US3825560A - N-monoacyl derivatives of arginine - Google Patents

Abstract

THE LOWER ALKYL OR BENZYL ESTERS AND AMIDES OF THE NMONOACYL DERIVATIVES OF THE BASIC AMINO ACIDS (A,Y-DIAMINOBUTYRIC ACID, ORTHININE, CITRULLINE, LYSINE, ARGININE, AND HISTIDINE) AND THEIR N-METHYL AND N-BENZYL DERIVATIVES IN WHICH ACYL IS THE ACYL RADICALOF AN ALIPHATIC MONOCARBOXYLIC ACID HAVING 6 TO 20 CARBON ATOMS HAVE BACTERIOSTATIC EFFECTS OF THE ORDER OF MAGNITUDE OF THAT OF HEXACHLOROPHENE. THEY MAY BE USED AS ANTIBAL AGENTS IN THE FORM OF THEIR WATER SOLUBLE SALTS WITH HYDROCHLORIC ACID, PYRROLIDONE CARBOXYLIC ACID, AND OTHER INORGANIC AND ORGANIC ACIDS.

Description

ABSORBANCE ABSORBANCE ABSORBANCE TADAOMI SAITO ET AL N-IONOACYL DERIVATIVES 0F ARGININE 3 Sheets-Sheet 1 July 23, 1974 Filed me 28. 1971 FIG. 1A FIG. 1B v 2 A Strain '8' Strain NO.1

u o 0.6-- 5 NO. 2 g o g 4 [N08, 3,4,5 l l T 10 2O 30 HOURS FIG. 1C '0' Strain u.| 0 Z m n: 0 (o :1: 1:

m g 0.20 m n: o 3 q 0.10-

HOURS y 1974 TADAOMI SAITO ETAL N-HONOACYL DERIVATIVES 0F ARGININE Filed June 28, 1971 3 Sheets-Sheet 2 FIG. IH

'H' Stroin FIG. IG

'0' Strain NOS.I,2v

IO 20 HOURS FIG. 28

FIG. 2A

5 4 1w m 3. N m m R w n .l H 1w 8 C ...U. 1 O w02 mm0mm 1w 2 S 3 m s I O 2 N s m m n .m [10' & HA 1 O w0z mm0mm FIG. 2D

'6' 81min NOS. L2

FIG. 2C

HOURS HOURS July 23, 1974 TADAONU SAITO ETAL N-HONOACYL DERIVATIVES 0F ARGININE 3 Sheets-Sheet 3 Filed June 28, 1971 FIGBB FIG. 3A

0' Strain 'A Strain u u 4 2 O HOURS HOURS FIG. 3C

'6' Strain United States Patent 3,825,560 N-MONOACYL DERIVATIVES 0F ARGININE Tadaomi Saito, 890 Fukaya-cho, Totsuka-kn, Yokohamashi; Masahiro Takehara, 1060 Ogura, Kawasaki-sin; Kazuhiko Yamada, 22-22, 3-chome, Fujigaoka, Fujisawa-shi; and Ryonosuke Yoshida, 1265-11 Kajiwara, Kamakura-shi, all of Kanagawa-ken, Japan; and Yukiko Sasaki, 28-6, 3-chome, Minami-cho, Kokubunji-shi, Tokyo, Japan Continuation-impart of abandoned application Ser. No. 99,768, Dec. 21, 1970. This application June 28, 1971, Ser. No. 157,584

Claims priority, application Japan, Dec. 30, 1969, 45/ 1,750 Int. Cl. C07d 27/08 US. Cl. 260-326.45 6 Claims ABSTRACT OF THE DISCLOSURE The lower alkyl or benzyl esters and amides of the N- monoacyl derivatives of the basic amino acids (any-diaminobutyric acid, orthinine, citrulline, lysine, arginine, and histidine)and their N-methyl and N-benzyl derivatives in which acyl is the acyl radicalof an aliphatic monocarboxylic acid having 6 to 20 carbon atoms have bacteriostatic effects of the order of magnitude of that of hexachlorophene. They may be used as antibial agents in the form of their water soluble salts with hydrochloric acid, pyrrolidone carboxylic acid, and other inorganic and organic acids.

This application is a continuation-in-part of the copending application Ser. No. 99,768, filed on Dee. 21,1970 and now abandoned.

This invention relates to antiseptic, disinfectant, medicinal, preservative, bactericidal, bacteriostatic, germicidal and fungicidal materials and compositions.

Corrosive sublimate, cresol, alcohol, invert soap and etc. have been used as disinfectants or antimicrobial detergents in food hygienic field, environment sanitary field and medicinal field. However, these disinfectants have the following unsatisfactory properties. Corrosive sublimate has powerful disinfectant action, but it has a strong irritation against skin and causes a serious problem in the disposal of waste matters owing to mercury containing material. And cresol has high toxicity to men and beasts, while alcohol has weak disinfectant action. Although invert soap such as Hyamine (Product of Rohm & Haas Co., Inc.) has a powerful disinfectant action, the effect is markedly decreased by co-existence of protein, fat, sodium chloride, or soap, and it has high toxicity to men and beasts (For example, the acute oral toxicity (LD value of Hyamine is 420:25 mg. per kg. of body weight of mice.)

Recently, a disinfectant incorporating soap or synthetic detergent, the so-called deodorant detergent is recommended. As disinfectants or deodorizers in these detergents, for example hexachlorophene, trichlorocarbanilide, tribromosalicyanilide, tetramethylthiuram disulfide and the like have been practically used. However, each of these deodorizers has irritation of the skin and is hardly soluble or insoluble in water, and hence it is necessary that these deodorizers are incorporated in a relatively large amount into detergent for purpose to retain their antimicrobial activity. And the use of such a large amount causes further strong irritation on the skin. Particularly, hexachlorophene or tetramethylthiuram disulfide incorporating detergent has been reported to cause eczematid, sensitization to the skin when exposed to sunlight after its use. Also, usual cationic surfactants which exhibit in general strong antimicrobial action are water soluble, but they cannot be used as deodorizers of detergents, since an insoluble precipitation is formed when they are incorporated in detergent such as soap and thereby antibacterial activity is reduced or disappeared as well as the detergency is markedly reduced.

