CA1076123A - Omega-guanidinoacyl-l-histidines - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A variety of substances are reported which alter host resistance to cocci and bacilli bacterial infections. Never-the less, because of the extreme difficulty of total eradication, and the frequent reappearance of the same strains, even after their apparently successful elimination, there is a continuing need for drugs for the treatment of coccic infections. Certain guanidinoacylhistidines are effective in inducing resistant to infections due to cocci and bacilli. These compounds are omega-guanidinoacyl-L-histidines of the formula

Description

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,.',':, BACKGROUND OF THE INVENTION -This invention per~ains of antimicrobials. In a particular aspect this invention relates to antimicrobials effective in protecting against cocci and bacilli bacterial infections.
8acteria such as cocci and bacilli are a unique group of organisms embodying within themselves an array of yet unanswered puzzles in biology, both fundamental and experimental. It is recognized that the significance of staphyolococcal infections is not so much in severity, except in a few instances, as in the subtleties of the infection due to the unpredictable vagaries of these organisms. The result is the disease continues to be a problem.
Treatment of staphylococcal diseases is complicated by the ability of the organisms to develop resistance. The magnitude of the problem is further amplified by the extreme difficulty of total , ~ eradication, and the frequent reappearance of the same strain even after - -~

-~ apparently successful elimination. The inability to eliminate the ;carrier state by any of the currently known methods and the prevalence of the new antibiotic resistant hospital strains have added a new :: :
dimension to the frustration situation. ~ -PenicilIin G (benzyl penicillin) is still the drug of choice ~for the treatment of infections caused by susceptible cocci strains.

However, numerous strains are known which elaborate an enzyme ,, pencillinase in response to the drug and thus remain insensitive.

This led to the development of semi-synthetic penicillins which are not ~;
activated by penicillinase~ and resistance of staphylococci to these : : .
newer penicillins has been reported. However, there is a seemingly never ending demand for anticoccic agents.

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A variety of substances are repo~ted which alter host resistance to coccic infections. However? because of the ubiquitous nature of cocci and bacilli, and the diversification of their biological and biochemical characteristics ? there is a continuing need for drugs for the trea-tment of these infections.
The existence of multiple antibiotic-resistant strains of the organism suggests the desirability of continuous investigation of drugs for combating the infection. This invention provides an antimicrobial for the treatment of staphylococcic and bacillic infections.

In accordance with this invention it has been found that ~ -selected omega-guanidinoacyl-L-histidines afford desirable degrees : .. . .. .
of protection against cocci and bacilli infections. In fact `

these histidine compounds possess an anticoccic activity superior ~ -'-::: ::: '. : .: ' to that of their corresponding omega-guanidino acids. ~
DETAILED DESCRIPTION OF THE INVENTION ` ~ ~-The processes of infection leading to disease are accepted ~ -to be a problem in the ecology of the parasite. It is being !;,. ; , 20 increasingly realized that the bacterial and host determinants ~.
are closely interrelated. Staphylococcal virulence derives from the combined action of several bacterial factors whose effectiveness is conditioned by the reactions of the host.
Perhaps the most striking feature of host-parasite relationships in staphylococcal infections is the relatively atypical ~
immunologic response. For this reason additional antimicrobials . ~ ;
are always in demand.
By the practice of the invention there is provided an additional method of protecting mammals against bacterial infections. In accordance with the invention an antibacterial amount of certain omega-guanidinoacyl-L-histidines is administered to mammals in need of an antimicrobial effec-tive in protecting ~; , -4- ~:

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against cocci and bacilli. These histidine compounds, admin-istered for the inhibition of bacterial infections, are comp-ounds having the formula (A~ H2N-C(=NH)-NH-(CH2)n-CO-His where n is an integer of 1 through 5, i. e. a whole number less than six, and His is histidine, i, e. alpha-amino-4-imidazole-propionic acid. Included are guanidinoacetyl-L-histidine (n=l), beta-guanidinopropionyl-L-histidine (n=2), gamma-guanidino-butyryl-L-histidine (n=3), delta-guanidinovaleryl-L-histidine -~
(n=4), and epsilon-guanidinohexanoyl-L-histidine (n=5).
The antistaphylococcal activities of the omega-guanidinoacyl-L-histidines will be apparent from the following test results. I-t has been found the amino acids disappear from the blood stream within six hours after their subcutaneous administration, In : - ~
the technique employed herein, therefore, a total 5 mg. of , each drug was given subcutaneously in equally divided doses two ~-.. :
hours before and four hours after the injection of StaphyIococcus aureus.
The strain of S. aureus used in the present investigations and termed "original" strain was isolated from an infected 20 tonsil and has been maintained in our laboratory in the ~ -lyophilized state. It is penicillin-resistant, is highly chromogenic, ferments a number of sugars, including mannitol, mannose, maltose, lactose, galactose, glucose ? and Pruc-tose, and produces coagulase, catalase, gelatinase, deoxyribonuclease, phosphatase, urease, and alpha-toxin. -Culture conditions were standardized, and the third subculture form the lyophilized mother culture was used. The ~-subcultures were grown at 37 C. for 24 hours on Staphylococcus Medium 110 (Difco). The organisms from the third subculture were twice washed and suspended in TC Tyrode Solution (Difco), and the concentration was adjusted turbidimetrically, with a nephelometer, for injection in-to animals. The transmi-ttance .
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levels on the scale of the instrument were taken as a ~e~erence' ,' of the density of the suspensions and were correlated with - ~
viable bacterial counts. Animals were inoculated subcutaneously ' '- ' ., -: .
with 0.5 ml. of a suspension having 70% transmittance or ,~-' ,,,,'

