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Title:
NOVEL DERIVATIVES OF THE OLIGOSACCHARIDE ANTIBIOTIC COMPLEX 13-384, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
Document Type and Number:
WIPO Patent Application WO/1987/002366
Kind Code:
A2
Abstract:
Novel derivatives of the oligosaccharide antibiotic complex 13-384, the derivatives being of general formula (I), wherein R is hydrogen or Z, where Z is a moiety of general formula (II) in which R1 is nitroso, N-hydroxylamino, N-acyl-N-hydroxyl-amino, N-acylamino, N-alkylamino or N,N-dialkylamino and their pharmaceutically acceptable salts. Also disclosed are processes for the preparation of the derivatives and pharmaceutical compositions containing them. The novel derivatives exhibit advantageous antibacterially properties.

Inventors:
Ganguly
Ashit
Kumar, Girijavallabhan
Viyyoor
Moopil, Sarre
Olga
Application Number:
PCT/US1986/002135
Publication Date:
April 23, 1987
Filing Date:
October 15, 1986
Export Citation:
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Assignee:
SCHERING CORPORATION.
International Classes:
C07H9/04; C07H13/08; C07H17/04; (IPC1-7): C07H19/01; A61K31/72; A61K31/73
Foreign References:
US4129720A1978-12-12
US4597968A1986-07-01
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Description:
NOVEL DERIVATIVES OF THE OLIGOSACCHARIDE ANTIBIOTIC

COMPLEX 13-384, THEIR PREPARATION AND

PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

This invention relates to novel antibacterially-active derivatives of the oligosaccharide antibiotic complex 13-384, to processes for the preparation of the derivatives and to pharmaceutical compositions containing them. More specifically, this invention relates to derivatives of antibiotic 13-384- component-5, in particular to derivatives at the amino function of antibiotic 13-384-component-5.

In U.S.P. 4,597,968 there is described and claimed an oligosaccharide antibiotic complex, designated antibiotic 13-384, produced by cultivation of a strain of Micromonospora carbonacea var africana. Samples of this novel variety of Micromonospora were deposited on June 25, 1982 at the Northern Utilization and Research Division Agriculture Research Service, U.S. Department of Agriculture in Peoria, Illinois under assession number 15099 and at the American Type Culture Collection in Rockville, Maryland under assession number ATCC 39149. Sub-cultures of these microorganisms are freely available to the public. USP 4,597,968 further describes and claims two components of the novel antibiotic 13-384 complex, these components being characterized by

specified physical data and being designated antibiotic 13-384 component-1 and component-5.

We have now found that certain novel derivatives of antibiotic 13-384-component-5 possess useful and advantageous antibacterial properties, the derivatives exhibiting antibacterial activity against gram positive and gram negative bacteria and being generally active against methicillin-resistant Staphylococcus aureus. Further, these derivatives are indicated as giving good blood levels at antibacterial dosages. Elucidation of the structure of the novel derivatives and of antibiotic 13-384 components -1 and -5 has shown that the novel derivatives of the invention are derivatives at the amino function of antibiotic 13-384- component-5. The novel derivatives are obtainable either from antibiotic 13-384-component-5 itself or from component-1, component-1 being the nitro analog of component-5.

In accordance with one of its aspects the present invention provides novel oligosaccharide compounds of the general formula I:

(I),

wherein R is hydrogen or Z, where Z is a moiety of the general formula II:

in which R^ is nitroso, N-hydroxylamino, N-acyl-N- hydroxylamino, N-acylamino, N-alkylamino or N,N- dialkyla ino, and the pharmaceutically acceptable salts of the foregoing.

The term "acyl" as used herein means acyl radicals derived from carboxylic acids having up to 12 carbon atoms which acids may be straight- or branched- chain aliphatic acids, which may be substituted by one or more amino, halo, hydroxy, mercapto, aryl (in particular phenyl) and heterocyclyl groups; cyclic carboxylic acids having 4 to 12 carbon atoms; heterocyclic carboxylic acids; and aromatic carboxylic acids which may be substituted by one or more alkyl, halo, nitro, tri luoromethyl and phenyl groups.

Exemplary of such acyl radicals derived from unsubstituted alkanoic acids are for yl, acetyl, propanoyl, n- and iso-butyroyl, valeroyl, pivalyl, heptanoyl, octanoyl, nonanoyl and dodecanoyl.

