GB1570986A - Derivatives of thienamycin and its isomers - Google Patents

Abstract

N-Acyl derivatives and phosphorus-containing N-acyl derivatives of the antibiotic thienamycin of the general formula (II) <IMAGE> in which R<1> and R<2>, independently of each other, are selected from hydrogen, acyl and phosphorus-containing acyl, with the proviso that R<1> and R<2> are not both hydrogen and that, when R<1> or R<2> denotes acetyl, R<2> or R<1> is different from hydrogen and their non-toxic, pharmaceutically acceptable salts, are obtained by treating thienamycin with an appropriate acylating agent and converting the resulting compounds of the formula (II), where appropriate, into their non-toxic, pharmaceutically acceptable salts. The compounds which are obtained, and their pharmaceutically acceptable salts, are suitable for use as antibiotics.

Description

(54) DERIVATIVES OF THIENAMYCIN AND ITS ISOMERS (71) We, MERCK & CO. INC., a corporation duly organized and existing under the laws of the State of New Jersey, United States of America, of Rahway, New Jersey, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- There is a continuing need for new antibiotics. For unfortunately there is no static effectiveness of a given antibiotic because continued wide scale usage of any such antibiotic selectively gives rise to resistant strains of pathogens. In addition, the known antibiotics suffer from the disadvantage of being effective only against certain types of microorganisms. Accordingly the search for new antibiotics continues.

This invention is concerned with N-acyl derivatives of the antibiotic thienamycin and its stereoisomers, processes for their preparation and pharmaceutical compositions comprising them. Such derivatives and their pharmaceutically acceptable salts have been found useful as antibiotics.

Thienamycin, one of the starting materials required for preparation of the compounds of the present invention, is disclosed and claimed in the specification of our copending application No. 48208/75 (1,498,087). Thienamycin is known to be a stereoisomer of the following structural formula:

The N-acyl derivatives of the present invention may be depicted by the following generic structural formula (II):

or more conveniently by the symbol (it):

In these formulae, "Th" symbolizes the bicyclic nucleus of thienamycin or a stereoisomer of it and the OH, amino and carboxyl groups of the thienamycin-type nucleus are illustrated.

M is a hydrogen atom or a pharmaceutically acceptable cation, e.g. of an alkali metal or alkaline-earth metal, such as sodium, potassium or calcium or of an amine or ammonium or quaternary ammonium salt, such as N(R)3 where R is alkyl having 1 to 6 carbon atoms.

R2 is an acyl group other than acetyl and R' is a hydrogen atom or an acyl group, or R1 and R2 are both acetyl. Preferably R' is hydrogen and R2 is acyl. The term "acyl" includes not only carboxylic acyl, but also its thio analogues, in which the carbonyl oxygen is replaced by sulphur, diacyl radicals, in which R1 and R2 are joined together, and acyl radicals from sulfur and phosphorus acids, such as substituted sulfonyl, sulfinyl and sulfenyl radicals and substituted P(III) and P(V) radicals such as the substituted phosphorous, phosphoric, phosphonous and phosphonic radicals.

Antibiotics of the present invention are active against a broad range of pathogens which representatively include both gram-positive bacteria such as S. aureus, Strop. pyogenes and B. subtilis and gram-negative bacteria such as E. coli, Proteus organic, Serratia and Klebsiella.

In the generic representation of the compounds of the present invention (II, above), the acyl radical represented by either R' and R2 can, inter alia, be a substituted or unsubstituted aliphatic, aromatic, heterocyclic, araliphatic or heterocyclylaliphatic carboxylic acid radical, a substituted or unsubstituted carbamyl radical or a carbothioic acid radical. One group of acyl radicals can be represented by the general formula:

where X is O or S and R" represents hydrogen; amino; substituted amino such as C16 alkylamino or di(C1~e alkyl)amino in which the alkyl radical(s) are substituted or unsubstituted; straight or branched-chain C16 alkyl; mercapto, substituted mercapto such as alkylthio, typically comprising 1 to 6 carbon atoms, or arylthio, typically comprising 6 to 10 carbon atoms; hydroxy; substituted hydroxy such as alkoxy, typically comprising 1 to 6 carbon atoms, or aryloxy, typically comprising 6 to 10 carbon atoms; alkenyl or alkynyl, typically comprising 2 to 6 carbon atoms; aryl, such as phenyl; aralkyl, such as benzyl; cycloalkyl, typically comprising 3 to 6 carbon atoms; or a heteroaryl or heteroaralkyl group (mono- and bicyclic) in which the alkyl residue (if any) typically comprises 1 to 3 carbon atoms and the heterocyclic ring typically comprises W10 atoms and the hetero atom or atoms is/are preferably 0, N and/or S; such above-listed groups can be substituted or can be substituted by radicals such as OH, SH, SR' (R8 is C18 alkyl or aryl such as phenyl), alkyl or alkoxy groups having 1 to 6 carbon atoms, halogens, viz. Cl, Br, F or 1, cyano, carboxy, sulfamino, carbamoyl, sulfonyl, azido, amino, substituted amino, such as (Ct~e alkyl)-substituted amino including quaternary ammonium, C16 halogenated alkyl such as trifluoromethyl, carboxy(C,~6 alkyl), carbamoyl(C16 aikyl), N-substituted carbamoyl(C,~, alkyl), amidino, guanidino, N-substituted guanidino or guanidino-(C,~8 alkyl). Representative examples of such acyl groups that might be mentioned are those in which R" is benzyl, p-hydroxybenzyl, 4amino-4-carboxybutyl, methyl, (in which case, when X is 0, the acyl radical is acetyl), cyanomethyl, 2-pentenyl, n-amyl, n-heptyl, ethyl, 3- or 4-nitrobenzyl, phenethyl, ss,ss-diphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, 3,5-dimethyl-4-isoxazolyl, 3butyl-5-methyl-4-isoxazolyl, 5-methyl-3-phenyl-4-isoxazolyl, 3-(2-chlorophenyl)-5methyl-4-isoxazolyl, 3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl, D-4-amino-4carboxybutyl, D-4-N-benzoylamino-4-carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, (3-pyridyl)methyl, 2-ethoxy- 1 - naphthyl, 3-carboxy-2-quinoxalinyl, 3-(2,6-dichlorophenyl)-5-(2-furyl)-4-isoxazolyl, 3-phenyl-4-isoxazolyl, 5-methyl-3-(4-guanidinophenyl)-4-isoxazolyl, 4-guanidinomethylphenyl, 4-guanidinomethylbenzyl, 4-guanidinobenzyl, 4-guanidinophenyl, 2,6-dimethoxy-4-guanidinophenyl, o-sulfobenzyl, p-carboxymethylbenzyl, pcarbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl, pmethoxybenzyl, I-naphthylmethyl, 3-isothiazolylmethyl, 4-isothiazolylmethyl, 5isothiazolylmethyl, guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4methoxy-5-isoxazolylmethyl, 4-methyl-5-isoxazolylmethyl, 1 -imidazolylmethyl, 2benzofuranylmethyl, 2-indolylmethyl, 2-phenylvinyl, 2-phenylethynyl, l-amino- cyclohexyl, 2- and 3-thienylaminomethyl, 2-(5-nitrofuranyl)vinyl, phenyl, omethoxyphenyl, o-chlorophenyl, o-phenylphenyl, p-aminomethylbenzyl, 1-(5cyanotriazolyl)methyl, difluoromethyl, dichloromethyl, dibromomethyl, 1-(3methylimidazolyl)methyl, (2 or 3)-(5-carboxymethylthienyl)methyl, (2 or 3)-(4carbamoylthienyl)methyl, (2 or 3)-(5-methylthienyl)methyl, (2 or 3)-(methoxythienyl)methyl, (2 or 3)-(4-chlorothienyl)-methyl, (2 or 3)-(5-sulfothienyl)methyl, (2 or 3)-(5-carboxythienyl)methyl, 3-( 1 ,2,5-thiadiazolyl)methyl, 3-(4-methoxy- 1,2,5thiadiazolyl)methyl, 2-furylmethyl, 2-(5-nitrofuryl)methyl, 3-furylmethyl, 2-thienylmethyl, 3-thienylmethyl, tetrazolylmethyl, benzamidinomethyl and cyclohexylamidinomethyl.

The acyl group can also be a radical of the formula:

where Xis O or S; n is0, 1, 2, 3 or 4; Z is oxygen, sulfur, carbonyl or imino; R" is as defined as above, except that when Z is oxygen R is not mercapto or hydroxy, when Z is sulfur R is not amino or hydroxy, and when Z is imino R is not mercapto; and R* is as defined below. Representative members of the substituent --(CH,),ZR" that might be mentioned are allylthiomethyl, phenylthiomethyl, butylthiomethyl, a-chlorocrotylthiomethyl, phenoxymethyl, phenoxyethyl, phenoxybutyl, phenoxybenzyl, phenoxyphenoxymethyl, (dimethylmethoxy)methyl, (dimethylbutoxy)methyl, (dimethylphenoxy)methyl, 4-guanidinophenoxymethyl, 4-pyridylthiomethyl, p-(carboxymethyl)phenoxymethyl, p-(carboxymethyl)phenylthiomethyl, 2 thiazolylthiomethyl, p-(sulfo)phenoxymethyl, 2-pyrimidinylthiomethyl, phenethylthiomethyl, 1-(5,6,7,8-tetrahydronaphthyl)oxymethyl, N-methyl-4-pyridylthio, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio, amino, methylamino, dimethylamino, a pyridinium methyl or trimethylammonium-methyl non-toxic salt, cyanomethylthiomethyl or trifluoromethylthiomethyl. R* is 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 3-imidazolylethyl, 3-imidazolylpropyl, 3-imidazolylbutyl, 1pyrrolylethyl, I-pyrrolylpropyl and l-pyrrolylbutyl. Alternatively, the acyl group can be a radical of the formula:

where R" is defined as above and R"' is azido, carbamoyl, guanidino, amidino, acyloxy, halo, namely Cl, F, Br, I, sulfamino, tetrazolyl, sulfo, carboxy, carbalkoxy, phosphono, alkoxy or arylthio. Representative members of the substituent

that might be mentioned are a-aminobenzyl, a-amino-(2-thienyl)methyl, a (methylamino)benzyl, a-amino-methylthiopropyl, a-amino-(3 or 4)-chlorobenzyl, a-amino-(3 or 4)-hydroxybenzyl, a-amino-2,4-dichlorobenzyl, a-amino- 3,4-di- chlorobenzyl, D(-)-&alpha;-hydroxybenzyl, &alpha;-carboxybenzyl, &alpha;-amino-(3-thienyl)- methyl, D(-)-&alpha;-amino-3-chloro-4-hydroxybenzyl, &alpha;-amino(cyclohexyl)methyl, &alpha;- (5-tetrazolyl)benzyl, 2-thienyl-carboxymethyl, 3-thienyl-carboxymethyl, 2-furyl carboxymethyl, 3-furyl-carboxymethyl, a-sulfaminobenzyl, 3-thienyl-sulfami no- methyl, -(N-methylsulfamino)benzyl, D(-)-2-thienyl-guanidinomethyl, D(-)-aguanidinobenzyl, &alpha;-guanylureidobenzyl, &alpha;-hydroxybenzyl, &alpha;-azidobenzyl, &alpha;- fluorobenzyl, 4-(5-methoxy-1,3-oxadiazolyl)-aminomethyl, 4-(5-methoxy-1,3-oxadiazolyl)-hydroxymethyl, 4-(5-methoxy-1,3,-sulfadiazolyl)-hydroxymethyl, 4-(5chlorothienyl)aminomethyl, 2-(5-chlorothienyl)-hydroxymethyl, 2-(5-chlorothienyl)carboxy-methyl, 3-(1,2-thiazolyl)-aminomethyl, 3-(1,2-thiazolyl)-hydroxymethyl, 3-(1,2-thiazolyl)carboxymethyl, 2-(1,4-thiazolyl)-aminomethyl, 2-(1,4-thiazolyl)-hydroxymethyl, 2-(1,4-thiazolyl)-carboxymethyl, 2-benzothienylaminomethyl, 2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl, &alpha;-sulfo- benzyl or a-phosphonobenzyl. The acyl radical can also be a-diethylphosphono or a-monoethylphosphono. Further acyl radicals of interest when X = oxygen are:

where R3' and R4' are as defined below. R3' represents hydrogen, chloro, fluoro, bromo, iodo, amino, guanidino, phosphono, hydroxy, tetrazolyl, carboxy, sulfo or sulfamino and R4' represents phenyl, substituted phenyl, a mono- or bicyclic heterocyclyl containing one or more oxygen, sulfur or nitrogen atoms in the ring, (such as furyl, quinoxalyl, thienyl, quinolyl, quinazolyl, thiazolyl, isothiazolyl, tetrazolyl, oxadiazolyl or thiadiazolyl), phenylthio, phenyloxy, alkyl of 1-6 carbon atoms, heterocyclic-thio or substituted heterocyclic-thio groups; or cyano. The substituents on the residues, R3' and R4,, can be halo, carboxymethyl, guanidino, guanidinomethyl, carboxyamidomethyl, aminoethyl, nitro, methoxy or methyl.

When R3' is hydrogen, hydroxy, amino or carboxy and R4' is phenyl or a (5 or 6)membered heterocyclic ring having one or two sulfur, oxygen or nitrogen hetero atom such as tetrazolyl, thienyl, furyl and phenyl, the following acyl radicals are representative: phenylacetyl, 3-bromophenylacetyl, p-aminomethylphenylacetyl, 4carboxymethylphenylacetyl, 4-carboxyamidomethylphenylacetyl, 2-furylacetyl, 5nitro-2-furylacetyl, 3-furylacetyl, 2-thienylacetyl, 5-chloro-2-thienylacetyl, 5methoxy-2-thienylacetyl, a-guanidino-2-thienylacetyl, 3-thienylacetyl, 2-(4-methylthienyl)acetyl, 3-isothiazolylacetyl, 4-methoxy-3-isothiazolylacetyl, 4-isothiazolylacetyl, 3-methyl-4-isothiazolylacetyl, 5-isothiazolylacetyl, 3-chloro-5-isothiazolylacetyl, 3-methyl- 1 ,2,5-oxadiazolylacetyl, 1 ,2,5-thiadiazolyl-4-acetyl, 3-methyl- 1,2,5thiadiazolylacetyl, 3-chloro-1,2,5-thiadiazolylacetyl, 3-methoxy-1,2,5-thiadiazolylacetyl, phenylthioacetyl, 4-pyridylthioacetyl, cyanoacetyl, 1-tetrazolylacetyl, &alpha;- fluorophenylacetyl, D-phenylgylcyl, 4-hydroxy-D-phenylglycyl, 2-thienylglycyl, 3thienylglycyl, phenylmalonyl, 3-chlorophenylmalonyl, 2-thienylmalonyl, 3-thienylmalonyl, &alpha;-phosphonophenylacetyl, &alpha;-amino cyclohexadienylacetyl, &alpha;-sulfamino- phenylacetyl, &alpha;-hydroxyphenylacetyl, &alpha;-tetrazolylphenylacetyl and &alpha;-sulfophenyl- acetyl.

The substituents R', R2 and if appropriate R3 may also be selected from sulphur (I) and phosphorus (2) radicals:

in which, with respect to (1), each of m and n isO or I and Y=0M&commat;, -N(R")2,-or R"; where M&commat; is hydrogen, an alkali metal cation, an alkaline-earth metal cation or an organic base; and R" is as defined above, e.g., alkyl, alkenyl, aryl or heteroaryl.

With respect to (2), X=0 or S; n=0 or 1; and each of Y' and Y", independently of the other, is 0eM&commat;, -N(R")2, -R" or -ZR" where all symbols are as defined above, e.g., R" and ZR" may be alkyl, alkenyl, aryl or heteroaryloxy; or Y' and Y", including Rt residues, are joined together to form cyclic ester, ester-amide and amide functions. Illustrative examples of compounds containing values of R' and/or R2 having the formula (1) are N-(methylsulfonyl)thienamycin, N-(o-nitrophenylsulfonyl)thienamycin, N-(p-chlorophenylsulfinyl)thienamycin, N-(o-nitrophenylsulfenyl)thienamycin, N-sulfamoylthienamycin, N-dimethylsulfamoylthienamycin and thienamycin N-sulfonic acid sodium salt. Illustrative examples of compounds containing values of R' and/or R2 having the formula (2) are N-(di methoxyphosphino)thienamycin, N-(dibenzyl-oxyphosphino)thienamycin, N-(dihydroxyphosphino)thienamycin disodium salt, N-(dimethoxyphosphinyl)thienamycin, N-(dimethoxyphosphinothioyl)thienamycin, N-(dibenzyloxyphosphinyl)thienamycin and N-(dihydroxyphosphinyl)thienamycin disodium salt.