2 X 108 organisms by count. This dosage was approximately 1.5 ,~'; --times the LD50 ;~
Swiss albino female mice maintained on the Rockland diet, ',~
ranging in age from 8 to 10 weeks and in weight from 20 to 25 grams were used in all tests. All mice were randomized for 10 individual experiments. These mice were propagated in our 1~' ,-, laboratory from stock originally obtained from Texas Inbred Mice Co., Houston, Texas. ' - ' The antis-taphylococcal effects of the omega-guanidinoacyl- ',' '~
L-hlstidine will now be given in tabular form. In the table the '~
compound number is the value of n in formula A given hereinbefore. ,' '' ;' These are compared with L-Arginine and gamma-aminobutyryl-L-his- ' ' ', tldine (homocarnosine). Percent protection is (mortality , ~'--' control-mortality treated) X 100/(mortality control) on the , , ' , fourth day after infection with S'ta`phylococcus aureus. '' ~ , 20 - - ~ Antistaphylococcal Activity of ' ;~'' -Omega-guanidinoacyl-L-histidines .. , , " ~ .. . .
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No. of Percent Protection Comp'ound Animals MeanS'td.' D'ev'iation ;
~, : , 1 30 45 9 ~, 2 30 58 18 ',' ',

3 30 78 10 .'

4 30 65 4 '~

Gabu-His* 71 6~15 11 Argine 22 6 5 ' . ' *Gamma-aminobutyryl-L-histidine ', '"

The desirable antistaphylococcal activities of the omega- '~'' ,' 30 guanidinoacyl-L-histidines of this invention are apparent from "

the table. Gamtna-guanidinobutyryl-L-histidine and epsilon- '',''''~:"'~'~
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guanidinohexanoyl-L-histidine af~or~ded unexpectedly high protec-' ' "~ ''' -6- '"'~; ' .':' ' ' ' ", ' ' " ' ' ' ' "', '; ' ' ", ' ' `' ',, ''1'' ' "' " ~'' ' ~' '" , " , ' .' ~

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tion, exhibiting an antistaphylococcal activity even greater ' than homocarnosine. Moreover, as can be seen from the following the omega-guanidinoacyl-L-histidines were generally more potent than their corresponding omega-guanidino acids, and always more potent than their corresponding omega-amino acids. i' Comparisons of Guanidino-histidines with Guanidino ' -- ' Acids and Amino Acids Percent Protection '''',':
Compound on 4th Day ;' Mean ' ' Histidine (n-l) 45 -;~
Glycine 12 ' ' Guanidinoacetic Acid 50 '-Histidine (n=2) 58 Beta-Alanine 12 -,-- beta-Guanidinopropionic Acid 32 ~ . . . .
Histidine (n=3) 78' Gaba 32 gamma-Guanidinobutyric Acid 44 ~ ''' Histidine (n=4) 65 Dava 51 delta-Guanidinovaleric Acid 66 ' ' ' Histidine (n=5) 71 ' Eaha 33 ' ' epsilon~Guanidinohexanoic Acid 57 -.. ..
Histidines are those of formula A having n values given in the '' ' '' table; Gaba, gamma-aminobutyric acid; Dava, delta-aminovaleric acid; Eaha, epsilon-aminohexonioc acid. The amino and guanidino acids were available comercially in part. Others were synthesized. TheC~-guanidinoacyl-L-histidines were prepared 'by treating the corresponding ~-aminoacyl-L-histidines with S-ethylisothiourea by the following procedure which is a modification of the one described by Takahashi'et al in Japanese patent 20564 (Chem.'Abs'tr. 60, 2786h'(1964) ). '' ' ~' Example 1 Guanidinoacetyl-L-histidine H2O (n=1) was prepared from ' glycyl-L-histidine and S-ethyliso-thiourea H2SO4. Solu-tions of , - ~ .
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1.50 g. of S-ethylisothiourea-H2SO4 in 5 ml of H2O and 1.74 g. ~ ~
of Gly-His HCl in 3 ml of H2O were adjusted to pH 8--9 with 4 ~`
N NaOH. They were then mixed and kept at room temperature for -a week. The cloudy suspension was filtered and the filtrate ;~
concentrated to dryness in vacuo. The residue, after addition ~ -of a small amount of EtOH, was dried in vacuo. A small amount of H2O was added to dissolve the dry residue and resulting solution was poured onto the column (2.5 cm i.d. X 30 cm ~ -Amberlite CG-120, 200--400 mesh, pyridine form). H2O, l.O M .