Exemplary of acyl radicals derived from substituted alkanoic acids are 2-aminoalkanoyl groups derived from the naturally occurring α-amino acids such as cystine, gluta ic acid, histidine, hydroxylproline, methionine, proline, tyrosine or threonine as well as corresponding D-α-amino acids such as D-ornithine, D-

methionine, D-lysine and D-alanine; hydroxyalkanoyl such as those derived from α-hydroxy acids such as glycolic, lactic, mandelic (phenylglycollic) , α-hydroxybutyric, o- hydroxyisobutyric, α-hydroxy-n-and-iso-valeric acids as well as α-hydroxylic acids such as malic (hydroxy- succinic) and tartaric (dihydroxysuccinic acid); hal- alkanoyl, especially α-haloalkalkanoyl such as chloro-, bromoacetyl, trifluoroacetyl and 2,2-difluoropropanoyl; thio-substituted alkanoyl such as thiolactic and thioglvcolic; arylalkanoyl such as phenylacetyl, γ- phenylbutyryl, phenylglycyl, and phenylalanyl.

Typical cyclic acyl groups are cyclopropyl- carbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, cycloheptylcarbonyl, adamantyl- carbonyl, while typical heterocyclic acyl groups are those derived from thiophenacetic acid, furoic acid (furan-2-carboxylic acid), furanacetic acid and tetrazylacetic acid.

Typical acyl groups derived from aromatic carboxylic acids include benzoyl and benzoyl substituted by one or more of alkyl, halo, nitro, trifluoromethyl and phenyl, such as 2-methylbenzoyl, 3-methylbenzoyl and 2,4,6-trimethylbenzoyl, 3-chlorobenzoyl, 4-trifluoro- methylbenzoyl, 4-nitro-3-trifluoromethylbenzoyl and 4- phenylbenzoy1.

The term "alkyl" as used herein means straight, branched-chain and cyclic hydrocarbon groups of up to 6 carbons including methyl, ethyl, n- and isoproyl, cyclopropyl, n-, iso-, tert-butyl, cyclobutyl, n-iso, sec- and tert-pentyl, cyclopentyl, n-, iso-, sec- and tert-hexyl, and cyclohexyl.

Preferred acyl groups for the substituent R^ include those derived from lower (C-^-Cg) alkanoic acids for instance acetic acid, those derived from cyclic carboxylic acids such as cyclohexane-carboxylic acid and

those derived from arylalkanoic acids such as phenylglycine. A preferred alkyl value for R-, is ethyl.

The compounds of the formula I contain 3 acidic phenolic hydroxy functions and therefore may be converted into a corresponding pharmaceutically acceptable salts. Typical pharmaceutically acceptable salts are metal salts such as alkali metal salts and alkaline earth metal salts, and ammonium or substituted ammonium salts such as those derived from trialkylamines (e.g. triethylamine) , procaine, dibenzylamine, N-benzylbetaphenylethylamine, N,N'-dibenzylethylenediamine, dehydroabietylamine, N,N'- bisdehydroabietylethylenediamine, N-alkylpipe idines (e.g., N-ethylpiperidine) and N-methylglucamine. The preferred substituted ammonium salt is the one derived from N-methylglucamine. Representative alkali metal and alkaline earth metal salts include the sodium, potassium, and calcium salts; preferred are sodium salts. Also included within the term pharmaceutically acceptable salts are acid addition salts typically formed with organic and inorganic acids.

The compounds of the present invention may be prepared from antibiotic 13-384-component-5 itself (of the general formula I except that R^ is amino) or from component-1 (of the general formula I except that R^ is nitro) using methods known per se.

