290 rng were obtained of a product having the following characteristics:
m.p. 214-5C;
J D -73.2 D (C= 1 in me thanol);

UV methanol 2a7 nm ( ~ 25.3);
lR (KBr): 3570, 3550,3530, 3460 (shoulder), 1745, 1735, 1465, 13B0, 1365, 1340, 1300, 1275l 1250, 1180, 1115, 1080, 1055, 1010, 1000 (shoulder), 985, 945, 935, 920, 895, 8553 840, 810 cm The analysi5 for C28~49FO1o gave the following values:
calculated (%): C 59.55; H 8.75; F 3.37;
found (%) : C 59.68; H 8.60; F 3.48 Preparation of 3-0-mycarosil-(85)-8-fluoroerythronolide B (IV) from 3-0-mycarosyl-(85)-8-fluoroerythronolide B-6,9; 9,11-acetal (Ill) A mixture formed by 6.560 9 (tl.012 m~les) of 3-0-mycarosil-(85)-8-fluoroerythronolide B-6,9; 9,11-acetal (III) and 3000 mls of a water solution (pH 3) of acetic acid was maintained at room temperature under stirring until completely dissolvecl and then 220 rnls of acetic acid were added.
The solution was left under stirring at room temperature until the starting compound (III) completely disappeared, (the control being effected by high pressure liquid chromatography), made neutral with NaHCO3, and extracted with ethyl acetaee.
~he ethyl acetate solution, after anhydrification over sodium sulphate, was evaporated to dryness under vacuum.
When the raw product was puri~ied by means of silica gel column chromatography (ratio I :50) with methylene chloride-methanol (95:5) as the eluant, a product was obtained having chemical and physical characteristics equal to those oF the compound (IV) isolated in the example 7.

Preparation of (8S)-8-tluoroerythronolide A (IV) from (8S)-8-fluoroerythronolide A-6,9;
9,11-acetal (III) A mixture formed by 5.020 g ( 0.012 moles ) of (85)-8-fluoroerythronolide A-6,9; 9,11-acetal (111) and 3000 mls of a water solution (pH 3) of acetic acid was refluxed at 110~C for 15 minutes under stirring and then 220 mls of acetic were added.
After one hour at 110~C all the starting material was dissolved.
The heating was continued for half a hour and the solution was thereafter cooled as much rapidly as possible to room temperature, made neutral with NaHCO3 and extracted with acetic acid.
The solution of e~hyl ace~ate, after anhydrification over sndium sulph~Le, was evaporated to dryness under vacuum.
The raw solid was then purified by means of silica gel column chromatography (ratio 1:10), preparated in methylene chloride.
The elution with increasing concentrations of methanol in rnethylene chloride g~ve fractions still containing starting compound (85)-8-fluoroerythronolide A-6,9; 9,11-acetal (Ill) and fractions containing only (85)-8-fluoroerythronolide A (IV).
13y repeating the reaction on the recovered starting compound and carrying out subsequently the chrornatographic purification other fractions containing (85)-8-fluoroerythronolide A were obtained.
By evaporation to dryness oF all the combined fractions and then ricrystallization of the resulting solid 0.725 9 o~ (85)-8-fluoroerythronolide ~ (IV) were obtained having the same characteristics reported in the example 3.
~:XAMPLE 10 Preparation of the antibiotics (85)-8-fluoroerythromycin C (P-80206) and (E35)-8-fluoroerythromycin A (P-80205).
A seeded culture of StreptomYces erythreus ATCC 31772, a mutant blocked in the synthesis of erythromycin, was prepared in a medium comprising (in grams per litre):
sucrose 30.0; cane molasses 8Ø soy bean oil 9.0; (NH4)25O~ S.0; CaC037.0 The oulture was incubated at 33UC for 48 hours on a rotary shakerO The seed was added at a level of 5% (V/V) into 250 ml Erlenmeyer flasks containing 30 mls of a ferrnentation medium having the Following composition (in grams per litre): corndextrins 30.n; raw corn starch 4û.0; soy bean meal 30.0; soy bean oil 20.0; (NH4)250L

2.0; and CaC03 6.û~
The fermentation flasks were incubated at 33C on a rotary shaker (220 rpm, 4 cmstroke) for 24 hours.
Fifteen milligrams of finely divided (8S)-~-fluoroerythronolide B (Ill) sterilized under UV light for 15 rninu~es were added to each flask, and the incubation with shaking was continued for 96 hours.
Treatrnent of the sarnples by thin layer chromatography and by hiqh pressure liquid .
phase chromatoqraohy 8~856 At the end of the ferm~ntation time a sample of fermentation broth assa~ing about 9Q0-1000 mcg/ml (titre expressed ~s e~ythromycin ~, ~as centrifu~ed and the Surna-tant lI~uid was clarified by addin~ equal volumes of a 10%
(W/Vl a~ueous solution of ~ SO4 and a 4% (W/V) aqueous solution of sod.iu~ hydroxide. After centrifugation the clear surnatant liquid was extracted by vortexing 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 developed in CH2C12-MetOH-H2O-conc. NH40H
(90:9.5:0.5:1) for 2 hours; the spots were located with the spray reagent comprising methanol-anisaldehyde-conc.sulfuric acid: acetic acid, t85:0,5:5:10~ and the active cornpounds were revealed by means of bioautography on plates seeded with Mi~^ococcus luteus (Sarcina lutea) ATCC 9341.

The results of the TLC showed the disappearancee of the added (8S)-8-fluoroerythronolide A (IV) and the appear-ance of the two active compounds the RSt values of which with respect to erythromycin A are resepctively 0.8.9 and 1.12 (0.087 and 1.040 with respect to the erythromycin B). Furthermore, the~ sh~" different chromatic reactions after application of a spray reagent and heating:dark brown ~hot) for the slowest com-pound (antibiotic P-80205) and dark violet colour (after cooling) for the other compound (antibiotic P-302

3 0 HPLC CONTROL

A sample of or~anic phase is evaporated to dryness/
taken with acetonit~ile and i~nje.~,ted in the column (RP~ 10~ m 25 c~; mobile ph,~se ph.osphate ~uffer 0.Ql M pH 7~acetonitrile 36:64; ~low 2 mls/m~n,; colu~n temperature 40C~. Two ~ea,ks are detected ha~ng retention tI,me with respect to erythromycin 1~39~

A o~ 0.68 (P-80205) and o~ 0.87 (P-80206).

EXAMRL~ 11 Purification of th~ antibiotics (8S)-8-fluoroerythro-m~cin C (P-8Q205) and (8S~-8-fluoroerythromycin ~ (P-80206).