On the other hand, cosmetics, leather goods, rubber goods, paints, foods and feed are easily attacked and deteriorated by microorganism and hence almost all of them does not stand long storage or use.

Various antiseptic agents or antifungal agents have been hitherto developed and employed in this field. However, there has not yet been proposed antiseptic agent which possesses low or non-toxicity, non-irritation in addition to the complete inhibition of the growth of microorganisms. For example, since cosmetics are composed mainly of water and oil and since they also contain glycerin, protein and other materials which are easily attacked by microorganisms, they are able to be changed in quality and appearance as well as foods. Hence, benzoic acid, its derivatives hexachlorophene and the like have been employed as antiseptic agent for cosmetics, but all of them have the following disadvantages: (l) The concentration in water phase which is easily attacked by microorganisms is low because of their oil-soluble properties. (2) Their antiseptic effect is greatly reduced by the action of surface active agents which are present in cosmetics. (3) They have skinirritation and high toxicity.

In addition, sorbic acid, sodium propionate, dehydro acetic acid, nitrofuran series compounds and etc. have been used as antiseptic agents for food but the amount added of them is restricted from the standpoint of toxicity and so, they can not exert sufiicient antiseptic effect within their allowance of addition. Recently, higher alkyl derivatives of peptide containing glutamic acid or basic amino acid have been used as antiseptic agents, but the former is hard to handle because of its very low water solubility, whereas the latter injures the taste of food because of very bitter taste in addition to high cost.

An object of the present invention is to provide deodorizers which are suitable to be incorporated into a detergent and which have the desired characteristic properties in that they exert excellent antimicrobial action, cause no irritation to the skin and are effective to enhance cleaning elfect.

It is another object of the present invention to provide an antiseptic material composition in which the disadvantages of the known antiseptic materials are reduced or obviated.

It has been found that mono-N-higher aliphatic acyl basic amino acid derivatives of u,'y-diaminobutyric acid, arginine, ornithine, citrulline, lysine and histidine, and salts thereof, hereinafter described in detail, have the desirable and satisfactory characteristic property as deodorizers of detergents, owing to their water soluble property, their excellent antimicrobial activity against microorganisms such as bacteria and fungi, their good surface active action and their mildness to the skin. Moreover, by the use of them, skin troubles such as irritation of the skin, exzernatid and sensitization are not entirely recognized, and even when incorporated in soap and other synthetic detergents, they greatly exert their excellent antimicrobial action without reducing cleaning effect. Also, in case where they are incorporated in synthetic detergents which usually roughen the skin, they have skin protecting action preventing the skin from chapping.

It has further been found that said mono-N-higher aliphatic basic amino acid derivatives antiseptic, medicinal, preservative, bactericidal, bacteriostatic, germicidal and fungicidal properties.

According to the present invention, there are provided antiseptic, disinfectant, medicinal, preservative, bactericidal, bacteriostatic, germicidal and fungicidal agents which are much secure to men and beasts, comprising at least one mono-N-higher aliphatic acyl basic amino acid derivatives having the following general formula (I), (H) and (III), and salts thereof:

/NH -NHC NHOONHz or I m r-r andn is 1, 2, 3 or4.

Compounds which are incorporated as deodorizers in detergent and which are also useful as antiseptic material or germicide according to the present invention may be easily prepared and are cheaply available. That is, among the compounds of the general formulas (I) and (H), ones wherein n is 2 are a-y-diaminobutyric acid derivatives, ones wherein n is 3 are ornithine derivatives and ones wherein n is 4 are lysine derivatives. And, the compounds of general formula (III) are arginine, citrulline and histidine derivatives. Each of these mono-N-higher acyl basic amino acid derivatives may be easily prepared by the reaction of corresponding basic amino acid with higher fatty acid chloride in alkaline aqueous medium.

Either of .optically active L- or D-form or racemic form of basic amino acid components are effective.

As salts of these N-higher aliphatic acyl basic amino acid derivatives, for example mineral acid salts such as bydrochloride and sulfate, salts with organic acid such as an optically active or inactive a-pyrrolidonecarboxylic acid, an optically active or inactive acidic amino acids (e.g. glutamic acid and aspartic acid), lactic acid, citric acid and acetic acid are taken. Especially, the use of hydrochloride and salt with DL- or L-u-pyrrolidonecarboxylic acid are suitable from a point of view of crystalline nature.

