N-substituted dimeric cyclopeptide derivatives and preparation thereof

The N-substituted dimeric cyclopeptide derivatives of formula ##STR1## in which A is a peptide residue having one to four amino acid residues; R.sup.1 is lower alkyl, phenyl or pheny(lower) alkylene; R.sup.2 is lower alkyl, cyclo(lower)alkyl or lower alkoxycarbonyl(lower)alkylene; R.sup.3 is a neutral amino acid side chain and a method for the preparation of the compounds of formula I are disclosed. The compounds of formula I are useful for treating microbial infections. Pharmaceutical compositions also are disclosed.

BACKGROUND OF THE INVENTION 
(a) Field of Invention 
The present invention relates to N-substituted dimeric cyclopeptide 
derivatives with antimicrobial activity and to a process for their 
preparation. 
(b) Description of the Prior Art 
A number of cyclic peptides have been either isolated from natural sources 
or prepared by classical synthetic methods, for example, see the review by 
E. Schrodes and K. L. Lubke, "The Peptides"; Vol. II; Academic Press, New 
York, 1966, pp 424-478. 
The present invention discloses novel dimeric cyclopeptide derivatives in 
which two nitrogen atoms of the peptide back bone are substituted. These 
compounds have been found to have the desirable attributes of useful 
antimicrobial activity coupled with a low order of toxicity. 
In addition, a novel process for preparing N-substituted dimeric 
cyclopeptide derivatives is disclosed. 
SUMMARY OF THE INVENTION 
The N-substituted dimeric cyclopeptide derivatives of this invention are 
represented as the compounds of formula I 
##STR2## 
in which A is a peptide residue having one to four neutral amino acid 
residues; R.sup.1 is lower alkyl, phenyl or phenyl(lower)alkylene; R.sup.2 
is lower alkyl, cyclo(lower)- alkyl or lower 
alkoxycarbonyl(lower)alkylene; and R.sup.3 is a neutral amino acid side 
chain. 
A preferred group of N-substituted dimeric cyclopeptide derivatives of this 
invention are represented by formula I in which A is a peptide residue 
having one to four neutral amino acid residues wherein said neutral amino 
acid residues have a neutral side chain selected from hydrogen or lower 
alkyl; R.sup.1 is lower alkyl, phenyl or phenyl(lower)alkylene; R.sup.2 is 
lower alkyl, cyclo(lower)alkyl or lower alkoxycarbonyl(lower)alkylene; and 
R.sup.3 is a neutral amino acid side chain selected from hydrogen or lower 
alkyl. 
Another preferred group of N-substituted dimeric cyclopeptide derivatives 
of this invention are represented by formula I in which A is a peptide 
residue having two neutral amino acid residues wherein said neutral amino 
acid residues have a neutral side chain selected from hydrogen or lower 
alkyl; R.sup.1 is lower alkyl, phenyl or phenyl(lower)alkylene; R.sup.2 is 
lower alkyl, cyclo(lower)alkyl or lower alkoxycarbonyl(lower)alkylene; and 
R.sup.3 is a neutral amino acid side chain selected from hydrogen or lower 
alkyl. 
Still another preferred group of N-substituted dimeric cyclopeptide 
derivatives of this invention are represented by formula I in which A is 
Gly-Gly; R.sup.1 is lower alkyl, phenyl or phenyl(lower)alkylene; R.sup.2 
is lower alkyl, cyclo(lower)alkyl or lower alkoxycarbonyl(lower)alkylene; 
and R.sup.3 is hydrogen. 
The compounds of formula 1 in which A, R.sup.1, R.sup.2 and R.sup.3 are as 
defined herein are prepared by a process which comprises condensing 
together an aldehyde of formula R.sup.1 CHO in which R.sup.1 is as defined 
herein, an isonitrile of formula R.sup.2 NC in which R.sup.2 is as defined 
herein and a peptide of formula H.sub.2 N--CH(R.sup.3)--CO--A--OH in which 
A and R.sup.3 are as defined herein. 
The compounds of formula I form a pharmaceutical composition which 
comprises a compound of formula I and a pharmaceutically acceptable 
carrier. 
The compounds of formula I are useful treating microbial infections in a 
mammal by administering to the mammal an antimicrobial effective amount of 
a compound of formula I. 
DETAILS OF THE INVENTION 
The term "lower alkyl" as used herein means straight chain alkyl radicals 
containing from one to six carbon atoms and branched chain alkyl radicals 
containing three or four carbon atoms and includes methyl, ethyl, propyl, 
isopropyl, butyl, isobutyl, pentyl, hexyl and the like. 
