GB1570987A - Thienamycin derivatives - Google Patents

Abstract

Novel thienamycin derivatives of formula <IMAGE> wherein the substituents are defined in Claim 1, are prepared by introducing the radical R<3> into thienamycin by acylation. The compounds obtained may be used as antibiotics.

Description

(54) THIENAMYCIN DERIVATIVES (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 derivatives of the antibiotic thienamycin, and with their preparation and pharmaceutical compositions comprising them. The compounds in accordance with the present invention, as defined below, have been found to be broad-spectrum antibiotics and to be useful in animal and human therapy and in inanimate systems.

Thienamycin (I), which is disclosed in U.S. Patent No. 3,950,357 and U.K.

Patent No. 1,498,087, may serve as a starting material for making the compounds of the present invention:

Another convenient starting material is the N-acylated carboxyl derivative of Thienamycin (Ia) which is described and claimed in the specification of our copending Application No. 48239/76: (Serial No. 1570989)

where X, R, R' and R2 are as defined below.

The compounds of the present invention are depicted by the following generic structural formula, in which the nucleus may have a different stereoisomeric configuration from that of thienamycin:

or, more conveniently, by the symbol

In these formulae, "Th" symbolizes the bicyclic nucleus of thienamycin type and the OH, amino, and carboxyl groups on the nucleus are illustrated.

Each of R' and R2 is a hydrogen atom, an acyl group, or a non-acyl blocking group, e.g. a substituted sulfenyl or silyl radical, or a radical of formula:

where Y' and Y" are as defined later. Preferred compounds are those in which R1 is hydrogen and R2 is acryl. The term "acyl" is by definition inclusive of the alkanoyls including derivatives and analogues thereof such as thio analogues, in which the carbon oxygen is replaced by sulphur, diacyl radicals, where R1 and R2 are joined together, and the sulphur and phosphorus acyl analogues such as substituted sulfonyl and sulfinyl radicals and substituted P (III and V) radicals such as the substituted phosphorous, phosphoric, phosphonous and phosphonic radicals.

X is oxygen, sulphur, NH or NR.

R is, inter alia, hydrogen, a conventional blocking group such as trialkylsilyl, acyl or a pharmaceutically acceptable salt, ester or amide residue known in the bicyclic p-lactam antibiotic art. The definition of R is given in greater detail below.

R3 is 1.) acyl (generically the group OR3 is classifiable as an ester); or 2.) R3 is a radical such that the group R3 is generically classifiable an an ether, (e.g. alkyl, aryl, or aralkyl). The term "acyl" includes those residues mentioned in connexion with R1 and R2. Such acyl radicals are further defined below, as are the radicals (2., above) that constitute the ethers of the present invention.

The compounds of the present invention (II) are conveniently and preferably prepared in the following sequence; first thienamycin (I) is N-acylated to form the N-acyl intermediate (1) which is esterified to form the carboxyl derivative (2); derivatization of 2 provides II;

where all symbols are as previously defined. Compounds of the present invention IIa are obtained when X is oxygen and R is an easily removable blocking group.

Similarly when R' and R2 are hydrogen or an easily removable N-blocking group, compounds IIb are obtained by selective deblocking.

It should be noted that the carboxyl group may, when desired, be derivatized first-prior to derivatization of the amino group.

The above preparation is described in greater detail below.

In the generic representation of the compounds of the present invention (II, above), the acyl radical represented by R1 or R2, or by R3, when it is an acyl radical, can, inter alia, by substituted or unsubstituted aliphatic, aromatic, heterocyclic, araliphatic or heterocyclylaliphatic carboxylic acid radical, a substituted or unsubstituted carbamoyl 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(C,~8 alkyl)amino in which the alkyl radicals(s) are substituted or unsubstituted; straight or branched-chain 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 410 atoms and the hetero atom or atoms is/are preferably O,N and/or S; such above-listed groups can be unsubstituted or can be substituted by radicals such as OH, SH, SR8 (R8 is C16 alkyl or aryl such as phenyl), alkyl or alkoxy groups having 1 to 6 carbon atoms, halogens, viz. Cl, Br, F or I, cyano, carboxy, sulfamino, carbamoyl, sulfonyl, azido, amino, substituted amino, such as (C1 alkyl)-substituted amino including quaternary ammonium, C16 halogenated alkyl such as trifluoromethyl, carboxy(C,~6 alkyl), carba moyl(C,~8 alkyl), N-substituted carbamoyl(C16 alkyl, amidino, guanidino, Nsubstituted 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, 4-amino-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 4nitrobenzyl, phenethyl, ,-diphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, 3,5dimethyl-4-isoxazolyl, 3-butyl-5-methyl-4-isoxazolyl, 5-methyl-3-phenyl-4-isoxazolyl, 3-(2-chlorophenyl)-5-methyl-4-isoxazolyl, 3-(2,6-dichlorophenyl)-5methyl-4-isoxazolyl, D-4-amino-4-carboxybutyl, D-4-N-benzoylamino-4-carboxy-n- butyl, p-aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, (3pyridyl)methyl, 2-ethoxy- I -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, 4guanidinobenzyl,4-guanidinophenyl, 2,6-dimethoxy-4-guanidinophenyl, o- sulfobenzyl, p-carboxymethylbenzyl, p-carbamoylmethylbenzyl, m-flurobenzyl, mbromobenzyl, p-chlorobenzyl, p-methoxybenzyl, l-naphthylmethyl, 3-isothiazolylmethyl, 4-isothiazolylmethyl, 5-isothiazolylmethyl, guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl, 4-methoxy-5-isoxazolylmethyl, 4-methyl-5-isoxazolylmethyl, I-imidazolylmethyl, 2-benzofuranylmethyl, 2-indolylmethyl, 2-phenylvinyl,- 2-phenylethynyl, I-aminocyclohexyl, 2- and 3-thienylaminomethyl, 2-(5nitrofuranyl)vinyl, phenyl, o-methoxyphenyl, o-chloro henyl, o-phenylphenyl, p-aminomethylbenzyl, l-(5-cyanotriazolyl)-methyl, difluoromethyl, dichloromethyl, dibromomethyl, 1-(3-methylimidazolyl)-methyl, (2 or 3)-(5-carboxymethyl thienyl)methyl, (2 or 3)-(4-carbamoylthienyl)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-l ,2,5-thiadiazolyl)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 X is O or S and n is 0, 1, 2, 3 or 4, Z represents oxygen, sulfur, carbonyl or nitrogen, R" is defined as above and R is as defined below. Representative members of the substituent (CH2)nZR" 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, Nmethyl-4-pyridylthio, benzyloxy, methoxy, ethoxy, phenoxy, phenylthio, amino, methylamino, dimethylamino, a pyridinium methyl or trimethylammonium-methyl non-toxic salt, cyanomethylthiomethyl or trifluoromethylthiomethyl. R is 4pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl, 3-imidazolylethyl, 3-imidazolylpropyl, 3-imidazolylbutyl, 1 -pyrrolylethyl, 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 &alpha;-aminobenzyl, &alpha;-amino-(2-thienyl)methyl, &alpha;- (methylamino)benzyl, &alpha;-amino-methylthiopropyl, &alpha;-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, &alpha;-(5- tetrazolyl)benzyl, 2-thienyl-carboxymethyl, 3-thienyl-carboxymethyl, 2-furyl-carboxymethyl, 3-furyl-carboxymethyl, a-sulfaminobenzyl, 3-thienyl-sulfaminomethyl, a-(N-methylsulfamino)benzyl, D(-)-2-thienyl-guanidinomethyl, D(-)-aguanidinobenzyl, cr-guanylureidobenzyl, a-hydroxybenzyl, a-azidobenzyl, a- fluorobenzyl, 4-(5-methoxy- 1 ,3-oxadiazolyl)-aminomethyl, 4-(5-methoxy- 1 ,3-oxadiazolyl)-hydroxymethyl, 4-(5-methoxy- 1 ,3-sulfadiazolyl)-hydroxymethyl, 4-(5 chlorothienyl)-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-(1,4thiazolyl)-hydroxymethyl, 2-(1,4-thiazolyl)-carboxymethyl, 2-benzothienylaminomethyl, 2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl, a-sulfobenzyl or a-phosphonobenzyl. The acyl radical can also be cr-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, carboxyl, 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, carboxamidomethyl, 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, 4-carboxymethylphenylacetyl, 4-carboxyamidomethylphenylacetyl, 2-furylacetyl, 5-nitro-2-furylacetyl, 3-furylacetyl, 2-thienylacetyl, 5-chloro-2-thienylacetyl, 5methoxy-2-thienylacetyl, a-guanidino-2-thienylacetyl, 3-thienylacetyl, 2-(4methylthienyl)acetyl, 3-isothiazolylacetyl, 4-methoxy-3-isothiazolylacetyl, 4-isothiazolylacetyl, 3-methyl-4-isothiazolylacetyl, 5-isothiazolylacetyl, 3-chloro-5-isothiazolylacetyl, 3-methyl-1,2,5-oxydiazolylacetyl, 1,2,5-thiadiazolyl-4-acetyl, 3methyl-1,2,5-thiadiazolylacetyl, 3-chloro-1,2,5-thiadiazolylacetyl, 3-methoxy-1,2,5thiadiazolylacetyl, phenylthioacetyl, 4-pyridylthioacetyl, cyanoacetyl, I-tetrazolylacetyl, a-fluorophenylacetyl, D-phenylglycyl, 4-hydroxy-D-phenylglycyl, 2-thienylglycyl, 3-thlenylglycyl, phenylmalonyl, 3-chiorophenylmalonyl, 2 thienylmalonyl, 3-thienylmalonyl, &alpha;-phosphonophenylacetyl, &alpha;-amino cyclohexadienylacetyl, &alpha;- sulfaminophenylacetyl, &alpha;-hydroxyphenylacetyl, &alpha;-tetrazolylphenylacetyl, and &alpha;- sulfophenylacetyl.

The substituents R1, R2 and if appropriate R3 may also be selected from sulfur (1) and phosphorus (2) radicals:

in which, with respect to (I), each of m and n is 0 or 1 and Y = OnMe, -N(R")2, or R"; where Me 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 heteroarvl.

In this case when R3 is acyl, sulfo and its salts (i.e., m = n = 1 and Y is OM) are especially preferred values, particularly when R' is hydrogen, R2 is acyl and X is oxygen. With respect to (2), X=O or S; n=1; and each of Y' and Y", independently of the other, is O-M+, -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 R" residues, are joined together to form cyclic ester, ester-amide and amide functions. Illustrative examples of compounds containing values of R1 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 (dimethoxyphosphino)thienamycin, N-(dibenzyloxyphosphino)thienamycin, N-(dihydroxyphosphino)thienamycin disodium salt, N-(dimethoxyphosphinyl)thiena- mycin, 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, ringsubstituent carbobenzyloxy such as o- and p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy, chloroacetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl, trifluoroacetyl, bromoethoxycarbonyl, 9-fluorenylmethoxycarbonyl, dichloroacetyl, 2,2,2-trichloroethoxycarbonyl, bromo-t-butoxycarbonyl, phenoxyacetyl. Non-acyl protective groups such as o-nitrophenylsulfenyl and tri(C,~ 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, propionyl, butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, phenylthiocarbamoyl, 3-aminopropionyl, 4aminobutyryl, N-methylaminoacetyl, N,N-dimethylaminoacetyl, an N,N,Ntrimethylammoniumacetyl salt, 3-(N,N-dimethyl)aminopropionyl, a 3-(N,N,N trimethyl)-aRlmonium propionyl salt, an N,N,N-triethylammoniumacetyl salt, a pyridiniumacetyl salt, guanylthioacetyl, guianidinoacetyl, 3-guanidinopropionyl, N3-methylguanidinopropionyl, hydroxyacetyl, 3-hydroxypropionyl, acryloyl, propynoyl, malonyl, phenoxycarbonyl, amidinoacetyl, acetamininoacetyl, amidinopropionyl, acetamidinopropionyl, guanylureidoacetyl, guanylcarbamoyl, carboxymethylaminoacetyl, sulfoacetylaminoacetyl, phosphonoacetylaminoacetyl, N3-dimethylaminoacetamidinopropionyl, ureidocarbonyl, dimethylaminoguanylthioacetyl, a 3-(1 -methyl-4-pyridinium)-propionyl salt, 3-(5-aminoimidazol l-yl)propionyl, 3-methyl- 1-imidazoliumacetyl salt, 3-sydnonylacetyl, o-aminomethylbenzoyl, o-aminobenzoyl,

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

Especially preferred acyl radicals (R and R 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:

and these radicals and the corresponding radicals in which the

group is omitted are also preferred values of R3, 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 C1-6 alkyl) or S, and Y is an amino or substituted amino radical of formula: -NR*)2 or -N(R*)3 an amidino or substituted amidino radical or 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 alkoxyl; (C1-6 alkoxy)-(C2-6 alkyl), C3-6 cycloalkyl or cycloalkyl-(C1-3alkyl) 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 (C3-6 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.

Such heterocycles are representatively illustrated by the following list of radicals (R' is H or C1-6 alkyl):

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

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.

The N-acylated intermediate compound (1 above) is 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 heterocyclicaliphatic 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 l,3-dicyclohexylcarbodiimide, and activated esters of a carboxylic acid such as a p-nitrophenyl ester.

The N-acylated thienamycin starting material is described and claimed in the specification of our copending Application No. 48236/76: (Serial No. 1570986) and its stereoisomers can also be used.

The acylation reaction may be conducted at a temperature in the range -20" to 100"C. but is preferably conducted at a temperature in the range from -80C. to 25"C. 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) hexamethylphosphoramide (HMPA), acetone, dioxane, tetrahydrofuran (THF), acetonitrile, heterocyclic amines such as pyridine, ethyl acetate, and 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.

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 are suitable for this purpose. Silylation of thienamycin proceeds rapidly to give the tris-triorganosilyl derivative, for example tristrimethylsilyl 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 atomosphere. The resulting NH4CI is removed by centrifugation and the solvent is removed by evaporation to provide the desired silyl derivative.

In the generic representation of the compounds of the present invention (II, above), the radical represented by -COXR, is, inter alia, -COOH (X is oxygen and R is hydrogen) and all radicals known to be effective as pharmaceutically acceptable ester, anhydride (R is acyl) and amide radicals in the bicyclic p-lactam antibiotic art, such as the cephalosporins and penicillins and the nuclear analogues thereof.

