FI96115B - Process for the preparation of peptides with T helper cell activity - Google Patents

Description

96115

A method for preparing peptides having T cell activity and activity

Divided by application FI 900267 5

The present invention relates to a process for the preparation of synthetic peptides capable of stimulating helper T cell activity. More specifically, the peptides of the present invention are pentapep-10-based tysplenin molecules.

Background of the invention

Immune regulatory proteins, thymopoietin and tys-plenin (formerly referred to as "spelin"), have been isolated from bovine and human thymus and 15 spleens, respectively. In addition, small peptides that mimic the biological activity of thymopoietin have been chemically synthesized and further modified to provide additional properties such as resistance to enzymatic action. See, e.g., U.S. Patent No. 20,450,853.

A large number of articles and patents on such proteins and synthesized peptides have now been published. U.S. Patent No. 4,190,646 discloses the pentapeptide thymopentin, which is the active site of thymopoietin-25 and has the sequence Arg-Lys-Asp-Val-Tyr, and peptide compositions in which the amino and / or carboxyl terminus of this pentapeptide is substituted with various groups. Both thymopoietin and thymopentin induce biological changes in two human T cell-30 lines, CEM and MLTLT-4, thus demonstrating their role in stimulating biological activities in T cells. None of the thymopentin analogs shorter than the pentapeptides (5 amino acids in the sequence) were found to be active in CEM cells.

96115 2 U.S. Patent 4,923,964 discloses a 48 amino acid immunoregulatory protein isolated from human spleen, splenin (hereinafter referred to as "tysplenin"). Bovine splenin stimulates helper T cell activity in vivo in mice. Humanizationplenin is thus expected to show analogous biological activity in humans. Human application planenin was described in the above-identified application to induce an increase in intracellular cGMPr in the human T cell line MOLT-4. The active site of bovine splenic-10, called SP-5, covers its amino acid residues 32 to 36 and has the sequence Arg-Lys-Glu-Val-Tyr. U.S. Patent 4,923,964 discloses Arg-Lys-Ala-Val-Tyr as the active site for a human sequence.

15 In contrast to thymopoietin, thysplenin does not alter the biological activity of CEM cells. Thus, tysplenin appears to be involved in the stimulation of T cell suppressor activity, not T cell suppressor activity. See also, for example, Goldstein, G., Na-20 ture (London) 247 (1974) 11-14; Basch, R.S., and Goldstein, G., Proc. Natl. Acad. Sci. U.S. 71 (1974) 1474-1478; Scheid, M.P., et al., J.Exp.Med. 147 (1978) 1727-1743; Scheid, M.P., et al. . Science 190 (1975) 1211-1213; Ranges, G.E., et al. . J. Exp. Med. 156 (1982) 1057-1064; T.

li 25 Audhya et al. Biochem. 20 (1981) 6195-6200; Venkatasubramanian, K., et al. . Proc. Natl. Acad. Sci. U.S. 83 (1986) 3171-3174; Malaise, M.G., et al., "Immuno-regulatory UCLA Symposium on Molecular and Cellular Biology", ed. Goldstein, G., et al., Liss, New York, 1986; 30 Sunshine, G.H., et al., J. Immunol. 120 (1978) 1594-1599, ”and E. Rentz et al. . Arch. Geschwulstforsch .. 54: 2 (1948) 113-118. See also U.S. Patent Nos. 4,190,646; 4,261,886; 4,361,673; 4,420,424; and 4,629,723. For a discussion of other background material and related biological methods related to the present invention, reference is made to the patent publications, application publications, and articles described above.

U.S. Patent 4,428,938 to Kisfaludy et al. and published January 31, 1984, 5 discloses certain peptides that affect immune regulation. These peptides include the following tetrapeptides:

Arg-Lys-Asp-Val Arg-Lys-Asn-Val 10 Arg-Lys-Ala-Val

Arg-Lys-Asp-Ala Arg-Lys-Asp-Ile Arg-Lys-Glu-Val Glp-Arg-Lys-Asp.

The '938 patent generally includes salts, amides, (lower alkyl) esters, and protected derivatives of these sequences, as well as methods of using these sequences to treat immunological disorders due to thymic deficiencies. In this patent, the activity of these peptides was tested in vitro by an E-rosette assay.

