US2631146A - Biguanide salts of penicillin - Google Patents

Description

latented Mar. 10, 1953 2,631,146 BIGUANIDE SALTS or PENICILLIN Joseph F. Weidenheimer, Pearl River, and Lawrence Ritter, Valley Cottage, N. Y., assignors to American Cyanamid Company, New York, N. Y., a corporation of Maine No Drawing.

9 Claims. 1

This invention relates to a new therapeutic composition which has as an essential ingredient a comparatively insoluble organic penicillin salt. More particularly the invention relates to organic salts of penicillin which areless soluble than procaine penicillin and which are non-toxic when injected whereby these compounds may be used for penicillin therapy, giving a blood level which is therapeutically effective over a prolonged period.

This application is a continuation-in-part of our application Serial No. 87,826 filed April 15, 1949, entitled Penicillin Derivatives, and of our application, Serial No. 268,933, filed January 29, 1952, entitled Penicillin Salts, now both abandoned in favor of this application.

' It is found that'thecompounds of this invention may readily be used for the injection of patients with such a quantity that a therapeutically useful concentration of penicillin is maintained in the blood of a normal patient for a period in excess of 48 hours so that additional injections are not required.

The therapeutic composition of this invention has as an essential element thereof a salt of penicillin with a blguanide or substituted biguanide which forms with the penicillin anon-toxic salt of such solubility characteristics that the material when administered to a subject is therapeutically available at such a rate as to build up suificient penicillin concentration to be effective against the particular organism for which the treatment is being given but yet which is not sufficiently soluble to permit all of the penicillin to be dissipated within a comparatively short period.

The biguanide salts of penicillin of this application may also be used in processes of refining penicillin as they form salts by which penicillin may be precipitated from solutions containing the penicillin. Also, certain of these salts are extremely insoluble and stable under adverse conditions so that the salts may be used as additives for foodstufis where the penicillin salt is distributed and where conventional salts or penicillin would be inactivated due to moisture present.

Among the biguanides, the para-halophenylbiguanides, the -'-nitrogen of which may be substituted by one or more 'alkyl groups, have been found to be particularly eflicacious. The prepae ration and certain characteristics of some of these biguanides are disclosed and set forth in a U. S. patent toDaniel E. Nagy, No. 2,455,896 entitled l-Aryl, 5-Alkyl-Biguanides a British Application November 13, 1952, Serial No. 320,334

Patent 577,843 to Francis H. S. Curd and Francis L. Rose, entitled Biguanide Derivatives; a U. S. patent to Francis H. S. Curd and Francis L. Rose, 2,467,371, entitled Biguanicle Derivatives and a U. S. patent to Daniel E. Nagy, 2,455,897, entitled 1,5-Diarylbiguanides from Aromatic Amines and Dicyanimide.

Certain of the biguanide penicillins are sufiiciently heat stable that the salts may be sterilized by the use of heat, as for example, C. for 10 hours, thereby enabling the sterilization of the penicillin salts at a later and more convenient stage in their manufacture.

Any of the penicillins may be used in this invention. At present the medical" profession prefers penicillin G, and accordingly the penicillin G salts are those which are normally prepared. From the standpoint of convenience and preparation, the availability of intermediates and the comparatively low cost of the materiaLthe l-(pchlorophenyl) -5 isopropyl biguanide compound of penicillin G is among those which are normally commercially preferable.

The chemistry appears to be that of the straight reaction between an acid and a base. Penicillin forms alkaline salts very readily and the herein used biguanides from acid salts.