According to the process descr~bed in the preceding example, several fermentations of a total volume of 2100 mls to which 1.00 y of (8S)-&-fluoroer~thronolide A had been added, were filtered under vacuum after addition under stirring of Hyflo Supercell (~a trademaxk) (4% ~V).

The solid was washed with water and the combined fil-trates were adjusted to pH 5.5 with acetic acid. The acidicaqueous solution was extracted three times with an e~ual volume of ethyl acetate. The aqueous solution was neutralixed with 2N NH40H and evaporated under reduced pressure to a volume of 1000 mls, adjusted to pH 8.8 with 2N NH40H and extracted with Z0 an equal volume of methylisobutylketone. The latter organic extracts were combined and washed two times with half a volume of 0.1 M KH2PO4 and then one time more with water.

After drying (Na2SO4) and removal of the solvent under vacuum, the residue was purified by silica gel column chromatography according to the method disclosed by N.L.
Oleinick in J. Biol, ChemO, Vol. 244, n.3, pag~ 727~1969).

The fractions ~0 to 174 containing only the anti-3Q biotic P-80206 were combined and evaporated to dryness at 40C.
The solId resid1ue by crystallization from absolute ethanol gave Z3Q ~ ~ C8~1-8-fluoroexythrom~cin A (~8Q206~ having the fol-lowin~ characteri5tics:
m.~: 183-4C; ~] 2Q _55o (C~l in ~ethanol~;
UY ~methanoll:283 nm C~17.91 IR (KBrl: 3520, 3480 (shoulder~, 3250 (broad), 1735, 1720, 1460, 1425, 1400, 1380, 13~5, 1330, 1305, 1280, 1190, 1170, 1120, lO9Q, lQ75, 1055, 1030, 1015, 1005, 980, 960 (shoulder~, 935, 890, 870, 855, 835, 800 cm (this spec~.ru~ is shown in Fi~. 11.

The analysis for C37H66FN.O13 gave the following values:
calculated (%):1 C 59.10; H 8.85; F 2.52; N 1.86.
found (~) ; C 5g.09/ H 8.89/ F 2.59/ N 1.88 The fractions 280 to 400 containing only the anti-10 ~iotic P-80205 were combined and evaporated to dryness at 40C
under vacuum. The solid residue by crystalli~ation from absolute ethanol gave 145 mg of C8S]-S-Lluoro~hra~cin C ~P-80205) having the m.p. 217-8C;
[~ ]D0 -42.35 (C = 1 in methanol~
15 UV (methanol~:284 nm (E23.2) IR ~KBr):3550,3500, 3440 (shoulder), 3300 (broad), 1730, 1455, 1425, 1410, 1380, 1360, 1340, 1330, 1305, 1280, 1270, 1245, 1200 (shoulder), 1170 (broad), 1115r 1090, 1075, 1060, 1030, 1010, 1000, 980 (shoulder), 965, 955, 945, 935, 920, 905, 895, 870, 840, 830, 810cm 1, (the related spectrum is shown in Fig. 2).

~ 18~ -- 19 ~ 9~35~;
36 64 13 9 he followiny values:
calculated (%): C 5~3.60j H 8.74; F 2.57; N 1.90;
found (%~ : C 58.47; H 8.87; F 2.60; N 1.82 Preparation of the antibiotic 3-0-oleandrosyl-5-0-desosaminyl-(85)-8-fluoroerythronoli-cle A (P-80207).
A preseed culture of S.antibioticus ATCC 31771, a mutant blocked in the biosynthesis of oleandomycin, was prepared in a medium comprising (in grams per litre of deionized water) soy bean meal 30.0; cerelose 15.0; yeast autoJysate 1.0; soy bean oil 30.0; MgSO4- 7H20 1.0; and CaC03 10.0, the pH of the medium being adjusted to 7.2 before the sterili~ation.
After 24 hours at 28C on a rotary shaker, this culture was utilized for the seeding of the same rnedium at a cuncentration of 2% (V/V) and W2S further incubated under the same conditions For 16 hours. This seed was added at the concentration of 3% (V/V) in 250 ml Erlenmeyer flasks containing 30 mls of a fermentation medium having the following composition (grams per litre):
cerelose 40.0; soy bean meal 20.û; maize meal 3.0; dried baker yeast 2.0; CaCO3 20.0 For the fermentation the flasks were incubated at 28C on a rotary shaker (240 rpm, 4 cm stroke) for 32 hours; 15 mg of finely divided (8S3-8-fluoroerythronolide A (IV) were added to each flask and the incubation with stirring was continued for 64 hours.~t the end of this period the titre of the culture, expressed as erythrolnycin A, was 100-120C mcg/ml. The treatment of the fermentation broth for the TLC analysis was carried out with the systern and under the conditions indicated in the example 10.
TLC CONTROL
According to the technique described in the example 10 a novel active compound (antibiotic P-80207) is revealed, indicating the disappearance of the added substrate.
The Rf thereof with respect to erythromycin A is 0.80 and with respect to elandomycin is 0.87.
HPL C CONT ROL
According to the technique described in the example 10, the novel active compound (antibiotic P-80207) has a retention time wil;h respect to erythromycin A of 0.6~ and with respect to oleandomycin of 0.95 . .

