Abstract:
There are disclosed 2(N-heterocyclyl) penems and their pharmaceutically acceptable salts and esters and their use as anti-bacterials.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to 2-(N-heterocyclyl) penems and their pharmaceutically acceptable salts and pharmaceutically acceptable esters, which compounds possess potent anti-bacterial activity. 
     There is a continuing need for new antibacterial agents because continued extensive use of effective anti-bacterials gives rise to resistant strains of pathogens. 
     SUMMARY OF THE INVENTION 
     This invention provides novel penems substituted in the 2-position by a heterocyclic group wherein a nitrogen of the heterocyclic ring is connected by a bond to the 2 carbon of the penem molecule. The compounds of this invention are anti-bacterial agents. 
     More particularly, this invention provides compounds represented by the formula ##STR1## and pharmaceutically acceptable salts and pharmaceutically acceptable esters thereof, in racemic or optically active form, wherein each X independently is ##STR2## with the proviso that at least one X is ##STR3## each R is independently selected from hydrogen, lower alkyl, hydroxy, amino lower alkyl, mono- and di-lower alkyl amino lower alkyl, hydroxy lower alkyl, carbamoyloxy lower alkyl, amino, mono- and di-lower alkyl amino, carboxy lower alkyl, carbamoyl lower alkyl, mono- and di-lower alkyl carbamoyl lower alkyl, cyano lower alkyl, halo lower alkyl, carboxy, cyano, hydroxyl imino-methyl, sulfo lower alkyl, lower alkoxy imino-methyl, lower alkoxy, 1(N,N-mono- or di-lower alkyl) hydrazino-2-ylidenyl methyl, carbamoyl amino, mono- and di-lower alkyl carbamoyl amino, lower alkanoyloxy, carbamoyloxy, lower alkanoyl amino, lower alkanoyl amino lower alkyl, N-lower alkylureido, lower alkyl sulfonylamino, sulfonic acid, nitro and 2 adjacent R groups can be connected to form, fused to the five membered heterocyclic ring, a six membered heterocyclic ring of the formula ##STR4## wherein X is as defined herein. 
     Contemplated within the scope of this invention are aromatic and non-aromatic heterocyclic rings, e.g., imidazoles, triazoles, tetrazoles, purines, imidazolines, and the like. 
     Preferred compounds of formula I are those in which the nitrogen-containing heterocyclic substituents are unsubstituted and substituted imidazoles, triazoles and tetrazoles, e.g., imidazol-1-yl, 1,2,4-triazol-4-yl, 1,2,4-triazol-1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-1-yl, 1,2,3,4-tetrazol-1-yl, 1,2,3,4-tetrazol-2-yl and pyrazol-1-yl. 
     Preferred R substituents are amino, hydroxy and lower alkyl. 
     The terms &#34;lower alkyl,&#34; &#34;lower alkoxy&#34; and &#34;lower alkanoyl&#34; as used herein means alkyl, alkoxy and alkanoyl groups of 1 to 6 carbon atoms in the alkyl portion and includes branched and straight chains, e.g., methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tertiary butyl, pentyl, hexyl, neopentyl, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, iso-butoxy, pentoxy, hexoxy, acetyl, propionyl, butyryl, pentanoyl and hexanoyl. &#34;Halo&#34; includes fluorine, chlorine and bromine. 
     As used herein, &#34;pharmaceutically acceptable salts&#34; means alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium, magnesium and aluminum salts; amine salts formed from a wide variety of suitable organic amines, i.e., aliphatic, cycloaliphatic, (cyloaliphatic)aliphatic or araliphatic primary, secondary or tertiary mono-, di- or polyamines, or heterocyclic bases, e.g., salts derived from triethylamine, 2-hydroxyethylamine, di-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, 4-aminobenzoic acid-2-diethylaminoethyl ester, 1-ethylpiperidine, bicyclohexylamine, N, N&#39;-dibenzylethylenediamine, pyridine, collidine, quinoline, procaine, dibenzylamine, 1-ephenamine and N-alkylpiperidine, acid addition salts formed from mineral acids such as hydrochloric, hydrobromic, hydroiodic, phosphoric or sulfuric acids, or formed from organic carboxylic or sulfonic acids such as trifluoroacetic, para-toluene sulfonic, maleic, acetic, citric, oxalic, succinic, benzoic, tartaric, fumaric, mandelic, ascorbic and malic acids. The compounds of this invention contain a 3-carboxylic group and a basic group (the heterocyclic group) which form an inner salt, i.e., a Zwitterion. 
