DK149595B - ANALOGY PROCEDURE FOR PREPARING FTS ANALOGUE TETRA AND PENTAPEPTID DERIVATIVES - Google Patents

Description

in 149595

The present invention relates to an analogous process for the preparation of novel FTS analog tetra and penta-peptide derivatives.

It is well known that many polypeptides have been isolated from various tissues and organs (including the blood) in animals.

Many of these polypeptides are related to the body's immune function, such as e.g. the various immune globulins, the thymus hormone thomopoietin and the like. The applicant has isolated and synthesized several of these polypeptides as described in U.S. Patents Nos. 4,002,602 and 4,002,740, as well as in several scientific articles.

Until approx. Ten years ago, you knew very little about thymus, while today it is understood that thymus is one of the organs that is primarily responsible for the immune function of mammals and birds.

15 Despite great interest in possible functions of thymus and early speculation and experimentation, until recently, little was known about thymus function. Today, however, it has been realized that the thymus is a composite organ with both epithelial (endocrine) and lymphoid (immunological) components and that 20 thymus is thus involved in the body's immunity functions. Thymus consists of an epithelial stroma derived from the third bronchial arch and lymphocytes derived from stem cells derived from Hanopoietic tissue, Goldstein et al, The Human Thymus, Heinemann, London, 1969. Lymphocytes differentiate within 25 thymus and leave it as mature thymus-derived cells, called T-cells, which circulate to the blood, lymph, spleen and lymph nodes. The induction of stem cell differentiation in the thymus itself appears to be mediated by secretions of thymus epithelial cells.

30 It has been known for some time that the thymus is related to the immunity properties of the body, and there has therefore been expressed great interest in substances that have been isolated from the thymus. In this regard, a relatively large number of articles have been published in recent years, based on scientific work on materials present in the bovine thymus. The applicant has himself published a number of articles relating to studies in this area. Relevant publications can eg. found in The Lancet, 20.

July 05, 1968, pages 119-122J Triangle, Volume II, no. 1, pages r 7-14, 1972. Annals of the New York Academy of Sciences, r

Volume 183, pages 230-240, 1971. and Clinical and Experimental i

Immunology, Volume 4, no. 2, pages 181-189, 1969. Nature, b

Volume 247, pages 11-14, 1974. Proceedings of the National 10 Academy of Sciences USA, Volume 71, pages 1474-1478, 1974.

Cell, Volume 5, pages 361-365 and 367-370, 1975. Lancet, Volume 2, pages 256-259, 1975. Proceedings of the National Academy of Sciences USA, Volume 72, pages 11-15, 1975. Biochemistry , f

Volume 14, pages 2214-2218, 1974. Nature, Volume 255, pages 15 423-424, 1975.

Another group of immune function lymphocytes are the B lymphocytes or B cells. These are differentiated in the bursa fabricii of birds and by a yet-unidentified mammalian organ. T cells and B cells cooperate in many aspects of immunity. See e.g. articles by Applicant in Science, 193, 319 (July 23, 1976) and Cold Spring Harbor Symposia on Quantitative Biology, Volume XLI, 5 (1977).

U.S. Patent No. 4,002,602 discloses a long-chain polypeptide described as "Ubiquitous Immunopoietic Polypeptide" (UBIP). This polypeptide was later named Ubiquitin. Ubiquitin contains 74 amino acids and is distinguished by its ability to induce, at nanogram concentrations in vitro, 30 differentiation of both T cell and B cell immunocytes from precursors present in bone marrow or spleen. Thus, ubiquitin is useful in therapeutic areas which include -thymus impairment or immune deficiency and the like.

35 A nonapeptide material has recently been isolated from pig serum by J.F. Bach, et al and identified as "facteur thymique serique" (FTS). The insulation of this material and its structure is described in C.R. Acad. Sc. Paris, page 283 (November 29, 1976), Series D-1605 and Nature 266, 55 (3.

3 1: 49595 March 1977). The structure of this nonapeptide has been identified as GLX-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN, where "GLX" represents either glutamine or pyroglutamic acid. The material in which GLX is glutamic or pyroglutamic acid 05 has been synthesized. In these articles, Bach stated that his nonapeptide FTS selectively differentiated T cells (and not B cells) using an E-rosette sample. Bach therefore concluded that his material was a thymus hormone.

Recently, a more in-depth study of the activity of this nonapeptide by the present applicant has shown that FTS differentiated both T-cells and B-cells and therefore resembled ubiquitin rather than thomopoeitin in activity. Fire,

Gilmour and Goldstein, Nature, 269: 597 (1977).

It has now unexpectedly been found that the tetrapeptide segment FTS

(2-5) and the pentapeptide GLN1-FTS (1-5) as well as their analogs possess the ubiquitin-like activity of the FTS nonapeptide.

The present invention is based on this disclosure and relates to an analogous method for preparing novel tetra and pentapeptide derivatives of the general formula RX-LYS-Y-GLN-R1 wherein X is selected from the group consisting of L-alanyl, D-alanyl and sarcosyl, Y is selected from the group consisting of D and L-seryl, sarcosyl, 2-methyl-alanyl and D-alanyl, R is H, GLN 25 or SAR, and R 'is OH or NH 2 or pharmaceutically acceptable acid addition salts thereof. , in the characterizing part of claim 1.