As a method of preparation it is normally most convenient to mix an alkali, or alkaline earth metal, or a suitable organic base, salt of the de-' sired variety of penicillin with a calculated quantity 0r slight excess of an acid salt of the desired biguanide thereby forming the desired complex. From the standpoint of cost and availability the salts most commonly used are the sodium, potassium, calcium or triethylamine salts of penicillin and a halide, usually hydrochloride, salt of the biguanide such as l-(p-chlorophenyl) -5 isopropyl biguanide hydrochloride. There is thereby formed the desired salt, and, sodium, calcium, potassium or triethylamine halide, such as chloride, which material is readily removed from the desired biguanide penicillin, although no harm is done if such a salt is injected with the penicillin. It may be that some type of complex other than that classed as a true salt is formed by the interaction of the specific'biguanides and penicillins herein mentioned, but for. purposes of con-' venience the reaction product will be referred to as a salt because it exhibits certain of the properties of saltsand probably is properly classed as a salt.

In the preparation of these salts the initial crystallization at times requires considerable stirring and cooling; but normally the crystal 3 habit is such that the crystals as formed are suitable for injection. In some instances recrystallization from suitable solvents may be desired.

For purposes of injection it is desired that the particular biguanide penicillin salt be suspended in a suitable medium such as distilled water, normal saline, 20% propylene glycol or other commonly used aqueous diluent or in an oil such as peanut oil, sesame oil, cottonseed oil or other assimilable triglyceride, together with such stabilizing agents, buiiers, thixatropic agents, viscosity modifiers and wetting agents as may be desired to cause the material to have a fluidit'y and stability such that it may be easily filledinto a hypodermic syringe and injected into a patient.

Additionally, it is frequently desirable to have mixed with this soluble penicillin salt a "water soluble penicillin type such as the sodium or potassium or calcium salt which will cause a high initial blood level of penicillin in a patient so that by the combination the patient receives a very rapid, high blood level caused by th soluble penicillin derivative and there is maintained in the patient a therapeutic level of penicillin over a prolonged period because of the low solubility and lowered rates of absorption of our specific biguanide derivatives of the penicillin.

Qertain of the biguanide derivatives of penicillin, particularly l (p-chlorophenyl) -5 isopropyl biguanide penicillin, may be used in lieu of procaine penicillin-in the various forms of ointments and suspending media now used for procaine penicillin and thereby will be obtained a product possessing superior stability and a greater period of therapeutic efficacy. I

In certain instances the 1-(p-chlorophenyl) -5 isopropyl biguanide will be iound to have a therapeutic effect of its own although normally patients requiring penicillin do not require treatment with l-(p-chlorophenyl) -5-isopropyl biguahide. The concentration of l-(p-chlorophenyl) 5 isopropyl biguanide which is built up in the patient is not sufliciently high to cause any complications and is less than that which is frequently used as a prophylactic in malaria control.

In some instances for patients who are particularly sensitive there may be-added to the mixture a-local anesthetic to prevent pain at the point of injection.

Becauseof -the:greater 'insolubility in water, if l-(p-chlorophenyl) -5 isopropyl biguanide is added to azprocaine penicillin solution there will be formed a I-(p chlorophenyD-k'; isopro'pyl biguanide penicillin and free procaine which may be as the hydrochloride. It may be convenient to have procaine or other anesthetic present either from this or other sources so that the sensitivity at the point of injection is diminished.

Whereas our compounds may be formed by the interaction of salts of penicillin and salts of'the specific biguanides, whichare to be used, they may also be-forfned by the reaction of free penicillin and the "free biguanide. They may be formedi'n-a watersolution, or in an'aqueous mediumsuch as a mixture of water and alcohol, in analcohol medium containing a large proporti'on'of alcohol, in water, or even an anhydrous alcohol, or any or the various organic solvents in "which the particular biguanides and penicillins are each somewhat soluble and the solubility characteristics of all of the compounds are such that/the desiredproducts are formed thereby, even, for-example, by-removal of the solvent 4 or other methods well known to those desiring to manufacture salts.

