ANTIBIOTICS AND PROCESS FOR PREPARATION FIELD OF THE INVENTION This invention is concerne with the preparation of a wide variety of antibiotics of new and novel structure and antimicrobial activity. The compound thus prepared are products from the linking of diverse antibiotic moities via difunctional organic compound such as diisocyanates, dianhydrides, diacidchlorides, diepoxides and carbodiimides, said antibiotics being drawn from the classes of compound sulfonamides, penicillins and relate, cephalosporins and relate, quinolones, chloramphenicol, erythromycins, metronidazole, tetracyclines and aminoglycocides.

BACKGROUND OF THE INVENTION The medical literature regarding antimicrobial agents is vast and describes a number of antimicrobials including naturally occurring compound as well as synthetic or semisynthetic compound produced in the organic laboratory.

These antimicrobial agents are classifie as noted above, and there are many classes in addition to the above-noted ones.

It has been realized that the linking of two antibiotic moities functioning in different fashions, as for example inhibiting cell-wall synthesis or protein synthesis or DNA synthesis, can be of value. Two antibiotic moities can also be linked in which one is known to attack Gram positive bacteria and another to attack Gram negative bacteria, and this new entity is of value.

Usually the synthesis of linked antibiotics requires an extended set of organic laboratory procedures in which prior to the linkage of diverse types, such as quinolones and lactams, certain groups in the molecule must be blocked, the blocked entity then linked to a second antibiotic, which may also require blocking of some functional groups, and also the blocking groups require removal. It has been found surprisingly that a number of difunctional reagents can effect an efficient linkage of very diverse antibiotic structures. Further, the progress of the rection can easily be followed via IR spectroscopy techniques, and the isolation of meaningful quantities achieved in facile fashion via liquid chromatography techniques.

SUMNLARY OF THE INVENTION This invention is concerne with simple methods of preparing a large number of new and novel structures possessing a wide range of antibiotic activity via linking together two antibiotic moities.

A-L-B wherein A has the structure drawn from the following classes of antibiotics: 1. sulfonamides and related 2. penicillins and related 3. cephalosporins and related 4. quinolones 5. chloramphenicol 6. erythromycin 7. metronidazole 8. tetracyclines 9. aminoglycosides and B, drawn from the same classes.

The classes may be further characterized by he following general formulas and particular examples. L is drawn from a group of difunctional linking reagents.

1. Sulfonamides and Related where R' is a variety of substituents.

The sulfonamides listed below are of particular interest: A. p-aminobenzenesulfonamide B. sulfamethoxyazole C. acetylsulfoxazole D. sulfamethoxypyridazine E. sulfadiazine 2. Penicillins and Related where R" is a variety of substituents. The penicillins listed below are of particular interest.

A. benzyl penicillin B. procaine penicillin G C. phenoxymethyl penicillin D. ampicillin E. amoxycillin F. methicillin G. oxacillin H. cloxacillin I. dicloxacillin J. flucloxacillin K. nafcillin L. carbenicillin M. ticaricillin N. talampicillin O.becampicillin P. pivampicillin Q. penamcarboxylic acid R. hydroxyethyl penem S. imipenem T. amdinocilin 3. Related:and where R3 and R4 are a variety of substituents. The cephalosporins listed below are of particular interest.

A. cephalosporin C B. cephalothin C. cephaloridine D. cephradine E. cephazolin F. cephalexin G. cefadroxil H. cefaclor I.cephamandole J. cefuroxine K. cefotaxime L. ceftizoxime M. ceftazidime MN.cefoperazone CO.cephamycin P. cefoxitin Q. moxalactam 4. u'n 1 e where R5 is a variety of substituents and the quinoline nucleus contains fluoro atom substitution. The quinolones listed below are of particular interest.

A. nalidixic acid B. norfloxacin C. enoxacin D. ciprofloxacin E. ofloxacin 5. Chloramphenicol 6. Erythromycin ' 0 . fro" Wo 4-N HO H H llreraeyelic letoa Mn9 H H p^Cldlnou HT-TH H OH 7. Metronidazole 8. Tetracyclines where the general formula given above is substituted to yield the particular compound listed below.