Representative examples of mono-N-higher aliphatic acyl basic amino acid drerivatives are the following N- cocoyl*-N",N-dimethyl-a,-y-diaminobutyric acid methyl ester hydrochloride, N-lauroyl-L-ornithine methyl ester hydrochloride, N"-lauroyl-N ,N"-dimethylornithine benzyl ester hydrochloride, N -palmitoyl-N,N",-N-trimethylornithine methyl ester hydrochloride, N-lauroylbenzyl-N- methyl-ornithine methyl ester hydrochloride, N"-cocoyl*- N-methylornithine methyl ester hydrochloride, N-palmitoyl-N",N-dimethylornithine benzyl ester hydrochloride, N-lauroy1-N-benzyl-N' -methyl ornithine methyl ester hydrochloride, N ,N-dimethyl-N -hydrogenated tallowyl** ornithine ethyl ester hydrochloride, N -lauroylcitrulline methyl ester pyrrolidonecarboxylate, Nlauroy1lysine methyl ester hydrochloride, N-myristoyllysine methyl ester hydrochloride, N-myristoyllysine amide hydrochloride, N"-palmitoyl-N,N -dimethyllysine methyl ester hydrochloride, N -lauroyl-N-benzyl-N'-methyllysine methyl ester hydrochloride, N-cocoyl* arginine ethyl ester pyrrolidonecarboxylate, N"-cocoyl* arginine methyl ester pyrrolidonecarboxylate, N-1auroylarginine methyl ester orpyrrolidonecarboxylate, N"-lauroylarginine methyl ester hydrochloride, N-stearoylarginine methyl ester hydrochloride, N-hydrogenated tall0wyl** arginine methyl ester hydrochloride, N-semihydrogenated tallowyl** arginine butyl ester hydrochloride, N-lauroylhistidine methyl ester hydrochloride and so on. The abbreviations used herein are as follows: cocoyl*, coconut oil fatty acid residue; hydrogenated tallowyl* hydrogenated beef tallow fatty acid residue; semihydrogenated tallowyl***, semihydrogenated beef tallow fatty acid residue. Of course, the compounds which may be employed in the present invention are not limited thereto.

The first characteristic feature is a fact that the mono- N-higher aliphatic acyl basic amino acid derivatives show a good inhibitory effect against microorganisms which possess relatively strong resistance not only to gram positive bacterium, such as Staphylococcus aureus and Bacillus subtilis, but also to gram negative bacterium, such as Pseudomonws aeruginosa, Escherichia coli and Proteus vulgwris. The bactericidal or inhibitory effect of the compounds of the present invention in comparison with that of Hyamine (product of Rohm & Haas Co., Inc.) and hexachlorophene are illustrated in the Table l. The phenol coeflicient of N-cocoyl-L-arginic ethyl ester pyrrolidonecarboxylate shows high value. For example, that phenol coeflicient toward Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus is 170, 85 and 250 respectively.

TABLE 1 Microorganism Compound 1 2 3 4 5 6 7 The present invention: N "-cocoyl-L-arginine ethyl ester pyrrohdone carboxylate 75-100 150-200 -100 50-100 400-500 100-150 2000-2300 N -cocoyl-L-arglnine methyl ester pyrrolidone carboxylate -100 150-200 50-100 50-100 400-500 -500 2,000-3,200 N -lauroyl-L-arginine methyl ester hydrochloride 75-100 150-200 50-100 50-100 400-500 100-150 2,000-2,300 N -lauroyl-L-citrulllne methyl ester pyrrolidone carboxylate 100-150 200-250 50-100 100-150 500-600 150-200 2000-2500 N -lauroly-L-ornithine methyl ester hydrochloride 100-150 150-200 50-100 50-100 400-500 100-150 2,000-2,300 N -lauroyl-L-lysine methyl ester hydrochloride 100-150 200-250 75-150 100-150 500-600 100-150 2000-2500 N -myrlstoyl-L-lysine methyl ester hydrochloride 75-100 150-200 50-100 50-100 400-500 100-150 2,000-2,300 N -myristoyl-L-lyslne amide hydrochlorifle 100-150 200-250 75-150 100-150 500-600 200-300 2,500-2,800 N -mylistoyl-L-histidlne methyl ester hydrochloride; 150-200 200-250 50-100 100-150 500-600 150-200 2,000-2,500 N -palmitoyl-N N -dimethyl-L-ornithine methyl ester hydrochloride -200 300-500 100-200 -200 400-500 200-300 2,500-2,800 The reference example:

Hyamlne (benzethonium chlon'de)--... 50-100 150-200 50-100 25-100 400-500 100-150 800-1,000 G-ll (hexachlorophene) 1,2001,500 1,2001,500 1,000-1,200 250-300 400-500 100-150 800-1,000

Norm-The numerical value in table 1 indicates inhibiting concentration of compound against the growth of microorganism lngy/cc. under contacting of each microorganism in those water solution for ten minutes.

The microorganisms, culture media and preparation of cultured cells employed in the test, and the assay method of bactericidal activity are as follows:

(a) Microorganisms employed:

. Eschericida coli (ATCC 3655) Pseudomonas aeruginosa IAM 1002) Proteus vulgaris (IAM 1025) Staphylococcus aureus (ATCC 6538) Bacillus subtilis (ATCC 6633) Candiad albicans (ATCC 10259) Aspergillus niger (ATCC 9642) (b) Culture media employed:

. Meat extract 1.0%, polypeptone 1.0%, NaCl 0.25% pH 7.0 (used for microorganisms 1-5).

. Yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%, glucose, 1.0%, pH 6.2 (used for microorganisms 6 and 7).

Preparation of cultured cells: Microorganisms 1-6 were cultured statically at at 31 C. for 20 to 24 hr. in test tubes into which are above-mentioned media were poured, while microorganism 7 was cultured at 31 C. for 4 days on the yeast-malt agar slant.

(d) Assay method of bactericidal activity: 0.5 ml. of the above-mentioned cultured cells was inoculated respectively to each test tube in which 10 ml. of sterilized water solution containing each concentration of various compounds was poured. After cells were contacted with the compound, one loopful of cell suspension of microorganismss 1 to 5 was spread on a nitrient-bouillon agar plate, while the yeast-malt agar plate was used in case of microorganisms 6 and 7. After cultivation at 31 C. for 48 hr., living or died of cells was judged.

The second characteristic feature in this invention is the fact that these non-N-higher alpihatic acyl basic amino acid derivatives possess a powerful antibacterial activity against microorganisms which possess relatively strong resistance to various known antiviral compounds, such as Bacillus subtilis, Candida albicans and Aspergillus niger. For example, the bacteristatic or inhibitory effect of the compounds of the present invention in comparison with that of nitrofuran series compound, palmitoyl-L-lysil-L- lysin methyl ester dihydrochloride, streptomycin, penicillin and sorbic acid lauroyl sarcosine are illustrated in the Table 2.