In particular, the tetra- and pentapeptides prepared according to the present invention are capable of inducing differentiation of both T lymphocytes and B lymphocytes, which is why they are believed to be valuable to the human and animal immune system.

A list of natural amino acids can be found ± many reference books, e.g. R.T. Morrison and R.N. Boyd, Organic Chemistry, 35 Allyn and Bacon, 1959, Chapter 33. In addition to the natural amino acids (which are those found in proteins), there are also a number of so-called "non-natural" amino acids which are not found in proteins, although they sometimes occur naturally as metabolic intermediates or the like. These non-natural amino acids may be the D-isomers corresponding to the optically active (L-forms) natural amino acids or they may be completely different chemical compounds such as sarcosine (N-methylglycine) or 2-methylalanine mentioned above. Enumerations of such non-natural amino acids are also found in many reference works.

The present tetra and pentapeptide derivatives are unusual in that they exhibit the same biological activity as the natural nonapeptide containing an amino acid sequence corresponding to the sequence of the tetrapeptide H-Ala-Lys-Ser-Gln-OH prepared according to the invention. Against this background, it is believed that the activity of the molecule is developed by its stereochemistry, i.e. the particular "folding" of the molecule. In this regard, it should be understood that polypeptide bonds are not rigid but flexible, and polypeptides can appear as surfaces, helices, and the like. As a result, the entire molecule is flexible and it will "fold" in a determined manner. In the context of the present invention, it has been found that the novel tetrapeptide segments probably "fold" in a similar manner to the natural tetrapeptide segment of the natural nonapeptide and therefore exhibit the same biological properties. For this reason, the tetrapeptide derivatives may be terminally substituted with said amino acids, since they do not significantly affect the biological activity or interfere with the natural "folding" of the molecule.

Whether a compound has the biological effect can be readily determined by testing the compound for differentiating Th-1 + T lymphocytes and Bu-1 + B lymphocytes in the chicken induction test described below. Compounds that are specifically active in nanogram (ng) / milliliter (ml) concentrations (about 1 ng / ml) or less in this sample are considered biologically active.

The ability of the molecule to maintain biological activity and natural folding is clearly illustrated by the fact that the tetrapeptide segments in question exhibit the same biological properties as the natural non-peptide described as FTS 35 by J.F. Bach in the above articles. In this nonapeptide, a tetrapeptide sequence made according to the invention can be identified within the molecule, but only in combination with the other amino acids described therein. Since the tetrapeptide derivatives of the invention provide the same biological activity as the non-peptide FTS, it is clear that the amino acids substituted on the one terminal amino acid residue of the tetrapeptide derivative O5 do not affect the biological properties.

Furthermore, within the scope of the invention, the pharmaceutically acceptable acid additions fall to all tetra and pentapeptides. Sam acids capable of forming salts with the polypeptides can be mentioned inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, phosphoric, etc. and organic acids such as formic, acetic, propionic, glycolic, lactic , pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid or e.g. sulfanilic.

For convenience, the amino acid components of the peptide are abbreviated herein. These abbreviations have the following meanings. The D-amino acids are indicated by affixing "D" before the abbreviation, e.g. D-alanine is indicated by "D-ALA".

20 Amino Acids Abbreviated designation

L-alanine ALA

L-serine SER

L-glutamine GLN

L-lysine LIGHT

25 Sarcosin SAR

2-methylalanine 2-Me-ALA

The aforementioned peptides have been shown, as mentioned, to exhibit properties similar to those of the non-peptide FTS isolated from pig blood as referred to in the above articles by Bach 30 et al. The peptides prepared according to the invention are particularly characteristic of their ability to induce the differentiation of both T precursor and B precursor cells. Some of the subject tetra and pentapeptides are active at a concentration of as little as 35 picogram (pg) / ml in the chicken induction test described below.

6 U9595

It has been found that the tetra- and. pentapeptides induce the differentiation of immunocyte precursor cells in vitro in the same way as the nonapeptides described by Bach. Thus, the polypeptides of this invention have been found to induce the 05 differentiation of both B precursor cells, as measured by the acquisition of the thymus differentiation antigen TH-1, as well as B precursor cells measured by the acquisition of the differentiation antigen Bu-1. Stated another way, the polypeptides in question have the ability to induce the differentiation of both Th-1 T lymphocytes and Bu-1 B lymphocytes.

It has also been found that the subject tetra and pentapeptides increase the ability to produce in vivo cytotoxic lymphocytes by stimulation with allogeneic antigens. I.e. to administer the tetra and pentapeptides of the present invention to e.g. rats promote the production of cytotoxic lymphocyte precursors, as measured by an in vitro sample of rat spleen cells. As the development of cytotoxic lymphocytes directly corresponds to the extent of transplant rejection by allogenic transplantation versus host reaction, the above discovery provides further substantiation for the immunological utility of the polypeptides in question.