A. tetracycline B. oxytetracycline C. chlortetracycline D. rolitetracycline E. methacycline F. doxycycline G. demeclocycline H. sancycline I.lymecycline J. clomocycline K. minocycline 9. Aminoglycosides NH NH H, NCNHH HNCNH, H m Str*ptidine H OU PU C H H O LStaptose Calo H HIC H OH Streptobiosemine H O HO RAl-Mothyl-l-glucosamine H H OH H Streptomycin (RCH NH) The general formula above is variously substituted to give the particular isomers listed below.

A. streptomycin B. tobramycin C. kanamycin D. amikacin E. gentamicin C I F. nitilimicin G. neomycin H. paromomycin I. spectinomycin The linking reagents are drawn from the type listed below.

Diisocyanates NCO-Y-NCO Diianhydrides Diacidchlorides Diepoxides Carbodiimides In the above general formulas Y can be aliphatic, alicyclic, aromatic and heterocyclic groups.

The particular formulas for each type are listed below.

Diisocyanates: 1,6-hexamethylenediisocyanate 2,4-tolyldiisocyanate 2,6-tolyldiisocyanate 4,4'-methylene bis phenylisocyanate 4,4'-isopropylidene bis phenylisocyanate 1,4-phenyldiisothiocyanate 1,4-phenyldiisocyanate Dianhydrides pyromellitic dianhydride bis maleic dianhydride 3,3,4,4'-benzophenonetetracarboxylic dianhydride 1,2,6,7- hexanetetracarboxylic dianhydride 1,2,4,5-naphthalenetetracarboxylic dianhydride Diacidchlorides terphthaloyl chloride isophthaloyl chloride phthaloyl chloride adipolyl chloride glutaryl chloride Diepoxides 1,3-butane diepoxide 1, 5-cyclooctatetraene diepoxide vinylcyclohexenediepoxide 1,4-divinylbenzene diepoxide Carbodiimides dicyclohexylcarbodiimide ditolylcarbodiimide onLinkingAgentsRulesBased Surprisingly only a few rules must be obeyed to take avantage of five different linking reagents applicable to linking two antibiotic molecules. The five linking reagents are: diisocyanates, dianhydrides, diacidchlorides, diepoxides and carbodiimides.

The types of antibiotics that can be linked are sulfonamides, trimethoprim, penicillins and related structures, cephalosporins and related structures, chloramphenicol, erythromycin, metronidazolc, quinolones, tetracyclines and aminoglycosides.

The linking rules are as follows: 1. Diisocyanates can react with all acid groups, all hydroxyl groups and all primary and secondary amino groups. Thus any antibiotic moiety, A, containing a carboxylic acid, hydroxyl or amine function can be linked to any other antibiotic moiety B containing a carboxylic acid, hydroxyl or amine function. When a single antibiotic moiety contains more than a single functional group, as C, the diisocyanate can be used to link with an antibiotic moiety containing a single reactive group, as A and B above, or with an antibiotic moiety containing two functional groups as D, carboxylic acid and amine.

When a diisocyanate is used to link antibiotic moities containing a plurality of groups, a mixture of products will be realized, but with chromatographic techniques the mixtures are easily separated.

Summarizing, the diisocyanate can be used to link any two antibiotics containing at least one carboxylic acid, alcohol or amino functional group, and will also effect linkage when each antibiotic moiety contains a plurality of groups.

2. Dianhydrides can be employed to link a wide variety of antibiotic moities containing hydroxy or primary or secondary amines. The reagent will also link antibiotic molecules where each antibiotic moiety contains a plurality of hydroxy, primary and secondary amine functional groups.

3. Diacidchlorides can be employed to link a wide variety of antibiotic moities containing hydroxyl and primary or secondary amine functional groups, and also where each moiety contains a plurality of said functions.

4. Diepoxides can be utilized to link a very wide variety of antibiotic moities where each contains carboxylic acid, alcool, and primary or secondary amine functional groups, or a plurality of such groups.