Suitable radicals (R) include conventional protecting or carboxyl blocking groups. Such blocking groups are used in the preparation of species IIa, above. The term "blocking group" is used herein in the same manner and in accordance with the teaching of U.S. Patent 3,697,515. Pharmaceutically acceptable thienamycin derivatives of the present invention falling in this class are given below. Suitable blocking esters thus include those selected from the following list which is representative and not intended to be an exhaustive list of possible ester groups, in which X is oxygen and the value of R is given: (i) R is CReRnR- where at least one of Ra, Rb and R is an electrondonor, e.g., p-methoxyphenyl, 2,4,6-trimethylphenyl, 9-anthryl, methoxy, CH2SCH3, tetrahydrofur-2-yl, tetrahydropyran-2-yl or fur-2-yl. The remaining Ra Rb and RQ groups may be hydrogen or organic substituting groups. Suitable ester groups of this type inlcude p-methoxybenzyloxycarbonyl and 2,4,6-trimethylbenzyloxycarbonyl .

(ii) R is CRa-Rb-Rc where at least one of Ra-, Rb- and Rc- is an electronattracting group, e.g. benzoyl, p-nitrophenyl, 4-pyridyl, trichloromethyl, tribromomethyl, iodomethyl, cyanomethyl, ethoxycarbonylmethyl, arylsulphonylmethyl, 2-dimethylsulphoniummethyl salt, o-nitro-phenyl or cyano. Suitable esters of this type include benzoylmethoxycarbonyl, p-nitrobenzyloxycarbonyl, 4-pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl and 2,2,2-tribromoethoxycarbonyl.

(ii) R is CRa-Rb-Rc- where at least two of Ra-, Rb-, and Rc- are hydrocarbon such as alkyl, e.g., methyl or ethyl, or aryl, e.g., phenyl and the remaining Ra-, Rb- or Rc-, if there is one, is hydrogen. Suitable esters of this type include t-butyloxycarbonyl, t-amyloxycarbonyl, diphenylmethoxycarbonyl and triphenylmethoxycarbonyl.

(iv) R is adamantyl, 2-benzyloxyphenyl, 4-methylthiophenyl or tetrahydropyran-2-yl.

Silyl esters, under this catagory of blocking groups may conveniently be prepared from a halosilane or a silazane of the formula: R34SiX'; R24SiX'2; R34Si.NR24; R34Si.NH.COR4; R34Si.NH.CO.NH.SiR34 R4NH.CO.NH.SiR34; or R4C(OSiR34); HN(SiR34)2 where X'is a halogen such as chlorine or bromine and the various group R4, which can be the same or different, represent hydrogen, alkyl, e.g. methyl, ethyl, n-propyl, iso-propyl; aryl, e.g. phenyl; or aralkyl, e.g., benzyl.

More generally stated, pharmaceutically acceptable carboxyl derivatives of the present invention are those derived by reacting an N-acylated thienamycin (I above) with alcohols, phenols, mercaptans, thiophenols, or acylating reagents that yield compounds 2 above, which are then derivatized to establish the R3 group of the compounds of the present invention (II, above).

For example, esters and amides of interest are the compounds of the formula II above having the following group at the 2-position: -COXR where X is oxygen, sulfur, NH or NR, and R (or each R) is alkyl having 1-10 carbon atoms, straight or branched, such as methyl, ethyl,. t-butyl, pentyl and decyl; carbonylmethyl including phenacyl, p-bromophenacyl, p-t-butylphenacyl, acetoxyacetylmethyl, pivaloxyacetylmethyl, carboxymethyl and its alkyl and aryl esters; a-carboxy-a- isopropyl; aminoalkyl including 2-methylaminoethyl, 2-diethylaminoethyl, 2acetamidoethyl, phthalomidomethyl and succinimidomethyl; (C,~10 alkoxy)-(C,~6 alkyl) in which the alkoxy residue can be branched, straight or cyclic, such as methoxymethyl, ethoxymethyl, isopropoxymethyl, decylopxymethyl, ethoxypropyl, decycloxypentyl or cyclohexyloxymethyl; (C1-6 alkanoyloxy)-(C1-6 alkyl in which the alkanoyloxy portion is straight or branched, such as acetoxymethyl, pivaloyloxymethyl, acetoxyethyl, propionyloxyethyl, or acetoxypropyl; halogenated straight or branched C16 alkyl in which the halogen is iodine, chlorine, bromine and/or fluorine, e.g. 2,2,2-trichloroethyl, tri-fluoroethyl, 2-bromopropyl, diiodomethyl, 2chloroethyl, or 2-bromoethyl, and also including C1-6 perhaloalkyl; alkenyl having 2-10 carbon atoms, either straight or branched, e.g., alkyl, 2-propenyl, 3-butenyl, 4-butenyl, 4-pentenyl, 2-butenyl, 3-pentenyl, 3-methyl-3-butenyl, methallyl and 1,4 cyclohexadien-l-yl-methyl; alkynyl having 2-10 carbon atoms, either straight or branched, e.g., 3-pentynyl, propargyl, ethynyl and 3-butyn-1-yl; alkanoyl, either straight or branched, having 1-10 carbon atoms, such as pivaloyl atoms in the alkyl chain, e.g. 2,2-dimethyl-5-coumaranmethyl, 5-indanylmethyl, 2thienylmethyl, 2-furylmethyl, 3-t-butyl-5-isothiazolmethyl, 6-pivaloyloxy-3 pyridazinylethyl or 5-phenlthio-1-tetrazolylmethyl; phthalidyl; phenylethyl, 2-(p methylphenyl)ethyl, and their arylthioalkyl analogues; aryloxy-(C,~ alkyl) where aryl is preferably a phenyl ring having 03 substituents, preferably 0 or I substituent in the ortho or para positions, e.g., (4-methoxy)phenoxymethyl, phenoxymethyl, (4-chloro)phenoxymethyl, (4-nitro)-phenoxymethyl, (4 benzyloxy)phenoxymethyl, (4-methyl)phenoxymethyI, (2-methoxy)phenoxymethyl, (I-phenoxy)ethyl, (4-amino)phenoxymethyl, (4-methoxy)phenylthiomethyl, (4 chloro)phenylthiomethyl or phenylthioethyl; phenyl, 5-indanyl, substituted phenyl having 1-3 substituents, preferably one substituent in the ortho or para position, e.g., (4-methyl)-phenyl, (4-hydroxy)phenyl, (4-t-butyl)phenyl, p-nitrophenyl, 3,5 dinitrophenyl or p-carboxyphenyl, the latter having either the free acid or the sodium salt form; phenyI-(C28 alkenyl), such as 3-phenyl-2-propenyl; benzyloxy (C13 alkyl) such as benzyloxymethyl, (4-nitro)benzyloxymethyl or (4-chloro) benzyloxymethyl; and alkylthioalkyl where the alkylthio residue has 1--10 and preferably 16 carbon atoms, and can be branched, straight or cyclic, and the alkyl portion has 16 carbon atoms, such as methylthioethyl, ethylthioethyl, cyclohexylthiomethyl, decylthiobutyl, methylthiopropyl, isopropylthioethyl or methylthiobutyl.

In addition to the esters and thio esters listed above, amides are also embraced R' by the present invention i.e., compounds where X in the -N- group.

Representative of such amides, Th-CONR'R, are those in which R' is hydrogen, methyl, ethyl, phenyl, p-methoxyphenyl, benzyl, carboxymethyl, methylthioethyl, or heteroaryl; also embraced by -COXR are anhydrides, in which R is, for example, benzyloxycarbonyl, ethozycarbonyl, benzoyl, or pivaloyl.

Preferred esters and amides are those in which X is oxygen, sulphur or NR' (R' is hydrogen or C18 alkyl) and R is aralkyl, aryloxyalkyl, aralkoxyalkyl, alkylthio alkyl, haloalkyl or alkenyl.

The particularly preferred compounds of the present invention are those in which (relative to Formula II, above) X is oxygen and R is C10 alkyl, C28 alkenyl, such as methallyl, 3-methylbutenyl or 3-butenyl; methylthio ethyl; benzyl; substituted benzyl, such as p-t-butylbenzyl, m-phenoxybenzyl, p-pivaloyloxybenzyl or p-nitrobenzyl; pivaloyloxymethyl, 3-phthalidyl, acetoxymethyl, propionyl oxymethyl, acetylthiomethyl, pivaloylthiomethyl, allyl, 4-pentenyl, 2-butenyl, 3 methyl-2-butenyl, phenacyl, acetoxyacetylmethyl, pivaloylacetylmethyl, diethylaminoethyl, dimethylaminethyl, methoxymethyl, p-acetoxybenzyl, p pivaloyloxybenzyl, p-isopropoxybenzyl, 5-indanylmethyl, 5-indanyl, benzyl oxymethyl, ethylthloethyl, methylthlopropyl, methoxycarbonyloxymethyl, ethoxy carbonyloxymethyl, dimethyaminoacetoxymethyl, crotonolacton-3-yl, or acetamidomethyl.

The preferred N-blocking groups for the starting material Ia are carbobenzyloxy, ring-substituted carbobenzyloxy such as o- and p-nitro carbobenzyloxy, p-methoxycarbobenzyloxy, chloroacetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl, trifluoroacetyl, bromoethoxycarbonyl, 9-fluorenyl methoxycarbonyl, dichloroacetyl, o-nitrophenylsulfenyl, 2,2,2-trichloroethoxy carbonyl, bromo-t-butoxycarbonyl and phenoxyacetyl; non-acyl protective groups such as alkylidenes, for example benzylidene and salicylidine, are also of interest.

As noted above, the intermediate starting material for the preparation of 44(above) is prepared according to the following scheme:

where all symbolism is as previously defined.

In general, the transformation 1 ) 2 is accomplished by conventional procedures. Such procedures include: (1) Reaction of compound I with a diazoalkane such as diazomethane, phenyldiazomethane or diphenyldiazomethane in a solvent sich as dioxane, ethyl acetate or acetonitrile at a temperature of from 0 C. to reflux for from a few minutes to 2 hours.

(2) Reaction of an alkali metal salt of compound 1 with an activated halide such as an alkyl halide (e.g. methyl iodide), benzyl bromide, m-phenoxybenzyl bromide, p-t-butylbenzyl bromide or pivaloyloxymethyl chloride. Suitable reaction conditions include solvents such as hexamethylphosphoramide at a temperature of from 0 C. to 600 C. for from a few minutes to 4 hours.

(3) Reaction of compound 1 with an alcohol such as methanol, ethanol or benzyl alcohol. This reaction may be conducted in the presence of a carbodiimide condensing agent such as dicyclohexyl carbodiimide. Suitable solvents, at a temperature of from 0 C to reflux for from 15 minutes to 18 hours, include CHCl3, CH3Cl and CH2C12.

(4) Reaction of an N-acylated acid anhydride of compound 1 prepared by reacting the free acid I with an acid chloride such as ethyl-chloroformate or benzylchloroformate with an alcohol such as those listed in (3) under the same conditions of reaction as given above for (3). The anhydride is prepared by reacting compound 1 and the acid chloride in a solvent such as tetrahydrofuran (THF) or CH2CI2 at a temperature of from 25"C. to reflux for from 15 minutes to 10 hours.

(5) Reaction of labile esters of compound 1 or thienamycin such as the trimethylsilyl ester or dimethyl-t-butylsilyl ester with RX' where Xis halogen such as bromine or chlorine and R is as defined above, in a solvent such as THF or CH2CI2 at a temperature of from 0 C to reflux for from 15 minutes to 16 hours, for example, according to the following scheme:

OTMS ~ OTMS Nucytation Th NR1 TMS ester Th NHTMS 1 fcat'ion Th TMS ç ThNR1 TMS NR1 TMS COOTMS - COOTMS - COOR 3 OH mild hydrolysis 2 They - NHR1 COOR where TMS is a triorganosilyl radical such as trimethylsilyl and all other symbolism is as previously defined.

The amides of the present invention are most conveniently prepared by reacting the acid anhydride of 2 (X =O, R = acyl) with ammonia or with the desired amine, e.g., the alkyl-, dialkyl, aralkyl- or hetero cyclic amines listed above.

The above-recited schemes of esterification are well-known in the related bicyclic p-lactam antibiotic art and indeed in all of general organic synthesis and it is to be noted that there is no undue criticality of reaction parameters in the preparation of the N-acylated carboxyl derivatives 2 useful as starting materials in the practice of the present invention.

Identification of R3 In the generic representation of the present invention, Structure II (above), the radical R3 is (1) acyl (generically the groupOR3 is classifiable as an ester); or (2) R3 is a radical (e.g. alkyl, aryl or aralkyl) such that the groupOR3 is classifiable as an ether. For the esters, (1), R3 is selected from the above defined acyl radicals (R1 and R2). In the ethers, R3 is selected from the above-identified acyl radicals in which the carbonyl residue,

or more generally

is omitted. Thus R3 is selected from the following radicals where all symbolism is as previously defined and p is 1 or 0.

For the ethers (2) as well as in the esters (1), the preferred radicals R3 are those that have a relatively low molecular weight and are hydrophilic. Thus, with respect to the ethers (2), the following radicals are especially preferred: methoxymethyl, hydroxyethyl, methoxyethyl, dimethylaminomethyl, dimethylaminoethyl, methylthioethyl, amidinoethyl, and guanidinoethyl.

For the esters (I), the following radicals are representative and preferred: sulfo, phosphono, carbamoyl, methylsulfonyl, sulfamoyl, dimethylsulfamoyl, 'Nmethyl carbamoyl, bromoacetyl, hydroxyacetyl, aminoacetyl, dimethylaminoacetyl, triethylammoniumacetyl, amidinoacetyl, guanidinoacetyl, methoxyacetyl, guanylacetyl, guanylthioacetyl, phosphamoyl, phosphonothioyl and thiocarbamoyl.

In general, the compounds of the present invention are prepared by any of a variety of well-known esterification or etherification reactions (2 ) II) upon the secondary alcoholic group of 2. Such procedures (2 ) Il) include:

(I) For the preparation of ethers of the present invention, the acid-catalysed reaction of 2 with a diazoalkane such as diazomethane, phenyldiazomethane or diphenyldiazomethane in an inert solvent such as dioxane, tetrahydrofuran (THF), a halohydrocarbon such as CH2CI2, or ethylacetate in the presence of a catalytic amount of a strong acid or Lewis acid such as toluenesulfonic acid, triflouroacetic acid, fluoboric acid or boron trifluoride at a temperature of from -78"C. to 250C. for from a few minutes to 2 hours.