8~3S~
PuriFica~ion of t~e antihiotic 3-0-oleandrosyl-5-0-desosaminyl-(85)-~-fluoloeryLhrorlol-ide A (P-90207).
The ~otal culture broth, deriving from 50 fermentations carried out in Erlenrneyer flasks, under stirring and effected according to the preceding exampJe 12, was treated with an equal volume of methanol. After the addition of Hyflo Supercell over 30 minutes under stirring, the mixture was filtered. The solid was washed with ~ater/methanol (1:1) and the combined filtrates were evaporated under reduced pressure to half the starting volume.
The pH of the solution was ad3usted to 8.2 by addition of KOH and the solution was extracted three times with amounts corresponding to one third of the volume of methylisobutylketone. The extracts with organic solvent, combined, were washed witl-0.1 M Na2HPO4 and then with water.
The organic solution, after anhydrification over Na25O4, was evaporated to dryness under vacuum. The residue was purified by chromatography on silica gel column according to the method described in example 11.
The fractions 45 to 80 containing only the antibiotic P-80207 were combined and brought to dryness under vacuurn at 40C. THe solid residue was crystallized from absolute ethanol to obtain 130 mg of 3-0-oleandrosyl-5-0-desosaminyl-585)-8-fluoroe-rythronolide A (P-E30207) having the following characteristics:
m.p.: 155-7C;
o4~ - 40.2 (C= 1 in me thanol);
UV (methanol): 283 nrn ( 20.3);
IR (KBr): 3480 (broad), 1730, 1510, 1380,1340, (broad), 1305, 1275, 1195, 1165, 1105, 1095, 1075, 105û, 1û30, 1010, 1000, 980, 960 (shoulder), 935, 915, 895, 875, 830 cm . ( this spectrum is shown in fig. 3).
The analysjs for C36H64FNO13 gavP the following values:
calculated (%): C 58.60; H 8.74; F 2.57; N 1.90;
found (%) : C 58.52; H 8.75. F 2.63; N 1.95;
Ex~\MpLE 14 Preparation of the antibiotics (8S)-8-fluoroerythromycin D (P-80202) and (8s!-8--fluoroerythromycin B (P-80203).
A seeded cul~ure of Streptomyces erythreus ATCC 31772, a mutant blocked in tl e - 2~ 9~5~
hiosynth~sis of eryLhromycin, wa~. prepared in a medium Co~nrpisin~1 (in gra~ per litre) sucrose 30.0; cane molasses 8.0; 50y bean oil 9.0: (NH4)2So22-o; CaCO3 7.0 The culture was incuba~ed at 33C for 48 hours on a rotary shaker.
The seed was added at a level of 5% (V/V) to 250 ml Erlenmeyer flasks containing 30 mls oF a fermentation medium having the following composition in grams per litre:
corn dextrins 30.0; raw corn starch 40.0; soy bean meal 30.0; soy bean oil 20.0; (NH4)25O4 2.0; CaCO3 6.0 The fermentation flasks were incubated at 33C for 24 hours on a rotary shaker (220 rmp9 4 cm stroke).
15 mg of (8S)-8-fluoroerythronolide B (IV) in a finely divided form and sterilized under ultraviolet light for 15 minutes were added to each flask" and the incubation with shaking was continued for 96 hours.
Treatment of the sample by thin layer chromatoqraphy (TLC) and hiqh pressure liquid phase chromatoqraphy (HPLC) At the end of the fermentation period a sample of the fermentation broth having an activity of about 900-1000 mcg/rnl (titre expressed as eryhtromycin A) was centrifugated and the surnatant liquid was clarified by adding equal volumes of 10%
(W/V) aqueous solution of ZnSO4 and of a 4% (W/V) aqueous solution of sodium hydrox ide.
After centrifugation the surnatant clear liquid was extracted by vortexing with orle third of its volume of ethyl acetate.
TLC CONTROL
A sample of the organic phase was deposited nnto a silica gel G plate and developed in CH2Cl2-MetOH-H20-conc. NH4OH (90:9.5 0.5 1~ for 2 hours; the spots were located hy means of a spray reactant comprising methanol-anisaldehyde-conc. sulfuric acid-acetic acid (85:0.5:5:10) and the active compounds were detectsd by bioautography on plates seeded with Microccus luteus (Sarcina lutea) ATCC 9341. The results of the TLC
showed the disappearance of the added (85)-8-fluoroeryhtronolide B (IV) and the appearance of two active compounds, the R t values of which with respect to erythromycin A are respectively 0.9 and 1.13 (D.85 and 1.06 with respect to erythromycin B). Furthemore they show different chromatic reactions afLer application of a spray reactant and heating; dark brown colour (under hot conditions) ~ 22~ 9~35~
for the slowest compound (antibiotic P-80202) ~nd dark violet colour (after cooliny) for the other compound (antibiotic P-802û3).
HPLC CONTROL
A sample of the organic phase is evaporated to dryness, taken with acetonitrile and injected in column (RP8 10 /um 25 cm; mobile phase phosphate buffer 0.01 M pH
7/acetonitrile 36:64; flDw 2 mls/ min.; column temperatùre 40C). Two peaks are revealed with a retention time with respect to erythromycin A of 0.79 (P-B0202) and 1.06 (P-80203).

Purification of the antibiotics (8S)-8-fluoroerythromycin D (P-80202) and (85)-8-fluoroerytrhomycin B (P-80203) According to the procedure described in the preceding example 14, several fermenta-tions for a total volume of 2100 mls, which had been supplemented with 1.0 g of (85)-8-fluoroerythronolide B were filtered under vacuum after addition under stirring of Hyflo Supercell (4% weight/volume). The solid was washed with water and the combined filtrates were adjusted to pH 5.5. with acetic acid. The acidic aqueous solution was extracted three times with an equal volume of ethyl acetate. The aqueous phase was l eutralized with 2N NH40H, evaporated at reduced pressure to a volume of 1000 mls, adjusted to a pH of 8.8 with 2N NH4oH and extracted with an equal volume of methylisobutylketone.
The latter organic extracts were combined and washed two times with half a volume of 0.1 M KH2PO4 and then a further time with water. After drying (Na2S04) and removal of the solvent under vacuum, the residue was purified by silica gel column chromaLography according to the procedure described by N.L. Oleinick in J. Biol.Chem. Vol. 244, n. 3 pag. 727 (1969?.
The fractions 18 to 32 containirrg only the antibiotic P-80203 were combined andbroùght to dryness under vacuum at 40C. The solid residue by crystallization from absolute ethanol gave 150 mg (of (8S)-8-fluoroerythromycin B (P-80203) having the following characteristics:
m.p.: 164-6C;
r ~20 L~JD -63 (C= 1 in methanol)7 UV (rnethanol) . 285 nm ( ~ 29.5) ~39~5~
lR (I<Br): 3480 (broad), 1735,1465, 1435, 13857 1375, 1330, ~305, 1280, 1170, 1115, 1090, I075, 1[)55, 1035, 1020, lono, 975, 940,890, a35, 805 cm~1(fig. 4) The analysis for C37H66FNO1 gavE the following values:
calculated (%): C 60.39; H 9.04; F 2.58; N 1.90 found (%) : C 60.31; H 9.09; F 2.60; N 1.E~8 The fractions 55 to 105 containing only the antibiotic P-80202 were combined andbrought to dryness under vacuum at 40C The solid residue by cristallization from absolute ethanol gave 150 mg of (85)-8-fluoroerythromycin D (P-802U2) having thefollowing characteristics:
m.p. 213-15C;
L~ ~D -60D (C= 1 in me thanol);
UV (rnethanol): 285 nm ( ~ 30.8) IR (KBr): 3~00, 3520, 3300 (broad), 1730, 1460, 1420, 1385, 1370, 1355, 1345, 1330, 1310, 1275j 1190, 1160, 1120, 1100, 1060, 104û, 1030, 1010, 1000, 995, 975, 960, 935, 920, 910, 890, 875, 84D, B259 810 cm (fig. 5).
The analysis for C36H6~FNO12 gave the following results calculated (%): C 59.8O; H 8.94; F 2.63; N 1.94;
found (%) : C 59.87; H 8.85; F 2.63; N 1.88 Preparation of the antibiotic 3-0-oleandrosyl-5-0-desosaminyl-(8S)-8-fluoroerythronoli-de B (P-80204?.
A preseed culture of S.antibioticus ATCC 3:L771, a mutant blocked in the biosynthqsis of oleandomycin, was prepared in a medium comprising (in grams per litre of deionized water) soy bean meal 30.0; cerelose 15.0; yeast autolysate 1.0; soy bean oil 30.0; MgSO4- 7H2O 1.0; CaC03 10.0; the pH of the medium being adjusted to 7.2 before the sterilization.
After 24 hours at 28C on rotary shaker, this culture was used for the seeding of the same medium at a concentration of 2% ~V/V) and was further incubated under the same conditions for 16 hours.
This seed was added at the concentration of 3% (V/V) to 250 ml Erlenmeyer flasl<s containing 30 mls oF a fermentation medium having the following composition (grams per litre): cerelose 40.0;soy bean meal 20.0; maize meal 3.0; dried baker yeast 2.0;