5. Carbodiimides can be utilized to link a wide variety of antibiotic moities where each moiety contains at least one of the following filnctional groups: carboxylic acid, alcool, and primary or secondary amine. This reagent differs from the four previously discussed since the reagent bonds the two antibiotic moities via the removal of the elements of water from the functional groups. Moities containing carboxylic acid groups can be linked with moities containing carboxylic acid groups to form anhydrides. Moities containing carboxylic acid groups can be linked to moities containing alcools or primary or secondary amines to form esters or amides. Moities containing hydroxyl groups can be linked to moities containing hydroxyl or primary or secondary amine groups to form ethers or substituted amines. Where pluralities of the carboxylic acid, hydroxyl or amine functional groups are contained in one or both antibiotic moities, linkage will occur but the products may be complex and require chromatographic separation.

DESCRIPTION OF THE INVENTION Section 1 The present invention describes methods for making a number of linked antibiotic molecules. The linked antibiotics are to be utilized in treating various infections in man and animals, without undue adverse side effects such as toxicity, inflammation and allergies.

There are several groups of these to-be linked compound which can be enumerated: sulfonamides, penicillins, cephalosporins, quinolones, chloramphenicol, erythryomycin, metronidazole, tetracyclines and aminoglycosides. With each case, the antibiotics to be linked will be taken two at a time from the above groups, thus: sulfonamide + sulfonamide sulfonamide + penicillin sulfonamide + cephalosporin sulfonamide + quinolone sulfonamide + chloramphenicol sulfonamide + erythromycin sulfonamide + metronidazole sulfonamide + tetracycline sulfonamide + aminoglycoside penicillin + penicillin penicillin + cephalosporin penicillin + quinolones penicillin + chloramphenicol penicillin + erythromycin penicillin + metronidazole penicillin + tetracyclines penicillin + aminoglycosides cephalosporin + cephalosporin cephalosporin + quinolone cephalosporin + chloramphenicol cephalosporin + erythromycin cephalosporin + metronidazole cephalosporin + tetracyclines cephalosporin + aminoglycoside quinolone + quinolone quinolone + chloramphenicol quinolone + erythromycin quinolone + metronidazole quinolone + tetracyclines quinolone + aminoglycoside chloramphenicol + chloramphenicol chloramphenicol + erythromycin chloramphenicol + metronidazole chloramphenicol + tetacyclines chloramphenicol + aminoglycoside erythromycin + erythromycin erythromycin + metronidazole erythromycin + tetracyclines erythromycin + aminoglycoside metronidazole + metronidazole metronidazole + tetracyclines metronidazole + aminoglycoside tetracyclines + tetracyclines tetracyclines + aminoglycosides aminoglycoside + aminoglycoside Within each of the above groups of antibiotics the members of each to be linked are defined as: 1. Sulfonamides: A. p-aminobenzenesulfonamide B. sulfamethoxyazole C. acetylsulfoxazole D. sulfamethoxypyridazine E. sulfadiazine F. trimethoprim t,'14 2- CD 2 c0 o cH3 ! U z- 2. Penicillins: A. benzyl penicillin B. procaine penicillin G C. penicillin D. ampicillin E. amoxycillin F. methicillin G. oxacillin H. cloxacillin I. dicloxacillin J.flucloxacillin K. nafcillin L. carbenicillin M. ticaricillin N. talampicillin O.becampicillin P. pivampicillin Q. penemcarboxylic acid R. hydroxyethyl penem S. imipenem T. amdinocilin 3. Cephalosporins: A. cephalosporin C B. cephalothin C. cephaloridine D. cephradine E. cephazolin F.cephalexin G. cefadroxil H. cefaclor I.cephamandole J. cefuroxine K.cefotaxime L. ceftizoxime M. ceftazidime N. cefoperazone CO.cephamycin P. cefoxitin Q. moxalactam 4. Quinolones: A. nalidixic acid B. norfloxacin C. enoxacin D. ciprofloxacin E. ofloxacin 5. Chloramphenicol: 6. Erythromycin: A. erythromycin 9 v a. o 'w H Desoeevie H H HO R 'N Mxrxycllc lactose ring H Itctott rino H, > 1 _w Cladinott H m ru 7. Metronidazole: 8. Tetracyclines: B. oxytetracycline C.chlortetracycline D. rolitetracycline E. methacycline F. doxycycline G. demeclocycline H. sancycline I. lymecycline J. clomocycline K. minocycline 9. Aminoglycosides: A. streptomycin w °"5a r o. N OU N N H O t3lr pn0 N NC H'. HX-Y'.. H O O H, C9H :. HN HXHOH N B. tobramycin C. kanamycin D. amikacin E. gentamicin C I F. nitilimicin G. neomycin CH2NH2 --- > v 2 O 9s w2 0 1112 HZN VH2 uH J nobtosamlne B NOHZC 0 (H HO 0/H HOU L O J I 4HZ CHZNH2 H. paromomycin I. spectinomycin The linking agents that will be used to link the antibiotic moities are drawn from several classes of organic molecules: I. Diisocyanates and related structures II. Dianhydrides III. Diacidchlorides IV. Diepoxides V. Dicyclohexylcarbodiimide and related structures The class I linking agents are drawn from the group consisting of the followingstructures: I. Diisocyanates and related structures: A. hexamethylene diisocyanate OCN (CH2) 6NCO B. 2,4-tolyldiisocyanate C. 2,6-tolyldiisocyanate D. 4,4'-methylene-bis-phenylisocyanate E. 4,4'-isopropylidene-bis-phenylisocyanate F. 1,4-phenyldiisothiocyanate G. 1,4-phenyldissocyanate II. Dianhydrides: A. pyromellitic dianhydride B. bismaleic dianhydride C. benzophenone, 3,3', 4,4 tetracarboxylic anhydride D. 1, 2, 6,7-hexane-tetracarboxylic anhydride E. 1,2,5,6-naphthalene tetracarboxylic anhydride III. Diacidchlorides: dichlorideA.terphthalolyl B. isophthaloyl dichloride C. pthaloyl dichloride D. adipolyl chloride E. glutaryl chloride IV. Diepoxides and related structures: A. 1,3-butadiene diepoxide B. cyclooctatetraene diepoxide, 1.5 C. vinyl cyclohexene D. divinylbenzene epoxide V. Carbodiimides and related structures: A. Dicyclohexylcarbodiimide B. Ditolylcarbodiimide DESCRIPTION OF THE INVENTION Section 2 Methods of Linking Antibiotic Moities: The structure of the two antibiotic moities being linked will determine the nature of the particular linking agent to be employed. Thus when sulfonamides listed above are to be coupeled the basic sulfonamide structure below shows that the group which will be linking the two sulfonamide structures is the aromatic amino group. Consideration of the entire group of sulfonamides listed above will show that the only reactive group is the aromatic amino group. Of the five linking reagents listed above, four may be employed: diisocyanates, dianhydrides, diacidcklorides, and diepoxides.