In accordance with another aspect of the present invention there is provided a process for the preparation of a compound of the general formula I set forth in claim 1, which process is selected from the following processes A to E:

A: acylation of an antibiotic 13-384-component-5 starting material or the hydroxylamino derivative thereof, at the amino function to give a compound of the general formula I wherein R is the moiety Z and R- j^ is N-acylamino or N-acyl-N-hydroxylamino;

B: reductive alkylation of an antibiotic 13-384- component-5 starting material at the amino function to give a compound of the general formula I wherein R is the moiety Z and R-^ is N-alkylamino or N,N- dialkylamino;

C: reduction of an antibiotic 13-384-component-l starting material at the nitro function to give a compound of the general formula I wherein R is the moiety Z and Ri is N-hydroxylamino;

D: oxidation of an N-hydroxylamino antibiotic 13-384- component-5 starting material at the N-hydroxylamino group to give the corresponding nitroso antibiotic 13-384-cpmponent-5 of the general formula I wherein R is Z and R-^ is NO; and

E: reductive cleavage of a nitroso antibiotic 13-384- component-5 starting material to give a corresponding desevernitrose antibiotic 13-384 component-5 of the general formula I wherein R is hydrogen,

said process selected from processes A to E being following by isolation of the so-obtained compound of the general formula I in free form or in the form of a pharmaceutically acceptable salt.

The acylation process A may be effected by reacting the antibiotic 13-384-component-5 starting material, suitably at room temperature, with the reguired acylating agent in an inert medium such as an ether solvent and then isolating the so-obtained N-acylamino compound of the formula I. Suitable acylating agents are acid anhydrides and mixed acid anhydrides formed from the desired carboxylic acid with p-toluene sulfonyl chloride or a chloroformate in the presence of a base. Where the acylation reaction is carried out in the absence of a base, acylation of the phenolic hydroxy groups, as well as the amino group, occurs to give di-, tri- and tetra- acyl derivatives. Partial hydrolysis of these derivatives under mild basic conditions effects removal of the O-acyl functions. Selective acylation of the amino function may be achieved by acylating in the presence of an alcohol such as a C^_g alcohol, for instance isopropanol.

Illustrative of the foregoing process A is the preparation of N-acetylamino antibiotic 13-384-component- 5 which may be effected by reacting antibiotic 13-384- component-5 with acetic anhydride in an ether solvent such as tetrahydrofuran, dioxan, dimethoxyethane or a C-, - Cg alcohol such as isopropanol, at room temperature, for example 25°C, for a period of about three hours. The progress of the reaction may conveniently be monitored by thin-layer chromatography and upon completion the resulting desired product recovered.

Where the desired acyl derivative is an N-acyl- N-hydroxylamino compound of the formula I, the reguired N-hydroxylamino starting material may conveniently be formed in situ and concurrently, or subseguently, subjected to acylation. Thus, the N-acyl-N-hydroxylamino compound of the formula I may be formed by subjecting the corresponding nitro analog, antibiotic 13-384-component-

1, to reductive acylation, with a suitable reduction catalyst such as active zinc powder in the presence of the appropriate acid anhydride. The active zinc powder catalyst may conveniently be prepared by washing zinc powder with agueous mineral acid and then finally removing water by conventional methods. The acid anhydride may suitably be present in an amount of four to six times the stoichiometric guantity. The reductive acylation is typically carried out in an inert anhydrous organic solvent such as tetrahydrofuran or dioxan under an inert atmosphere and at room temperature for a period of time sufficient to produce a crude reaction mixture of the N-acyl-N-hydroxylamino and N-acylamino products. The crude reaction mixture is treated with methanolic ammonia to hydrolyze the acylphenolic esters as well as to destroy the excess acid anhydride. The N-acyl-N- hydroxylamino compounds are then recovered and purified by conventional technigues e.g. extraction and column chromatography.

The alkylation process B may be effected by subjecting an antibiotic 13-384-component-5 starting material to reactive alkylation. This may be carried out by reacting the starting material in an inert solvent with the reguired amount of the appropriate aldehyde under a hydrogen atmosphere in the presence of a suitable hydrogenation catalyst. We have used Raney nickel as catalyst in the preparation of N-alkyl derivatives and platinum oxide in the preparation of N,N-dialkyl derivatives. Any suitable inert solvent may be used such as 2-methoxyethanol, dimethoxyethane, 2-methoxyethylether or a C-^-Cg alkanol. Alternatively, the desired N-alkyl product may be prepared by reductive alkylation of the appropriate Schiff's base, the Schiff's base being firstly formed by reacting the reguired aldehyde with antibiotic 13-384-component-5 in a suitable alkanol

solvent and then reducing the Schiff's base in situ using for example sodium cyanborohydride.