The term "lower alkoxy" as used herein means straight chain alkoxy radicals 
containing from one to six carbon atoms and branched chain alkoxy radicals 
containing three or four carbon atoms and includes methoxy, ethoxy, 
isopropoxy, butoxy, hexanoxy and the like. 
The term "cyclo(lower)alkyl" as used herein means saturated cyclic 
hydrocarbon radicals containing from three to six carbon atoms and 
includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 
The term "(lower)alkylene" as used herein means a divalent organic radical 
derived from both straight and branched chain aliphatic hydrocarbons 
containing from one to six carbon atoms by removal of two hydrogen atoms 
and includes methylene, ethylene, butylene, 2-methyl-propylene, and the 
like. 
The term "organic proton acceptor" as used herein includes triethylamine, 
N-ethylmorpholine, N-ethyldiisopropylamine and the like. 
The terms "amino acid" and "amino acid residue" as used herein means the 
common amino acids and amino acid residues having a neutral side chain and 
includes alanine, asparagine, cysteine, glycine, tryptophan, methionine, 
serine, tyrosine, valine, leucine, phenylalanine, isoleucine, proline, 
threonine and the like. The preferred amino acids and amino acid residues 
are selected from the common amino acids and amino acid residues having a 
neutral side chain selected from hydrogen or lower alkyl, and includes 
glycine, alanine, valine, leucine, isoleucine and the like. 
In general the abbreviations used herein for designating the amino acids 
and the protective groups are based on recommendations of the IU-IUB 
Commission on Biochemical Nomenclature, see Biochemistry, 11, 1732(1972). 
For instance Ala, Leu and Gly represent "residue" of L-alanine, L-leucine 
and glycine, respectively. The term "residue" means a radical derived from 
the corresponding L-amino acid by eliminating the hydroxy portion of the 
carboxy group and a hydrogen of the .alpha.-amino group. The term "amino 
acid side chain" is that part of a common neutral amino acid exclusive of 
the --CH(NH.sub.2)COOH portion, as defined by K. D. Kopple, "Peptides and 
Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 
and 33. Examples of neutral amino acid side chains are --CH.sub.2 
CH(CH.sub.3).sub.2 (the side chain of leucine), --H(glycine), --CH.sub.3 
(alanine), --CH.sub.2 CONH.sub.2 (asparagine), --CH.sub.2 SH(cysteine), 
3-indolylmethylene(tryptophan), --CH.sub.2 CH.sub.2 SCH.sub.3 
(methionine), --CH.sub.2 OH(serine), 4-hydroxybenzyl(tyrosine), 
--CH(CH.sub.3).sub.2 (valine), benzyl(phenylalanine), 
--CH(CH.sub.3)C.sub.2 H.sub.5 (isoleucine), --CH(OH)CH.sub.3 (threonine) 
and the like. Note, therefore, that the term "amino acid side chain" 
includes hydrogen. The preferred neutral amino acid side chains are 
selected from hydrogen or lower alkyl and includes --H(the side chain of 
glycine), --CH.sub.3 (alanine), --CH(CH.sub.3).sub.2 (valine), --CH.sub.2 
CH(CH.sub.3).sub.2 (leucine), --CH(CH.sub.3)C.sub.2 H.sub.5 (isoleucine) 
and the like. 
The amino acids and amino acid residues are all of the L configuration. It 
will be noted that the structures of the compounds of this invention 
include asymmetric carbon atoms. It is to be understood accordingly that 
the isomers arising from such asymmetry are included within the scope of 
this invention. Such isomers are obtained in substantially pure form by 
classical separation techniques and by sterically controlled synthesis and 
have arbitrarily been named as isomers L or M, respectively. 
