US3627884A - Controlling bacteria with l-lysine-n-dodecylester, l-lysine-a-tetradecylester and acid addition salts thereof - Google Patents

Abstract

A method of inhibiting and controlling the growth of bacteria which comprises contacting said bacteria with an antibacterially effective amount of a long chain aliphatic ester selected from the group consisting of L-lysine-n-dodecylester L-lysine-ntetradecylester and acid addition salts thereof is described.

Description

United States Patent [72] Inventor Karl Vogler Riehen, Switzerland [21] Appl. No. 57,361 [22] Filed July 22, 1970 [45] Patented Dec. 14, 1971 [73] Assignee Hoffman-La Roche Inc.

Nutley, NJ. [32] Priority Oct. 14, 1963 [33] Switzerland [31 12600/63 Continuation-impart of application Ser. No. 777,502, Nov. 20, 1968, now abandoned which is a continuation-impart 01' application Ser. No. 399,329, Sept. 25, 1964, now Patent No. 3,433,779. This application July 22, 1970, Ser. No. 57,361

[54] CONTROLLING BACTERIA WITH L-LYSINE-N- -DODEC YLESTER( E-LYSINE-N- TETRADECYLESTER AND ACID ADDITION SALTS THEREOF 3 Claims, No Drawings [52] U.S. Cl 424/177,

[51] Int.Cl A61k 27/00 [50] Field oi Search 424/177; 260/1 12.5

[56] References Cited UNITED STATES PATENTS 2,063,987 12/1936 Dreyfus 260/112.5 X

2,290,174 7/1942 Epstein et a1. 424/177 2,932,635 4/1960 Amiard et al.. 260/1 12.5 X

3,004,021 10/1961 Selle et a1. 260/112.5 X

3,215,684 11/1965 Strojny et al... 260/1 12.5 X

3,281,378 10/1966 Garber et al 260/1 12.5 X

Primary Examiner-Shep K. Rose Anomeys-Samuel L. Welt, Jon S. Saxe, Bernard S. Leon,

William G. Isgro and Margaret C. Bogosian ABSTRACT: A method of inhibiting and controlling the growth of bacteria which comprises contacting said bacteria with an antibacterially effective amount of a long chain aliphatic ester selected from the group consisting of L-lysinen-dodecylester L-lysine-n-tetradecylester and acid addition salts thereof is described.

CONTROLLING BACTERIA wr'ru L-LYSINE-N- DODECYLESTER, L-LYSINE-A-TETRADECYLESTER AND ACID ADDITION SALTS THEREOF CROSS-REFERENCE RELATED APPLICATIONS This is a continuation-in-part of U.S. Pat. application Ser. No. 777,502, filed Nov. 20, 1968 now abandoned, which in turn is a continuation-in-part of Ser. No. 399,329, filed Sept. 25, I964, now issued as US. Pat. No. 3,433,779.

BRIEF SUMMARY OF THE INVENTION This invention relates to antibacterial compounds of the formula Ac-OR wherein R is an aliphatic hydrocarbon moiety containing at least eight carbon atoms and Ac is selected-from the group consisting of the acyl residue of a basic a-amino monocarboxylic acid and an acyl residue of dipeptide or tripeptide containing at least one basic a-amino monocarboxylic acid moiety, and their pharmaceutically acceptable acid addition salts.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel compounds, methods and intermediates useful in their preparation, and uses therefor. The novel compounds of this invention are long chain aliphatic acid esters of amino carboxylic acids. More particularly, the novel compounds of this invention are of the formula Ac-OR I wherein R is an aliphatic hydrocarbon moiety containing at least eight carbon atoms and Ac is selected from the group consisting of the acyl residue of a basic a-amino monocarboxylic acid and an acyl residue of a dipeptide or tripeptide, containing at least one basic a-amino monocarboxylic acid moiety; and their phannaceutically acceptable acid addition salts.

The aliphatic hydrocarbon moiety containing at least eight carbon atoms, which is represented by the symbol R, can be straight or branched chain, saturated or unsaturated. Said aliphatic hydrocarbon moiety preferably has a chain length of from 10 to 20 carbon atoms. Especially preferred are those having a chain length of from 10 to l6 carbon atoms. Exemplary of the aliphatic hydrocarbon moieties defined by R are the following long chain .alkyl groups: n-decyl, n-dodecyl, noctadecyl, n-eicosyl, n-tetradecyl and n-hexadecyl.