(2) For the preparation of ethers of the present invention , the reaction of 2 with an alkylating agent such as an active halide, for example methyl iodide, benzyl bromide or m-phenoxybenzyl bromide, or an alkyl sulphate such as dimethyl sulphate or diethyl sulfate or methylfluorosulphonate in the presence of a strong base capable of forming the alcoholate anion of compound 2. Suitable bases include alkali and alkaline-eath metal oxides and hydroxides, alkali metal alkoxides such as potassium tertiary-butoxide, tertiary amines such as triethylamine, alkali metal alkyls and aryls such as phenyllithium, and alkali metal amides such as sodium amide. Suitable solvents include any inert anhydrous solvent such as tbutanol, dimethylformamide (DMF), THF, hexamethylphosphoramide (HMPA) and dioxane at a temperature of from 780C. to 25"C., for from a few minutes to 4 hours.

(3) For the preparation of esters of the present invention, the reaction of compound 2 with any of the above-listed acyl radicals in their acid form. This reaction may be conducted in the presence of a carbodiimide condensing agent such as dicyclohexylcarbodiimide. Suitable solvents include any inert solvent such as CHCl3, CH2CI2, DMF, HMPA, acetone or dioxane at a temperature of from 0 C. to 600C. for from 15 minutes to 2 hours.

(4) For the preparation of esters of the present invention, the reaction of compound 2 with an acyl halide or an acid anhydride, in which the acyl residue is as described above. When the above-described acylating reaction involves an acid halide (suitable halides are chloroides, iodides and bromides) or an acid anhydride, the reaction is usually conducted in an anhydrous organic solvent such as acetone, dioxane, methylene chloride, chloroform or DMF in the presence of a suitable acceptor base such as NaHCO3, MgO, triethylene or pyridine at a temperature of from 0 C. to 400C. for from 1 to 4 hours.

Suitable acyl halides and anhydrides include acetic anhydride, bromoacetic anhydride, propionic anhydride, benzoylchloride, phenylacetyl chloride, azidoacetyl chloride, 2-thienylacetyl chloride, 2-, 3- and 4-nicotinyl chloride, pnitrobenzoyl chloride, 2,6-dimethoxybenzoyl chloride, 4-guanidinophenylacetyl chloride, methanesulfonyl chloride, dibenzylphosphorochloridate, dimethylthiophosphorochloridate, 2-furoyl ethyl carbonic anhydride, methylchloroformate and bis(p-nitrobenzyl)phosphorochloridate.

(5) For the preparation of esters of the present invention, the reaction of compound 2 with a suitably substituted ketene or isocyanate such as ketene, dimethyl ketene, methylisocyanate, methylisothiocyanate or chlorosulfonyl isocyante. Suitable solvents include dioxane, tetrahydrofuran and chloroform at a temperature of from -70"C. to 600C. for from 15 minutes to 18 hours.

Compounds of the present invention designated as lIb (above) are also preferred and are conveniently prepared by establishing either R' or R2 as an easily removable N-blocking, or protecting, group; when compounds Ilb are desired the other N-substituent is hydrogen. The N-deblocking procedure for the preparation of llb is accomplished by any of a variety of well known procedures which include hydrolysis and hydrogenation; for hydrogenation, suitable conditions involve a solvent such as a C16 alkanol in the presence of a hydrogenation catalyst such as palladium or platinum or oxides thereof:

wherein all symbolism is as previously defined.

Suitable N-blocking groups for the preparation of Ilb are carbobenzyloxy, ring-substituted carbobenzyloxy such as o- and p-nitrocarbobenzyloxy, pmethoxycarbobenzyloxy, chloroacetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl, trifluoroacetyl, bromoethoxycarbonyl, 9-fluoroenylmethoxycarbonyl, dichloroacetyl, o-nitrophenyl sulfenyl, 2,2,2-trichloroethoxycarbonyl, bromo-tbutoxycarbonyl, phenoxyacetyl; non-acyl protective groups such as tri(C,~ alkyl)silyl, for example, trimethylsilyl and t-bytyldimethylsilyl, are also of interest.

Preferred N-blocking groups Rl and RZ are bromo-t-butoxycarbonyl and those in which R' is hydrogen and R2 is substituted or unsubstituted carbobenzyloxy radical:

wherein n is 0, 1 or 2 and R' is C16 alkoxy or nitro.

Compounds of the present invention designated as IIa (above) are conveniently and preferably obtained when X is oxygen and R is a readily removable carboxyl protecting or blocking, group (see above). Compounds IIa are prepared by deblocking according to any of a variety of well known procedures which include hydrolysis and hydrogenation; when the preferred carboxyl-blocking groups are used (below), the preferred deblocking procedure is hydrogenation, in which the intermediate species II, in a solvent such as C16 alkanol, is hydrogenated in the presence of a hydrogenation catalyst such as palladium, platinum or oxide thereof:

In this connection, it is noted that suitable "blocking groups" R include the sub-generic groups defined above as aralkyl, haloalkyl, alkanoyloxyalkyl, alkoxyalkyl, alkenyl, substituted alkyl, or aralkoxyalkyl, and also including alkylsilyl, where alkyl has 1--10 carbon atoms. For example, suitable "blocking groups R include benzyl, phenacyl, p-nitrobenzyl, methoxymethyl, trichloroethyl, trimethyl silyl, -tributyltin, p-methoxybenzyl and benzhydryl. These blocking groups are preferred since they are generally recognized as easily removable blocking groups in the cephalosporin and penicillin art.

The preferred carboxyl blocking groups, R, are benzyl and substituted benzyl:

where n is 0, 1 or 2 and R' is C16 alkoxy or nitro.

The Compounds of the present invention have been found to be valuable antibiotics active against various gram-positive and gram-negative bacteria and, accordingly, find utility in human and veterinary medicine and in inanimate systems. Such compounds can therefore be used as antibacterial drugs for treating infections caused by gram-positive and gram-negative bacteria, for example against Staphylococcus aureus, Streptomyces pyogenes, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Salmonella (e.g.) Salmonella typhosa, Pseudomonas and Bacterium proteus. The antibacterial compounds of the invention may further be utilized as additives to animal feedingstuffs, for preserving foodstuffs and as disinfectants.

For example, they may be used in aqueous compositions in concetrations 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 waterbased paints and in the white water of paper mills to inhibit the growth of harmful bacteria.

The products of this invention may be used alone or in combinations as an active ingredient in any one of a variety of pharmaceutical preparations. These antibiotics and their corresponding pharmaceutically acceptable salt, ester and amide detivatives may be used in capsule form or as tablets, powders or liquid solutions or as suspensions, elixirs. They may be administered orally, intravenously or intramuscularly. Such pharmaceutically acceptable forms are prepared according to well known procedures.

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 or silica gel; 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 by 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 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 acohol 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 ampoules, or in multidose containers with an additive preservative. The compositions may take such forms as suspensions, solutions. 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 composition 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 or liquid sprays or inhalants, lozenges or 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, or powders.

Also, in addition to a carrier, the compositions may include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubrictors, 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 anitibiotic activity.

For veterinary medicine the composition may, for example, be formulated 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 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 compositions will generally contain from 15 mg. to 1500 mg. of 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 or a soluble powder intended for solution.

Of the following Examples only Examples 1 to 7, Parts 1 and 2 of Example 10, and Examples 31 to 38 (with certain exceptions specifically noted) illustrate the present invention. In the Examples, the compounds of the present invention will be designated by the previously introduced symbol:

In which the three functional groups are illustrated, the words "Dowex", "Nujol", "Teflon", "Supercel", "Celite" and "Difco" are trade marks, "mmol" means "millimo1e", the proportions of liquid mixtures are given on a volume basis, and screen and capsule sizes are U.S. standards. Example 1

TMS =trimethylsilyl Preparation of Silylated-Thienamycin Thienamycin (80.0 mg.) is suspended in 40 ml. tetrahydrofuran (THF) under a N2 atmosphere, the solution is concentrated to 10 ml. and hexamethyldisilazane (1.0 ml.) and trimethychlorosilane (300,us) 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 2 Preparation of O-Methyl-N-(p-Nitrobenzyloxycarbonyl)-Thien amycin-p- Nitrobenzyl Ester

Step A 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 p-nitro benzylchloroformate 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, 50V/cm., 20 minutes) shows no free Thienamycin present, the aq. extract is adjusted to pH 2.2 with IM H3PO4 solution and extracted three times with EtOAc. The EtOAc ectract is dried over MgSO4, filtered and reextracted 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%).

Step B N-(p-Nitrobenzyloxycarbonyl-Thienamycin-(p-nitrobenzyl Ester A mixture of p-nitrobenzyloxycarbonyl-Thienamycinlithium salt (295 mg.) and 0.4 g. of p-nitrobenzyl bromide in 3 ml. of hexamethyl phosphoramide is stirred for 3 hours at 250C. The solution is diluted with 50 ml. of ethyl acetate and extracted successively with water (3 portions), pH 7 phosphate buffer and saturated sodium chloride solution. The organic phase is dried over magnesium sulfate and evaporated to 5 ml., causing the solvent to crystallize. The crystals are collected and washed with ethyl acetate, yield 160 mg. of N-p-nitrobenzyloxycarbonyl Thienamycinp-nitrobenzyl ester, M.P. 168--170"C. NMR (CDCl3) T 1.35 (d), (J = 6 Hz,CH3), 4.04.3 and 2.8-3.5(m); 5.16 (s), carbamate OCH2; 5.24 (AB quartet J= 14); 7.42 (d), 7.56 (d), 8.16 (d) J=9Hz aromatic. IR 5.65y (lactam C=O).

O-Methyl-N-(p-Nitrobenzyloxycarbonyl)-Thienamycin-(p-Nitrobenzyl Ester To a solution of 135 mg. of N-p-nitrobenzyloxycarbonyl-Thienamycin-(p-nitrobenzyl)ester in 50 ml. of methvene chloride at OOC. is added with vigorous stirring 0.5 ml. or 0.006 M fluoboric acid a 3:1 mixture of ether and methylene chloride, immediately followed by 10 ml. of a cooled solution of 0.6 M diazomethane in methylene chloride. The diazomethane is decolorized on one minute. Tb; solution is extracted with 10 ml. of 0.1N pH 7 phosphate buffer, dried and evaporated to a small volume. The solution is applied to two 8" x 8" 1000y silica plates which are developed with 3:1 ethylacetate-chloroform. The band at 3--4.5 cm yields 12 mg. of recovered starting material. the band at 6--8 cm. yields 20 mg. of crystalline 0methyl N-p-nitrobenzyloxycarbonyl thienamycin p-nitrobenzylester, MP 172--174"C. MS m/e 600 (M+), 568, 542, 500, 447, 389, 304 and 59.

Example 3

Preparation of O-Acetyl-N-(p-Nitrobenzyloxycarbonyl)-Thienamycin-(p-Nitro- benzyl)Ester To a solution of 50 mg. of p-nitrobanzyloxycarbonyl thienamycin p-nitrobenzylester in 0.5 ml. of pyridine is added 0.16 ml. of acetic anhydride. The mixture is allowed to react at room temperature for three hours, then pumped to dryness under vacuum. The solid residue is dissolved in chloroform and chromatographed on an 8" x 8 lOOO,t silica gel plate in a 3:1 mixture of ethyl acetate and chloroform. the band at Rf 0.55 is isolated yielding 36 mg. of O-acetyl N-p-nitrobenzyloxycarbonyl Thienamycin p-nitrobenzyl ester, M.P. 172--175"C. NMR (CDCl3) , 1.41, ((d) J = 6 Hz CH3), 2.04 ((s) COCH3), 2.7-3.6 ((m) 4.14 (t of d) J = 3 and 9 Hz), 5.17 (s) carbamyl CH2; 5.35 (AB quartet) J = 14m Hz; 7.45 (d) 7.60 (d), 8.18 (d) J = 9 Hz, aromatic H. Mass Spec M+ m/e 628.

Example 4 Preparation of O-Acetyl-N-Azidoacetyl-Thienamycin-B enzyl Ester

Step A Preparation of N-Azidoacetyl-Thienamycin-Sodium (I) and Lithium (II) Salts

(I) Thienamycin (48 mg., 0.18 mmol) is dissolved in 10 ml. cold water and is kept at 00. To the solution is added sodium bicarbonate (147 mg., 17.6 mmol) and dioxane (10 ml.). Azidoacetyl chloride (60 mg., 0.50 mmol) is added to the solution during a period of 2 min. The reaction mixture is stirred for 15 min and then neutralized to pH 7.0 with 30% phosphoric acid and transferred into a separatory funnel. The solution is extracted with 2 x 50 ml. The aqueous layer is concentrated to 5 ml, and then is charged to a Dowex AG-50 x 8 (sodium form) ion exchange column monitored by UV. The desired fractions are combined and lyophilized to give 21 mg. of the product. The electrophoresis (50 V/cm., 20 min.) of the product in pH 7.0 phosphate buffer shows a single bio-active band, which moves 40 mm. towards the anode. UV A H20 300 nm; NMR (100 MHz, D2O):8 1.26 (d, CH3CH); max 2.92-3.43 (m, 3CH2 and Cs-H), 4.01 (s, CH2N3) and 4.20 ppm (m, C5-H and C7-H).

(II) Thienamycin (76.2 mg., 0.28 mmol.) is dissolved inlO 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 1 min. 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 charied to the Dowex AG--50 x 8 (lithium form) ion-exchange column. The desired fractions are combined and l

(b) N-Azidoacetyl-Thienamycin-sodium salt (30 mg., 0.08 mmol.) is stirred with HMPA (3 ml.) and benzyl bromide (120 mg., 0.70 mmol.) at room temperature for 30 min. The product (30 mg.) is isolated by the procedure described above.

Step C Preparation of O-Acetyl-N-Azidoacetyl-Thienamycin-lBenzyl Ester

N-Azidoacetyl-Thienamycin-benzyl ester (30 mg., 0.067 mmol.) is dissolved in 0.5 ml. of pyridine. To the solution is added acetic anhydride (0.2 ml.). The mixture is kept at room temperature for 40 min. The solution is diluted with I ml. ethyl acetate and washed with ice-water. The organic layer is separated and dried over sodium sulfate. The desired product (10 mg.) is isolated by silica gel TLC (Rf = 0.49 in ethyl acetate). IR (CHCI3): 2121(N3), 1777 (p-lactam), 1738 (OAc), and 1683 cm-' (ester and amide): NMR (CDCI3, 60 MHz): 1.38 (d, CH3CH), 2.05 (s, OAc), 3.96 (s, CH2N3), 5.20 (s, CH2C"H ), and 7.40 ppm (m, CH)- Example 5 Preparation of O-Acetyl-N-Glycyl-Thienamycin

O-Acetyl-N-Azidoacetyl-Thienamycin-benzyl ester (5.0 mg., 0.006 mmol.) is dissolved in 0.3 ml. dioxane. The solution is added to a hydrogenation flask containing 20 mg of palladium (from palladium oxide) and 0.5 ml. 50% dioxane in water. The mixture is stirred for 10 min., then is filtered from the catalyst. After extraction with ether, the solution is lyophilized to give the desired product.