More particularly, our invention includes the salts of penicillin such as penicillin G F. X and other penicillins, hereinafter called penicillin, with a basic compound from the group consisting of biguanide and compounds containing a biguanide nucleus of the formula in R;

where R1, R2, R3 and R4 may be either hydrogen or such radicals as alkyl, alkenyl, alkynyl, cycloalkyl, monocycli'c aryl, polycyclic aryl, monocyclic aroyl, 5 membered monocyclic heterocyclic radicals and 6 'membered monocyclic heterocyclic radicals, which radicals may either be the simple radicals or substituted. It is found that such radicals as are mentioned above may have on the aliphatic portions such substituents as :hydroxy, alkoxy, aryl and nitrile groups and on the aromatic portions such groups as alkyl, aryi, halogen, nitro, alkoxy, aryloxy, nitrile, hydroxy and carbethoxy groups. We prefer that not more than two of these substituent groups be aromatic radicals as otherwise the basicityof the biguanide is less than is preferred for the formation of the biguanide salts. For penicillin salt formation R5 and Rs may be either hydrogen or lower alkyl groups, but it is normally more convenient to prepare the biguanides where R5 and Rs represent hydrogens. Where R2 is hydrogen, R1 and R5 are equivalent, as the hydrogen is labile. Similarly, where R4 is hydrogen, R3 and Rs are equivalent.

To list all biguanides with which our salts may be formed would unduly lengthen this disclosure, but by way of example, certain typical ones are listed. Among the new salts of our invention are the salt of penicillin with:

i-ethylbiguanide 1,1-diethylbiguanide 1,2-diethylbi'guanide 1 gfi-diethylbiguanide l -ethyl -'5 -propylbiguanide 1,1-diethyl-2 ethylbiguanide 1,1-diethyl-5-ethylbiguanide 1 ,1,2 triethyl-5 prop ylbiguanide 1 beta-hydroxyethylbiguanide l -'beta methoxyethylbiguanide 1,1 -"dibutylbiguanide 1 -betacyanoethylbiguanide 1 -betaorthochlorophenyl) -ethylbiguanld 1-beta-phenylethylbiguanide phenylbiguanide o-tolylbiguanide 1-octadecyl 5-phenylbiguanide 1-butyL5-octylbiguanide l -benzenesulfony1-5 phenylbiguanide 1 -'butyl-5, phenylbiguanide 1,'4-diphenyl-'3;5-diisopropylbiguanide LB-diphenylbiguanide l,5 di p chlorophenylbiguanide 1 dodecyl-5-phenylbiguanide 1-dodecyl-5-p-sulfonamidobiguanide 1,1-dibutyl-5-phenylbiguanide 1,5-dimethyl-Lfi-diphenylbiguanide 1,5-di beta-naphthylbiguanide 1,5-di-o-chlorophenylbiguanide 1,5 -"di--mchlorophenylbiguanide 1,5-di-p-methoxyphenylbiguanide 1 ,5 di-p-bromophenylbiguanide 1,5-dip-nitrophenylbiguanide 1- (o'-biphenyl) biguanide 1 m-methoxyphenylbiguanide 1-m-phenoxyphenylbiguanide l-m-cyanophenylbiguanide l-p-hydroxyphenylbiguanide l-p-carbethoxyphenylbiguanide 1,1-dibutyl-5-methyl-5-phenylbiguanide 1-butyl-5-methyl-5-m-tolylbiguanide 1,1-di-betaehydroxyethyl-5-p-tolylbiguanide 1-allyl-5-alpha-naphthylbiguanide l n hexyl 5 ethyl-5-p-tert-amylphenylbi-.

guanide 1-tert-amyl-5eoeanisylbiguanide 1.1epentamethylenee5-p-nitrophenylbiguanide 1-cyclohexyl-Beethyl-5-p-chlorophenylb-iguanide 1,1+diallyl-5ep-cyanophenylbiguanide 1;5,5-tributylbiguanide L Any of the above listed penicillin salts may be formed by reaction of-penicillin and the biguanide. As illustrative examples showing the production of suchsalts'in accordance with this invention are: c l