To understand the importance of the differentiating biological properties of the tetra- and. pentapeptides, it should be noted that the function of thymus in relation to immunity can be broadly stated as the production of thymus-derived cells or lymphocytes called T cells. T cells make up a large portion of the total amount of recirculating small lymphocytes. T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homo-transplantation response) as effector cells. However, T cells do not secrete humoral antibodies. These antibodies are secreted by cells (called B cells) that are directly directed by the bone marrow independently of the action of the thymus. However, for many antigens, B cells require the presence of appropriate responsive T cells before they can produce antibodies. The mechanism of this process of 05 cell cooperation is not yet fully understood.

In the light of the above explanation, it can be said in practice that thymus is necessary for the development of cellular immunity and many humoral antibody responses and it affects these systems by inducing within the thymus the differentiation of haemopoietic stem cells into T cells. This inductive effect is mediated by secretions of the epithelial cells of the thymus, i.e. Thymus Hormones 15 To further understand the effect of the thymus and lymphocyte cell system as well as the circulation of lymphocytes in the body, it should be noted that stem cells arise in the bone marrow and reach the thymus by blood flow. Inside the thymus, stem cells are differentiated into immunologically competent T-20 cells that migrate to the bloodstream and, together with B cells, circulate between tissue, lymph, and bloodstream.

The body cells secreting antibody (B cells) are also developed from hemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they are differentiated into an organ analogous to the thymus, called bursa fabricii.

In mammals, no equivalent organ has been found and these cells are believed to differentiate into the bone marrow. They are therefore referred to as bone marrow derived cells or B cells. The 30 physiological agents that dictate this differentiation remain completely unknown.

As stated above, the tetra and pentapeptides of the invention are therapeutically valuable in the treatment of humans and animals. As the novel tetra-35 and pentapeptides are capable of inducing the differentiation of lymphopoietic stem cells from hemopoietic tissue into both thymus-derived lymphocytes (T cells) and immunocompetent B cells capable of participate in the immune response of the drug, the compounds of this invention are believed to have numerous therapeutic uses. Since the pre-05 binders are capable of performing certain of the stated functions of the thymus, they primarily have utility in various areas of thymus function and immunity. A primary area of application lies in the treatment of DiGeorge syndrome, a condition in which there is an innate deficiency of thy-10 mice. Injection of one of the subject tetra and pentapeptides described below will overcome this deficiency. Another use is in agammaglobulinemia, which is caused by a defect in the body's putative B-cell differentiating hormone. Injection of one of the subject tetra and pentapeptides will overcome this defect. Because the tetra and pentapeptides of the present invention are extremely active at low concentrations, they are valuable for enhancing the body's collective immunity by increasing therapeutic stimulation of cellular immunity and humoral immunity and are thereby valuable in the treatment of. diseases involving chronic infection in vivo such as fungal or mycoplasma infections, tuberculosis, leprosis, acute and chronic viral infections and the like. Furthermore, the present peptides are believed to be valuable in any area where cellular or humoral immunity is important and especially where there are immunity deficiencies such as in DiGeorge syndrome discussed above. Furthermore, because of the properties of the tetra and pentapeptides, they have in vitro utility in inducing the development of surface antigens of B cells, in inducing the development of the functional ability to achieve responsiveness to mitogens and antigens, collaboration by improving the ability of B cells to produce antibodies. They have utility in vitro by inducing the development of B cells as measured by the development of surface receptors for complement. The present peptides are also valuable for inhibiting the uncontrolled proliferation of lymphocytes responsive to ubiquitin 1495959 (U.S. Patent No. 4,002,602). An important feature of the subject tetra and pentapeptides is their in vivo ability to rebuild cells with the characteristics of T cells and, moreover, their in vivo ability to rebuild cells with the property of B cells 05. They are therefore valuable for treating relative or absolute B-cell deficiencies as well as relative or absolute T-cell deficiencies, whether due to deficiencies in either the tissue-differentiating B cells or in thyroid mice or for a completely different reason. .

10

Another important property of the tetra and pentapeptides treated herein is their high activity at very low concentrations. Thus, it has been found that the tetra and pentapeptides are generally active at concentrations of ca. 1 ng / ml, while certain particularly potent tetrapeptides (H-SAR-LYS-D-ALA-GLN-NH 2 and H-SAR-LYS -SAR-GLN-NE 2) are active at concentrations ranging from about

0.1 µg / ml. The carrier may be any of the well known carriers for this purpose, including normal saline solutions, preferably with a protein diluent such as bovine serum albumin to prevent loss of adsorption to glass objects at these low concentrations. The tetra- and pentapeptides treated are generally active in the range of about 1 µg / kg body weight, while some particularly potent tetrapeptides are active fraca.

1 ng / kg body weight. For the treatment of DiGeorge syndrome, tetra-2β and pentapeptide can be administered in an amount of approx. 1 to approx.

100 µg / kg body weight, while the particularly potent tetrapeptides can be administered in an amount of approx. 1 to approx. 100 ng / kg body weight. Generally, the same dose ranges can be used to treat the other conditions or diseases mentioned. Although the discussion above concerns parenteral administration, oral administration is also possible in dose ranges which are generally approx. 100 to 1000 times higher than injection.