Acyl groups R' and R2, of Formula II, of particular interest are conventionally known N-acyl blocking or protective groups such as carbobenzyloxy, ring- substituted carbobenzyloxy such as o- and p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy, chloroacetyl, bromocetyl, phenylacetyl, t-butoxycarbonyl, trifluoroacetyl, bromoethoxycarbonyl, 9-fluorenylmethoxycarbonyl, dichloroacetyl, 2,2,2-trichloroethoxycarbonyl, bromo-t-butoxycarbonyl, phenoxyacetyl. Non-acyl protective groups such as o-nitrophenylsulphenyl and tris,~ alkyl)silyl, for example, trimethylsilyl and t-butyldimethylsilyl, are also of interest.

The following radicals, according to the foregoing definition of acyl (radicals R1 and R2 of Formula II, above), are preferred: formyl, pro jonyl, butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, et oxycarbonyl, methylcarbamoyl, ethylcarbamoyl, phenylthiocarbonyl, 3-aminopropionyl, 4-aminobutyryl, N-methylaminoacetyl, N,N-dimethylamino-acetyl, an N,N,N-trimethylammoniumacetyl salt, 3-(N,N-dimethyl)aminopropionyl, a 3-(N,N,Ntrimethyl)ammonium propionyl salt, an N,N,N-triethylammoniumacetyl salt, a pyridiniumacetyl salt, guanylthioacetyl, guanidinoacetyl, 3-guanidinopropionyl, N3-methylguanidinopropionyl, hydroxyacetyl, 3-hydroxypropionyl, acryloyl, propynoyl, malonyl, phenoxycarbonyl, amidinoacetyl, acetamidinoacetyl, amidinopropionyl, acetamidinopropionyl, guanylureidoacetyl, guanylcarbamoyl, carboxymethylaminoacetyl, sulfoacetylaminoacetyl, phosphonoacetyl-aminoacetyl, N3-dimethylaminoacetamidinopropionyl, ureidocarbonyl, dimethylaminoguanylthioacetyl, a 3-( 1 -methyl-4-pyridinium)propionyl salt, 3-(5-aminoimidazol-1- yl)propionyl, 3-methyl-I -imidazoliumacetyl salt, 3-sydnonlyacetyl, o-aminomethylbenzoyl, o-aminobenzoyl,

where M is as defined above, and is in particular sodium.

Especially preferred acyl radicals (R' and R2 of Formula II, above) are terminally substituted acyls in which the substituent is one of certain basic groups.

Such preferred substituted acyls may be represented by the following formula:

a'nd these radicals and the corresponding radicals in which the

group is omitted are also preferred values of R . In the formula q is 0 or 1; each of m and n, independently of the other, is 0, 1, 2, 3, 4 or 5; A is 0, -NR - (where R is hydrogen or C1-5 alkyl) or S, and Y is an amino or substituted amino radical of formula: -N(R*)2 or -N(R*)3 ; an amidino or substituted amidino radical of formula:

a guanidino or substituted guanidino radical of formula:

or a guanyl or substituted guanyl radical of formula:

where each R, independently of the other, is hydrogen; N(R )2 (where R is hydrogen or C1-6 alkyl); C1-6 alkyl, C1-6 alkyloxyl; (C1-6 alkoxy)-(C-6 alkyl), C3-6 cycloalkyl or cycloalkyl-(C1-3 alkyl) or the two R groups are joined to form, together with the N atom to which they are attached, a ring having 3 to 6 atoms; R* is a radical as defined for R except that if cycloalkylalkyl, it must be (C36 cycloalkyl)-C,~3 alkyl; and R** is a radical as defined for R or a (C,~ alkoxy)methyl radical; or Y is a monocyclic or bicyclic heterocyclic aromatic or non-aromatic radical having 4 to 10 nuclear atoms and in which the hetero atom or atoms are nitrogen and optionally oxygen or sulfur.

Such heterocycles are representatively illustrated by the following list of radicals (R' is H or C16 alkyl):

The following specific carboxylic acyl radicals are additionally representative and are preferred:

where Q is an anion.

However, it is to be understood that any acyl radical may be used in the practice of the invention and is to be considered within the scope of the invention.

In general the compounds of the present invention are prepared by treating thienamycin (I) with an acylating agent, for example, an acyl halide or acyl anhydride such as an aliphatic, aromatic, heterocyclic, araliphatic or heterocyclic aliphatic carboxylic acid halide or anhydride. Other acylating agents may also be used, for example, mixed carboxylic acid anhydrides and particularly C16 alkyl esters of mixed carboxylic-carbonic anhydrides, i.e. compounds of formula R'-CO-O-CO-OR"; also carboxylic acids in the presence of a carbodiimide, such as 1,3-dicycylohexylcarbodiimide, and activated esters of a carboxylic acid such as a p-nitrophenyl ester.

The acylation reaction may be conducted at a temperature in the range -200C. to 100 C. but is preferably conducted at a temperature in the range of from -8 C. to 250C. Any solvent in which the reactants are soluble and substantially inert may be used, for example, polar solvents such as water, alcohols and polar organic solvents in general such as dimethylformamide (DMF), hexamethyl phosphoramide (HMPA), acetone, dioxane, tetrahydrofuran (THF), acetonitrile, heterocyclic amines such as pyridine, ethyl acetate, aqueous mixtures of the above, as well as halogenated solvents such as methylene chloride and chloroform. The reaction is conducted for a period of time of from five minutes to a maximum of three hours, but in general, a reaction time of 0.5 to one hour is sufficient. The following equation illustrates this process using a carboxylic acid halide; however, it is to be understood that by substituting a carboxylic acid anhydride or other functionally equivalent acylating agent similar products may be obtained.

OH OH 11SCH2CH2NH2 XSCH2CH2NR 0NCOOH acyt -C00'rl COSH acvl hG li de O w v Generally when the above-described acylating reaction uses an acid halide (suitable halides are chlorides, iodides and bromides) or anhydride the reaction is conducted in water or an aqueous mixture of a polar organic solvent such as acetone, dioxane, THF, DMF or acetonitrile in the presence of a suitable acceptor base such as NaHCO3, MgO, NaOH or K2HPO4.

In carrying out the reactions described herein, it is generally not necessary to protect the 2-carboxy group or the l'-hydroxy group; however, in cases where the acylating reagent is exceedingly water-sensitive it is sometimes advantageous to perform the acylation in a non-aqueous solvent system. Triorganosilyl (or tin) derivatives of thienamycin or its enantiomers are suitable for this purpose.

Silylation of thienamycin proceeds rapidly to give the tris-triorganosilyl derivative, for example tris-trimethylsilyl thienamycin, Th < TMS)3:

Such derivatives, which are readily soluble in organic solvents, are conveniently prepared by treating thienamycin with an excess of hexamethyldisilazane and a stoichiometric amount of trimethylchlorosilane at 250C. with vigorous stirring under a N2 atmosphere.

The products of this invention (II) form a wide variety of pharmacologically acceptable salts with inorganic and organic bases; these include, for example, metal salts made by using alkali metal or alkaline-earth metal hydroxides, carbonates or bicarbonates and salts derived from primary, secondary or tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, Cie alkanolamines, di-(C,~e alkanol)amines, C28 alkylenediamines, N,N-diaralkyl (C26 alkylene)diamines, aralkylamines, amino-substituted C18 alkanols, N,N-di (C", alkyl)amino-substituted C16 alkanols, amino-, polyamino- and guanidinosubstituted C16 alkanoic acids and nitrogen-containing heterocyclic amines.

Representative examples include salts made by using sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzyl amine, ethylenediamine. N,N'-dibenzylethylenediamine, diethanolamine, piperazine, ditnethylaminoethanol, 2-amino-2-methyl- I -propanol, theophylline or N-methylglucamine. Acid-addition salts, e.g., with hydrochloric, hydrobromic, sulfuric, nitric, toluene-p-sulphonic and methane sulphonic acids may also be used, in such instances where the acyl radical contains a basic group.

The salts can be mono-salts such as the monosodium salt, which can be obtained by treating one equivalent of sodium hydroxide with one equivalent of the product (II), also mixed di-salts. Such salts may be obtained by treating one equivalent of a base having a divalent cation, such as calcium hydroxide, with one equivalent of the product (II). The salts of this invention are pharmacologically acceptable non-toxic derivatives which can be used as the active ingredient in suitable unit-dosage pharmaceutical forms. Also, they may be combined with other drugs to provide compositions having a broad spectrum of activity.

Compounds of the present invention, including free acid and its salts such as metal salts, particularly the alkali metal and alkaline-earth metal salts, and salts with organic bases, particularly primary, secondary and tertiary amines, are useful bactericides and are valuable antibiotics active against various gram-positive and gram-negative bacteria and, accordingly, find utility in human and veterinary medicine. Compound, of this invention are active against, and can therefore be used as antibacterial drugs for treating infections caused by, gram-positive or gramnegative bacteria, for example against Stapkviococcus aureus. Escherichia coli., Strep. pyogenes. Klebsiella pneumoniae, Bacillus subtilis, Salmonella schottmuellerie, Sahnonella ttphosa, Pseudomonas, Proteus vulgaris and Bacterium proteus. For this latter purpose pharmacologically acceptable salts with inorganic and organic bases such as those known in the art and used for the administration of penicillins and cephalosporins can be utilized. These salts can be combined with pharmaceutically acceptable liquid and solid vehicles to form suitable dosage unit forms such as pills, tablets, capsules, suppositories, syrups and elixirs, which can be prepared in accordance with well known procedures.

The antibacterial compounds of the invention may further be utilized as additives to animal feedstuffs, for preserving food and as disinfectants. For example, they may be used in aqueous compositions in concentrations ranging from 0.1 to 100 parts of antibiotic per million parts of solution in order to destroy and inhibit the growth of harmful bacteria on medical and dental equipment and as bactericides in industrial applications, for example in water-based paints and in the white paper of paper mills to inhibit the growth of harmful bacteria.

The products of this invention may be used alone or in combination as an active ingredient in any one of a variety of pharmaceutical preparations. These antibiotics and their corresponding salts may be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. They may be administered orally, intravenously or intramuscularly.

The compositions are preferably presented in a form suitable for absorption by the gastro-intestinal tract. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example, lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine: lubricants, for example, magnesium stearate, talc, polyethylene glycol, silica; disintegrants, for example, potato starch or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to well known methods. Oral liquid preparations may be in the form of aqueous or oily suspension, solution, emulsions, syrups or elixirs, or may be presented as a dry product, for reconstitution with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible oils, for example almond oil, fractionated coconut oil, oily esters, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoates or sorbic acid. Suppositories will contain conventional suppository bases, e.g., cocoa butter or other glycerides.

Compositions for injection may be presented in unit dose form in ampules, or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compositions may also be prepared in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently take the form of powder, liquid sprays, inhalants, lozenges and throat paints. For medication of the eyes or ears, the preparations may be presented as individual capsules, in liquid or semi-solid form, or as drops. Topical applications may be formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints and powders.

Also, in addition to a carrier, the compositions may include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricators, suspending agents, viscosity agents or flavoring agents. In addition, there may also be included in the composition other active ingredients to provide a broader spectrum of antibiotic activity.

For veterinary medicine the composition may, for example, be fomulated as an intramammary preparation in either long acting or quick-release bases.

The dosage to be administered depends to a large extent upon the condition of the subject being treated and the weight of the host, the route and frequency of administration, the parenteral route being preferred for generalized infections and the oral route for intestinal infections. In general, a daily oral dosage consists of from 15 to 600 mg. of active ingredient per kg. of body weight of the subject in one or more applications per day. A preferred daily dosage for adult humans lies in the range of from about 80 to 120 mg. of active ingredient per kg. of body weight.

The compositions may be administered in several unit dosage forms, for example, in solid or liquid orally ingestible dosage form. The compositions per unit dosage, whether liquid or solid, may contain from 0.1% to 99% of active material, the preferred range being from 1060%. The composition will generally contain from 15 mg. to 1500 mg. of the active ingredient; however, in general a dosage in the range of from 250 mg. to 1000 mg. is preferred. In parenteral administration the unit dosage is usually the pure compound in a slightly acidified sterile aqueous solution or a soluble powder intended for solution.

In the following Examples, which further illustrate the present invention, the compounds of the present invention will be designated by the previously -introduced symbol:

in which the three functional groups are illustrated.

In the Examples, the worms "Dowex", "Nujol", "Teflon", "Celite", "Supercel", "Linde", "Branson", "Sonifer" and "Difco" are trade marks; "mmol" and "mmole" mean "millimole", the proportions of liquid mixtures are given on a volume basis, and screen and capsule sizes are U.S. standards.

EXAMPLE 1.

Preparation of N-(O-formyl)-D-mandeloylthienamycin Sodium Salt To thienamycin (40 mg.) in 10 ml water is added successively at OOC., 124 mg.

NaHCO, 8 ml. dioxane and then with stirring 1.2 equivalents of N-(O-formyl)-lmandelolyl chloride over a period of one minute. After six minutes total reaction time, the mixture is extracted three times with cold diethyl ether. Electrophoresis of an aqueous portion (0.05M, pH 7, aqueous phosphate buffer, 50 V/cm., 20 minutes) shows 67% conversion to desired product. The pH is adjusted to 2.2 with IM H3PO4 solution and the solution is extracted three times with ethyl acetate. The ethyl acetate (EtOAc) solution is dried over MgSO4 and extracted twice with two equivalents of NaHCO3 solution.

The aqueous extract, lyophilized, contains 164 optical density units (ODU), at 302 nm by uv analysis at pH 7.0, of which 95 Ó is extinguished after treatment with hydroxylamine for one hour. The yield is 53%. Electrophoresis as before shows one spot by bioautograph, 4 cm towards the anode. NMR (8 D2O) 1.30 (d, 5=6 Hz), CH3CH; 2.8-3.7 (m, CH2), 4.04.5 (m, CH p-lactam), 4.73, HDO, 5.97 (s, C3HsCHOCHO), 7.53 (s, C6H5), 8.30 (s, CssHsCHOCHO).

EXAMPLE 2.

Preparation of N-D-Mandeloylthienamycin Sodium Salt The title compound is made following the procedure of Example 1, but before EtOAc extraction the aqueous extract is allowed to stand at 250C. for one hour.

Electrophoresis (50 V/cm., 20 min., pH 7, aqueous phosphate, 0,05M) shows one spot by bioautograph, 4 cm. towards the anode. NMR (#. D2O) 1.50 (d, J=6 Hz, CH3CH); 2.8-3.8 (m, CH2), 4.2-4.6 (m, CH ss-lactam); 4.96 (s, HDO); 5.40 (s, OH C6H5CH).

EXAMPLE 3.

N-Propionylthienamycin Sodium Salt To thienamycin, (25 mg. in 6 ml. water at 0 C.) is successively added 38.6 mg.

NaHCO3, 5 ml dioxane and then with stirring one equivalent of propionic anhydride over a period of 3 min. After 10 min., the mixture is extracted three times with cold ethyl ether. Electrophoresis of the aqueous (aq.) extract (0.05M, pH 7, phosphate buffer, 50 V/cm., 20 min.) shows no free thienamycin present. The aq. extract is adjusted to pH 6.8 and contains 600 ODU at 302 nm by uv analysis which is 95% extinguished after treatment with hydroxylamine for one hour. NMR (#, D2O) 1.42 (m CH2CH3, CH3CH); 2.48 (q, CH2CH3); 2.86-2.90 (m, CH2), 4.30-4.70 (m, CH ss-lactam), 4.86 (HDO).

EXAMPLE 4.

N-(Methoxyacetyl)thienamycin Sodium Salt To thienamycin, (55 mg. in 6 ml. water at 0 C.), is added successively 68 mg.

NaHCO3, 6 ml dioxane and with stirring 1.1 equivalents of methoxyacetylchloride over a period of 1.5 minutes. The mixture is stirred for an additional 10 minutes.

The mixture is extracted three times with cold ethyl ether. Electrophoresis of aq. extract (0.05 M, pH 7 phosphate buffer, 50 V/cm., 20 min.) shows no free thienamycin present. The aq. extract, adjusted to pH 6.8, contains 105 ODU at 302 nm. by uv analysis, which is 95% extinguished after treatment with hydroxylamine for one hour. NMR (#, D2O) 1.56 (m, CHCH3), 2.84-3.60 (CH2), 3.72 (s, OCH3), 4.29 (s, OCH2), 4.98 (s HDO).

EXAMPLE 5.