As stated above, the symbol Ac represents members selected from the group consisting of the acyl residue of a basic a-amino monocarboxylic acid and an acyl residue of a dipeptide or tripeptide, containing at least one basic a-amino monocarboxylic acid moiety. The term the acyl residue of a basic a-amino monocarboxylic acid, as used in the above definition, comprehends acyl residues of a-amino monocarboxylic acids which, in addition to the a-amino group, contain at least one further basic group such as, for example, an imino group. an amino group or a guanidino group. Thus, preferred as the acyl residue of basic a-amino monocarboxylic acids are moieties ofthe formula:

ginyl-L-arginine-n-decylester,

wherein B is imino are moieties of acids containing imino groups such as the imidazol-S-yl group, for example, histidine and the like. Moreover, exemplary of a-amino monocarboxylic acids of formula II wherein B is guanidino are moieties of acids such as guanidino-(a-amino)-lower alkanoic acids, for example, arginine and the like. Especially preferred a-amino monocarboxylic acid moieties are moieties of naturally occurring a-amino monocarboxylic acids, i.e., those basic amino acids which are the component acids of proteins.

The basic a-amino monocarboxylic acid moieties can be present in racemic (D,L) or optically active (D- or L-) form. Preferred are moieties of naturally occurring a-amino monocarboxylic acids having the L-configuration.

Preferred compounds of formula I wherein Ac is the acyl residue of a basic a-amino monocarboxylic acid are L-lysinen-dodecylester and L-lysine-n-tetradecylester.

Compounds of formula I which are esters of basic dipeptides, i.e., compounds of formula I wherein Ac is the acyl residue of a dipeptide containing at least one basic a-amino monocarboxylic acid moiety, form a second group of novel compounds within the scope of the present invention. Exemplary of dipeptide moieties represented by Ac in formula I are such dipeptide moieties as seryl-lysine, lysyl-serine, lysyllysine, and the like. As stated above, at least one of the amino acid moieties of the dipeptide group must be that of a basic 0:- amino monocarboxylic acid. The second amino acid moiety in the dipeptide chain can be the moiety of either a neutral aamino monocarboxylic acid or can be the moiety of a basic aamino monocarboxylic acid. Exemplary of neutral a-amino monocarboxylic acid moieties are those of neutral naturally occurring (i.e., component acids of proteins) a-amino acids, such as alanine, phenylalanine, cysteine, cystine, methionine, glycine, leucine, isoleucine, valine, norvaline, proline, serine, threonine and tyrosine. Such neutral amino acid moieties having an asymmetric center, can be used not only in their racemic (D,L) form but also in their optically active (D or L- form. Cystine, though not a monocarboxylic acid, is classed in the above group, since it contains an a-amino group for each carboxylic acid moiety, and moreover, it is conventionally classed as a neutral naturally occurring amino acid.

Dipeptides synthesized from one neutral and one basic 0:- amino monocarboxylic acid can be classed in two subgroups, the first being those dipeptides in which the terminal carboxyl function forms part of the moiety of the neutral a-amino monocarboxylic acid (such as, for example, in lysyl-serine). The second subgroups are those dipeptides in which the terminal carboxyl function is part of the basic a-amino monocarboxylic acid moiety (such as,-for example, in seryl-lysine). The latter classes can be further subdivided, i.e., into those dipeptides which'are linked in an a-amide fashion and those which are linked in an m-amide fashion; exemplified by N'-seryllysine and N--seryl-lysine, respectively. Similarly, dipeptides containing two basic a-amino monocarboxylic acid moieties can be subgrouped according to the latter differentiation, i.e., as to whether the peptide linkage is a-amide or m-amide, exemplified by N-lysyl-lysine and N-lysyl-lysine, respectively.

Exemplary compounds of formula I wherein Ac is the acyl residue of a dipeptide containing at least one basic a-amino monocarboxylic acid moiety are compounds such as: N -L-ar- -D-seryl-L-lysine-n-hexadecyl ester, NQ-L-phenylalanyl-li sine-n tetradecylester, L-

and N -L-lysyl-L- same lines as discussed above for the compounds of formula I containing a dipeptide moiety. For example, besides one basic a-amino monocarboxylic acid moiety, the second and third amino monocarboxylic acid moieties can each be derived from either a basic or a neutral a-amino monocarboxylic acid. Furthermore, the various tripeptide moieties can be differentiated into subgroups according to whether the esterified carboxy group is part of a basic a-amino monocarboxylic acid moiety or part of a neutral a-amino monocarboxylic acid moiety. Moreover, the tripeptide moieties are to be distinguished according to whether the peptide linkages are aamide and/or w-amide linkages.Thus, as is apparent, the

. number of subgroups of compounds of formula I containing a tripeptide moiety is even greater than the number of subgroups of compounds of formula I containing a dipeptide moiety.

Exemplary compounds of formula I containing a tripeptide moiety are L-lysyl-blysyl-L-lysine-n-hexadecylester, N -L- lysyl-(N-L-lysyl)-L-lysine-n-tetradecylester and glycyl-L- lysyl-L-lysine-n-dodecylester.

In one embodiment of this invention, a compound of formula l is prepared by a process which comprises esterifying a compound of the formula:

Ac-OH (Ill) wherein Ac has the same meaning as above, or a reactive derivative thereof, with an alcohol of the formula:

HO-R (IV) wherein R has the same meaning as above, or a reactive derivative thereof.

