New peptides and a process for their preparation

What are disclosed are pentapeptides, useful for influencing the maturing of T-lymphocytes, of the formula EQU G--K--Q--X--M wherein PA1 G is arginine, lysine, ornithine, or homoarginine, or is an unsubstituted or substituted .omega.-aminoalkanoyl, .omega.-guanidinoalkanoyl, or .omega.-dimethylaminoalkanoyl; PA1 K is a basic amino acid such as lysine, arginine, homoarginine, or ornithine; PA1 Q is L- or D-glutamic acid, d-aspartic acid, or D-.alpha.-aminoadipic acid; PA1 X is L-valine or L-isoleucine; and PA1 M is an L- or D-aminoacid having a hydrophobic side chain, or an ester or amide of such an acid.

Several peptides have been isolated from thymus extracts and their 
structure has been elucidated, for example thymosin .alpha..sub.1, 
thymopoietine and the "facteur Thymique serique" (FTS), which inter alia 
contribute to the differentiation ("maturing") of thymus-dependent 
lymphocytes (T cells). 
In the course of investigations into the dependence of this action [which 
is, for example, detectable in vitro in its effect on autologous 
rosette-forming cells from the spleen of mice without thymus analogously 
to the method of Proc. Natl. Acas. Sci. U.S.A., 72 (1975), page 3201] on 
the structure of low-molecular weight peptides, it has now been found that 
suitable peptides which, in accordance with the general properties of 
their units, can be characterized as follows: 
basic-basic-acid-hydrophobic-hydrophobic are all effective in the test 
mentioned and in other tests. 
The invention relates, accordingly, to peptides of the formula 
EQU G--K--Q--X--M 
in which G denotes arginine, lysine, ornithine or homoarginine, in each 
case in the L- or D-configuration or .omega.-amino-alkanoyl, 
.omega.-guanidino-alkanoyl or .omega.-dimethylamino-alkanoyl having 3 to 6 
C atoms and optionally an .alpha.-amino group, in the D- or 
L-configuration, which in turn can carry alkanoyl having 1 to 6 C atoms, 
aroyl having 7 to 11 C atoms, cycloalkanoyl having up to 2 alkyl C atoms 
and 5 to 7 cycloalkyl C atoms, aralkanoyl having up to 9 C atoms 
altogether, wherein a --CH.sub.2 -- group can be replaced by --O-- or 
--S--, alkyloxycarbonyl or aralkyloxycarbonyl having up to 7 C atoms or 
succinoyl, succinamoyl, glutaroyl, glutaminyl, pyroglutamyl, phthaloyl, 
phthalamidyl or 2-carboxybenzoyl; K denotes a basic aminoacid, preferably 
L-lysine, L-arginine, L-homoarginine or L-ornithine; Q denotes L-glutamic 
acid, D-glutamic acid, D-aspartic acid or D-.alpha.-aminoadipic acid; X 
denotes L-valine or L-isoleucine and M denotes an aminoacid having a 
hydrophobic side chain, in the L- or D-configuration, or an ester, amide, 
alkylamide or aralkylamide thereof, the alkylamide or alkyl ester having 1 
to 6 C atoms or the aralkylamide or aralkyl ester having 7 to 10 C atoms. 
The acid function in the characterization shown above is taken care of, in 
accordance with the invention, by L- and/or D-glutamic acid and by 
D-aspartic acid and D-.alpha.-aminoadipic acid; the hydrophobic sector can 
comprise 1 to 2 aminoacids or amides or esters thereof, Q-I/e or Q-Val as 
the central sector imparting a particularly advantageous quality of action 
to the peptides. 
The substituent at the .alpha.-amino group of G and the unit M are not 
critical for the action, but they affect it quantitatively. Thus, 
particularly in G, the alkanoyl radical can be a formyl to hexanoyl 
radical, the aroyl radical can be benzoyl, optionally substituted by 
methyl, methoxy or chlorine, and the aralkanoyl radical can be phenacetyl, 
cinnamoyl, dihydrocinnamoyl, phenoxyacetyl or phenylthioacetyl, and the 
aryl radical can be unsubstituted or substituted by methyl, methoxy or 
chlorine. 
Alkyloxycarbonyl or aralkyloxycarbonyl is preferably ethyloxycarbonyl, 
isobutyloxycarbonyl, tert.-butyloxycarbonyl, benzyloxycarbonyl, 
4-methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl or 
4-chlorobenzyloxycarbonyl. 
The aminoacid which has a hydrophobic side chain and is represented by M 
can be, in particular, Ala, Val, Leu, Ile, Met, Phe, Pro, Tyr, or Phg 
(C-phenylglycine) and can also be an aminoacid having hydrophobic 
substituents, such as Ser(Bu.sup.t), Thr(Bu.sup.t), Cys(Bu.sup.t), 
Cys(Et), Cys(Bzl), Glu(OBu.sup.t), Asp(OBu.sup.t), Glu(NH-Bu.sup.t), 
Glu(NH-Et), Lys(Boc), Orn(Boc), Tyr(Bu.sup.t), Tyr(Me), Phe(Cl) or 
Tyr(Cl), and also tryptophan, substituted by alkyl, halogen or methoxy. 