Time for preculti- Num- Culture vation, ber Microorganism conditions hours Medium 1 Escherichea colz 37 0., 3 24 (ATCC 3655). days. 2 Staphylococcus do 24 aureus (ATCC 6538). Nutrient- 3 Bacillus subtilis do 24 bouillon, pH 7.

(ATCC 6633). 4 Pseudomonas 30 0., 3 24 aeruginosa days. IAM 1002). 5 Candida albt'cane 25 0., 3 24 Koji added (ATQC 10259). days. glucose and 6 Aspergtllus mger d0 24 yeast, pH 5.8.

(ATCC 9642).

The third characteristic feature in this invention is the fact that they possess, only a small, or nontoxicity and do not cause any significant skin irritation. For example, in an acute oral toxicity test (LD carried out on mice, the respective LD values of N-lauroyl-L-arginine methyl ester DL-a-pyrrolidone carboxylate and N-cocoyl- L-arginine ethyl ester pyrrolidone carboxylate are 2-3.0 g.kg., 10.75 g./kg. body weight. Accordingly, injuring to men and beasts are scarcely considered. Moreover, after this amino acid derivative was well mixed with polyethylene glycol, it was spread on the cloth of a sticking plaster for a patch test used in the wet state, and then applied to the skin for 24 hours. As the result of this patch test, almost no irritation against the skin Was observed. And since this effective materials are water soluble cationic surface active agents, they have a remarkable detergent effect owing to strong foaming action.

Mono-N-higher aliphatic acyl basic amino acid derivatives or salts thereof can be used in any form of liquid, paste, powder, solid and etc. for the purpose of disinfection and sterilization in food sanitary field, environment public sanitary field, industrial field and farm and gardening field. As not only usual disinfectant agents but also toilet agents, deodorizer, rinsing agents for shampoo, detergents for vegetables and fruits and disinfectant agents for beasts they are also used.

Moreover, as they have strong emulsifying power toward cosmetics and are water-soluble, the solubility in TAB LE 2 Microorganism Compound 1 2 3 4 5 6 1 N -lauroyl-L-lysine methyl ester hydrochloride. 1, 000 1, 000 100-1, 000 100-1, 000 1, 000 1, 000 2 N -lauroyl-L-ornithine methyl 100-1, 000 10 100 10-100 100-1, 000 1, 000 10-100 ester hydrochloride. 3 N -palmitoyl-N -dimethyl-L- 100-1, 000 10-100 10 1, 000 10-100 10-100 ornithine methyl ester hydrochloride. 4 N"-myristoy1-N ,N -dimethyl 1,000 10 10-100 1,000 10-100 10-100 L-lysine methyl ester hydrochloride. 5 N -lauroyl-L-arginine methyl 10-100 10-100 10-100 100-1, 000 10-100 10-100 ester pyrrolidone carboxylate. 6 N -lauroyl-L-citrulline methyl 1, 000 1, 000 1, 000 1, 000 1, 000 1, 000

ester. 7 N -layroyl-L-histidine methyl 100-1, 000 100-1, 000 1, 000 1, 000 1, 000 1, 000

ester hydrochloride. 8 AF-2 10-100 10-100 10-100 1, 000 1, 000 1, 000 9 Palmitoyl L lysil L lysine 10-100 10 10 100-1, 000 1, 000 1, 000

methyl ester dihydroehloride. Streptomycin... 10 100 1, 000 1, 000 1, 000 Lauroyl sarcosine 1, 000 100 100-1, 000 1, 000 1, 000 1, 000 Penicillin 10-100 10 1, 000 1, 000 1, 000 Sorbic aci 1, 000 1, 000 1, 000 1, 000 1, 000

Figures in Tables represent the concentration ('y/ml.) of compounds which causes the growth inhibition of microorganisms.

Microorganisms tested, culture conditions and culture medium are as follows:

water phase, which is easily attacked by microorganisms, is higher than that in oil phase and they show appreciable antiseptic effect even when added in a small amount to cosmetics. And they possess strong penetrating power to a fiber. Accordingly, they may be applied to wide use.

7 The following shows the water solubilities of various mono-N-higher aliphatic acyl basic amino acid derivatives:

Mono-N-higher aliphatic acyl basic amino acid derivatives or salts thereof can be compounded, applied or sprayed according to the shape of soap, synthetic detergent, food, feed, cosmetics, fiber good, leather goods and paint in any form of liquid, powder, or emulsion.

In addition, we want to refer to a few following concrete use of the above-mentioned amino acid derivatives. Namely it was found that antiseptics comprising one or more than two kinds of these esters or salts as an eifective component prevent brewage from hiochi putrefaction rather completely.

The phenomenon of hiochi putrefaction means that sake (Japanese wine), synthetic sake which contains partially brewing alcohol, or the products of brewing alcohol such as mirin (a sweet kind of sake) becomes impossible to eat and drink because of white muddy and rancidity during the storage, or after the bottling.

Generally, hiochi putrefaction is caused by Lactobacillus heterohiochi, Lactobacillus japonicus or Lactobacillus homohiochi which comes from brewage.

The example 9 and 10 indicate the antibacterial activity of N"-lauroyl-L-arginine-methyl ester pyrrolidone carboxylate in comparison with salicylic acid which has been used habitually for prevention of hiochi putrefaction.

Moreover it is confirmed that these compounds adhered to a mucosa of oral cavity and were disinfectant in it for a long time. From these findings these compounds have marked antibacterial activity against both a bacterium belonging to genus Lactobacillus, a main pathogene of dental caries, and a bacterium belonging to genus Staphylococcus, a main pathogene of alveolar pyorrhea.