In process C for the preparation of the N- hydroxylamino compound of the formula, the reduction of the corresponding nitro-co pound starting material, antibiotic 13-384-component-l, may be carried out using any suitable reduction catalyst, for instance sodium amalgam, or zinc in the presence of a proton source such as ammonium chloride, tartaric acid or ammonium carbonate. The reaction is effected in an inert solvent such as tetrohydrofuran, dioxan, dimethoxyethane or 2- methoxyethylether under an inert atmosphere and at room temperature.

Preparation of the nitroso compound of the formula I by the oxidation process D may be achieved by reacting the N-hydroxylamino antibiotic 13-384-component- 5 starting material with activated carbon in an inert solvent such as a lower alkanol, for instance methanol or ethanol, until reaction is complete, typically about 5 to 10 hours.

Preparation of the compound of the formula I where R is hydrogen by the reductive cleavage Process E may be achieved by treating the nitroso-antibiotic 13-384 starting material with an alkoxy phosphorous compound such as a trialkyl phosphite, for instance triethyl phosphite or tributyl phosphite, or a triaryl phosphine, for instance triphenyl phosphine and then isolating the so-obtained desevernitrose product. The reaction may be effected in an inert solvent such as tetrahydrofuran under an inert atmosphere and at an elevated temperature up to the boiling point of the reaction mixture.

The compounds of the formula I obtained by the aforementioned processes A to E may be isolated in free form or in the form of a pharmaceutical acceptable salt.

Preparation of the pharmaceutically acceptable salts may be carried out according to conventional procedures for forming salts. Salts may be formed, for example, by treating with metal hydroxide compounds such as sodium hydroxide, calcium or magnesium hydroxide, or with ammonia or a suitable organic a ine or N- methylglucamine, wherein at least about a stoichiometric amount of the salt-forming agent is used. Acid addition salts of compounds of the formula I are obtainable in the usual manner, for example, by treating with an acid or a suitable anion exchange reagent.

The following examples illustrate the preparation of the compounds of the present invention. In the examples the following abbreviations are used in the sections giving physical data: Infrared Spectroscopy (IR), Proton Magnetic Resonance (PNMR), Cl AJ _ Magnetic

Resonance (CNMR) , and High Resolution Fast Atom

Bombardment Mass Spectrometry, (MS/FAB).

EXAMPLE I

N,N-DIETHYLAMINO-13-384-COMPONENT-5

A solution of 200 mg of antibiotic 13-384- component-5, as isolated and purified in U.S. Patent 4,597,968 in 8 ml of 2-methoxyethanol and 2 ml of freshly distilled acetaldehyde was stirred under hydrogen in the presence of 40 mg of sodium bicarbonate and 200 mg of platinum oxide at 25 psi for 48 hours. The catalyst was filtered off and the filtrate concentrated to dryness, under vacuum, to yield crude title compound. Chromatography on a silica gel column, eluting with 5% v/v of methanol in chloroform, yielded pure title compound. Anal: found; C, 50.13; H,6.33; N, 0.74%; C 74 H 107°36 NC1 2* C ' *-3 reguires C, 50.69; H,6.13; N, 0.79%; MS: (FAB) [m+H] + 1656; Rotation: [ ] D 26 -45.9°

(MeOH); CNMR: δ 119.357 ppm and 6 120.529 ppm (Ortho Esters) .

EXAMPLE II N-ETHYLAMINO-13-384-COMPONENT-5

A solution of 150 mg of antibiotic 13-384- component-5 as isolated and purified in U.S. Patent 4,597,968 in 3 ml of methanol and 3 ml of freshly distilled acetaldehyde was stirred under hydrogen in the presence of 35 mg of sodium bicarbonate and Raney nickel (about 1.5 ml) at 25 psi for 48 hours. The catalyst was filtered off and the solvent was removed under reduced pressure to yield the title compound. Chromatography on a silica gel column, eluting with 10% v/v of methanol in chloroform, yielded pure title compound. Anal.: Found: C, 51,96; H,6.24; H,0.97; C^H-^O^NC^ l / 2 CHCI3 reguires: C,51.60; H,6.18; N,0.83%; MS/FAB (M+H) + 1628; Rotation: [α] D 26 -45.3° (MeOH); CNMR: δ 119.277 ppm and δ 120.488 ppm (Ortho Esters).