A number of procedures or techniques for the preparation of peptides have 
hitherto been well established and found in general textbooks of peptide 
chemistry; for example K. D. Kopple, supra, pp. 33-51 and E. Schroder and 
K. L. Lubke, "The Peptides"; Vol. 1; Academic Press, New York, 1965, pp. 
3-128. For instance, the functional groups which are not involved in the 
peptide bond formation reaction are optionally protected by a protecting 
group or groups prior to the condensation reaction. Examples of protecting 
groups for an amino function of a peptide or amino acid not involved in 
the peptide bond formation are: the alkoxycarbonyls which include 
benzyloxycarbonyl (represented by Z), t-butoxycarbonyl (represented by 
Boc), .alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl (represented 
by Ddz), 2-(p-biphenyl)-isopropyloxycarbonyl (represented by Bpoc), 
p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, 
isopropyloxycarbonyl, or ethoxycarbonyl; the acyl type protecting groups 
which include formyl, trifluoroacetyl, phthalyl, acetyl (Ac), or 
toluenesulfonyl; the alkyl type protecting groups which include 
triphenylmethyl or trityl (represented by Trt) or benzyl; the preferred 
protecting groups used in the process of this invention are 
benzyloxycarbonyl, t-butoxycarbonyl, triphenylmethyl and 
.alpha.,.alpha.-dimethyl-3,5-dimethoxy-benzyloxycarbonyl. The carboxylic 
acid function of a peptide or amino acid can be considered protected by a 
lower alkyl or lower aralkyl ester which includes methyl (represented by 
OMe), ethyl (OEt), benzyl (OBzl) or tert-butyl (OBu.sup.t) ester. 
A peptide or amino acid is coupled with another peptide or amino acid to 
form a new peptide by the elimination of water (i.e. dehydrative 
coupling). More specifically, the OH portion of a free carboxyl group of a 
peptide or amino acid and the H portion of a free amino group of a peptide 
or amino acid are eliminated to form a new amide bond joining the peptide 
or amino acid starting materials. To promote facile condensation of a 
peptide free carboxyl group with a free amino group of another peptide to 
form a new peptide bond, the free carboxyl group must be activated. 
Descriptions of such carboxyl activating groups are included in the 
general textbooks of peptide chemistry by Kopple, or Schroder and Lubke, 
cited above. Examples of the activated form of a carboxyl are acid 
chloride, anhydride, azide, imidazolide, activated ester or O-acyl urea of 
a dialkylcarbodiimide (i.e. cyclohexylcarbodiimide). The following 
activated esters have proved to be particularly suitable in the process of 
this invention: 2,4,5-trichlorophenyl (represented by OTcp), 
pentachlorophenyl (OPcp), p-nitrophenyl(ONp), or 1-benzotriazolyl; the 
succinimido derivative also is useful for this purpose. 
The coupling of a peptide or amino acid having the activated carboxyl with 
the peptide or amino acid having a free amino group is conducted in an 
inert organic solvent at a temperature from -30.degree. C. to about 
50.degree. C. For coupling to occur, the amino group must not be 
protonated. A sufficient amount of an organic proton acceptor is added to 
the above reaction mixture until the amino group is no longer protonated 
(usually pH 7.2 to 8.0). 
The terms "peptide, dipeptide, tripeptide, and the like" used herein are 
not limited to refer to the respective parent peptides but also are used 
in reference to modified peptides which are functionalized or having 
protecting groups. The term "peptide" as used herein can be used in 
reference to a peptide with one to ten amino acid residues. 
ANTIMICROBIAL ACTIVITY 
The compounds of formula I exhibit utility as antimicrobial agents against 
a number of microorganisms, for example, bacteria, fungi and protozoa. The 
antibacterial and antifungal activity is demonstrated in standard tests, 
for example in those described in "Antiseptics, Disinfectants, Fungicides 
and Sterilization", G. F. Reddish, Ed., 2nd. ed., Lea and Febiger, 
Philadelphia, 1957 or by D. C. Grove and W. A. Randall in "Assay Methods 
of Antibiotics", Med. Encycl. Inc., [New York] 955. The antiprotozoic 
activity is demonstrated in standard tests, for example, see R. J. 
Schnitzer in "Experimental Chemotherapy," Vol. I. R. J. Schnitzer and F. 
Hawking, Ed., Academic Press, N.Y., 1963. p. 289. 
The compounds are useful as antibacterial agents against pathogenic 
bacteria, for example, Klebsiella pneumoniae and Serratia marcescens and 
as antifungal agents against pathogenic fungi, for example, Candida 
albicans and Microsporum gypseum. In addition, the compounds exhibit 
utility as antiprotozoic agents against parasitic protozoa, for example, 
Trichomonas vaginalis. 
For example, by employing a test like the serial broth dilution, see Grove 
and Randall, cited above, in which dilutions of the compounds of this 
invention in nutrient broth are inoculated with the microorganisms or 
fungi, described above, incubated at 37.degree. C. for 2 days, 
respectively, and examined for the presence of growth, it may be shown 
that the preferred compounds 
N'N'-dicyclohexyl-.alpha.,.alpha.'-diisopropyl-2,5,8,11,14,17-hexaoxo-1,4, 
7,10,13,16-hexaazacyclooctadecane-1,10-diacetamide, isomer L, (Example 1), 
N'N'-dicyclohexyl-.alpha.,.alpha.'-diisopropyl-2,5,8,11,14,17-hexaoxo-1,4, 
7,10,13,16-hexaazacyclooctadecane-1,10-diacetamide, isomer M, (Example 1), 
and 
.alpha.,.alpha.'-diphenyl-N'N'-dicyclohexyl-2,5,8,11,14,17-hexaoxo-1,4,7,1 
0,13,16-hexaazacyclooctadecane-1,10-diacetamide, isomer L, (Example 1) are 
able to inhibit growth totally in this system of Klebsiella pneumoniae and 
Serratia marcescens at a concentration of 100 mcg/ml. 