In another embodiment of this invention, compounds of formula I wherein Ac is a dipeptide or tripeptide moiety can be prepared via acylation wherein an ester of the formula:

Ac'OR (V) wherein Ac is the acyl residue of a neutral or basic a-amino monocarboxylic acid or of a dipeptide, the components of which are neutral or basic a-amino monocarboxylic acid moieties, and R has the same meaning as above, is treated with a compound of the formula Ac -OH (Vl) or with a reactive derivative thereof,

wherein Ac is an acyl moiety of neutral or basic a-amino monocarboxylic acid or the acyl moiety of a dipeptide, the components of which are neutral or basic a-amino monocarboxylic acid moieties; and wherein at least one of Ac and Ac contains at least one basic a-amino monocarboxylic acid moiety. Thus, as outlined above, all compounds of formula I, i.e., not only the esters of the amino acids but also the esters of the dipeptide and tripeptides, can be prepared by esterification of the corresponding unesterified compounds. Moreover, the dipeptide and tripeptide esters can be obtained from amino monocarboxylic acid and dipeptide esters by lengthening the chain thereof with one or two amino acid residues by means of N-acylation.

The preparation of a compound of formula I by esterification of a corresponding unesterified starting compound can be effected by conventional esterification means according to known methods. Thus, for example, an N-protected basic 0:- amino monocarboxylic acid or an N-protected dipeptide or tripeptide of formula III containing the moiety of at least one basic a-amino monocarboxylic acid can be reacted in the presence of a tertiary base (such as a trilower alkylamine, e.g., triethylamine) with a reactive derivative of an alcohol of formula IV. The halogenides (e.g., the chlorides, bromides or iodides) are especially suitable as reactive derivatives of the alcohols offormula IV.

The free amino group of the starting material compounds can be protected with the usual protecting groups, conventional in peptide synthesis. Such N-protecting groups are, for example, carbobenzoxy, tosyl, phthalyl, trityl, formyl, trifluoroacetyl, tert. butyloxycarbonyl and the like. Also, as is conventional, the nitro group can be utilized as the protecting group for the guanidino group of arginine. The solvent or reaction medium in which the esterification of this invention can be conducted are suitably organic compounds conventionally used as solvents and inert to the reactants, for exampie ethers such as dioxane, dilower alkyl formamides, such as dimethylformamide, and the like. The'reaction can con-.

veniently be effected at elevated temperatures, for example, at the reflux temperature of the reaction medium. For example, the n-hexadecyl ester of L-lysine can be prepared by heating to boiling under reflux, a solution of N", N-dicarbobenzoxylysine in dioxane in the presence of triethylamine, together with approximately one mole equivalent of l-bromohexadecane, and subsequently removing both of the carbobenzoxy protecting groups by hydrogenolys is. Esterified dipeptide or tripeptides of formula I can be prepared in the corresponding manner, for example L-lysyl-L-lysine or L- lysyl-L-lysyl-L-lysine can be esterified in a corresponding manner by reacting the peptide (with prior protection of the amino groups) with a long chain alkyl or alkenyl halogenide, and subsequently splitting off the protecting groups.

The esterification reaction can also be efi'ected by reacting reactive derivatives of the acids of formula Ill with a long chain alcohol of formula IV. Thus, for example, the anhydride or a mixed anhydride of an acid of formula III (suitably with protected amino groups) can be reacted with a long chain alcohol (such as cetyl alcohol).

Moreover, the esterification can also be effected by the acid catalyzed reaction of an acid of formula III with an alcohol of formula IV. Exemplary of suitable acid catalysts is, for example, p-toluenesulfonic acid. For the purposes of such acid catalyzed esterification, it is not necessary to protect the amino groups in the starting material compounds.

In addition to the esterification reactions above-described, the long chain esters of dipeptides can also be prepared from long chain esters of basic or neutral a-amino monocarboxylic acids by aor w-amide combining with a second a-amino monocarboxylic acid. Starting from a long chain ester of a basic a-amino monocarboxylic acid (such as the n-dodecyl ester of ornithine), then the second a-amino monocarboxylic acid moiety inserted by the peptide synthesis can be either a basic or a neutral a-amino monocarboxylic acid (such as, for

, example, ornithine or threonine). On the other hand, starting acid moiety). Thus, for example, an a-amino monocarboxylic acid can be reacted with a long chain ester of an a-amino monocarboxylic acid in the presence of a suitable condensation agent (such as a carbodiimide), for example, dicyclohexylcarbodiimide or carbonyldiimidazole or 2- ethyl-5-m-sulfonato-phenylisoxazole, or the like). The reaction is preferably effected at a low temperature, for example, in the range of about 0-20 C. in the presence of a solvent such as dimethylformamide, chloroform, methylene chloride, ethyl acetate, tetrahydrofuran or the like Moreover, for the purposes of this reaction, the acylation of the preexisting ester component can be effected by using as the acylating agent, an a-amino monocarboxylic acid which is modified in the carboxyl group, for example, an azide, a halogenide (such as the chloride), an activated ester (such as the p-nitrophenyl ester, the thiophenyl ester or the cyanomethyl ester), a mixed anhydride with an inorganic acid (such as carbonic acid, sulfuric acid or phosphoric acid), or the like. The reaction can be effected at or below room temperature.

