GB1589896A - Thienamycin and its isomers - Google Patents

Description

(54) THIENAMYCIN AND ITS ISOMERS We, MERCK & CO. INC., a corporation duly organised 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 since, unfortunately, there is no static effectiveness of any given antibiotic because continued wide-scale usage selectively give 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.

The antibiotic 3 - (2 - aminoethylthio) - 6 - ( I - hydroxyethyl) - 7 - oxo - I azabicyclo[3.2.0]hept - 2 - ene - 2 - carboxylic acid, otherwise known as thienamycin, which has the formula

is disclosed and claimed in our prior U.S. Patent No. 3,950,357 issued April 13, 1976 and in our prior U.K. Patent No. 1,498,087. These patents disclose the utility of thienamycin as an antibiotic in animal and human therapy and in inanimate systems. The present invention provides certain novel isomeric forms of I, which isomeric forms, individually and as mixtures, are useful as antibiotics.

The absolute configuration of thienamycin is 5R, 6S, 8R. Two other thienamycin isomers are known. They are useful as antibiotics and are isolated as natural products of fermentation. These isomers, known as "desacetyl 890At" and "desacetyl 890A3" are disclosed and claimed in the specification of our copending Patent Application No. 48233/76, (Serial No. 1561108). The corresponding N-acetyl fermentation product derivatives, which are known as 890A, and 890A3, respectively, are disclosed and claimed in the specification of our copending Patent Application No. 48235/76, (Serial No. 1561109). Desacetyl 890A3 has been assigned the absolute configuration 5R,6S,8S; desacetyl 890A1 has been assigned the absolute configuration 5R,6R,8S. Thus'three of the possible eight thienamycin isomers are known. The remaining five isomers may be designated, relative to formula I, by absolute configurations 5R,6R,8R; 5S,6S,8S; 5S,6S,8R; 5S,6R,8S; and 5S,6R,8R. These five isomers, and their pharmaceutically acceptable salts, constitute embodiments of the present invention. By practice of the total synthesis disclosed below, which constitutes another embodiment of the present invention all eight isomers of I are made available as a mixture of the four diastereoisomers.

This mixture possesses antibacterial activity and is amenable to resolution by conventional techniques to provide any desired isomer in substantially pure form.

Thus, for example, when resolving the mixture, the 4 diastereoisomers (2 cis, 2 trans) are separable by chromatography; and resolution of any given d/I pair with optically active acids or bases proceeds according to conventional techniques.

Alternatively, the total synthesis may be conducted in a stereo-selective manner to provide any given isomer.

It has been found that compounds of the present invention are broad-spectrum antibiotics, which are useful in animal and human therapy and in inanimate systems. These antibiotics are active against a broad range of pathogens which representatively include both gram-positive bacteria such as S. aureus; Strep.

pyogenes, and B. subtilis and gram-negative bacteria such as E. cold, Proteus morgan ii, Serratia, Pseudomonas and Klebsiella.

The compounds of the present invention (1, above) may unambiguously be identified by absolute configuration when referenced against the numbering system of structure I. The following projection drawings may be helpful in visualizing the compounds (isomers) in question and the three known isomers:

I desacetyl 890A11

natural fermentation product. ' thienamycin"

(desacetyl 890A3) The total synthesis of the present invention starts from Compound 17 in the following reaction diagram, which also indicates how Compound 17 is prepared from simple starting materials:

In words relative to the above diagram, the 4 - (2 - substituted vinyl)azetidine - 2 - one, 4, starting material is prepared by reacting an R' oxybutadiene, Q, with chlorosulfonyl isocyanate 2. The reaction is conducted without a solvent or may be run in a solvent such as diethyl ether, ethyl acetate, chloroform or methylene chloride at a temperature of from -78 C to 250C for from a few minutes to 1 hour to provide 3. The radical R1 is an easily removable acyl blocking group such as an alkanoy or aralkanoyl group which bears no functional group or groups which might interfere with the desired course of the reaction (1+2 - > 4). Intermediate compound 2 is converted to the sulfinamide by reduction and Is then hydrolysed to 4 at pH 6--8. Typically the reaction solution comprising ,Q, is contacted (5-30 minutes) with an aqueous solution (at 0.250C) of a reducing agent such as sodium sulfite or thiophenol at pH 6-8 to provide 4.

The reaction 4,5 is a reduction, and is preferably achieved by hydrogenation in a solvent such as ethyl acetate, ether, dioxane, tetrahydrofuran (THF) F) or ethanol at 0 to 250C for from 5 minutes to 2 hours under 1 to 10 atmospheres of hydrogen in the presence of a hydrogenation catalyst such as platinum metal or an oxide thereof or 10% Pd/C.

The de-blocking reaction 5 - > 6 is usually desirable when R' is acyl to permit the later alkylation, 7 - > 8. The preferred de-blocking procedure is by alcoholysis in which the solvent is a lower alkanol such as methanol or ethanol in the presence of the corresponding alkali metal alkoxide, such as sodium methoxide. Typically, the reaction is conducted for from 5 minutes to 1 hour at a temperature of from -10" to 25"C.

Blocking groups R3 and R2 are established (t,) to provide a suitably protected species for alkylation (D,). There is no criticality in the choice of blocking groups, provided only that they do not interfere with the intended alkylation. R3 may be hydrogen, a triorganosilyl group such as trimethylsilyl, or a cyclic ether group such as 2 - tetrahydropyranyl; R2 may also be a cyclic ether group such as 2 - tetrahydropyranyl: alternatively R3 and R2 may be joined together to form a protected compound such as

For example, compound 7a is conveniently prepared by treating 6 with 2,2 dimethoxypropane in the presence of a catalyst such as boron trifluoride etherate or toluene sulphonic acid in a solvent such as methylene chloride, ether, chloroform or dioxane at a temperature of from -10 C to 35 C for from a few minutes to 1 hour.

The alkylation (7- > 8) is preferably conducted by treating 1. with a strong base such as lithium diisopropylamide, sodium amide or potassium hydride in a solvent such as THF, glyme, ether, dimethylformamide (DMF) or dimethylsulfoxide (DMSO) at a temperature of from -78 C to OOC. The resulting anion is then treated with excess acetaldehyde to provide 8.

The reaction .2 establishes the blocking group R4 and is typically accomplished by treating ,Q, with a base such as an alkali metal hydroxide, lithium diisopropyl amide, 4 - dimethylaminopyridine, or n - butyllithium in a solvent such as ether, THF, dioxane, DMF or DMSO, followed by treatment with an acyl halide of choice such as an alkanoyl, aralkanoyl or nuclear-substituted aralkanoyl or an (alkyl, aryl or aralkyl) - substituted (aralkyl or aryl) haloformate such as p nitrobenzyl chloroformate at a temperature of from -78 C to 250C for from 1-24 hours.

The deblocking reaction 9- > 10 is typically conducted by acid hydrolysis such as with aqueous acetic acid at a temperature of from 25 C to 750C for from 5 minutes to 3 hours.

The aldehyde intermediate compound 11 is prepared by treating 10 with an oxidizing agent such as CrO3 . 2 (pyridine) in CH3CN, 1:1 mixture of dimethylsulfoxide and acetic anhydride, or cyclohexylcarbodiimide in DMSO at a temperature of from 0-25 C for from 5 minutes to 1 hour. The resulting compound 11 in a solvent such acetonitrile, methylene chloride, or chloroform at a temperature of from -10 to 250C is treated with an excess of N-blocked cysteamine, HSCH2-CH2NHR4, in the presence of an acid catalyst such as boron trifluoride etherate or p - toluene sulphonic acid to provide 12. Typically, the reaction requires from 1 to 60 minutes.

There is no criticality as to the identity of the N - protecting group, R4, of the cysteamine reagent and suitable groups are p - nitrobenzyloxycarbonyl, o- nitrobenzyloxycarbonyl, t - butyloxycarbonyl, 2,2,2 - trichloroethoxycarbonyl, or phthaloyl.

The vinyl sulphide 14 is obtained via intermediate 1 by treating 12 with a halogen such as chlorine or bromine (X=Cl or Br), usually in a solvent such as ether, methylene chloride, tetrahydrofuran or glyme and at a temperature of from -78 to 300C for from I to 30 minutes, followed immediately by treating with an olefin such as cyclohexene or isobutylene in the presence of a base such as triethylamine or sodium hydride, in a solvent such as DMF, glyme, THF or hexamethylphosphoric triamide (HMPA). The solution is held at -200C to 25 C for from 1 to 8 hours to yield 14.

The vinyl sulphide 14 is reacted with a diester of oxomalonic acid (or its monohydrate) to provide t . There is no criticality as to the identity of the esterforming residue, R5, of the oxomalonic acid. R5 may be a conventional, easily removable blocking group or it may be a pharmaceutically acceptable esterforming residue. Suitable ester-forming radicals R5 are p-nitrobenzyl, benzyl, o- nitrobenzyl, t-butyl and 2,2,2-trichloroethyl. The reaction 1i is typically conducted in a high-boiling organic solvent such as benzene, toluene, cyclohexane or a haloaromatic compound at a temperature of from about 50 C to reflux for from 0.5 to 6 hours.

The halogenation reaction 15- > 16 is typically conducted in a solvent such as THF, glyme, ether, methylene chloride or chloroform in the presence of a halogenating agent such as thionyl chloride or phosphorus pentachloride in the presence of a base such as pyridine at a temperature of from -20eC to 250C for from 5 minutes to 3 hours. The selective reduction of 15- > 17 via 16 is completed by treating 1 with tributylphosphine or triphenylphosphine in aqueous DMF or a similar aqueous system involving dioxane, THF, glyme, DMSO, or acetone at a temperature of from 0-50 C for from 10 minutes to 5 hours.

Compound 17 is halogenated by the previous procedure (1 i), but omitting the addition of the cyclohexene or other olefin, to provide the di alo compound 18.

This is treated with a base such as triethylamine, sodium hydride or potassium hydride, usually in a solvent such as DMF, acetonitrile, methylene chloride, chloroform or glyme and at a temperature of from -78 C to 250C for from 1 to 5 hours to provide compound 19. Compound 19 is converted to 20 on treatment with a strong base such as 1,5 - diazabicyclol5.4.Olundec - 5 - ene(DBU) or 1,5 diazabicyclol3.4.0]non - 5 - ene(DBN) preferably in a solvent such as DMSO, acetone, chloroform, DMF, THF or glyme or on treatment with AgF in pyridine, and at a temperature of from (00C for from 1/4 to 24 hours. The reaction 20- > 21 is conducted by treating 20 with an aromatic base such as pyridine, s-collidine or lutidine, in the presence of a displacing agent such as lithium iodide, lithium bromide or sodium bromide, conveniently at a temperature of from 80-150 C for from 15 minutes to 2 hours. An aqueous work-up of the resulting reaction mixture provides Compound 21. Isomerization of the double bond 21e22 is accomplished by treating 21 in a solvent such as DMF, DMSO, ethyl ether, THF, glyme or methylene chloride with a strong base such as diisopropylamine, DBU or DBN usually at a temperature of from 0 to 250C for from a few minutes to 2 hours or until equilibrium has been established, as determined by examination of sample aliquot portions by ultraviolet absorption or by thin-layer chromatography. The final reaction 22l (by hydrogenolysis of the blocking groups) is accomplished by treating 22 in a solvent such as dioxane, ethanol or THF or an aqueous mixture thereof in the presence of a platinum-group metal catalyst such as Pd/C. The hydrogenolysis is suitably carried out under a hydrogen pressure of from 14 atmospheres for from 0.5 to 8 hours at a temperature of from 0--25"C.

