DE2804566C2 - Arginyl-Lysyl-Aspartyl-Valyl-Tyrosine and its derivatives, process for their preparation and their use - Google Patents

Description

a) the specified pentapeptide sequence from the relevant, optionally functional groups of the Rare chain protected amino acid derivatives via the covalent bond of the tyrosyl residue to an insoluble polymer resin gradually builds up the ester obtained,

b) with the intermediate product obtained in the case of the linking of protected amino acid residues Formula. _ .......

R ¹ R ² R ³ R ⁴

R ⁵ -ARG-LYS-ASP-VAL-TYR resin

wherein R to R ⁵ are conventional protective groups for the respective functional group In the rare chain or a-Amlnogruppe are ¹ of the Arglnlns, either ⁵ alkanoyl Derlvate having 1 to 7 C atoms is prepared in the alkanoyl after previous selective Abspaliung of R and / or carries out the splitting off of the other protective groups and the polymeric carrier and

c) the pentapeptide prepared in this way, if appropriate, into the specified C-terminal derivatives or salts convicted.

3. Use of the compounds according to claim 1 in the differentiation of T lymphocytes, but not of K.complementreceptor (CR ⁺ ) -B lymphocytes.

The invention relates to the pentapeptide mentioned in claim ^ and its derivatives, processes for their Manufacture and its use in the differentiation of T lymphocytes.

Numerous polypeptides are known to have been isolated from various organs of animals. About until However, over the past decade there was very little about the thymus, an organ involved in the human body Birth makes up about 0.8% of his body weight, although previously believed to be one neuromuscular blocking substance was present in the thymus. Despite very strong interest in possible Functions of the thymus and early speculation and experimentation was until recently little about function of the thymus known. We know today, however, that the thymus is a connecting organ with both eplthelial (endocrine) and lymphoid (immunological) connections 1st and in the immunity functions of the body plays a role. The thymus is known to be a composite organ that consists of one of the third branchial arches Eplthellal stroma and lymphocytes derived from stem cells derived from your Originate in hemopoietic tissues; see Goldstein and co-workers "The Human Thymus", Heinemann, London 1969. Lymphocytes are differentiated within the thymus and appear as mature, dated Thymus-derived cells, so-called T cells, which go to the blood, to the lymph gland, to the MIIz and to the lymph nodes circulate. The induction of the differentiation of stem cells within the thymus appears to be through secretions of the thymus eplthelial cells to be triggered, however, prevented difficulties with biological So far, attempting the complete isolation and structural characterization of any hormones that may be present.

To understand the importance of the differentiating biological properties of the polypeptides according to the invention To make it understandable, it should be noted that the function of the thymus in relation to immunity is roughly as Formation of cells or lymphocytes originating from the thymus, so-called T cells, can be indicated. The T cells make up a large proportion of the total amount of circulating small lymphocytes. the T cells exhibit immunological specificity and are directly involved in cell-mediated immune responses (e.g. Homografi-Reaktlonen) involved as nerve endorganzellen (effector cells). However, the T cells do does not produce humoral antibodies as these antibodies are secreted by cells that are directly from the

6fl bone marrow originate regardless of the influence of the thymus. These latter cells are called B cells designated. For many antigens, however, the B cells require the presence of correspondingly reactive T-

Cells before they can make antibodies. The mechanism of this process of cell cooperation Has not yet been fully clarified. On the basis of this explanation it can be stated that, in functional terms, the thymus

necessary for the development of cellular immunity and numerous reactions of humoral antibodies Is and affects these systems. By having the differentiation of hemopoietic stem cells in the thymus to trigger T cells. This inductive influence is mediated by secretions of the epithelial cells of the Thymus, d. H. the thymus hormones exercised.

About the function of the thymus and the cell system of the lymphocytes as well as the circulation of lymphocytes To understand in the body, it must also be pointed out that the stem cells arise in the bone marrow and reach the thymus through the bloodstream. Within the thymus, the stem cells become immunological differentiated competent T cells, which migrate to the bloodstream and along with the B cells between the Tissues, lymph vessels and the bloodstream circulate.

The cells of the body that secrete antibodies also develop from hemopoietic stem cells, however, their differentiation is not determined by the thymus. They are therefore considered to be from the bone marrow termed derived cells or B cells. In birds they become in an organ analogous to the type differentiated, which is called the bursa of Fabriclus. In mammals no equivalent organ was found and it is believed that the B cells differentiate within the bone marrow. The physio- ίο logical substances that dictate this differentiation remain completely unknown.