One method of forming the dipeptide chain which not only utilizes an acylating agent possessing an activatedcarboxyl group, but also an component containing an activated amino group, is the method of Anderson (J. Am. Chem. Soc. 74 (1952), 5304 and 5309) in which the carboxyl group is converted into a COOP(OC H group and the amino group into a NHP(OC HB) group by means of tetraethylpyrophosphite [i.e., (C,H,,O) =POP (OC,H

Esters of basic a-amino monocarboxylic acids, such as the a,w-diamino-monocarboxylic acids can be acylated at the 0:-

position can be effected by blocking the wor a-amino group, respectively. Suitable for the blocking of such amino groups are the protecting groups already mentioned above, such as the carbobenzoxy or formyl group. If neither of the two amino groups of the basic ester component group is protected there can thus be obtained N, N"'-diacyl derivatives (i.e., tripeptide esters).

Long chain esters of tripeptides can be obtained by the process described immediately above, and also by condensation of an a-amino monocarboxylic acid ester with a dipeptide or by condensation of a dipeptide ester with an a-amino monocarboxylic acid. Thus, for example, the n-decyl ester of lysyl-lysyl-lysine can be obtained by acylating the N'-protected N-decylester of lysine with lysyl-lysine azide, all the amino groups of which are protected, and subsequently splitting off said protecting groups. Alternatively, the same tripeptide ester can also be prepared by N -acylation of lysyllysine n-decylester, the e-amino groups of which are protected, with lysine (the amino group of which may be protected), and then subsequently splitting off all protecting groups.

The various protecting groups discussed above can be split off according to methods conventional in the art. After having effected esterification or N-acylation, for example, the protecting groups can be split off by hydrogenolysis or hydrolysis. Thus, the carbobenzoxy protecting group can be split off by means of catalytically activated hydrogen, for example by using palladium as the catalyst, or by means of HBr/glacial acetic acid. The formyl protecting group can be split off with mineral acids at low temperatures. Other protecting groups can similarly be split off by methods known in the art.

The products of formula 1, obtained according to the above described processes, which are obtained as bases can be converted into acid addition salts by methods known per se. Moreover, bases of formula I can be liberated from such acid addition salts according to methods known per se, and, if desired, the so-liberated bases can be converted into other acid addition salts. Thus, bases of formula 1 form acid addition salts with both inorganic and organic acids, for example, sulfuric acid, phosphoric acid, hydrohalic acid (such as hydrochloric acid, hydrobromic acid), oxalic acid, acetic acid, citric acid, tartaric acid, sorbic acid, p-toluene sulfonic acid, and the like.

TABLE I.TOIAL INHIBITION AGAINST PATHOGENIC BACTERIA 'y/ml. OVER 24 HOURS The compounds of formula 1, including acid addition salts thereof, are useful as disinfectants and bactericidal agents. Thus, the compounds of formula 1 and the acid addition salts thereof have a high antibacterial activity against gram-positive bacteria (such as Pneumocci, Streptocci, Anthrax bacilli, Staphylococci, Enterococci) and gram-negative bacteria (such as Escherichia coli, Salmonella typhi murium, Shigella, Klebsiella pneumoniae as well as, in particular, pseadomonas aeruginasa). Moreover,the compounds of formula I, as well as their acid addition salts, are characterized by a very low level of toxicity. Such compounds can, for example, be used as disinfectants for rooms, apparatus and utensils in dairy farms. Furthermore, they can be used as antiseptics for warmblooded animals.

The following compounds show a particularly high antibacterial activity: L-lysine-n-dodecylester, L-lysine-ntetradecylester and their acid addition salts.

The aforementioned antibacterial activity can be demonstrated, for example, utilizing the following procedure:

For each ,of the micro-organisms used in the test, ten dilutions (2500 'y/ml. to 5 'y/ml.) of the test compound are prepared in culture tubes containing a culture medium. A brain-heart infusion serves as the culture medium for Pneumococci, Streptococci and Emerococci, and for the other bacteria to be tested a nutrient-broth serves as the culture medium. The inoculum for each culture tube comprises one drop of a 24- hour preliminary culture of the test bacteria. Pneumococci, streptococci, and Enterococci are cultured in liver bouillon; the other bacteria to be tested are cultured in a dextrose medium. After 24-hour incubation at 37 C., the inoculated samples are inspected for bacterial growth and simultaneously one loopful of material is taken from each culture tube and streaked across nutrient-agar (subculture) for the purpose of ascertaining whether the bacteria are killed by the test compound. The subcultures for Pneumococci, Streptococci and Enterococci are prepared in blood agar, the subcultures for the remaining bacteria is Difco-blood-agar base. Kill observations are made after 24-hour incubation at 37 C. When, for

example, L-lysine-n-dodecylester dihydrochloride, which has L-lysine-n- L-lysine-n- L-lysine-n- L-lysine-tetra- L-lyslne-rroctaoctylesterdeeylesterdodecylesterdecylesterdecylestertartrate tartrate dihydrochloride dlhydrochlorlde dlhydrochloride Gram-positive:

78 19.6 19.6 78 19. 5 10 10 78 S. typhi mun. 39 6 10 78 Shin flez'neri. 39 10 10 78 Paeud pyocyan. 78 39 156 78 Kleba pneuzn 39 10 10 78 1 Not tested.