The above-described total synthesis may also advantageously start with 4 vinyl - azetidinone[(23), below; E. J. Moriconi, W. C. Meyer, J. Org. Chem. 36, 2841 (1971)] rather than the enol acylate azetidinone (4, above). This variation in the total synthesis has the advantage of conveniently imparting stereo-seleclivity to the process at an early stage. The stereo-chemistry of the present invention is discussed fully below. The following scheme illustrates this 4 - vinyl - azetidinone embodiment of the present invention; it ties into the above scheme at compound IA.

In words relative to the above reduction diagram, 4 - vinyl - azetidinone 2,3 is silylated to provide the N - silyl compound 24. The groups R' on the silyl radical are alkyl having from 1-6 carbon atoms; especially preferred triorganosilyl groups are trimethylsilyl and t-butyl-dimethylsilyl. Typically, the silylation (2~ < 24) is achieved by treating 23 in a solvent such as DMF, DMSO or HMPA with the silylating agent of choice, e.g. dimethyl - t - butylsilyl chloride, and a base such as Et3N, pyridine or N,N - dimethylaniline at a temperature of from -10" to 30 C for from 1 to 8 hours. Compound 24 is alkylated to form 2, by treatment with acetaldehyde in the presence of base. This reaction 24~2 is conducted exactly as described above for the alkylation 7 - > 8. The 0- protecting group is established in the reaction 25- > 26. The protecting group R4 is as previously defined and the reaction 25- > 26 is exactly analogous to the above described reaction .2 It should be noted here, and will be developed below, that the reactions (242~) and (25- > 26) represent convenient opportunities to separate compounds 25 and 26 into the four racemic diastereoisomers. The removal of the N - tnorganosilyl group is accomplished in reaction 26- > 27 by mild acid-catalysed solvolysis. The halo sulfide compound 28 is obtained Irom 22 by treating 27 in a solvent such as methylene chloride, T#F, or glyme with the reagent XSCH2CH2NHR4 where R4 is as previously defined and X is halogen such as chlorine or bromine at a temperature of from -50 to 500C for from 1 to 16 hours. The sulfide intermediate compound 14 which is common to the above illustrated scheme of total synthesis, is obtained from 28 by elimination of HX on treatment of 2,0 with a base such as 1,5 diazabicyclo[5.4.0]undec - 5 - ene (DBU), 1,5 - diazabicyclo[4-3-0]non - 5 - ene, (DBN), 1,4 - diazabicyclo[2.2.2loctane, (DABCO), or silver fluoride in a solvent such as DMSO, pyridine DMF or HMPA at a temperature of from 200 to 50"C.

for from 1/4 to 16 hours.

Another procedure for the total synthesis employs an azide radical rather than the protected amino group, which route is illustrated above. The following diagram illustrates the azide route; the starting material is the previously defined compound ll:

X --hatogen

X= halogen

x = halogen

In words relative to the above diagram, the aldehyde intermediate compound 1=1 in a solvent such as acetonitrile, ethylene chloride or chloroform at a temperature of from -10 to 250C. is treated with an excess of 2 - mercaptoethanol in the presence of an acid catalyst such as boron trifluoride etherate or toluene sulphonic acid to provide I Typically, the reaction requires from I to 30 minutes.

The diazide compound 1,J' is formed from 12' by treating I in a solvent such as THF, chloroform, methylene chloride or ether with an esterifying agent such as mesyl chloride, tosyl chloride or benzenesulfonyl chloride in the presence of base such as triethylamine, pyridine, N,N - dimethylalanine or potassium carbonate.

The resulting diester, which may optionally be isolated in a solvent such as DMSO or DMF, is treated with an excess of sodium azide at a temperature of from 0 35 C for a period from 1--24 hours to provide 1~'.

The diazide compound 1~' is reacted with a diester of the monohydrate of oxomalonic acid to provide 1,Q'. There is no criticality as to the identity of the esterforming residue R4 of the oxomalonic acid. R4 may be a conventional easily removable blocking group or it may be a pharmaceutically acceptable esterforming residue, suitably p - nitrobenzyl. The reaction 1~'+1~'1,Q' is typically conducted in a high boiling organic solvent such as toluene, cyclohexane or a haloaromatic compound at a temperature of from 50 C to reflux for from 0.5 to 6 hours.

The halogenation reaction 1~' 16' is typically conducted in a solvent such as THF, glyme, ether, methylenechloride or chloroform in the presence of a halogenating agent such as thionyl chloride or phosphorus pentachloride in the presence of base such as pyridine at a temperature of from -20" to 0 C for from 5 minutes to 3 hours. The selective reduction of 15' 1,J' via 1,' is completed by treating I' with triphenylphosphine in aqueous D F or similar aqueous systems involving dioxane, THF, glyme, DMSO, or acetone at a temperature of from 0 50 C for from 10 minutes to 5 hours.

The halogenation reaction 17'--t18' is conducted by treating 12' with bromine or chlorine at a temperature of from about -20 to 0 C in a solvent such as diethyl ether/pentane, THF, F, glyme, methylene chloride, or chloroform. Intermediate compound 18', in a solvent such as DMF, acetonitrile, methylene chloride, chloroform glyme or THF in the presence of an acceptor such as cyclohexene and a base such as sodium hydride, potassium hydride or a trialkylamine at a temperature of from 200 to 10 C for from 0.5 to 6 hours yields compound 1,2' which is halogenated by the previous procedure to provide the dihalo compound 20'. This is treated with a base such as sodium hydride or potassium hydride, preferably in a solvent such as DMF, acetonitrile, methylene chloride, chloroform or glyme at a temperature of from 780 to 10 C, to provide 2"!'. Compound 21' is converted to 22' on treatment with a strong base such as 1,5 diazabicyclol5 4 undec - 5 - ene (DBU) or 1,5 - diazabicyclo[3.4.0]non - 5 - ene (DBN) in a solvent such as DMSO, acetone, chloroform, DMF, THF or glyme, preferably at a temperature of from (00C for from 1 to 24 hours. The reaction 22'- > 23' is conducted by treating 22' with an aromatic base such as pyridine, s collide, lutidine or DMF in the presence of a displacing agent such as lithium iodide, lithium bromide or sodium bromide, preferably at a temperature of from 80-150 C for from 15 minutes to 2 hours. An aqueous work-up of the resulting reaction solution provides 2,J'. Isomerization of the double bond 23'24' is accomplished by treating 2 in a solvent such as DMF, DMSO, ethyl ether, THF, glyme or methylene chloride with a strong base such as DBU or DBN, preferably at a temperature of from 0 to 250C for from a few minutes to I hour or until equilibrium has been established, as determined by examination of sample aliquot portions by ultraviolet absorption or by thin-layer chromatography. The final reaction 2,4'I (by hydrogenolysis of the blocking group and reduction of the azide) is accompashed by treating 24' in a solvent such as dioxane, ethanol or THF or an aqueous mixture thereof in the presence of a platinum-group metal catalyst such as PtO2, usually under a hydrogen pressure of from 1-50 atmospheres for from 0.5 to 8 hours at a temperature of from 250C.

Separation of Isomers In the following schemes of resolution, which impart stereoselectivity to the above-defined total synthesis, the intermediate compounds will be numbered as shown in the above reaction diagrams. The stereo configuration of any given compound will be designated relative to formula I and its numbering system.

Scheme 1 Central to this scheme is the preparation of 4, from an acyloxy butadiene (1) possessing an optically active acyl residue, 1+, > [,Q,1 < 4, in which R1 is menthyloxyacetyl or monomethylcamphoryl, for example. This early resolution and ensuing steps are summarized by the following diagram: SCHEME 1

Scheme 2 Central to this scheme is the acylation of the 2'-hydroxyethyl side chain of 4 with an optically active acid such as menthoxyacetic or camphoric acid. Resolution of the resulting species may be carried out at several stages. For example, hydrogenation of the double bond of the optically active enol acylate may be followed by resolution:

Alternatively, a optically inactive enol acylate 4 (e.g., enol acetate) can be hydrogenated, saponified and re-esterified with an optically active acid and then resolved as shown above. Another alternative is to delay resolution until formation of compound 10

where R* is optically active. Resolution of this intermediate compound provides: lO(5R,6R,8S); l0(5S,6S,8R); 10(5R,6R,8R); 10(5S,6S,8S); lQ(5R,6S,8S); l?k5S,6R,8R); IQ(SR,6S,8R); and 1,9(5S,6R,8S). Once any of the above resolutions has been achieved, the acyl group is removed by saponification and the synthesis proceeds.

Scheme 3 The vinyl-azetidinone starting material 2 is a known compound and is resolvable into its optical isomers by known procedures. See, for example, British Patent No. 1,273,278. The following diagram outlines a stereo-selective synthesis employing as starting material a pure optical isomer of vinyl-azetidinone.

In words relative to the above diagram, isomers 23(5S) and 2 (SR) are obtained according to known procedures IBritish Patent 1,273,278]. Silylation, followed by alkylation, provides 2(5R,6R), 2 (5R,6S), 2(5S,6R), and 2(5S,6S) via compound 24. Compounds 2 are separable by physical or chemical methods such as chromatography. Following establishment of the protecting group R4, compounds 26 are separable into: 26(5R,6R,8R); 26(5R,6R,8S); 26(5R,6S,8R); 26(5R,6S,8S); 26(5S,6R,8R); 26(5S,6R,8S); 2(5S,6S,8R); and 26(S,6S,8S) by chromatography. The synthesis may now proceed as illustrated above to provide l(5R,6R,8R); I(5R,6R,8S); 1(5R,6S,8R); I(5R,6S,8S); I(5S,6R,8R); I(5S,6R,8S); 1(5S,6S,8R); and l(5S,6S,8S), each in substantially pure form.

The products of this invention (I) form a wide variety of pharmaceutically acceptable salts with inorganic and organic bases; these include, for example, metal salts derived from alkali or alkaline-earth metal hydroxides, carbonates or bicarbonates and salts derived from primary, secondary or tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-loweralkanolamines, lower alkylenediamines, N,N - diaralkyl - lower alkylenediamines, aralkylamines, amino - substituted lower alkanols, N,N - di - lower alkylamino substituted lower alkanols, amino-, polyamino- and guanidino - substituted lower alkanoic acids and nitrogen - containing heterocyclic amines. Representative examples include salts derived from sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N' - dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2 - amino - 2 - methyl - 1 - propanol, theophylline and N- methylglucamine. Acid - addition salts e.g. with hydrochloric, tartaric, hydrobromic, sulfuric, nitric, toluene - p - sulphonic and methanesulphonic acids may also be used.