It has been known for some time that relationships between the thymus and immunity characteristics of the body so that great interest has been shown in substances which have been isolated from the thymus became. In this regard, there have been a relatively large number of articles based on of the scientific work published which concerns substances present in the bovine thymus. Of the A number of articles relating to research in this area have been published to applicants. Relevant publications appeared, for example, in "The Lancet", July 20, 1968, pages 119-122; "Triangle", Vol. 11, No. 1 (1972) 7-14; Annals of the New York Academy of Sciences 183 (1971) 230-240; Cilnlcal and Experimental Immunology 4, No. 2 (1967) 181-189; "Nature" 247 (1974) Π-14; Proceedings of the National Academy of Sciences USA 71 (1974) 1474-1478; "Cell" 5 (1975) 361-365 and 367-370, and "Lancet" 2 (1975) 256-259.

In the publication by Goldstein and Manganaro In "Annals of the New York Academy of Sciences" 183 (1971) 230-240 the presence of a thymic polypeptide is reported, which is a myasthenic induces neuromuscular block in animals analogous to the human disease myasthenia gravls. This article also states that two different effects are caused by separate polypeptides Im Bovine thymus are produced. It was believed that one of these polypeptides, called "thymotoxin" referred to causing myositis, but it was further suggested that this polypeptide is not isolated Although it was found to be a polypeptide of about 7,000 molecular weight, it had become a strong pure had a positive charge and was retained on the ion exchange resin "CM-Sephadex" at pH 8.0.

In the publication In "Nature" 247 (January 4, 1985) 11 are identified as Thymln I and Thymln H Described products that were found to be novel polypeptides isolated from bovine thymus and have specific uses in various therapeutic areas. More similar due to usage Names for other products that had "jerelts been isolated from the thymus" become these products Thymine I. and Thymin II now referred to as Thjmopoletln I and Thymopoletln II. These products and processes for your Manufacture are described in US Pat. No. 20,951.

The US-PS 4002 602 describes long-chain polypeptides, which are known as sublqultäre immunopoetlsche polypeptides (UBiP). This peptide was later renamed Ubiquiiin. This polypeptide is a 74-amino acid polypeptide, which is characterized by its ability. In vitro In concentrations of Nanograms the differentiation of both T-cell and B-cell immunocytes from precursors, which are present in the bone marrow or in the spleen. The polypeptide is thus valuable in the Therapy of deficient function of the thymus or of immunodeficiency.

The US-PS 40 02 740 describes synthesized Trldecapeptldmlschungen that have the ability to differentiate by L-lymphocytes, but not by complementary receptor B-lymphocytes. This Polypeptld thus exhibited many of the properties of the long chain polypeptides produced according to the above US-PS 41 20 951 isolated and referred to as Thymopoletln.

The invention relates to the synthesized pentapeptide according to claim 1 with a very specific one Active site sequence which was found to have many of the properties of the isolated long chain polypeptide, that in the publications mentioned above and in US-PS 41 20 951 as "Thymopoietln" and the Tridecapeptlds described in US-PS 40 C2 740, as well as the claims 1 specified derivatives of this pentapeptide. The pentapeotld and its derivatives are hereinafter referred to as "pentapeptides" designated.

The invention accordingly sets itself the task of creating new pentapeptides which are biologically important and which Have ability. The differentiation of bone marrow cells into T cells in concentrations of nanograms to effect and are therefore extremely valuable in the immune system of humans and animals do.

The invention further relates to methods of making the novel pentapeptides according to the invention as well their use in differentiating T lymphocytes but not complement receptor (CR *) B lymphocytes directed.

The above objects and advantages are achieved according to the invention by new pentapeptides realized, which have the following sequence as an active site: μ

-ARG-LYS-ASP-VAL-TYR-

Is linked in the ARG with R and TYR with R 'according to claim 1.

The invention further relates to methods of making the pentapeptides according to the invention Peptide synthesis in the solid phase and directed to the use of the peptides according to claim 3. In Medicines that contain the pentapeptides and administered to humans and animals are biological Wlrkuneen to exercise on it.