35 or 149595

The present tetra and pentapeptides have been prepared using similar principles as described by Merrifield in the Journal of the American Chemical Society, 85, pages 2149-2154, 1963. The synthesis involved the stepwise addition of protected 05 amino acids to a growing peptide chain containing covalent bonds were bound to a solid resin particle. By this method, reagents and by-products were removed by filtration and the recrystallization of intermediates was avoided. The general principle of this process depends on attaching af0 of the C-terminal amino acid in the chain to a solid polymer by a covalent bond and adding the subsequent amino acids one at a time until the desired sequence is collected. Finally, the peptide is removed from the solid support and protective groups are removed. This method provides a growing peptide chain attached to a completely insoluble solid particle so that it is in a convenient form for filtration and leaching of reagents and by-products. The amino acids can be attached to any suitable polymer, which alone must be easily separable from the unreacted reagents. The polymer may be insoluble in the solvents used or soluble in some solvents and insoluble in others. The polymer should have a stable physical form which allows easy filtration. It should contain a functional group to which 25 can be attached to the first protected amino acid by a covalent bond. Various insoluble polymers suitable for this purpose are such as cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene, but in the synthesis of the invention a chloromethylated copolymer of styrene and divinylbenzene was generally used. Polymers which are soluble in organic solvents but insoluble in aqueous solvents may also be used. One such polymer is polyethylene / glycol having a molecular weight of approx. 20,000 which is soluble in methylene chloride but insoluble in water. Use of this polymer in peptide syntheses is described by F. Bayer and M. Mutter in Nature 237, 512 (1972) and the references cited therein.

11 149596

The various functional groups on the amino acid which were active but which were not to be included in the reactions were protected throughout the reaction by conventional protecting groups used in the polypeptide technique. Thus, the functional group on lysine was protected by protective groups which could be removed after completion of the sequence without adversely affecting the final tetra or pentapeptic product. During the synthesis, fluorescence was used to determine whether the coupling was complete at 10 indicates positive fluorescence (see Felix, et al., Analit, Biochem., 52, 377, 1973). If the sample did not show complete coupling, the coupling was repeated with the same protected amino acid before deprotection.

The C-terminal amino acid can be attached to the polymer in a variety of well-known ways. Summaries of methods for attachment to halomethyl resins are given in Horiki, et al, Chem.

Letters, pages 165-168 (1978) and Gisin, Helv. Chim. Acta, 56, 1476 (1973) and the references cited therein.

20

The general method first involved esterification of L-glutamine, protected on the α-amino group to the resin in absolute alcohol containing an amine. The coupled amino acid resin was then filtered, washed with alcohol and water and dried. The protecting group on the d-amino group of the glutamine amino acid (e.g., t-BOC, i.e., t-butyloxycarbonyl) was then removed.

The resulting coupled amino acid resin with the free amino group was then also reacted. with a protected L-serine, preferably alpha-t-BOC-O-benzyl-L-serine for coupling the L-serine. The reactions were then repeated with protected L-lysine and e.g. L-alanine until the complete molecule was prepared. The sequence of reactions was carried out as follows:

resin

A-R1-Gln-OH

α-R ^ -Gln-resin

Removal of α-amino protecting group Ί / H-Gln resin 9 R

'A-R1-L-Ser-OH

R ^ 1 'α-R -Ser-Gln resin

Removal of α-amino 0 protecting group R ^ in H-Ser-Gln resin R3

A-R1-Lys-OH

'32 RJ R ^ ι ι α-R -Lys-Ser-Gln resin

Removal of α-amino.39 protecting group RJ R ^ 5 I Φ * H-Lys-Ser-Gln resin

A-R1-Ala-r OH

3 2

R RZ

T I '•' 'l α-R -Ala-Lys-Ser-Gln resin

Removal of all protective groups and resins

H-Ala-Lys-Ser-Gln-OH

In the above reaction sequence, Rx represents a protective group for the α-amino group and R and R are protecting groups on the reactive side chains of L-serine and L-lysine, respectively, that are not affected or removed when R ^ is removed to allow 5 further reaction. In the above tetrapeptide resin intermediate, the term stands for a protected group such as t-butyloxycarbonyl, R stands for benzyl or 3 substituted benzyl (e.g., 4-chlorobenzyl) and R stands for substituted benzyloxycarbonyl (e.g., 2, 6-dichlorobenzyloxycarbonyl). The resin may be any of the resins mentioned above as useful in the process.

After preparation of the last intermediate, the peptide resin was cleaved to remove the protecting groups R1, R2 and R3 as well as the resin. The cleavage of the protecting groups occurred, for example, by treatment with anhydrous hydrogen fluoride, and the resulting free peptide was then recovered.

As stated above, in the practice of the process, it is necessary to protect or block the amino groups to control the reaction and obtain the desired products. Suitable amino protecting groups which may be used advantageously include salt formation to protect highly basic amino groups, or urethane protecting substituents such as p-methoxybenzyloxycarbonyl and t-butyloxycarbonyl. It is preferred to use t-butyloxycarbonyl (BOC) or t-amyl-oxycarbonyl (AOC) to protect the α-amino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since they are BOC and AOC- (t amyloxycarbonyl) protecting groups are readily removed after such reactions and before the subsequent step (where such α-amino group itself undergoes reaction) by relatively mild action of acids (e.g. trifluoroacetic acid), which treatment does not otherwise

14959S

In this way, the groups used to protect other responsive side chains affect. Thus, it will be appreciated that the α-amino groups can be protected by reaction with any material that will protect the amino groups from the subsequent reaction (s), but which can later be removed under conditions that would not otherwise affect the molecule. Examples of such materials are organic carboxylic acid derivatives which acylate the amino group.