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

As already mentioned, compounds of the present invention are valuable antimicrobial substances which are active against various gram-positive and gramnegative pathogens, for example against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Salmonella typhosa, Pseudomonas and Bacterium proteus, some of which have already been mentioned, and can therefore be used as antibacterial drugs for treating infections caused by such bacteria. Thus the free acid, free base, and especially the salts thereof such as those known and used for administration of penicillins and cephalosporins, e.g. primary, secondary and tertiary amine salts and metal salts, particularly alkali metal and alkaline-earth metal salts, are useful bactericides. The compounds of Formula I, and more particularly their pharmaceutically acceptable salts, and mixtures thereof, can be combined with pharmaceutical carriers, such as acceptable liquid and solid vehicles, to form antibiotic pharmaceutical compositions, which may be suitable dosage unit forms such as pills, tablets, orally administrable powders, capsules, suppositories, solutions, suspensions, emulsions, syrups and elixirs which can be prepared in accordance with well known procedures. They may be administered orally, intravenously presented in optic or opthalmic solution, e.g. as individual capsules or as drops, or in semi-solid form. Topical formulations may be in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints and powders.

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

For veterinary medicine the composition may, for example, be 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 5 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 15 to 240 mg of active ingredient per kg of body weight.

The present 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 0.10/n to 99% of active material, the preferred range being from 1-60. The composition will generally contain from 15 mg to 1500 mg of the active ingredient; however, in general, it is preferable to use a dosage in the range of from 250 to 1000 mg. In parenteral administration the unit dosage is usually the pure compound in a slightly acidified sterile water solution or in the form of a soluble powder intended for dissolution.

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

The following examples illustrate but do not limit the present invention.

Examples I to 11 and 14 are concerned with the preparation of starting materials and intermediate compounds; Examples 12, 13 and 15 with the preparation of compounds in accordance with the present invention, and Example 16 with pharmaceutical compositions containing such compounds. The words "Teflon", "Supercel", "Linde", "Celite" and "Amberlite" are trade marks, mesh and capsule sizes are U.S. standards, and temperatures are on the Centrigrade scale.

The abbreviation "mmole" means "millimole".

EXAMPLE I Preparation of 4-(2-acetoxyvinyl)azetidinone-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 (CSI) solution is added dropwise to the acetoxybutadiene solution by means of a Teflon tube immersed in the CSl solution and pressurized with N2. The addition takes 10 minutes. Little or no color is seen and the reaction mixture is stirred at -200C for 30 minutes. 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 30-minute reaction time and is cooled in an ice bath; 20 ml of ether is added and the mixture is vigorously stirred in an ice bath. At the end of the 30-minute reaction time, the reaction mixture is transferred, again using N2 pressure and the Teflon tube, from the reaction flask, which is maintained in the -200C 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 W8, preferably pH 8.

The phases are separated, leaving a yellowish-orange gum with the aqueous phase. The ethereal 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 ethereal 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 with ether is then begun. 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 4-6, 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.

EXAMPLE 2 Preparation of 4-(2-Acetoxyethyl)-2-Azetidinone

A solution of 4 - (2 - acetoxyvinyl) - 2 - azetidinone (10.0 g, 0.065 mole) in 200 ml ethyl acetate containing 100 mg of 10% Pd/C is hydrogenated on a Parr shaker at 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.

447 ; ir (CHCl ) 5 66 5.74; nmr (CDCl2) T 3.44 (broad s, 1, NH), 5.82 (m, 2, CH2OCOCH2), 6.29 (m, 1, C-4H), 6.87 (1/2 AB pattern further split in four by C-4H and NH, 1 J =12.8Hz, J=4.5 H JNH=1.9 Hz, 7.38 (1/2 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 (son m, total 5, OCOCHI and CH2CH2OCOCH3, respectively).

EXAMPLE 3 Preparation of 4-(2-Hydroxyethyl)-2-Azetidinone

Under nitrogen at 0 , a solution of 4 - (2 - acetoxyethyl) - 2 - azetidinone (2.24 g, 0.014 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 being stirred 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% MeO/CHCl2,to give 1.55 g of the alcohol: m.p. 500; ir (CHCI3) ,u 5.67; nmr (CDCl2) t 3.20 (broad s, 1, NH), 6.24 and 6.28 (m on t, total 3, C-4H and CH2OH respectively), 6.90 (broad s on 1/2 AB pattern further split in four by C-4H and NH, total 2, OH and C-3H respectively J rn=13.0Hz, J,,,=4.2Hz, J,,=1.6Hz), 7.42 (1/2 AB pattern further split in four by C 4H and NH, 1, C-3H, Jg6rn=13.0Hz, J,,,=2.2Hz, JNH=1.1Hz) 8.16 (m, 2, CH2CH2OH).

EXAMPLE 4 Preparation of 8-Oxo-2,2-dimethyl-3-oxa-1-azabicyclo[4.2.0]octane

A solution of 4 - (2 - hydroxyethyl) - 2 - azetidinone (1.87 g, 0.016 mole) and 2,2 - dimethoxypropane (1.69 g, 0.016 mole) in 25 ml anhydrous methylene chloride is treated with boron trifluoride etherate (0.201 ml, 0.002 mole) at 250 C.

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 - I - azabicyclo[4.2.0]octane (1.59 g) as a crystalline solid.

Recrystallization from ether/hexane gives product of m.p. 60-1 .

ir (CHCIl),u: 5.73 (p-lactam) nmr (CDC13)T: 6.026.28, m, 2H, C-4 methylene 6.22-6.62, m, 1H, C-6 methine 6.90, dd, 1H, J7,=14Hz, J6.7=4.5Hz C-7 proton cis to C-6H 7.47, dd, 1H, J77=14Hz, Jg,7=2Hz C-7 proton trans to C-6H 7.82-8.68, m, 2H, C-S methylene

8.23, s, 3H # C-2 methyls 8.57, s, 3H C-2 methyls EXAMPLE 5 Preparation of 8-oxo-2,2-dimethyl-7ce and A-(I-hydroxyethyl)- 3-oxa- 1 -azabicyclo[4.2.0]octane

To a solution of 1.1 equivalents of freshly prepared lithium diisopropylamide in anhydrous tetrahydrofuran under a nitrogen atmosphere at -78 is added a solution of 8 - oxo - 2,2 - dimethyl - 3 - oxa - 1 - azabicyclo[4.2.0]octane in anhydrous tetrahydrofuran which has been cooled to -780C. After two minutes, the resulting lithium enolate is treated with excess of acetaldehyde. The solution is stirred for 30 minutes at -78 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 - (I - hydroxyethyl) - 3 - oxa - I - azabicyclo[4.2.0]octane.

Data for 8 - oxo - 2,2 - dimethyl - 7 - ( I - hydroxyethyl) - 3 - oxa - I - azabicyclo[4.2.0]octane: ir (CH2CI2)8: 5.72 ( -lactam) nmr (CDCI3)r: 5.53-6.43, m, 4H, C-4 methylene+C-6 methine+C-9 methine 6.90, dd on broad s, 2H, J79=9Hz J6,7=5.5Hz, C-7 methine + OH 7.70-8.83, m, 2H, C-S methylene

8.27, s, 311 C2 methyl 8.60. s. 311 8.78, d, 3H, J9,o=6.5Hz, C-10 methyl Data for 8 - oxo - 2,2 - dimethyl - 7a - (I - hydroxyethyl) - 3 - oxa - I azabicyclo[4.2.0]octane: ir (C11Cl3)p: 2.9 broad O-H 5.73 -lactam nmr (acetone-d6)#: 4.23-3.33, m, C-9 methine+C-4 methylene+C-6 methine 3.33, broad s, OH

2.83, dd, J=2Hz, 6Hz l C 7 th 2.67, dd, J=2Hz, 8Hz J ~ me Ine 1.93-1.63, m, C-S methylene

1.63, s C-2 methyls 1.40, s 1.23, d, J=6.5Hz, C-10 methyl EXAMPLE 6 Preparation of 8-Oxo-2,2-dimethyl-7a-( I -p-nitrobenzyloxy- carbonyloxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane

Under anhydrous conditions at 0 C. a solution of 8 - oxo - 2,2 - dimethyl 7ez - ( I - 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 1M pH 7 phosphate buffer and more ether. The ether phase is washed with water and brine, dried over magnesium sulfate and filtered. Evaporation of the filtrate under reduced pressure gives 67 mg of a colorless oil. Purification by preparative thick-layer chromatography on silica gel developing with 1:9 ethyl acetate/benzene gives 8 oxo - 2,2 - dimethyl - 7a - (I - p - nitrobenzyloxycarbonyloxyethyl) - 3 - oxa 1 - azabicyclo[4.2.0]octane (40 mg) as a mixture of diastereomers.

ir (CH2C12) 5.68 (p-lactam and carbonate), 6.19 and 6.54 (nitro) nmr (CDC3): 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, C-4 methylene 6.25-6.62, m, 1H, C-6 methine 6.75-7.12, m, 1H, C-7 methine 7.75-8.83, m, 2H, C-S methylene 8.22, s, 3H, C-2 methyl 8.50-8.58, m, 5H, C-2 methyl+CH3CH The 7P - diastereoisomers or the 7a and ,B- - mixture is obtained in an analogous manner.

EXAMPLE 7 Preparation of Cis and Trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl)- 4-(2-hydroxyethyl)-2-azetidinone

8 - Oxo - 3 - oxa - 2,2 - dimethyl - 7a - (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 650C for 1.25 hours. The acetic acid and water are removed under reduced pressure and the residue is taken up in benzene and evaporated to give trans - 3 - (I - p nitrobenzyloxycarbonyloxyethyl) - 4 - (2 - hydroxyethyl) - 2 - azetidinone as a mixture of diastereoisomers.

ir (CH2CI2): 5.67 (p-lactam), 5.72 shoulder, 6.20 and 6.57 (nitro) nmr (CDCi3"): 1.73, d, 2H, J=8.5 Hz, ArH 2.43, d, 2H, J=8.5 Hz, ArH 3.63, broad s, IH, NH 4.37513, m, 1H, CH3CH 4.72, s, 2H, ArCH2 6.076.53, m, 1H, C-4 methine 6.23, t, 2H, J=5.5 Hz, CH2OH 6.736.93, m, 1H, C-3 methine 7.63-8.97, m, 3H, CH2CH2OH 8.53, d, J=6.5 Hz, CH3CH The cis diastereoisomers or the cis-trans mixture is obtained in an analogous manner.