EXAMPLE 1 p In a mixture of 2500 milliliters of distilled water and 750 milliliters of ethyl alcohol were dissolved 125 grams of l-(p- -chlorophenyll-5 iso propyl biguanide acetate. In an equal volume of a similar solution were dissolved 175 grams of the triethylamine salt of penicillin. The l-(pchlorophenyl)- 5 isopropyl biguanide acetate solution was "filtered through a sterilizing filter into a previously sterilized reaction vessel equipped with mechanical stirrer, inlet tubes and a. filter foot. The triethylamine penicillin was sterile filtered and slowly introduced into the reaction vessel with stirring. After about of the penicillin solution had been introduced, the'1'e' action mixture wassterilely seeded to induce crystallization. The'remainder of the penicillin solution was added over a period of about 1 hours, the l-(p-ohlorophenyD-5 isopropyl bi: guanide Penicillin .crystallizing as formed. The mixture was 'placed'in'the chill room at approximately C. for two hours to insure complete precipitation. "The solvent was removed through the filter foot and the residual l-(p-chlorophenyl) isopropyl biguanide .penicillin crystals washed with approximately 1 liter. of sterile .dis-' tilled water anddried for 48 hours. The entire:

procedure, after the sterilefiltrations, was carried out; under; Sterile; conditions, so that the material as formed was sterile. There was obtained a yield of 163 grams-or 69% of the theoretical yield. Theoretically the material should assay 1015 units per milligram; on actual bioanalysis it was found to show 1043 units per milligram. The l-(p-chlorophenyD-S isopropyl biguanide penicillin as thus formed was found to have a melting point of approximately 161-162 C. and was found to be soluble tothe extent of approximately 0.257% in water. 200 milligrams of, the thus obtained l-j(p-chlorophenyl)-5 iso propyl biguanide penicillin dry was mixed with sufiicient sesame oil to form 1 cc. of a suspension,

thereby forming an injectable therapeuticgdose EXNEAPLE 2 To a solution of 125 grams of l-(p-chloro phenyl)-5 isopropyl biguanide acetate in 2500.

milliliters ofjdistilled water and 750 milliliters,

of alcohol was added slownwitustirrinea some; 1 mixedwit a n rmal salineiiiluent. a. therapeutic.

6 tion-of 137 Tgrams of sodium'penicillinin 1000 milliliters of distilled water. After approxi mately of the penicillin had been run in, the addition was stopped and the material seeded with vl-(p-chlcrophenyl)-5 isopropyl biguanide penicillin crystals. As the solution became opalescent indicating that crystallization had commenced, theremainder of the penicillin was added over a period of approximately l hours with continuous stirring. Strict conditions of cleanliness were maintained but the materials were not sterile nor sterilized. The suspension was placed in a chill room at approximately 0 C. overnight to allow complete precipitation, and the solvent removed. The residual crystals of 1-'(p-chlorophenyl)--5 isopropyl biguanide penicillin were washed with distilled water and dried at-room temperature in a desiccator for 48 hours givinga yield of 195 grams or 87% of the theoretical amount. The l--(p-chlorophenyl)-5'isopropyl-biguanide penicillin was filled into ampoules containing the desired dosages and the ampoules were sterilized in an oven at C. for 3 hours. Thereby were obtained sterile ampoules containing dry 1-(p-chlorophenyD-5 isopropyl biguanide penicillin in thedesired dosage units. For injection, to such an ampoule may be added a sufficient quantity of a sterile aqueous 20% propylene glycol diluent to form a suspension having the desired concentration for injection into the patient.