N-(p-Nitrobenzyloxycarbonyl) thienamycin Lithium Salt To thienamycin (220 mg. in 60 ml. water at 0 C.), is added successively, 679 mg. NaHCO3, 60 ml. dioxane and then with stirring 1.1 equivalents of pnitrobenzylchloroformate over a period of 1.5 minutes. The mixture is allowed to react for 10 minutes, and is then extracted three times with cold ethyl ether.

Electrophoresis (0.05 M, pH 7, phosphate buffer, 50 V/cm., 20 minutes) shows that no free thienamycin is present. The aqueous extract is adjusted to pH 2.2 with IM H3PO4 solution and extracted three times with EtOAc. The EtOAc extract is dried over MgSO4, filtered and reextracted with 0.1 N LiOH, to pH 8.2. The final pH is adjusted to 7.0 with 1M H3PO4 and the sample lyophilized. The yield is 205 mg.

(54%).

EXAMPLE 6.

Preparation of N-(o-Nitrobenzyloxycarbonyl)thienamycin Sodium Salt To thienamycin (43 mg. at OOC.) is added 1:1 tetrahydrofuran-water (10 ml.).

The mixture is rapidly stirred while 132 mg. NaHCO3 (10 equiv.) is added, and then, dropwise with stirring over 2 min., four equivalents of o-nitrobenzylchloroformate is added. After thirty minutes, the pH is adjusted to 7 with 25% H3PO4 and the solution extracted three times with ether. The aqueous portion is evaporated in vacuo at 250C. temperature and is then brought to pH 2.2 at 0 C. Solid NaCI is added and then the cold acidic solution is extracted three times with cold EtOAc.

The EtOAc extracts are combined and quickly back-washed with cold brine, dried with anhydrous MgSO4, filtered and back-extracted with 10 ml. of water containing 1.7 equivalents of solid NaHCO,. The extract is lyophilized in vacuo at ambient temperature and contains 366 ODU at 303 nm by uv analysis in H2O at pH 7.0, which is 95% extinguished after treatment with hydroxylamine for 1.5 hr.

Electrophoresis (50 v/cm, 20 min., pEt 7 0.05M phosphate buffer) shows one spot by bioautograph (MB--108 staph. aureus), 3.7 cm towards the anode. IR (u, film) 5.62 (p-lactam); 5.78 broad, (urethane) NMR consistent with the structure.

EXAMPLE 7.

Preparation of N-(Trichloroethoxycarbonyl)thienamycin Lithium Salt

To thienamycin (40 mg. in 18 ml. 1:1 THF-H20 at 0 C.) is added while stirring 225 mg. (15.2 equiv.) NaHCO3, and then, dropwise with stirring over 2 min., 1.8 equivalents of trichloroethylchloroformate dissolved in 0.6 ml. THF. After 6 minutes the pH is adjusted to 7.2 with 25% H3PO4 and the solution is extracted with ether. The aqueous portion after removing any entrained ether in vacuo is then brought to pH 2.5 at OOC. and extracted with cold EtOAc. The ethyl acetate extracts are combined, quickly back-washed with cold brine, dried with anhydrous MgSO4, filtered and back-extracted with 0.01M LiOH to pH 6.8. The aqueous extract is freed from any EtOAc fn vacuo and lyophilized. The residual solid contains 936 ODU (39.7%) by uv analysis at 302 nm, which is 90% extinguished after treatment with hydroxylamine for one hour in 0.05M phosphate buffer (pH 7).

The yield is 32 mg. Electrophoresis (50 v/cm, 20 min., pH 7 0.05M phosphate) exhibits one zone by bioautograph (MB 108, staph. aureus), 2.4 cm toward the anode. Liquid chromatography on C18 Bondapak (Waters Assoc.) in aq. 10% THF exhibits one main peak free of any unreacted thienamycin.

EXAMPLE 8.

Preparation of N-Bromoacetylthienamycin To a cooled solution of thienamycin (28.8 mg) and sodium bicarbonate (0.3 g.) in 10 ml. of water and 8 ml. of dioxane is added with stirring a solution of 0.25 g. of bromoacetic anhydride in 2 ml. dioxane over a period of 20 min. The pH is maintained at 8-8.3. The mixture is stirred for an additional 10 minutes, layered with ether, and neutralized to pH 7 by the addition of 8% phosphoric acid. The ethereal layer is separated and the aqueous layer is extracted twice again with ether. The aqueous layer is evaporated under reduced pressure to 0.5 ml., diluted to 2 ml. with water and put on 50 ml. of XAD-2 resin. The column is eluted with water. The first 80 ml. is discarded, then the next 100 ml. is collected. The solvent is changed to 10% TIllF and an additional 100 ml. collected. The combined eluates are adjusted to pH 7, evaporated to 5 ml. under reduced pressure and freeze-dried to give the sodium salt of N-bromoacetylthienamycin in 60% yield. UVAmax 302 my.

EXAMPLE 9.

( T MS=tri methylsilyl ) Preparation of Silylated thienamycin Thienamycin (80.0 mg.) is suspended in 40 ml. tetrahydrofuran (THF) under a N2 atmosphere and is concentrated to 10 ml.; hexamethyldisilazane (1.0 ml.) and trimethylchlorosilane (300 ,ul) are added. The mixture is reacted for 20 mins. at 250C. with vigorous stirring. The suspension is then centrifuged to remove ammonium chloride. The supernatant is evaporated to an oil under a nitrogen stream for future reaction.

EXAMPLE 10.

Preparation of N-(o-nitrobenzylcarbonyl)thienamycin via Th(TMS)3 To Th(TMS)3 (24 mg.) in 0.8 ml. dry TIllF is added 23 mg. of onitrocarbobenzyloxy chloride followed by 0.015 ml. of triethylamine. After vibromixing for 30 minutes at 250C., the mixture is concentrated to dryness in a stream of dry N2. The pasty residue is washed three times with petroleum ether. The residue is stirred with a mixture of 1.5 ml. THF and 10 ml. of pH 7 phosphate buffer for 20 mins., then charged to a column (30 ml.) of Dowex 50 (Na+) resin. The column is eluted with water and the effluent monitored by UV absorption at 302 nm. The fractions containing the product are combined and freeze-dried.

EXAMPLE 11.

Preparation of N-(p-methoxybenzyloxycarbonyl)thienamycin Sodium Salt (I) and Lithium Salt (II) To thienamycin (20 mg. in 5 ml. water at 0 C.) is added 105 mg. NaHCO3 (20 equiv.), 5 ml dioxane, and then, dropwise with stirring over 1 min., ten equivalents of p-methoxybenzyl chloroformate. After 15 min., the pH is adjusted to 7.5 with IM H3PO4 and the solution extracted three times with ether. The aqueous portion is then adjusted to pH 2.2 at OOC. and extracted three times with EtOAc. The EtOAc is dried quickly with MgSO4, filtered, and extracted with a few ml. water containing 6.3 mg. NaHCO3. The first extract, lyophilized, contains 172 ODU at 303 nm by UV analysis in H2O at pH 7.0, which is 95% extinguished after treatment with hydroxylamine for one hour. The yield of I is 16 mg. Electrophoresis (50 v/cm, 20 min., pH 7 phosphate buffer) shows one spot by bioautograph, 4 cm towards the anode. NMR (#, D2O): 1.49 (d, J=6 Hz, CH3CH); 2.8-3.7 (m, CH2); 3.99 (s, OMe); 4.0-4.6 (m, ss-lactam CH); 4.92 (s, HDO); 5.20 (s, OCH2); 7.13 (d, J=8 Hz, C6H4).

Extracting the EtOAc solution with 0.1NLiOH to pH 7.8 and lyophilizing gives the Lithium Salt II. The spectral and electrophoretic properties of II are the same as those of I.

EXAMPLE 12.

Preparation of N-Azidoacetylthienamycin Sodium (I) and Lithium (II) Salts

Thienamycin (48 mg., 0.18 mmol) is dissolved in 10 ml. cold water and is kept at 0 C. To the solution is added sodium bicarbonate (147 mg., 17.6 mmol) and dioxane (10 ml.). Azidoacetyl chloride (60 mg., 0.05 mmol) is added to the solution during a period of 2 min. The reaction mixture is stirred for 15 min. and then is neutralized to pH 7.0 with 30% phosphoric acid and is transferred into a separatory funnel. The solution is extracted with 2x50 ml. ether. The aqueous layer is concentrated to 5 ml. and then is charged to a Dowex AG-50x8 (sodium form) ion-exchange column. The desired fractions, monitored by U.V., are combined and lyophilized to give 21 mg. of the product (I). Electrophoresis (50 V/cm., 20 min. in pH 7.0 phosphate buffer) of the product shows a single bio-active band which moves 4 cm. towards the anode.

UV#maxH2O 300 nm; Pmr (100 MHz, D2O): 1.26 (d, CH3CH), 2.92-3.43 (m, 3CH2 and C6-H), 4.01 (s, CH2N) and 4.20 ppm (m, C5-H and C7-H).

Thienamycin (76.2 mg., 0.28 mmol) is dissolved in 100 ml. of cold water and is kept at 0 C. To the solution is added 0.6 ml. of 1.0 N lithium hydroxide solution and 10 ml. dioxane. After stirring for 1 min., azidoacetyl chloride (33.6 mg., 0.28 mmol) is added during a period of 2 min. The reaction mixture is stirred for an additional one minute then is neutralized to pH 7.0 with 30% phosphoric acid. After extraction with.ether, the aqueous solution is concentrated to 5 ml. and is charged to the Dowex AG-50wx8 (lithium form) ion exchange column. The desired fractions, monitored by U.V., are combined and lyophilized to give 38 mg. of the product (II).

UV#maxH2O 300 nm.

EXAMPLE 13.

Preparation of N-Glycyl-thienamycin Platinum oxide (60 mg.) and 2 ml. water are placed in a hydrogenation flask and stirred under 1 atm. hydrogen at 25 C. for 20 minutes. To the flask is added Nazidoacetyl thienamycin sodium salt (6.0 mg. 0.02 mmol in 4 ml. water). The reaction mixture is stirred at 250C. under I atm. hydrogen for 30 minutes. The resulting mixture (pH 8.7) is adjusted to pH 7.0 with 30% phosphoric acid and filtered from the catalyst. The aqueous solution is concentrated to 2 ml. and then is charged to the Dowex AG-50wx8 (sodium form) ion exchange column. The desired fractions, monitored by U.V., are combined and lyophilized to give 3.8 mg. of the product. The electrophoresis (50 v/cm, 20 min.) of the product in pH 7.0 phosphate buffer shows one single bio-active band at the origin.

UV#maxH2O 300 nm; Pmr (100 MHz, D2O): # 1.27 (d, CH3CH), 2.96-3.37 (m, 3CH2 and C6-H), 3.70 (s, COCH2NH2), and 4.20 ppm (m, C5-H and C7-H).

EXAMPLE 14.

Preparation of N-Benzyloxycarbonylthienamycin A solution of 14 mg. of Thienamycin in 4 ml. of 0.05M pH 7 phosphate buffer and 2 ml. of dioxane in a 3-necked flask fitted with a stirrer, thermometer, pH electrode and the delivery tip of an automatic titrator is cooled to -80C. in a methanol-ice bath. The pH is brought to 8.2 by the addition of 0.2N sodium hydroxide in 50 aqueous dioxane and a solution of 0.015 ml. of carbobenzyloxy chloride in 2 ml. of dioxane is added. The mixture is stirred at -60C., pH 8.2, for ten minutes, then layered with ether and adjusted to pH 7 by the addition of hydrochloric acid. The layers are separated by centrifugation and the aqueous phase is extracted twice again with ether. The aqueous phase is layered with ethyl acetate and acidified to pH 2. The ethyl acetate is separated and the aqueous layer is extracted again with ethyl acetate. The combined ethyl acetate layer is washed with saturated sodium chloride solution, dried over magnesium sulfate and filtered.

The filtrate is stirred with water and the pH brought to 7 by the addition of dilute sodium bicarbonate solution. The aqueous phase is separated and freeze dried giving the sodium salt of N-benzyloxycarbonylthienamycin, 10 mg. (46%). The UV spectrum, imax 303 m,u, E ,/o 147 (E 6,290) indicates about 80% purity.

Electrophoresis at 50 V/cm for 20 min. at pH 7 followed by bioautograph on S. aureus gives a zone of inhibition at +2.5 cm.

EXAMPLE 15.

Preparation of N-(bromo-t-butoxycarbonyl)thienamycin STEP A Preparation of N-(bromo-t-butoxycarbonyl)-O-Trimethylsilylthienamycin Trimethylsilyl Ester

Th(TMS)3 (16 mg.) obtained as above is dissolved in 0.4 ml. of dry tetrahydrofuran to which is added 20 ,al (28 mg., 0.13 mmol) of bromo-t-butyl- chloroformate (b.p. 350/0.9 mm) and 8 ,ul (5.67 mg, 0.057 mmol) of triethylamine (redistilled from BaO). The mixture is shaken at 250C. for 20 min. Evaporation of solvent and excess of reagents gives desired product.

EtOAc UVAmax 320 nm (E 9,000).

STEP B Preparation of N-(bromo-t-butoxycarbonyl)thienamycin N-bromo-t-butoxycarbonyl-O-trimethylsilyl-thienamycin trimethylsilyl ester (3 mg.) is dissolved in 0.1 ml. of tetrahydrofuran to which is added 0.5 ml. of 0.1 M pH 7.0 phosphate buffer. The solution is left at 250C. for 20 minutes and is then passed down a column (5 ml.) of Dowex 50x8 (Na+ form) with the eluate monitored by U.V. The correct fractions are combined and freeze-dried to yield the desired product. UVAbmuaffXer 304 nm (E 9,000); electrophoresis at 50 V/cm for 20 min. in pH 7.0 buffer shows a single bioactive zone which moves 31.5 mm towards the anode.

EXAMPLE 16.

N-(Methylcarbamoyl)thienamycin O,N-Bistrimethylsilylthienamycin trimethyl silyl ester, [Th(TMS)3 prepared as in Example 91 from 66 mg (0.24 mmole) of thienamycin is dissolved in 5 ml. of dry tetrahydrofuran. The solution is stirred at 25"C. and 80 mg. (1.1 mmoles) of redistilled methyl isocyanate is added. After 1 hour the IR spectrum of the tetrahydrofuran solution shows bands at 6.0 y (-NH-CO-NH-) and 6.5 y (-NH-). The reaction solution is evaporated to dryness to remove the excess of methyl isocyanate. The residue is dissolved in 5 ml. of dry tetrahydrofuran and again evaporated to dryness. The residue is dissolved in 5 ml. of0.lNpH 7.0 sodium phosphate buffer and chromatographed on a column of 80 ml. of Dowex 50Wx8 (Na+) using deionized water as eluant. Fractions of 8 ml. are taken at a rate of 8 ml./5 min. and the column effluent is monitored by UV absorption and refractive index. The product is located in fractions 4 to 9 which are combined and concentrated to a volume of 12 ml. The concentrate is chromatographed on a column of 80 ml. of XAD-2 resin using deionized water as the eluant. Fractions of 8 ml. are taken every 5 min. and the column is monitored by UV absorption and refractive index. Portions (size based on UV absorption) of fractions 11, 15, 17, 20, 22, 25 and 30 are subjected to paper electrophoresis at 50 v/cm for 15 mins. using 0.5 M pH 7.0 phosphate buffer. The paper strips are evaluated using agar plates containing S. aureus. Zones characteristic of the starting material, thienamycin, .which show virtually no electrophoresis mobility at pH 7.0 are absent from all fractions tested whereas zones for N-(methylcarbamyl)thienamycin are observed in fractions 11, 15; 17, 20, 22, 25 and 30 at about 3.5 cm from the origin toward the anode. Fractions 14 to 35 are combined (158 ml.) and concentrated to a volume of about 20 ml. A sample of the 158 ml. of solution diluted 1:4 with water gives a UV max at 303 nm; A=1.38. The solution is freeze-dried to yield 29.8 mg. of the desired derivative. A Max 303 nm E%=285.

The nmr spectrum (D2O) of N-(methylcarbamoyl)thienamycin shows, in addition to resonances expected for the other functions, a singlet at a 2.88 for the CONHCH3 moiety.

EXAMPLE 17.