TABLE II.TOTAL KILL AGAINST PATHOGENIC BACTERIA 'y/ml. OVER 24 HOURS L-lyslne-n- L-lysine-n- L-lyslne-n- L-lyslne-tetra- L-lysine-n-octaoctylesterdecylesterdodeeylesterdecylesterdecylestertartrate tartrate dihydrochloride dihydrochloride dihydrochloride, Gram-positive:

Pneumococei 1) 39 5 5 78 Stre t0cocci 312 19.6 5 10 166 Ant rax t) 312 19.5 39 2, 500 Staphylococci 625 312 19. 5 39 78 Enterococci 78 10 5 156 Gram-negative:

a coli 78 19. 5 19. 5 625 s emerlt 0-- 78 19.5 19.5 9 6 S. typhi mur... 39 10 10 J12 Silly flezneri 39 10 10 312 Pseud. prion an" 1,250 156 30 166 626 lflebu. pneum 78 10 Ill. 5 500 1 Not tested.

The novel compounds of formula I, as well as their acid addition salts, can be used as medicaments in the form, for example, of pharmaceutical preparations containing a compound of formula I or a pharmaceutically acceptable acid addition salt thereof in admixture with a pharmaceutical organic or inorganic pharmaceutical carrier suitable for, preferentially, enteral administration, such as, for example, water, gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, gums, polyalkylene glycols, Vaseline, etc. Moreover, they can be submitted to conventional pharmaceutical expedients such as sterilization. Also, the pharmaceutical preparations can be administered in conventional solid forms, e.g., tablets, dragees, suppositories, capsules, or the like, or in conventional liquid forms, e.g., solutions, suspensions or emulsions; and can contain additive materials such as preserving, stabilizing, wetting or emulsifying agents, salts for the adjustment of osmotic pressure, or buffers. Furthermore, they can contain other therapeutically activeiifaterialsfl or nonpharmaceutical uses, such as for room disinfection, a compound of formula l or an acid addition salt thereof (which, of course, need not be pharmaceutically acceptable) can be used by merely dissolving or suspending an amount thereof sufficient to exhibit an antibacterial effect in a suitable liquid carrier, for example, water, ethanol, or the like. An effective antibacterial amount may be in the range of between about 0.05 percent to about l percent. However, higher or lower concentrations may also be effectively utilized.

The following examples are illustrative, but not limitative, of this invention. All temperatures are stated in degrees centigrade. The symbol Z represents the carbenzoxy group.

EXAMPLE 1 41.4 g. N-Z(N-Z)-L-lysine, 200 ml. of dioxane, l4.7 ml. of triethylamine and 30.5 g. of l-bromo-hexadecane are heated under reflux at 95-l00 for 20 hours. The separated triethylamine hydrobromide is filtered off under suction, the filtrate evaporated in vacuo, the residue taken up in ethyl acetate and the ethyl acetate extract washed neutral with lN hydrochloric acid, 5 percent NaCl solution, IN ammonia and 5 percent NaCl solution. After drying over Na SO the solu tion is then evaporated in vacuo and the so-obtained N-Z(N -Z)-L-lysine-n-hexadecylester is crystallized from ether/petroleum ether. Melting point 70-72, [a],,=-9.0 (c=2 in methanol).

The so-obtained N-protected amino acid ester is decarbobenzoxylated in glacial acetic acid with 5 percent palladium/carbon and hydrogen. The catalyst is then separated off, the filtrate evaporated in vacuo, the residue dissolved in 4N hydrochloric acid/methanol, the so-obtained solution evaporated in vacuo and the residue then mixed with acetone. The so-obtained L-lysine-n-hexadecylester dihydrochloride is filtered off under suction, washed with acetone and recrystallized from ethenol. Melting point l08-l 10 (decomposition); [a],,=-+6.9 (c=2 in methanol).