The pentapeptides according to the invention have therapeutic value in various fields.
As a main embodiment, the invention comprises the pentapeptide with the amino acid sequence I as the active site:

I. ARG-LYS-ASP-VAL-TYR

As a further embodiment, the invention is directed to pentapeptides containing the above Have sequence as active site and can be described by the following general formula II:

«° II. R-ARG-LYS-ASP-VAL-TYR-R '

Here, R stands for hydrogen or alkanoyl radicals with 1 to 7 carbon atoms and R 'for a hydroxy radical, Alkoxy radicals with 1 to 7 carbon atoms or an amino radical. Both residues are substituents on the pentapeptide sequence, which do not materially affect the biological activity of the basic active sequence. Under This information is to be understood as modifying the terminal amino acids on this pentapeptide chain can be if functional groups (R and R ') are attached to these terminal amino acids, without significantly affecting the biological activity of the molecule. It must therefore be stated that the terminal amino and carboxylic acid groups for biological activity as is the case with some polypeptides of the Pentapeptide are essential. The scope of the invention therefore includes pentapeptides that are terminal are unsubstituted (R = H; R '= OH) and are terminated by one or more functional groups R and / or R '. which do not significantly affect the biological activity disclosed here are substituted.

This statement is to be understood in such a way that these functional groups take part in a normal process such as acylation on the free amino group and amidation on the free carboxylic acid group. Of these Aspects of the pentapeptides according to the invention prove to be exceptional, since the pentapeptides the

"have the same biological activity as long-chain natural peptides in which this pentapeptide sequence forms a part or is present therein. It is therefore assumed that the activity requirements of the Molecule by stereochemistry of the molecule, i. H. the special "folding" of the molecule can be generated. In In this context it should be noted that polypeptide connections are not rigid, but flexible and in flat shape, as a screw or helix and the like. As a result, the entire molecule is flexible and "folds" in very definite ways. According to the invention it has been found that the pentapeptide is In of the same catfish as the long-chain natural polypeptide "folds" and therefore the same biological properties having. For this reason, the pentapeptide can be substituted with various functional groups as long as the substituents do not significantly impair the biological activity or the natural ones Do not interfere with the "folds" of the molecule.

In a more specific embodiment, the invention encompasses novel Psntapeptides having the following Sequence:

II. R-ARG-LYS-ASP-VAL-TYR-R ' H '^ rln stands for R z. B. for hydrogen or acetyl or proplonyl, and R 'stands for OH or NH ₂ . Particularly

pentapeptides in which R is hydrogen and R 'is OH are preferred.
The pharmacologically acceptable salts of the pentapeptides also fall within the scope of the invention. as

Examples of acids which can form salts with the Pentapeptlden are: inorganic acids, z. B.

Hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thlocyanic acid, sulfuric acid and phosphoric acid, and organic acids, e.g. B. formic acid, acetic acid, propionic acid, glycoic acid, lactic acid, pyruvic acid. Oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranyl acid, cinnamic acid, naphtha

linsulfonic acid and sulfanic acid.
In the above structures, the amino acid components of the peptide are used for convenience

identified by abbreviations: The following abbreviations are used:

·

Amino acid Abbreviated designation

L-arginine AI \ G L-lysine LYS L-aspartic acid ASP L-valine VAL

L-tyrosine TYR

The pentapeptide sequence of the invention is a five amino acid peptide which has been found to have properties similar to the 49 amino acid celiac thymus isolated polypeptide described in Applicants' US Pat. No. 4,120,951. The peptides according to the invention are distinguished in particular by their ability, the selective differentiation of Thy-1 * T cells (but not CR * ¹ ^ -B-cells) in concentrations of 1 ng to 10 ug / ml trigger. Thy-1 is a differential alloantigen present on T cells but not on B cells, while CR is a complement receptor present on B cells but not on T cells.

Investigations of these synthetic peptides in the induction test in vitro showed that they have the same induction

have special fluids such as thymopolyin II, d. H. they caused the differentiation of ΤΗΥ-Γ cells to Thy-I * - T cells, but not the differentiation of CR ~ cells into CR * cells. True, were numerous substances Identified that thymopolyols can mimic in vitro and increase T-cell termination by enhancing them of the intracellular cyclic AMP, but it should be emphasized that only a few substances in such low concentrations are effective; the peptides according to the invention selectively effect the difference in reduction from T cells but not from CR * B cells. It has also been shown that these are synthetic Peptides also affect neuromuscular transmission like thymopolytes themselves. That was 24 hours after a single injection of 10 to 100 mg / mouse there was a marked impairment of the neuromuscular Transmission detectable by electromyograph.