Electrophoresis of the product in pH 7.0 buffer shows one bio-active band at the origin (50 V/cm., 20 min.).

Example 6 Preparation of O-Acetyl-N-Azidoacetyl-Thienamycin-3-Methyl-2-Buten-l-yl Ester

Step A Preparation of N-Azidoacetyl-Thienamycin-3-Methyl-2-Buten-1-yl Ester N-Azidoacetyl-Thienamycin-sodium salt (11.0 mg., 0.029 mmol.) is stirred with HMPA (1 ml.) and l-bromo-3-methyl-2-butene (39 mg., 0.26 mmol.) at room temperature for 30 min. The mixture is then diluted with 10 ml. ethyl acetate and washed thoroughly with water. The desired product (10 mg.) is isolated by silica gel TLC (Rf=0.18 in ethyl acetate). IR (CHCl3: 2121 (N3), 1777 (ss-lactam) and 1685 cm-1 (ester and amide): NMR (CDCl3, 100 MHz): 1.34 (d, CH3CH), 1.73

2.803.80(m, 3 CH2 and C6-H), 3.98 (s, CH2N3), 4.20 (m, C5-H and C7-H), 4.72 (d, CH2CH = ), 5.40 (t, CH2CH=), and 6.70 (broad, NH); Mass Spect. (E. I.): e/m 423 (molecular ion), 405 (M±H2O), 395 (M+8N2), and 337 (M±86).

Step B Preparation of O-Acetyl-N-Azidoacetyl-Thienamycin-3-Methyl-2-Buten-l-yl Ester N-Azidoacetyl-Thienamycin-3-methyl-2-buten-1-yl ester (5.0 mg.) is dissolved in pyridine (0.5 ml.). To the solution is added acetic anhydride (0.1 ml.). The mixture is kept at room temperature for 2 hours and then evaporated to dryness.

The residue is worked up by silica gel TLC to give the desired product (Rf=0.52 in ethyl acetate). NMR (CDCl3, 100 MHz): 1.38 (d, CH3CH), 1.74

2.03 (s,OAc), 2.90-3.80 (m, 3CH2 and C6-H), 3.98 (s, CH2N3), 4.20 (m, C5-H), 4.74 (d, CH2CH =), and 5.32 ppm (t, CH2CH =).

Example 7 Preparation of O-Acetyl-N-Glycyl-thienamycin-3-Methyl-2-B uten- 1 -yl Ester

O-Acetyl-N-Azidoacetyl-Thienamycin-3-methyl-2-buten-1-yl ester (10 mg.) is dissolved in ethyl acetate (1 ml). This solution is added to the hydrogenation flask containing 10 mg. of palladium (from palladium oxide) and 0.5 ml. 50% methanol in ethyl acetate. The mixture is stirred under 1 atm hydrogen at 250C for I hr. The mixture is filtered from the catalyst. The desired product is isolated by silica gel TLC (20% methanol/chloroform). Electrophoresis of the product at pH 7.0 buffer (50V/cm., 20 min.) shows one bio-active band, which moves towards the cathode.

Example 8 Preparation of N-(p-nitrobenzyloxycarbonyl)-Thienamycin p-t-butyl benzyl Ester N-(p-nitrobenzyloxycarbonyl)Thienamycin Li salt, the product of Example 2, (205 mg) in 2 ml. hexamethylphosphoramide (HMPA) is treated for 2.5 hrs. with 0.1625 ml. p-t-butylbenzyl bromide. The starting material is insoluble in HMPA but goes into solution after 30 minutes.

The reaction mixture is diluted with ethyl acetate (EtOAc), washed successively with water, aqueous K2HPO4, water and saturated aqueous NaCI, dried over MgSO,, filtered, evaporated and subjected to preparative thin-layer chromatography on silica gel; eluting with 1:2 CHC13:EtOAc. Yield 160 mg (58%), Rf 0.38, IR ( film) 2.98 NH and OH 5.63, ss-lactam; 5.86 broad ester and urethane; NMR (#, CDCl3), 1.24 (s, CHCH3, t-butyl), 2.59-3.27 (m, CH2) 3.83-4.47 (m, CH ss-lactam), 5.15 (s OCH2C6H4NO2), 5.22 (s OCH2C6H4 t-butyl (7.45 and 8.12 (AB quartet, J = 8Hz CeH4NO2), CeH4 t-butyl).

Example 9 Preparation of N-(p-Nitrobenzyloxycarbonyl)Thienamycin m-Phenoxybenzyl. Ester Following the procedure of Example 8, the title compound is prepared with an equivalent amount of m-phenoxybenzyl bromide is substituted for the p-t butylbenzyl bromide of Example 8. Yield 11%, IR (u film) 3.0 NH2, and OH 5.63 P- lactam; 5.86 broad peak ester and urethane; nmr (8 CHCI3) 1.33 (d CHCH3 J = 6); 2.603.62 (m, CH2), OCH2C8H40Cf,H5); 7.45 and 8.13 (AB quartet, J = 8, C ,H4NO2); 7.26 (s CsH40CeHs) M.S. m/e 589, 559, 547, 183.

Example 10 Preparation of: 1.) N-(O-Formyl-D-mandeloyl) Thienamycin p-t-butylbenzyl ester 2.) N-(O-Formyl-D-mendeloyl) Thienamycin m-phenoxybenzyl ester 3.) N-D-Mandeloyl Thienamycin p-t-butylbenzyl ester 4.) N-D-Mandeloyl Thienamycin m-phenoxybenzyl ester 5.) N-Propionyl Thienamycin p-t-butylbenzyl ester 6.) N-Propionyl Thienamycin m-phenoxybenzyl ester 7.) N-Methoxyacetyl Thienamycin p-t-butylbenzyl ester 8.) N-Methoxyacetyl Thienamycin m-phenoxybenzyl ester.

Following the procedure of Examples 8 and 9, title compounds 1., 3., 7 and 2., 4., 6., 8 are prepared respectively when the appropriate N-acyl thienamcyin starting materials (prepared in exact analogy to the above illustrated N-acyl thienamycin) replace, in equivalent amount, the N-(p-nitrobenzyloxycarbonyl) thienamycin starting material of Examples 8 and 9, respectively.

Example 11 Preparation of N-(p-Methoxybenzyloxycarbonyl)thienamycin p-t-B utylbenzyl Ester Step A: 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 equivalents) 5 ml. dioxane, and then, dropwise with stirring over 1 min., ten equivalents of p-methoxybenzyl chloroformate. After 15 min. the pH is adusted to 7.5 with IM H3PO4 and the solution extracted 3X with ether. The aqueous portion is then adjusted to pH 2.2 at OOC. and extracted 3X with ethylacetate (EtOAc). The EtOAc is dried quickly with MgSO4, filtered and extracted with a few ml. water containing 6.3 mg. NaMCO3. 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 is 16 mg. Electrophoresis (50 v/cm., 20 min., pH 7 aqueous phosphate, 0.05M) shows one spot by bioautograph, 4 cm towards the anode. NMR (#, D2O); 1.49 (d, J = 6Hz 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 = 8Hz CeH4) The lithium salt II is made in the same way, but extracting the EtOAc solution with 0.1N LiOH to pH 7.8 (instead of aqueous NaHCO3), and lyophilizing. The spectral and electrophoretic properties of II are the same as those of I.

N-(p-methoxybenzyloxycarbonyl)thienamycin-p-t-benzyl ester The lithium salt of Step A (37 mg.) in 0.4 ml. hexamethylphosphoramide (HMPA), is treated for 2 hours 12 minutes with 0.033 ml. p-t-butylbenzyl bromide.

The lithium salt is insoluble in HMPA but goes into solution after 15 minutes reaction time.

The reaction mixture is diluted with EtOAc, washed successively with water twice, aqueous K2HPO4, water and brine, dried with MgSO4, filtered, evaporated and subjected to preparative thin-layer chromatography on silica gel, eluting with 1:2 CHCl3-EtOAc, affording 47 mg. pure II, Rf=0.3. IR (u, film): 3.0, 5.63, p- lactam; 5.87 broad, ester and urethan. NMR (8, CDCI3): 1.21 (s Me and t-butyl); 2.6-3.6 (m, CH2); 3.72 (s, OMe); 3.8-4.4 (m, p-lactam CH); 4.97

6.84 and 7.20 (AB quartet, C8H4OMe); 7.32 (s, C6H4-t-Bu)..

MS: 582, 538, 496.

Example 12 Preparation of N-(p-methoxybenzyloxycarbonyl)thienamycin Benzhydryl Ester To Thienamycin (23.5 mug.) in 5 ml. water is added successively 4 ml. dioxane, 62 mg. NaHCO3, and then, in portions at OOC. with stirring, 4 equivalents p methoxybenzyl chloroformate over 4 minutes. After ten minutes total reaction time, the pH is adjusted to 7.0 with 1M H3PO4 and the mixture extracted three times with ether. Electrophoresis of the aqueous portion (0.05 M pH 7 aqueous phosphate buffer, 50V/cm, 20 minutes) shows 50% conversion to N-(p methoxybenzyloxycarbonyl)thienamycin.

The aqueous solution is brought to pH 2.2 with 1M H3PO4 at OOC. and extracted 3X with EtOAc. The EtOAc solution is treated with 50 mg. diphenyl diazomethane, evaporated and taken up in CH3CN. More diphenyldiazomethane is added to a persistent purple color. After 0.5 hour the solution is evaporated and chromatographed on silica gel, eluting with 1:2 CHCl2-EtOAc, affording 10 mg. pure title compound Rf 0.25. IR (,u, film): 3.0, NH; 5.63, p-lactam; 5.85, 5.89, ester and urethan. NMR (8, CDC13); 1.23 (s, OH); 1.30 (d, J = 6Hz, CH3CH); 2.63.6 (m, CH2); 3.78 (s, OMe); 5.02 (s 0CM2); 3.84.4 (m, p-lactam CH); 6.9 and 7.35 (AB quartet, J = 9Hz, C8H4), 7.3s CMPh2.

Example 13 Preparation of N-(o-Nitrobenzyloxycarbonyl)Thienamycin B enzyl Ester Step A: N-(o-Nitrobenzyloxycarbonyl)thienamycin Sodium Salt To Thienamycin (43 mg.) at 0 C is added 10 ml. 1:1 tetrahydrofuran (THF:Water). The mixture is rapidly stirred while 102 mg. NaHCO3 (10 equivalents) is added, and then, dropwise with stirring over 2 minutes, four equivalents of o-nitrobenzylchloroformate is added. After 30 minutes, the pH is adjusted to 7 with aqueous 25% H3PO4 and the solution extracted three times with ether. The aqueous layer is evaporated at 250C., in vacuo and is then adjusted to pH 2.2 at 0 C. Solid NaC1 is added, and the cold acidic solution is extracted 3x with cold EtOAc. The EtOAc extracts are combined and quickly back-washed with cold brine; dried with MgSO4, filtered and back extracted with 10 ml. of water containing 1.75 equivalents of solid NaMCO3. The extract is lyophilized in vacuo at 25"C. to provide the title compound.

Step B: N-(o-Nitrobenzyloxycarbonyl)Thienamycin Benzyl Ester The product of Step A in 7.5 ml. EtOAc (from the pH 2.2 extraction) is treated with an excess of phenyldiazomethane (4 ml. of solution comprising 20 mg./ml. ether) at 4 C. for 2.3 hours. The mixture is concentrated to wet residue at 20 C under reduced pressure. The desired compound is isolated by thin layer chromatography, EtOAc ether (9:1) to afford 17.5 mg. of N-(o-Nitrobenzyloxycarbonyl)thienamycin benzyl ester.

Example 14 Preparation of N-(o-Nitrobenzyloxycarbonyl)Thienamycin methoxy Benzyl Ester To N-(o-Nitrobenzyloxycarbonyl)thienamycin (70 mg.) in 8 ml. of EtOAc is added 4 ml. of p-methoxyphenyldiazomethane (Y mg/ml. acetonitrile) at 40C. The mixture is stirred for 1.5 hours at 40C. and is then concentrated to a wet paste under reduced pressure at 200 C. The title compound (42 mg.) is isolated by thinlayer chromatography on silica gel, eluting with EtOAc:ether (9:1).

Example 15 Preparation of N-(o-nitrobenzyloxycarbonyl)thienamycin p-Bromo-phenacyl Ester

[wherein TMS = trimethylsilyl] To Th (TMS3, I, (24 mg.), which is prepared according to Example 1, in 0.8 ml. dry TMF is added 23 mg. o-nitrocarbobenzyloxy chloride, followed by 0.015 ml. of triethylamine. After vibro-mixing for 30 minutes at 250C., the mixture is concentrated to a pasty residue in a stream of dry N2, and is washed 3x with petroleum ether. The residue is suspended in 1 ml. of dry THF, and p-bromophenacylbromide (14 mg.) is added, followed by 0.03 ml. triethylamine. After vibromixing for 30 minutes at 250C., the mixture Is evaporated to dryness in vacuo at 20"C. The residue is dissolved in EtOAc (2 ml) and shaken with 0.3 ml. of pH 4 buffer for 5 minutes. The organic layer is dried over MgSO4, filtered and evaporated to a pasty residue, and the desired product is ioslated (44 mg.) by preparative thinlayer chromatography on silica gel, eluting with EtOAc:CHCI3 (7:3).

Example 16 Preparation of N-(Trichloroethoxycarbonyl)thienamycin Benzyl Ester Step A: N-(Trichloroethoxycrbonyl)Lithium Salt To Thienamycin (40 mg.) in 18 ml. 1:1 TMF-M2O at OOC is added while stirring 225 mg. (15.2 equivalents) of NaMCO3, 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 aqueous 25% H3PO4 and the solution extracted with ether. The aqueous portion after removing any extrained ether in vacuo is then brought pH 2.5 at OOC. and extracted with cold EtOAc. The ethyl acetate extracts are combined, quickly backwashed with cold brine, dried with anhydrous MgSO4, filtered and back-extracted with 0.01 M LiOH to pH 6.8. The aqueous extract is freed from ay EtOAc in vacuo and lyophilized. The residual product 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 volts/cm. 20 min., pH 7 aq. phosphate 0.05 M) exhibits one zone by bioautograph (MB 108, staph. aureus)., 2.4 cm towards the anode. Liquid chromatography Cl8 Bondpak (Waters Assoc.) in aqueous 10% TMF exhibits one main peak free of any unreacted Thienamycin.