EXAMPLE 3 7 116 grams of l-(p-chlorophenyD-S isopropyl biguanide hydrochloride were dissolved in a solution of 2500 milliliters of distilled water and 750 milliliters of alcohol. Thereto was added grams of potassium penicillin G in a solution of 2500milliliters of distilled water and 750' millilit'ers of alcohoL- The penicillin was added slowly with stirring; the mixture being seeded to insure crystallization, after part of the penicillin-containing solutionwas added.- The mixture was stirred thoroughly and permitted to stand overnight! The crystals were removed from the solvent thereby being obtained l-(p-chlorophenyl)-5 isopropyl biguanide penicillin in an unsterile condition. The thus prepared l-(pchlorophenyD-5 isopropyl biguanide penicillin was sterilized by heating to 110 C. for 10 hours; after which it was ready for blending, mixing and filling under sterile conditions for therapeutic EXAMPLE4 '125 grams of 1-(p-chlorophenyll-5 isopropyl biguanide acetate was powdered and suspended in 1 liter of distilled water. Thereto was added a solution of 1'75 gramsof triethylaminepenicillin dissolved in 1 liter of water. The suspension was stirred for 5 hours with a small quantity of crystalline 1-(p-chlorophenyl) -5 isopropyl biguanide penicillin added as seed during the first hour. The desired 1- (p-chlorophenyl )-5 iso propyl biguanide penicillin was separatedfrom the aqueous layer, washed with water to remove salt -anddried under high vacuum for 48'hours. The dried material was sterilizedby heating to 110 C. for 8 hours. The thus prepared dry sterile l-(p-chlorophenyD-B isopropyl biguanide penicillin wasmixed with sterile potassium penicillin and the mixture filledinto vials, each vial containing a sufi'icient quantity of; each of the 1- (p-cholorop-henyll-B isopropyl biguanide penin. and potassium. pen c l n to prov de. wh

7 dose giving high initial levels and prolonged -levels in accordance with the desire of the user.

EXAMPLE '5 5 grams of 1-(p-chlorophenyll-5 isopropyl biguanide acetate were dissolved ina mixture of 100 milliliters of water and 30 milliliters of ethyl a1- cohol. Thereto were added 7.15 grams of the triethylamine salt of penicillin G dissolved in 100 milliliters of water and .30 milliliters of alcohol over a period of one hour. A very small quantity of seed crystals were added, the crystallization beginning almost immediately, and at the end of the mixture was quite pasty. The mixture was stirred for an additional hour at C., the crystals collected, washed in distilled water, .anddried in vacuo. There was obtained a yield of 86% of the theoretical analyzing 1040 units per milligram and melting at 161-162 C. and showing an EXAMPLE 6 960 grams of para-chlorophenyl-biguanide hydrochloride dissolved in a mixture of 25 liters of water and liters of ethyl alcohol were treated with 2.2 kilograms of the triethylamine salt of penicillin dissolved in 25 liters of water and 5 liters of alcohol. The penicillin solution was added to the biguanide solution and when an opalescent solution was obtained, the mixture was seeded and stirred until crystallization began, the remainder of the penicillin solution then being slowly added. The crystals formed, clusters of thin rods, were collected, washed with water and dried in vacuo. There was obtained a yield of 63% of the theoretical; the crystals melted .at 12l-l22 C. (uncorrected), the material was soluble to the extent of 0.3% in water. Because the crystals melt so close to the temperatures :required for heat sterilization, as a matter of convenience the product is prepared under sterile conditions .to obviate the necessity of heat sterilization after its preparation. The material was found to have an activity of 1079 units per milligram, and on analysis showed:

Percent Carbon 52.180 Hydrogen 5.14 Nitrogen 17.99

EXAMPLE 7 EXAMPLE 8 1 gram of 1,1-dibutyl-5-(para-iodophenylibiguanide hydrochloride was dissolved in milliliters of water and 15 milliliters-of alcohol. The clear solution was'then treated with a solution of 1 gram of potassium penicillin dissolved in a mixture of 17.5 milliliters of water and "7.5

milliliters of alcohol. An oil was formed which upon storage overnight at 4 C. yielded crystals in hexagonal plates which were collected, dried and found to assay 751 units per milligram.

EXAMPLE 9 Phenylbiguanide penicillin 1 gram of phenylbiguanide hydrochloride dissolved in 20 milliliters of water is added with stirring :to a solution of 2 grams of triethylamine penicillin G dissolved in 20 milliliters vof water. An oil is deposited which does not crystallize readily on standing. The supernatant liquid is decanted to yield a gummy residue which upon drying in a vacuum oven yields :a dry semicrystalline powder. The power analyzes-917 units per milligram.