- 24 - ~389~
.
CaCO3 20Ø
The'flasksfor the Fermentation were incubated at 28UC on a rotary shaker (24û rlnp, 4 cm stroke) for 32 hours; 15 mg of finely divided (85)-8-fluors~erythronolide B were added to each flask and the incubation under stirring was continued for 64 hours.
At the end of this period the titre of the culture expressed as erythromycin A was 100-120 mcg/ml. The treatment of the fermentation broth for the TLO analysis was carried out with the system and under the conditions indicated in the example 14.
TLC CONTROL
According to the technique indicated in the example 1~ a novel active compound is detected (antibiotic P-8û204), which follows the disappearance of the added substrate.
The R t thereof wlth respect to the erythromycin B is 0.82 and with respect to oleandomycin is 0.90.
HPLC CONTROL
According to the technique indicated in the example 14 the novel active compound (P-~û204) has a retention time with respect to erythromycin B of 0.62 and with respect to the oleandomycin of 1.06 . _ _ Purification of the antibiotic 3-0-oleandrosyl-5-û-desosaminyl-(8S)-8-fluoroerythronol-ide B (P-80204).
The total culture broth deriving from fifteen fermentations, carried out in Erlenrneyer flaOks) stirred and under the conditions described in the preceding example 16, was treated with an equal volLIme of methanol. After the addition of the Hyflo Supercell in '30 Inls under stirring the mixture was filtered. The solid was washed with water-rnethanol (1:1) and the combined filtrates were evaporated under reduced pressure to half of the starting volume.
The pH o f the solution was adjusted to 8.2 by adding KOH and the solution was extracted three times with an amount of one third of the volume of methylisobutylke-tone.
The combined extracts with organic solvent were washed with 0.1 M NaH2PO4 and then with water. The organic solution after anhydrification over l\la254 was evaporated to dryness under vacuum. The residue was purified by means of silica gel column chromatography according to the technique indicated in the exarnpie 15.

- 25- ~ 5~
- Tlle fractinrls 17 to 42 contairliny orlly the antibiotic P-~30204 were comi~ e(l and brought to dryness urlder vacuum at 40C. The solid residue was further purified in a Sephande~ LH-20 (a trademark) column (2 x 98 cm) preparated in hexane-chloro-form (1:1) and eluted wi-th the san~ solvent.
The frac~ions containing onIy the novel antibiotic P-30204 were combined snd concentrated to dryrless uncler vacuum at 40UC to give, a fter crystallization from acetone-hexane, 170 rng of 3-0-oleandrosyl-5-û-desosaminyl-(85)-8-Fluoroerythronolide B (P-80204) having the FoIlowing characteristics:
rn.p.: ~95-6C;
L~ 48 (C= 1 in methanol);
UV (methanol): 2~35 nm (~- 29) IR (KBr): 3600, 34ll0 (hroad), 3250, 1730, 1455, 1400, 1380, l365, l350, 1325, l305, 1270, 1255, IlBO) 1160, 1145, 1100, 1060, lO40, 1010~ 995, 975, 960, 935, 915, E390, EJ75, 855, 845, 830, 820 crn (fig. 6) The analysis for C36H64FN012 gave the following values:
calculated (n~o): C 59.91); 1-1 i3.94; F 2.63; N 1.94;
found (/0) : C 59.94; H 9.06; F 2.69; N 1.83 EXA MPL[- 1 ~
Preparation of the antibiotic (85)-8--fluoroerythromyciIl D (P-B0202 and (85)-8-fluoroerythrornycirl B (P-80203) A see(Ied cuItlJre of Streptornvces erythreus ATCC 31772, a rnutant blocl<ecl in the hiosynthesis t3~ erythromycin, was prepared in a medium comprising (in grarns per litre) sucrose 30.0; cane rnolasses 8.0; soy bean oil 9.0; (NH4)2So~ 2.0; CaC03 7.0 rhe cuIt(Jre was incubated for 48 hours at 33C on rotary shaker~
The seed was added at a level of 5% (V/V) to 250 rnl ErIenmeyer flasl<s containing 30 mIs of a ferlr)( lltatiur) medium having the following composition in grarns per litre: raw corn starch 40.0; corn dextrins 30.0; soy bean meal 30.0; soy bean oil 20.0; (NH~l)250~
2.0; Cr~C03 ~-- , The; fermentation flasks were incubated at 33C on ro-tary shaker (220 rpm, 4 cm stroke) for 24 hours.
15 mg of finely divided 3-0-mycarosy1-(8S)-f1uoroeryhtronolide B (IV) sterilized under ul-traviolet light for 15 minutes were added to each f lask and the incubation with J ~