Table 3 shows the result of antibacterial test of these compounds against genus Lactobacillus, i.e., Lactobacillus fermenti-36 AT CC 9338, and Streptococcus faecalis ATCC 8083, and genus Staphylococcus, Staphylococcus aureus ATCC 65389.

As shown in Table 3, the antibacterial activity of generally used sodium N-lauroyl-sarcosinate was a control.

l The growth of Lactobacillus and Streptococcus was measured after 2% gi tanding culture at 37 0., and Staphylococcus was after 48 hr. a.

Fatty acid residue of coconut oil.

3 DL-Pyrrolidone carboxylic acid.

Figures in Table 3 represent the concentration ('y/ml.) of compounds which causes the fifty percents inhibition of bacterial growth.

It is evident from the Table 3 that any of them is more effective than a control compound.

They possess nearly the same foaming power, which is a desirable surface active action as dentifrice, is the same as a control, sodium-N-lauroyl sarcosinate as shown in Table 4. The foaming power of 0 minute value are illustrated in the method of JISK 3362. As the results, above mentioned each compound possesses many extremely satisfactory properties as combining component of dentifrice.

TABLE 4 Name of compound: Foaming power (mm) N -cocoyl-L-arginine methyl ester PCA salt 190 N-cocoyl-Larginine ethyl ester PCA salt 192 N-lauroyl-L-arginine methyl ester hydrochloride Sodium-N-lauroyl sarcosinate 200 Also, these active compounds are incorporated in soap or other synthetic detergents in 01-50% by weight, preferable 0.1-5% by weight based thereon. As synthetic detergents, for example, detergents containing predominantly at least one of N-higher acyl glutamate, N-higher acyl aspartate, alkylbenzenesulfonate, higher alkylsulfonate, a-olefin sulfonate and alkylethersulfate are taken. The shape of detergent may be bar, tablet, powder, paste or liquid according to the appearance of a product required.

In the appended drawing, FIGS. 1A to 1H diagrammatically illustrate the bacteriostatic effects of N -lauroylarginine methyl ester a-pyrrolidonecarboxylate of this invention against respective strains of microorganisms when added to culture media for the eight strains together with soap;

FIGS. 2A to 2D illustrate the bacteriostatic effects of the compound of FIGS. 1A to 1H in cooperation with a synthetic detergent when added to culture media of the strains illustrated in FIGS. 1A, 1C, 1E, and 1G respectively.

FIGS. 3A to 3C diagrammatically illustrate the bacteriostatic effects of a milled soap containing the same compound of the invention when added to culture media of the strains illustrated in FIGS. 1A, 10, and 1G.

The drawing is explained in more detail in Examples 14, 15, and 1-6 hereinbelow.

EXAMPLE 1 Effect of 0.1% -cocoyl-L-arginine ethyl ester-pyrrolidone earboxylate (CAE-P) on fingers disinfection and washing was examined.

Various kinds and numbers of bacteria were found to exist on the fingers of fifteen persons selected as a panel.

Each member immersed his hands and forearms by 35 cm. length from the tip of the middle finger in a washbowl containing 2 l. of tap water, and then repeated the same thing for a minute in 0.1% N-cocoyl-L-arginine ethyl ester pyrrolidone earboxylate solution. Finally be washed them in 2 l. of sterilized water.

The number of viable cells in these washings was counted after the performance of all members. On the other hand, in another group consisting of fifteen members the same experiment was carried out as control by using tap water instead of 0.1% N -cocoyl-L-arginine ethyl ester pyrrolidone earboxylate solution.

Number of viable cells (in 0.1 ml.)

After washing Before washing With 0.1% CAE-P 9 EXAMPLE 2 The Bath Preparations were prepared by mixing such components as written in the following.

EXAMPLE 3 A Preventive Cleansing agent was prepared by mixing such components as shown in the following table.

Percent by weight N-lauroyl-L-arginine ethyl ester DL-a-pyrrolidone carb oxylate 3 .0 Triethanol amine 2.0 Perfume 0.3

Water 94.7

EXAMPLE 4 The O/W type cold cream was prepared as follows.

Fifty grams of mineral oil, 7 g. of beeswax, 2 g. of tween 40 (a registered trademark), 8 g. of Atlas G-1726 (a registered trademark), were emulsified with 33 ml. of aqueous 1% N-myristoy1-NN-dimethyl-L- lysine hydrochloride at 70 C.

On the other hand, the control cold cream containing 33 ml. of water was also prepared in the same way.

The newly prepared Cream Containing Lysine derivative caused a good emulsion and any change of the quality could not be found on storage at 30 C. and at moisture 90% during two months.

The control cream showed the growth of mold and resulted in coloration.

EXAMPLE 5 The toilet water was prepared as follows.

Ten part of Ethanol, 0.05 part of gum tragacanth, 5 part of propylene glycol, 1 part of N-lauroyl-L-arginine methyl ester-DL-u-pyrrolidone carboxylate were mixed with 85 part of water.

On the other hand, the control toilet water containing polyoxyethylene sorbitan monolaurate instead of arginine derivative was prepared in the same Way.

In the newly prepared toilet water containing arginine derivative any change of the quality could not be found on storage at 30 C. and at the moisture 90% during one month in a room.

The control toilet water showed the growth of mold.

EXAMPLE 6 0.5 g. of N-parmitoyl-NN -dimethyl-L-ornithine amide was mixed with 1 kg. of assorted feed.

In the newly prepared assorted feed containing ornithine derivative any change of the quality could not be found on storage at room temperature and during two weeks.

The control assorted feed showed the growth of mold and a putrid smell.

EXAMPLE 7 0.5 ml. of aqueous 3% N"-lauroyl-L-arginine ethyl ester DL-a-pyrrolidone carboxylate was sprayed per 100 cm? of surface of dressed oxhide.