Step B: N-(Trichloroethoxycarbonyl)thienamycin Benzyl Ester The compound of Step A (32 mg.) in 2 ml. dry distilled DMF containing 7% HMPA (dry, pH 6.3), is treated with 0.015 ml. benzyl bromide for 2 hours at 150 C.

(allowing the contents to warm up to 250C. during the course of the reaction). The reaction mixture is diluted with EtOAc, washed successively with cold H2O, 1% aqueous NaHCO3, water and cold saturated aqueous NaC1, dried with MgSO4, filtered, evaporated and subjected to preparative thin-layer chromatography on silica gel, eluting with 1% CH3CN in EtOAc to afford 10 mg. of the title compound, Rf= 0.63; IR (u CHC13) 5.63, p-lactam; 5.78 and 5.88 broad ester and urethane.

NMR (8 CDCI3) 1.35 (d, Me); 2.8-3.7 (m CH2); 3.51 and 4.27 (dd, J=6Hz, A- lactam CH); 4.79 (s, OCH2CCI3) 5.42 s(OCH2C6Hs); and 7.41 (m, C6H5).

Example 17 Preparation of N-Bromoacetyl thienamycin Methyl and Benzyl Esters Step A: N-Bromoacetyl thienamycin 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 minutes. The pH is maintained at 8.0. The mixture is stirred for an additional 5 minutes then layered with 10 ml. of ether and the pH adjusted to 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 collocted. The solvent is changed to 10% THF and an additional 100 ml. collected. The combined eluates are adjusted to pH 7, evaporated to 5 ml. under reduced pressure, then freeze-dried to give the sodium salt of N-bromoacetyl thienamycin in 60% yield. UV AmaX 302 m,a.

Step B: N-Bromoacetyl thienamycin Methyl and Benzyl Esters An aqueous solution of the sodium salt is layered with ethyl acetate at OOC. and adjusted to pH 2. The ethyl acetate phase is separated and the aqueous phase is extracted with ethyl acetate. The combined ethyl acetate solutions are dried over MgSO4 and then treated with a solution of diazomethane. The solvents are evaporated and the residue chromatographed on silica gel plate Rf 0.11 in a 2:1 mixture of ethyl acetate and chloroform. m.p. 1 18-l200C. Mass spectrum shows M+ at m/e 406 and significant fragments at m/e 362, 320, 183 and 164.

The corresponding benzyl ester is prepared in a similar way from phenyldiazomethane. m.p. 142--30C.

Ir: 5.65,u, 5.89y and 6.1,u. Mass spec. M+ m/e 482 also m/e 438, 396, 316, 259 and 164.

Example 18 Preparation of N-(Guanylthioacetyl)-Thienamycin Methyl and Benzyl Esters Hydrobromide Salts A solution of 36 mg. of N-bromoacetyl-Thienamycin methyl ester and 150 mg. ofthioureain 4 ml. of dioxane is kept at230C. for 18 hours. The addition of 50 ml of ether gives a precipitate of N-(guanylthioacetyl)-thienamycin methyl ester as the hydrobromide which is recovered by filtration. The corresponding hydrobromide of N-(guanylthioacetyl)-thienamycin benzyl ester is prepared by following the same procedure except that the benzyl ester is substituted for the methyl ester.

Example 19 Preparation of N-(Bromo-t-Butoxycarbonyl)thienamycin p-Bromophenacyl Ester Step A: Preparation of N-Bromo-t-B utoxyvarbonyl-O-TMS-Thienamycin-TMS Ester Th(TMS)3 (16 mg.) is dissolved in 0.4 ml. of dry tetrahydrofuran to which is added 20 l (28 mg., 0.13 mmole) of bromo-t-butylchloroformate (b.p. 35 /0.9mm) and 8y1 (5.67 mg., 0.057 mmole) or triethylamine (redistilled from BaO). The mixture is shaken at 250C. for 20 min. Evaporated of solvent and excess of reagents gives the crude desired product. UV . CH3C02CH2CH3 320 nm (E9,000). max Step B: Preparation of N-(Bromo-t-Butoxycarbonyl) thienamycin The product of Step A (3 mg.) is dissolved in 0.5 ml. of pH 7 phosphate buffer and 0.1 ml. of tetrahydrofuran and the solution left at 250C., for 20 minutes. The solution is then passed down a column (5 ml.) of Dowex 50 x 8 (Na+ form) and the eluate fractions monitored by U.V. The correct fractions are combined and freezedried to yield the desired product. UV A b er 304 nm (e = 9,300); electrophoresis at 50V/Cm. 20 min in pH 7.0 buffer shows a single bioactive zone which moves 31.5 mm towards the anode.

Step C: Preparation of N-(Bromo-t-Butyoxycarbonyl) thienamycin p-Bromophenacyl Ester The product of Step B (13 mg. 0.022 mmol) is dissolved in 0.4 ml. of tetrahydrofuran. To this solution is added p-bromophenacyl bromide (9.6 mg., 0.035 mmol) and 20 l (14.4 mg., 0.14 mmol) of triethylamine. The mixture is shaken at 25 C. for 30 min. and then evaporated to dryness. Ten ml. of ether is added to the residue and the mixture treated with 0.2 ml. of 0.1M pH 7.0 phosphate buffer.

The organic layer is separated, dried over sodium sulfate, concentrated to 0.5 ml. and applied to two 20 x 20 cm. 250 y silica gel GF tic plates which are developed with 20% ethyl acetate in chloroform (Rf = 0.65). The desired product (6.7 mg.) is isolated in 42% yield.

Example 20 Preparation of N-Glycyl thienamycin 3-Methyl-2-buten-l-yl Ester

N-Azidoacetyl thienamycin 3-methyl-2-buten-l-yl ester (10 mg.) is dissolved in ethyl acetate (1 ml.). This solution is added to the hydrogenation flask containing 10 mg. of palladium (from palladium oxide) and 0.5 ml. 50% methanol in ethyl acetate. The mixture is stirred under I atm hydrogen at 250C. for 1 hour, then the mixture is filtered from the catalyst. The desired product is isolated by TLC (Rf = 0.16, in 200,o methanol/chloroform). Electrophoresis of the product at pH 7.0 buffer (50V/cm, 20 min.) shows one bio-active band which moves 34 mm towards the cathode Uv # maxethanol 315 nm, IR (CHCl3): 3300 (NH2), 1777 ( -lactam), and 1672 cm-1 (ester and amide).

Example 21 Preparation of N-Azidoacetyl thienamycin p-t-Butylbenzyl Ester N-Azidoacetyl thienamycin sodium salt (136 mg., 0.36 mmole) is stirred with HMPA (5 ,ml.) and p-t-butylbenzyl bromide (180 mg., 0.79 mmole) at 250C. for 30 min. The solution is diluted with 10 ml. ethyl acetate and washed thoroughly with water. The organic layer is separated and dried over sodium sulfate. The desired product (80 mg.) is isolated by silica gel TLC (Rf=0.18 in ethyl acetate IR (CHCl3): 2121 (N3) 1777 ( -lactam) and 1684 cm-1 (ester and amide); Pmr (CDCl3, 60 MHz): 1.32 (s, t-Bu), 1.34 (d, CH3CH), 2.60-3.75 (m, 3CH2 and C6-H), 3.90 (s, CH2N3), 4.30 (m, C5-H and C7-H), 5.21 (s,

6.90 (broad, NH), and 7.33 ppm (s, aromatic protons).

Example 22 Preparation of N-Glycyl thienamycin p-t-Butylbenzyl Ester N-Azidoacetyl thienamycinp4-butylbenzyl ester (10 mg.) is dissolved in 0.5 ml. ethyl acetate. The solution is added to the hydrogenation flask containing 50 mg. pf palladium (from palladium oxide) and 0.5 ml. ethyl acetate. the mixture is stirred under 1 atm hydrogen at 250C. for 10 minutes. TLC indicates that all the starting material has been consumed. The reaction mixture is filtered from the catalyst and evaporated to dryness to give the crude product. IR (CHC13): 1776 ( -lactam) and 1675 (ester and amide); Uv A ethxn ' 320 nm and 275 nm; Pmr (CHCl3 60 MHz):1.32 (s, t-Bu), 1.34 (d, CH3CH), 5.23

and 7.33 ppm (s, aromatic protons).

Example 3 23 Preparation of N-Azidoacetyl thienamycin Pivaloxymethyl Ester

N-Azidoacetyl thienamycin sodium salt (11.0 mg., 0.04 mmole) is stirred with HMPA (1 ml.) and chloromethyl pivalate (36 mg., 0.24 mmole) at 250C. for 30 min.

The mixture is diluted with ethyl acetate and washed with water. The desired product is isolated by silica gel TLC (Rf=0.18 in ethyl acetate)IR (CHCl3): 2121 (N3), 1777 cm-1 ( -lactam); Pmr (CDCl3, 60 MHz); 1.22 (s,t-Bu), 1.32 (d, CH3CH), 3.98 (s, CH2N3), and 5.83 ppm (dd, CO2CH2O-).

Example 24 Preparation of N-Azidoacetyl thienamycin 2-Methyl-2-Propene-l-yl Ester

N-Azidoacetyl thienamycin lithium salt (20 mg., 0.055 mmole) is stirred with HMPA (1 ml.) and 3-chloro-2-methylpropene (27 mg., 0.30 mmole) for 30 min. at 25 . The mixture is diluted with ethyl acetate and washed with water. The desired product is isolated by silica gel TLC Rf=0.18 in ethyl acetate) IR (CHCl3): 2121 (N3), 1777 ( -lactam), and 1684 cm-1 (ester and amide).

Example 25 Preparation of N-Glycyl thienamycin Esters

Compounds I-Il are prepared respectively, when the corresponding starting material is substituted in equivalent amount in the reduction process of Example 20, for the preparation of N-azidoacetyl thienamycin 3-methyl-2-butene-l-yl ester.

Example 26 Preparation of thienamycin, of N-benzyloxycarbonyl thienamycin and N benzyloxycarbonyl Benzylcarbonic Acid Anhydride

A solution of 16.6 mg. of Thienamycin in 4 ml. of0.05MpH 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 chloroform is added. The mixture is stirred at 60 C., pH 8.2, for ten minutes, then layered with ether and the pH adjusted to 7 by the addition of N 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-benzyloxycarbonyl Thienamycin Weight 10 mg.

(464d) The UV spectrum, AmaX 303 m,u, E% 147 (E 6,290) indicates about 80% purity. Electrophoresis at 50 V/cm. for 20 minutes at pH 7 followed by bioautographic on S. aureus gives a zone of inhibition at +2.5 cm.

The ethereal extracts of the reaction mixture contain the desired product Nbenzyloxycarbonyl thienamycin benzyl carbonic acid anhydride. UV;1,,, 335 m,u.

Example 27 Preparation of N-Benzyloxycarbonyl thienamycin Benzyl Ester The N-Benzyloxycarbonyl thienamycin [Example 26], in EtOAc is carried through the procedure of Example 26, except that an equivalent amount of phenyldiazomethane is added to the dried EtOAc solution from the pH 2 extraction and the solution left at 40 for 2 hours. Evaporation to dryness yields crude Nbenzyloxycarbonylthienamycin benzyl ester, which is isolated by thin layer chromatography R, 0.24 in a 3:1 mixture of ethyl acetate and chloroform: It crystallizes from ether. IR 5.63 y (lactam carbonyl); shoulder 5.8 ,u (ester); 5.88 y (urethane carbonyl). UV, dioxane, ARmaX 318 m,u, E% (E = 10,900). m/c M+ 496.

Example 28 Preparation of N-Carbomethoxy thienamycin p-pivaloyloxy-benzyl Ester Step A: N-Carbomethoxy thienamycin Thienamycin (49 mg., 148 cm mol) is dissolved in 0.05M pH 7 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 l, 600 mol) in pdioxane (580 l) is added at once to give a homogeneous solution. Subsequently. the pH is mainta Step B: N-Benzenesulfonyl thienamycin 2-Methyl-2-propen- 1 -yl Ester Following the procedure of Example 21 and substituting in equivalent amounts N-Benzenesulfonyl thienamycin (as its sodium salt) and 2-methyl-2-propen-1-yl chloride for the N-azidoacetyl thienamycin sodium salt and p-t-butylbenzyl bromide respectively, the title compound is obtained.

Example 29a Preparation of N-[N'-Acetimidoyl-p-alanyl]Thienamycin p-t-Butylbenzyl Ester Step A: N-[ss-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 NaMCO3, 30 ml of dioxane and 163 mg of p- azidopropionyl chloride. The mixture is stirred for 15 minutes, 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 50W x 8 (Na form) ion-exchange column (1" x 10"). The column is eluted with H2O to give 81 mg of the desired product which shows uv absorption at A mMa2Ox 306 nm.

Electrophoresis of the product at 2 KV in 0. IM pH 7.0 phosphate buffer for 2 hours shows a single bioactive zone which moves 30 mm towards anode.

Step B: N-(p-Alanyl)Thienamycin

The aqueous solution of N-(A-azidopropionyl)-Thienamycin (40 mg in 20 ml water) is hydrogenated under 1 atm of hydrogen in the presence of 200 mg of palladium at 250C., for 40 minutes. The resultant solution of pH 9.0 is neutralized with 30% H3PO4 and filtered from the catalyst. The mixture is chromatographed on a Dowex 50W x 8 (Na form) ion-exchange column (1" x 10") and the column is eluted with water to give 20 mg of the desired product, which shows uv E #H2O302 max nm. Electrophoresis of the product at 2 KV in 0.1M pH 7.0 phosphate buffer for 20 min. shows a single bioactive zone which moves 10 mm toward cathode.

Step C: N-[N'-Acetimidoyl-ss-alanyl]Thienamycin

The aqueous solution of N-(ss-alanyl)thienamycin (125 mg. in 15 ml. water) is kept at 0CC and maintained at pH 8.5 by adding 2.5N NaOH while O-ethyl- acetimidate 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 HCI and concentrated to 15 ml. The crude product so obtained is chromatographed twice on Dowex 50W x 8 (Na form) column (I" x 10") to yield 25 mg. The product is eluted with water and the solution lyophylized. Recrystallization of the product from water gives a crystalline solid which shows ir (Nujol mull): 1769 cm-1 (p- lactam); nmr (D2O, 100 MHz): 2.20 ppm (s, acetimidoyl CH2); uv #maxH2O302 nm. max Electrophoresis of the product at 2 KV in 0.1M pH 7.0 phosphate buffer for 20 min shows a single bio-active zone which moves 10 mm toward cathode.