Such tetrapeptides were reported by the same investigators in Kisfaludy et al. . Hoppe-Sevler's Z. Phvsiol. Chem. 364 (1983) 933-940. In this article, it was criticized that the sequence Arg-Lys-Glu-Val was a very active analog in an in vitro E-rosette experiment, and that the sequences Arg-Ala-Asp-Val and Arg-Lys-Ala-Val have a very strong decreased activity.

There is still a need in the art for other peptides that would be useful in stimulating the human immune system in a variety of T cell deficiency conditions.

FI patents 86,860, 70,905 and 79,329 all relate to pentapeptides whose amino acid sequences at position 3 are Asp.

96115 4 EP-25 897 is directed to pentapeptides of the formula

G-K-Q-X-M

wherein G may be Arg, K may be Lys, Q may be Glu, Asp or 2-aminoadipic acid; X may be Val or Ile and M is a hydrophobic amino acid or an ester or amide thereof.

There is no description or suggestion in said references for a pentapeptide in which Ala is at amino acid position 3. Although both asparagine and alanine are hydrophilic, the two amino acids differ in their acidity. Asp is a basic amino acid. Instead, Ala is a neutral amino acid.

The activity of a small peptide, such as a peptide prepared according to the present invention, is very al-15 tis for amino acid changes. The choice of a particular amino acid can alter the relative acidity or basicity of the amino acids incorporated. Whether the amino acids were hydrophilic or hydrophobic, the amino acids cannot be interchanged. Due to the relative size and relative acidity of amino acids, such amino acid substitutions can cause significant changes in activity or even loss of activity. Therefore, it was not foreseeable that replacement of amino acids with amino acids of different size and acidity would result in a peptide having the biological activity of the peptides of the present invention.

SUMMARY OF THE INVENTION

The present invention describes a series of tysplenin peptide analogs capable of inducing biological activity in a MOLT-4 T cell line. In contrast to the previously reported thymopentin nianal analogs of less than five amino acids in length and tysplenin-related, peptides are active in inducing T cell adjuvant activity in MOLT-4 cells, as described herein.

Il 96115 5

The present invention relates to a process for the preparation of a therapeutically useful peptide having the amino acid sequence of the formula R2-Arg-X'-Lower-Y'-Z'-R3 wherein R2 is H, lower alkyl, acetyl, formyl, lower alkanoyl or des-amino; X 'is Pro, dehydro-Pro, hydroxy-Pro, D-Lys, Aib or Lys; Y 'is an amino acid in D or L form which is selected from Val, Ile, Leu, Ala, Asp, Glu, Gln, Lys; Z 'is an amino acid in D or L form selected from Tyr, Val, Leu, His, Ala and Trp; R3 is OH or NR4RS, wherein R4 and R5 are hydrogen or straight-chain or branched alkyls or alkenyls having 1 to 6 carbon atoms, or R4 and R5 together form a cyclic methylene group of 3 to 7 carbon atoms.

These new pentapeptides are characterized by the ability to increase cGMP activity in the human T cell line MOLT-4.

Surprisingly, these peptides and compositions containing these peptides retain the biological activity of humanizing plenin. Many of these peptides are also characterized by improved resistance to attacks by endo- and exopeptidases as well as trypsin-like enzymes in the gastrointestinal tract and serum. Thus, these peptides offer significant advantages in the treatment of immune deficiencies. In particular, said Penta peptides in which X 'is Pro or Aib have a surprising resistance to degradation by enzymes such as serum mipeptidases.

Other aspects and advantages of the present invention are disclosed in the following detailed description, which includes examples of presently preferred embodiments.

96115 6

Brief description of the drawings

Figure 1 is a graphical representation of a MOLT-4 cGMP assay showing cGMP levels (picograms / ml) as a function of peptide concentration (micrograms / ml) and comparing 5 thymopentin (TP-5) and peptides prepared in accordance with this invention. activity to controls.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a series of pentapeptides characterized by the ability to induce activity in MLTLT-4 cells of the formula R2-Arg-X'-Ala-Y '-Z' -R3 or a pharmaceutically acceptable acid or base thereof. -addition salt, wherein in the formula R2, X ', Y', Z 'and R3 are as defined above.