When the compounds of this invention are employed as antimicrobial agents 
in a mammal they are used administered alone or in combination with 
pharmacologically acceptable carriers. The amount of the compound is 
determined by the solubility and chemical nature of the compound, chosen 
route of administration and standard biological practice. For example, the 
compounds may be administered orally in solid form containing such 
excipients as starch, milk sugar, certain types of clay and so forth. They 
may also be administered orally in the form of solutions or they may be 
injected parenterally. For parenteral administration they may be used in 
the form of a sterile solution containing other solutes, for example, 
enough saline or glucose to make the solution isotonic. 
The dosage of the present therapeutic agents as antimicrobial agents will 
vary with the form of administration and the particular compound chosen. 
Furthermore, it will vary with the particular host under treatment. 
Generally, treatment is initiated with small dosages substantially less 
than the optimum dose of the compound. Thereafter, the dosage is increased 
by small increments until the optimum effect under the circumstances is 
reached. In general, the compounds of this invention are most desirably 
administered at a concentration level that will generally afford 
antimicrobially effective results without causing any harmful or 
deleterious side effects and preferably at a level that is in a range of 
from about 1.0 mg to about 500 mg per kilogram body weight per day, 
although as aforementioned variations will occur. However, a dosage level 
that is in the range of from about 10 mg to about 200 mg per kilogram body 
weight per day is most desirably employed in order to achieve effective 
results. 
In addition, the compounds may be employed topically. For topical 
application they may be formulated in the form of solutions, creams, or 
lotions in pharmaceutically acceptable vehicles containing 0.1-5 percent, 
preferably 2 percent, of the agent and may be administered topically to 
the infected area of the skin. 
Also the antibacterial properties of the compounds of this invention may be 
utilized for washing equipment in hospitals, homes and farms, instruments 
used in medicine and bacteriology, clothing used in bacteriological 
laboratories, and floors, walls and ceiling in rooms in which a background 
free of bacteria is desired. When employed in this manner the compounds of 
this invention may be formulated in a number of compositions comprising 
the active compound and an inert material. In such compositions, while the 
compounds of formula I of this invention may be employed in concentrations 
as low as 500 p.p.m., from a practical point of view, it is desirable to 
use from about 0.10 percent by weight, to about 5 percent by weight or 
more. 
The formulations that may be used for antiseptic wash solutions of the 
compounds of this invention are varied and may readily be prepared by 
standard techniques, see for example, "Remington's Practice of Pharmacy," 
E. W. Martin et al., Eds., 12th ed., Mack Publishing Company, Easton, Pa., 
1961, pp. 1,121-1,150. In general, the compounds may be made up in stock 
solutions. They can also be formulated as suspensions in an aqueous 
vehicle. These make useful mixtures for decontaminating premises. Also, 
aqueous vehicles containing emulsifying agents, such as sodium lauryl 
sulfate, and relatively high concentrations, e.g., up to about 5 percent 
by weight, of the compounds may be formulated by conventional techniques. 
A typical antiseptic preparation useful for disinfecting floors, walls, 
ceiling, and articles in a contaminated room may be prepared by adding 5 
to 25 g of a compound of this invention to a mixture of 150 to 300 g of 
polyethylene glycol 1,540 and 150 to 300 g of polyethylene glycol 300. The 
resulting mixture is stirred while a solution of 1 to 10 g of sodium 
lauryl sulfate in 300 to 400 ml of water is added portionwise. The article 
to be disinfected is coated or immersed in the preparation for a prolonged 
time, for example, one hour, and then rinsed with sterile water. 
PROCESS 
The starting materials required for the preparation of the compounds of 
formula I are aldehydes, isonitriles and peptides. These starting 
materials are either known or commercially available. 
The aldehydes of formula R.sup.1 CHO are known and most are commercially 
available, for example, isobutyraldehyde and benzaldehyde, or are prepared 
by known methods, for example, see P. Karrer, "Organic Chemistry", 2nd. 
ed., Elsevier Publishing Co. Inc., New York, 1946, p. 149. 