EXAMPLE III

DESEVERNITROSE-13-384-COMPONENT-5

a) N-HYDROXYLAMINO-13-384-COMPONENT-5

To a solution of 1.0 g of antibiotic 13-384 component-1, as isolated and purified in U.S. Patent 4,597,968 in 30 ml of peroxide-free tetrahydrofuran was added 600 mg of zinc powder. To the stirring slurry was added, dropwise, 2 ml of a 10% aqueous solution of ammonium chloride. The suspension was stirred under nitrogen at room temperature for 35 minutes. The progress of the reaction was monitored by thin layer chromatography using 8% v/v methanol in chloroform as eluent. The filtered reaction mixture was washed twice with brine solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the title compound.

b) NITROSO-13-384-COMPONENT-5

To a solution of 960 mg of crude hydroxylamino- 13-384-component-5, in 30 ml of methanol was added 2 g of 12 x 40 mesh active carbon (Specifically, that sold under the trade designation "Nuchar" was used; Westvaco Chemical Division U.S.A.). The suspension was stirred at room temperature for 7 hours. The progress of the reaction was followed by thin layer chromatography using 8% v/v methanol in chloroform as eluent. The active carbon was filtered off on a buchner funnel using a filter pad, and thoroughly washed with methanol. The clear filtrate was concentrated to dryriess under reduced pressure. The active carbon was further extracted with tetrahydrofuran to give crude title compound.

c) DESEVERNITROSE-13-384-COMPONENT-5

Under nitrogen atmosphere, to a solution of 500 mg of crude nitroso-13-384-component-5, in 16 ml of peroxide free dioxane was added 1.6 ml of triethyl- phosphite. The reaction solution was heated in a 100°C oil bath, with stirring, until thin layer chromatography analysis indicated the absence of starting compound (about 2 hours). The reaction mixture was evaporated to a residue under vacuum. The residue was dissolved in a minimal amount of tetrahydrofuran and slowly precipitated with hexane. The precipitate was filtered and thoroughly washed with hexane. The crude title compound was chromatographed through a silica gel column, eluting with 3% v/v of methanol in chloroform, to give pure title compound, which may be further purified by crystallization from acetone. Anal.: found: C,51.45; H,5.97%; C 2 H 84 θ34Cl2 reguires: C,51.56; H,5.86%; M.P. 198-202°C; MS/FAB: (M+DEAH) + 1548; M+1442; Rotation: [α] D 26 -20.7°(MeOH) ; PNMR and IR were consistent with the assigned structure.

EXAMPLE IV

ACETAMIDO-13-384-COMPONENT-5

To a solution of 200 mg of antibiotic 13-384- component-5, in 13 ml of peroxide free tetrahydrofuran was added 85 mg of sodium bicarbonate and 0.1 ml of acetic anhydride. The reaction mixture was stirred at room temperature until thin layer chromatography analysis in 18% v/v of methanol in chloroform, indicated the absence of starting compound (about 3 hours). The reaction mixture was filtered and the clear filtrate was concentrated to a residue under reduced pressure. Titration of the residue gave solids of crude multiacetylated 13-384-component 5. The solids were dissolved in 2.5 ml of 5% ammonium hydroxide in methanol and allowed to stand at room temperature for about 40 minutes. The progress of the reaction was monitored by thin layer chromatography, using 10% v/v of methanol in chloroform. The reaction solution was concentrated to a low volume, under reduced pressure, diluted with ethyl acetate and again concentrated to a lower volume. The residue was then partitioned between ethylacetate and water, and separated. The agueous portion was extracted with ethylacetate. The combined ethylacetate extracts were washed once with brine, dried over sodium sulfate, and evaporated to a residue, under reduced pressure, to give crude title compound. Chromatography of the crude title compound on four preparative thin layer silica gel plates (1,000 microns thick) using 10% v/v of methanol in chloroform as eluent, and extraction of the product off of the silica gel with distilled tetrahydrofuran gave the title compound. Anal.: found: C,52.14; H,6.25; N,0.69%; C 72 H 101°37 NC1 2 requires: C,52.62; H,6.19 and N,0.85%; MS/FAB (M+DEAH) + 1747; M + 1641; [ ] D 26 -47.3° (MeOH). IR was consistent with the assigned structure.