EXAMPLES 811 Examples 8, 9, 10, and 11 as alternative to Examples 4, 5, 6, and 7 for the Preparation of 3-(1-p-nitrobenzyloxycarbonyloxyethyl)-4- (2-hydroxyethyl)azetidinone

EXAMPLE 8 Preparation of 1 -(2-Tetrahydropyranyl)-4-[2-(2-tetrahydropyranyl)- oxyethyll-2-azetidinone

Under nitrogen and at 250 C, a solution of 4 - (2 - hydroxyethyl) - 2 azetidinone (62 mg, 0.539 mmole) in 0.5 ml of anhydrous p - dioxane is treated with 2,3 - dihydropyran (0.98 ml, 1.08 mmoles) and p - toluenesulfonic acid monohydrate (19 mg, 0.10 mmole). The resulting solution is stirred for a period of 60 minutes and then partitioned between 10 ml of 0.5M 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 I - (2 tetrahydropyranyl) - 4 - 12 - (2 - tetrahydropyranyl)oxyethyl] - 2 - azetidinone as an oil.

nmr (CDCI3)T: 5.13-5.60, m, OCH 5.83-6.85, m, C-4H+OCH2

6.95, dd, J=SHz and 15Hz t C 3 methylene 7.35, dd, J=3Hz and 15Hz 7.62-8.95, m, CHCH2CH2CH2CH2+CHCH2CH2O EXAMPLE 9 Preparation of Cis and Trans-1-(2-tetrahydropyranyl)-3-(1-hydroxyethyl)-4-[2-(2- tetrahydropyranyl)oxyethyl]-2-azetidinone

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

EXAMPLE 10 Preparation of Cis and Trans-1-(2-tetrahydropyranyl)-3- (1 -p-nitrobenzyloxycarbonyloxyethyl)-4-[2-(2-tetrahydro- pyranyl)oxyethyl]-2-azetidinone

Following the procedure described for the preparation of 8 - oxo - 2,2 dimethyl - 7a - (1 - 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 - I - (2 - tetrahydropyranyl) - 3 - (1 - hydroxyethyl) - 4 - [2 - (2 tetrahydropyranyl)oxyethyl] - 2 - azetidinone there is obtained trans - 1 - (2 tetrahydropyranyl) - 3 - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4 - [2 - (2 tetrahydropyranyl)oxyethyl] - 2- azetidinone. The cis diastereoisomers are obtained in an analogous manner.

EXAMPLE 11 Preparation of Cis and Trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl) 4-(2-hydroxyethyl)-2-azetidinone

A solution of trans - 1 - (2 - - tetrahydropyranyl) - 3 - ( I - p nitrobenzyloxycarbonyloxyethyl) - 4 - [2 (2 - tetrahydropyranyl)oxyethyl] - 2 azetidinone in methanol at 250C. is treated with 0.1 molar equivalent of p toluenesulfonic acid monohydrate. The solution is stirred for a period of 2 hours and then neutralized with 1M pH7 phosphate buffer. The product is extracted into ethyl acetate. The ethyl acetate solution is washed with brine, dried over magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give trans - 3 - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4 - (2 hydroxyethyl)- 2 - azetidinone. The cis diastereoisomers are obtained in an analogous manner.

EXAMPLE 12 Preparation of (SR,6S,8S)- and (SS,6R,8R)-3-(2-aminoethylthio)-6- ( 1 -hyd roxyethyl)-7-ox o- 2 -azabicyclo[3.2.0]hept- 2-ene-2-carboxylic acid Step A

To 6.75 ml anhydrous pyridine (mw-79; p=0.98?; 83.9 mmole) in 350 ml anhydrous acetonitrile is added 5.05 g anhydrous powdered chromium trioxide (mw=100; 40.5 mmole). After 30 minutes, stirring at room temperature (250 C), 9.6 g dried Supercel is added and stirring is continued for 5 additional minutes. A solution of 3.21 g trans - 3 - (I - p - nitrobenzyloxycarbonyloxyethyl) - 4 - (2 hydroxyethyl) - 2 - azetidinone (mw=338; 9.5 mmole) in 30 ml anhydrous acetonitrile is added all at once. The reaction mixture is stirred under anhydrous conditions at room temperature (250C) for one hour. Addition of 9.6 g NaHSO2 is followed by 5 minutes of stirring after which the reaction mixture is filtered through a mixed packed bed of 40 g silica gel and 40 g anhydrous magnesium sulfate. The bed is washed repeatedly with acetonitrile (total volume of filtrate 600 ml).

The filtrate is concentrated under a N2 stream to 130 ml total volume.

To this solution containing crude aldehyde at 0 C under N2 is added 9.64 g 2 (p - nitrobenzyloxycarbonylamino)ethanethiol (mw=256; 37.7 mmole) as prepared below (Example 12, Step B). To the stirred reaction mixture is added 8.0 ml boron trifluoride etherate (mw=142; p=1.125; 63.4 mmole). After 1.5 hours at 0 C., the reaction mixture is poured into a stirred ice-cold mixture of 69 g K211PO4-S00 ml H2O and 700 ml ethyl acetate (EA). The layers are separated, and the aqueous one is saturated with NaCI and re-extracted with additional EA. The combined organic layers are washed twice with brine, dried over anhydrous MgSO4 and filtered. The filtrate is concentrated under a N2 stream and then pumped on high vacuum to give 14.5 crude 1.

The material is chromatographed on 450 g silica gel (column height=48 cm; diameter=5.5 cm) packed and applied in CHCI3 and eluted with increasing percentages of MeOH in CHCI3 (04 /" MeOH/CHCI3). Those fractions containing the desired product are combined, concentrated under a N2 stream; and pumped on high vacuum to give 5.09 g of j (65% yield).

NMR (d6-acetone) 8.17-7.47 (aromatic protons), 7.25 (active hydrogen), 6.69 (active hydrogen), 5.404.97 (-Cll2pNO2,s & C113-CH-O), 4.12 (t, J=7Hz,

3.80 (m, C4-H), 3.35 (m, -CH2-NH- & C3-H), 2.82 (m, -S-CH2-), 2.15 (m,

1.42 (d, J=6.5 Hz, CH3-CH-O) in ppm downfield from TMS.

IR (CHCI3 solution) carbonyls 1770 cm-' & 1725 cm-l.

Step B Preparation of 2-(p-Nitrobenzyloxycarbonylamino)ethanethiol

To 600 ml diethyl ether (Et2OF75 ml H2O in an ice bath with stirring is added 3.2 g cysteamine hydrochloride (mw=114; 28.1 mole). A solution of 7.14 g NaHCO3 (mw=84; 85 mmole) in 75 ml H2O is added. The ice bath is removed, and at room temperature a solution of 6.75 g p - nitrobenzylchloroformate (mw=216; 31.3 mmole) in 270 ml Et2O is added dropwise over a period of one hour. After 10 additional minutes, the layers are separated. The ethereal layer is extracted with 150 ml 0.25 N HCI, and then with 200 ml brine. Each aqueous layer is then backwashed successively with 100 ml Et2O. The combined Et2O layers are dried over anhydrous MgSO4, filtered, and concentrated under a N2 stream. The crystalline residue is slurried in a small amount of ether, filtered, and the pale yellow crystals are dried under high vacuum to give 4.7 g 2 - (p - nitrobenzyloxycarbonylamino)ethanethiol (65% yield).

NMR (CDCl3) 8.18 (d, J=8Hz, aromatic protons ortho to nitro), 7.47 (d, J=8Hz, aromatic protons meta to nitro), 5.27 (-NH-), 5.20 (s, -CH2- #-pNO2), 3.40 (m, -CH2-NH-), 2.67 (m, -CH2-SH), 1.35 (t, J=8.5Hz, -SH) in ppm downfield from TMS.

IR (CHCI3 solution) carbonyl- 1725 cm-1.

M.S.-molecular ion-256, (M-47) at 209, (M-136) at 120, +CH2pNO2 at 136.

Step C

To 14.2 ml pentane (dried over 4A Linde molecular sieves) is added 0.5 ml Br2 (mw=160; 9.75 mole). To S g of 1 (mw=830; 6.02 mmole) in 58 ml tetrahydrofuran (THF) (freshly distilled from lithium aluminium hydride) (LAH) and 65 ml Et2O (dried over 3A 1/16 Linde molecular sieves) at 0 C under N2 with stirring is added dropwise 10 ml of the above 0.66M Br2 solution (6.6 mmole). After 10 minutes at OOC, 0.67 ml cyclohexene (mw=82; p=0.81; 6.6 mmole) is added.

After 5 minutes at 0 C., 1.7 ml triethylamine (mw=l0l; p=0.729; 12.3 mmole) is added immediately followed by 40 ml ice-cold dimethylformamide (DMF) (distilled from anhydrous CaSO4 at 40 mm and stored over 4A Linde molecular sieves). The ice bath is removed, and stirring is continued for 2+ hours at room temperature. The reaction mixture is poured into a stirred ice-cold mixture of 12.6 ml IMKH2PO4 160 ml H2O500 ml (EA). After separation of the layers, the aqueous layer is saturated with sodium chloride and re-extracted with EA. The combined organic layers are extracted once with brine, dried over anhydrous MgSO4, filtered and concentrated under a N2 stream followed by pumping under high vacuum to provide crude 2.

The material is chromatographed on 250 g silica gel (height=45 cm; diameter=4.5 cm), packed and applied in CHCl3, and eluted with increasing percentages and MeOH in CHCI? (03% MeOH/CHCI3). Those fractions containing clean product are combined, concentrated under a N2 stream, and pumped on high vacuum to give 2 g of 2. Contaminated fractions are rechromatographed on silica gel using increasing percentages of EA in CHCI3 (0 25% EA/CHC13) to give an additional 0.645 g of 2 (total yield=77%).

NMR (d,-acetone) 8.40-7.54 (aromatic protons), 7.30 (active hydrogen), 6.72 (active hydrogen), 6.44 (d, J=lSHz, -CH=CH-S- (trans)), 6.34 (d, J=9Hz, -CH--CH-S- (cis)), 5.77 (dd, J=15,7Hz -CH--CH-S- (trans)), 5.74 (dd, J=9, 8Hz, -CH--CH-S- (cis)), 5.44-4.92 (-C112- #-p-NO2's & CH3-CH-O), 4.34 (dd, J=8,3Hz, C4-H of cis thioenolether), 4.09 (dd, J=7, 3Hz, C4-H of trans-thioenolether), 3.35 (m, -C112-NH & C2-H's), 2.90 (m, -S-CH2-), 1.42 (d, J=6.5Hz, CH2- CH-O) in ppm downfield from TMS.

IR (CHCl3 solution) carbonyls - 1767 cm-' & 1730 cm-'.

Step D

To a stirred solution of 2.48 g di(p - nitrobenzyl) oxomalonate (from Example 12, Step E) (mw=388; 6.39 mmole) in 400 ml hot anhydrous toluene is added a solution of 2.52 g of 2 (mw=574; 4.39 mmole) in 20 ml THF (distilled from LAH) and 40 ml anhydrous toluene. After some of the solvent has boiled off, additional anhydrous toluene is added, and the azeotropic drying process is repeated three times. The solution is then refluxed under N2 for 30 minutes. Additional toluene is then allowed to boil off yet the volume is not allowed to diminish so much that precipitation occurs. Total heating time is approximately 2+ hours. The clear yellow reaction mixture is removed from the oil bath and placed under CHCL3 (height=43 cm; diameter=4.5 cm). Elution with 500 ml 0.5 /O MeOH/CHCI3 is followed by continued elution with 1% MeOH/CHCI3 for the remainder of the chromatography. After the emergence of excess of reagent, those fractions containing pure .3. are combined, concentrated under a N2 stream and then on high vacuum to give 1.22 g of 3.