In general, any of the amino groups can be protected by reaction with a compound containing a group of the formula:

ISLAND

4 "ROC-15 wherein R4 is any group which will prevent the amino group from engaging in subsequent coupling reactions and which can be removed without destruction of the molecule. Thus, R4 may be a straight or branched alkyl group which may be unsaturated, preferably with 1 -10 carbon atoms, and preferably halogen or cyano-substituted, aryl, preferably with 6-15 carbon atoms, cycloalkyl, preferably with .5-8 carbon atoms, aralkyl, preferably with 7-18 carbon atoms, alkaryl, preferably with 7-18 carbon atoms or heterocyclic, e.g. isonicotinyl, the aryl aralkyl and alkaryl groups may also be further substituted such as with one or more alkyl groups having from 1 to about 4 carbon atoms. Preferred meanings for R include t-butyl, t-amyl, tolyl, xylyl and benzyl. Particularly preferred specific amino protecting groups include 30 benzyloxycarbonyl, substituted benzyloxycarbonyl, wherein the phenyl ring is substituted by one or more halogen atoms, for example Cl or Br, and furthermore nine belief, lower alkoxy, e.g. methoxy, lower alkyl, t-butyloxycarbonyl, t-amyloxycarbonyl, cyclohexyloxycarbonyl, vinyloxycarbonyl, adamantyl oxycarbonyl, biphenylisopropoxycarbonyl and the like. Other protecting groups which may be used include isonicotinyloxy-carbonyl, phthaloyl, p-tolylsulfonyl, formyl and the like.

149595 15

In practicing the general method of the invention, the peptide is built up by reacting a compound with a free amino group with a compound having a protected amino group. For reaction or coupling, the compound to be attacked is activated by the carboxyl group so that the carboxyl group can then react with the free amino group on the peptide chain attached to the resin. To obtain the activation, the carboxyl group can be converted to any reactive group such as an ester, anhydride, azide, acid chloride or the like. Alternatively, can a suitable cup ·? Addition reagent is added during the reaction. Suitable coupling reagents are e.g. disclosed in Bodanszky, et al., Peptide Synthesis, Interscience, 2nd Edition, 1976, Chapter 5, including carbodiimides (e.g., dicyclocarbodiimide), carbonyl diimidizole and the like.

It should also be noted that during the coupling reaction, the amino acids contain both an amino group and a carboxyl group, and usually one group is included in the reaction while the other is protected. Prior to the next coupling step, the protecting group on the α-amino group or the terminal amino group of the attacked peptide is removed under conditions that do not significantly affect other protective groups, e.g. the group on the epsilon amino of the lysine molecule. The preferred method for performing this step is mild acidolysis, such as reaction at room temperature with trifluoroacetic acid.

As will be appreciated, the above-described series of process steps results in the preparation of e.g. the tetrapeptide of formula III, as follows: III. H-ALA-LYS-SER-GLN-OH 35

14959S

16

This tetrapeptide has the indicated biological activity. Substitution with a natural or non-natural amino acid residue in place of either L-alanyl or L-seryl or both or with a dipeptide in place of L-alanyl can be done by replacing either 05 alanine or serine or both with the appropriately protected nature equally. or non-natural amino acid or the suitably protected dipeptide in the above synthesis scheme to form the tetrapeptide of the following formula:

IIIa. R-X-Lys-Y-Gln-OH

10 wherein X and Y have the above meanings.

The identity and purity of the subject peptides were determined by well known methods such as thin layer chromatography, electrophoresis, amino acid analysis and the like.

The invention is further illustrated in the following examples, in which all parts are parts by weight unless otherwise stated.

149596 17

Example 1

To prepare a tetrapeptide of the formula H-ALA-LYS-SER-GLN-OH, the following materials were purchased commercially: Increase Alpha-BOC-L-glutamine-o-nitrophenyl ester

Alpha-BOC-e-2-chloro-benzyloxycarbonyl-L-lysine Alpha-BOC-O-benzyl-L-serine Alpha-BOC-L-alanine.

* 10 In these reagents, BOC means t-butyloxycarbonyl. Sequential grade reagents for amino acid sequence assays, dicyclohexylcarbodiimide, fluorescamine and the resin were also commercially purchased. The resin used was a polystyrene divinylbenzene resin, 200-400 sieve size containing 15% of 1% divinylbenzene and 0.75 mM chloride per gram of resin.

To prepare the tetrapeptide, two mmoles of α-BOC-L-glutamine were esterified to 2 mmoles of chloromethylated resin in absolute alcohol containing 1 mM triethylamine for 24 hours, at 80 ° C. The resulting amino acid resin ester was filtered, washed with absolute alcohol and dried. Then, the other α-BOC amino acids were similarly coupled to the deprotected α-amino group in the peptide resin in the correct order to form the subject tetrapeptide resin intermediate, using equivalent amounts of dicyclohexylcarbodiimide. After each coupling reaction, a portion of the resin was tested with fluorescamine and if positive fluorescence was found, the coupling was assumed to be incomplete and repeated with the same protected amino acid. Due to the series of coupling reactions, the tetrapeptide resin intermediate was prepared.