     &#34;Pharmaceutically acceptable esters&#34; means physiologically cleavable esters, i.e., metabolizable esters known in the penicillin, cephalosporin and penem arts to be easily cleaved within the body to the parent acid. Examples of such esters are indanyl, phthalidyl, methoxymethyl, glyclyoxymethyl, phenylglycyloxymethyl, thienylglycyloxymethyl, acetoxymethyl and pivaloyloxymethyl. 
     Preparation of the foregoing salts and esters may be carried out according to conventional procedures for forming salts of beta-lactams such as penicillins, cephalosporins and penems. Salts of the compound can be formed, for example, by treating with metal compounds such as alkali metal salts of suitable carboxylic acids, or with ammonia or a suitable organic amine, wherein preferably stoichiometric amounts or only a small-excess of the salt-forming agent is used. Acid addition salts of the compound are obtained in the usual manner, for example, by treating with an acid or a suitable anion exchange reagent. Inner salts of the compounds of formula A, i.e., a zwitterion, may be formed by neutralizing salts such as acid addition salts to the isoelectric point. The esters are preparable in a manner analogous to the preparation of the corresponding esters of penicillins and cephalosporins. 
     Salts may be converted in the usual manner into the free carboxy compounds. 
     At the 5, 6 and 8 positions of the penem nucleus having chiral atoms, the compounds of this invention may possess 5R, 6S, 8R or 5R, 6R, 8S stereochemistry at those chiral atoms. The preferred absolute stereochemistry for the compounds of this invention at those positions is 5R, 6S, 8R. 
     DETAILED DESCRIPTION 
     When tested in standardized microbiological assays, the compounds of this invention are active against such gram-positive organisms as Staphylococcus epidermis and Bacillus subtilis, and such gram-negative organisms as E. coli and Salmonella at test levels of 0.1 to 2.0 micrograms/ml. Additionally, they show activity against organisms which produce beta-lactamase, e.g., penicillinase and cephalosporinase, indicating a stability toward those enzymes. For instance, (5R,6S,8R)-2-(imidazol-1-yl)-6-(1-hydroxyethyl) penem-3-carboxylic acid is active against E. coli 74081501 TEM-1 (a beta-lactamase producing organism) at 0.5 microgram/ml. 
     When tested against over sixty organisms, the mean test level against gram-negative organisms was 1.54 micrograms/ml and against gram-positive organisms was 0.27 micrograms/ml. 
     The compounds of this invention and their metabolites have little or no unpleasant odor. 
     As anti-bacterial agents, the compounds of this invention are conventionally formulated for oral, parenteral, topical and transdermal use. Thus, this invention includes within its scope pharmaceutical compositions comprising the compounds of this invention in admixture with a pharmaceutically acceptable carrier thereof. In addition, the present invention also provides a method of treating bacterial infections in animals, particularly warm-blooded animals having a susceptible bacterial infection which comprises administering to said animal an anti-bacterial effective amount of a compound of this invention, or a pharmaceutical composition thereof. In the foregoing compositions, the compounds of this invention can be used as the sole active anti-bacterial agent or in combination with other anti-bacterial agents and/or enzyme inhibitors. 
     For oral administration, the compounds of this invention are typically formulated in the form of tablets, capsules, elixirs, or the like. For parenteral administration, they may be formulated into solutions or suspensions. Typical topical formulations are those such as lotions, creams, ointments, sprays, and mechanical delivery devices, e.g., transdermal. Parenteral administration is preferred. 