EXAMPLE o-iolylbiyu uide penicillin 1 gram of o-tolylbiguanide hydrochloride dissolved in 20 milliliters of water is added with stirring to a solution of 1.9 grams of triethylamine penicillin G in 20 milliliters of water. A gum is deposited which upon drying in a vacuum oven yields a dry powder having a potency of 9.30 units per milligram.

EX M E.

1-octaclecyl-S-phenylbiguanide penicillin A solution is prepared'of 0.5 gram of l-octadecyl-fi-phenylbiguanide hydrochloride, as the free base, in 15 milliliters of ether. A solution of 0.4 gram of the free acid form of penicillin (penicillinic acid) in '30 milliliters of ether is added with stirring. The resulting clear ethereal solution is evaporated to dryness, yielding the amorphous l1octadecyl-5-phenylbiguanide penicillin.

EXAMPLE 1'2 1,5-dzfphcnylbiyuanide penicillin A solution is prepared of 4.1 grams of 1,5-dlphenylbiguanide hydrochloride dissolved in a mixture of 50 milliliters of ethyl alcohol and 25 milliliters of water. This solution is added to a solution of 5 grams of sodium penicillin in 25 milliliters of water. The combined mixtures are stirred and on cooling, the desired L-S-diphenylbiguanide penicillin salt separates as crystals.

EXAIVIPLE 13 1,5-di p-chlorophenylbi uanide penicillin .5igra-ms of 1,5-di-p-chlorophenylbiguanide hydrochloride is suspended in '25 milliliters of ethyl alcohol and 50 milliliters of water. To this is added dropwise with stirring a solution containing '5 grams of crystalline sodium penicillin G in the same solvent. The biguanide hydrochloride dissolvesand the desired 1,5-di-p-chlorophenylbiguanide penicillin precipitates in crystalline form. The precipitate is collected, washed with water, and dried. The yield is 8.7 grams of material assaying 861 units per milligram. The melting point is approximately "161 C. and the material is soluble to the extent of 0.01459; in water. The low solubility ,makes this salt particularly useful as an additive for poultry feed. A sampleof this salt, upon being heated at 110 C. for 11 hours, is found to have a potency of 833 units per milligram, showing that the salt 'may beheatsterilized.

9 EXAMPLE 14 1,5-di-alpha-naphthylbiguanide penicillin grams of 1,5-di-alpha-naphthylbiguanide hydrochloride is mixed with 50 milliliters of alcohol and 50 milliliters of water. Thereto is added 5 grams of sodium penicillin in 25 milliliters of water. On cooling and standing, the amorphous 1,5-di-alpha-naphthylbiguanide pencillin separates out.

Other biguanides acid salts such as the sulfate, the nitrate, the phosphate or the acetate may be used instead of the hydrochloride salts. A wide variety of salts of penicillin may be used but the alkali or ammonium or alkylamine salts are particularly convenient. The reaction may be carried out in water, methanol, ethanol, propanol, butanol, 2-ethoxyethanol, 2-methoxyethanol, other alkoxy ethanols, other alcohols, dioxane, formamide, dimethylformamide, acetone, methyl ethyl ketone, or methyl isobutyl ketone, or mixtures of two or more of these solvents or other similar solvents.

Having thus set forth by description and example certain aspects thereof, as our invention we claim:

1. The salt of penicillin and a compound of the formula where R1 represents a member selected from the group consisting of alkyl, monocyclic aryl, halomonocyclic aryl, and dicyclic aryl radicals; R2 represents a member selected from the group consisting of hydrogen, alkyl, monocyclic aryl, chloromonocyclic aryl, and dicyclic aryl radicals; and R3 represents a member selected from the group consisting of hydrogen and alkyl radicals.

2. A salt of a penicillin and a compound of the formula No references cited.

Claims (1)

1. THE SALT OF PENICILLIN AND A COMPOUND OF THE FORMULA