In the sprayed dressed oxhide any change of the quality could not be found on storage at 30 C. and at moisture 90% during a month.

l 0 The control dressed oxhide showed the growth of mold.

EXAMPLE 8 The grams of N N -dimethyl N"-lauroyl-DL-ornithine ethyl ester was dissolved homogeneously in 500 g. of water emulsion type paints consisting of part of 50% vinyl chloride emulsion, 70 part of titanium white, 10 part of silicic anhydride, part of calcium carbonate, 30 part of aqueous 10% polyvinyl alcohol, 1 part of ammonium polyacrylate, 1 part of tween 40, and 100 part of water.

(i) In the newly prepared paint containing ornithine derivative any change of the quality could not be found on storage at 30 C. and at moisture 90% during three months in a room. The control paint oceured to phase separation by putrefaction.

(ii) Above paint was applied on the cedar board. In the film formed from the newly prepared paint containing ornithine derivative any change of the quality could not be found on storage at 30 C. and at moisture 90% during three months in a room. The control paint film showed the growth of mold.

EXAMPLE 9 (LD orally in rat: 0.9 g./kg.) was used as a control in the example.

TABLE 5 Hioehi-baeteria Lacto- Lacto- Lactobacillusbacillusbacillusheterojaponic'us homohioch" hi ch" n o 1: Concentration 107 50 10 50 10 507 N -lauroyl-L-arginine methylester pyrrolidone carboxylate Salicylic acid 1 Cell growth was not detected. 2 Cell growth was clearly observed.

EXAMPLE l0 Sake A (17% as spirits) added with 0.001% of N-cocoyl-L-arginine ethel ester pyrrolidone carboxylate and Sake B (17% as spirits) added with 0.001% of Na-salicylate were stored at 30 C.:2 C., and the flavor and taste were compared each other.

Sake A did not turn sour at all even after six months, while Sake B began to turn sour virtually after a month.

EXAMPLE ll Kamoboko was prepared according to a known method by employing the following components; 70.0% of frozen fish meat, 2.6% of sodium chloride, 0.3% of HI-ME (Product of Ajinomoto Co., Inc., Mixture of sodium inosinate and sodium glutamate), 5.5% of egg albumin, 2.1% of sucrose, 13.8% of starch and 7.0% of cold water.

Kamaboko A added with 0.012% of L-citrulline methyl ester hydrochloride together with 0.012% of AF-2 (Veno Pharmaceutical Co., Nitrofuran compounds) and Kamaboko B added with only 0.012% of AF-2 were stored at 30 C. for 3 days in a petri dish with a cover. The result was that colonies of fungi conceived as Penicillium occurred on the Kamaboko B. However, any spoilage with bacterial slime or infection with fungi was not detected on the Kamaboko A in addition to no change in taste.

1! EXAMPLE 12 The tooth paste were prepared by mixing such components as written in the following. Percent by Weight Dicalcium phosphate 2H O 44.5 Gum tragancanth 2.0 Glycerol 18.5 N -cocoyl-L-arginine ethyl ester DL-ix-pyrrolidone carboxylate 3.0 Saccharin 0.4 Flavoring material 1.0 Water 30.6

EXAMPLE 13 The wet dentifrice was prepared by mixing such components as shown in the following table.

Percent by weight Calcium carbonate precipitate 70.2

Sodium carboxymethyl cellulose 22.0

N-lauroyl-L-arginine methyl ester DLu-pyrrolidone carboxylate 3.0

Saccharin 0.5

Flavoring material 1.3

Water 3.0

EXAMPLE 14 N -Lauroyl L arginine methyl ester DL-oc-pyrrolidone-carboxylate (referred to as DL-I) was added to 2.0 g. of fatty acid soap material in an amount given in Table 6 and the mixture was dissolved in 100 ml. of the culture medium shown in Table 7, and then was adjusted to a pH of between 6.2 and 7.3. 4 ml. of the respective solutions was inoculated with each of the eight kinds of microorganisms shown in Table 2 and cultivated under the condition shown in Table 2. The growth of microorganisms was estimated by increased turbidity of the culture solution and was verified by measuring at times absorbance (optical density) at 560 m with a spectrophotometer. The results were as shown in FIG. 1 from which the culture mediums (Nos. 4 and 5) added with DL-I were recognized to exhibit marked inhibitory eifect to the growth of microorganisms as compared with the culture medium alone (No. 1) and with the culture medium (No. 2) added with fatty acid soap material alone, and also their inhibitory effect was recognized to be approximately equal with that of hexachlorophene.

TABLE 6 Amount of soap Type and material amount of Number (g deodorizer None None.

2. 0 Do. 2. 0 (3-11 1 0.041 g.

2 0 DL-I 0.039 g. 2 0 DL-I 0.025 g. 1 (3-11: hexachlorophene.

TABLE 7 Cultivation pH of tempera- Culture culture Microorganism 1 ture, 0. medium 1 medium Cultivation A. Lactabaciillug3C 37 1 7.2-7.3

casei T 7409. flggg B. Lg ecigqligzlilus 37 1 7.2-7.3 cultivation ATCC 0338. C. Staphylococcus 30 2 7. 0-7. 2

aureus, ATC'C D. Escherichia coli, 30 2 7. c-v. 2

ATCC 10798. Shaking E. Pseudomonas 30 2 7. 0-7. 2 liquid aeruginosa, cultivation. ATCC 1263. F. Bacillus sub- 30 2 7.0-7.2

tilts. ATCC 6633. G. Candidcgz ih-O 30 3 6.2

0071.8, 10259. fi g g H. Aspergtllus so a 6.2 mjltivafiom niger, AT 0 G 9642.