35 149595 18

This peptide resin was cleaved and the protecting groups removed in a "Kel-F" cleavage apparatus (Peninsula Laboratories / Inc.) using anhydrous hydrogen fluoride 05 at 0 ° C for 60 minutes with 1.2 ml of anisole per gram of peptide resin as a detergent . The peptide mixture was washed with anhydrous ether and extracted with aqueous acid. The extract was lyophilized and the peptide chromatographed on "P-6 Bio-Gel" in 1N acetic acid. The resulting tetrapid time was determined to be 94% pure and determined to have the following sequence:

H-ALA-LYS-SER-GLN-OH

For identification, thin layer chromatography and electrophoresis were performed as follows:

Thin layer chromatography was performed on a 30 µg sample on silica gel (Brinkman Silica gel with fluorescent indicator, 20 x 20 cm, 0.1 mm thick) using the following eluents: n-butanol: pyridine: acetic acid: water. 30: 15: 3: 12 r / 2 R 2: ethyl acetate: pyridine: acetic acid: water. 5: 5: 1: 3 3 R ^ :. ethyl acetate: n-butanol: acetic acid: water. 1: 1: 1: 1 <S> 20 Electrophoresis was performed on a 100 µg sample of Whatman 3 mm paper (11.5 x 56.5 cm) using a pH 5.6 pyridine acetate buffer solution and 1000 V voltage for one hour.

Spray reagents for both thin layer chromatography and electrophoresis were Pauly and Ninhydrin.

The following results were obtained: R 2 = immobile, R 2 = immobile and R 3 = 0.336. Electrophoresis resulted in a migration of 9.4 cm toward the cathode.

149596 19

Example 2

To determine the activity and properties of the tetrapeptide prepared in Example 1, the following chicken induction test was used. This sample is described in more detail 05 in Brand, et al., Science, 193 319-321 (July 23, 1976) and the references contained therein.

Bone marrow from newly hatched chickens was selected as the source of inducible cells because it lacks a significant number of Bu-1 + or Th-1 cells. Collected femur and tibiotar 10 cells of five newly hatched chickens of the strain SC (HY-line) were fractionated by ultracentrifugation on a 5-layer discontinuous bovine serum albumin (BSA) gradient. Cells from the two lightest layers were pooled, washed and suspended for incubation at a concentration of 5 x 10 6 cells per milliliter with the appropriate concentration of test polypeptide in RPMI 1630 medium supplemented with 15 mM N-2-hydroxyethyl polypeptide. perazine N'-2-ethanesulfonic acid (hepes), 5 percent γ-globulin-free fetal calf serum, deoxyribonuclease (14-18 units / ml), heparin (5 units / ml), penicillin (100 units / ml) ml) and streptomycin (100 µg / ml). Control preparations were incubated with BSA (1 µg / ml) or medium alone. After incubation, the cells were tested in the cytotoxicity sample using chicken C1 and guinea pig C2 to C9 complement fractions as described in the reference article. The percentage of 25 Bu-1 + or Th-1 + cells in each layer was calculated as a cytotoxicity index, 100 (a-b) / a, wherein a and b are the percentages of living cells in the complement control and test preparation, respectively. The percentage amount of induced cells was obtained by subtracting the mean values in the control incubations without inducers (usually 1-3%) from the values of the test inductions.

The efficacy specificity of the test tetrapeptide and its similarity to ubiquitin was demonstrated by inhibiting the induction of Bu- + + 1 B cells and Th-1 τ cells by the test polypeptide 35 after the addition of ubiquitin at a concentration of 100 µg / ml. ml. This high dose of ubiquitin inactivates the ubiquitin receptors and thus prevents the induction of cells by any agent acting through these receptors.

20 143595

As a result of this study, it was discovered that the tetrapeptide of Example 1 exhibited biological activity similar to ubiquitin by inducing the differentiation of both + + TH-1 T and Bu-1 B lymphocytes in ng / ml concentrations.

Example 3 A. The sample of Example 2 was repeated using one of the following as test pentapeptide:

H-GLN-ALA-LYS-SER-GLN-OH

H-SAR-LYS-ALA-SER-GLN-OH

In each case, biological activity corresponding to ubiquitins was observed.

B. The sample of Example 2 was repeated using one of the following as test tetrapeptide: h-sar-lys-d-ala-gln-nh2 h-sar-lys-sar-gln-nh2 HD-ALA-LYS-D -ALA-GLN-NH 2 In each case, biological activity similar to ubiquitins was observed. First, of these tetrapeptides, this activity was observed in the concentration range of ca. 1 pg / ml to approx. 100 µg / ml. Second, tetrapeptide activity was observed at a concentration as low as 0.1 µg / ml.

Examples 4-6

Using the reaction methods described herein to prepare substituted tetra peptides, tea peptides of the following general formula were prepared: R-X-LYS-Y-GLN-R1

Example No. R X Y R / _ 4 H SAR D-ALA NH2 4 A H SAR SAR NH2 05 5 H D-ALA D-ALA NH2 5 A H D-ALA SAR NH2 6 H SAR 2-Me-ALA NH2

6 A H SAR SAR OH

These peptide amides were prepared on a benzhydrylamine resin 10 by solid phase synthesis known in the art.