Later fractions containing ,Q, and the corresponding cis thioenol ether are rechromatographed on silica gel to give an additional 0.5 g of 2, (total yield=40%).

NMR (d6-acetone) 8.28-7.45 (aromatic protons), 6.65 (br.s., active hydrogen), 6.48 (d, J=lSHz, -CH=CH -S-), 5.78 (dd, J=15, 8Hz, -CH=CH --s-), 5.45-5.07 (-CH2-#-pNO2's & CH3-CH-O), 4.65 (dd, J=8, 3Hz, C4-H), 3.40 (m, -CH2-NH- & C3-H), 2.88 (m, -CH2-), 1.42 (d, J=7Hz, CH3-CH-O) in ppm downfield IR (CHCl3 solution) carbonyls - 1758 cm-', shoulder at 1725 cm-1 Step E Preparation of di-p-Nitrobenzyl Oxomalonate

KOH C112(CO2H)2+2B rCH2pNO2 EtOH SeO2 CH2(CO2CH2NO2)2 H p O=C(CO2CH2pNO2) 1' 2' z A H=benzene A mixture of 100 g p-nitrobenzyl bromide (0.46 mole), 28.6 g malonic acid (0.275 mole) and 750 ml ethanol (EtOH) is stirred and warmed on the steam bath until solution is achieved. A solution of 33 g KOH ( > 85% purity; --0.6 mole) in 200 ml of water is added carefully with swirling. An additional 200 ml of water is added, and the two-phase system is refluxed for 1.8 hours. The lighter-colored homogeneous solution is cooled in ice for 1 hour and the crude product isolated by filtration, washed twice with a minimum of cold EtOH, and dried by pulling dry N2 through the cake; 33.7 g of solid is obtained. If, during the refluxing stage the reaction mixture is slowly concentrated to ca. half volume by allowing refluxing solvent to distill off, the crude product yield rises to 77 g. The material is recrystallized from methanol to give pure di-p-nitrobenzyl malonate 1'.

A mixture of 23.4 of ', 10 g SeO2, and 3040 ml of xylene is stirred in a flask immersed in an oil bath. The bath temperature is raised over I hour to 130-135 .

A gradual darkening of the reaction mixture is noted, and after a total of 4 hours at 130-135 , most of the insoluble residue is black Se. The mixture is cooled, MgSO4 is added to remove the water, and Celite is added to aid in filtration. The mixture is filtered through Celite and the cake washed with xylene and a small portion of EtOAc. Final volume: 60 ml. A 100 g column of Baker Silica Gel is prepared in benzene and 10 ml of filtrate applied, then eluted with increasing amounts of EtOAc in benzene, 500 ml fractions being collected. After one 2% ethyl acetate (EtOAc)/H, and two 10% EtOAc/H fractions, the third 10% and first 20% EtOAc/H provide the bulk of the product (-1.6 g from 10 ml filtrate) as judged by tIc (20% EtOAc/CHCl3; silica gel GF). Recrystallization from benzene, (1 g in ca.

50 ml concentrated to 1/3 volume and "spiked" with 1 ml of H2O saturated benzene): provides 0.24 g '; mp 121-122 .

Step F

A solution of 1.468 g of 3 (mw=962; 1.53 mmole) in CH2CI2 is dried over anhydrous MgSO4, filtered, concentrated under a N2 stream, and dried further under high vacuum just prior to the following reaction. To a solution of 2, in 24 ml THF (freshly distilled from LAH) at -200C is added 0.206 ml anhydrous pyridine (mw=79; p=0.982; 2.56 mmole). With stirring under N2, 294 mg of freshly distilled thionyl chloride (mw=ll9; 2.47 mmole) in 5 ml T11F is added dropwise. The reaction mixture is stirred for 10 minutes at -200C., half an hour at 0 C and finally I hour at 25 C. The pyridine hydrochloride is filtered under N2 and washed with 20 ml THF. The filtrate is concentrated under N2 stream followed by pumping on high vacuum. The resulting yellow foam is swirled in 25 ml anhydrous THF, and a small amount of orange-red insoluble material is filtered off under N2. The filtrate is re concentrated as above to a yellow foam.

To this freshly prepared chloro compound is added with stirring a freshly shaken suspension of 678 mg tributylphosphine (mw=202; 3.36 mmole) in 36.5 ml 9:1 DMF-H2O followed by 294 mg K2HPO4 (mw=174; 1.69 mmole). The reaction mixture is stirred at 25 C., for 35 minutes. After dilution with 120 ml EA and 60 ml brine, the layers are separated, and the aqueous one is extracted two times 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 give crude .

The material is chromatographed on 100 g silica gel (height=28.5 cm; d=4 cm) packed and applied in CHCI3 and eluted with 0.5 /O MeOH in CHCl3. Those fractions containing clean product are combined, concentrated under a N2 stream and then on high vacuum to give 0.786 g of 4. Contaminated fractions are re chromatographed on silica gel thin layer plates (eluant=50% acetone/hexane; extraction of desired u.v. band with CHCl2 and EA) to provide 0.203 g additional 4 (total yield=64%).

NMR (d8-acetone) 8.30-7.50 (aromatic protons), 6.68 (active hydrogen), 6.53 (d, J=lSHz, -CH=CH-S), 5.68 (dd, J=15, 9Hz, -CH=CH-S), 5.45- 5.03 (-CH2pNO2,s, C112-CH-O, & -CH(-CO2CH2pNO2)2), 4.53 (dd, J=9, 2.5Hz, C4-H), 3.41 (m, -CH2-N11- & C3-H), 2.85 (m, - S-CH2-), 1.41 (d, J=7Hz, C113-CH-O) in ppm downfield from TMS.

IR (CHCl3 solution) carbonyls 1750 cm-', shoulders at 1725 cm-' & 1775 cm-1.

Step G

To 8.5 ml pentane (dried over 4A Linde molecular sieves) is added 0.2 ml Br2 (mw=160; 3.9 mmole). To 0.706 g of 4 (mw=946; 0.746 mmole) in 18 ml T11F (freshly distilled from LAH) and 5.7 ml Et2O (dried over 3A 1/16" Linde molecular sieves) at 0 C under N2 with stirring is added dropwise 1.8 ml of the above 0.45M Br2 solution (0.81 mmole). After 15 minutes at 0 C., 0.42 ml triethyl amine (mw=l0l; p=0.729; 3.03 mmole) is added immediately followed by 10.5 ml ice-cold DMF (distilled from CaSO4 at 40 mm and stored over 4A Linde molecular sieves).

The ice-bath is removed, and stirring at room temperature is continued for 2 hours.

The reaction mixture is poured into a stirred ice-cold mixture of 3.1 ml 1M KH2PO4-70 ml H2O100 ml EA. The layers are separated, and the aqueous layer is saturated with NaCI and re-extracted with EA. The combined organic layers are washed once with brine, dried over anhydrous MgSO4, and filtered. The filtrate is concentrated under a N2 stream and then pumped on high vacuum to give crude ,Q,.

The material is chromatographed on 60 g silica gel (diameter=2.8 cm) packed and applied in CHCl3 and is eluted with 100 ml-2% EA/CHCl3; 100 ml-4% EA/CHCI3 and then 5% EA/CHC13 for the remainder of the chromatography. The fractions containing pure 5 are combined, concentrated under a N2 stream, and pumped on high vacuum to give 0.385 g of 5 (50% yield).

NMR (d6-actone) 8.32-7.45 (aromatic protons). 6.55 (active hydrogen), 5.45-5.14 (-CH2pNO2,s & CH3-CH-O), 5.07 (dd, J=6, 5Hz, -CHBr), 4.49 (d, J=6Hz, -CHS-), 4.29 (dd, J=5, 3Hz, C5-H), 3.85 (dd, J=4.5, 3Hz, C8- H), 3.42 (m, -CH2-NH-) 2.89 (-S-CH2-), 1.45 (d, J=6.5Hz, CH3- CH-O) in ppm downfield from TMS.

IR (CHCI3 solution) -lactam-~1783 cm-1, carbonyl 1742 cm-l.

Step H

To 29 mg anhydrous silver fluoride (mw=127; 0.23 mmole) is added a solution of 146 mg of (mw=1024; 0.14 mmole) in 3.5 ml anhydrous pyridine. The stoppered reaction mixture is stirred at room temperature in the dark for one hour and then poured into 20 ml cold water-30 ml EA. After separation of the layers, the aqueous layer is extracted twice with EA and once with CHCl3. Each organic layer is extracted once with H2O and once with brine. The combined organic layers are dried over anhydrous MgSO4, filtered, and concentrated under a N2 stream followed by pumping on high vacuum to give crude .

Preparative thin-layer chromatography (eluant=40% acetone/hexane; repeated extraction of desired u.v. band with a large volume of CHCl3) yields slightly contaminated t. Re-chromatographing on silica using EA in CHCl3 as an eluting system gives 95 mg pure 6 (700/, yield).

NMR (CDCl3) 8.32-7.35 (aromatic protons), 6.18 (br.d, J~ 1.5Hz, vinyl proton), 5.50-5.05 (-CH2pNO2,s & CH2-CH-O), 4.57 (dd, J=3,~1.5Hz, C5-H), 3.47 (m, -CH2-NH- & C6-H), 3.03 (m, -S-CH2-), 1.47 (d, J=6.5Hz, CH2-CH-O) in ppm downfield from TMS.

IR (CHCI3 solution) -lactam-~1790 cm-', carbonyls- ~ 1745 cm-1.

Step I

A solution of 77 mg of . (mw=944; 0.082 mmole) in 0.9 ml S-collidine (distilled from powdered KOH 30 mm pressure) is added to 13.4 mg anhydrous Lii (dried for few hours at 100 C over P205 under vacuum) (mw=134; 0.1 mmole). With stirring under N2, the reaction mixture is heated in an oil bath at 120 C. After a total of 30 minutes, the reaction mixture is cooled to 250C., diluted with CH2Cl2, and transferred to a round-bottom flask for concentration under a N2 stream and then on high vacuum. Partitioning the residue between EA-H2O and I ml IM KH2PO4 is followed by extraction of the aqueous layer twice more with EA and once with CHCl3. Each organic layer is then backwashed with brine. The combined organic layers are dried over anhydrous MgSO4, filtered, concentrated under a N2 stream and then on high vacuum to give crude 7.

Preparative thin-layer chromatography on silica gel (plate is eluted two times with 40% acetone/hexane; repeated extraction of desired u.v. bands with large volume of CHCl3) yields 18 mg of starting material and 28 mg of 1 (44 /n yield).

NMR (CDCl3) 8.30-7.50 (aromatic protons), 6.00 (m, vinyl proton), 5.38- 5.10 (-CH2-#-pNO2's, C113-CH-O, & C2-H), 4.53 (m, C5-H), 3.44 (m, -CH2-NH-), 3.35 (dd, J=5, 3Hz, C"-H), 3.00 (m, -S-CH2-), 1.50 (d, J=6.5Hz, CH2-CH-O) in ppm downfield from TMS.