N-(a-Azidophenylacetyl)thienamycin A solution of 39 mg. (0.14 mmole) of thienamycin in 10 ml. of dioxane-water (1:1) is treated with 176 mg. (2.1 mmoles) of NaHCO3 and cooled to OOC: A l00-,vl portion of a solution of 295 mg. of a-azidophenylacetylchloride in dioxane (total volume 0.5 ml.) is added with rapid stirring. After 15 min., two additional 50by1 portions of the a-azidophenylacetyl chloride solution are added at 15 min. intervals. Fifteen minutes after the final addition of the acid chloride, the reaction mixture is extracted with two 5-ml. portions of ether; 15 ml. of ethyl acetate is added to the aqueous phase, which is rapidly stirred and acidified to pH 2.2-2.5 with phosphoric acid. The ethyl acetate layer is separated and the aqueous phase is washed with 5 ml. of ethyl acetate. The combined ethyl acetate solution is dried (MgSO4) and, after removal of the drying agent, 20 ml.of water is added. The N-(a- azidophenylacetyl)thienamycin is extracted into the aqueous layer by adding 103 mg. of NaHCO3 at 0 C. with stirring. The aqueous layer is separated and the pH is adjusted to 7.2 by adding small amounts of NaHCO3.

A 0.5 ml. sample is diluted with 2.5 ml. of D2O and freeze-dried for nmr spectral analysis. Resonances at a 5.0 and 7.48 are characteristic of the

and CeHs residues.

EXAMPLE 18.

N-(a-Aminophenylacetyl)thienamycin A water solution (6 ml.) of 6 mg. of N-(a-azidophenylacetyl)-Thienamycin is stirred with 60 mg. of 10% Pd/C catalyst at 250C. for 30 min. in an atmosphere of hydrogen. The reaction mixture is filtered through Supercel which is washed with nine l-ml portions of water. The filtrate and washings are combined (15 ml.) and a 4-1 sample is subjected to electrophoresis on paper in pH 7 buffer at 50 v/cm for 15 mm. The electrophoresis is evaluated by bioautograph on MB--108 agar plates and shows bioactive zones at +0.5 cm (dia. 32 mm) for N-(a-aminophenylacetyl) Thienamycin; +3.0 cm (dia. 16 mm) forN-(a-azidophenylacetyl)Thienamycin; and +4.5 cm (dia. 22 mm) for a by product.

EXAMPLE 19.

N-(Thiobenzoyl)thienamycin A solution of 55.8 mg. (0.2 mmole) of thienamycin in 18 ml. of dioxane-water (1:1) is treated with 263 mg. (3.1 mmoles) of NaHCO3 and cooled to OOC. Two 200 ,vl portions of a solution of 100 mg. of thiobenzoyl chloride in 0.6 ml. of dry dioxane are added to the rapidly stirred reaction solution at 15 min. intervals. Each portion of acid chloride solution contains 0.2 mmole of thiobenzoyl chloride. Fifteen minutes after the second addition, the reaction solution is washed with two 8-ml. portions of ether. Ethyl acetate (8 ml.) is added to the aqueous phase, which is adjusted to pH 2.2 at OOC. with rapid stirring using 20% H3PO4. The layers are separated and the aqueous layer is washed with 3 ml. of ethyl acetate. The combined ethyl acetate layers are dried (MgSO4). After separation of the drying agent, 10 ml. of water is added to the ethyl acetate solution and the product is extracted into the aqueous phase by adding 50 mg. (0.62 mmole) of NaHCO3 with stirring at OOC. (pH 7.4). The layers are separated and an aqueous phase containing N-(thiobenzoyl)thienamycin sodium salt is obtained and freeze dried. Nmr. in D2O: a 7.3-7.9 characteristic of the

S C8H5O-moiety.

EXAMPLE 20.

N-(Chloroacetyl)thienamycin A solution of 94 mg. (0.34 mmole) of thienamycin in 15.75 ml. of water and 6.75 ml. of distilled tetrahydrofuran is treated with 420 mg. (5.2 mmoles) of NaHCO3 and cooled to OOC. Three 100-y1 portions of a solution of 390 mg. of chloroacetyl chloride in 1 ml. of dry tetrahydrofuran are added to the reaction solution at 15-min. intervals. Each portion of acid chloride solution contains 0.34 mmole of chloroacetyl chloride. Fifteen minutes after the third addition, the reaction solution is washed with three 10-ml portions of ether. The aqueous layer is concentrated to a volume of about 10 ml. to remove last traces of organic solvents.

The residual solution is chromatographed on 75 ml. of XAD-2 resin using water as the eluant at a rate of 8 ml./5 min. Electrophoresis of sample from selected fractions followed by bioautography indicates that product is present in fractions 16 to 47. These same fractions contain in addition variable amounts of unreacted Thienamycin. Fractions 16 to 47 are combined (250 ml.) and concentrated to a volume of 20 ml. The concentrate is chromatographed on 150 ml. of Ag 50x2 (Na+) resin using deionized water as eluant at a rate of 8 ml./4 min. Samples of selected fractions are subjected to paper electrophoresis followed by bioautography against S. aureus MB 108 organisms. Product, uncontaminated by starting Thienamycin, is located in fractions 9 to 12. These fractions are combined to give 37 ml. of an aqueous solution of N-(chloroacetyl)-thienamycin.

A 2 ml. sample is concentrated to 1 ml., diluted with 5 ml. of D2O and freezedried for determination of the nmr spectrum. A resonance at 4.22 a is characteristic of the CICH2CO residue.

EXAMPLE 21.

N-(Methylthiocarbamoyl)thienamycin O,N-Bistrimethylsilylthienamycin trimethylsilyl ester, [TH(TMS)3 prepared as in Example 9] from 66 mg. (0.28 mmole) of thienamycin is dissolved in 5 ml. of dry tetrahydrofuran and to the solution is added 30 mg. of bis-trimethylsilyt trifluoroacetamide followed by 50 mg. (0.68 mmoles) of methylisothiocyanate.

Samples (10 ,us) of the reaction solution are added to pH 7.0 phosphate buffer (90 y1, 0.1N) at intervals for evaluation by electrophoresis (at 50 v/cm for 15 min. at pH 7 phosphate buffer and the strips are evaluated by bioautography using S. aureus agar plates).

The zones observed at 4.2 cm. toward the anode are characteristic of N (methylthiocarbamoyl)thienamycin

EXAMPLE 22.

Preparation of N-Trimethylammoniumacetylthienamycin

N-Bromoacetylthienamycin Sodium salt (20 mg.) is dissolved in water (2 ml.) containing trimethylamine hydrochloride. Dipotassium hydrogen phosphate is added to bring the solution to pH 8 and the mixture is kept at 40C. for 20 hours.

The mixture is evaporated under vacuum to a semisolid. The residue is redissolved in 10 ml. of water and the pH is readjusted to 8 by the addition of sodium hydroxide solution. The process of evaporation, which serves to remove excess of trimethylamine, is repeated. The solution is applied to a column containing 25 ml. of a sulfonated polystyrene resin (Dowex 50) in the sodium cycle and the column is eluted with water. The first 20 ml. of eluant is discarded and the next 100 ml. is concentrated and freeze dried, yielding 5 mg. of a light powder consisting of Ntrimethylammoniumthienamycin. UVAmrX 30.1 nm (E% 117). Electrophoresis at 50 v/cm, pH 7, for 20 minutes, followed by bioautography, shows a major zone of inhibition moving 1 cm. toward the cathode.

EXAMPLE 23.

Preparation of N-(Sodiumthiosulfatoacetyl)thienamycin

A solution of sodium N-bromoacetylthienamycin (84 mg.) in 20 ml. of water is treated with 20 ml. of 0.1N sodium thiosulfate and the mixture is allowed to stand at 4"C. for 18 hours. The solution is concentrated to 10 ml. and applied to a column (230 ml.) of XAD-2 resin. The column is eluted with water and the fractions are monitored by U.V. absorbance and refractive index. The correct fractions are combined and lyophilized to give the desired product: (75 mg. U.V. ;tmax 302 nm E% sus).

Electrophoresis of a sample (at 50 V/cm., pH 7, 20 minutes) shows a bioactive zone which moves 9 cm. toward the anode.

EXAMPLE 24.

Preparation of N-o-nitrobenzenesulfenylthienamycin

To a cooled solution of thienamycin (2.4 mg.) in 1.5 ml. of 0.05M pH 7 phosphate buffer and 0.75 ml. of dioxane is added to a solution of onitrobenzenesulfenyl chloride (10 mg.) in 0.75 ml. dioxane. The pH is maintained at 8.2 by the addition of 0.1 N sodium hydroxide. After 10 mins. the solution is adjusted to pH 7 with dilute hydrochloric acid and is extracted 3 times with ether.

The aqueous solution is freeze dried to yield the sodium salt of Northonitrobenzenesulfenylthienamycin. Electrophoresis at 50 v/cm, pH 7 for 20 minutes followed by bioautography gives a single zone of inhibition at 2.5 cm toward the anode.

EXAMPLE 25.

Preparation of p-Bromobenzoylthienamycin Thienamycin (45.6 mg.) is dissolved in a mixture of 0.05M pH 7 phosphate buffer (4 ml.) and dioxane (2 ml.) and the solution is brought to pH 8.2 with 0.2N NaOH in 1:1 dioxane water. The solution is cooled to -80C. and p-bromobenzoyl chloride (45 mg.) in dioxane (2 ml.) is added during 3 min. The pH is maintained between 7.6 and 8.4 by the addition of 0.2N NaOH for an additional 10 mins. The solution is neutralized to pH 7 and extracted 3 times with 8-ml. portions of ether.

The aqueous phase is freeze dried to provide the sodium salt of N-pbromobenzoylthienamycin, UVAm x 303 nm, 866 ODU. Electrophoresis at 50 v/cm, pH 7, for 20 mins, followed by bioautography shows a 22-mm. diameter zone of inhibition at -0.5cm corresponding to Thienamycin and a 41-mm. diameter zone at +2.5 cm corresponding to the title compound.

EXAMPLE 26.

Preparation of N-p-Guanidinophenylacetyl Thienamycin

Thienamycin (60 mg.) is dissolved in pH 7.0 phosphate buffer (0.05N, 6 ml.) and thoroughly cooled in an ice bath. The solution is then adjusted to qH 8.3 using a dispensing autoburette with 1.ON NaOH. To the stirred solution is added pguanidinophenylacetyl chloride hydrochloride (64 mg., 259 mol) in several portions so as to allow the autoburette to maintain a pH near 8.3. After a few minutes the reaction solution is acidified (10% H3PO4 solution) to pH 7.0 and chromatographed on 60 g. XAD-2 resin. The column Is eluted with water followed by 10% aqueous tetrahydrofuran solution, which elutes the product. The product fractions are combined and freeze-dried to give a white fluffy solid product (45 mg., Amax. 301 nm (E 7, 020)). The product has an electrophoretic mobility of 2.5 cm towards the anode at 50V/cm for 25 min. The nmr is consistent with that expected for product. The ir (Nujol mull) shows a band at 1775 cm-' (p-lactam).

EXAMPLE 27.

Preparation of N-Pyridiniumacetyl Thienamycin A mixture of 10 mg. of N-bromoacetyl thienamycin sodium salt and 0.5 ml. of pyridine in 1 ml. of HMPA is stirred for four hours at 250C. The excess of pyridine is removed under reduced pressure and the residue is taken up in 3 ml. of water and applied to a column of 20 ml. of XAD-2 resin. After elution with water the product is eluted with 10% aqueous THF. The solution has an optical density of 93 at A max 261 nm and 71 at A max. 297 nm. Electrophoresis and bioautography shows a major zone of inhibition at cm corresponding to the desired product, Npyridiniumacetyl Thienamycin.

EXAMPLE 28.

Preparation of N-Methoxycarbonyl Thienamycin Thienamycin (49 mg.) is dissolved in 0.05M pH 7.0 phosphate buffer (14 ml.) and cooled in an ice bath. With stirring the pH is adjusted to 8.2 using an automatic burette. A solution of methyl chloroformate (46 ,ul, 600,umol) in p-dioxane (580 L1) is added at once to give a homogenous solution. Subsequently, the pH is maintained at 8.2 using the automatic burette. After 10 min., the solution is adjusted to pH 7.0 using dilute phosphoric acid solution and washed three times with an equal volume of diethyl ether. The aqueous solution is then concentrated to 4.5 ml. and chromatographed on an XAD-2 resin column. The product is eluted (after water elution) with an aqueous 5% tetrahydrofuran solution and is freezedried to give 28.9 mg. of product. UV (pH 7.0 phosphate buffer 0.in) A max 303 nm (E 6,450) Electrophoresis (20 min., 0. IN pH 7.0 phosphate buffer 50 v/cm) mobility 3.5 cm to cathode.

EXAMPLE 29.

Preparation of N-Benzenesulfonyl Thienamycin

Thienamycin (52 mg.) is dissolved in pM 7.01N phosphate buffer (25 ml.) and magnetically stirred in an ice bath. The pH is adjusted to 8.2 with 2.5NNaOH using an automatic dispensing burette and benzenesulfonyl chloride (227,us, 226 jumol) in 500 ml. pdioxane) added at once. The pH is maintained at 8.2 (using the automatic burette) for 30 min. and then adjusted to pH 7.0 using dilute aqueous phosphoric acid. The reaction solution is concentrated to 15 ml. and chromatographed on XAD-2 resin (50 cc). The column is eluted with water, then 10% aqueous tetrahydrofuran, which elutes product. The 10% aqueous tetrahydrofuran eluate is concentrated to 1/3 volume and freeze-dried to give 28 mg of product.

Electrophoretic mobility of the product (50 v/cm, 20 min., pE 7 0.05N phosphate buffer) is 3.5 cm towards the cathode. A max 303 (E 3,650) in pH 7 0. IN phosphate buffer).

EXAMPLE 30.

Preparation of N-Guanylthioacetyl Thienamycin

A solution of 90 mg. of sodium N-bromoacetyl Thienamycin in 40 ml. of water is adjusted to pH 6 and 36 mg. of thiourea is added. The solution is kept at 40C. for five days. The solution is chromatographed on 80 ml. of XAD-2 resin. Elution with 400 ml. water removes thiourea, a small amount of the starting bromoacetyl derivative and Thienamycin. Elution with 120 ml. of 10% tetrahydrofuran gives a solution containing the N-guanylthioacetyl derivative of Thienamycin. High pressure liquid chromatographic analysis on Bondapak C18 Porasil with 10% THF solvent shows a major peak of > 90% purity. The solution is evaporated to 15 ml.

(pH 5.5) and freeze-dried. Yield, 40 mg. of white powder. U.V. A max 299 E% 173.

EXAMPLE 31.

Preparation of Thienamycin N-(S-Phenylcarbothioate) Sodium Salt

To thienamycin (37 mg., 0.14 mmole) in 24 ml. 1:1 dioxane-water at 0 C., is added while stirring 150 mg. (1.78 mmoles, 12 eq) of NaHCO3, and then, dropwise with stirring over 4 min., 1.5 equivalents (39 mg., 0.22 mmole) of phenyl chlorothioformate in 0.6 ml. dioxane. After 15 min. the pH is adjusted to 5.9 with aqueous 25% H3PO4 and the solution extracted with ether. The aqueous layer after removing any entrained ether in vacuo is then brought to pH 2.5 at OOC. and extracted with EtOAc. The ethyl acetate extracts are combined and quickly backwashed with cold brine, dried over MgSO4, filtered and back-extracted with water containing 1.5 equivalents of NaOH to a final pH of 6.8. The aqueous layer is freed from any EtOAc in vacuo and lyophilized. The aqueous solution contains 486 ODU at 302 nm (UV analysis in H2O at pH 7.0), which is 95% extinguished after treatment with hydroxylamine for one hour. The yield is 17.7 mg. (29%).

Electrophoresis (50 v/cm for 18 min., pH 7.0 aq phosphate, 0.05M) shows one spot by bioautograph, 3.6 cm toward the anode.

EXAMPLE 32.

Preparation of Thienamycin N-(O-Phenylcarbothioate) Sodium Salt To Thienamycin (46.5 mg., 0.17 mmole) in 26 ml. 1:1 dioxane-H2O at OOC., is added while stirring 220 mg. (2.62 mmoles, 15 equiv.) of NaHCO3, and then dropwise over 4 min., 1.5 equivalents of phenoxythiocarbonyl chloride in 0.6 ml. dioxane. After 8 min. the pH is adjusted to 7.0 and then the solution extracted with ether. The aqueous layer, after removing any entrained ether in vacuo, is then brought to pH 2.5 at OOC. and extracted with EtOAc. The ethyl acetate extracts are combined, quickly backwashed with cold brine, dried over MgSO4, filtered and back-extracted with water containing 1.5 eq. of 0.1M NaOH to a final pH of 6.8.

The aqueous layer is freed from any EtOAc in vacuo and lyophilized. The aqueous solution contains 572 ODU at 302 nm (UV analysis in H2O at pH 7.0) which is 95% extinguished after treatment with hydroxylamine for 1 hour. The yield is 19 mg.