The tetrapeptides prepared in Examples 4-6 retain the biological activity described herein for the active polypeptide segment.

For identification, thin layer chromatography and electrophoresis were performed as follows:

Thin layer chromatography was performed on 20 µg samples on silica gel (silica gel 5 x 20 cm) using as eluent of n-butanol: acetic acid: ethyl acetate ligand at 1: 1: 1: 1 (R 1) and on cellulose 6064 (Eastman, 20 x 20 cm) using 20 as the eluent of n-butanol: pyridine: acetic acid: water in a ratio of 15: 10: 3: 12 (Rf2).

Electrophoresis was performed on 50 µg samples on Whatman No. 3 paper (5.7 x 55 cm) using a pH 5.6 pyridine acetate buffer solution and 1000 V voltage for one hour. The connectors migrate toward the cathode.

Spray reagents for both thin layer chromatography and electrophoresis were Pauly and Ninhydrin.

The following results were obtained (both Rf values and electrophoresis are reported relative to H-ARG-LYS-ASP-VAL-TYR-OH): 149595 22, 5 Electrophoresis

Example R, R_ Migration Purity f f cm toward the cathode 4 0.44 0.68 2.07 98% 4 A 0.88 0.60 1.78 98% 05 5 0.56 0.71 2.10 98%

After thin-layer chromatography and electrophoresis as in Example 1, the following results were obtained for the compound of Example 6: 1 ° R 1 = (0.155), Rf 2 = immobile, Rf 3 = 0.265 and electrophoresis migrations 13.1 cm toward the cathode.

Examples 7-8

Using the appropriate dipeptides in the coupling reaction as above, the following pentapeptides containing the active amino acid sequence are substituted on the terminal amino group of formula: R-ALA-LYS-SER-GLN-R 'which is substituted with the 20 amino acids listed in the following table.

Example No. R R

7 GLN OH

8 SAR OH

The tetra and pentapeptide derivatives prepared in Examples 4-8 retain the biological activity as described herein for the active polypeptide segment.

After thin layer chromatography and electrophoresis as described in Example 1, the following results were obtained for the compounds of Examples 7 and 8.

149595 23

Example Rp1 Rf2 Rf3 Electrophoresis 1 migration toward cathode_ η Immobilized immobile 0/303 7f4 cm 8 immobilized immobilized 0f186 8.3 cm

Example 9

To further elucidate the utility of the subject tetra and pentapeptides, this example describes a microculture sample for estimating the frequencies of the cytotoxic lymphocytes produced by stimulation with halogen antigens. The frequencies of cytotoxic precursors between control animals and animals injected with different concentrations of the test compound were compared by a boundary dilution test.

Materials and Methods Mice

Inbred C57 BL / 6J (females, 8 weeks) was obtained from Jackson Laboratory, Bar Harbor, Maine.

Inbred DBA / 2J (males or females) were also obtained from Jackson Laboratory, Bar Harbor, Maine.

Media

Phosphate buffered saline (PBS), "RPMI 1640¾ fetal calf serum (FCS) - (portion # R776116), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer was obtained from Gibo,

Grand Island. 2-mercaptoethanol from Eastman Kodak, Rochester, N.Y. Cells were washed with PBS and grown in RPMI containing 10% FCS, 10 mM HEPES buffer and 5x10 M 2 mercaptoethanol.

149595 2 4

Treatment with test compounds

The test animals (C57 BL / 6J) were injected (iv or ip) with different concentrations of H-SAR-LYS-SAR-GLN-N ^ (test compound. Identification number GO40) in 0.2 ml volume 24 for 05 hours before are sacrificed for the trials.

Cellepraeparater

Spleen cell suspensions in C57 BL / 6J or DBA / 2J mice were prepared by cutting the organ and pressing through a wire tissue (# 60) with the plunger of a 5 cm syringe into a "falcon" petri dish (falcon 3002 , 15x60 mm). The cell suspensions were allowed to stand at room temperature for 10 minutes to allow insertion of the larger tissue pieces. The cell suspensions were then transferred to 15 ml Corning centrifuge tubes (Corning 25310) and centrifuged for 10 minutes at 15 1500 OPM in a Beckmen TJ-6 centrifuge. All cell suspensions were washed at least three times more with PBS. After the third washout, the response cells (C57 BL / 6J) were resuspended in culture medium and counted on Coulter counter. DBA / 7 2J (stimulator cells) were resuspended in RPMI to 10 cells / ml per ml. 30 µg mitomycin C was added to each ml of DBA / 2J spleen cells and the mixtures were incubated at 37 ° for 30 minutes.

After treatment with mitomycin C, the spleen cells were washed three times with PBS to remove any excess mitomycin C. The DBS cells were then resuspended in the culture medium and counted on the Coulter counter.

Mixed Lymphocyte Cultures (MLC) MLC was placed in microtiter trays (Linbro Chemicals, New Haven, Conn., IS-MVC-96). Each tray contained 96-V bottom sources. The outermost sources surrounding the edge of the plate were not used for cell culture but were filled with PBS to avoid evaporation from the culture sources. 60 samples were placed in each V-bottom tray. Usually, each tray contained three response cell concentrations (20 repeats of each) and one stimulator cell concentration. The response cells were usually suspended to concentrations of 7.5x10, 5x105 and 2.5x10 per ml and 0.1 ml was added to each source.