     Typical pharmaceutically acceptable carriers for use in the formulations described above are exemplified by: sugars such as lactose, sucrose, mannitol and sorbitol; starches such as corn starch, tapioca starch and potato starch; cellulose and derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium suflate; polyvinyl pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates such a magnesium stearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; non-ionic, cationic and anionic surfactants; ethylene gylcol polymers; betacyclodextrin; fatty alcohols; hydrolyzed cereal solids; water; polyalkylene gylcols; gums; and petrolatum; as well as other non-toxic compatible fillers, binders, disintegrants and lubricants commonly used in pharmaceutical formulations. The compositions may also contain preservatives, aerosol propellants and coloring, thickening, suspending, dispensing, emulsifying, wetting, stabilizing and buffering agents. 
     The dosage of the compounds of this invention which is administered is dependent, on the judgment of the attending clinician taking into account a variety of factors, i.e., the age and weight of the individual being treated, the mode of administration, and the type and severity of the bacterial infection being prevented or reduced and the potency of the specific compound administered. Typically, the dosage administered per day will be in the range of from about 1 to 250 mg/kg and preferably from about 5 to 20 mg/kg in divided dosages. Typically, the dosage will be administered is dosage units containing convenient amounts, for example, 250, 500, 1,000 or 2,000 mg of active ingredient combined with a suitable physiologically acceptable carrier or diluent. 
     The preferred compounds of this invention are prepared by reacting an azetidinone represented by the formula ##STR5## with bis(trimethylsilyl) acetamide or other known readily removable hydroxy protecting group to protect the hydroxyl group, then with a thiocarbonyl compound represented by the formula ##STR6## wherein X is as defined for formula I, to obtain a compound represented by the formula ##STR7## wherein X is as defined above. 
     The above reactions take place in an organic solvent which is inert to the reaction, e.g., methylene chloride. 
     The compound of Formula IV is then cyclized by heating in an organic solvent such as toluene and the hydroxy group is deprotected by conventional means, e.g., heating with methanol in acetic acid. To obtain a compound represented by the formula ##STR8## wherein X is as defined above. 
     Removal of the allyl group from compound V is effected by the addition of the above allyl ester to a solution containing palladium (zero) and an alkali alkylcarboxylate, carboxylic acid or aqueous carbonate. This is described by McCombie in U.S. Pat. No. 4,314,942 which is incorporated herein by reference. Under these conditions, removal of the allyl group and formation of the alkali salt or the free acid occurs. 
     The thiocarbonyl intermediates are known compounds or can be made by known methods disclosed in Larson et. al., J. Org. Chem., 43, 337 (1978). Analogous heterocyclic substituents and substituted heterocyclic substituents can be made by using as the starting material the corresponding substituted silylated derivative. For example, the N-trimethylsilyl derivative of the heterocyclic compound is converted to the corresponding thiocarbonyl by reaction with CSCl 2  (thiophosgene) in a carbontetrachloride solvent as shown in the following reaction scheme. ##STR9## wherein X is as defined for formula I. 
     The azetidinone intermediate can be prepared, for example, by the following reaction scheme ##STR10## wherein OP is a protected hydroxyl group. 
     The acetoxy group of compound A is displaced with --SC(C 6  H 5 ) 3  by reaction with trityl thiol. The resulting compound B is converted to compound C by reaction with the allyl ester of glyoxylic acid to obtain a compound represented by the formula ##STR11## 
     The hydroxy group is converted to the corresponding chloro derivative by treatment with a chlorinating agent in an inert organic solvent in the presence or absence of a base. 
     The chloro compound is then reacted with triphenyl phosphine in an inert organic solvent in the presence of a base to produce the corresponding phosphorane compound represented by the formula ##STR12## 
     The hydroxy protecting group of compound E is removed by conventional means known in the lactam art and the resulting compound is reacted with a reactive silver salt to form compound C. 
    