(26% of theory). Electrophoresis (50 v/cm for 18 min., pH 7 aq phosphate, 0.05M) shows one spot by bioautography, 3.3 cm toward the anode.

EXAMPLE 33.

Preparation of N-(Dimethoxyphosphinothioyl)Thienamycin Sodium Salt

To the trimethylsilyl derivative, Th(TMS)3, prepared from 214 mg. of Thienamycin in 25 ml dry THF is added 227 mg dimethylphosphorochloridothionate in 2 ml TIllF followed by 0.18 ml (130 mg) of triethylamine. After stirring under dry nitrogen for approximately 2 hours, the contents are concentrated to a viscous residue in vacuo. The pasty residue is suspended in 40 ml ethylacetate containing 5 ml deionized water and the pH brought to 3.5 with dilute aqueous phosphoric acid and stirred for 10 minutes. The aqueous layer is separated and extracted with 2x20 ml ethylacetate (EtOAc). The EtOAc extracts are combined, quickly dried with anhydrous MgSO4 and filtered. To the cold EtOAc extract is added 20 ml of water and the pH is brought to 7 with dilute NaOH. The aqueous layer is separated and concentrated in vacuo to a smaller volume (7 ml) and placed on a 35x4 cm column containing 500 ml of an XAD-2 resin. The column is eluted with H2O followed by 6% aqueous TIllF and finally 10% aqueous THF. The fraction eluted with 10% TIllF (tubes 99-150,14 tube) has a Amax 304 nm and contains the desired product. Electrophoresis (50 v/cm, 16 min. pH 7 aq. phosphate, 0.05M) exhibits one zone only by bioautograph (MB--108 Staph. aureus) 3 cm towards the anode. Lyophilization gives 39 mg (10.5% yield) of N (dimethoxyphosphinothioyl)thienamycin Sodium Salt.

NMR (D2O) 4.0 (d, J=10 Hz.P-OCH3) EXAMPLE 34.

Preparation of N-(Dimethoxyphosphinyl)Thienamycin Sodium Salt

To the trimethylsilyl derivative Th(TMS)3 prepared from 93 tng of thiena mycin in 2 ml dry THF is added 88 mg dimethylphosphorochloridate in two portions followed by 45 g (0.06 ml) of triethylamine. After stirring vigorously for 1 hour the contents are concentrated to a viscous residue in vacuo. The residue is suspended in 10 ml EtOAc, 3 ml of pH 3 phosphate buffer is added and the mixture is stirred for 10 min. The aqueous layer is separated and extracted with 2x3 ml EtOAc. The EtOAc extracts are combined, dried with anhydrous MgSO4 and filtered. To the cold EtOAc extract is added 6 ml H2O and the pH is brought to 7 with dilute NaOH. The aqueous layer is separated and concentrated in vacuo to provide 18 mg (13% yield) of N-(dimethoxyphosphinoyl)thienamycin sodium salt; AmaX 303 nm; electrophoresis (50 v/cm, 20 min pH 7 aq. phosphate 0.05M) exhibits one zone 3.2 cm towards the anode (bioautograph MB-108 staph. aureus.) NMR (a, D2O)4.0 (d 5=10 Hz POCH3).

EXAMPLE 35 Preparation of N-(Bis(methylthio)phosphinothioyl) thienamycin Sodium Salt

To the trimethylsilyl derivative Th(TMS)3 prepared from 213 mg of thienamycin in 20 ml of dry THF is added 300 mg of dimethylphosphordchloridotri- thioate in 2 ml of TIllF followed by 0.18 ml (130 mg) of triethylamine. After stirring under dry nitrogen for 3 hours, the contents are concentrated to a viscous residue in vacuo. The pasty residue is suspended in 25 ml EtOAc containing 5 ml of H2O and the pH brought to 3.5 with dilute aqueous phosphoric acid and stirred for 10 minutes. The aqueous layer is separated and extracted with 2x 10 ml EtOAc. The EtOAc extracts are combined, dried with anhydrous MgSO4 and filtered. To the cold EtOAc extract is added 5 ml of H2O and the pH is brought to 7 with dilute NaOH. The aqueous layer is separated and concentrated in vacuo to a smaller volume (7 ml) and placed on a 30x3 cm column containing 150 ml of XAD-2- resin. The column is washed with H2O followed by aq. 9% THF which removes the product N-(Bis(methylthio)phosphinothioyl)thienamycin sodium salt (max 306 nm) which is then lyophilized to give 69 mg of a yellow-light orange solid (19.6%).

Electrophoresis (50 V/cm, 20 min. pH 7 aq phosphate, 0.05M) exhibits one zone 3 cm towards the anode (bioautograph My~108 Staph aureus).

EXAMPLE 36.

Preparation of N-Sulfamoyl thienamycin Sodium Salt

To the trimethylsilyl derivative Th(TMS)3, prepared from 210 mg of thienamycin in 20 ml THF, is added 223 mg of sulfamoyl chloride in 5 ml of TIllF followed by 0.25 ml (180 mg) of triethylamine. After stirring under dry nitrogen for 2 hours, the mixture is concentrated to a viscous residue, in vacuo. The pasty residue is suspended in 50 ml EtOAc containing 5 ml H2O the pH brought to 3.5 and the mixture is stirred for 10 min. The aqueous layer is separated and extracted with l-x10 ml EtOAc. The EtOAc extracts are combined dried with anhydrous MgSO4 and filtered. To the cold EtOAc extract is added 18 ml H2O and the pH adjusted to 7.0 with dilute NaOH. The aqueous layer is separated and concentrated in vacuo to a smaller volume and placed on a 33x4 cm column containing 500 ml XAD-2 resin. The column is eluted with H2O to provide N-Sulfamoyl thienamycin sodium salt. #max 303 nm Electrophoresis (50 V/cm 20 min pH 7 aq phosphate buffer 0.05M) exhibits one zone 3.2 cm toward the anode (bioautography MB 108, Staph aureus). Lyophilization of the above solution gives 90 mg (41%) of N-sulfamoyl thienamycin sodium salt as a yellow solid. IR Nujol A- lactam C=0 1750 shoulder 1770 (5.71+5.65 ) and 1150 cm-' (8.65 ) for HNSO2NH2.

EXAMPLE 37.

Preparation of N- [P-Azidopropionyl] Thienamycin

Thienamycin (184 mg) is dissolved in 30 ml of water and is kept at OOC. To the solution is added 0.52 g of NaHCO3, 30 ml of dioxane and 163 mg of p- azidopropionyl chloride. The mixture is stirred for 15 minutes and then neutralized with 30% H3PO4 and extracted with ether. The aqueous layer is separated and concentrated to 5 ml. The crude product is chromatographed on a Dowex 50Wx8 (Na form) ion-exchange column (I"x 10"). The column is eluted with H2O to give 81 mg of the desired product which shows uv absorption at HO 306 nm.

Electrophoresis of the product at 2 KV in 0.1MpH 7.0 phosphate buffer for 2 hours shows a single bioactive zone which moves 30 mm towards the anode.

EXAMPLE 38.

Preparation of N-(p-Aianyl)Th ie namycin

An aqueous solution of N ( -azidopropionyl)Thienamycin (40 mg in 20 ml water) is hydrogenated under 1 atm of hydrogen in the presence of 200 mg of palladium at 25 C., for 40 minutes. The resultant solution (pH 9.0) is neutralized with 30% H3PO4 and filtered from the catalyst. The mixture is chromatographed on a Dowex 50Wx8 (Na form) ion-exchange column (I"x10") and the column is eluted with water to give 20 mg of the desired product which shows uv #maxH2O 302 nm.

Electrophoresis of the product at 2 KV in 0.1M pH 7.0 phosphate buffer for 20 mins shows a single bio-active zone which moves 10 mm towards the cathode.

EXAMPLE 39.

Preparation of N-[N'-Acetimidoyl- -Alanyl]Thienamycin

An aqueous solution of N-(A-alanyl)thienamycin (125 mg in 15 ml water) is kept at 0 C and maintained at pH 8.5 by adding 2.5N NaOH while 0ethylacetimidate hydrochloride (350 mg) is added portionwise to the solution during a period of 10 min. The mixture is stirred for 1 hour then is neutralized with 2.5N HCl and concetrated to 15 ml. The crude product so obtained is chromatographed twice on Dowex 50Wx8 (Na form) column (1"x10") to yield 25 mg. The product is eluted with water and the solution lyophilized. Recrystallization of the product from water gives a crystalline solid which shows ir (Nujol mull): 1769 cm-1 ( -lactam); nmr (H2OD2O, 100 MHz): 2.20 ppm (s, acetimidoyl CH3); uv #maxH2O 320 nm. Electrophoresis of rheproduct at 2 KV in 0.1M pH 7.0 phosphate bufferfor 20 min shows a single bio-active zone which moves 10 mm towards the cathode.

EXAMPLE 40.

Following the procedures described in Examples 37, 38 and 39 except that: 1.) O-ethyl acetimidate hydrochloride is replaced by an equivalent amount of O-methylformimidate hydrochloride in Example 39; 2.) p-azidopropionylchloride is replaced by an equivalent amount of azidoacetyichloride in Example 37; 3.) N-(,-azidopropionyl)thienamycin is replaced by an equivalent amount of N-(azidoacetyl)thienamycin in Example 38; 4.) N-(A-alanyl)thienamycin is replaced with an equivalent amount of N glycyl thienamycin in Example 39; 5.) O-ethyl acetimidate hydrochloride is replaced with an equivalent amount of O-methyl formimidate hydrochloride and N-(c-alanyl) thienamycin is replaced with N-glycyl thienamycin in Example 39; 6.) O-ethyl acetimidate hydrochloride is replaced with an equivalent amount of methyl sulfate in Example 39.

7.) O-ethyl acetimidate hydrochloride is replaced with an equivalent amount of methyl sulfate and N-(fi-alanyl)thienamycin is replaced with N-glycyl thienamycin in Example 39; and 8.) N-(p-azidopropionyl)thienamycin is replaced with an equivalent amount of N-(2-azidoethoxycarbonyl)thienamycin in Examples 38; There are obtained the compounds enumerated in the table below:

Compound m n A Y NH 1.) 1 1 - -NH2C-Ill 2.) t 0 - -N3 3.) 1 O -NH, -NH3 NH o ii 4.) 1 O - -NH2-C-CH3 NH 53 1 O -NH,-C-H 0 0 7.) 1 0 --N(CH, - -N(CH3)3 0 8.) l 2 Oxygen - NH3 EXAMPLE 41.

Preparation of N-phenoxyacetyl thienamycin Sodium Salt To a 250-ml flask containing thienamycin (190 mg) is added 30 ml of water, 0.6 g of sodium bicarbonate and 30 ml of dioxane. While the mixture is stirred and kept at 00C., phenoxyacetyl chloride (170 mg) is added dropwise to the flask during a period of 10 minutes. The solution is stirred for an additional 10 minutes and then acidified with 30% phosphoric acid to pH 4.5. The acidified solution is quickly extracted with 50 ml of ether to remove excess of reagent and its hydrolysed product phenoxyacetic acid. The aqueous layer so obtained is further acidified with 30% phosphoric acid to pH 2.0, and extracted with 50 ml of ethyl acetate. The organic layer, which contains the free acid form of N-phenoxyacetyl thienamycin, is separated and back-extracted with 30 ml of aqueous solution containing 60 mg of sodium bicarbonate. The aqueous layer is freeze-dried to yield 120 mg of Nphenoxyacetyl thienamycin sodium salt. Electrophoresis (0.5M, pH 7.0, phosphatebuffer, 2KV for 20 min): single bioactive zone which moves 45 mm toward anode.

H20 UV: AmaX 302 nm.

EXAMPLE 42.

N-p-Guanidinopropionyl thienamycin STEP A: A-Guanidinopropionyl chloride hydrochloride p-guanidinopropionic acid (130 mg) is added in portions to I ml of thionyl chloride in a centrifuged tube under nitrogen. The pasty reaction mixture is rubbed with a glass rod until it becomes crystalline. The excess of thionyl chloride is evaporated in a stream of nitrogen leaving a solid residue of p-guanidinopropionyl chloride hydrochloride. I.R. 5.6 ,u

broad band 64.3 L (guanidino).

STEP B: Thienamycin (54 mg) is suspended in I ml of anhydrous dimethylformamide.

The mixture is cooled to 0 and p-guanidinopropionyl chloride hydrochloride (37 mg) is added with stirring, immediately followed by a solution of 25.8 mg of diisopropylethylamine in 0.1 ml of DMF. After an additional 5 minutes the product is precipitated with ether. The ether is decanted and the residue is dissolved in 2 ml of water, adjusted to pH 6.8 with sodium bicarbonate and chromatographed on 75 ml of XAD-2 resin. After elution with water the product is eluted with 10% aqueous THF, concentrated and freeze-dried. UV ,,x 303 m,u E% 157.

Electrophoresis (1 hr, 50 V/cm, pH 7) shows a single bioactive spot which moves 4.5 cm towards the cathode and gives a positive Sakaguchi color test.

EXAMPLE 43.

Preparation of N-Guanylcarbamoyl Thienamycin Thienamycin (253 mg) is dissolved in 0.1N pH 7 phosphate buffer (11 ml), cooled in an ice bath, and adjusted to pH 8.5 using IN NaOH dispensed from an autoburette. To this solution is added a cold solution (2.5 ml) of l-amidino-semicarbazide dihydrochloride (354 mg) and sodium nitrite (128 mg). The pH drifts to 9.0 and after 10 minutes the pH is adjusted to and maintained at 8.2. After 30 minutes, a precipitate forms and the pH is adjusted to 7.0 using IN HCI. The mixture is filtered and the filtrate chromatographed on a column of XAD-2 resin (120 ml.), which is eluted with water, followed by aqueous 10% tetrahydrofuran.

The N-guanylcarbamoyl thienamycin derivative elutes in aqueous tetrahydrofuran and is lyophilized to a solid. UV (pH 7 0.1N phosphate buffer) Amax 298 nm (E 5,500) HPLC C18 Bondapak, aqueous 10% tetrahydrofuran) Retention time 2.0 times that of thienamycin. Electrophoresis (40 v/cm, pH 7 0.1 N phosphate buffer, 20 min) mobility of 2 cm towards the anode.

EXAMPLE 44.

Following the procedures set out in the foregoing Examples and text, the following compounds of the present invention are prepared:

Compound M Rl R2 1.) Na H -C-CH-CH2 0 2.) Na H -C-CH2O-C2 H5 0 3.) Na H -C-NH2 il 0 4.) H H -C-CH2 N-CH3 II I o H 5.) H H -C-CH2N(CH3)2 II 0 H 6.) H H -C-CH2-N-C-NH2 It II O N H 7.) Na H -C-CH2-S-CH3 II 0 8.) Na H -C-CH(-OCH3)2 II 0 9.) Na H -C-CH,OH 1 0 10.) Na H -C-C- CH 0 11.) H H -C-CH2 N-C-N-C-NH2 n II || H O O N H 12.) Na H -C-CHOHCH3 II o 13.) K H -C-CH(CH3)2 ! o 14.) NH4 H S-CF3

Compound M R1 R2 15.) K H -C-H 0 H N 16.) Na H - C - CH2- C N Ii II o N-N H C 17.) Na H -CCH2N N II I ti O H-N-N H N 18.) Na H C-CH-C N II II II N- N 19.) Na H -CCH2 SCH2 CN 0 20.) Na H -CCH(NH2)CH3 21.) Na H SO2 CF3 0 22.) Na H > SCF3 EXAMPLE 45.

Preparation of Pharmaceutical Compositions One such unit dosage form consists in mixing 120 mg. of N-glycylthienamycin with 20 mg. of lactose and 5 mg of magnesium stearate and placing the 145 mg. mixture into a No. 3 gelatin capsule. Similarly, by using more of the active ingredient and less lactose, other dosage forms can be put up in No. 3 gelatin capsules and, should it be necessary to mix more than 145 mg. of ingredients together, larger capsules and compressed tablets and pills can also be prepared.

The following Examples are illustrative of the preparation of pharmaceutical formulations: TABLET PER TABLET N-glycylthienamycin 125 mg.

Cornstarch, U.S.P. 6 mg.

Dicalcium Phosphate 192 mg.

Lactose, U.S.P. 190 mg.

The active ingredient is blended with the dicalcium phosphate, lactose and about half of the cornstarch. The mixture is then granulated with 15% cornstarch paste (6 mg.) and rough-screened. It is dried at 45"C. and screened again through No. 16 screens. The balance of the cornstarch and magnesium stearate is added and the mixture is compressed into tablets, approximately 0.5 inch in diameter, each weighing 800 mg.