IR (CHCl3 solution) -lactam-~1783 cm-', carbonyl-~1750 cm-' & 1735 cm-'.

Step J

To 49 mg of 2 (mw=765; 0.064 mmole) in 0.7 ml DMSO (distilled from CaH2 at 8 mm and stored over 4A Linde molecular sieves) is added 100 ,ul diisopropylamine (distilled from NaH under N2 and stored over 4A Linde molecular sieves) (mw=101; p=0.722; 0.71 mmole). The stoppered reaction mixture is stirred for a few minutes and then allowed to stand for 2 hours. The amine and most of the DMSO are then concentrated off under high vacuum with no external heating. The residue is passed quickly through a column of silica gel (packed, applied, and eluted with EA) to remove residual DMSO. After concentration under a N2 stream of all fractions having u.v. absorbance, the material is chromatographed on a thin layer silica gel plate (eluant=S0% EA/CHCl3; repeated extraction of desired u.v.

bands with a large volume of chloroform). The starting material band yields 35 mg.

The Product band yields 6 mg of . Starting material is re-submitted to the reaction conditions and isolation procedure twice more to yield 9 mg additional 8 (total yield=30%).

NMR (CDCI3) 8.27-7.47 (aromatic protons), 5.53-5.13 (-CH2-#-pNO2's & CH3CH-O-), 4.17 (m, C5-H), 3.50 (dd, J=5,3Hz, C,-H), 3.43 (m, - CH2-NH-) 3.03 (m, -S-C112- & C4-H's), 1.50 (d, J=6Hz, CH3- CH-O), in ppm downfield from TMS.

IR (CHCl3 solution) -lactam-~1783 cm-l, carbonyls-~1743 cm-l & 1725 cm Step K

To 5.2 mg a is added 0.60 ml dioxane, 0.05 ml ethanol, 0.35 ml deionized water and 0.01 ml of l.0M K2HPO4. To the resultant clear solution is added 5 mg of 10% Pd/C. The suspension is flushed with N2, then 5-6 times alternately with 50 psi H2 and vacuum. Finally, it is shaken under a 50 psi H2 atmosphere for 30-40 min. After centrifugation, the Pd/C is washed and centrifuged 2-3X with 0.5 ml portions of deionized water. The combined centrifugates are extracted 5x1-2 ml ether. Residual ether is removed under vacuum and the aqueous solution applied to an Amberlite XAD-2 column (20x 140 mm). Fractions of 100 drops (6-7 ml) are collected, with continuous UV monitoring, by elution with deionized water. Emergence of strongly UV absorbing material begins around fractions 3-5 and is usually complete by fractions 25-30.

Early fractions are examined by UV to exclude those few deemed too strongly absorbing in the 270280 my region. The remaining fractions are combined and lyophilized. The residue is evaluated by dissolving in 10.0 ml of deionized water and measuring the UV absorption at 298 my indicating a l30'/n yield of desired product. The nmr spectrum in D2O shows, amongst other peaks, a multiplet with prominent peaks at 4.24, 4.18, 4.13 and 4.06 a (Cs and C8 protons), a doublet of doublets at 3.49 a (C6-proton J5 -25Hz; J68=4.9Hz), two strong broad peaks at 3.20 and 3.14 a and a broader multiplet between 2.8 and 3.0 a due to the three sets of methylene protons, and a sharp doublet at 1.31 a (J89=6.4Hz) for the side chain methyl.

Step L Resolution of the Racemic Mixture The racemic mixture comprising the desired (5R,6S,8S)- and (5S,6R,8R) - 3 (2 - aminoethylthio) - 6 - ( I - hydroxyethyl) - 7 - oxo - I - azabicyclo[3.2.0]hept - 2 - ene - 2 - carboxylic acid in 20% ethanolic water is treated with an equimolar quantity of three S- p- nitrophenyl- 2aminopropane - 1,3 - diol, warmed to 500, and allowed to cool. When crystallization appears complete, the mother liquors are carefully pipetted away and the crystals washed on a filter with the minimum amount of ice-cold 20% ethanolic water. The combined filtrates and mother liquors, upon standing in the refrigerator for 18 hrs, afford the salt of the other enantiomer, which is isolated by filtration and washed with a minimum of ice-cold 20% ethanolic water. The salts are separately taken up in water and passed through an Amberlite XAD-2 column, eluting with water, and monitoring the eluate by U.V. The aromatic amine is retarded by the column, providing the desired isomer in the eluate, which upon evaporation yields, respectively, the (5R,6S,8S) and (5S,6R,8R) isomers.

The following Example specifically illustrates a preferred stereo-selective process embodiment of the present invention: As described above in detail, the starting material is a pure optical isomer of4 - vinyl - 2 - azetidinone (2,J, above).

These isomers are identified as U (5R) and 22 (5S). In the following Example, all intermediate compounds in the chain of synthesis are named according to the previously introduced stereochemical nomenclature (see the above chart and text).

In addition to the stereo-chemical symbol, such compounds are also named in a conventional manner in the Example.

EXAMPLE 13 Step A Preparation of 2~4(5S) [I-(t-b,utyidimethylsilyl)-4-vinyl-2-azetidinone]

A solution of 2,3(5S) [4 - vinyl - 2 - azetidinone] (1.153 g, 11.89 mmoles) and triethylamine (1.82 ml, 13.08 mmoles) in anhydrous N,N - dimethylformamide is placed under a nitrogen atmosphere, cooled to OOC and treated with t butyldimethylchlorosilane (1.885 g., 12.48 mmoles) resulting in the immediate appearance of a heavy white precipitate. This mixture is stirred for one hour while gradually warming to room temperature. The mixture is partitioned between 30 ml.

methylene chloride and 90 ml cold IM potassium dihydrogen phosphate. The aqueous phase is extracted with 20 ml methylene chloride. The combined organic phases are washed four times with 30-ml portions of water and finally with 50 ml brine. The methylene chloride solution is dried over anhydrous magnesium sulfate and filtered. The filtrate is evaporated under reduced pressure to give 2.25 g of 2 > 4(5S) [1 - (t - butyldimethylsilyl) - 4 - vinyl - 2 - azetidinone] as a colorless liquid NMR (CDCI3)a: 6.23-5.10, m, CH=CH2 4.07, 7-line m, J=8,6 and 3Hz, C-4H 3.35, dd, J=15 and 6Hz, C-3H cis to C-4H 2.73, dd, J=15 and 3Hz, C-3H trans to C-4H .98, s, (CH3)3CSi .23, s .18, s (CH3)2S; Following the above procedure, but making the indicated substitution, the 24(5R) isomer is obtained.

Step B Preparation of 25(5R,6S,8R and S) and 2, (5R,6R,8R and S) [l-(t- butyldimethylsilyl)-3-( 1 -hydroxyethyl)-4-vinyl-2-azetidinone]

To a solution of freshly prepared lithium diisopropylamide (7.82 mmoles) in 36 ml anhydrous tetrahydrofuran under a nitrogen atmosphere at -750C is added a solution of 24(5S) [I - (t - butyldimethylsilyl) - 4 - vinyl - 2 - azetidinone] (1.50 g, 7.11 mmoles) in 10 ml anhydrous THF. The resulting yellow solution of the lithium enolate is, after 16 minutes, treated with acetaldehyde (1.59 ml, 28.4 mmoles). In 10 minutes, the reaction mixture is quenched by adding 30 ml of a saturated aqueous ammonium chloride solution. This mixture is extracted with 50-ml and 25-ml portions of ethyl acetate. The combined ethyl acetate solutions are washed with 50 ml of brine and dried over anhydrous magnesium sulfate. The drying agent is removed by filtration and the filtrate is evaporated in vacuo to give the crude product as a yellow oil Purification by chromatography on silica gel eluting with 10% ethyl acetate/chloroform gives 2 (SR,6S,8R and S) and 2 (5R,6R,8R and S) [I - (t - butyldimethylsilyl) - 3 - (I - hydroxyethyl) - 4 - vinyl - 2 - azetidinone].

Following the above procedure, except making the indicated substitution, the 2~(5S,6R,8R and S) and 2(5S,6S,8R and S) isomers are obtained.

Step C Preparation of 2~(5R,6S,8R) and 26(5R,6S,8S) [l-(t-butyldimethylsilyl)- 3-(1 -p-mtrobenzyloxycarbonyloxyethyl)-4-vinyl-2-azetidinone]

Under nitrogen at -780C a solution of (5R,6S,8R) and 2(5R,6S,8S) (56 mg, 0.220 mmole) in 1 ml of anhydrous tetrahydrofuran is treated with 2.4M n butyllithium in hexane (101 y1, 0.242 mmole). To this solution is added, in five minutes, a solution of p - nitrobenzyl chloroformate (52 mg, 0.242 mmole) in anhydrous tetrahydrofuran. After stirring at -780C for a period of 55 minutes, 10 ml of a saturated aqueous ammonium chloride solution is added and the product extracted into ethyl acetate. The combined ethyl acetate solutions are washed with brine and dried over anhydrous magnesium sulfate. The drying agent is removed by filtration, and the filtrate is evaporated in vacuo to give 91 mg of a yellow oil.

Purification by preparative thick-layer chromatography on silica gel developing with 5% ethyl acetate/chloroform gives, respectively, 2~(5R,6S,8R) and 2(5R,6S,8S) in 54% overall yield.

In a similar manner, the diastereoisomers 2~(5R,6R,8S) and 2,(5R,6R,8R) are obtained when the indicated substitution is made, i.e., an equivalent amount of 2(5R,6R,8R & S) replaces the 2, (5R,6S,8R & S) of Step C.

Following the above procedure, but making the indicated substitution, the following diastereoisomers are obtained: 26(SS,6R,8R); 2,(5S,6R,8S); 2(5S,6S,8R); and -26(5S,6S,8S) Step D Desilylation of 26(5R,6S,8R) to provide 22 (SR,6S,8R) 13-(1-p-nitrobenzyloxy- carbonyloxyethyl)-4-vinyl-2-azetidinone]

A solution of 26(SR,6S,8R) [I - (t - butyldimethylsilyl) - 3 - (1 - p nitrobenzyloxycarbonyloxyethyl)- 4- vinyl- 2 - azetidinone] (61 mg, 0.141 mmole) in 2 ml of 0.5N HCVMeOH is stirred at room temperature (250C) for a period of 3 hours. The solution is then cooled to 0 C and neutralized by the addition of 5 ml of 5% aqueous sodium bicarbonate. The product is extracted into ethyl acetate (10 ml, 2xS ml). The combined ethyl acetate solutions are washed with water (2x5 ml) and 10 ml brine and then dried over anhydrous magnesium sulfate. The drying agent is removed by filtration, and the filtrate is evaporated in vacuo to give an oil. Preparative thick-layer chromatography of this material on silica gel developing with 10% ethyl acetate/chloroform gives 44 mg of 27(5R,6S, & R) [3 - (1 - p - nitrobenzyloxycarbonyloxyethyl) - 4 - vinyl - 2 azetidinonei.