The same stimulator-cell concentration was used throughout the plate. Control plates containing only response cells without stimulator cells were also designed to assess the background 05 stimulation due to the medium. The cells were cultured for 6 days at 37 ° C in a humidified incubator containing 5% CC

Attack-cells

The attack cell used in the cytotoxic sample was a DBA mastocytoma cell line P815. The cell line was maintained at 10 g by routine passage through DBA / 2J mice. 5x10 8 P815 cells were used for each passage and the tumor cells from the peritoneal cavity of the bases were used 4 to 5 days after passage. The tumor cells from the peritoneal cavity were washed three times with 51 PBS and then labeled with Cr at a concentration of 7 15 100 µl per 10 cells. The labeling was done for one hour at 37 ° C in a humidified incubator. The labeled attack cells were then washed three times with PBS to remove any excess labeling.

Cytotoxic Sample 20 After 6 days of culture, 0.1 ml of the medium was removed from each source without disturbing the cell stain. Next, pipette using an automatic micropipette (MLA pipette) 4 51 100 µl of attack cells containing 2.5x10 Cr-labeled attack cells in each source, resuspending the cell stain during the process (a new pipette tip must be used for each source). Microtiter trays were then centrifuged at room temperature at 1000 rpm for seven minutes on "Sorvall GLC-2B". The trays were then incubated for 4 hours at 37 ° C. 100 microliters of the above liquid was then transferred into "gamma counting tubes. ("Amershen 196271") «The tubes were then counted on Beckman® gamma counter (Beckman 310). The tubes are usually counted for one minute.

Determination of the frequencies of the precursors of cytotoxic lymphocytes (CLP) 149595 26

The boundary dilution assay is an all or nothing response sample described using the Poisson probability distribution. The probability of a non-response is given by the zero-order expression Po = e 'where δ = the frequency of CLP and N = 05 the number of lymphocytes per source. A plot of the logarithm of the amount of non-responding cultures to the cell dose should thus give a straight line with a slope of - 6, which is the frequency of CLP.

In the example, the mean background chromium release (spontaneous release) was calculated from 20 sources containing only response cells (C57 BL / 6J) without stimulator cells. The test sources were marked as positive if their counts were greater than the mean spontaneous value at more than 2.07 standard deviations (P <0.05). The spontaneous lysis usually ranged from 9-15% of the total counts incorporated

-6N

in the attack cells. According to Poisson's equation Po = e, when Po = e ^ = 0.37 (corresponding to 37% non-responding cultures), δ = 1 / N such that the reciprocal value of the responding cell number corresponding to 37% non-responding responding to 20 cultures is the CLP frequency. Usually, the number of cells per source and their corresponding value for percent non-response were entered into the computer, which calculates the most appropriate line of regression through these points and the number of cells per source corresponding to 37% non-responding cultures, whereby the reciprocal value of which is the frequency of CLP.

results

The frequencies of cytotoxic lymphocyte precursors from each stimulator cell concentration were plotted as a function of the dose of the test compound. The mean and standard deviation 30 at the 20 repetitions were also calculated for comparison. The 5 three stimulator cell concentrations used were 10 (sub-optimal stimulation) 2.5x10 (optimal stimulation) and 5x10 (over-optimal stimulation). It was found that the test compound promoted the production of cytotoxic lymphocyte precursors at concentrations ranging from ca. 1 pg / mouse to approx. 100 ng /

Claims (1)

149595 mice (equivalent to about 50 µg / kg to about 5 µg / kg body weight) in the presence of sub-optimal stimulator cell concentrations. Therefore, the test compound acts as an immunoregulator at these concentrations to enhance the cellular 05 immune response of the treated mice. An analogous method for preparing FTS analog tetra or pentapeptide derivatives of the general formula RX-LYS-Y-GLN-R 'wherein X is selected from the group consisting of L-alanyl, D-alanyl and sarcosyl, Y is selected from the group consisting of D- and L-seryl, sarcosyl, 2-methyl-alanyl, and D-alanyl, R is H, GLN or SAR, and R 'is OH or NH 2 or pharmaceutically acceptable acid addition salts thereof, esterifies L-glutamine protected on the amino group to an insoluble resin polymer by covalent bond, removes the α-amino protecting group from the L-20 glutamine moiety, reacts with the Y-amino acid protected on the amino group , for coupling the Y-amino acid to the L-glutamine resin, removes the α-amino protecting group from the Y-amino acid moiety, converts with «-amino-protected L-lysine to link L-lysine to Y-amino acid-L- the glutamine resin, removes the cr-25 amino protecting group from the L-lysine moiety, reacts with the necessary X-amino acid or is the required RX dipeptide protected on the α-amino group for coupling amino acid or dipeptide to the L-lysine-Y-amino acid L-glutamine resin, wherein all reactive side chains of the incoming amino acids are protected during the reaction, whereupon the resin and all protecting groups of the peptide are cleaved with an acid or ammonia, and then, if desired, a prepared sequence is converted into a pharmaceutically acceptable acid addition salt thereof. 35