àhaking Wcl~; cootinu~d for 9G hours.
Treatment of the samples by thin l~er chromatoqraphy (TLC3 an l ~ y hiqh pres~ur~
liquid phase chromatoqra~hy (HPLC) At the end of the fermentation period a sample oF the fermentation broth having an activity of about 900-1000 mcglml (titre expressed as erythromcin A) was centrifuga-ted and the surnatant liquid was clarified by adding equal volumes of a 10% (W/V) aqueous solution of ZnSO4 and of a 4% (W/V) aqueous solution of sodium hydroxide.
After centrifugation the clear surnatant liquid was extracted by vortexing with one third of its voiume of ethyl acetate.
TLC control A sample of the organic phase was deposited on a silica gel G plate and developed in CH2C12-MetOH-H20-conc. NH40H (90:9.5:0.5:1) for 2 hours; the spots were located wih a spray reagent comprising methanol - anisaldehyde - conc. sulfuric acid - acetic acid (85:û.5:5:10) and the active compounds were revealed by means of bioautography on plates seeded with Microccus luteus (5arcina lutea) ATCC 9341.
The results of the TLC showed the disappearance of the added 3-0-mycarosyl-(BS)-8-fluoroerythronolide B (IV) and the appearance of the two active compounds having R t with respect to erythromycin A o-F 0.9 and 1.13 respectively (0.85 and 1.06 with respect to erythromycin B)o Furth^rmore they show different chromatic reactions after application of a sprayrea~ent and heating; dark brown colour (hot) for the slowest compound (antibiotic P-802û2~ and dark violet colour (after cooling) for the second compound (antibiotic P-80203).
HPLC CONTROI
A sample cf the organic phase wase evaporatecl to dryness taken with acetonitrile and injected in column (RP8 10 /um 25 crn; mooile phase phosphate buffer 0.01 M
pH7/acetonitrile 3G:64; flow 2 mls/min.; column temperature 40C). Two peaks arerevealed having a retention time with respect to erythromycin A of 0.79 (P-80202~ and .06 (P-80203).

Purification of the antibiotics (8S)-8-fluoroerythromycin D (P-80202) and (8S)-8-fluoroerythromycin B (P-80203).

~ 3~

Accqrding to the ~roceduxe descxibed in the preced-ing example 18, seve~al fermentatIons for a total volume of 2100 mls, which has been added with 1.0 g 3-0-m~caros~1-(8S)-fluoroery~
thronol~de B we~e filtered under ~acuu~ after addition under stirXlng o~ Hyflo Supercell (4% weight/volume).

The solid was washed with water and the combined fil-trates were ad~usted to pH 5.5 with acetic acid.

The acidic water solution was extracted three times with an equal volume of ethyl acetate. The aqueous phase was neutralized wtth 2N NH4OH and evaporated under reduced pressure to a volume of 1000 mls, adjusted to pH 8.8 with 2N NH4OH and extracted with an equal volume of methylisobutylketone.
The latter organic extracts were combined and washed two times with half a volume of 0.1 ~ KH2PO4 and then a further time with water. After drying (Na2SO4) and removal of the solvent under vacuum, the residue was purified by means of silica gel column chromatography according to the technique described by N.L. Oleinich, in J. Biol. Chem., Vol, 244, n. 3, pag 727(1969~.

The fractions 23 to 38 containing only the antibiotic P-80203 were combined and brought to dryness under vacuum at 40C. The solid residue, by crystallization from absolute ethanol, gaVe 165 ~g of (8S)-8-fluoroerythromycin B (P-80203) having the ~ollowing characteristics:
m.p.- 164-6~C;
[~]2-63~ (C~ 1 ~n me-thanol);
UV (methanol):285 nm ( 29.52 IR (K~X2; 3480 (Pxoad~, 1735, 1465, 1435, 1385, 1375, 1330, 1305, 1280, 1170, 1~5~ lQ9Q, 107S~ 1055~ 1035, 1~2Q, 1000, 975, 940, 890, 835, 805 cm The analysis ~or C37~6FNG12 gaVe the following values:

~P~ 56 calculated (%~: C 60.39; H 9.04; F 2.58; N 1.90 found (~ : C 60.31; H 9.09; F 2.6Q; N 1.88 The ~ractions 65 to 120 containing only the antibiotic p-8Q2Q2 were combined and brou~ht to dryness under vacuum at 49C. The solid residue ~y crystallization from absolute eth-anol gave 125 m~ of (8S)-8-fluoroerythromycin D(P-80202) having the following characteristics:
m.p. 213-5C;

- 27a -2-60~ (C= 1 in meLhanol);
UV (rnethanol): 285 nrn ( ~ 30.8) IR (KBr); 3600, 3520, 3300 (broad), 1730, 1460, 1420, 138$, 1370, 1355, 1345, 1330, 1310, 1275, 1190, 1160, 112û~ 1100, 1060, 1040, 1030, 1010, 1000, 99S, 975, 960, 935, 920, 910, ago, ~7S, 840, 825, 810 cm The analysjs for C36H64FN012 gave the following values;
calculated (%): C 59.85; H 8.94; F 2.63; N 1.94;
found (%) : C 59.87; H 8 8S. F 2.G3; N 1.88 Preparation of (85)-8-fluoroerythromycin A acetate To a solotinn of 7.520 9 (0.010 moles) of (85)-8-fluoroerythromycin A in 30 mls of anhydrous acetone containing 3.760 g of sodium bicarbonate 1.23 mls (0.013 moles) of acetic anhydride were added.
The mixture was maintained under stirring at 25~C for 2 hours and then poured inwater-ice. After 2 hours it was extracted three times with chloroform, rapidly washed with a saturated solution of sofium bicarbonate and then with water.
The chloroformic solution was dried over anhydrous Na25O4 and evaporated under vacuum to dryness to give 7.545 9 of solid residue.
By crystallization of the solid from ethyl ether-hexane-n 6.325 9 of (85)-B-fluoroerythromycin A acetate were obtained haviny the following characteristics:m.p. 130-5"C;
-52~ (C= 1 in acetone);
IR (KE~r): 3480, 1740, 1455, 1370, 1340, 1280, 1235, 1160, 1110, 1085, 105071030,1010, 995, 975, 955, 930, 890, B70, 830, 800 cm The analysis for C39H68FNO1~ gave the following values:
calculated (%): C 59.00, H 8.63; F 2.39, N 1.76 found (%) : C 59.32; H 8.75; F 2.32; N 1.79 Preparation of (85~-8-fluoroerythromycin A propionate Using the general method of the example 20 (85)-8-fluoroerythromycin A was converted into (85)-8-fl~oroerythromycin propionate by esterification with propionic anhydride. The final product had the following characteristics:

- 29 ~ 856 m.p. 115-20DC (eLhyJ ether/n-hexane);
[,~,]2-56.50 (C= 1 in acetone) IR (KBr): 3480, 1735, 1455, 1375, 1340, 1180,(shoulder), 1160, 108û, 1050, 1030, 995, 975, 955, 930, 890, 800 cm The ana~ysis for C40H70FNO14 yave the following values calculated (%): C 59.46; H, 8.73; F 2.35; N 1.73 found(%) C 59.75; H 8.65; F 2.42; N 1.69 I~XAMPLE 22 Preparation of (85)-8-fluoroerythromycin A butyrate Using the general method of the example 20 (85)-8-fluoroerythromycin A was converted to (85)-8-fluoroerythromycin A butyrate by esterification with butyraldehy-de. The final product had the following characteristics:
m.p. 120-5DC (ethyl etlier/n-hexane);
[d~-49D (C=1 in acetone) ~R (KBr): 3490, 1740, 1455, 1370, 1340, 1180 (shoulder), 1160, 1û85, 1050, 1030, 1010, 995, 975, 955, 930, 890, 865, 830 cm The analysis for C41H72FNO1~ gave the following results:
calculated (%): C 59.91; H 8.83; F 2.31; N 1.70 ruund (%) : C 60.13; H 8.71; F 2.27; N 1.75 EXAMPLE ?3 Pr~paration of (85)-8-fluoroerythromycin A ethyl succinate.
Using the general process of the example 20 (8S)-8-fluoroeryLhromycine A was converted ~o (85)-8-fluoroerythromycin A ethyl succinate by esterification with ethyl succinyl chloride. The final product showed the following characteristics:
m.p. 80-85C (ethyl ether/n-hexane);
[1]D-52.7 (C=1 in acetoe) IR (KBr) 3480, 1735, 145D, 1370, 1345, 1190 (shoulder), llSO, 1050, lO30, 1010, 995, 975, 955, 89û, 800 cm The analysis for C43~17~FNO16 gave the following values:
calculated (%): C 58.69; H 8.48; F 2.16; N 1.59 found (%) : C 58.81; H 8.57; F 2.07; N 1.65 . _ _ - 30 - ~8~85~
Preparation of (85)-8-fluoroeythromycin A succinate A solution containing 7.5ZD 9 (0 010 moles) of (85)-8-fluoroerythromycin A and 1 9 (0.010 moles) of succinic anhydride in 37.5 mls of anhydrous acetone was heated to 80C for 15 minutes9 cooled and maintained at room ternperature for 2 hours.
Then the procedure of the example 20 was adopted until a solid residue of 7.565 9 was obtained. By crystallization oF the solid from ethy~ ether 6.450 9 of (85)-8-fluoroerythromycin A succinate werr~ obtained having the follwoing characteristics:
m.p. 150-55C:
[~ ~D-52.7U (C= 1 in acetone);
IR (KBr): 3450, 1730, 1575, 1455, 1370, 1340, 119D (shoulder), 1160, 1050, 990 975, 950, 930, 885, 860, 825, 800 cm The analysis for C41H70FNO16 gave the following results:
calculated (%): C 61.25; H 8.77; F 2.36; N 1.74 found (%) : C 61.52; H 8.65; F 2. 32; N 1.78 Preparation of (8S)-8-fluoroerythromycin A lactob}onate.
A solution of 3.4 g (û.010 moles) of ~-lactone of the lactiobionic acid in 20 mls of distilled ~ater was added to a solution of 7.520 9 (0.010 moles) of (85)-8-fluoroerythromycin A in 40 mls of acetone.
The resulting solutinn was evaporated under vacuum at 40nc until a gummy residuewas obtained. The residue was then dissolved in 50 mls of distilled water and the res-llting solution was Iyophilized. 10.6 9 of (85)-8-fluoroerythrornycin A lactobionate were thus obtained having the following characteristics:
m.p. 145-5SC;
IR (I<Br) 3400 (broad), 1725, 1605, 1455 (shoulder), 1370, 1340 (shoulder), 1160, 1070, 1040, 1000, 950, 885 cm The analysis for C49H88FNO25 gave the following values:
calculated (%): C 53.01; H 7.99; F 1.71; N 1.26;
found (%) : C 52.72; H 7.67; F 1.65; N 1.21 Preparation of (8S)-8-fluoroerythromycin A stearate A solution of 2.85 9 (0.010 moles) oif stearic acid in 20 mls of acetone-distilled water 35~

(1:1) was added to a solution of 7.520 9 ( U-010moles) of (85)-8-fluoroerythromycin A in 40 mls of acetone.
The resulting solution was then evaporated under vacuum until a solid residue was obtained which by crystallization from acetone/n-hexane gave 10.2 9 of (85)-8-fluoroerythromycin A stearate characterized by:
m.p. 100-105C:
IR (KBr): 3470, 1730, 1455, 1375, 1340, 1160,1150, 1105, 1050, 1û30, 1010, 990, 975, 9509 930, B90, B30, 800 cm The analysis for C55 102 15 9 calculated (%): C 63.74; H 9.92; f 1.B3; N 1.35 found (%) : C 63.37; H 9.B1; F 1.69; N 1.27 .
Preparation of (BS)-8-fluoroerythromycin A propionate lauryisulphate A solution of 2.8B5 9 (0.010 moles) of lauryl sulphate sodium salt in 50 mls of distilled water was added to a solution of 8.080 9 (0.010 moles) of (85)-B-fluoroerythromycin A
propionate(prepared in the example 21) in 75 mls of acetone. Under electromagnetic stirr~ng the thus obtained solution was then supplemented with 20 mls of a 5% aqueous solutiG~l of acetic acid. The resulting salt was filtered, washed several times with water and then drizd at 50C under vacuum. 10.25 9 of (85)-8-fluoroerythromycin A
propionate laurylsulphate were thus obtained.
ThP ~nalysis for C52H96FN018S gave the following results:
calculateo (%): C 5a.13; H 9.01; F 1.77; N 1.30; S 2.98 found (%) : C 57.89; H 8.92; F 1.72; N 1.26; S 2.94 Preparation of (85)-8-fluoroerythromycin A carbonate A solution of 7.045 9 (0.080 moles) of ethylene carbonate in 20 mls of anhydrousbenzene, already heated, was dropwise added (over about 1 hour) to a mixture of 7.520 9 (0.010 moles) of (flS)-8-fluoroerythromycin A, 3.760 9 of potassium carbonate and 20 mls of benzene, vigorously stirred and heated to reflux temperature~ At the end of the addition the reaction rnixture was heated to refluxing for further 15 rninutes and cooled to roorn temperaturP. ~here were then added under stirring 40 mls of water.
~he henzene phase was then separated, washed three times with watern dried over 32 3~
anhydrous Na2504 and then evaporated to dryness under vacuum at 5nDc.
The residue was dissolved in ethyl ether and brought to dryness under vacuum. The latter operation was repeated some times and the resulting residue was then purified by chromatography in a silica gel column. The fractions containing only (85)-B-fluoroerythromycin A carbonate were combined (the control being carried out by high pressure liquid phase chromatography).
The combined fractions 16 to 22, evaporated under vacuum to dryness and crystalli~ed from ethyl ether, gave 0.98û 9 of (85)-8-fluoroerythromycin A carbonate, having the following characteristics:
m.p. 234-5C;
_ 20 [ D~4.2~ (C= 1 in rnethanol).
IR (KBr); 3500, 3450, 1795, 1745, 1455, 1380, 1365, 1345, 1325, ~295, 12B0, l235, 116(), 1û9û, 1070, 1040, 1015, 1000, 990, 94û, 930, 900, 875, 865, 830, 815,800cm The analysis for C38H64FNO14 gave the following values:
calculated (%): ~ 58.67; H 8.29; F 2.44; N 1.80;
found (%) : C 58.41; H 7.80; F 2.40; N 1.71 According to the same procedures there are prepared the salts, esters, and salt-esters corresponding to the other macrolide antibiotics of the invention.
The novel antibiotics of the present invention as well as the above n-lentioned related rl~r!vatjves are used for the preparation of pharrnaceutical compositions, mainly for tht oral administration, which are obtained with the conventional pharmaceuticaltechniques and with the usual excipients, vehicles, fillers etc.
There are thus contemplated tablets, pills, capsules9 suspensions and solutions containing from :L0 to 1000 mg of active ingredient per dose, whereas the daily dosages are those norrnally adopted for the analogous antibiotic, namely the erythromycin.
The following examples illustrate the preparation of compositions and formulations containing (85)-8-fluoroerythromycin A, it being meant that it is likewise foreseen for the other macrolide antibiotcs of the present invention and that these exarnples shall not unduly limit the invention.