Following the procedure of Step D, but making the indicated substitution, the following isomers are obtained by desilylation: 26(5R,6R,8R)- > 27(5R,6R,8R) 26(5R,6R,8S)- > 27(5R,6R,8S) 26(5R,6S,8S)- > 27(5R,6S,8S) 26(5S,6R,8R)- > 27(5S,6R,8R) 2~(5S,6R,8S)e21(5S,6R,8S) 2~(5S,6S,8R)e27(5S,6S,8R) 26(5S,6S,8S)- > 21(5S,6S,8S) Step E Preparation of 1,2(5S,6S,8R) via 2~(5S,6S,8R) by sulfenyl halide addition and dehydrohalogenation

A solution of the N -p - nitroCBZ cysteamine disulfide, 96 mg (0.19 mmole) in 1.5 ml THF (freshly distilled from LiAIH4) is cooled to -25 C and treated dropwise with stirring with 0.5 ml of a solution of 135 mg Br2 in sieve-dried CCl4 (2.2 ml final volume; portion added is equivalent to 0.19 mmoles of Br2). The resultant orange solution is stirred at -200C for 5 min. then treated with 54.0 mg of the vinyl azetidinone, 27(5R,6S,8R) in 0.5 ml sieve-dried CH2CI2. The color lightens to yellow. The mixture is allowed to come to 0 C over 5-10 minutes. Examination by tic (silica 5% MeOH in CH,CI2 or 20% EtOAc in CH2CI2) shows a main spot with R, and Ce'V+/H+/heat characteristics different from either disulfide or starting 4 vinyl - 2 - azetidinone. The reaction mixture is concentrated to 0.5 ml under N2, streaked directly on two 8"x8" 1000 8 silica GF plates, and developed with 20% EtOAc in CH2Cl2. The main band under U.V., is scraped off, and extracted with EtOAc to give 2~(5S,6S,8R).

The bromosulfide, 28(5S,6S,8R), 77.0 mg (0.162 mmole) is dissolved in 1.0 ml.

sieve-stored DMSO (distilled from CaH2), and stirred under nitrogen while 25R DBU (0.19 mmole) is added. After 3 hours, the mixture is poured into water/KH2PO4 and extracted repeatedly with EtOAc. The combined extracts are washed twice with water, dried with anhydrous MgSO4 and evaporated under nitrogen. The crude product, 42 mg, is streaked on an 8x8" 1000 silica GF plate and developed with 20% EtOAc in CH2Cl2 to give 14(5S,6S,8R).

Following the above procedure but making the indicated substitution, the following isomers are obtained: 1,(5R,6R,8R) 1,(5 R,6R,8S) 1,(5R,6S,8S) 1,2(5S,6R,8R) 14(5S,6R,8S) 14(5R,6S,8R) 1,(5S,6S,8S) Step F Preparation of 1(5R,6R,8R); I(SR,6S,8S); I(5R,6R,8S); I(SS,6S,8R); 1(5R,6S,8R); I(SS,6R,8S); 1(5S,6S,8S); and I(5S,6R,8R).

Following the exact procedure described in Example 12, Steps D-K, but making the indicated substitutions, all isomers 14 of Example 13, Step E, are converted to the corresponding isomeric form of I:

I(5R,6R,8R) I(5S,6S,8S) I(5R,6R,8S) I(5S,6S,8R) 1(5R,6S,8R) I(SS,6R,8S) I(SR,6S,8S) I(SS,6R,8R) EXAMPLE 14 Preparation of Bis[2-(p-Nitrobenzyloxycarbonylamino)ethyl]disulfide

Under nitrogen at -200C, bromine (1.21 ml, .022 mmole) is added to a solution of 2 - (p - nitrobenzyloxycarbonylamino)ethanethiol (11.28 g, 0.044 mole) in 100 ml of anhydrous tetrahydrofuran. The cooling bath is removed, and the cold solution is stirred for 15 minutes. The solution is then diluted with 400 ml ethyl acetate and washed with 200 ml IM pH 7 phosphate buffer, 200 ml 1M dibasic potassium phosphate, water (2x200 ml, 100 ml) and 200 ml brine. It is dried over anhydrous magnesium sulfate and filtered. The filtrate is evaporated in vacuo giving a yellow solid residue. This material is chromatographed on silica gel eluting with 5% ethyl acetate/chloroform to give 10.5 g of crystalline bis[2 - (p nitrobenzyloxycarbonylamino)ethyl]disulfide: IR (CH2CI2) y: 3.04NH 5.96 carbonyl 6.22, 6.61 nitro NMR (CDCl,)s:

8.24 d, J=8.SHz, ArH 5.37, broad s, NH 5.26, s, ArCH2O 3.60, q, J=6Hz and 6Hz, NHCH2CH2 2.86, t, J=6Hz, NHCH2CH2S EXAMPLE 15 Step A: Preparation of Cis and Trans-3-(1-p-nitrobenzyloxycarbonyloxyethyl)- 4-[2,2-bis(2-hydroxyethyl)thioethyll-2-azetidinone

Under nitrogen at 250C, a mixture of anhydrous pyridine 0.146 ml, 1.81 mmoles) and anhydrous, powdered chromium trioxide (92 mg, 0.916 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 - (I - p - nitrobenzyloxycarbonyloxyethyl) - 4 - (2 - hydroxyethyl) - 2 azetidinone (186 mg, 0.550 mmole) in I ml anhydrous acetonitrile. After being stirred for a period of I hour, the reaction mixture is filtered through a mixed, packed bed of 2 g each of silica gel and magnesium sulfate. The bed is washed repeatedly with a total of 30 ml of additional acetonitrile. The filtrate is concentrated under reduced pressure at 250C. to a volume of 3 ml. By thin-layer chromatography (silica gel; ethyl acetate/benzene 2:1) this solution contains a product (R,=.38) less polar than the starting material (R,=.21).

The acetonitrile solution of trans - 3 - (I - p nitrobenzyloxycarbonyloxyethyl) - 4 - (2 - oxoethyl) - 2 - azetidinone prepared above is, under nitrogen and at 00, treated with 2 - mercaptoethanol (0.386 ml, 5.5 mmoles) followed immediately by boron trifluoride etherate (0.176 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 - ( I - p - nitrobenzyloxycarbonyloxyethyl) - 4 - 12,2 - bis(2 hydroxyethyl)thioethyl] - 2 - azetidinone as a colourless oil.

ir (CH2CI2)y: 5.75 (5.79 shoulder) p-lactam and carbonate 6.20, 6.55 nitro nmr (acetone-d)T: 1.70, d, J=8.5Hz, 2H, ArH 2.28, d, J=8.5Hz, 2H, ArH 2.48-2.88, m, 1H, NH

4.63, s, ArCH,3H 4.63-5.12, m, CH3CH I

5.80--7.45, m, C-4H+C-3H+SCH2CH2OH 7.63-8.33, m, 2H, CH2CH 8.53, d, J=6.5Hz, 3H, CH3CH The cis diastereoisomers are obtained in an analogous manner. Alternatively, the mixed diastereoisomers are obtained when the starting materials comprise a mixture of the diastereoisomers.

Step B: Preparation of Trans-3-( I -p-nitrobenzyloxycarbonyloxyethyl)- 4-12,2-bis(2-azidoethyl)thioethyl]-2-azetidinone

To a solution of 211 mg (mw=474; 0.445 mmole) trans - 3 - (I - p nitrohenzyloxycarbonyloxyethyl) - 4 - 12,2 - bis(2 - hydroxyethyl)thioethyll - 2 azetidinone in 5 ml tetrahydrofuran (THF) (distilled from lithium aluminium hydride) at OOC is added 103 mg mesylchloride (mw=114; 0.904 mmole) in 1 ml T11F followed immediately by 134 yI triethylamine (mw=10l; p=0.729; 0.967 mmole). The reaction mixture is stirred for I hour under N2. The triethylamine hydrochloride is filtered under N2, washing with a few milliliters of 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 NaN2 (mw=65; 5.34 mmole). The reaction mixture is stirred overnight under N2 and 10 ml H2O and 20 ml ethyl acetate (EA) are then added. The layers are separated, and the aqueous layer is washed three times with 10 ml EA, each organic layer then being backwashed with 10 ml H2O and 10 ml brine. The combined EA layers are dried over anhydrous MgSO4, filtered and concentrated under a N2 stream to give the crude diazide.

Preparative thin layer chromatography on silica gel yields trans - 3 - (I - p nitrobenzyloxycarbonyloxyethyl) - - [2,2 - bis(2 - azidoethyl)thioethyl] - 2 azetidinone. The cis diastereoisomers and the cis - trans mixture are obtained in an analogous manner.

Step C:

A freshly prepared (H. Davies and M. Schwarz, J.O.C., 30, 1242 (1965)) solution of p - nitrophenyldiazomethane (29 mmole) in 150 ml of ether is added with stirring to a solution of 1.0 g oxomalonic acid monohydrate (mw=136; 7.35 mmole) in 50 ml ethylacetate (EA) at OOC. After 2+ hours the yellow solution is concentrated on a rotary evaporator with mild heating to approximately half the volume, dried over anhydrous sodium sulfate, filtered and concentrated as above to an oil. To the crude p - nitrobenzyl ester in 50 ml toluene (Tol.) is added 3.54 g of trans - 3 - (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 azeotropic drying process is repeated three additional times. The solution is then refluxed under N2 for one hour, the azeotropic drying process repeated a final time, and refluxing continued for an additional hour. Concentration of the resulting solution under a stream of N2 yields crude 1 The crude material is chromatographed on silica gel to give 1. The cis diastereoisomers and the cis - trans mixture are obtained in an analogous manner.

Step D

To a solution of 2.80 g j (mw=912; 3.07 mmole) in 35 ml THF (distilled from lithium aluminium hydride) at -200C is added 0.3 ml pyridine (mew=79, p=.982; 3.73 mmole) (distilled from NaH and stored over 4A Linde Molecular sieves). With stirring under N2, 0.438 g thionyl chloride (mw=l 19; 3.68 mmole) in I 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 1/16" Linde Molecular sieves). The combined filtrate and washings are concentrated under a N2 stream, slurried in a small volume of benzene with anhydrous MgSO4, filtered under N2 and then concentrated under a N2 stream. Pumping on high vacuum for half an hour yields an oil. To this freshly prepared chloro compound is added with stirring 0.885 g triphenyl phosphine (mw=262; 3.38 mmole) in 66 ml 9:1 dimethylformamide (DMF)/H2O followed by 550 mg K2HPO4 (mw=174; 3.16 mmole). The reaction mixture is stirred at 25"C for 35 minutes. After dilution with EA and brine, the layers are separated and the aqueous layer 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 and the cis-trans mixture are obtained in an analogous manner.

Step E

To 7.8 ml pentane (dried over 4A Linde molecular sieves) is added 0.2 ml Br2 (mw=160; p=3.12; 3.9 mmole). To a solution of 950 mg 2, (mw=896; 1.06 mmole) in 15 ml Et2 (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 KH2PO4-40 ml H2O-75 ml EA. After separation of the layers, the aqueous layer 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 ,Q,. Preparative thin-layer chromatography on silica gel yields ,Q,. The cis diastereolsomers and the cis-trans mixture are obtained in an analogous manner.