    
     The following examples illustrate the preparation of the compounds and compositions of this invention. 
     EXAMPLE 1 
     Silver 3S,4R)-3-(1-Hydroxyethyl)-1-!Allyl(Triphenylphosphoranylidene Acetate)1-Azetidin-2-One-4-Thiolate 
     Add 1.15 ml of a 1 molal solution AgNO 3  /1:1 pyridine/MeOH to a solution of 0.8 gm of compound E (with the hydroxy deprotected) in 5 ml methanol and 1.5 ml CH 2  Cl 2  in an ice-bath at 0° C. Stir for 1 hour until thin layer chromatography (TLC)(75% EtOAc/hexane) shows no starting material. Remove the solvent, add 25 ml CH 2  Cl 2 , wash with water and dry over Na 2  SO 4  to obtain the title compound as a yellow solid. 
     EXAMPLE 2 
     1,1&#39; Thiocarbonyl-Di-Pyrazole 
     Add a solution of 1.43 gm (0.0125 mole) CSCl 2  in 5 ml of CCl 4  during 1.5 hours with cooling in an ice-bath to a solution of 3.5 gm trimethyl silyl pyrazole in 20 ml CCl 4 . Continue stirring for 6 hours. 
     Remove the solvent under vacuum and crystallize the resulting oil by adding seed crystals (prepared by cooling a small amount of the oil in dry ice/acetone) to obtain title compound. 
     EXAMPLE 3 
     1,1&#39; Thiocarbonyl-Di-1,2,4 Triazole 
     Add a solution of 1.43 gm (0.0125 mole) CSCl 2  in 5 ml CCl 4  during 1.5 hours to an ice-bath cooled and stirred solution of 3.5 gm (0.025 mole) trimethyl silyl- 1,2,4 triazole in 20 ml CCl 4 , a yellow solid begins precipitating after about 45 minutes. Stir for 8 hours at room temperature after addition is complete. Isolate the title compound as a yellow solid. 
     EXAMPLE 4 
     (5R,6S,8R)2-(Imidazol-1-yl)-6-(1-Hydroxyethyl)-Penem-3-Carboxylic Acid 
     (a) Add 0.12 ml (0.0994 gm, 0.00049 mole) bis(trimethylsilyl)acetamide to a solution of 100 mg (0.00016 mole) of the compound of Example 1 in 5 ml CH 2  Cl 2 . Stir the resulting solution for 0.5 hour at room temperature and remove the CH 2  Cl 2  solvent under vacuum. Dissolve the resulting residue in 10 ml CH 2  Cl 2 , add 0.057 gm (0.00032 mole) 1,1&#39;-thiocarbonyldiimidazole (available from Aldrich Chemical Co., Milwaukee, Wisc.). Stir the mixture at room temperature for 1 hour. The resulting product, (3S,4R)-3-(1-trimethylsilyloxy ethyl)-1-[allyl(triphenylphosphoranylidene acetate)]-4-imidazol-1-yl thiocarbonyl-azetidin-2-one, was recovered. 
     (b) Heat a solution of 0.5 gm of the product of part (a) of this example in 50 ml toluene under reflux for three hours. Remove the solvent under vacuum and recover the product, allyl(5R,6S,8R)-2(imidazol-1-yl)-6-(1-trimethylsilyloxyethyl)-penem-3-carboxylate. 
     (c) Dissolve about 200 mg of the compound made in part (b) of this example in methanol. After about three hours, add acetic acid dropwise until the reaction is complete as determined by TLC (50% ethylacetate/CH 2  Cl 2 ) and recover allyl(5R,6S,8R)-2-(imidazol-1-yl)-6-(1-hydroxyethyl)-penem-3-carboxylate (formula V). 
     NMR: (CDCl 3 ), δ7.8(1H, S), 7-7.2 (2H, M), 5.7-6.0(1H, M), 5-5.3(2H, M), 4.55(2H, d), 4.15(1H, M), 3.75(1H, d of d), 1.3(3H, d). 
     (d) Add 0.8 ml of 2-ethyl hexanoic acid and 250 mg triphenylphosphine to a solution the compound produced in part (c) of this example. Add to the resulting solution a solution of 100 mg Pd[P(C 6  H 5 ) 3  ] in 3 ml CH 2  Cl 2 . The reaction is completed within one hour. Recover the product and obtain the title compound as a white solid. 
     NMR: D 2  O: δ8.85(1H, broad singlet), 7.57(1H, broad s), 7.4(1H, broad s), 5.8(1H, d), 1.25 (3H, d), between 4 &amp; 4.6 masked by spinning bands of DHO. 
     MS [M+H] +  282. 
     EXAMPLE 5 
     (5R,6S,8R) 2-(Pyrazol-1-yl)-6-(1-Hydroxyethyl)-Penem-3-Carboxylic Acid 
     (a) Add 1.2 ml bis-(trimethyl silyl) acetamide to a solution of 1.0 gm of the compound of Example 1 in 25 ml CH 2  Cl 2 . Stir the solution at room temperature for 0.5 hour and then remove the CH 2  Cl 2  under vacuum. Dissolve the resulting residue in 40 ml CH 2  Cl 2  and add 0.57 gm 1,1&#39;-thiocarbonyldipyrazole. Stir at room temperature for 2 hours and then recover the product as a yellow oil, (3S,4R)-3-(1-trimethylsilyloxyethyl)-1-[allyl(triphenylphosphoranylidene acetate)]-4-pyrazol-1-yl thiocarbonylazetidin-2-one. 