15 As used herein, the term "lower alkyl" includes branched and straight chain saturated hydrocarbons having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, pentyl, hexyl, and the like, while the term "lower alkanoyl" refers to

20 a group O is tried

n (lower alkyl) -C-,

Throughout this application, the amino acid moieties of the peptides and certain materials used in their preparation are identified by abbreviations for convenience.

»9 25 Most of the three-letter abbreviations used for amino acids are quite well known. Other lesser known abbreviations are Asu, from aminosuccinimidyl, and Glp, from pyroglutamyl (also p-Glu). Unless otherwise stated, all amino acids are in the L-isomer configuration. For the D-isomer configuration, it is labeled.

Certain preferred pentapeptides to be prepared in accordance with the present invention are those pentapeptides of the formula wherein X 'is Pro or Aib. More preferred peptides are those peptides of the preceding formula wherein X 'is Pro, Y' is Val and Z 'is Tyr. Even more preferred pentapeptides are the following peptides: li 96115 7

Arg-Pro-Ala-Val-Tyr

Arg-Pro-Ala-Val-Tyr-NH2 acetyl-Arg-Lys-Ala-Val-Tyr-NH2.

However, acids capable of forming salts with these peptides include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and the like. Organic acids can also be used to form the salts of the invention, e.g. formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, anthric acid, succinic acid, maleic acid, fumaric acid, fumaric acid, fumaric acid, similar.

A non-exhaustive list of bases capable of forming salts with peptides having acidic proportions include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like. Organic bases for such use include, but are not limited to, mono-, di-, and tri-alkyl and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine).

The peptides described herein can generally be prepared according to known techniques. The method according to the invention is characterized by what is stated in claim 1. The synthetic preparation of the practically described polypeptide can be performed according to the solid phase synthesis method described by Merrifield in A.A.C.S. 85 (1963) 2149-2154. This technique is well understood and is a common method for preparing peptides. In the solid phase synthesis method, protected amino acids are added step by step to a growing peptide chain covalently linked to a solid resin moiety. In this procedure, reagents and by-products are removed by filtration to avoid purification of intermediates. The general guideline for this method depends on the attachment of the first amino acid of the chain to the solid polymer by a covalent bond.

The following protected amino acids are added stepwise one at a time, or in groups, until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved off.

The amino acids can be attached to any suitable resin polymer. The resin should contain a functional group to which the first protected amino acid can be firmly attached by a covalent bond. Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethyl methacrylate and polystyrene. Suitable protecting groups for use in such a synthesis include t-butyloxycarbonyl (BOC), benzyl (BZL), t-amyloxycarbonyl (AOC), tosyl (TOS), o-bromophenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (BZLC12) and phenyl-20-lymethoxycarbonyl (Z or CBZ). Additional protecting groups are identified in the preceding text as well as in J.F.W. McOmie, "Protective Groups in Organic Chemistry," Plenum Press, New York, 1973. Both of these books are incorporated herein by reference.

*.! In a general procedure for preparing peptides in accordance with the present invention, an initially protected C-terminal amino acid is attached to a resin in accordance with the present invention. After attachment, the resin is filtered, washed, and the protecting group (preferably t-butyloxycarbonyl) containing the alpha-amino group of the C-terminal amino acid is removed. The removal of this protecting group must, of course, be carried out without breaking the bond between that amino acid and the resin. The resulting resin peptide is then coupled with the second last C-terminal amino acid protected. This coupling is accomplished by forming an amide bond between the free carboxyl group of the second amino acid 961195 and the amino group of the first amino acid attached to the resin. This sequence of steps is repeated with successive amino acids until all amino acids are attached to the resin. Finally, the protected peptide is cleaved from the resin and the protecting groups removed to obtain the desired peptide. The cleavage procedures used to separate the peptide from the resin and to remove the protecting groups depend on the choice of resin and protecting groups, and are familiar to those skilled in the art of peptide synthesis.

Alternative techniques for peptide synthesis are described in "Peptide Synthesis", Bodanszky et al. . 2.

ed., John Wiley & Sons, 1976. For example, the peptides described herein can also be synthesized using conventional peptide synthesis in solution procedures in which amino acids or peptide fragments are coupled either 15 steps at a time or in groups using chemical or enzymatic methods to form an amide bond. These solution synthesis methods are known in the art.