Possible alkylamides are preferably n-alkyl alkylamides or branched 
alkylamides, such as isopropylamide, isobutylamide, tert.-butylamide, 
3-methylbutylamide or 3-ethylbutylamide; examples of possible 
aralkylamides are benzylamide or phenethylamide, which can be substituted 
by 1 to 2 methyl groups in the nucleus and/or in the side chain. The same 
alkyl, aryl or aralkyl radicals can also be present in the ester linkage. 
The peptides according to the invention have a similarity to a pentapeptide 
Arg-Lys-Asp-Val-Tyr which has recently been described in Science 204 
(1979), page 1309, a part sequence of thymopoietin, which is regarded as 
the sector of this peptide which is supposed to be responsible for its 
biological action. It is interesting that this sequence corresponds to the 
succession basic-basic-acid-hydrophobic-hydrophobic mentioned above, which 
thus appears to be an overriding principle for peptides having the thymus 
activity mentioned. 
In contrast with this peptide, however, the peptides according to the 
invention contain L- or D-glutamic acid and also D-aspartic acid or 
D-.alpha.-aminoadipic acid as the acid aminoacid. Peptides of glutamic 
acid and .alpha.-aminoadipic acid are considerably more stable than 
aspartic acid peptides in a weakly acid medium, in which these compounds 
are mostly used. Aspartic acid peptides undergo a rearrangement via 
aspartimide peptides, mainly into isoasparagine peptides, so that, for 
example, it is not possible to heat-sterilize aspartic acid peptides. 
However, the rearrangement still takes place at a noticeable rate at room 
temperature and even at refrigerator temperature. If Q is represented by 
the acid D-aminoacids, the stability of the peptides towards enzymes is 
increased. 
In addition, the compounds according to the invention also include 
tripeptide and tetrapeptide derivatives, the action of which is in many 
cases the same as, or even greater than a pentapeptide, prepared in 
accordance with the invention, having the sequence Lys-Lys-Glu-Val-Val. 
The invention also relates to a process for the preparation of the said 
peptides, which comprises synthesizing, in accordance with methods of 
peptide syntheses, aminoacid sequences of the formula 
EQU G--K--Q--X--M 
in which G, K, Q, X and M have their earlier meaning. 
The synthesis of the compounds according to the invention follows the known 
methods of peptide chemistry, such as are described in detail, for example 
in Houben-Weyl, Methoden der Organischen Chemie (Methods of Organic 
Chemistry), Volume 15. The examples shown on the following pages 
illustrate the synthetic processes, which are in themselves known. 
The abbreviations customary in peptide chemistry are used, in particular 
the following: 
Boc tert.-butoxycarbonyl 
Z benzyloxycarbonyl 
Adoc adamantyloxycarbonyl 
Me methyl 
Bzl benzyl 
Bu.sup.t tert.-butyl 
Ipr isopropyl 
Tcp 2,4,5-trichlorophenyl 
HONSu N-hydroxysuccinimide 
HOBt 1-hydroxybenzotriazole 
DCC dicyclohexylcarbodiimide 
Glu pyroglutamyl 
TLC also stands for thin layer chromatography and HPLC stands for high 
performance liquid chromatography. In the aminoacid analyses, Glu is taken 
as 100. 
In vitro and in the presence of liver homogenates, the peptides according 
to the invention have a considerably prolonged life, compared with natural 
thymus peptides. The compounds in which Q is represented by an acid 
D-aminoacid, such as D-glutamic acid or D-.alpha.-aminoadipic acid, should 
be singled out particularly. Their action can be demonstrated, for 
example, in vitro by their effect on T-lymphocytes which form SRBC 
rosettes, from the blood of patients deficient in immunity or human 
umbilical cord blood analogously to the methods of J. Exptl- Med. 136 
(1972) page 207; Anm. N.Y. Acad. Sci. 249 (1975) page 308 and Int. Archs. 
Allergy appl. Immun. 53 (1977) page 242, and also their effect on the 
PHA-induced lymphoblast transformation of human and animal lymphocytes 
analogously to the methods of J. Exptl. Med. 131 (1970), page 1049, and 
Cell. Immunol. 16 (1975), page 413. (SRBC=sheep red blood cell; 
PHA=phytohaemagglutinin). 
The compounds according to the invention can be used for treating 
deficiencies of immunity, viral and fungoid, and also chronic bacterial 
infections and autoimmunity diseases, and also for the therapy of 
illnesses caused by cells having immunologically relevant changes in the 
cell membrane characteristics (for example tumor cells). 
In this sense, the invention also relates to the use of the said peptides 
very generally for influencing the maturing of T-lymphocytes.