     (b) Heat under reflux for 3 hours a solution of 0.5 gm of the product of part (a) of this example in 50 ml toluene. Remove the solvent under vacuum and recover the product, allyl (5R,6S,8R)-2-(pyrazol-1-yl)-6-(1-trimethylsilyl-oxyethyl)penem-3-carboxylate. 
     (c) Dissolve 0.1 gm of the compound made in part (b) of this example in 15 ml methanol. After 8 hours add 0.25 ml glacial acetic acid to complete the reaction as determined by TLC. Recover the product, allyl (5R,6S,8R)-2-(pyrazol-1-yl)6-(1-hydroxyethyl)penem-3-carboxylate) as a yellow solid. 
     NMR: cDd 3  : d 8.6(1H, d), 7.7(1H, d), 6.4(1H, t), 5.7-6.2(1H, M), 5.6(1H, d), 5.1-5.5(2H, M), 4.7(2H, M), 4.2(1H, quin), 3.8(1H, d of d), 2.8(1H, broad S), 1.3(3H, d). 
     (d) Add 0.12 ml 2-ethyl hexanoate and 40 mg triphenylphospine to a solution of 80 mg of the compound made in part (c) of this example in 10 ml of CH 2  Cl 2 . Add to the resulting solution a solution of Pd[P(C 6  H 5 ) 3  ] 4  in 1 ml CH 2  Cl 2 . The reaction was completed after 1 hour as determined by TLC. Recover the title compound as a white solid. 
     MS[M +  H +  ]282. 
     EXAMPLE 6 
     (5R,6S,8R)-2(1,2,4 Triazol-1-yl)-6-(1-Hydroxyethyl) Penem-3-Carboxylic Acid 
     (a) Add 0.24 ml bis(trimethylsilyl) acetamide to a solution of 0.2 gm of the compound made in Example 1 in 5 ml dry CH 2  Cl 2 . After 0.5 hour concentrate on a rotary evaporatos and then under high vacuum. Dissolve the resulting oily residue in 5 ml CH 2  Cl 2  and add to the solution a solution of 116 mg 1,1&#39; thiocarbonyldi-1,2,4 triazol-1-yl in 1 ml CH 2  Cl 2 . When the reaction is completed as determined by TLC (10% ethylacetate/CH 2  Cl 2 ), recover the product as a yellow solid. 
     (b) Dissolve 50 mg of the compound made in part (a) of this example in 10 ml toluene and heat under reflux for 1 hour to complete the reaction as determined by TLC (10% ethylacetate/CH 2  Cl 2 ). Store the recovered product overnight in a refrigerator to cause deprotection of the hydroxy group and obtain allyl(5R,6S,8R)-2(1,2,4-triazol-1-yl)-6(1-hydroxyethyl)penem-3-carboxylate. 
     NMR: cDd 3  : δ9.1(1H, S), 8.0(1H, S), 5.7-5.1(1H, M), 5.65(1H, d), 5.15-5.4(2H, M), 4.7(2H, M), 4.25(1H, M), 3.82(dH, d of d), 2.6(1H, broad S), 1.35(3H, d). 
     (c) Add 0.03 gm Pd[P(C 6  H 5 ) 3  ] 4  to a solution of 0.23 gm of the compound made in part (b) of this Example, 0.1 gm triphenylphosphine and 0.4 ml ethylhexanoate in 10 ml CH 2  Cl 2 . Stir the resulting solution at room temperature for 2 hours until TLC (H 2  O, reverse phrase) shows the reaction is complete, to obtain the title compound as a yellowish solid. 
     MS. [M+H] +  283. 
     The following are representative compounds of this invention that can be prepared following the procedures of Examples 3, 4 and 5 by substituting equivalent amounts of the appropriate thiocarbonyl compound of formula III for those in Examples 3, 4 and/or 5, it being understood that all of the compounds of formula I can also be prepared in the same manner. 
     1. (5R,6S,8R) 2-(1,2,3,4-tetrazol-1-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     2. (5R,6S,8R) 2-(1,2,3,4-tetrazol-2-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     3. (5R,6S,8R) 2-(1,2,4-triazol-4-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     4. (5R,6S,8R) 2-(purin-9-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     5. (5R,6S,8R) 2-(5-hydroxymethylimidazol-1-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     6. (5R,6S,8R) 2-(4-hydroxymethylimidazol-1-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     7. (5R,6S,8R) 2-(2-hydroxymethylimidazol-1-yl)-6-(1-hydroxyethyl)-penem-3-carboxylic acid. 
     In the following examples Active Ingredient is (5R,6S,8R) 2-(imidazol-1-yl)-6-(1-hydroxyethyl)penem-3-carboxylic acid or an equivalent amount of any of its pharmaceutically acceptable salts and esters. 
     It will be appreciated that this compound can be replaced by equivalent amounts of the other compounds of formula I. 
     EXAMPLE 7 
     