The peptides of this invention have been found to exhibit biological activity similar to that of humanized plenin, as disclosed in the aforementioned U.S. Patent 4,923,964 and the aforementioned articles. This biological activity is primarily demonstrated by an assay measuring the induction of cyclic GMP production in the human T cell line MOLT-4 compared to humanizationplenin and human thymopentin. Induction of cGMP production by a peptide of the invention in this assay demonstrates the ability of that peptide to bind to the humanizationplenin receptor center on the cell surface and to induce humanizationplenin-like biological activity.

Many of these pentapeptides offer another significant advantage over humanized splenin. Many of the peptides 35 of the present invention are characterized by resistance to enzymatic degradation by either digestive or serum enzymes. Thus, they show a prolonged half-life in vivo when injected into a biological subject. Another advantage of many of these peptides is that they can be administered orally. Humanizationplenin itself is a molecule that is too large to be effectively administered orally and to be digested in the gastrointestinal tract.

Prior to testing the peptides 10 of the invention, it was not expected that pentapeptide analogs of humanizing splenin with the same biological specificity could be prepared because Arg-Lys-Ala-Val-Tyr, which is a bovine pentapeptide SP-5 (Arg-Lys-Glu-Val- Tyr) analogue in humans, is inactive in MOLT-4 cells. Thus, it was unexpected that pentapeptide analogs of humanityplenin were found that showed the same biological activity as the intact humanityplenin molecule.

Due to their immunoregulatory properties, these peptides are therapeutically useful in the treatment of humans, and possibly animals, because of their ability to induce T cell differentiation and maturation, which (T cells) are able to participate in the body's immune response. As a result, these peptides are considered to have a variety of therapeutic uses.

Peptides prepared in accordance with this invention are considered useful in assisting the body's collective immunity in the sense that they increase or assist in the therapeutic stimulation of cellular immunity. They are thus useful in the treatment of diseases associated with chronic inflammation, such as fungal and mycoplasma infections, tuberculosis, leprosy, acute or chronic viral infections, and the like.

Pharmaceutical compositions containing said peptides or said peptides or their acid or base salts are generally considered useful in any region of cellular immunity, and in particular in the case of immunodeficiency. Thus, in cases of excessive antibody production due to unbalanced T cells, these peptides are able to correct this condition by stimulating T cell function. Thus, they are expected to be therapeutically useful in certain autoimmune diseases in which harmful antibodies are formed, such as systemic rubella, rheumatoid arthritis or the like.

In its most comprehensive application, said peptides or pharmaceutical compositions containing them are useful for regulating the immune system of a subject, human or animal, in need of such regulation. As used herein, the term "regulate" means that the compounds in question cause the immune system to return from an abnormal, diseased condition to a normal, equilibrium state, and although there may be many applications for this regulation in the correction of immunological deficiencies (e.g. -20 list for the repair of conditions with immunological activity (e.g. autoimmune diseases).

The disclosed peptides are useful for treating conditions resulting from relative or absolute deficiencies in a subject's immune system, particularly in the ΤΙ:25 cell helper function, wherein said subject is administered a therapeutically effective amount of at least one peptide of this invention.

As used herein, the term "therapeutically effective amount" means an amount effective to treat the aforementioned conditions. Peptides can also be used to induce T cell differentiation and maturation, wherein an effective inducing amount of at least one peptide is administered to a subject.

Pharmaceutical compositions may further be prepared for the practice of the methods of treatment. To prepare pharmaceutical compositions, the peptide prepared in accordance with this invention is combined as an active ingredient into a pharmaceutical mixture with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. 5, this carrier may take a wide variety of forms depending on the desired form of the preparation administered, e.g. oral, sublingual, rectal, nasal, or parenterally administrable form.

The pentapeptide of the invention is generally active when administered in amounts in excess of about 1 microgram / kg body weight, and is preferably in amounts from about 0.001 to about 10 mg / kg body weight.

In general, the same dosage range can be used to treat said diseases or conditions in which immunodeficiency is to be treated. Large amounts (e.g., about 10 to 100 mg / kg body weight) are useful in suppressing excessive immune activity.