Step D: N-[N'-Acetimidoyl-ss-alanyl]Thienamycin-p-t-Butylbenzyl Ester Following the procedure of Example 21, the title compound is obtained when the indicated substitutions are made.

Example 29b Preparation of N-Bromo-t-butyloxycarbonyl Thienamycin Sodium Salt Method A: Thienamycin (190 mg.) dissolved in 15 ml 0.1M pH 7.0 phosphate buffer and 15 ml. dioxane is kept at 0CC. The solution is adjusted and maintained between pH 8.5-9.0 with IN NaOH while 480 mg. of bromo-t-butyl chloroformate is added to the solution during a period of 5 minutes. The mixture is stirred for 30 min., then is neutralized to pH 7.0 with 1N HCl and extracted with ether. The aqueous layer is separated, concentrated to 10 ml. and chromatographed on a Dowex-50 x 8 (Na form) column (1.5" x 10") which is eluted with 1120 to give 113 mg of the desired product. Lyophilization of the solution gives solid product.

Method B: Thienamycin (95 mg) in 10 ml 0. IM phosphate buffer and 10 ml dioxane is kept at OOC. The solution is adjusted and maintained between 8.5-9.0 while 240 mg. of bromo-t-butyl chloroformate is added. The mixture is stirred for 30 minutes, then is acidified to pH 2.0 with H3PO4. The acidified solution is extracted with 2 x 25 ml. ethyl acetate. The organic layer is separated and backextracted with 10 ml NaHCO3 solution which contains 30 mg of NaHCO3. The aqueous layer, which contains 30 mg of the desired product, is lyophilized to give solid product. Nmr (60 MHz, D2O): X 1.26(d), 1.60(s), 2.65-3.50(m) 3.70(s), and 3.90-4.20(m). UV #maxD2O303 nm.

Example 29c Preparation of N-Bromo-t-butyloxycarbonyl Thienamycin p-Nitrobenzyl Ester The lyophilized N-bromo-t-butyloxycarbonyl-thienamycin sodium salt (100 mg) is stirred at 250C. with p-nitrobenzyl bromide (300 mg.) in 2 ml. hexamethylphosphoramide for 1 hour. The mixture is duluted with 10 ml ethyl acetate and then washed thoroughly with water. The organic layer is separated, dried over Na2SO4 and chromatographed on two 250y silica gel GF TLC plates using ethyl acetate as solvent (R, 0.45) to give 50 mg of the desired product. IR (CDCl3): 1777 (p-lactam) and 1711 cm-1 (ester): UV #maxEtOH270 nm and 322 nm; NMR (CDCl3, 60 MHz): # 1.38(d), 1.58(s), 2.603.80(m), 3.78(s), 3.90-4.20(m), 5.30(s), 7.55(d) and 8.30 ppm (d).

Example 30 Preparation of O-Dibenzylphosphoryl-N-(p-nitrobenzyloxycarbonyl) Thienamycin p-nitrobenzyl Ester

To a solution of N-(p-nitrocarbonbenzyloxy)-Thienamycin-(p-nitrobenzyl)- Ester (50 mg) in 5 ml. TMF at 30C. is added 30 mg. of dibenzyl phosphorochloridate followed by 14 ,al of triethylamine. The mixture is stirred at 250C for 2 hours, whereupon the TMF is removed in vacuo. The residue is taken up in methylene chloride and washed with water. The methylene chloride solution is dried over magnesium sulfate and evaporated. The residue is chromatographed on silica gel yielding 0 - dibenzylphosphoryl - N - (p - nitrobenzyloxycarbonyl Thieliamycin pnitrobenzyl ester.

Example 31 Preparation of O-(Methylcarbamoyl)-N-p-nitrobenzyloxycarbonyl-Thienamycin-p nitrobenzyl Ester

A solution of N-(p-nitroenzyloxycarbonyl)-thienamycin-(p-nitrobenzyl)ester (20 mg.) and methylisocyanate (20 mg.) in methylene chloride (5 ml.) is stirred at 23"C for 18 hours. The solvent is evaporated and the residue is extracted with hexane. The hexane insoluble residue is chromatographed on silica gel giving substantially pure O-(Methylcarbamoyl)-N-p-nitrobenzyloxycarbonyl-Thienamycin-p-nitrobenzyl ester.

Example 32 Preparation of O-(Methoxymethyl-N-(p-nitrobenzyloxycarbonyl-Thienamycin-p nitrobenzyl Ester

A solution of 58 mg. of p-nitrobenzyloxycarbonyl-Thienamycin-p-nitrobenzyl ester in 5 ml. of 1.0 ml. of THF and HMPA is cooled to -78 C. To this solution is added with stirring a 2N solution of phenyllithium (0.1 ml) immediately followed by the addition of 0.2 ml. of methylchloromethyl ether. The mixture is allowed to warm to 250C. during a period of one hour. Methylene chloride (25 ml.) is added and the solution is extracted with 0.1N pH 7 phosphate buffer (25 ml.) and water (4 x 25 ml.). The methylene-chloride solution is evaporated and the residue is triturated with hexane. The hexane insoluble residue is chromatographed on silica gel yielding O-methoxymethyl-N-(p-nitrobenzyloxycarbonyl-Thienamycin-(pnitrobenzyl)ester.

Example 33 Preparation of O-Methyl-N-Benzyloxycarbonyl-Thienamycin-B enzyl Ester A solution of 5 mg. of N-carbobenzyloxy thienamycin benzyl ester in 0.3 ml of methylene chloride is cooled to OOC. and 0.1 ml. of a .006M solution of fluoboric acid in a 5:1 mixture of ether and methylene chloride is added, followed immediately by 0.5 ml. of 0. lM diazomethane in methylene chloride. The solution is decolorized in 1 minute. The mixture is stirred with ether and pH 7 phosphate buffer and the ethereal phase is evaporated. The residue is chromatographed on 2 x 8" 250,u silica plates in 35% ethyl acetate in chloroform. The band at Rf 0.5 is eluted with ethyl acetate. Measurement of the U.V. absorbance at 318 nyl shows a 13% recovery of the initial optical density. Mass spectrum analysis shows m/e M+ 510 and fragments at m/e 410, 59, 333 and 478 characteristic of O-methyl-N-benzyloxycarbonyl-Thienamycin benzyl ester.

Alternate Preparation O-Methyl-N-B enzyloxycarbonyl-Thienamycin-B enzyl Ester solution of N-benzyloxycarbonyl-Thienamycin-benzyl ester (5 mg.) in 0.2 ml. dry THF is cooled to -78 C. To this solution is added 5 l of phenyllithium solution (2N) followed immediately by 1 l of methylfluorosulfonate. The mixture is stirred at -78"C. for 20 minutes and is then diluted with 2 ml. of ester and extracted with 2 ml. of 0.01N pH 7 phosphate buffer. The ethereal layer is evaporated and the residue is chromatographed on a 2D x 8" thin-layer silica gel plate in 1:1 ethylacetate-chloroform.. The band at Ref =0.63 is isolated giving the desired product, O-Methyl-N-benzyloxcarbonyl-Thienamycin-benzyl ester.

Example 34 Preparation of O-Acetyl-N-(p-Nitrobenzyloxycarbonyl)-Thienamycin-(p Nitrobenzyl)-Ester

R1 p-nitrobenzytoxycarbonyt R2=p-nitrobenzyl To a solution of 50 mg. of N-(p-nitrobenzyloxycarbonyl)-Thienamycin-(p- nitrobenzyl)ester in 0.5 ml. pyridine is added 0.16 ml. of acetic anhydride. The mixture is allowed to react at 250"C., for 3 hours, then pumped to dryness under vacuum. The solid residue is dissolved in chloroform and chromatographed on an 8" x 8" silica gel plate in a 3:1 mixture of ethyl acetate and chloroform. the band at Rf 0.55 is isolated yielding 36 mg. of O-acetyl-N-(p-nitrobenzyloxycarbonyl) thienamycin-(p-nitrobenzyl)ester; m.p. 172--175"C. NMR (CDCl3) 8, 1.41, (d J = 6 HZ CH3); 2.04 (s COCH3), 2.7-3.6 (m); 4.14 (t of d) (J = 3 and 9 Hz); 5.17 (s, carbamyl CH2); 5.35 (AB quartet J = 14 Hz), 7.45 (d), 7.60 (d), 8.18 (d) (J = 9Hz, aromatic H) Ms. M+ m/e 628.

Example 35 Preparation of N-(p-Nitrobenzyloxycarbonyl)Thienamycin O-methanesulfonyl p nitrobenzyi ester

R = p-nitrobenzyloxycarbonyl R = p-nitrobenzyl Methanesulfonyl chloride (34 mg.) in CH2CI2 (0.8 ml.) is added dropwise over 5 minutes to an ice-cold, stirred solution of N-(p-nitrobenzyloxycarbonyl) Thienamycin-p-nitrobenzyl ester (117 mg.) and triethylamine (40 mg.) in anhydrous CH2Cl2 (6 ml.). After stirring for 1.5 hours more, the reaction mixture is diluted with cold CH2Cl2 (20 ml.), washed with water (3"C., 10 ml.), 0.1M pH 3 phosphate buffer (3C., 5 ml.), and 2% aqueous NaHCO3 (3 C), dried with MgSO4 and filtered. Evaporation of the solvent in vacuo leaves p-nitrobenzyl-6-(1-methanesulfonyloxyethyl) - 3-[2 - (p-nitrobenzyloxycarbonylamino)ethylthio - 7 -oxo 1 - azabicyclo[3.2.0]-hept - 2 - ene - 2 - carboxylate (otherwise referred to as N (p-nitrobenzyloxycarbonyl)Thienamycine O-methanesulfonyl p-nitrobenzyl ester, for convenience) an an oil.

Example 36 When the N-acyl thienamycin carboxylic esters of Examples 8-29b are substituted in equivalent amounts for the N-acyl thienamycin carboxylic ester starting materials of Examples 2-7 and 3035, the corresponding 0--, N- and carboxyl thienamycin derivatives of the present invention are obtained.

Example 37 Following the procedures set out in the foregoing Examples and text the following compounds are obtained:

N.B. Compounds 14, 23 and 28 are not compounds in accordance with the present invention; the others all are.

Compound R3 R R2 X R1 1.) -C-M - CH24 H U H l! o 2.) -C-NH2 -CH2-0-C-C(CH2 )a H O H II II O 0 3.) C-S-CHa -CH2 CM2-CM3 -C-M O H O 0 4.) -C-O-CH2 -CM2-CH2-N(CM3 )2 -C-O-C113 0 H II o 0 5.) -C-CH, Br -CH2 CM2 CH2 -C-CM2 Br O H (I II o 0 6.) -C-CM2 NH2 H -C-CM2 NH2 0 H li 11 o 0 H .7.) -C-CM2-N(CH3 )2 H -C-CM2-N-C = NH2 O H II I 0 0 CM3.MCl 8.) -C-CH,OH -CH,-C-CH, -C-N-CM3 0 H II I H O CH2 0 9.) -C-S-CM3 -CM2-S-C=CH3 -C-CM2 Cl O H II II S O 0 10.) -C-CH2-0-CH3 CH2-C-;6 -C-CH2CH2NM2 O H ii Ii o 0 0 /NH 11.) C\ . MCI -CH2-CM2 = C-CM3 -C-CH2C - CH2 0 H NH2 CH3 O 12.) -C-CM2 N = C-NH2 H C-CH2-N= C-NH. H2 SO4 0 H II I 1! O H O o II H H 13.) C CH22NNCIINH2 . H3 PO4 -CH2-CH2-C - CH2 H O H N H

Compound R3 R R2 X R1 14.) -C-CH2-C0()H -CH,-O-C-O-CH, -C-NH2 O H II II 0 O NH 15.) -S03H - CH2O-C-CH3 H o H - II o 16.) )2 -cH,-o-c-c, + 16.) -CH2-0-C-C2H3 C-CH3N(CH3)3Cl O H N(CH3)2 0 0 17.) SO2 NH2 oM) C-0-CH2 0 M O oil 0 18.) SO2N(CH3)2- CH 2O CH(CH3)2 {-0-CM2,3'-NH2 H 19.) -CH2-N-(CM3 )3 -CH2-S-C-CH(CM3 )2 C-0-C-(CM2 Br) (CM3 )2 O H II II 0 0 20.) -CH2 CH2-OM -CH2-0-CH3 C-CF3 O H 0 21.) -CM2CH2-N(C2H5)2 -C2H5 C-0-CH2CF3 S H 0 22.) -CH2-C-CM3 !I Na C-CH2-0- S H 0 0 23.) -CH2-C- K - K H NOz o M 24.) -CH2-S-CH3 CH2S-CM3 -C-CH2CM2-N-C "'NH HCI O H o CH3

Compound R3 R R2 X Rl ~ 25.) -Si(CH3)3 -CH3 eocH3 -C-N-CM3 O H M 0 ONa 26.) C0CH2o-CH3 -CH2 -P 0 II 26.) '7.) -C-CM2CCl3 -CH2NO2 -C-C-CM H 0 0 0 Ij X8.) COC(CH2 Br) (CH3 )2 0-CH2-0-C-N-(CM3 ) -S-CF3 O H 0 29.) - C- O- CH2t 0-CM2-N-C-CM3 -C-S-CM3 O M oll , H oIl /0-CH, 30.) -CH,-O-C-CH, -CH,-O-C-CH, O O H il II ii\ 0 0 O OCH3 Example 38 Preparation of Pharmaceutical Compositions One such unit dosage form consists in mixing 120 mg. O-methanesulfonyl-N glycyl-thienamycin-(3-methyl-2-butene- l-yl) ester 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 O-methanesulfonyl-N-glycyl thienamycin -(3-methyl-2-butene- I -yl) 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 450C. and screened again through No. 16 screens. The balance of the cornstarch and the magnesium steatate is added and the mixture is compressed into tablets, approximately 0.5 inch in diameter, each weighing 800 mg.

PARENTERAL SOLUTION Ampoule: PER TABLET O-methanesulfonyl-N-glycyl-thienamycin (3-methyl-2-butene I -yl) 500 mg.

Diluent: Sterile Water for Injection 2 cc.

OPHTHALMIC SOLUTION O-methanesulfonyl-N-glycyl-thienamycin (3-methyl-2-butene- l-yl) 100 mg.