Capsules: doses for 1000 units (85)-8fluoroerythrornycin A 9 100 39~5~
_ 33 --magnesium stearate 9 3 Preparation -The above indicated substances are homogeneously mixed and capsules of hard gelatin are filled according to the usual tecllnique.
~he contents for capsule is lû3 mg. Each capsule contains 100 mg of active ingredient.

Capsules: doses for 1000 units (BS) 8-fluoroPrythromycin A 9 250 magnesium stearate 9 7,5 Preparation The same technique disclosed in the preceding example is repeated. Each capsule,containing 257 mg of powder mixture, corresponds to 250 mg of active ingredient.EXAMPLE 31 Gapsules: doses for lûO0 units (85)-8-fJLoroerythromycin A 9 50û
magnesium stearate 9 15 The same technique described in the preceding exarnple is repeated.
Each capsule, containing 515 mg of powder mixture, correspond to 500 mg of active substance.

Tablets: doses for lûO0 units (85)-8-fluoroerythromycin A 9 100 corn starch 9 50 lactose 9 30 talc 9 8 magnesium stearate 9 2 hydroxypropylmethylcellulose 9 6 ethylcellulose 9 4 Preparation (85)-8-f~uoroerythromycirl A, part of the starch and the lactose are homogeneously mixed and the granulation is effected, according to the ~echnique of the moist gran(Jiation, using as the bonding agent the remaining amount of starch in form oF

_ 34 --starch-water.
The dried granulate is mixed with lubricants and the compression is carried out.Tablets are obtained having 190 mg weight. Each tablets contains 100 mg of active substance.
The tablets can be charged in a coating basin and film coated by means of a solution of hydroxypropylmethylcellulose and ethylcellulose. The weight of the finished tablets is 200 mg.

, Tablets: doses for 1000 units (85)-8fluoroerythromycin A 9 250 corn starch 9 70 lactose 9 40 talc ; g ` 16 magnesium stearate 9 4 hydroxypropylmethylcellulose 9 12 ethylcellulose 9 8 Preparation ~he teci nique described in the preceding example is repeated.
Each tablet of the weight of 4ûO mg contains 250 mg of active substance.

Tablets: doses for lOOD units (8S)-8-fluoroerythromycin A 9 50û
corn starch 9 140 Iactose 9 85 talc 9 37 magnesium stearate 9 8 hydroxypropylmethylcellulose 9 18 ethylcelluJose 9 12 The same technique described in the preceding example is repeated.
Each tablet of the weight of 800 mg contains 500 mg of active substance.

Extemporary suspension: 60 mls of suspension _ 35 --(85~-8-fluoroerythromycin A 9 0.6 sodium carbuxymethylcellulose g O.û10 methyl p-hydroxybenzoate 9 0.04B
propyl p-hydroxybenzoate 9 0.012 flavoring agerIts 9 0.600 sucrose powderto a total weight of 9 30 Preparation The ingredients are intimately admixed and charged in a 60 ml calibrated bottle.Before the use the bottle is filled with water to obtain 60 mls of suspension, which are well stirred before the use. The reformed suspension contains 10 mg/ml of active substance.

Extemporary suspension: doses for 60 mls of suspension (85)-~-fluoroerythromycin A 9 3 carboxymethylcellulose 9 0.010 methyl p-hydroxybenzoate 9 0.048 - prnpyl p-hydroxybenzoate 9 0.012 fla~Dring agents 9 0.600 sucroseto form a total weight of 30 g Pren3ration The same technique described in the preceding example is repeated. The suspension reconstituted contains 50 m9tml of active substance.

C)rops: dose.s for 1n mls (8S)-8-fluoroerythromycin A 9 0.5 methyl p hydroxyben70ate 9 0 09 propyl p-hydroxybenzoate 9 0.01 hydroxyethylcellulose 9 3.050 glycerine 9 0.400 sweetening and flavoring agents 9 0.100 depurated water to form lD mls In a suitable container provided with a mechanical stirrer 90% of the water needed for the preparation is charged and heated to 80C.

8~856 -- 3~ --Thereinto are dissolved the parasepticals and subsequently the hydroxyethylcellulose.
Under stirring the othEr components are added and 10 ml vials are filled. The thus obtained suspension has a content of 5û mg/ml of active substance.

Drops: doses for 10 mls ~85)-8-fluoroerythromycin A 9 2.500 methyl p-hydroxybenzoate 9 0.009 propyl p-hydroxybenzoate g 0.001 hydroxyethylcellulose g 0.050 glycerine 9 0-400 sweetening and flavoring agents g 0.100 depurated water to form 10 mls Preparation The same technique described in the preceding example is repeated. Each ml of the suspension contains 250 mg of active substance.