On the basis of these properties, the pentapeptides according to the invention are therapeutically valuable for the treatment of humans and animals, since they have the ability to trigger the differentiation of the lymphopoietic stem cells formed in the hemopoietic tissues into cells or T cells derived from the thymus able to interfere with the immune response of the body. As a result, find the products gpnyäß the invention multiple therapeutic applications. Since the compounds have the ability to exercise some c ^ r mentioned functions of the thymus, they found primarily in various Thymusfunktions- ^ι ς and immunity areas Application. A primary field of application is the treatment of DlGeorge syndrome, a condition in which the lack of the thymus is congenital. This deficiency is overcome by injection of the pentapeptides. Due to their biological properties, the pentapeptides, which are more active with low KotUcuiraiionen: active are therefore valuable that they support the collective immunity of the body by increasing or supporting the therapeutic stimulation of cellular immunity and thereby become valuable for the treatment of diseases in which chronic infections in vivo, e.g. B. fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and the like. Occur. Furthermore, the peptides are believed to be of value in any area where cellular immunity is controversial. Especially when there is a lack of immunity! as present, for example, in the above-mentioned DlGeorge syndrome. Furthermore, if there is excessive antibody formation due to an imbalance of 2> T cells and B cells, the compounds can correct this condition by stimulating the formation of T cells. They can thus be of therapeutic value in certain autoimmune diseases in which harmful antibodies are present, e.g. B. in systemlsc'ism lupus erythematosus. Due to the properties of the pentapeptides, they are also valuable in vitro by dissolving the development of surface antigens of T cells, by triggering the development of functional ability, sensitivity to mltogens and antigens, and by helping the cells to increase the ability of B cells To produce antibodies. The pentapeptides are also valuable in that they inhibit the uncontrolled proliferation of lyniphocytes that respond to thymopoletin.

An important property of the pentapeptides is their ability in vivo. Cells with the properties of Restore T cells. The Pentapeptlde according to the invention are therefore as a result of their ability to Boosting the body's immune response is effective in numerous areas. Since they are neuromuscular transmission affect, very high doses of the peptides according to the invention are valuable for treatment of diseases with excessive neuromuscular transmission, e.g. B. Spastlzltät.

Another important property of the peptides according to the invention is that they are at very low Concentrations of 1 ng / ml are effective and maximal activity at concentrations of about 100 ng demonstrate. Suitable carriers are all carriers known for this purpose, including normal saline solutions, preferably with a protein as a diluent, e.g. B. bovine serum albumin to reduce losses To prevent adsorption on glassware at these low concentrations The peptides according to the invention are effective at a dosage above about 1 mg / kg body weight. For the treatment of DlGeorge syndrome the Pentapeptlde can be administered in a dose of about 1.0 to 10 mg / kg body weight will. In general, doses can be in the same range for treating the other mentioned Conditions or diseases are applied.

The pentapeptides according to the invention were prepared by methods similar to those of Merrlfleld In Journal of the American Chemical Society 85 (1963) 2149-2154. The synthesis passed from the gradual addition of protected amino acids to a growing peptide chain, which is caused by covalent Bonds was attached to a solid resin particle. By doing this, reagents et al By-products were removed by filtration and the recycling of intermediates was superfluous. The general concept of this process is based on the attachment of the first amino acid in the chain to a solid polymer through a covalent bond and the individual, gradual addition of the subsequent Amino acids until the desired sequence is put together. Finally, the solid is removed Carrier and the removal of the protecting groups. In this process a growing peptide chain is obtained, which is bound to a completely insoluble solid particle so that it is in a convenient form for the Filtration and removal of reagents and by-products by washing is present.

The amino acids can be bound to any suitable polymerizate which is only used in the Insoluble in solvents and in a persistent physical form that allows easy filtration, must have. The polymer must contain a functional group to which the first is protected Amino acid can be firmly bound by a covalent bond. The various polymers, e.g. B. cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene, however, a chloromethylated copolymer of styrene and was used for the synthesis according to the invention D! V! Ny! Benzene is used.

The various functional groups on the amino acid that were active but not in the reactions should participate were through conventional protecting groups as used in the field of polypeptides are protected throughout the reaction. For example, the functional groups, e.g. B. the

Rare chain of arginine, lysine, aspartic acid and tyrosine, protected by protective groups when completed the sequence could be removed without adversely affecting the pentapeptide formed as the end product. The synthesis was carried out after a modification of the solid synthesis, as Fluorescamln was used to determine by a sign of positive fluorescence whether the coupling was completely

s dig was [see Felix et al. In Anaiyt. Blochem. 52 (1973) 377]. If full clutch fails indicated, the coupling was repeated with the same protected amino acid before the protecting groups the ct-amino group were removed.