Step F:

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 0.42MBr2 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+ hours at OOC., then poured into a stirred ice-cold mixture of 1.6 ml IM KH2PO4, 20 ml H2O, and 20 ml EA. The layers are separated and the aqueous layer saturated with NaCI and re-extracted with additional EA.

The combined organic layers are washed once with brine, dried over anhydrous MgSO4 and filtered. The filtrate is concentrated under a N2 stream and then pumped on high vacuum to give crude 4. Preparative thin-layer chromatography on silica gel gives 4. The cis diastereoisomers and the cis-trans mixture are obtained in an analogous manner.

Step G:

To 210 mg 4 (mw=87l 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 250C. with stirring 40 mg 1,5 - diazabicyclo[5.4.0] undec - 5 - ene (distilled at 800C./2 mm) (mw=152; 0.263 mmole) in 0.7 ml dimethyl sulfoxide (DMSO). The solution is stirred under N2 for 4 hours, and then added to a stirred ice-cold mixture of 0.48 ml IM 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 once with brine, dried over anhydrous MgSO4, filtered, and concentrated under a N2 stream followed by pumping on high vacuum, to provide crude . Preparative thinlayer chromatography on silica gel yields ,Q,. The cis diastereoisomers and the cistrans mixture are obtained in an analogous manner.

Step H:

oCoOPNB 5-coidine SCH,CH,N, LiI (CO2PNB)2 5 OCOOPNB SeH2CH2N2 H CO2PNB 6 A solution of 187 mg Q (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 Lil (dried for a few hours at 100"C over P205 under vacuum) (mw=134; 0.336 mmole). With stirring under N2, the reaction mixture is heated in an oil bath at 1200C. After a total of 25 minutes, the reaction mixture is cooled to 250C., diluted with CH2CI2 and transferred to a round-bottom flask for concentration under a N2 stream and then on high vacuum. Partitioning of the residue between 10 ml EA and 1.8 ml I M KH2PO4 in 10 ml H2O is followed by extraction of the aqueous layer twice more with EA. The combined organic layers are extracted with brine, dried over anhydrous MgSO4, filtered and concentrated under a stream of N2 to give crude Preparative thin layer chromatography on silica gel yields 6. The cis diastereoisomers and the cis-trans mixture are obtained in an analogous manner.

Step I:

To a solution of the mixed diastereoisomers, " (34 mg; mw=612; 0.055 mmole) in 0.2 ml DMSO (distilled from CaH2 at 8 mm and stored over 4A Linde Molecular sieves) with stirring is added 9.5 yl 1,5 - diazobicyclo[5.4.0]undec - 5 ene (distilled at 800C/2mm), (mw=152; p=l; 0.0625 mmole). The solution is stirred under N2 for 15 minutes at 250C, diluted to 1 ml total volume with CHCI3 and applied immediately to 2-1000 silica gel plates. The product band yields 1, as a mixture of cis and trans distereoisomers.

Step J:

In the presence of 61 mg PtO2, 61 mg of 7 (mw=612; 0.1 mmole) in 6 ml dioxane, 6 ml THF and 3 ml H2O is hydrogenated at 40 p.s.i. 112 for 4 hours, The reaction mixture is then filtered through Celite, washing with 2 ml 0.1N pH7 phosphate buffer. After concentration in vacuo to the cloud point, the aqueous mixture is extracted with ethyl acetate. The aqueous layer is concentrated to a small volume and applied to a column of 100 g Amberlite 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.

EXAMPLE 16 Preparation of Pharmaceutical Compositions One unit dosage form consists in mixing an equimolar mixture of (SR,6S,8S)and (SS,6R,8R) - 3 - (2 - aminoethylthio) - 6 - (I - hydroxyethyl) - 7 - oxo - I azabicyclo[320]hept - 2 - ene - 2 - carboxylic acid [I(16)] with 20 mg of lactose and 5 mg of magnesium stearate and placing the 145 mg mixture in 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 capsulses 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 other pharmaceutical formulations: Tablet Per Tablet 1(16) 125 mg.

Cornstarch, U.S.P. 6 mg.

Dicalcium Phosphate 192 mg.

Lactose, U.S.P. 190 mg.

Magnesium Stearate Balance/800 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 magnesium stearate is added and the mixture is compressed into tablets, approximately 0.5 inch in diameter, each weighing 800 mg.

Parenteral Solution Ampoule: Per Tablet 1(16) 500 mg.

Diluent: Sterile Water for Injection 2 cc.

Ophthalmic Solution I(16) 100 mg.

Hydroxypropylmethyl Cellulose 5 mg.

Sterile Water to I ml.

Otic Solution 1(16) 100 mg.

Benzalkonium Chloride 0.1 mg.

Sterile Water to 1 ml.

Topical Ointment 1(16) 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, gentamicin, neomycin, colistin and kanamycin, or with other therapeutic agents such as probenecid.

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

which has the absolute configuration SS,6S,8R and the pharmaceutically acceptable salts thereof.

2. A compound having the structural formual:

which has the absolute configuration 5R,6R,8R and the pharmaceutically acceptable salts thereof.

3. A compound having the structural formula:

which has the absolute configuration SS,6S,8S and the pharmaceutically acceptable salts thereof.

4. A compound having the structural formula:

which has the absolute configuration SS,6R,8R and the pharmaceutically acceptable salts thereof.

5. A compound having the structural formula:

which has the absolute configuration SS,6R,8S and the pharmaceutically acceptable salts thereof.

6. A mixture comprising two or more isomers of a compound having the structural formula:

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (31)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   Topical Ointment 1(16) 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, gentamicin, neomycin, colistin and kanamycin, or with other therapeutic agents such as probenecid.

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

   which has the absolute configuration SS,6S,8R and the pharmaceutically acceptable salts thereof.
2. 2. A compound having the structural formual:

   which has the absolute configuration 5R,6R,8R and the pharmaceutically acceptable salts thereof.
3. 3. A compound having the structural formula:

   which has the absolute configuration SS,6S,8S and the pharmaceutically acceptable salts thereof.
4. 4. A compound having the structural formula:

   which has the absolute configuration SS,6R,8R and the pharmaceutically acceptable salts thereof.
5. 5. A compound having the structural formula:

   which has the absolute configuration SS,6R,8S and the pharmaceutically acceptable salts thereof.
6. 6. A mixture comprising two or more isomers of a compound having the structural formula:

   which isomers, in terms of their absolute configuration relative to the above structure are 5R,6R,8S; 5S,6S,8R; 5R,6R,8R; 5S,6S,8S; 5R,6S,8S; 5S,6R,8R; 5R,6S,8R; or 5S,6R,8S, at least one of the isomers in the mixture being a compound as claimed in any one of claims 1 to 5.
7. 7. A protected form of a compound as claimed in any one of claims I to 5 or of a mixture as claimed in claim 6, in which the three groups --OH, --NH, and COOH are blbcked by O-protecting, N-protecting and esterifying groups respectively.
8. 8. A process for preparing a compound having the formula:

   and its pharmaceutically acceptable salts; comprising halogenating, and then cyclizing in the presence of a base, a compound of formula:

   to form a compound of formula:

   dehydrohalogenating the resulting intermediate compound in the presence of a strong base to form a compound of formula:

   heating this compound with an aromatic base in the presence of a displacing agent to form a compound of formula:

   isomerizing the position of the double bond of the last mentioned compound by treating it with a strong base in a solvent to form a compound of formula:

   and deblocking the latter by treatment in a solvent with hydrogen in the presence of a metal of the platinum group as catalyst to form a compound of formula:

   where R4 and R5 are blocking groups, and X is a halogen atom.
9. 9. A modification of a process as claimed in claim 8, in which the deblocking step is omitted and a product as claimed in claim 7 is produced.
10. 10. A process for preparing a compound having the formula:

    comprising cyclizing in the presence of a base a compound of formula:

    to form a compound of formula:

    dehydrohalogenating this intermediate compound in the presence of a strong base in a solvent to form a compound of formula:

    treating this compound with an aromatic base in the presence of a displacing agent to form a compound of formula:

    isomerizing the position of the double bond of the last mentioned compound by heating it with a strong base in a solvent to form a compound of formula:

    and hydrogenolysing the latter in a solvent in the presence of a metal of the platinum group to deblock and reduce the acid to form a compound of formula:

    where R4 is a blocking group, and X is halo.
11. 11. A process for preparing a compound having the formula:

    substantially as hereinbefore described in any one of Examples 12, 13 and 15.
12. 12. A compound of formula:

    when prepared by a process as claimed in claim 8, 10 or 11.
13. 13. A pharmaceutically acceptable salt of a compound as claimed in Claim 12.
14. 14. An antibiotic pharmaceutical composition comprising a compound according to claim I and a pharmaceutical carrier.
15. 15. An antibiotic pharmaceutical composition comprising a compound according to claim 2 and a pharmaceutical carrier.
16. 16. An antibiotic pharmaceutical composition comprising a compound according to claim 3 and a pharmaceutical carrier.
17. 17. An antibiotic pharmaceutical composition comprising a compound according to claim 4 and a pharmaceutical carrier.
18. 18. An antibiotic pharmaceutical composition comprising a compound according to claim 5 and a pharmaceutical carrier.
19. 19. An antibiotic pharmaceutical composition comprising a mixture according to claim 6 and a pharmaceutical carrier.
20. 20. An antibiotic pharmaceutical composition comprising a mixture or compound according to claim 12 or 13 and a pharmaceutical carrier.
21. 21. A composition as claimed in any one of claims 14 to 20 in the form of a pill, tablet, orally administrable powder, capsule, solution, syrup, suspension, emulsion or elixir.
22. 22. A composition as claimed in any one of claims 14 to 20 in the form of a suppository.
23. 23. A composition as claimed in any one of claims 14 to 20 in the form of an ampoule or a multidose container containing an injectable liquid medium.
24. 24. A composition as claimed in any one of claims 14 to 20 in the form of a powder or liquid spray or inhalant, a lozenge or a throat paint,
25. 25. A topical composition as claimed in any one of claims 14 to 20 in the form of an ointment, cream, lotion, paint or powder.
26. 26. A composition as claimed in any one of claims 14 to 20 in the form of an otic or ophthalmic solution.
27. 27. A disinfectant containing as an antibacterial ingredient a compound or mixture as claimed in any one of claims 1 to 6, 12 and 13.
28. 28. An animal feedingstuff or an additive for an animal feedingstuff containing as an antibacterial ingredient a compound or mixture as claimed in any one of claims I to 6, 12 and 13.
29. 29. A water-based paint containing as an antibacterial ingredient a compound or mixture as claimed in any one of claims 1 to 6, 12 and 13.
30. 30. A paper mill white water containing as an antibacterial ingredient a compound or mixture as claimed in any one of claims 1 to 6, 12 and 13.
31. 31. A pharmaceutical composition substantially as hereinbefore described in Example 16.