Capsules: dcnes for 1000 units t85)-B-fluoroerythromycin A propionate lauryl sù~phate (corresponding to 100 9 of (BS)-B-fluoroerythromycin A) 9 142.9 magnesium stearate 9 4.1 Preparation The sarne technigue described in the preceding example 29 is repeated. The contents of each capsule is 147 mg. Each capsule contains (85)-8-fluoroerythromycin A propionate laurylsulphate corresponding to 100 mg of ~8S)-8-fluoroerythromycin A.

Capsules: doses for 1000 units (85)-B-fluoroerythromycin A propionate lauryl sulphate (corresponding to 250 9 of (85)-8-fluoroerythromycin A) 9 357.2 magneisum stearate 9 10.8 Preparation The same technique described in the preceding example is repeated. Each capsule, containing 36EI mg of powder mixture, contains 357.2 mg of (85)-8-fluoroerythromycin A propionate lauryl sulphate corresponding to 25û rng of (85)-8-Fluoroerythromycin A.

Tablets: doses for 1000 units (BS)-8-fluoroerythromycin A stearate (corresponding to 100 9 of (85)-8-fluo-roerythromycin A) 91~7.83 corn starch g 55.00 lactose 9 32.50 talc g 10.00 magnesium stearate 9 2.17 hydroxypropylmethylcellulose g 7.50 ethyl cellulose 9 5.00 Preparation The same technique described in the preceding example 32 is used. Tablets are obtained having a weight of 240 mg. Each tablet contains 137.83 rng o-f (85)-8-fluoroerythromycin A stearate, corresponding to lOû mg of (85)-8-fluoroerythromycin A.
The tablets are charged in a shallow basin and Film coated by means of a solution of hydroxypropylmethylcellulose and ethyl cellulose. The weight oF the finished tablets is 250 mg.

Tabiets: doses per 1000 units (85)-8-fluoroerythromycin A stearate (corresponding to 500 9 of (85)-8-fluo-roerythromycin A) ~ 9 689.17 corn starch 9 150.00 lactose 9 80.00 talc 9 40.83 magnesium stearate g lO.Dû
hydroxypropylrnethylcellulose 9 18.ûO

5~

ethylcellulose 9 12.00 Pre aration p The same technique described in the preceding example 32 is used. Each tablet of the weight of 1 9 contains 689.17 mg of (85)-B-fluoroerythromycin A stearate, correspon-ding to 500 mg of (BS)-B-fluoroerythrornycin A.

Ex~emporary suspension: 60 mls of suspension (85)-8-fluoroerythromycin A ethylsuccinate - (corresponding to 0.6 9 of (85)-8-fluoroery-thromycin A) 9 0.7û2 sodium carboxymethylcellulose 9 0.010 methyl p-hydroxybenzoate 9 0.048 propyl p-hydroxyben~oate 9 0.012 flavoring agents 9 0.600 sucrose to form 30 9 Preoaration The same technique described in the example 35 is used,. The reconstituted suspension con ~ins (85~-8-fluoroerythromycin A ethylsuccinate correspording to 10 mg/ml of(8S)-8-fluoroerythromycin A.

Vials for i.v. use: doses for 1000 units 738 9 of (85)-B-fluoroerythromycin A lactobionate, corresponding to 500 9 of (85)-S-fluoroerythromycin A, are dissolved in water and after sterile filtration, are Iyophilized. The obtained product is subdivided in a sterile environnment in glass vials each containing 738 mg (corresponding to 5ûO mg of (8S3-8-fluoroerythromycin A).The macrolide antibiotics of the present invention have been investigated by pharmacological tests aiming to determine:
- the bacteriostatic power, expressed as the minimun inhibiting concentration (MIC) against both aerobic and anaerobic, Gram positive and Grarn negative, bacterial stocks: the related results are reported in the tables 2 and 3.
- the serum concentrations relating to (85)-8-fluoroerythromycin A and to the erythromycin A (table 4).

- the baclerial power, expressed as the minimun bactericidal power (MBC) againstsorne Gram positive aerobic stocks ttable 5).

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Claims (17)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLU-SIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of the compounds of the formulae:

which comprises a) reaction of a compound selected among 8.9-anhydroerythronolide A-6,9-hemiacetal, 8.9-anhydroerythronolide B-6,9-hemiacetal and 3-0-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal with a compound capable of generating elec-trophilic fluorine in the presence of an organic inert sol-vent and at low temperature to form the corresponding (8S)-8-fluoro acetalic derivative in admixture with the related (8S)-8-fluoro derivative, and b) reaction of (8S)-8-fluoro acetalic derivative in admixture with the related (8S)-8-fluoro acetalic derivative with an acid and in aqueous solu-tion.

2. A process according to claim 1, in which said reagent capable of generating electrophilic fluorine is selected in the class of the fluoroxy-perfluoroalkanes.

3. A process according to claim 2, in which said reagent generating electrophilic fluorine is fluoroxy-tri-fluoromethane.

4. A process according to claim 1, in which said reagent generating electrophilic fluorine is perchloryl fluoride.

5. A process according to claim 1, in which said reagent capable of generating electrophilic fluorine is selected from fluoroxy-sulphur-pentafluoride, molecular fluorine and lead tetraacetate-hydrogen fluoride.

6. A process according to claim 1, in which said solvent is selected from chlorinated hydrocarbons, tetrahydrofuran and their mixtures.

7. A process according to claim 6, in which said solvent is selected from trichlorofluoromethane, chloroform and methylene chloride.

8. A process according to claim 1, in which the reaction temperature is of between -75 and -85°C.

9. A process according to claim 1, in which said acid is selected from among acetic acid and hydro-chloric acid.

10. A process according to claim 1, in which said reaction with the acid is carried out at a temperature of between 30°C and 150°C.

11. A compound selected from those of the formulae:

whenever prepared or produced by the process claimed in claim 1, 2 or 3 or an obvious chemical equivalent thereof.

12. A process according to claim 1, in which 8,9-anhydroerythronolide A-6,9-hemiacetal is used.

13. A process according to claim 1, in which 8,9-anhydroerythronolide B-6,9-hemiacetal is used.

14. A process according to claim 1, in which 3-0-mycarosyl-8,9-anhydroerythronolide B-6,9-hemiacetal is used.

15. (8S)8-fluoroerythronolide A, having the for-mula:

whenever prepared or produced by the process claimed in claim 12 or an obvious chemical equivalent thereof.

16. (8S)-8-fluoroerythronolide B, having the formula:

whenever prepared or produced by the process claimed in claim 13 or an obvious chemical equivalent thereof.

17. 3-0-mycarosyl-(8S)-8-fluoroerythronolide B, having the formula:

whenever prepared or produced by the process claimed in claim 14 or an obvious chemical equivalent thereof.