         ______________________________________CapsulesNo.  Ingredient        mg/tablet  mg/tablet______________________________________1.   Active Ingredient 250        500.02.   Lactose USP       100        50.03.   Corn Starch, Food Grade                  50         43.54.   Microcrystalline  95         50.0Cellulose NF5.   Magnesium Stearate NF                  5          6.5Total             500        650.0______________________________________ 
    
     Method of Manufacture 
     Mix Items Nos. 1, 2, 3 and 4 in a suitable mixer for 10-15 minutes. Add Item No. 5 and mix for 1-3 minutes. Fill the mixture into suitable two-piece hard gelatin capsules using encapsulating machine. 
     EXAMPLE 8 
     
         ______________________________________TabletsNo.  Ingredient          mg/tablet mg/tablet______________________________________1.   Active Ingredient   250       5002.   Lactose USP         57        1143.   Corn Starch, Food Grade 10%                    20        40paste in Purified Water4.   Corn Starch, Food Grade                    18        395.   Magnesium Stearate NF                    5         7Total               350       700______________________________________ 
    
     Method of Manufacture 
     Mix Items Nos. 1 and 2 in a suitable mixer for 10-15 minutes. Granulate the mixture with Item No. 3. Pass the wet granulation through a coarse screen (e.g., 1/4&#34;), if needed, and dry the wet granules. Mill the dried granules. Combine Item No. 4 and the dried granules and mix for 10-15 minutes. Add Item No. 5 and mix for 1-3 minutes. Compress the mixture to appropriate size and weight on a suitable tablet machine. 
     EXAMPLE 9 
     
         ______________________________________Injectable Powder           g/vial                 g/vial______________________________________Active Ingredient 0.5     1.0______________________________________ 
    
     Add sterile water for injection or bacteriostatic water for injection for reconstitution. 
     EXAMPLE 10 
     
         ______________________________________Injectable SolutionIngredient              mg/ml______________________________________Active Ingredient       500Methylparaben           1.8Propylparaben           0.2Sodium Bisulfite        3.2Disodium Edetate        1.5Sodium Sulfate          2.6Water for Injection q.s. ad                   1.0    ml______________________________________ 
    
     Method of Manufacture 
     1. Dissolve parabens in a portion (85% of the final volume) of the water for injection at 65°-70° C. 
     2. Cool to 25°-35° C. Charge and dissolve the sodium bisulfite, disodium edetate and sodium sulfate. 
     3. Charge and dissolve the active ingredient. 
     4. Bring the solution to final volume by adding water for injection. 
     5. Filter the solution through 0.22 micron membrane and fill into appropriate containers. 
     6. Terminally sterilize the units by autoclaving. 
     EXAMPLE 11 
     
         ______________________________________Injectable Powder          g/vial______________________________________Active Ingredient            1.00Sodium Citrate   0.05______________________________________ 
    
     pH is adjusted to 6.2 using 0.1N citric acid solution. Add sterile water for injection or bacteriostatic water for injection for reconstitution.