EXAMPLE 2 18.2 g. of cetyl alcohol, 14.25 of p-toluenesulphonic acid monohydrate, 9.1 g. of L-lysine monohydrochloride and 200 ml. of benzene are boiled at reflux for hours. The water which fonns is continuously removed with a water-separator. The reaction mixture is evaporated in vacuo. The residue is extracted with a mixture of water and ethyl acetate for the purpose of removing the excess cetyl alcohol, the aqueous phase is then adjusted to a pH of 9-10, extracted with ethyl acetate, the ethyl acetate solution dried over Na SO and evaporated. The residual oil is neutralized with 1N HCl/methanol, the solution evaporated, the residue mixed with acetone and the crystal-paste filtered off under suction. Finally, the so-obtained L-lysine-n-hexadecylesterdihydrochloride is recrystallized from ethanol. Melting point l08-220 (decomposition); [a],, =+6.9 (c=2 methanol).

EXAMPLE 3 29.8 g. of n-eicosanol, 200 ml. of absolute tetrahydrofuran and 1 15 mg. of powdered sodium are boiled under reflux for 3 hours under a calcium chloride trap. The n-eicosanol/Na-neicosanolate mixture is then decanted off from the remaining small amount of sodium. Meanwhile, 41.4 g. of N-Z(N-Z)- L-lysine are dissolved in 200 ml. of tetrahydrofuran, cooled down to l0, treated with 16.7 g. of carbonyl-diimidazole and stirred for one hour at l0. .Then the above neicosanol/Na-n-eicosanolate mixture is added dropwise at a temperature of 0 while stirring. The mixture is stirred for 30 minutes at 0 and then for a further 24 hours at 20. The resultant solution is then evaporated in vacuo. The residue is dissolved in ethyl acetate, washed neutral with 1N hydrochloric acid, 5 percent NaCl solution, lN ammonia and 5 percent NaCl solution, dried over Na sO evaporated and the so-obtained residue crystallized from ether/petroleum ether. The

jso-oblained N-Pm jniw sstq [Neitbelkk lysine-n-eicosylester] is hydrogenolyzed in glacial acetic acid with 5 percent palladium/carbon and hydrogen gas. The catalyst is then separated off, the filtrate evaporated in vacuo, the residue dissolved in 4N HCl/methanol, and again evaporated in vacuo, and the residue is then mixed with acetone. The so-obtained L-lysinen-eicosyl ester dihydrochloride is filtered off under suction and precipitated several times from methanol with the dropwise addition of acetic ester. Melting point l07-l09; [a],, =+7.0 (c-=2 in methanol).

EXAMPLE 4 The following compounds can be manufactured according to the esterification method described in the foregoing exam- EXAMPLE 5 Heals-three. .SL1Q1$-- f ..NfirZiNfZkL-lfi -Z)- i lysine is reacted according to the procedure of example I with l 1.4 g. of l-bromohexadecane in the presence of ml. of dioxane and 5.2 ml. of triethylamine. The so-obtained N-protected dipeptide ester [N-Z-(N-Z)-L-lysyl-(NZ)-L-lysine n-hexadecyl ester] is recrystallized several times from ethyl acetate/petroleum ether (melting point l0O-l02) and then decarbobenzoxylated by hydrogenation in glacial acetic acid using 5 percent palladium/carbon. After separation of the catalyst, the glacial acetic acid is distilled off, the residue evaporated with 4N HCl/methanol and precipitated several times from ethanol/acetone. The thus obtained L-lysyI-L- lysine n-hexadecylester trihydrochloride melts at 234" (decomposition); lal =+3 (c=2 in methanol).

EXAMPLE 6 liquified by a short heating, rapidly cooled down and the resulting dicyclohexyl-urea separated off by filtration. The filtrate is then precipitated in IN hydrochloric acid, reprecipitated from dimethylformamide /lN ammonia, filtered under suction and dried. The so-obtained crude N-protected dipeptide ester [Z-L-phenylalanyl-(N-Z)-L-lysyl-nhexadecyl ester] is recrystallized from ethanol and decarbobenzoxylated by hydrogenation in 33 percent HBr/glacial acetic acid for 3 hours under a calcium chloride trap. The soobtained V L-phenylalanyl-l-lysine n-hexadecylester dihydrobromide is precipitated with absolute ether, filtered off under suction, washed with ether, dried and recrystallized from ethanol. Melting point l45-l47, [a],, =3 (c=2 in methanol).

The (N -Z)-L-lysine-n-hexadecylester used as the starting material can be obtained as follows:

61.6 g. of N-formyl-(N-Z)-L-lysine, 200 ml. of dioxane, 29 ml. of triethylamine and 61 g. of l-bromo-hexadecane are heated under reflux at 95l00 for 20 hours. The triethylamine hydrobromide is filtered ofi' under suction, the filtrate evaporated in vacuo, the residue, in ethyl acetate, washed neutral with percent acetic acid, 5 percent NaCl solution, 1N ammonia and S percent Naif] solution, dried over Na SO evaporated and crystallized from ethanol. The so-obtained N-formyl-(N-Z)-L-lysine n-hexadecylester melts at 86-88.

This Nprotected amino acid ester is deformylated by standing for 16 hours in 2N HCl/methanol, after which the solution is evaporated and the residue then crystallized from methanol/ether. The so-obtained (N-Z)-L-lysine N-hexadecylester hydrochloride melts at 88-90; [a],,=+6.5 2 in methanol).