Hydroxypropylmethyl Cellulose 5 mg.

Sterile Water to I ml.

OTIC SOLUTION O-methanesulfonyl-N-glycyl-thienamycin (3-methyl-2-butene- 1 -yl) 100 mg.

Benzalkonium Chloride 0.1 mg.

Sterile Water to 1 ml.

TOPICAL OINTMENT O-methanesulfonyl-N-glycyl-thienamycin (3-methyl-2-butene- I -yl) 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 thereapeutic agents such as probenecid.

Preparation of Alternative 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 dll 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. K2HPO4 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 minutes 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 a N2 stream. The product crystallizes from fractions 46, 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. ether acetate containing 100 mg. of 10% Pd/C is hydrogenated on a Parr shaker at 250C. 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) # 3.44 (broad 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= 12.8Hz, J = 4.5 H JNH = 1.9 Hz, 7.38 ( AB pattern further split in four by C-4H and NH, 1, Jgem = 12.8Hz, J = 2 3Hz, JNH = 1.0Hz), 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 0 , 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 1 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) # 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 AB pattern further split in four by C-4H and NH, total 2, OH and C-3H respectivelyt, 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-oxo-1-azabicyclo [4.2.0]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- 1azabicyclo[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 ( -lactam) nmr (CDCl3)#: 6.02-6.28, m, 2H, C-4 methylene 622-6.62, m, 1H, C-6 methine 6.90, dd, 1H, J77 = 14Hz, J6,7 = 4.5Hz C-7 proton cis to C-6H 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, 3M C-2 methyls 8.57, s, 3H Step E: Preparation of 8-oxo-2,2-dimethyl-7&alpha; and P-(l -hydroxyethyl)-3 -oxa-l-azabi- cyclo[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 anydrous tetrahydrofuran which has been cooled to - 780C. 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]ocatane. Data for 8-oxo-2,2 dimethyl - 7 - (1-hydroxyethyl) - 3 - oxa - 1 - azabicyclo[4.2.0]octane: ir (CH2Cl) :5.72 ( -lactam) nmr (CDCl3)#: 5.53-S.43, m, 4H, C-4 methylene + C-6 methine + C-9 methine 6.90, dd on broad s, 2H, J7.9 = 9Hz J67 = 5.5Hz, C-7 methine + OH 7.0-8.83, m, 2H, C-S methylene

8.27, s, 3H C-2 s, 3HC--2 methyl 8.60, s, 3M 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 azybicyclo[4.2.0]octane: ir (CHCl3) : 2.9 broad O-H 5.73 ss-lactam nmr (acetone-d6)#: 4.23-3.33, m, C-9 methyine + C-4 methylene + C-S methine 3.33,broad s, OH

2.83, dd, j = 2Hz, 6Hz) C-7 dd, J = Hz, P--7 methine 2.67, dd, J=2Hz, 8Hz 1.93-1.63, m, CL5 methylene l.63,s C-2 C2 methyls 1.40, s 1.23, d, J = 6.5Hz C-l0 methyl Step F: Preparation of 8-Oxo-2,2-dimethyl-7a-( 1 -p-nitrobenzyloxy-carbonyl oxyethyl)-3 oxa- 1 -azabicyclo[4.2.0]octane

Under anhydrous conditions at 0 C. a solution of 8-oxo-2,2-dimethyl-7c-(1- hydroxyethyl)-3-oxa - I - 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 250C for an additional 15 hours. The mixture is partitioned between 1 M 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 nitrobenzyloxycarbonyloxyethyl) - 3 - oxa - I - azabicyclo[4.2.0Joctane (40 mg.) as a mixture of diastereomers. ir (CH2CI) : 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 5.98-6.25, m, 2H, c4 methylene 6.25-S.62, m, IH, C-S 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 7ss-diastereoisomers 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 - I - azabicyclo[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 - (i - p - nitrobenzyloxycarbonyloxyethyl) - 4 - (2 - hydroxyethyl) - 2 azetidinone as a mixture of diastereoisomers. ir (CH2Cl2) :5.67 (a-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 6.07-S.S3, m, IH, C--I methine 6.23, t, 2H, J = 5.5 Hz, CH2OH 6.73-6.93, 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-(1 -p-nitrobenzyloxycarbonyloxyethyl) - 4 - (2-hydroxyethyl)azetidinone

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

Under nitrogen and at 25 C, 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., 1.09 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-tetrahydro-pyranyl)oxyethyl] - 2 - azetidinone as an oil. nmr (CDCl3)#: 5.13-5.60, m, OCH

5.83-S.8S, m, C-4H + OCH2 6.95, dd, J = 5Hz 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 - I - (2 - tetrahydropyranyl) - 3 - (I - hydroxyethyl) 4 - [2 - (2 - tetrahydropyranyl)oxyethyi] - 2 - azetidinone

Following the procedure described for the preparation of 8-oxo-2,2-dimethyl-7a and 5-(1-hydroxyethyl)-3-oxa-1-azabicyclo [4.2.0] octane from 8-oxo-2,2-dimethyl3-oxo-l-azabicyclo [4.2.0] octane and using l-(2-tetrahydropyranyl)-4-[2-(2-tetra hydropyranyl)oxyethyl]-2-azetidinone one obtains a diastereomeric mixture of both cis and trans - 1 - (2 - tetrahydropyranyl) - 3 - (I - hydroxyethyl) - 4 - [2 - (2tetrahydropyranyl) - oxyethyl] - 2 - azetidinone.

Preparation of Cis and Trans - I - (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 - dimethyl7a - (I - p - nitrobenzyloxycarbonyloxyethyl - 3 - oxa - I - azabicyclo[4.2.0]- octane from 8 - oxo - 2,2 - dimethyl - 7a - (I - hydroxyethyl) - 3 - oxa - 1 - azabicyclo [4.2.0]octane and using trans - 1 - (2 - tetrahydropyranyl) - 3 - (I - hydroxyethyl) - 4 [2 - (2 - tetrahydropyranyl)oxyethyl] - 2 - azetidinone there is obtained trans - 1 (2 - tetrahydropyranyl) - 3 - (I - p - nitrobenzyloxycarbonyloxyethyl) - 4 - [2 - (2tetrahydropyranyl)oxyethyl] - 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 - 1 - (2 - tetrahydropyranyl) - 3 - (I - p - nitrobenzyloxycarbonyloxyethyl) - 4 - [2 - (2 - tetrahydropyranyloxyethyl] - 2 - azetidinone in methanol at 250C. 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 - (I p - nitrobenzyloxycarbonyloxyethyl) - 4 - (2 - hydroxyethyl) - 2 - azetidinone.

The cis diastereoisomers are obtained in an analogous manner.

Step H: Preparation of Cis and Trans - 3 - (I - 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 trioxlde (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 -1-p - nitrobenzyloxycarbonyloxyethyl) - 4 - (2 - hydroxyethyl) - 2 azetidinone (186 mg, 550 mmole) in 1 ml. anhydrous acetonitrile. After stirring for a period of 1 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 starting material (Rf = .21).

The acetonitrile solution of trans - 3 - (1 - p - nitrobenzyloxy - carbonyloxyethyl) - 4 - (2 - oxoethyl) - 2 - azetidinone prepared above is, under nitrogen and at 00, 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 - (1 - p - nitrobenzylozycarbonyloxyethyl) - 4 - [2,2 - bis(2 - hydroxyethyl)thioethyl] - 2 - azetidinone as a colorless oil. ir (CH2CI2) L 5.75 (5.79 shoulder) p-lactam and carbonate 6.20, 6.55 nitro nmr (acetone-d6)#: 1.70, d, J = 8.5 Hz, 2H, ArH 2.28, d, J = 8.5Hz, 2H ArH 2.48-2.88, m, 1H NH

4.63, s, ArCH2 # 3H 4.63-5.12, m, CH3CH S # 5.80, t, J = 7Hz, CH2CH 13H S 5.80-7.45, m, C-4H + C-3H + SCH2CH2OH 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 mixture diastereoisomers are obtained when the starting materials comprise a mixture of the diastereoisomers.

Step I: Preparation of Trans - 3 - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4 - [2,2 bis(2 - azidoethyl)thioethyl] - 2 - azetidinone

To a solution of 211 mg (mw = 474; 0.445 mmole) trans - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4 - [2,2 - bis(2 - hydroxyethyl)thioethyl] - 2 - azetidinone in 5 ml. tetrahydrofuran (THF) (distilled from lithium aluminium hydride) at OOC. is added 103 mg. mesyl chloride (mw = 114; 0.904 mmole) in 1 ml. THF followed immediately by 134 yI triethylamine (mw= 101, p=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 N2O stream to give the crude diazide. Preparative thin layer chromatography on silica gel yields trans 3 - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4[2,2 - bis(2 - azidoethyl)thioethyl] - 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. Schwartz, 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. tolune (Tol.) is added 3.54 g. of trans - 3 - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4 - [2,2 - bis(2 azidoethyl)thioethyl] - 2 - azetidinone (mw = 524; 6.75 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 aeodrying process is repeated three additional times. The solution is then refluxed 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 The crude material is chromatographed on silica gel to give 1. The cis diastereoisomers 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 -200C 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. 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 -200C, then 1/2 hour at OOC. and finally I hour at 250C.

The pyridine hydrochloride is filtered under N2 and washed twice with benzene (dried over 3A 1/1 6" Linde Molecular sieves). The combined filtrate and washlngs 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 0.885g triphenyl phosphine (mw = 262; 3.38 mmole) in 66 ml. 9:1 dimethylformamide (DMF)/H20 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:

Te 7.8 ml pentane (dried over 4A Linde molecular sieves) is added 0.2 ml. Br (mw = 160; p = 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 OOC. 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 ,ul 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 sieves). 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. 1M KH2PO4--I0 ml. M2O-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 an hydros MgSO4, filtered and concentrated under a N2 stream followed by pumping on a high vacuum pump to provide crude I Preparative thin layer chromatography on silica gel yields 3 The cis diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

Step M:

To 9.16 ml. pentane (dried over 4A Linde Molecular sieves) 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 sieves) 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 0 C, 33 mg 57% NaH (57% 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 sieves). The reaction mixture is stirred for 1 1/2 hours at OOC., then poured into a stirred icecold mixture of 1.6 ml. 1M KH2PO4-20 ml. H2O, and 20 ml. EA. The layers are separated and the aqueous one saturated with NaCI and re-extracted with additional EA. The combined organic layers are washed 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 8. Preparative thin layer chromatography on silica gel gives 4. The cis diasteroisomers or the cis-trans mixture is obtained in an analogous manner.

Step N:

To 210 mg. R(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 250 C. with stirring 40 mg 1,5-Diazolicyclo[5.4.0]undec-5-ene (distilled at 800 C./2 mm) (mw= 152; 0.263 mmole) in 0.7 ml dimethylsulfoxide (DMSO). The solution is stirred under N22 for 4 hours, and then added to a stirred ice-cold mixture of 0.48 ml.

'lM KH2PO4, 7 ml. H2O, and 10 ml. EA. After separation of the layers, the aqueous layer is again extracted with EA. The combined organic layers are washed one time with brine, dried over anhydrous MgSO4, filtered, and concentrated under a N2 stream followed by pumping on high vacuum, to provide crude4 Preparative thin layer chromatography on silica gel yields 5. The cis diasteroisomers or the cis-trans mixture is obtained in an analogous manner.

Step O:

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 P2O5 under vacuum) (mw = 134; 0.336 mmole). With stirring under N2, the reaction mixture is heated in an oil bath at 120 C. After a total of 25 minutes, the reaction mixture is cooled to 25 C., diluted with CH2Cl2 and transferred to a roundbottom falsk for concentration under a N2 stream and then on high vacuum. Partitioning of the residue between 10 ml EA and 1.8 ml. 1M KH2PO4 in 10 ml. H2O is followed by extraction of the aqueous layer two additional times with EA. Thecombined 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 disasteroisomers or the cix-trans mixture is obtained in an analogous manner.

Step P:

To a solution of the mixed diastereoisomers, 4, (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 pi 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-l000,u silica gel plates. The product band yields 7 as a mixture of cis and trans diastereoisomers.

Step Q:

In the presence of 61 mg. PtO,, 61 mg. of 7 (mw=612: 0.1 mmole in 6 mi dioxane, 6 ml. THF, 3 ml. H2O is hydrogenated at 40 p.s.i. -H2 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 is applicable for all isomers of thienamycin-particularly the distinct N-acetyl isomers 890A, and 890AS, which are described below.

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

1 M Phosphate Buffer, pH 7.5* 10 ml.

Distilled H2O 1000 ml.

*1M 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 1M K2HPO4.

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

One-ml. portions of the stock suspension or 1 ml. portions of the 10x, 100x and 1,000x dilutions are added to 2-ml. 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 7.0 g, MgSO4 0.1 g.

Distilled H2O 1000 ml.

N-Acetylethanol-amine solution* 8.5 ml.

*N-acetylethanolamine Solution N-acetyl ethanolamine is diluted 10x 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 28"C. 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 280C 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 Pro tam inobacter 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 H20 1000 ml.

Corn Steep Liquor (wet basls) 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,ul portion of the sonicate is mixed with 25 ,ul of a solution containing 840 g/ml. of N-acetylthienamycin in 0.01M 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 28cC., 2 ,ul quantities are applied on cellulose coated TLC plates, which are developed in EtOH:H2O, 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 essay 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 37"C. 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 cm x 22.5 cm. petri plates, and these plates are chilled and held at 40C. until used (5-day maximum).

The TLC plate is removed and the assay plate incubated over-night at 370 C. In addition to the unreacted bioactive N-acetyl thienamycin spot at R, 0.74.89; a bioactive spot is observed at R, 0.440.47 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 0.440.47.

Antibiotics 890A, and 890A3 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 then be treated to cleave the N-acetyl group as above described to provide the corresponding free base.