PARENTERAL SOLUTION Ampoule: N-glycylthienamycin 500 mg.

Sterile Water 2 ml.

*OPHTHALMIC SOLUTION N-glycylthienamycin 100mg.

Hydroxypropylmethyl Cellulose 5 mg.

Sterile Water to 1 ml.

OTIC SOLUTION N-glycylthienamycin 100 mg.

Benzalkoniurn Chloride 0.1 mg.

Sterile Water to 1 ml.

TOPICAL OINTMENT N-glycylthienamycin 100 mg.

Polyethylene Glycol 4000 U.S.P. 400 mg.

Polyethylene Glycol 400 U.S.P. 1.0 gram The active ingredient in the above formulations may be administered alone or in combination with other biologically active ingredients as, for example, with other antibacterial agents such as lincomycin, a penicillin, streptomycin, novobiocin, gentamycin, neomycin, colistin and kanamycin, or with other therapeutic agents such as probenedid.

PREPARATION OF ALTERNATE STARTING MATERIALS In addition to the thienamycin, one skilled in the art will recognize that its various isomers, alone or as mixtures, may serve as starting materials in the preparation of the compounds of the present invention. Some of these isomers are obtainable from natural products of fermentation (see below). However, by total synthesis all isomers are made available (below) as a mixture of 4 diastereoisomers which possess antibacterial activity and which are amenable to resolution by conventional techniques. The 4 diastereoisomers (2 cis, 2 trans) are separable by. chromatography. Resolution of any given d/l pair with optically active acids or bases proceeds according to conventional techniques. It should be noted that the absolute configuration of the first-identified starting material (I) is 5R 6S 8R.

PREPARATION OF THIENAMYCIN BY TOTAL SYNTHESIS

STEP A: Preparation of 4-(2-acetoxyvinyl)azetidine-2-one

A solution of 1.0 ml distilled chlorosulfonylisocyanate (1.65 g; 11.7 mmoles) in 2.5 ml anhydrous diethyl ether is cooled under N2 in a -20 C bath.

A solution of 2.5 g l-acetoxybutadiene (22 mmoles) in 2.5 ml anhydrous ether is similarly cooled under N2 in a -20"C bath.

The chlorosulfonylisocyanate solution is added dropwise to the acetoxybutadiene solution by means of a Teflon tube immersed in the CSI solution and pressurized with N2. The addition takes 10 minutes. Little or no color is seen and the reaction is stirred at -200C for 0.5 hour. The solution is clear and has a light yellow color.

A solution of 2 g sodium sulfite and 5 g K2HPG4 in 20 ml H2O is prepared during the above 0.5 hour reaction time and is cooled in an ice bath; 20 ml of ether is added and the mixture is vigorously stirred in an ice bath. At the end of the 30 minute reaction time, the reaction mixture is transferred, again using N2 pressure and the Teflon tube, from the reaction flask which is maintained in the -20"C bath, to the vigorously stirred hydrolysis mixture. Rapid dropwise addition is completed in 5 minutes. The hydrolysis is allowed to continue for 5 additional minutes. The hydrolysis mix has a pH of 6-8, preferably pH 8.

The phases are separated, leaving a yellowish-orange gum with the aqueous phase. The ether phase is dried directly with MgSO4. The aqueous/gum phase is extracted three more times with 50 ml portions of ether, each being added to the initial ether/MgSO4.

The dried extracts are filtered and concentrated under a N2 stream to 5 ml; a portion of the product is crystalline at this stage.

A column of 10 g Baker silica gel, packed in ether is prepared, and the ether concentrate is applied to the top and run in. The flask/solids are rinsed three times with 2 ml ether, each being pipetted off and run into the column. Elution is then begun with ether. The first 25 ml is primarily void volume. The next five 10 ml fractions are collected followed by three 50 ml fractions, and all are reduced in volume under aN2 stream. The product crystallizes from fractions W6, with traces in 3 and 7. Fractions 1-3 contain a yellowish sharp-smelling material which resinifies on standing. Yield: 100 mg as a mixture of the cis and trans isomers.

STEP B: Preparation of 4-(2-Acetoxyethyl)-2-Azetidinone

A solution of 4-(2-acetoxyvinyl)-2-azetidinone (10.0 g, 0.065 mole) in 200 ml ethyl acetate containing 100 mg of 10% Pd/C is hydrogenated on a Parr shaker at 25 C under 40 psi hydrogen for 15 minutes. The mixture is filtered through a bed of Supercel and washed with additional ethyl acetate. The combined filtrate is evaporated in vacuo to give 4-(2-acetoxyethyl)-2-azetidinone (10.0 g) as a crystalline solid. Recrystallization from ether affords white crystals: M.P. 44-7 ; ir (CHCl3) 5.66, 5.74; nmr (CDCl3) &gamma; 3.44 (broads s, 1, NH), 5.82 (m, 2, CH2OCOCH3), 6.29 (m, 1, C-4H), 6.87 ( AB pattern further split in four by C-4H and NH, 1,Jgem=12.8 Hz, J=4.5 H JNH=1.9 Hz, 7.38 ( AB pattern further split in four by C-4H and NH, 1, Jgem=12.8 Hz, J=2.3 Hz, JNH=1.0 Hz), 7.93 and 8.02 (s on m, total 5 OCOCH3 and CH2CH2OCOCH3, respectively).

STEP C: Preparation of 4-(2-Hydroxyethyl)-2-Azetidinone

Under nitrogen at OOC, a solution of 4-(2-acetoxyethyl)-2-azetidinone (2.24 g, .014 mole) in 25 ml anhydrous methanol is treated with a solution of sodium methoxide (77 mg, 1.4 mmoles) in 5 ml anhydrous methanol. After stirring for I hour, the solution is neutralized with glacial acetic acid. Removal of the methanol in vacuo gives crude 4-(2-hydroxyethyl)-2-azetidinone as an oil. The product is purified by chromatography on silica gel eluting with 10% MeOH/CHCl3 to give 1.55 g of the alcohol: m.p. 50 ; ir (CHCl3) 5.67; nmr (CDCl3) &gamma; 3.20 (broad s,1, NH), 6.24 and 6.28 (m on t, total 3, C-4H and CH2OH respectively), 6.90 (broad s on 1/2 AB pattern further split in four by C=4H and NH, total 2, OH and C-3H respectively, Jgem=13.0Hz, Jvic=4.2Hz, JNH=1.6Hz), 7.42 ( AB pattern further split in four by C-4H and NH, 1, C-3H, Jgem=13.0Hz, Jvic=2.2Hz, JNH=1.1Hz), 8.16 (m, 2, CH2CH2OH).

STEP D: Preparation of 8-Oxo-2,2-dimethyl-3-oxa- 1 -azabicyclol4.2.0] octane

A solution of 4-(2-hydroxyethyl)-2-azetidinone (1.87 g, .016 mole) and 2,2dimethoxypropane (1.69 g, .016 mole) in 25 ml anhydrous methylene chloride is treated with boron trifluoride etherate (.201 ml, .002 mole) at 250C. The resulting solution is stirred for ten minutes. Removal of the solvent under reduced pressure gives an oil (2.5 g). Chromatography of the crude product on silica gel using 2:1 ethyl acetate/benzene as eluting solvent gives 8-oxo-2,2-dimethyl-3-oxa- 1- azabicyclo[4.2.0]octane (1.59 g) as a crystalline solid. Recrystallization from ether/hexane gives product of m.p. 60-1 . ir (CHCl3) : 5.73 (ss-lactam) nmr (CDC13)A: 6.02-6.28, m, 2H, C=4 methylene 6.226.62, m, 2H, C-6 methine 6.90. dd, 1H, J77=14Hz, J67=4.5Hz C-7 proton cis to CH 7.47, dd, 1H, J7.7=14Hz, J6.7=2Hz C-7 proton trans to C-6H 7.82-8.68, m, 2H, C-S methylene

8.23, s, 3H 8.57, s, 3H,#C-2 methyls STEP E: Preparation of 8-oxo-2,2-dimethyl-7cr and -( 1 -hydroxyethyl)-3-oxa- I - azabicyclo[4.2.0]octane

To a solution of 1.1 equivalents of freshly prepared lithium diisopropylamide in anhydrous tetrahydrofuran under a nitrogen atmosphere at -78 is added a solution of 8-oxo-2,2-dimethyl-3-oxa-1-azabicyclo[4.2.0]octane in anhydrous tetrahydrofuran which has been cooled to -78 C. After two minutes, the resulting lithium enolate is treated with excess acetaldehyde. The solution is stirred for 30 minutes at 780 and then poured into water. The aqueous phase is saturated with sodium chloride and extracted with ethyl acetate. The combined ethyl acetate solutions are dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give the crude product. Purification by chromatography on silica gel using ethyl acetate/benzene gives 8-oxo-2,2-dimethyl-7a and p-(1- hydroxyethyl)-3-oxa- 1 -azabicyclo[4.2.0]octane.

Data for 8-oxo-2,2-dimethyl-7ss-(1-hydroxyethyl)-3-oxa-1 azabicyclo[4.2.0]octane: ir (CH2)Cl2) : 5.72 y (p-lactam) nmr (CDCl3)#: 5.53-6.43, m, 4H, C-4 methylene+ C6 methine+C-9 methine 6.90, dd on broad s, 2H, J7.9=9Hz J6.7=5.5Hz, C-7 methine+OH 7.70-8.83, m, 2H, C-S methylene

8.27, s, 3H 8.60, s, 3H#C-2 methyl 8.78, d, 3H, J9.10=6.5Hz, C-10 methyl Data for 8-oxo-2,2-dimethyl-7&alpha;-(1-hydroxyethyl)-3-oxa-1- azabicyclo[4.2.0]octane: ir (CHCl3) : 2.9 broad O-H 5.73 p-lactam nmr (acetoned8)a: 4.23-3.33, m, C-9 methine+C-4 methylene+C-6 methine 33.3, broad s, OH

2.83, dd, J=2Hz, 6Hz # C-7 methine 2.67, dd, J=2Hz, 8Hz 1.93-1.63, m, C-S methylene

1.63, s # C-2 methyls 1.40, s 1.23, d, J=6.5Hz, C-10 methyl STEP F: Preparation of 8-Oxo-2,2-dimethyl-7a-( l-p-nitrobenzyloxy-carbonyloxyethyl)-3- oxa- 1 -azabicyclo [4.2.0] octane

Under anhydrous conditions at 0 C. a solution of 8-oxo-2,2-dimethyl-7&alpha;-(1- hydroxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane (60 mg, .302 mmole) in 0.6 ml ether is treated with powdered potassium hydroxide (19 mg, .332 mmole). After a period of 15 minutes, p-nitrobenzyl chloroformate (65 mg, .302 mmole) is added to the reaction mixture. Stirring is continued at 25 C for an additional 15 hours. The mixture is partitioned between 1M pH 7 phosphate buffer and more ether. The ether phase is washed with water and brine, dried over magnesium sulfate and filtered. Evaporation of the filtrate under reduced pressure gives 67 mg of a colorless oil. Purification by preparative thick-layer chromatography on silica gel developing with 1:9 ethyl acetate/benzene gives 8-oxo-2,2-dimethyl-7&alpha;-(1-p-nitro- benzyloxycarbonyloxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane (40 mg) as a mixture of diastereomers. ir (CH2CI2)u: 5.68 (p-lactam and carbonate), 6.19 and 6.54 (nitro) nmr (CDCl3): 1.67, d, 2H, ArH 2.37, d, 2H, ArH 4.67, s, 2H, ArCH2 4.67-5.22, m, CH3CH 5.986.25, m, 2H, C=4 methylene 6.256.62, m, 1H, C6 methine 6.75-7.12, m, 1H, C-7 methine 7.75-8.83, m, 2H, C-S methylene 8.22, s, 3H, C-2 methyl 8.50-8.58, m, 5H, C-2 methyl+CH3CH The 7p-didstereoisomers or the 7a and mixture are obtained in an analogous manner.

STEP G: Preparation of Cis and Trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl)-4-(2- hydroxyethyl)-2-azetidinone

8-Oxo-3-oxa-2,2-dimethyl-7&alpha;-(1-p-nitrobenzyloxycarbonyloxyethyl)-1-aza- bicyclo[4.2.0]octane (1.0 g) is dissolved in 8 ml acetic acid and 2 ml water and heated at 65 C for 1.25 hours. The acetic acid and water are removed under reduced pressure and the residue is taken up in benzene and evaporated to give trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl)-4-(2-hydroxyethyl)-2-azetidinone as a mixture of diastereoisomers. ir (CH2CI2)y: 5.67 (p-lactam), 5.72 shoulder, 6.20 and 6.57 (nitro) nmr (CDCl3): 1.73, d, 2H, J=8.5 Hz, ArH 2.43, d, 2H, J=8.5 Hz, ArH 3.63, broad s, 1H, NH 4.37-5.13, m, IH, CH3CH 4.72, s, 2H, ArCH2 6.07v.53, m, - 1H, C=4 methine 6.23, t, 2H, J=5.5 Hz, CH2OH 6.736.93, m, 1H, C-3 methine 7.63-8.97, m, 3H, CH2CH2OH 8.53, d, J=6.5 Hz, CH3CH The cis diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

STEPS D', E', F' AND G' as alternative to STEPS D, E, F, AND G for the preparation of 3-(l-p-nitrobenzyloxycarbonyloxyethyl)-4-(2-hydroxy ethyl)azetidinone

STEPS D', E', F' AND G' Preparation of 1-(2-Tetrahydropyranyl)-4-[2-(2-tetrahydropyranyl)oxyethyl]-2azetidinone

Under nitrogen and at 250C, a solution of 4-(2-hydroxyethyl)-2-azetidinone (62 mg, .539 mmole) in .5 ml of anhydrous p-dioxane is treated with 2,3-dihydropyran (.98 ml, 1.08 mmoles) andp-toluenesulfonic acid monohydrate (19 mg, .10 mmole).

The resulting solution is stirred for a period of 60 minutes and then partitioned between 10 ml of SM pH7 phosphate buffer and 10 ml of ethyl acetate. The aqueous phase is extracted a second time with ethyl acetate. The combined ethyl acetate solutions are washed with brine, dried over magnesium sulfate and filtered.

The filtrate is evaporated under reduced pressure to give 216 mg of crude product.

Purification by preparative thick-layer chromatography developing with ethyl acetate gives 80 mg of 1-(2-tetrahydropyranyl)-4-[2-(2-tetrahydropyranyl)oxyethyl]-2-azetidinone as an oil. nmr (CDCl3)#: 5.13-5.60, m, OCH 5.83-6.85, m, C-4H+OCH2

6.95, dd, J=SHz and 15 Hz) C-3 methylene 7.35, dd, J=3Hz and 15 Hz 7.62-8.95, m, CHCH2CH2CH2CH2+CHCH2CH2O Preparation of Cis and Trans-1-(2-tetrahydropyranyl)-3-(1-hydroxyethyl)-4-[2-(2- tetrahydropyranyl)oxyethyl]-2-azetidinone

Following the procedure described for the preparation of 8-oxo-Z,2-dimethyl- 7a and p-(1-hydroxyethyl)-3-oxa- 1-azabicyclo [4.2.0] octane from 8-oxo-2,2 dimethyl-3-oxa-l-azabicyclo [4.2.0] octane and using 1-(2-tetrahydropyranyl)-4-[2- (2-tetrahydropyranyl)oxyethyl]-2-azetidinone one obtains a diastereomeric mixture of both cis and trans-1-(2-tetrahydropyranyl)-3-(1-hydroxyethyl)-4-[2-(2-tetra- hydropyranyl)-oxyethyl]-2-azetidinone.

Preparation of Cis and Trans-1-(2-tetrahydropyranyl)-3-(1-p-nitrobenzyloxy- carbonyloxyethyl)-4-[2-(2-tetrahydropyranyl)oxyethyl]-2-azetidinone

Following the procedure described for the preparation of 8-oxo-2,2-dimethyl 7&alpha;-(1 -p-nitrobenzyloxycarbonyloxyethyl)-3-oxa- 1 -azabicyclo[4.2.0] octane from 8 oxo-2,2-dimethyl-7&alpha;-(1-hydroxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane and using trans-1-(2-tetrahydropyranyl)-3-(1-hydroxyethyl)-4-[2-(2-tetrahydropyranyl)oxyethyl]-2-azetidinone there is obtained trans-1-(2-tetrahydropyranyl)-3-(1-p-nitro benzyloxycarbonyloxyethyl)-4-[2-(2 - tetrahydropyranyl)oxyethyll - 2- azetidinone.