The free ester-base is obtained by partitioning the hydrochloride between chloroform and ammonia, washing the chloroform phase with water, drying over Na,SO, and concentrating in vacuo. The free (N-Z)-L-lysine n-hexadecyl ester then separates as an oil which solidifies upon cooling.

EXAMPLE 7 6.8 g. of L-phenylalanine n-hexadecyl ester and 7.2 g. of N -Z-( N -Z) -L-lysine are dissolved in 40 ml. of dimethylfonnamide, treated at 0 with 3.55 g. of dicyclohexylcarboniimide and left to stand for 20 hours at 0. The solid mass is heated for a short period of time until liquification occurs, rapidly cooled down, and the dicyclohexyl-urea filtered off under suction. The filtrate is then diluted with ethyl acetate, washed neutral with 1N hydrochloric acid, 5 percent NaCl solution, 1N ammonia and 5 percent NaCl solution, dried over Na,SO evaporated and the residue crystallized from ethyl acetate/petroleum ether. The so-obtained N-protected dipeptide ester is decarbobenzoxylated by hydrogenation in glacial acetic acid in the presence of 5 percent palladium/carbon, the catalyst separated off, the filtrate evaporated, the residue evaporated with 4N HCl/methanol and mixed with acetone. The so-obtained L-lysyl-L-phenylalanine n-hexadecyl ester dihydrochloride is filtered off under suction and recrystallized from ethanol. Melting point l60-l62; [a] +20.4 (c=2 in methanol).

EXAMPLE 8 Vacu There is thus obtained, N -formyl-L-a,y-diamino-butyric acid n-hexadecyl ester as a viscous oil which crystallizes upon cooling.

A solution of l5 g. of this n-hexadecyl ester and 15.7 g. of N :Z-(N -Z)-D-a,'y-diamino-butyric acid in 70 ml. of dimethylformamide is treated at 0 with a solution of 8.4 g. of dicyclohexylcarbodiimide and left to stand for 20 hours at 0. Then the separated dicyclohexyl-urea is filtered off under suction, the filtrate diluted with 500 ml. of ethyl acetate, washed tained Q.., neutral with [N acetic acid, water and 1N ammonia, and the ethyl acetate solution dried over Na SO and thereafter evaporated in vacuo. The so-obtained N -formyl-N -lN -Z- N -Z)-D-a,y-diamino-butyryl]-L-a,y-diamino-butyric acid nv hexadecyles ter is crystallized 'riar'n' ethyl acetate/petroleum ll4-l l6 [a],, =6.9 (C=2 in ether. Melting point dimethylformamide).

Five grams of the so-obtained N-protected dipeptide n-hexadecyl ester is decarbobenzoxylated by hydrogenation in glacial acetic acid in the presence of 5 percent palladium/carbon. The catalyst is then filtered off under suction, the filtrate evaporated in vacuo and the residue deformylated with 30 ml. of 4N HCl/methanol for 5 hours at 20. The solution is then evaporated in vacuo, the residue mixed with acetone, filtered off under suction, reprecipitated from methanol/acetone and methanol/ethyl acetate and dried in vacuo at 60. The so-ob- 7 Nf-gQ-otg diamim-butyryl)-L-az,y-diamino-butyric acid n-hexadecyl ester (decomposition); [a] =-l 5.7. (=3 in methanol).

EXAMPLE 9' Thirty-two grams of N -formyl-(N -Z)-L-lysine-n-hexadecyl ester are decarbobenzoxylated by hydrogenation in glacial acetic acid with palladium/carbon. The catalyst is then filtered off under suction, the filtrate evaporated in vacuo, the residue dissolved in a mixture of water and ethyl acetate, the solution adjusted to a pH of 9-10 with concentrated ammonia, then extracted with ethyl acetate, the ethyl acetate extract dried over Na SO and evaporated in vacuo. The N -formyl- L-lysine n-hexadecyl ester obtained as the residue solidifies upon cooling.

Twenty-two grams of the so-obtained amino acid hexadecyl ester and 22.9 g. of N-Z-(N-Z)-L-lysine are dissolved in 50 ml. of dimethylformamide, treated at 0 with a solution of l l .3 of dicyclohexylcarbodiimide and left to stand for 24 hours at 0. The resultant thick gel is liquified by heating, then the dicyclohexyl-urea is filtered off under suction and the filtrate precipitated in IN acetic acid. The precipitate is filtered off under suction, and is reprecipitated from dimethyl-formamide/ lN ammonia. The so-obtained N"-fonnyl-N-[N"-Z-( N -Z)-L-lysyl]-L-lysine n-hexadecyl ester is filtered off under suction, washed with water, dried in vacuo and recrystallized from ethyl acetate/petroleum ether. Melting point l02-l 04; [a],,=l0.2 (c=2 in methanol).