Claims (36)

WHAT WE CLAIM IS:

1. A compound having the structural formula:

where X is oxygen, sulphur or NR' (R' = H or R); R is hydrogen; acyl; trialkylsilyl; C1-10 alkyl, phenacyl, nuclear-substituted phenacyl in which the substituent is chrolo, bromo, fluoro, or C1-6 alkyl; (C1-10 alkoxy)-(C1-6 alkyl) in which the alkoxy residue is open-chain or cyclic; C2-12 alkanoyloxyalkyl; halogenated C1-6 alkyl in which the halo is chlorine, bromine and/or fluorine; C2-10 alkenyl; C3-14 alkoxycarbonylalkyl; C4-21 dialkylaminoacetoxyalkyl; C2-13 alkanamidoalkyl; aralkyl in which the alkyl residue has 1 to 3 carbon atoms and the aryl residue 6 to 10 carbon atoms; monocyclic and bicyclic heteroaralkyl having 4 to 10 ring atoms and 1 to 6 carbon atoms in the alkyl residue, the hetero atom or atoms being oxygen, sulphur and/or nitrogen; nuclear-substituted aralkyl and heteroaralkyl in which the substituent is chlorine, fluorine, bromine, iodine, C16 alkyl, C16 alkanoyloxy or C18 alkoxy; mono- and bicyclic heterocyclylalkyl in which the heterocycle comprises 4 to 10 atoms and the hetero atom or atoms is oxygen, sulfur and/or nitrogen and the alkyl residue comprises 1 to 6 carbon atoms; aryl and nuclear-substituted aryl comprising 6 to 10 ring atoms and in which the nuclear substituent is hydroxy, alkyl comprising 1 to 6 carbon atoms, chloro, fluoro or bromo; (C1-10 alkylthio)- (C,~6 alkyl), cycloalkylthioalkyl comprising 4 to 12 carbon atoms; acylthioalkyl in which the acyl residue comprises 2-10 carbon atoms and the alkyl residue comprises 1 to 6 carbon atoms; each of R and R is hydrogen or acyl; and R is an acyl radical as hereinbefore defined or a radical such that the group -OR is classifiable as an ether; R, R and R are not all hydrogen at the same time; and the pharmaceutically acceptable salts thereof.

2. A compound according to Claim 1 in which R3 is a radical of formula:

in which Xis O or S; p is 0 or 1; n is 0, 1, 2, 3 or 4; Z is O.

is hydrogen, amino, mercapto, hydroxy, substituted amino, substituted mercapto, substituted hydroxy, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl or heteroaralkyl, provided that R" is not mercapto or hydroxy unless Z or R"' is present, that R" is not mercapto or hydroxy if Z is oxygen, that R" is not amino or hydroxy if Z is sulfur, that R" is not mercapto if Z is nitrogen, that when @ is O R" in the forst formula is not hydrogen, amino, mercapto, hydroxy, substituted amino, substituted mercapto or substituted hydroxy, and that in the second formula p + n is not 0; and R"' is azido, carbamoyl, guanidino, amidino, acyloxy, halo, sulfamino, sulfo, tetrazolyl, carboxy, alkoxycarbonyl, phosphono, alkoxy or arylthio.

3. A compound according to Claim 2 in which R" is hydrogen, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-6 alkyl, C1-6 alkylthio, C6-10 aryltio, C1-6 alkoxy, C6-10 aryloxy, C2-6 alkenyl, C2-6 alkynyl, phenyl, benzyl, C3-6 cycloalkyl, or da monocyclic or bicyclic heteroaryl or heteroaryl(C1-3 alkyl) radical in which the heterocyclic ring(s) contains(s) from four to ten atoms, including at least one heteroatom from among oxygen, nitrogen and sulfur, the above mentioned hydrocarbon and heterocyclic radicals optionally having one or more hydroxy, mercapto, C1x6 alkylthio, arylthio, C1-6 alkoxy, C1-6 alkyl, halogen, cyano, carboxy, sulfamino, carbamoyl, sulfonyl, azido, amino, (C1-6 alkyl)-substituted amino, quaternary ammonium, C1-6 halogenated alkyl, carboxy-(C1-6 alkyl), carbamoyl-(C1-6 alkyl), N-substituted carbamoyl-(C1-6 alkyl), amidino, guanidino, N-substituted guanidino and/or guanidino-(C1-6 alkyl) substituents.

4. A compound according to Claim 1 in which R3 is a radical of formula:

in which X is O or S; each of m and n, independently of the other, is 0 or 1; Y is oe M+, -NR2* or R*; where M+ is a hydrogen atom, an alkyli metal or alkaline-earth metal cation, or an organic base; and R* is hydrogen, amino, mercapto, hydroxy, alkylamino, dialkylamino, alkyl, alkylthio, arylthio, alkoxy, aralkoxy, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, or heteroaralkyl; and each of Y' and Y", independently of the other, is on Mn, -NR*2, or R* where M and R* are as defined above; or Y' and Y" are joined to form, together with the phosphorus atom to which they are attached, a cyclic ester or amide.

5. A compound according to Clain 1 in which R is a radical formula:

in which each of p and q, independently of the other, is 0 or 1; each of m and n, independently of the other, is 0, 1, 2, 3, 4 or 5; A is O, -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 an amidino or substituted amidino radical of formula:

a guanidino or substituted guanidino radical or 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 alkoxy; (C1-6 alkoxy--(C2-6 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)-C1-3 alkyl; and R** is a radical as defined for R or a (C1-6 alkoxy)methyl radical; or Y is a monocyclic or bycyclic 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.

6. A compound according to Claim 1 in which R' is hydrogen and R3 is a radical of formula:

in which p is 0 or 1 and R is benzyl, p-hydroxybenzyl, 4 - amino - 4 - carboxy butyl, methyl, 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, 3 - butyl - 5 - methyl - 4 - isoxazolyl, 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, D - 4 - N - benzoylamino - 4 - carboxy - n - butyl, p - aminobenzyl, 0 - amino- benzyl, m - aminobenzyl, p - dimethylaminobenzyl, (3 - pyridyl)methyl, 2 ethoxy - 1 - napthyl, 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 - guanidino- methylbenzyl, 4 - guanidinobenzyl, 4 - guanidinophenyl, 2,6 - dimethoxy - 4 guanidinophenyl, o - sulfobenzyl, p - carboxymethylbenzyl, p - carbamoylmethylbenzyl, m - fiuorobenzyl, m - bromobenzyl, p - chlorobenzyl, p - methoxybenzyl, 1 - naphthylmethyl, 3 - isothiazolylmethyl, 4 - isothiazolylmethyl, 5 isothiazolylmethyl, guanylthiomethyl, 4 - pyridylmethyl, 5 - isoxazolylmethyl, 4 methoxy - 5 - isoxazolylmethyl, 4 - methyl - 5 - isoxazolylmethyl, 1 - imidazolylmethyl, 2 - benzofuranylmethyl, 2 - indolylmethyl, 2 - phenylvinyl, 2 - phenylethynyl, 1 - aminocyclohexyl, 2- and 3 - thienylaminomethyl, 2 - (5 - nitrofuranyl)vinyl, phenyl, o - methoxyphenyl, o - chlorophenyl, o - phenylphenyl, p aminomethylbenzyl, 1 - (5 - cyanotriazolyl)methyl, difluoromethyl, dibromomethyl, 1-(3-methylimidazolyl)methyl, (2 or 3) - (5 - carboxymethylthienyl)methyl, (2 or 3) - (4 - carbamoylthienyl)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) - (carboxythienyl)methyl, 3 (1,2,5 - thiadiazolyl)methyl, 3 - (4 - methoxy - 1,2,5 - thiadiazolyl)methyl, 2 - furylmethyl, 2 - (5 - nitrofuryl)methyl, 3 - furylmethyl, 2 - thienylmethyl, 3 - thienyl- methyl, tetrazolylmethyl, benzaminomethyl, cyclohexylamidinomethyl, 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 trimethylammoniummethyl, non-toxic salt, cyanomethylthiomethyl, trifluoromethylthiomethyl, 4 - pyridylethyl, 4 - pyridyipropyl, 4 pyridylbutyl, 3 - imidazolylethyl, 3 - imidazolylpropyl, 3 - imidazolylbutyl, 1 - pyrrolylethyl, 1 - pyrrolylpropyl, 1 - pyrrolylbutyl, &alpha; - aminobenzyl, &alpha; - amino - (2 - thienyl)methyl, &alpha; - (methylamino)benzyl, &alpha; - amino - methylthiopropyl, &alpha; amino - (3- or 4-)chlorobenzyl, a - amino - (3 or 4) - hydroxybenzyl,. a - amino 2,4 - dichlorobenzyl, &alpha; - amino - 3,4 - dichlorobenzyl, D(-) - &alpha; - hydroxybenzyl, &alpha; - carboxybenzyl, &alpha; - amino - (3 - thienyl)methyl, D(-) - &alpha; - amino - 3 - chloro4 - hydroxybenzyl, &alpha; - amino(cyclohexyl)methyl, &alpha; - (5 - tetrazolyl) - benzyl, 2 - thienyl - carboxymethyl, 3 - thienylcarboxymethyl, 2 - furyl - carboxymethyl, 3 - furyl - carboxymethyl, &alpha; - sulfaminobenzyl, 3 - thienyl - sulfaminomethyl, &alpha; -(N - methylsulfamino) - benzyl, D(-) - 2 - thienyl - guanidinomethyl, D(-) - &alpha;- guanidinobenzyl, &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 sulfodiazolyl) - hydroxymethyl, 4 - (5 - chlorothienyl) - aminomethyl, 2 - (5 chlorothienyl) - hydroxymethyl, 2 - (5 - chlorothienyl) - carboxymethyl, 3 - (1,2thiazolyl) - 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;-sulfobenzyl, &alpha;- phosphonobenzyl, &alpha; - diethylphosphono, &alpha; - monoethylphosphono or a radical of formula:

where Q- is a non-toxic anion.

7. A compound according to Claim 6 in which R2 is formyl, propionyl, butyryl, chloroacetyl, methoxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl, methylcarbamoyl, ethylcarbamoyl, phenylthiocarbonyl, 3-aminopropionyl, 4 aminobutyryl, N - methylaminoacetyl, N,N - dimethylaminoacetyl, an N,N,Ntrimethylaminoacetyl non-toxic salt, 3 - (N,N - dimethyl)aminopropionyl, an 3 (N,N,N - trimethyl)amino propionyl, N,N,N - triethylaminoacetyl, or pyridiniumacetyl non-toxic salt, guanylthioacetyl, guanidinoacetyl, 3 - guanidinopropionyl, N3 - methylguanidinopropionyl, hydroxyacetyl, 3 - hydroxypropionyl, acryloyl, propynoyl, malonyl, phenoxycarbonyl, amidinoacetyl, acetamidinoacetyl, aminopropionyl, acetamidopropionyl, guanylureidoacetyl, guanylcarbamoyl, carboxymethylaminoacetyl, sulfoacetylaminoacetyl, phosphonoacetylaminoacetyl, N3- dimethylaminoacetamidinopropionyl, ureidocarbonyl, dimethylaminoguanylthioacetyl, a 3 - (1 - methyl - 4 - pyridiniumpropionyl non-toxic salt, 3 - (5 - aminoimidazol - 1 - yl)propionyl, a 3 - methyl - 1 - imidazoliumacetyl non toxic salt, 3 - sydnonylacetyl, o - aminomethylbenzoyl or o - aminobenzoyl.

8. A compound according to Claim 1 in which R3 iS

where M is hydrogen or an alkali metal or alkaline-earth metal cation.

9. A compound according to Claim 6 in which X is oxygen and R is hydrogen, methyl, t-butyl, phenacyl, p-bromophenacyl; pivaloyloxymethyl, 2,2,2-trichloroethyl, allyl, 3-methyl-2-butenyl, 2 - methyl - 2 - propenyl, benzyl, benzhydryl, p t-butylbenzyl; phthalidyl, phenyl, 5 - indanyl, acetylthiomethyl, acetoxymethyl, propionyloxymethy, methallyl, 3 - butenyl, 4 - pentenyl, 2 - butenyl, acetoxyacetylmethyl, pivaloylacetylmethyl, diethylaminoethyl, dimethylaminoethyl, methoxymethyl, p-acetoxybenzyl, p - pivaloyloxybenzyl, p - isopropoxybenzyl, 5 indanylmethyl, benzyloxymethyl, methylthioethyl, dimethylaminoacetoxymethyl, crotonolaction - 3 - yl, acetamidomethyl, acetylthioethyl, pivaloylthiomethyl or methyithiomethyl.

10. A compound according to Claim 1 in which R3 is -SO3H and X is oxygen.

11. A process for preparing a compound according to Claim 1 that comprises reacting a compound of the formula:

with an acylating alkylating or arylating reagent capable of introducing the radical R3.

12. A process as claimed in Claim 10 in which X is oxygen.

13. A process as claimed in Claim 11 or 12 in which the said reagent is an activated acyl, alkyl or aryl halide.

14. A process for making a compound as claimed in Claim I, substantially as hereinbefore described in any one of Examples I to 7 and 31 to 36.

15. A compound according to Claim 1 when prepared by a process as claimed in any one of Claims 11 to 14.

16. A pharmaceutical composition comprising a compound according to Claim 1 and a pharmaceutical carrier.

17. A pharmaceutical composition comprising, in unitary dosage form, a therapeutically effective amount of a compound according to Claim I and a pharmaceutical carrier.

18. A pharmaceutical composition as claimed in Claim 15, in the form of a capsule, tablet, powder, elixir, aqueous or oily solution or suspension, emulsion or syrup.

19. A composition as claimed in Claim 16, in orally administrable form.

20. A composition as claimed in Claim 16, in intravenously administrable form.

21. A composition as claimed in Claim 16, in intramuscularly administrable form.

22. A composition as claimed in Claim 16, in the form of a suppository.

23. A composition as claimed in Claim 16, in a form suitable for absorption through the mucous membranes of the nose and throat or bronchial tissues.

24. A composition as claimed in Claim 23, in the form of a liquid spray or inhalant, a lozenge or a throat paint.

25. A composition as claimed in Claim 16, in aurally or optically administrable form.

26. A composition as claimed in Claim 16, in topically administrable form.

27. A composition as claimed in Claim 26, in the form of an ointment, cream, lotion, paint or powder.

28. An antibacterial composition comprising as active ingredient a compound as claimed in Claim 1 together witrh a material in respect of which antibacterial action is desired.

29. A composition as claimed in Claim 28, in which the said material is a human or aninial foodstuff.

30. A disinfectant comprising as active ingredient a compound as claimed in Claim 1 together with a suitable diluent or carrier.

31. A composition as claimed in Claim 28, in which the said material is a waterbased paint.

32. A composition as claimed in Claim 28, in which the said material is the white water of a paper mill.

33. A veterinary composition comprising as active ingredient a compound as claimed in Claim 1 together with a non-toxic base material.

34. A composition as claimed in Claim 33, in the form of an intramammary preparation.

35. A composition as claimed in any one of Claims 15 to 34. in which the said compound is a compound as claimed in any one of Claims 2 to 10.

36. A composition as claimed in Claim 16 substantially as hereinbefore described in Example 38.