The cis diastereoisomers are obtained in an analogous manner.

Preparation of Cis and Trans-3-( I -p-nitrobenzyloxycarbonyloxyethyl)-4-(2- hydroxyethyl)-2-azetidinone

A solution of trans-I -(2-tetrahydropyranyl)-3-( 1 -p-nitrobenzyloxy- carbonyloxyethyl)-4-[2-(2-tetrahydropyranyl)oxyethyl]-2-azetidinone in methanol at 25"C. is treated with .1 molar equivalent of p-toluenesulfonic acid monohydrate.

The solution is stirred for a period of 2 hours and then neutralized with I M pH7 phosphate buffer. The product is extracted into ethyl acetate. The ethyl acetate solution is washed with brine, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give trans-3-(1-lp-nitrobenzyloxy- carbonyloxyethyl)-4-(2-hydroxyethyl)-3-azetidinone. The cis diastereoisomers are obtained in an analogous manner.

STEP H: Preparation of Cis and Trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl)-4-[2,2-bis(2 hydroxyethyl)thioethyl] -2-azetidinone

Under nitrogen at 250C, a mixture of anhydrous pyridine (.146 ml, 1.81 mmoles) and anhydrous, powdered chromium trioxide (92 mg, .916 mmole) in 8 ml anhydrous acetonitrile is stirred for a period of 30 minutes. To the resulting dark brown solution is added 250 mg of dry Supercel followed by a solution of trans-3-(l p-nitrobenzyloxycarbonyloxyethyl)-4-(2-hydroxyethyl)-2-azetidinone (186 mg, .550 mmole) in I ml anhydrous acetonitrile. After stirring for a period of I hour, the reaction mixture is filtered through a mixed, packed bed of 2 g each of silica gel and magnesium sulfate. The bed is washed repeatedly with a total of 30 ml of additional acetonitrile. The filtrate is concentrated under reduced pressure at 250C. to a volume of 3 ml. By thin-layer chromatography (silica gel; ethyl acetate/benzene 2:1) this solution contains a product (Rf=.38) less polar than the startng material (Rf=.21).

The acetonitrile solution of trans-3-( I -p-nitrobenzyloxycarbonyloxyethyl)-4-(2 oxoethyl)-2-azetidinone prepared above is, under nitrogen and at 0 , treated with 2-mercaptoethanol (.386 ml. 5.5 mmoles) followed immediately by boron trifluoride etherate (.176 ml, 1.43 mmoles). After stirring for a period of 15 minutes, this solution is partitioned between aqueous dipotassium hydrogen phosphate (1.5 g. in 4 ml of water) and 12 ml of ethyl acetate. The aqueous phase is extracted a second time with ethyl acetate. The combined ethyl acetate solutions are washed with brine, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give 229 mg of an oil. The product is purified by preparative thick-layer chromatography on silica gel developing with ethyl acetate to give 118 mg of trans-3-(l -p-nitrobenzyloxycarbonyloxyethyl)-4- [2,2-bis-(2- hydroxyethyl)thioethyl]-2-azetidinone as a colorless oil. ir (CH2Cl2),u: 5.75 (5.79 shoulder) p-lactam and carbonate 6.20, 6.55 nitro nmr (acetone-d)T: 1.70, d, J=8.5 Hz, 2H, ArH 2.28, d, J=8.5Hz, 2H, ArH 2.48-2.88, m, 1H, NH

7.63-8.33, m, 2H, CH2CH 8.53, d, J=6.5 Hz 3H, CH3CH The cis diastereoisomers are obtained in an analogous manner. Alternatively, the mixed diastereoisomers are obtained when the starting materials comprise a mixture of the diastereoisomers.

STEP I: Preparation of Trans-3-(l -p-nitrobenzyloxycarbonyloxyethyl)-4- [2,2-bis(2- azidoethyl)thioethyl] -2-azetidinone

To a solution of 211 mg (mw=474; 0.445 mmole) trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl)-4-[2,2-bis(2-hydroxyethyl)thioethyl]-2-azetidinone in 5 ml tetra hydrofuran (THF) (distilled from lithium aluminium hydride) at 0 C is added 103 mg mesyl chloride (mw=114; 0.904 mmole) in 1 ml THF followed immediately by 134 ,ul triethylamine (mw=l0l; E=0.729; 0.967 mmole). The reaction mixture is stirred for 1 hour under N2. The triethylamine hydrochloride is filtered under N2 washing with a few milliliters additional THF. The clear colorless filtrate is concentrated under a stream of N2 followed by pumping under high vacuum for 10 minutes. The dimesylate is immediately dissolved in 5 ml DMSO (distilled from CaH2 at 8 mm and stored over 4A Linde Molecular sieves) in the presence of 347 mg NaN3 (mw=65; 5.34 mmole). After stirring overnight under N2, 10 ml H2O and 20 ml ethyl acetate (EA) are added. The layers are separated, and the aqueous one is washed three times with 10 ml EA, each organic layer then being backwashed with 10 ml H2O and 10 ml brine. The combined EA layers are dried over anhydrous MgSO4, filtered and concentrated under a N2 stream to give the crude diazide.

Preparative thin layer chromatography on silica gel yields trans-(l-p-nitro benzyloxycarbonyloxyethyl)-4-[2,2-bis(2-azidoethyl)thioethyll-2-azetidinone. The cis diastereoisomers or the cis-trans mixture are obtained in an analogous manner.

STEP J:

A freshly prepared (H. Davies and M. Schwarz, J.O.C., 30, 1242 (1965)) solution of p-nitrophenyldiazomethane (29 mmole) in 150 ml of ether is added with stirring to a solution of 1.0 g oxomalonic acid, monohydrate (mw=136; 7.35 mmole) in 50 ml ethylacetate (EA) at OOC. After 2 1/2 hours the yellow solution is concentrated on a rotary evaporator with mild heating to approximately half the volume, dried over anhydrous sodium sulfate, filtered and concentrated as above to an oil. To the crude p-nitrobenzyl ester in 50 ml toluene~(Tol.) is added 3.54 g of trans-3-( I -p-nitrobenzyloxycarbonyloxyethyl)-4-[2,2-bis(2-azidoethyl)thioethyli- 2-azetidinone (mw=524; 6.57 mmole). With stirring the reaction mixture is heated in an oil bath allowing approximately 1/3 of the toluene to boil off. Toluene (dried over 3A 1/16" Linde Molecular sieves) is added to again bring the volume to 50 ml, and the azeo-drying process is repeated three additional times. The solution is then reffuxed under N2 for one hour, the azeodrying process repeated a final time, and refluxing continued for an additional hour. Concentration of the resulting solution under a stream of N2 yields crude 1. The crude material is chromatographed on silica gel to give I. The cis diastereolsomers or the cis-trans mixture is obtained in an analogous manner.

STEP K:

To a solution of 2.80 g 1 (mw=912; 3.07 mmole) in 35 ml THF (distilled from lithium aluminium hydride) at -20 C is added 0.3 ml pyridine (mw=79; #=.982; 3.73 mmole) (distilled from NaH and stored over 4A Linde Molecular sieves). With stirring under N2, 0.438 g thionyl chloride (mw=119; 3.68 mmole) in 1 ml THF is added dropwise. The reaction mixture is stirred under N2 for 5 minutes at -20 C, then hour at 0 C, and finally 1 hour at 25 C. The pyridine hydrochloride is filtered under N2 and washed twice with benzene (dried over 3A 1/16" Linde Molecular sieves). The combined filtrate and washings are concentrated under a N2 stream, slurried in a small volume of benzene with anhydrous MgSO4, filtered under N2 and then concentrated under a N2 stream. Pumping on high vacuum for 1/2 hour yields an oil. To this freshly prepared chloro compound is added with- stirring 0.885 g triphenyl phosphine (mw=262; 3.38 mmole) in 66 ml 9:1 dimethylformamide (DMF))/H2O followed by 550 mg K2HPO4 (mw=174; 3.16 mmole). The reaction mixture is stirred at 250C for 35 minutes. After dilution with EA and brine, the layers are separated and the aqueous one extracted three times with EA. The combined EA layers are washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under a stream of N2 to give crude 2. The material is chromatographed on silica gel to give 2. The cis diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

STEP L:

To 7.8 ml pentane (dried over 4A Linde molecular sieves) is added 0.2 ml Br2 (mw=160; #=3.12; 3.9 mmole). To a solution of 950 mg 2 (mw=896; 1.06 mmole) in 15 ml Et2O (dried over 3A 1/16" Linde molecular sieves) at 0 C. under N2 with stirring is added dropwise 2.3 ml of the above 0.49 M Br2 solution (1.13 mmole).

After stirring for 10 minutes at 0 C., 114 ,xI cyclohexene (mw=82, p=.81; 1.13 mmole) is added. After 5 minutes at OOC, 53 mg 57% NaH (57% of 53 mg=30.2 mg, mw=24, 1.26 mmole) in mineral oil is added to the stirred reaction mixture. This is followed immediately by the addition of 14 ml ice cold DMF (distilled from anhydrous CaSO4 at 40 mm and stored over 4A Linde molecular seives). Stirring at 0 C under N2 is continued for 3 hours. The reaction mixture is poured into a stirred ice-cold mixture of 2.5 ml IM KH2PO4=40 ml H,O--75 ml EA. After separation of the layers, the aqueous one is saturated with NaCI and re-extracted with EA. The combined organic layers are extracted once with brine, dried over anhydrous MgSO4, filtered and concentrated under a N2 stream followed by pumping on a high vacuum pump to provide cride 3. Preparative thin layer chromatography on silica gel yields 3. The cis diastereoisomers or the cis-trans mixture is obtained in an analogous manger.

STEP M:

To 9.16 ml pentane (dried over 4A Linde Molecular seives) is added 0.2 ml Br2 (mw=160, 3.9 mmole). To 474 mg 3 (mw=793; 0.598 mmole) in 13 ml Et2O (dried over 3A 1/16" Linde Molecular seives) at 0 C under N2 with stirring is added dropwise 1.52 ml of the above 0.42 M Br2 solution (0.63 mmole). After 15 minutes at OOC, 33 mg 57% NaH (57 4 of 33 mg=18.8 mg; mw=24; 0.78 mmole) is added followed immediately by the addition of 6.35 ml ice-cold DMF (distilled from anhydrous CaSO4 at 40 mm and stored over 4A Linde Molecular seives). The reaction mixture is stirred for 1 1/2 hours at 0 C., then poured into a stirred icecold mixture of 1.6 ml IM KH2PO420 ml H2O, and 20 ml EA. The layers are separated and the aqueous one saturated with NaC1 and re-extracted with additional EA. The combined organic layers are washed once with brine, dried over anhydrous MgSO4 and filtered. The filtrate is concentrated under a N2 stream and then pumped on high vacuum to give crude 4. Preparative thin layer chromatography on silica gel gives 4. The cis diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

STEP N:

To 210 mg 4 (mw=871; 0.241 mmole) dissolved in 0.5 ml DMSO (distilled from CaH2 at 8 mm and stored over 4A Linde Molecular sieves) is added at 25 C. with stirring 40 mg 1,5-diazobicyclo[5.4.0]undec-5-ene (distilled at 80 C./2 mm) (mw=152; 0.263 mmole) in 0.7 ml dimethylsulfoxide (DMSO). The solution is stirred under N2 for 4 hours, and then added to a stirred ice-cold mixture of 0.48 ml lMKH2PO4, 7 ml H2O, and 10 ml EA. After separation of the layers, the aqueous layer is again extracted with EA. The combined o

STEP 0:

OCOOPN9 SCH2CHZ&num;3 + LiI s-collidine 0' + Lii (C02Pn8)2 ~ OCOOPNO nSCH2CH2N3 H CO2PN8 t A solution of 187 mg 5 (mw=791; 0.236 mmole) in 2.5 ml s-collidine (distilled from powdered KOH at 30 mm pressure) is added to 45 mg anhydrous LiI(dried for a few hours at 100 C over P2Os under vacuum) (mw=134; 0.336 mmole). With stirring under N2, the reaction mixture is heated in an oil bath at 1200C. After a total of 25 minutes, the reaction mixture is cooled to 250C., diluted with CH2CI and transferred to a round bottom flask for concentration under a N2 stream and then on high vacuum. Partitioning of the residue between 10 ml EA and 1.8 ml IM KH2PO4 in 10 ml H20 is followed by extraction of the aqueous layer two additional times with EA. The combined organic layers are extracted with brine, dried over anhydrous MgSO4, filtered and concentrated under a stream of N2 to give crude 6.

Preparative thin layer chromatography on silica gel yields 6. The cis diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

STEP P:

To a solution of the mixed diastereoisomers, (34 mg; mw=612; 0.055 mmole) in 0.2 ml DMSO (distilled from CaH2 at 8 mm and stored over 4A Linde Molecular sieves) with stirring is added 9.5 l 1,5-diazobicyclo[5.4.0]undec-5-ene (distilled at 800C/2 mm), (mw=152; p=l; 0.0625 mmole). The solution is stirred under N2 for 15 minutes diluted to 1 ml total volume with CHCI3 and applied immediately to 2-1000 - silica gel plates. The product band yields 7 as a mixture of cis and trans diastereoisomers.

STEP Q:

In the presence of 61 mg PtO2, 61 mg of 7 (mw=612; 0.1 mmole) in 6 ml dioxane, 6 ml THF, 3 ml H2O is hydrogenated at 40 p.s.i. 112 for 4 hours. The reaction mixture is then filtered through Celite washing with 2 ml 0.1N pH7 phosphate buffer. After concentration in vacuo to the cloud point, the aqueous mixture is extracted with ethyl acetate. The water layer is concentrated to a small volume and applied to a column of 100 g XAD-2 resin. Upon elution with H2O and discarding the initial fractions, those fractions containing product are lyophilized to give 8 as a mixture of cis and trans diastereoisomers.

The following procedure for the enzymatic N-deacylation of thienamycin, which forms part of the invention claimed in the specification of our copending application No. 48233/76 (Serial No. 1561108) is applicable for all isomers of thienamycin-particularly the distinct N-acetyl isomers 890A, and 890A3, which are described below.

Deacetylation of N-Acetyl Thienamycin A 1% (w/v) suspension of fertile lawn soil is prepared by suspending 1 gm. of lawn soil in 100 ml. of sterile phosphate-buffer-saline solution of the following composition: Phosphate-Buffer-Saline Solution NaCI 8.8 g.

IM Phosphate Buffer, pH 7.5* 10 ml.

Distilled H2O 1000 ml.

*IM Phosphate Buffer, pH 7.5 16 ml. of 1M KH2PO4 is mixed with 84 ml. 1M K2HPO4. The pH of the phosphate buffer is adjusted to 7.5 by adding small quantities of either 1M KH2PO4 or IM K2HPO4.

Aliquot portions of this 1% stock soil suspension are used to prepare 10x, 100x and l,000x dilutions.

One-ml. portions of the stock suspension or 1 ml. portions of the 10x, 100x and l,000x dilutions are added to 2-mi. portions of sterile, 1.0% agar solutions at 48 C. The mixtures are quickly poured over the surface of sterile petri dishes of 85 mm. diameter containing 20 ml. of Medium A. Medium A has the following composition: Medium A KH2PO4 3.0 g.

K2HPO4 7.0g.

MgSO4 0.1 g.

Distilled H2O 1000 ml.

N-Acetylethanolamine solution* 8.5 ml.

N-acetylethanolamine Solution N-acetylethanolamine is diluted lOx in H2O and membrane sterilized.

This solution is added after autoclaving.

For solid media: Add 20 g. agar The petri dishes are incubated for 18 days at 280C. A well-isolated colony is picked and streaked on a petri dish containing Medium B. Medium B has the following composition: Medium B Tomato paste 40 g.

Ground oatmeal 15 g.

Distilled H2O 1000 ml. pH: adjust to 6 using NaOH For solid media: Add 20 g. agar An individual clone is selected and grown for 2 days at 280 C. on a slant of Medium B. A portion of the growth on this slant is streaked on the surface of six slants prepared from Medium B. These slants are incubated for 2 days at 280C.

This culture was identified as Protaminobacter ruber and has been designated MB-3528 in the culture collection of Merck & Co.. Inc., Rahway, New Jersey, U.S.A. and a sample deposited with the Agricultural Research Service, U.S.

Department of Agriculture, Accession No. NRRL B-8143.

A portion of the growth on the slant of Protaminobacter ruber MB-3528 is used to inoculate a 250 ml. Erlenmeyer flask containing 50 ml of Medium C.

Medium C has the following composition: Medium C Dextrose 20 g.