In the general procedure, the one protected at its amino and hydroxyl groups was first L-Tyrosln Esterified to resin in absolute alcohol containing an amine. The coupled amlnosate resin

It was then filtered off, washed with alcohol and water, and dried. The protecting group on the amino group of the protected tyrosine (e.g. t-benzyloxycarbonyl, abbreviated t-BOC) was then removed without to influence other protecting groups. The thus obtained coupled amino acid resin containing the free amino group was then reacted with protected L-Valln, preferably cr-t-BOC-L-Valln, to give the L-Valln to couple to the amlnosate resin. The reactions were then protected with! .- aspartic acid,

υ protected L-lysine and L-arglnine repeated until the complete molecule was formed. This method was used to generate the basic five amino acid active sequence.

The sequence of reactions to produce the active site formed from five aminosuras performing sequence can be performed as follows:

fj

resin

4th

R4

H-Tyr resin

R4

i-BOC-Val-Tyr resin
R4

H-Val-Tyr resin

R ₃

! -BOC-Asp-Val-Tyr-Resin

σ-BOC-Tyr-OH

Removal of the protective group of the o-amino group

a-BOC-L-valine Removal of the protecting group
the α-amino group

Rj

a-BOC-Asp-OH Removal of the protecting group
the o-amino group

H-Asp-Va! -Tyr resin
R ₃ R ₃ R «

I I!

a-BOC-Lys-Asp-Val-Tyr resin

R2

o-BOC-Lys-OH Removal of the protective group
the o-amino group

R ₂ R ₃

I I

H-Lys-Asp-Val-Tyr-Harz _R)

I ι

I a-AOC-Arg-OH

Ri Ri R3 R »

III I.

o-AOC-Arg-Lys-Asp-Val-Tyr resin Removal of all protecting groups

H-Arg-Lys-Asp-Val-Tyr-OH

In the above sequence of reactions slni Ri, Rj, Rj and R «protecting groups on the various reactive groups on the amino acid rare chains that are not affected or removed when the protecting group on the ar-amino group is removed to allow further reaction allow. Vorzugswelse stands in the above, formed as an intermediate pentapeptide resin R, for tosyl (Tos), Rj for ben2yloxycarbonyl (Z), Rj for benzyl (BzI), R ₄ for 0-2,6-chlorobenzyl and aR _s for T-amyloxycarbonyl UAOC) . All resins which have been mentioned above as being suitable for the process are suitable as resins.

The peptide resin is cleaved in order to free the peptide from the resin and the protective groups R ₁ , R 2, Rj, R 1 and u? -R ₅ , with the pentapeptide desired as the end product being obtained at the same time. The protecting groups and the resin are by conventional methods, e.g. B. by treatment with anhydrous hydrogen fluoride, sat ^. ¹ - ten, and the peptide is isolated.

While the preferred method of making the pentapeptides according to the invention using an insoluble, solid polymer takes place, as it is described in the method of Merrlfleld, one can Of course, you can also use other manufacturing methods. For example, a different solid support can be used, such as B. an N-methyl-benzhydrylamine resin, which is advantageous under certain conditions is. In this process, the amino acid attached to the terminal carbon atom is bound directly to the resin, and the finished peptide can be cleaved from the substrate In HF to form terminal C amides. Methods of using an n-methyl-benzhydrylamine resin are described by Monahan et al. In Biochemical and Biophysical Research Communications, 48, 1100-1105 (1972). Method of using a Bc ~ iih "dr"! Sni'nhsrzcs are before. J. PJv! He st 2! Ip. Jcums! cf. Medicinal Chemistry! 973, Vol. 16, pp. 545-549 decide on.

Vt.-Schledene derivatives of the basic pentapeptide can also be prepared using those known to those skilled in the art Methods are made. It is obvious that in the preparation of such derivatives it is necessary to to block functional groups that can enter into the competition with the reaction to the to manufacture desired product. For example, the ar-carboxylic acid group of aspartic acid or the or-Amino group can be blocked by lysine during the manufacture of these derivatives.

As already mentioned. Is it necessary to protect the amino groups when carrying out the process? or to block in order to control the reaction and to obtain the desired products. To the appropriate Protective groups for the amino groups, which can be suitably used, belong to the Saizblldung Protection of strongly basic amino groups or protective urethane substances, e.g. B. benzyloxycarbonyl and t-butyloxycarbonyl. T-Butyloxycarbonyl (tBOC) or t-Amyloxycarbonyl (tAOC) are preferred for protection of the ar-amino group used in the reacting amino acids at the carboxyl end of the molecule that contains the Protective groups BOC and AOC are formed after this reaction and before the subsequent stage (in which the a-Amlnognippe itself) through relatively mild action of acids (e.g. trifluoroacetic acid) can be easily removed. Incidentally, this treatment does not affect any protective groups on the aforementioned Chains. The ar-amino group can thus be protected by reaction with any material that protects the ar-amino group for the subsequent reaction or reactions, but later can be removed under conditions which do not otherwise affect the molecule. As examples such materials include organic carboxylic acid derivatives which acylate the amino group.