1 Eight kinds of microorganisms are respectively referred to as A-H microorganisms hereinafter.

1 Culture me um:

TABLE 7Continued Amount of the Composition Grams final solution Peptone 1. 0 Malt extract 0.5 Yeast extract- 0. 5 Beef extract 0. 2

1 Casamino acids- 0. 5 100 ml. of aqueous Glucose 0. 2 solution. Glycerin 0. 8 Magnesium sulfate. 0. 1 Tween Beef extract" 1. 0

2 Peptone 1. 0 Do Sodium chloride 0.3 Yeast extract. 0. 3

3 Malt extract-.-" 0.3 Do.

Peptone 1. 0 Glucose 1. 0

1 1 drop.

EXAMPLE 15 N -Lauroyl L (or DL) arginine methyl ester L- a-pyrrolidone-carboxylate (referred to as L-I or DL-I respectively) was added to 2 g. of monosodium N-cocoyl- L-(or DL)-glutamate (referred to as L-II or DL-II respectively) in an amount given in Table 8. Subsequently, inhibitory tests against various microorganisms were carried out according to the similar manner in Example 14.

TABLE 8 Type and amount of Grams deodorizer Control:

1 Soap material 2 None. 2--. DL-II 2 D0. 3 DL-II 2 G-ll 1 0.041 g. The present t' 3??? DL-II 2 L-l 0.040 g.

5 L-II 2 DL-I 0.224 g.

l G-ll: hexachlorophene.

EXAMPLE 16 2 Parts of N-lauroyl DL arginine methyl ester L-apyrrolidone carboxylate (referred to as DL-I) was sufficiently compounded with parts of soap material with a small roller. The mixture was warmed to 50-60 C. and extruded into a bar of soap having 25 mm. in diameter with a prodder for making soap and then moulded in a metal mould installed to an impact type moulding machine. The appearance of the bar soap thus prepared was equal with that of the bar soap prepared similarly from soap material alone. Also, there is recognized no diiference between both the products in solubility by rubbing which was measured according to the Japanese Industrial Standards M-3304, and in disintegration in water.

Next, 2.0 g. of each of samples was scraped from the respective products, i.e., the bar soap added with DL-I and one added with no DL-I, and dissolved respectively in 100 ml. of culture solution as in the Example 14. 4 ml. of the solution was inoculated with the microorganisms shown in Table 2 and cultivated under the same conditions. The growth of microorganisms was estimated from the turbidity of the culture solution, which was measured using a spectrophotometer according to the similar manner as in Example 14.

As the result, the culture medium added with DL-I was recognized to exhibit an inhibitory effect against all of the A-H strains. Only the results of inhibitory test to the 13 A, C and G strains were shown in FIG. 3 wherein a dotted line means the case where DL- I was added and a line means the case where DL-I Was not added. It may be seen that the soap containing DL-I is elfective in inhibiting the growth of microorganisms as compared with the soap not containing DL-I and especially it exhibits a marked inhibitory eflect against A microorganism.

EXAMPLE 17 10 Parts of sodium sulfate was mixed with 90 parts of sodium dodecyl sulfate (referred to as SDS) and N- lauroyl-N, 'N-dimethyl-DL-ornithine methyl ester lactate (referred to as DL-II'I) was added thereto in an amount given in Table 9 to prepare shampoo composition. Each sample was dissolved in 100 ml. of the culture medium shown in Table 6 and 4 ml. of the solution was inoculated with each one of A-H microorganisms and cultivated. Subsequently, the growth of microorganisms was measured according to the similar manner as in Example 1. As the results, in case of the culture medium (No. 1) not added with DL-HI, symptom of the growth of microorganisms was recognized in one hour after cultivation, while the culture medium (Nos. 4 and added-,with DL-III was recognized to inhibit the growth of microorganisms for -25 hours after cultivation. This was approximately equal to the inhibitory effect of the culture medium (No. 2) added with hexachlorophene.

TABLE 9 Type and Amount Amount of amount to Number of SDS, g. NazS04, g. deodorizer 1. 8 0.2 None. 1.8 0.2 G-11 1 0.040 g. 1.8 0.2 DL-III0.041g. 1.8 0. 2 DL-I1I0.225g.

1 G-ll: bexachlorophene.

EXAMPLE 18 5 Parts of each of the eleven 'kinds of mono N-higher aliphatic acyl basic amino acid derivatives shown in Table 10 was sutficiently compounded together with 100 parts of soap material and with 1-3 parts of water using a roller. The mixture was warmed to 50-55 C. and extended into a bar soap having 25 mm. in diameter using a prodder, and the bar soap was stamped.

The result of having measured the solubility by rubbing according to the Japanese Industrial Standards K-3304 on the bar soap prepared in the above and the bar soap similarly prepared from soap material alone showed that any of the bar soaps containing the compounds of the present invention has a value within 20% from the measured value of the bar soap of control. Also, there was no appreciable difierence between both the products in disintegration in water.