EXAMPLE V

N-CYCLOHEXYLCARBONYLAMINO-13-384-COMPONENT-5

.Dissolve 1.3 g of cyclohexane carboxylic acid and 1.05 g of triethylamine in 20 mL of anhydrous tetrahydrofuran. Cool the reaction mixture to about 5°- 10°C and add 1.1 g of ethyl chloroformate with agitation. Continue agitating the reaction mixture for an additional 10 min. and filter. Add to the clear filtrate 300 mg of antibiotic 13-384-component-5 and continue agitating the reaction mixture. Monitor the progress of reaction by thin layer chromatography as described in Example IV. When the reaction is complete, treat the reaction mixture with sodium bicarbonate and extract with ethyl acetate. Concentrate the solution containing the acylated product mixture to a residue, dissolve the residue in methanol and hydrolyze the phenolic acylated hydroxyl moieties using a 20% solution of tetraethylammonium hydroxide in methanol to obtain the title compound.

Other N-acylamino compounds of the invention may be prepared by the procedure of this example by substituting for cyclohexane carboxylic acid other carboxylic acids such as propionic, valeric, dodecanoic pivalic, isopropylacetic, adamantanecarboxylic, cyclobutylcarboxylic, cycloheptylcarboxylic, benzoic, phenylacetic, and γ-phenylbutyric acid to give the corresponding N-acylamino derivatives of antibiotic 13- 384-component-5.

EXAMPLE VI

N-PHENYLGLYCYLAMINO DERIVATIVE OF ANTIBIOTIC 13-384-COMPONENT-5

To a solution of 600 mg of N- carbobenzyloxyphenylglycine in anhydrous methylene chloride add 220 mg of triethylamine followed by 250 mg of ethyl chloroformate while stirring at -20°C. Stir the reaction mixture for 2 hour and add thereto 250 rag of antibiotic 13-384-component-5 and raise the temperature to 25°C. Monitor the reaction by thin layer chromatography by the procedure described in Example IV. When reaction is complete, add ethyl. acetate and wash the resulting organic layer with agueous sodium bicarbonate solution and concentrate the ethyl acetate layer to a residue. Dissolve the residue in ethanol and hydrogenate at 30 psi, in the presence of palladium for about 20 hours to remove the N-carbobenzyloxy protecting group. Remove the catalyst by filtration, basify the filtrate with 20% tetraethylammonium hydroxide to hydrolyze the phenolic acylated hydroxyl moieties. Concentrate the filtrate to a residue to obtain the title product..

In a like manner, by replacing N- carbobenzyloxyphenylqlycine with an eguivalent guantity of other -aminoacids bearing reductively removable amino protecting groups and by following the procedure of this example, the corresponding α-aminoalkanoyl amino derivatives of antibiotic 13-384-component-5 may be produced.

EXAMPLE VII

N-ACETYL-N-HYDROXYL DERIVATIVE OF

ANTIBIOTIC 13-384 COMPONENT-5

Treat a solution of 200 mg of antibiotic 13- 384-component-l and 0.2 ml of acetic anhydride in 5 ml of anhydrous tetrohydrofuran with 300 mg of activated zinc dust (added in portions) at room temperature for 4 hours.Add 1 ml of cone, agueous ammonia and stir the mixture for 2 hour. Adjust the pH of the reaction mixture to 6.5 with tartaric acid and extract the aσueous solution with ethyl acetate. Dry the organic layer. Remove the solvent and subject the residue to silica gel column chromatography to obtain the title compound.

As previously stated the compounds of the present invention exhibit useful and advantageous antibacterial activity. Representative compounds of the formula I have been found unexpectedly to exhibit enhanced antibacterial activity as compared with the parent antibiotic 13-384-component-5. This is shown by Table I below in which in vitro activity data are given expressed in terms of a geometric mean Minimum Inhibitory Concentration (mcg/ml; 24 hrs) against 33 strains of Staphylococci and against 36 strains of Streptococci, the tests being performed using conventional agar dilution methods in Mueller-Hinton agar supplemented by 5% sheepsblood for the Streptococci.

Table I In Vitro Activity

Compound of the formula I Mean MIC (mcg/ml)

R Staph. Strep.