In general, the amino groups can be obtained by reaction with a compound which is a group of

formula

Il

R — O — C—

_ ...

contains where R is a group which prevents the amino group from subsequent coupling reactions and can be removed without destroying the molecule. For example, if R is a straight-chain or branched alkyl radical which can be unsaturated and preferably contains 1 to 10 carbon atoms, an aryl radical preferably having 6 to 15 carbon atoms, a cycloalkyl radical preferably having 5 to 8 carbon atoms Aralkyl radical preferably having 7 to 18 carbon atoms, an alkaryl radical preferably having 7 to 18 carbon atoms or a Heterocycle, e.g. B. Isonicotinyl. The aryl, aralkyl and alkaryl components can also, for example be further substituted by one or more alkyl radicals having 1 to 4 carbon atoms. Preferred as groups for R are, for example, t-butyl, t-amyl, phenyl, tolyl, xylyl and benzyl. Particularly preferred special Protective groups for the amino group are, for example, benzyloxycarbonyl, substituted benzyloxycarbonyl, wherein the phenyl ring is replaced by one or more halogen atoms, e.g. B. Cl or Br, is substituted, nitro, lower Alkoxy radicals, e.g. B. methoxy, lower alkyl radicals, tertiary butyloxycarbonyl, tertiary amyloxycarbonyl, cyclohexyloxycarbonyl, Vinyloxycarbonyl, adamantyloxycarbonyl and blphenylsopropoxycarbonyl. Furthermore can used as protecting groups, for example, ison / cotinyloxycarbonyl, phthaloyl, p-tolylsulfonyl and formyl will.

«1 In carrying out the general method according to the invention, the peptide is converted by reaction of the free ar-amino group with a compound containing a blocked ar-amino group. For the Reaction or coupling becomes the carboxyl component of the compound being attacked on its carboxyl group activated so that the carboxyl group then reacts with the free ar-amino group on the peptide chain can. To achieve activation, the carboxyl group In can be any reactive group, e.g. B.

an ester, anhydride, an AzId or acid chloride. It should also be noted that during these reactions the amino acid components contain both amino groups and carboxyl groups and that usually one group enters into the reaction while the other is protected. Prior to the coupling step, the protecting group at the ar-Amlnogruppe od er en dständigen amino group of the resin to the

bound peptide removed under conditions that other protecting groups, e.g. B. the groups at the ε-amino group of the lysine molecule, not: significantly impair. Preferred as a method of implementation a mild acidic hydrolysis, for example by reaction with trifluoroacetic acid, is used at this stage Room temperature.

It is evident that the series of process steps described above lead to the formation of the particular: Pentapeptide's eradicating formula results in:

H-ARG-LYS-ASP-VAL-TYR-OH

The substituted pentapeptide of the formula II in which the terminal ARG and TYR amino acid groups ic can be substituted by substituents R and R 'in the Welter described above, then throw by reacting the basic pentapeptide with those suitable for the preparation of the desired derivatives Reagents made. The reactions of this type, e.g. B. acylation, esterification and amidation are dem Known to those skilled in the art.

The invention is further illustrated by the following examples. In these examples the i are: Parts are parts by weight unless otherwise specified.

example 1

For the preparation of the pentapeptide according to the invention, the following materials are commercially available gene:

a-AOC-Ng-Tos-L-arginine

α-BOC-2-chloro-benzyloxycarbonyl-L-lysine

(Z-BOC-O-Benzyl-L-Aspartic Acid a-BOC-L-valine

α-BOC-2,6-dichlorobenzyl-L-tyrosine

In these reagents, BOC means t-butyloxycarbonyl, AOC means t-amyloxycaroonyl and tosyl. Sequenalw-grade reagents for amino acid sequence determination, cyclohexylcarbodiamide, Flurescamin x, and the resin were also obtained commercially. The resin used was a polystyrene-divinylbenzene resin which had a particle size of 37-74 μm and contained 1% divinylbenzene and 0.75 mmol of chloride per g of resin.