The structures which result from coupling sulfonamides with sulfonamides are shown below.

When identical sulfonamides are linked only a single product will result, but obviously when two nonidentical sulfonamides species are linked, three products will result as shown below.

Since, in the experimental section below, equimolar quantities are used in all rections the mixed products will predominate where the two reacting moities have diverse structures. The modem methods of liquid chromatography render the separation of such simple mixtures, as above, to be quite simple, thus adequate material can be separated for microbial evaluation tests. Commercial quantities can be separated via preparative scale HPLC (high performance liquid chromatography).

The linking of two antibiotic moities by utilizing dianhydrides follows a course identical to that described for diisocyanate linking agents, i. e., when a single type moiety is employed, a single product nJtill result, but when two dissimilar moities are employed, three products will result. As noted with diisocyanates, separation and evaluation of these products is not difficult.

The linking of two antibiotic moities utilizing diacid chlorides will pursue a course analagous to the rections of diisocyanates. When a single antibiotic entity reacts with a diacid chloride a single entity results, but when two different entities react, three products are formed.

Symbolically, the three products can be seen as: The rection of two antibiotic moities with a diepoxide follows a course similar to the diisocyanate rection with a single moiety resulting in a single product and two moities resulting in three products, as shown below.

The above comments with respect to product mix apply to all antibiotic linking rections occurring with the linking agents diisocyanates, dianhydrides, diacid chlorides, and diepoxides.

The use of the dicarbodiimides with antibiotic moities follows a different pattern. The situation when linking antibiotic moities via carbodiimides is the result of removing the elements of water from two moities.

Thus the linking of two antibiotic moities depends upon the presence of the following groups: carboxyl, amino and hydroxyl and requires a minimum total of two such groups, but with a further plurality may also be utilized. The possible combinations of the three reactive groups are five.