The isonitriles of formula R.sup.2 NC, are either known, namely, ethyl 
isocyanoacetate is described by R. Appel et al., Angew. Chem. Int. ed., 10 
132 (1971) or are easily prepared by known methods, for example, by the 
methods described by P. Hoffmann, et al. in "Isonitrile Chemistry", 
Organic Chemistry, Vol. 20, I. Ugi. Ed., Academic Press, New York, 1971, 
p. 9. 
The peptides of formula H.sub.2 N--CH(R.sup.3)--CO--A--OH are either known 
or commercially available, for example, glycyl-glycyl-glycine, or are 
prepared by known methods used in peptide chemistry. 
The compounds of this invention are prepared by the following description 
of a preferred embodiment. 
The practice of the preferred embodiment of the process of this invention 
involves the condensation of the following three starting materials; (1) 
an aldehyde of formula R.sup.1 CHO in which R.sup.1 is lower alkyl, phenyl 
or phenyl(lower)alkylene; (2) an isonitrile of formula R.sup.2 NC in which 
R.sup.2 is lower alkyl, cyclo(lower)alkyl or lower 
alkoxycarbonyl(lower)alkylene and (3) a peptide of formula H.sub.2 
N--CH(R.sup.3)--CO--A--OH in which A is a peptide residue having one to 
four amino acid residues and R.sup.3 is an amino acid side chain to obtain 
the corresponding compound of formula I 
##STR3## 
in which A, R.sup.1, R.sup.2 and R.sup.3 are as defined herein. 
The preferred N-substituted dimeric cyclopeptide derivatives of this 
invention are prepared by condensing the following three starting 
materials: (1) an aldehyde of formula R.sup.1 CHO in which R.sup.1 is as 
defined herein, (2) an isonitrile of formula R.sup.2 NC in which R.sup.2 
is as defined herein and (3) a peptide of formula H.sub.2 
N--CH(R.sup.3)--CO--A--OH in which A is Gly-Gly and R.sup.3 is hydrogen to 
obtain the corresponding compound of formula I in which R.sup.1 and 
R.sup.2 are as defined herein, A is Gly-Gly and R.sup.3 is hydrogen. 
Although not critical, it is preferable to use approximately equimolar 
amounts of the isonitrile of formula R.sup.2 NC and the peptide of formula 
H.sub.2 N--CH(R.sup.3)--CO--A--OH and about one to five molar equivalents, 
preferably two to four molar equivalents, of the aldehyde of formula 
R.sup.1 CHO for this condensation. The condensation is effected most 
conveniently in a dry inert organic solvent, for example, 
dimethylformamide, dimethyl sulfoxide or in an aliphatic alkanol which 
includes methanol, can be selected from methanol and ethylene glycol. 
The temperature and duration of the condensation are also not critical. The 
reaction may be performed at temperatures ranging from -20.degree. to 
100.degree. C.; however, a range from 10.degree. to 40.degree. C. is most 
convenient. The reaction time can be varied, and depends on the reactivity 
of the various starting materials; however, reaction times from one hour 
to several days are employed generally, with ten hours to ten days being 
preferred. 
The above condensation of the three starting materials requires the use of 
dilute solutions to counteract undesirable polymerization. Suitable and 
preferred concentrations of the reaction solution with respect to the 
isonitrile or peptide starting materials can range from 0.1 mmole per ml 
of solvent to 1.0 mmole per ml of solvent. 
Some of the peptides of formula H.sub.2 N--CH(R.sup.3)--CO--A--OH may not 
be sufficiently soluble in the inert organic solvent selected for the 
condensation to give the corresponding compound of formula 1 in sufficient 
yield. A useful method to increase the solubility of the peptide starting 
material is to prepare the acid addition salt of the peptide starting 
material, for example, salts formed with one molar equivalent of 
hydrochloric acid or trifluoroacetic acid. The acid addition salt of the 
peptide starting material is employed in the condensation along with a 
corresponding amount of an organic proton acceptor, for example, 
triethylamine, N-methyl morpholine and the like. 
Thereafter, the compound of formula I is isolated and purified according to 
standard procedures. For instance the product can be precipitated with a 
di(lower)alkyl ether or water and, if needed, purified by chromatography 
and crystallization. 
It should be noted that the product formed is a mixture of two isomers. 
Separation of the isomers can be effected by chromatography. For example, 
chromatography using silica gel as the absorbent has been found to be 
effective for the separation. 
The following examples illustrate further this invention.