(33 strains) (36 strains)

1 ) Z -NH 2 2.74 0.93

2 ) H - 0.20 0.08

3 ) Z -NHCOCH 0,66 0.07

4 ) Z -NH . Et 1.52 0.54

5 ) z -N . Et 2 0.50 0.20

6 ) z -NHOH 0.04 0.03

Compound 1 is the parent antibiotic, antibiotic 13-384-component-5 and compounds 2 to 6 are representative compounds of the invention. As will be seen, Table I indicates that the compounds of the invention process significantly enhanced activity in comparison with antibiotic 13-384-component-5. The compounds of the invention are non-toxic at the therapeutic dose.

In another of its aspects the present invention provides pharmaceutical compositions comprising, as active ingredient, a compound of the formula I together with a pharmaceutically acceptable carrier. The pharmaceutical composition may be in dosage form suitable for oral, parenteral or typical administration.

For oral administration, the antibiotics of this invention may be compounded in the form of tablets, capsules, elixirs or the like. Tablets and capsules may contain such excipients as starch or lactose; liguid forms may contain coloring or flavoring agents. Topical preparations may be in the form of creams, hydrophobic

and hydrophilic ointments, or agueous, non-agueous or emulsion-type lotions. Typical carriers for such formulations are water, oils, greases, polyesters and polyols. Parenteral formulations, e.g., injectable dosage forms, are usually liguids such as solutions or suspensions, with typical carriers being distilled water and saline solution.

Typical pharmaceutically carriers for use in the pharmaceutical formulations of the compounds of this invention are exemplified by sugars such as lactose, sucrose, mannitol and sorbitol; starches such as corn starch, tapioca starch and potato starch; cellulose and derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates such as dicalcium phosphate and tri-calcium phosphate; sodium sulfate; calcium sulfate; polyvinyl pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates such as magnesium stearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn set; non-ionic, cationic and anionic surfactants, ethylene glycol polymers; betacyclodextrin; fatty acids, hydrolyzed cereal solids; water; polyalkylene glycols; gums; and petroleum; as well as other non-toxic compatible fillers, binders, disintegrants and lubricants commonly used in pharmaceutical formulations. The compositions may also contain preservatives, aerosol propellants and coloring, thickening, suspending, dispensing, emulsifying, wetting, stabilizing and buffering agents.

The dose to be administered in any particular dosage form will depend upon various factors, such as the susceptibility of the infecting organism to the antibiotic and the stage and severity of the infection. Generally, the dosage administered is from about 1.0 mg to about 25 mg per kilogram of body weight per day, in

divided dosages, the specific dosage being left to the discretion of the practitioner. In treating certain patients with the compounds of this invention, it is possible to include other pharmaceutically active ingredients in the same dosage unit.

The following examples illustrate pharmaceutical compositions of the present invention.

Example VIII

Parenteral Solution (Quantities per 1 ml. of solution

Mg

Hydroxylamino antibiotic 13-384- 58.14 component-5 sodium salt.

Sodium chloride 5.0

Sodium bisulfate 1.625 Sodium hydroxide (IN to pH 8.7)

Methylparaben 1.8

Propylparaben 0.2 Purified Water, q.s. L-m-l-

Procedure

Purge with nitrogen about 90% of the reguired amount of water and heat the water to a temperature of about 60- 70°C. Add the methylparaben and propylparaben, stir until dissolved, then cool the solution to 25-30°C. Change and dissolve the sodium chloride and sodium bisulfite. Add the hydroxylamino compound of the invention and with stirring add IN sodium hydroxide until the solution reaches a pH of 8.7. Add water to the reguired total volume and filter through a sterilizing membrane and then fill into sterile vials.

Example IX

Parenteral Suspension (Quantities per 2 ml. of suspension)

Mg. Hydroxylamino antibiotic

13-384-component-5 100

Sodium σarboxymethylcellulose 3

Polysorbate 80 U.S.P. 1

Methylparaben 3.6

Propylparaben 0.4 Water, q.s. 2.0 ml.

Procedure

Prepare a solution of the sodium carboxylmethylcellulose Polysorbate 80, U.S.P., methylparaben and propylparaben. Aseptically, slurry hydroxylamino- antibiotic 13-384 component-5 with a portion of the above vehicle and pass through a colloid mill. Mix the milled slurry with the remainder of the vehicle and then fill into sterile vials.