To prepare the pentapeptide, 2 mmol of ar-BOC-O ^ .o-Dlchlorobenzyl-L-tyrosine were added at 80 ° C. for 24 hours Esterified in absolute alcohol containing 1 mmol of triethylamine to give 2 mmol of chloromethylated resin. That Coupled amino acid resin obtained was filtered off, washed with absolute alcohol and dried. Then throw the cr-BOC or ar-AOC amino acids in the same catfish to those of the protective group freed a-amino group of the peptide resin Coupled in the correct order, with the pentapeptide according to the invention using equivalent amounts of di-cyclohexylcarbodiamide. To an aliquot of the resin was tested with fluorescamine for each coupling reaction. When positive fluorescence was found to litter the clutch as incomplete and protected with the same Amino acid repeated. As a result of the various coupling reactions, the following pentapeptide resin was obtained obtain:

Tos Z BzI BzI (CI ₂ )

a-AOC-ARG-LYS-ASP-VAL-TYR resin

Here AOC stands for amyloxycarbonyl, Tos for tosyl, Z for benzyloxycarbonyl, BzI for benzyl and BzI (Gj) for O-2,6-dichlorobenzyl. ^{S ()}

This Peptide resin was cleaved and the protective groups were cleaved in a Kelf splitting apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0 ° C for 60 minutes with 1.2 ml anisole per g Removed Peptld resin as a radical catcher. The peptide mixture was freeze-dried and washed with anhydrous ether washed. The peptide was extracted with glacial acetic acid and water and then on P-6 bio-gel in in-acetic acid Chromatographies. The pentapeptide obtained was found to have a purity of 94% and that had the following sequence:

H-ARG-LYS-ASP-VAL-TYR-OH

Example 2 w>

To determine the activity and properties of the pentapeptide, determinations were made on healthy, 5th to 6 week old nu / nu mice of both sexes performed on a BALB / c basis cultured (with thymocytes expressing the Thy-1,2 surface antigen) and under normal conditions were held. Antl-Thy-1,2 sera in Thy-1 mice of both sexes (Thy-I « congenlc mice).

For triggering the differentiation of Thy-T-T cells or CR'B cells in vitro, the induction of the thymocyte gap in prothymocytes carried out in vitro as described by Komuro and Boyse.

ben [Lancet 1 (1973) 740], the occurrence of Thy-1,2 served as an indication of the differentiation of the T cells. The induction of differentiation of CR ⁺ B-ZeUen of CR'B cell precursors in vitro was performed under similar conditions using as criterion the assay served the capacity of CR ⁺ B cells, sheep erythrocytes, the nlcht with subagglutinating quantities of rabbit antibody and -lytian complement were coated to bind. Spleen cell populations from healthy nu / nu mice, fractionated on discontinuous marginal serum albumen, were used as the source of both types of precursors (Thy-1 "and CR") because they have very few or no Thy-1 * cells and low levels of CR ⁺ Cells.

As a result of this determination, the pentapeptide was found to have a selectivity of effects similar to that of thymopoletin II in inducing the differentiation of T lymphocytes and of

ίο Complement receptor (CR *) - B lymphocytes showed. The Pentapeptld led the differentiation of Thy-1 * - T cells at concentrations of 1 ng to 10 mg / ml. In concentrations from 0.01 ng to IQ Mg / m! did it not cause differentiation of CR'-B cells.

Example 3

To determine the effect of this peptide on neuromuscular transmission, mice received 10 to 100 US peptide in 1 η saline by intraperitoneal injection. 24 hours later, the nj. Omuscular transmission was determined by electromyography, as described in "Lancet" 12 (1975) 256-269. A detectable neuromuscular disorder in the mice injected with the peptide was detectable.

Example 4

The pentapeptide of Example 1 was made on a substrate containing a benzhydrylamine resin, the method of J. Rlvler et al, Journ. Med. Chem. Vol. 16, No. 5, pp. 545-548 (1973). In the synthesis, cyclohexylcarbodiamide was the coupling agent and the coupling was carried out in CHiCIi. The peptide was then cleaved from the substrate with hydrogen fluoride to release the peptide, whereupon the protecting groups were removed and the following derivative was obtained:

H-ARG-LYS-ASP-VAL-TYR-NH,

Identification was done by thunderstorm chromatography and electrophoresis, with the following Values were obtained: Thin-layer chromatography: Sample: 30 mg silica gel (Brinkman glass plate, 5 × 20 cm, 0.25 mm thick) ._