Pharmamedia 8 g.

Corn Steep Liquor (wet basis) 5 g.

Distilled H2O 1000 ml. pH: adjust to 7 with NaOH or HCI N-acetylethanolamine solution 8.5 ml.

N-acetylethanolamine Solution N-acetylethanolamine is diluted with 10x in H2O and membrane sterilized.

This solution is added after autoclaving.

The flask is shaken at 280C on a 220 rpm (2" throw) shaker for 4 days. A 25 ml. portion from the flask is centrifuged for 15 minutes at 8,000 rpm. The supernatant is removed and the cells on the surface of the media solids scraped off into 0.5 ml.

0.05M potassium phosphate buffer, pH 7.4. The resulting suspension is subjected to ultrasonic disruption using a Branson Instrument Model LS-75 Sonifier with a 1/2-inch probe at setting 4 for four, 15-second intervals, while chilling the suspension in ice water during and between disruption. A 10 zl portion of the sonicate is mixed with 25 y1 of a solution containing 840 ,ug/ml. of Nacetylthienamycin in 0.01H potassium phosphate buffer, pH 7 and incubated overnight at 280C. Controls containing antibiotic and buffer alone and sonicated cells and buffer without antibiotic are also run. After incubation overnight at 28"C., 2 ,ul quantities are aplied on cellulose coated TLC plates, which are developed in EtO11:112O, 70:30. After air drying, the TLC plate is placed on a Staphylococcus aureus ATCC 6538P assay plate for 5 minutes.

The assay plates are prepared as follows: An overnight growth of the assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth, plus 0.2% yeast extract to a suspension having 60% transmittance at a wavelength of 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g./l. Difco yeast extract at 370C. to 480C., to make a composition containing 33.2 ml. of the suspension per liter of agar. 40 ml. of this suspension is poured into 22.5 cmx22.5 cm. petri plates, and these plates are chilled and held at 4 C. until used (5-day maximum).

The TLC plate is removed and the assay plate incubated overnight at 37 C. In addition to the unreacted bioactive N-acetyl thienamycin spot at Rf 0.7--0.89, a bioactive spot is observed at Rf 0.44-0.47 due to thienamycin. Control incubation mixtures of antibiotic plus buffer, cell sonicate plus buffer, and antibiotic plus buffer to which cell sonicate is added just prior to TLC application produce no bioactive material at Rf 0.44-0.47.

Antibiotics SDA1 and 8903 can be obtained by the processes described in Examples 6 and 7 of the specification of our copending application No. 48235/76 (Serial No. 1561109). They can be treated to cleave the N-acetyl group as above described to provide the corresponding free base

Claims (37)

1. WHAT WE CLAIM IS: 1; A compound having the structural formula:

   or a non-toxic pharmaceutically acceptable salt thereof; in which R1 is a hydrogen atom or an acyl group and R2 is an acyl group other than acetyl, or Rl and R2 are both acetyl.
2. 2. A compound according to Claim 1 in which the acyl radical has the formula:

   where X is O or S and R is H, amino, C1-6 alkylamino, di(C1-6 alkylamino, C1-5 alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, or heteroaralkyl.
3. 3. A compound accoridng to Claim 1 in which the acyl radical has the formula:

   where X is O or s, R is amino, mercapto, hydroxy, C1-6 alkylamino, di(C1-6 alkyl)amino, alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, or heteroaralkyl; n is 0, 1, 2, 3, or 4; and Z is O, S, NH or carbonyl; except that when Z is oxygen R is not mercapto or hydroxy; when Z is sulphur R is not amino or hydroxy; and when Z is imino R is not mercapto.
4. 4. A compound according to Claim I in which the acyl radical has the formula:

   where X is O or S; R is H, amino, mercapto, hydroxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-6 alkyl, alkylthio, arylthio, alkoxy, aryloxy, alkenyl, alkynyl, aryl, araikyl, cycloalkyl, heteroaryl, or heteroaralkyl; and R' is azido, carbamoyl, guanidino, amidino, acyloxy, halo, sulfamino, tetrazolyl, sulfo, carboxy, carbalkoxy, phosphono, alkoxy or arylthio.
5. 5. A compound according to Claim 1 in which the acyl radical has the formula:

   where X is O or S; each of m and n, independently of the other, is 0 or 1; each of Y, Y' and Y", which are the same or different, is O-M+,-NR2 or R or Y' and Y" may be joined together to form, with the phosphorus atoms to which they are attached, a cyclic ester or amide, M is hydrogen, an alkali metal or alkaline-earth metal cation, or an organic base; and R is H, amino, mercapto, hydroxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-6 alkyl, alkylthio, arylthio, alkoxy, aralkoxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl or heteroaralkyl.
6. 6. A compound according to Claim 1 in which the acyl radical has the formula:

   in which q is 0 or 1; each of m and n, independently of the other, is 0, 1, 2, 3, 4 or 5; A is 0, -NR - (where R is hydrogen or C1-6 alkyl) or S, and Y is an amino or substituted amino radical of formula: + -N(R*)2 or -N(R*)3 an amidino or substituted amidino radical of formula:

   a guanidino or substituted guanidino radical of formula:

   or a guanyl or substituted guanyl radical of formula:

   where each R independently of the other, is hydrogen; N(R )2 (where R is hydrogen or C16 alkyl); C18 alkyl, C16 alkoxy; (C16 alkoxy)-(C2~e alkyl), C36 cycloalkyl or cycloalkyl-(C1~3 alkyl) or the two R groups are joined to form together with the N atom to which they are attached, a ring having 3 to 6 atoms; R* is a radical as defined for R except that if cycloalkylalkyl, it must be (C36 CyclOalkyl)-c1-3 alkyl; and R** is a radical as defined for R or a (C16 alkoxy)methyl radical; or Y is a monocyclic or bicyclic heterocyclic aromatic or non-aromatic radical having 4 to 10 nuclear atoms and in which the hetero atom or atoms are nitrogen and optionally oxygen or sulfur.
7. 7. A compound according to Claim 2 in which R1 is hydrogen and R2, the acyl radical. is:

   where R is benzyl, p-hydroxybenzyl, 4-amino-4-carboxybutyl, methyl, cyanomethyl, 2pentenyl, n-amyl, n-heptyl, ethyl, (3 or 4)-nitrobenzyl, phenethyl, ss,ss-diphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, 3,5-dimethyl-4-isoxazolyl, 3-butyl-5-methyl-4isoxazolyl, 5-methyl-3-phenyl-4-isoxazolyl, 3-(2-chlorophenyl)-5-methyl-4-isoxazolyl, 3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl, D-4-amino-4-carboxybutyl, D4-N-benzoylamino-4-carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, (3-pyridyl)methyl, 2-ethoxy- I -naphthyl, 3carboxy-2-quinoxalinyl, 3-(2,6-dichlorophenyl)-5-(2-furyl)-4-isoxazolyl,3-phenyl-4isoxazolyl, 5-methyl-3-(4-guanidinophenyl)-4-isoxazolyl, 4-guanidinomethylphenyl, 4-guanidinomethylbenzyl, 4-guanidinobenzyl, 4-guanidinophenyl, 2,6dimethoxy-4-guanidinophenyl, o-sulfobenzyl, p-carhoxymethylbenzyl, pcarbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl, pmethoxybenzyl, 1 -naphthylmethyl, 3-isothiazolylmethyl, 4-isothiazolylmethyl, 5isothiazolylmethyl, guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4methoxy-5-isoxazolylmethyl, 4-methyl-5-isoxazolylmethyl, I-imidazolylmethyl, 2benzofuranylmethyl, 2-indolylmethyl, 2-phenylvinyl, 2-phenylethynyl, I-aminocyclohexyl, 2- and 3-thienylaminomethyl, 2-(5-nitrofuranyl)vinyl, phenyl, omethoxyphenyl, o-chlorophenyl, o-phenylphenyl, p-aminomethylbenzyl, 1-(5 cyanotriazolyl)methyl, difluoromethyl, dichloromethyl, dibromomethyl, 1-(3methylimidazolyl)methyl, (2 or 3)-(5-carboxymethylthienyl)methyl, (2 or 3)-(4carbamoylthienyl)methyl, (2 or 3)-(5-methylthienyl)methyl, (2 or 3)-(5-methoxythienyl)methyl, (2 or 3)-(4-chlorothienyl)methyl, (2 or 3)-(5-sulfothienyl)methyl, (2 or 3)-(5-carboxythienyl)methyl, 3-(1,2,5-thiadiazolyl)methyl, 3-(4-methoxy-1,2,5thiadiazolyl)methyl, 2-furylmethyl, 2-(5-nitrofuryl)methyl, 3-furylmethyl, 2thienylmethyl, 3-thienylmethyl tetrazolylmethyl, benzamidinomethyl, or cyclohexylamidinomethyl.
8. 8. A compound according to Claim 3 in which R1 is hydrogen and R2 is:

   where X is oxygen or sulfur and R* is allylthiomethyl, phenylthiomethyl, butylthio methyl, a-chlorocrotylthiomethyl, phenoxymethyl, phenoxyethyl, phenoxybutyl, phenoxybenzyl, phenoxyphenoxymethyl, (dimethylmethoxy)methyl, (dimethylbutoxy)methyl, (dimethylphenoxy)methyl, 4-guanidinophenoxymethyl, 4pyridylthiomethyl, p-(carboxymethyl)phenoxymethyl, p-(carboxymethyl)phenylthiomethyl, 2-thiazolylthiomethyl, p-(sulfo)-phenoxymethyl, 2-pyrimidinylthiomethyl, phenethylthiomethyl, 1 -(5,6,7,8-tetrahydronapthyl)oxymethyl, N-methyl-4pyridylthio, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio, amino, methylamino, dimethylamino, a pyridinium methyl or trimethylammonium-methyl non-toxic salt, cyanomethyltliiomethyl, trifluoromethylthiomethyl, 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 3-imidazolylethyl, 3-imidazolylpropyl, 3-imida- zolylbutyl, I-pyrrolylethyl, l-pyrrolylpropyl or l-pyrrolylbutyl.
9. 9. A compound according to Claim 4 in which R1 is hydrogen and R2 is:

   where --CHRR' of the acyl radical is a-aminobenzyl, a-amino-(2-thienyl)-methyl, a-(methylaniino)benzyl, a-amino-methylthiopropyl, a-amino-3- or 4-chlorobenzyl, &alpha;-amino-3 or 4-hydroxybenzyl, &alpha;-amino-2,4-dichlorobenzyl, &alpha;-amino-3,4- dichlorobenzyl, D-(-)-&alpha;-hydroxybenzyl, &alpha;-carboxybenzyl, &alpha;-amino-(3- thienyl)methyl, D-(-)-&alpha;-amino-3-chloro-4-hydroxybenzyl, &alpha;- amino(cyclohexyl)methyl, a-(5-tetrazolyl)benzyl, 2-thienyl-carboxymethyl, 3thienyl-carboxymethyl, 2-furyl-carboxymethyl, 3-furyl-carboxymethyl, a sulfa minobenzyl, 3-thienyl-sulfaminomethyl, a-(N-methylsulfamino)benzyl, D-(-)- 2-thienyl-guanidinomethyl, D(-)-a-guanidinobenzyl, a-guanylureidobenzyl, ahydroxybenzyl, a-azidobenzyl, a-flaorobenzyl, 4-(5-methoxy- 1,3- oxadiazolyl)aminomethyl, 4-(S-methoxy- I ,3-oxadiazolyl)hydroxymethyl, 4-(5- methoxy-l ,3-sulfadiazolyl)hydroxymethyl, 4-(5-chlorothienyl)aminomethyl, 2-(5chlorothienyl)hydroxymethyl, 2-(5-chlorothienyl)carboxymethyl, 3-(1,2thiazolyl)aminomethyl, 3-(1,2-thiazolyl)hydroxymethyl, 3-(1 ,2-thiazolyl)carboxy- methyl, 2-(1 ,4-thiazolyl)aminomethyl, 2-(1 ,4-thiazolyl)hydroxymethyl, 2-(1,4-thiå- zolyl)-carboxymethyl, 2-benzothienylaminomethyl, 2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl, a-sulfobenzyl or a-phosphonobenzyl.
10. 10. A compound according to Claim 4 in which R' is hydrogen and R2 is

    where R is &alpha;-diethylphosphono or &alpha;-monoethylphosphono.

    11. A compound according to Claim 6 where R is hydrogen and R, the acyl radical, has the formula:

    where Q (i) is a non-toxic anion.
11. 11. A compound according to Claim 1 in which R' is hydrogen and R2 is formyl, propionyl, butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, ethylcarbamoyl, phenyithiocarbonyl, 3-aminopropionyl, 4-aminobutyryl, N-methylaminoacetyl, N,N-dimethylaminoacetyl,, N.N,N-trimethylaminoacetyl, 3-(N,N-dimethyl)aminopropionyl, 3-(N,N,N-trimethyl)amino propionyl, N,N,N-triethylaminoacetyl, pyridiniumacetyl, guanylthioacetyl, guanidinoacetyl, 3-guanidinopropionyl, N3-methyiguanidinopropionyl, hydroxyacetyl, 3-hydroxypropionyl, acryloyl, propynoyl, malonyl, phenoxycarbcnyl, amidinoacetyl, acetamidinoacetyl, amidinopropionyl, acetamidinopropionyl, guanylureidoacetyl, guanylcarbamoyl, carboxymethylaminoacetyl, sulfoacetylaminoacetyl, phosphonoacetylaminoacetyl, N3-di-methylaminoacetamidinopropionyl, ureidocarbonyl, dimethylaminoguanylthioacetyl, a 3 (1 -methyl-4-pyridinium)propionyl non-toxic salt, 3-(5-aminoimidazol-1- l)propionyl, a 3-methyl-l-imidazoliumacetyl non-toxic salt, 3-sydnonylacetyl, oaminomethylbenzoyl, o-aminobenzoyl,
12. 12. A process for preparing a compound according to Claim 1 which comprises treating a compound of the structure:

    with an acylating agent calculated to provide N-substituents R1 and R2.
13. 13. A pharmaceutical composition comprising a compound according to Claim 1 and a pharmaceutical carrier.
14. 14. A pharmaceutical composition comprising, in unitary dosage form, a therapeutically effective amount of a compound according to Claim 1 and a pharmaceutical carrier.
15. 15. A pharmaceutical composition as claimed in Claim 13, in the form of a capsule, tablet, powder, elixir, aqueous or oily solution or suspension, emulsion or syrup.
16. 16. A composition as claimed in Claim 13, in orally administrable form.
17. 17. A composition as claimed in Claim 13, in intravenously administrable form.
18. 18. A composition as claimed in Claim 13, in intramuscularly administrable form.
19. 19. A composition as claimed in Claim 13, in the form of a suppository.
20. 20. A composition as claimed in Claim 13, in form suitable for absorption through the mucous membranes of the nose and throat or bronchial tissues.
21. 21. A composition as claimed in Claim 20, in the form of a liquid spray or inhalant, a lozenge or a throat paint.
22. 22. A composition as claimed in Claim 13, in aurally or optically administrable form.
23. 23. A composition as claimed in Claim 13, in topically administrable form.
24. 24. A composition as claimed in Claim 23, in the form of an ointment, cream, lotion, paint or powder.
25. 25. An antibacterial composition comprising as active ingredient a compound as claimed in Claim I together with a material in respect of which antibacterial action is desired.
26. 26. A composition as claimed in Claim 25, in which the said material is a human or animal foodstuff.
27. 27. A disinfectant comprising as active ingredient a compound as claimed in Claim 1 together with a suitable diluent or carrier.
28. 28. A composition as claimed in Claim 25, in which the said material is a waterbased paint.
29. 29. A composition as claimed in Claim 25, in which the said material is the white water of a paper mill.
30. 30. A veterinary composition comprising as active ingredient a compound as claimed in Claim 1 together with a non-toxic base material.
31. 31. A composition as claimed in Claim 30, in the form of an intramammary preparation.
32. 32. A process for preparing a compound as claimed in Claim 1 substantially as hereinbefore described in any one of Examples 1 to 33.
33. 33. A process for preparing a compound as claimed in Claim 1 substantially as hereinbefore described in any one of Examples 34 to 44.
34. 34. A compound as claimed in Claim 1 when prepared by a process as claimed in Claim 12 or 32.
35. 35. A compound as claimed in Claim I when prepared by a process as claimed in Claim 33.
36. 36. A composition as claimed in any one of Claims 13 to 31 in which the said compound is a compound as claimed in any one of Claims 2 to 11, 34 and 35.
37. 37. A composition as claimed in Claim 13 substantially as hereinbefore described in any one of the formulations set out in Example 45.