Fifteen grams of the dipeptide hexadecyl ester thus obtained, is decarbobenzoxylated by hydrogenation in glacial acetic acid with palladium/carbon. The catalyst is then filtered off under suction, the filtrate evaporated in vacuo and the residue deformylated with ml. of 2N HCl/methanol for 5 hours at 20. Upon evaporation there results a crystalline residue which is recrystallized from ethanol, yielding N-L- lysyl-L-lysine n-hexadecyl ester trihydrochloride which melts at 220222 (decomposition); [a],,=-l-l8.9 (c=2 in methanol).

EXAMPLE 10 (N-Z)-L-lysine n-hexadecyl ester obtained is crystallized from ethanol. This N-protected dipeptide ester obtained is then decarbobenzoxylated by hydrogenation in glacial acetic acid with 5 percent palladium/carbon. The catalyst is separated off, the filtrate evaporated and the residue evaporated with 4N HCl/ethanol. The so-obtained D-seryl-L- lysine n-hexadecyl ester dihydrochloride is mixed with acetone, left to stand for 3 hours at 0, filtered off under suctrihydrochloride melts at T50 193"- tion and crystallized from ethanol. Melting point 120; [al l5 (e -=2 in methanol).

EXAMPLE 1 l Seventeen grams of L-lysine n-hexadecyl ester and 34.6 g. of N -l-(N -Z)-L-lysine are dissolved in 150 ml. of dimethylformamide, treated at l with 17 g. of dicyclohexylcarbodiimide and left to stand for 24 hours at 0. The resultant thick oil is liquified by heating, rapidly cooled down and freed from dicyclohexyl-urea by filtration under suction. The filtrate is diluted with 800 ml. of ethyl acetate and washed neutral with 1N hydrochloric acid, water, 1N ammonia and water. After drying over Na SO the ethyl acetate is distilled off in vacuo and the residue recrystallized, first from ethanol, then from methanol. The thus-obtained N-protected N -l-(N -Z)-Di-L- lysyl-L-lysine N-hexadecyl ester melts at 13 l-l 32.

This N-protected tripeptide ester is decarbobenzoxylated by hydrogenation in glacial acetic acid with percent palladium/carbon, the catalyst is separated ofi and the ethyl acetate is distilled off in vacuo. The residue is then evaporated with 4N HCl/methanol in vacuo, mixed with acetone, filtered off under suction, dried and reprecipitated several times from ethanol/acetone. The so-obtained N -L-lysyl-(N -l..-lysyl)-L- lysine n-hexadecyl ester tetrahydrochloride melts at 240 (decomposition); [a1 =+-l 2.9 (c--2 in methanol).

example I 2 with l50 ml. of ethyl acetate and cooled down to 1 0. At this temperature a solution of 2.2 g. of sodium nitrite in 10 ml. of water is added dropwise to the reaction mixture with stirring, the azide extracted after 20 minutes at 0 with ISO ml. of ethyl acetate, the ethyl acetate extract is washed with water, 1N sodium bicarbonate solution and dried at 0 over sodium sulfate. The azide solution, which is still acidic, is treated portionwise at 0 while stirring with a solution of 15.6 g. of (N- Z)-L-lysine hexadecyl ester in 50 ml. of ethyl acetate and left to stand for 48 hours at 0. The so-obtained N-protected tripeptide ester, [N"-Z(N-Z)-L-lysyl-(N-Z)-L-lysyl-(N-Z)- L-lysine n-hexadecyl ester], is filtered off under suction, washed with ether, precipitated from dimethylformamide in IN hydrochloric acid and reprecipitated from dirnethylf orr'nav EXAMPLE 13 The amino acid esters obtained according, to the methods of this invention can be converted into various application forms. It is especially advantageous for this purpose to use the amino acid esters in the form of their water soluble acid addition salts, e.g., hydrochlorides. Suitable application forms are, for example:

a. Aqueous solution (0.05; 0.]; l percent):

Active material 0.05 or O.l or 1 g. Distilled water ad ml. b. Tincture Active material 0.05 or I g. Distilled water 2 ml. Ethanol (94 percent ad 100 ml. c. Mucllnge-sslve Active material (H g. Methylcellulole 1.2 g. Distilled water ad l00.0 g.

(0.05 or I percent):

lclaim:

l. A method of inhibiting and controlling the growth of bacteria which comprises contacting said bacteria with an antibacterially effective amount of a long chain aliphatic ester selected from the group consisting of L-lysine-n-dodecylester, L-lysine-n-tetradecylester and acid addition salts thereof.

2. A method in accordance with claim 1 wherein the long chain aliphatic ester is L-lysine-n-dodecylester.

3. A method in accordance with claim 1 wherein the long chain aliphatic ester is L-lysine-ntetradecylester.

Claims (2)

1. 2. A method in accordance with claim 1 wherein the long chain aliphatic ester is L-lysine-n-dodecylester.
2. 3. A method in accordance with claim 1 wherein the long chain aliphatic ester is L-lysine-n-tetradecylester.