R ^ : n-BuOH: pyridine: HOAc: H ₂ O 30:15: 3:12

r / : EtOAc: pyridine: HOAc: H ₂ O 5: 5: 1: 3

r /: EtOAc: n-BuOH: HOAc: H ₂ O 1: 1: 1: 1
Spray reagent: Pauly, Ninhydrin, Sakapuchi and J ₂

Thin layer chromatography
Rf Rf V

Electrophoresis: peptide migrated to the cathode

H-Arg-Lys-Asp-Val-Tyr-NH ₂

0.53

0.68 0.26 7.5 cm

Electrophoresis: Whatman Papler 3 mm (11.5 cm 56 cm)

Sample: 100 mg pH 5.6, pyridine acetate buffer solution 1000 V, 1 hour

Spray reagent: ninhydrin and sakaguchl

This Pentapeptide amld showed when used in a concentration of 1 mg / ml In 89% 1ger Twomey solution a maximum activity of 105% and a minimum activity of 70% compared to that basic Pentapeptide according to Example 1.

Example 5

The pentapeptide produced according to Example 1 is esterified in order to produce the ethyl ester. In the preparation of of this ester it is necessary to protect the acid groups on the aspartic acid with a slightly acid-sensitive protective group during manufacture, with the preferred protecting group being t-butyl. The ct-amnoblocking group Is sensitive to a light base and is preferably fluorenylmethoxycarbonyl. If such a sr-aminoblocking group is used, it can be used for the addition of any amino acid ester can be removed without disturbing the acid-sensitive protective group on the aspartic acid residue. After manufacture of the protected pentapeptide resin removes the treatment with a mild base and then the

10

Treat these two protecting groups with a mild acid. Transesterification with ethyl formate using Sulfuryl chloride as a catalyst cleaves the pentapeptide from the resin to selectively remove the terminal C-ester to build. The esterification does not take place on the free acid group of the aspartic acid ester. The remaining Protective groups are removed and the peptide of the following formula is isolated:

H-ARG-LYS-ASP-VAL-TYR-OC ₂ Hs

Amino acid analysis: Arg Lys Asp VaI Tyr calculated: 1.00 1.00 1.00 1.00 1.00 found: 1.00 1.01 1.00 0.99 0.98 Thin layer chromatography (silica gel GF, 250 μΓΠ) Solvent system 3: 1: 1 n-BuOH: 0.17 4: 2: 3: 1 n-BuOH; : Pyr 0.72 : HOAC: H ₂ O ; HOAC: H ₂ O

Arg Lys Asp VaI Tyr 1.00 1.00 1.00 1.00 1.00 0.98 1.00 1.02 0.87 1.02

- ■ - · - ■

Example 6

The pentapeptide prepared according to Example 1 was by reaction with Acetylchlorld according to known Process acyllert, whereby the following acyllert derivative was obtained:

CHjCO-ARG-LYS-ASP-VAL-TYR-OH ² ' Optical rotation: - 30.8 °

(C = 1.32; 0.1 N-HOAC;)

w Amino acid analysis:

calculated:
found ':

Peptide content: 63%
Thin layer chromatography (silica gel 60, 250

Solvent system R ₁

4: 1: 1 n -BuOH: H ₂ O: HOAC 0.20

5: 5: 1: 3 EtOAC: Pyr: HOAC: H ₂ O 0.55

3: 2: 1 Pyr: HOAC: H ₂ O 0.73

Example 7-

The pentapeptide prepared according to Example 6 was also known by reaction with ammonia Process amldlert, whereby the following derivative was formed:

CHjCO-ARG-LYS-ASP-VAL-TYR-NH,

The Pentapeptldd ^ rlvate prepared according to Examples 5 to 7 preserve the hler for the basic amino acid sequence biological activity described.

Amino acid analysis: Arg Lys Asp VaI Tyr calculated: 1.00 1.00 1.00 1.00 1.00 found: 0.99 0.98 1.00 0.92 0.99

Claims (2)

Patent claims: 1. Arginyl-Lysyl-Aspartyl-Valyl-Tyrosln and its derivatives of the general formula II R-ARG-LYS-ASP-VAL-TYR-R ' In which R stands for hydrogen or alkanoyl radicals with 1 to 7 carbon atoms and R 'stands for a hydroxy radical, alkoxy radicals with 1 to 7 carbon atoms or an amino radical, as well as their pharmacologically acceptable salts. 2. Process for the preparation of the compounds of general formula II according to claim 1, characterized characterized in that one in each case in known catfish