TABLE 10 1. N-Myristoyl-L-lysine methyl ester hydrochloride 2. -Myristoyl-L-lysine amide hydrochloride 3 N-Palmitoyl N,N dimethyl-Irlysine methyl ester hydrochloride N Lauroyl-N;N ,N"-trimethyl-DL-ornithine methyl ester hydrochloride N-cocoyl-N"-methyl-DI.-ornithine methyl ester hydrochloride N -Palmitoyl-N",N"-dimethy1 DL ornithine benzylester hydrochloride N"-Lauroyl-N-benzyl-N -methyl-DL-ornithine methyl ester hydrochloride 8. N-cocoyl-N",N"-dimethyl DL a,'y-diaminobutyric acid methyl ester hydrochloride N,N"-dimethyl-N -hydrogenated tallowyl DL ornithine ethyl ester hydrochloride N-hydrogenated tallowyl arginine methyl ester L-apyrrolidone carboxylate 11. -semihydrogenated tallowyl arginine butyl ester hydrochloride 2 g. of the each sample was scraped from the respective bar soaps containing the above compounds and the bar soap for control, and dissolved in ml. of culture mediums shown in Table 6. 4 m1. of the each solution was inoculated with each microorganism of A and E microorganisms and cultivated. The growth of the microorganisms was estimated from the turbidity of the culture solution according to Example 14. As the result of carrying out the standing cultivation for 22 hours with respect to A microorganism, the absorbance of the culture solution added with the bar soap for control in 10 hours after cultivation was 0.80, while the absorbance of any culture solutions added with the bar soaps containing the additives in the that time was below 0.10. Also, as the result of carrying out the shaking cultivation for 12 hours with respect to E microorganism, the absorbance of the culture solution added with the bar soap for control in 8-10 hours after cultivation was 0.20, while that of any culture solutions added with the bar soaps containing the additives in that time was 0.05. As apparent from these results. Mono-N-higher aliphatic acyl basic amino acid derivatives were recognized to exhibit deodorant eflFect.

SUPPLEMENTAL EXAMPLE (i) Preparation of N "-lauroyl-L-lysine methyl ester hydrochloride 66.2 g. (0.2 m.) of N-carbobenzoyl-I .-lysine methyl ester hydrochloride were dissolved in 200 ml. of tetrahydrofuran and then added with 58.5 ml. (0.42 m.) of triethylamine and 43.7 g. (0.2 m.) of lauroyl chloride under stirring and cooling. The resulted mixture was allowed to stand overnight at room temperature. The resulting reaction mixture was neutralized with 800 ml. of 1N HCl.

The precipitate was filtered and it was dissolved in 400 ml. of ethanol. The ethanol solution was added slowly into 800 ml. of aqueous ammonia under cooled to 0 C. The precipitate of separates out was filtered and dried.

The thus obtained 59.8 g. of N-lauryl-N -carbobenzoyl- L-lysine methyl ester were dissolved in 600 m1. of ethanol and then with 12.7 ml. of cone. hydrochloric acid and 23 g. of 10% palladium-carbon. Thereafter, the reaction solution was reduced by hydrogen under stirring and atmospheric pressure. The catalyst was filtered off. The reaction mixture concentrated under reduced pressure to remove solvent and water completely. The residue were dissolved in methanol and then added with ethyl acetate. Whereby white crystals were precipitated and filtered out. The crude crystals were recrystallized from ethanol and ethyl acetate to obtain 13.3 g. of the purified product of N- lauroyl-L-lysine methyl ester hydrochloride. Yield: 88.0%, m.p. 9698 C.

(ii) Preparation of N-cocoyl-L-arginine ethyl ester DL-u-pyrrolidone carboxylate 35.0 g. (0.2 m.) of L-arginine were dissolved in 200 ml. of acetone and ml. of water and then added dropwise under cooled to 10-20 C., stirring and adjusting to pH 11.5-12.0 with 8N sodium hydroxide and 40 g. (0.18 m.) of cocoyl chloride (coconut oil fatty acid chloride). The reaction mixture was neutralized with 6 N HCl to pH 5.0 and it was added into 300 ml. of cold water.

The precipitate of separates out, was filtered and dried, 0.5 g. of crude crystalline Na-cOc0yl-L-a1'ginine was obtained. Yield: 77.9%, m.p. 230-235 C.

35.6 g. (0.1 m.) of above compound was saturated with 200 ml. of ethanol solution containing hydrogen chloride and allowed to stand overnight at room temperature. The insoluble material of resulting reaction mixture in filtered off. The filtrate was concentrated under reduced pressure. The residue was dissolved in 200 n11. of ethyl acetate and then added with triethyl amine under cooling. Organic solvent layer was washed with water and then added with 12.9 g. of DL-a-pyrrolidone carboxylic acid under heating at 30 C. Theerafter, the reaction mixture was filtered to remove a small amount of insoluble material. The filtrate was concentrated under reduced pressure. The residue was recrystallized from ethanol. 12.4 g. of white crystal of Nu-cocoyl-L-arginine ethyl ester DL-a-pyrrolidone carboxylate was obtained. m.p. 181-184 C. (dec.).

What we claim is:

1. A compound which is the lower' alkyl or benzyl ester of an N-monoacyl derivative of arginine or of the N -methyl or N -benzyl derivative of said arginine, or a water soluble salt of said ester with hydrochloric acid, sulfuric acid, a-pyrrolidonecarboxylic acid, glutamic acid, aspartic acid, lactic acid, citric acid, or acetic acid, said ester having the formula wherein R-CO is the acyl radical of a fatty acid having 6 to 20 arbon atoms, R is hydrogen, methyl or benzyl, and X is lower alkoxy having up to four carbon atoms or benzyloxy.

2. A compound as set forth in claim 1 which is an ester of N-monoacyl arginine or a salt of said ester.

3. A compound as set forth in claim 2, wherein acyl is lauroyl.

4. A compound as set forth in claim 3 wherein said ester is a lower alkyl ester of N-lauroyl arginine.

5. A compound as set forth in claim 4, wherein said compound is a salt of a lower alkyl ester of N-lauroyl arginine with hydrochloric acid or a-pyrrolidonecarboxylic aqid.

6. N-Lauroylarginine ethyl ester a-pyrrolidonecarboxylate.

References Cited GAF, Chem Abs. vol. 58: 11375-77 (1963) Abs of Germ. Pat. 1,139,738.

Virogradova 'et al.: Chem. Abs. vol. 74: 54165d (1970).

JOSEPH A. NARCAVAGE, Primary Examiner US. Cl. X.R.

260-471 A, 482 R, 404.5, 404, 561 A, 309; 424--273, 274, 311

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