4. Y-OH-YOH-- Y-O-Y The linking of two antibiotic moities via the groups above produces the followingproducts: 1. anhydride 2. ester 3. amide 4. ether Y-O-Y 5. amine Y-NH-Y When two antibiotics are linked each containing a single reactive group, as one COOH and one NH2, only a single product will result ; see the example below of the rection of dicyclohexylcarbodiimide with p-aminobenzene- sulfonamide with benzylpenicillin.

The amide product is easily separated from the by-product dicyclo- hexylurea by crystallization or liquid chromatography techniques.

When more than two active groups are present on a single moiety as penicillin type, carbenicillin, two products will result when linked with p- aminobenzenesulfonamide via dicyclohexylcarbodiimide, as shown below.

Rules are developed in the"Rules"section below to account for all products with all linking reagents linking the many antibiotic moities.

Linking Rules The linking rules developed below are based on the interactions of the five linking agents with a large number of eight classes of antibiotics.

DESCRIPTION OF THE INVENTION Section 3 The rules for linking the antibiotic moities are developed by considering all of the above data just concluded for linking all of the individual members of the said eight groups of antibiotics.

The linking rules are as follows: 1. Diisocyanates can react with all acid carboxyl groups, all hydroxyl groups and all primary and secondary amino groups. Thus any antibiotic moiety containing a carboxylic acid group, a hydroxyl group or an amine group, can be linked to any other antibiotic moiety also containing a carboxylic acid group, hydroxyl or amine function.

When a single antibiotic moiety contains a plurality of groups, as a carboxyl group and a hydroxyl group, or a carboxyl group and an amino group, this moiety can be linked by rection with a diisocyanate to a second antibiotic moiety containing a plurality of groups, as a carbovyl group and a hydroxyl group, or a carboxyl group and an amino group.

When a diisocyanate is utilized to link antibiotic moities containing a plurality of carboxyl acid, alcohol and amino groups, a mixture of products will be realized, but with chromatographic techniques the mixtures are easily separated.

Summarizing: the diisocyanate reagent can be used to link any two antibiotic moities each containing at least one carboxylic acid, alcohol or amino functional group, and can also be used when each antibiotic contains a plurality of said groups.

2. Dianhydrides can be utilized to link a wide variety of antibiotic moitieties in which each moiety contains at least one hydroxyl or primary or secondary amine group. The dianhydride reagent can also be utilized to link antibiotic moities in which each moiety contains a multiplicity of hydroxyl or amine groups. In cases involving the linking of antibiotic moities containing a multiplicity of groups, a mix of products will be realized but can be separated easily via chromatographic techniques.

3 Diacidchlorides as a linking agent are covered by rules identical to those for dianhydrides. Diacidchlorides can be used to link a wide variety of antibiotic moities in which each moiety contains at least one hydroxyl or amino group. The aciddichloride reagent can also be used to link antibiotic moities where each moiety contains a multiplicity of hydroxyl or and amino groups. In cases involving a multiplicity of groups, a mixture of products will be realized which can be separated easily via chromatographic techniques.

4. Diepoxides as linking agents can be used to link antibiotic moities where each moiety contains a carboxyl, hydroxyl, or amino group, or where each moiety contains a plurality of said groups. When antibiotics possessing a plurality of such groups react with the epoxy linking agents a complex mix of products will be formed which can be separated via chromatographic techniques.

5. Carbodiimides as linking agents can be utilized to link a very large number of antibiotic types. Antibiotic moities in which each moiety contains a single carboxyl group yield anhydrides. Antibiotic moities in which one moiety contains a carboxyl group only will react with a moiety containing a single hydroxyl group to form a single ester. Antibiotic moities containing a plurality of carboxyl and hydroxyl groups will form a complex mixture of esters when reacted with carbodiimides. Antibiotic moities containing a single carboxyl group will react with antibiotic moities containing a single amino group to form a single product containing an amide group. When antibiotic moities containing a multiplicity of carboxyl, hydroxyl and amino groups are linked via carbodiimides, a mixture of esters and amides will be forr : e. When antibiotic moities containing singular hydroxyl groups or a multiplicity of hydroxyl groups are linked, the products will be singular or multicomponent ethers. The linking of antibiotic moities containing singular or multiple hydroxyl and amino groups leads to the formation of a single substituted amine, or a multiplicity of amines. All of the mixtures generated by the above said rections can be separated via chromatographic techniques.