10 pyridine, 2.0 M pyridine, and l.O M pyridine--0.5 M NH40H were used as the effluent solutions. Each fraction was tested for ninhydrin, Pauli, and Sakaguchi reactions. The fractions -containing guanidinoacetyl-L-histidine were pooled and con-:,~ :: -centrated to dryness in vacuo. Cryst from H2O--EtOH; yield, ~ ~ -'. ~.
69.2%; mp, 108--111C., decomp. Hydrolysis (6 N HCl, 110, 24 hours.) gave guanidinoacetic acid and histidine, as confirmed i ~ ~ ;

be tlc. (thin layer chromatography).

Example 2 `~

To prepare beta-guanidinopropionyl-L-histidine H2O, (n=2) 20 S-ethylisothiourea H2S04 (5.0 g) and ~5Ala-His t5.3 g) were reacted by the procedure described in Example 1: yield1 38,8%;

mp, 121-124 C., decomp. Hydrolysis as described in Example 1 gave beta-guanidinopropionic acid and histidine (tlc).

ExampIe 3 ~-Guanidinobutyryl-L-histidine H2SO4 (n=3) -- S-.
Ethylisothiourea H2SO4 (10.0 g.) and ~Abu-His (11.0 g.) were ~ ~
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treated as described in Example 1 except for the following changes. For the final crystn, the aq solution was adjusted -to pH 5 with 2 N H2SO4 and abs EtOH was added to form fine white cryst; yield, 51.7%; mp, 119--124q~, decomp. Hydrolisis as described yielded ~-guanidinobutyric acid and histidine (tlc), . .

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" ' .~ . ' "'~ , Example 4 ' -Guanidinovaleryl-L-histidine.H2S04-H2O (n=4~ -- S-Ethylisothiourea H2SO4 (0.8 g.) and ~Avl-His (1.0 g,) were treated in the same way as Example 3; yield, 50.6%; mp, 80--82 C., decomp. Hydrolysis as above yieldedd~-guanidinovaleric acid and histidine (tlc).

Example 5 -Guanidinohexanoyl-L-histidine (n=5) -- S-Ethylisothiourea-H2SO4 (1.4 g.) and ~Ahx-His (1.7 g.) were treated in the~same '~
~, way as Example 2; yield, 56.1%; mp, 102--103 C., decomp. ,-Hydrolysis as above gave ~-guanidinohexanoic acid and histidine (tlc).
The yields and physical and analytical data for -the compounds :
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nificant new antimicrobials. It is contemplated that they will be taken during periods where contact with staphylococci ?
streptococci or Salmonella typhi infections are likely? such ~'~
as on entering and during a hospital exposure to the infection t ,'~,;
The guanidinoacyl-L-histidine can be taken orally in 250 and 500 mg. tablets or as injections of, say, 150 to 500 mg. The W-guanidinoacyl-L-histidine per se or combined with an aqueous, vegetable oil, monoglyceride or diglyceride vehicle for injection can be administered, sodium chloride being used if necessary to render the solution isotonic. The solution ~ . ~
will contain O.l to 15. percent by weight, of the ~-guanidino-,, acyl-L-histidine.
In the case of tablets, the ~-guanidinoacyl-L-histidine ,-can be combined with suitable colorants, adhesives, and lubri-cants, along with a solid pharmaceutical diluent, for instance, starches, lactose, sucrose, and other pharmaceutical diluents. .
These tablets will contain 0.08 percent to 5 weight percent of the 6~-guanidinoacyl-L-histidine, preferably 0.08 percent to 1.3 weight percent. Capsules can also be made.
A process is thus provided for the control of infections in humans and other mammals due to cocci and bacilli, which involves administering to the mammal æuffering from the infection an effective amount of the Cv-guanidinoacyl-L-histidine. In addition other variations and modifications will occur to those skilled in the art. Such ramifica-tions are deemed to ;~
be within the scope of this invention.
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Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1, A process for preparing an omega-guanidinoacyl-L-histidine having the formula wherein n is a whole number of 1 to 5 which comprises reacting a corresponding omega-aminoacyl-L-histidine with S-ethylisothiourea or a salt thereof.

2. An omega-guanidinoacyl-L-histidine having the formula H2N-C(=NH)-NH-(CH2)n-CO-Histidine where n is a whole number of 1 to 5 whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.