DESCRIPTION OF THE INVENTION Section 4 Experimental Procedures General Comments-The procedures outlined and discussed below describe the experimental procedures necessary to carry out the linking procedures with the many antibiotic moities previously described in this application.

Procedures for each linking ragent.

Coupling rections using: 1. Diisocyanates: Solvents pyridine 50 ml anhydrous DMAC 50 ml anhydrous DMF 50 ml anhydrous N-methylpyrrolidone 50 ml anhydrous Temperature 0 to 50°C Time 5-10 hours Quantities 0.01 mole each antibiotic moiety, 0.005 mole of linking reagent Monitor via IR spectroscopy for-NCO group, 4.45p Work-up add water to ppt. product achieve separation of products via chromatog- raphy; TLC, column or HPLC.

Evaluation apply to streal : ed plate of several cultures--E. coli, Strep. Group A, P. aruginosa Equipment 250 mi round bottom 3-neck flask equipped with glascol mantle for heating, thermometer, reflux condenser and teflon stirring bar energized by magnetic stirring.

Coupling rections using: 2. Dianhydrides Solvents Same as"1".

Temperature Same as"1".

Time Same as"I".

Quantities Same as"1".

Monitor IR via 5.50 and 5.80 anhydride band Work up See 1.

Evaluation See 1.

Coupling rections utilizing: 3. Diacid chlorides* All same as 1, but IR monitor via 5.80 acid chloride band in IR *Prior to the addition of water to terminate the rection g. (0.25 ml) of sodium bicarbonate is added in small portions to neutralize all hydrochloric acid.

Coupling rections utilizing: 4. Diepoxides All same as in 1, but rection time may be extended to 24 hours to complete rection. IR monitor via epoxide band at 9.5 tFl.

Coupling rections utilizing: 5. Carbodiimide All same as 1, but rection time may be as short as 1 hour. IR monitor is via carbodiimide band at 4.50F.

Exemple procedure 1. The rection p-aminobenzene sulfonamide with sulfapyridine.

The dry pyridine solvent, 50 ml., is psaced in the 250 ml round bottom flask and 1.72 g. (0.01 mole) of p-aminobenzenesulfonamide and 1.68 g.

(0.01 mole) of hexyldiisocyanate is added, the temperature raised to 40°C by means of the variac controlling the heating of the glascol mantle. The heating and stirring are continued for 4 hours, and at the end of each hour a small sample is withdrawn from the flask by means of a pipette and examine by means of IR sectroscopy. The IR spectrocopy scan is determined from 2.5 microns to 15.0 microns, and the concentration of diisocyanate is determined from the intensity of the absorption band at 4.45 microns, a band due to the-NCO group. A steady drop in the concentration of the-NCO group indicates progress of the rection. At the end of 4 hours at 40°C the concentration of the isocyanate group has dropped by 80 per cent. The rection is forced to conclusion by raising the temperatllre to 50°C for two heurs, at the end of which time the-NCO group is not detectable by IR spectroscopy.

The rection is terminated by the addition of 50 ml of water and the precipitated rection product dried in a vacuum oven at 25°C for 2 heurs to yield 3.0 g., 8.8 per cent. The product was evaluated for biological activity via applying a 1 per cent solution in pyridine to TLC (thin layer chromatography) plates. The developing solvent used was a 10-90 mixture of acetone and methanol, and the progress was monitored by a UV light.

The spots on the chromatogram were evaluated via mechanical removal and the absorbent was separated from the product fraction by dissolving in pyridine, and the pyridine solution was dried onto filter paper. Tabs of the filter paper were applied to ager culture plates streaked with standard bacterial cultures of S. aureus, E. coli and P aureginosa. Standard antibiotics, as p-aminosulfonamide and penicillins were used for comparison. All products showed modes inhibition zones in the vicinity of the filter paper tabs containing the product fractions.

Larger quantities of products are obtainable for animal testing most simply via prep scale liquid chromatography.