The following examples are provided to illustrate the invention, in which the invention is not particularly limited thereto. In the examples and throughout the specification, parts are by weight unless otherwise indicated. The following abbreviations are used in the examples: TFA from trifluoroacetic acid; HOAc acetic acid; CH2Cl2 from methylene chloride; CH3CN from acetonitrile; DMF from dimethylformamide; NH40Ac ammonium • · «·: 25 from acetate; NUjOH from ammonium hydroxide; n-PrOH from n-propanol; n-BuOH n-butanol; pyridine; DCC from dicyclohexylcarbodiimide; HOBt 1-hydroxybenzotriazole list; DMAP from dimethylaminopyridine; HF hydrogen fluoride; TCA from trichloroacetic acid; BHA from benzhydrylamine; and 30 MeOH from methanol.

Comparative Example 1

Synthesis of the tetrapeptide acetyl-Arg-Pro-Ala-Val-NH2

The above tetrapeptide was synthesized using solid phase synthesis by step-by-step couplings. The alpha-amino group of all amino acids

II

96115 13 was protected with a t-butyloxycarbonyl (BOC) group. Tosyl (TOS) was used to protect the Arg side chain. 2.5 equivalents of protected amino acids were used in excess of each substitution equivalent in hart-5. All couplings were performed with DCC / HOBt using equivalents corresponding to protected amino acids. All amino acids were dissolved in CH 2 Cl 2 except Arg, which was dissolved in DMF.

The washing, deprotection and coupling steps were as follows: 1. CH 2 Cl 2 2x3 min 2. 50% TFA - CH 2 Cl 2 1x3 min 3. 50% TFA - CH 2 Cl 2 1 x 30 min 4. CH 2 Cl 2 1x3 min 15 5. 40% isopropanol - CH 2 Cl 2 2x3 min 6. CH 2 Cl 2 2x3 min 7. 10% diisopropylethylamine 2 x 10 min 8. CH 2 Cl 2 1x3 min 9. amino acid 1x3 min 20 10. HOBt 1x3 min 11. DCC 1 x 120 min 12. CH 2 Cl 2 2x3 min 13. 40% isopropanol - CH 2 Cl 2 2x3 min 14. DMF 1x3 min 25 15. CH 2 Cl 2 2x3 min a. Synthesis

The protected peptide was synthesized using a Beckman Model 990 peptide synthesizer. The obtained tetrapeptide resin was 0.67 meg / g and the synthesis started with 5.0 g (3.35 mmol) of 30 resin. The resulting tetrapeptide was then acetylated by treating the resin with: 1. CH 2 Cl 2 2x3 min 2. 50% TFA - CH 2 Cl 2 1x3 min 3. 50% TFA - CH 2 Cl 2 1 x 30 min 35 4. CH 2 Cl 2 1x3 min 96115 14 5. 40% isopropanol - CH 2 Cl 2 2x3 min 6. CH 2 Cl 2 2x3 min 7. 10% diisopropylethylamine 2 x 10 min 8. CH 2 Cl 2 1x3 min 5 9. 50% acetic anhydride - CH 2 Cl 2 + 1x 120 min catalytic amount of DMAP 10. CH2Cl2 2x3 min 11. 40% isopropanol - CH2Cl2 2x3 min 12. DMF 1x3 min 10 13. CH2Cl2 2x3 min

The peptide-resin was dried under reduced pressure after first washing with ethanol (2 x 50 mL).

b. Cleavage with headset

The crude tetrapeptide was obtained by digestion of resin-peptide-15 with HF (25 mL) and anisole (25 mL) for 4 hours at 0 ° C. The HF was removed under reduced pressure. Diethyl ether (150 ml) was added to the mixture. The solution was stirred and transferred to a sintered glass funnel and further washed with diethyl ether (3 x 100 mL). The peptide was extracted from the resin with 10% HOAc (3 x 100 mL) and a lyophilized aqueous solution gave 1.20 g of the peptide product.

c. Purifying

The product was purified by preparative high performance liquid chromatography (HPLC) under the following conditions: • Whatraan column Partisil M20 10/50 ODS3; 300 mg sample using an isocratic 15% CH 3 CN / 0.01 M NH 4 OAc system (pH is adjusted to 5 with HOAc); flow rate 15 ml / min; Detection wavelength at 220 nm. All pure fractions were collected. The organic solvent was evaporated, after which the aqueous solution was freeze-dried to give 258 x * mg of a white solid.

Amino acid analysis: Pro, 1.00; Ala, 1.00; Vai, 1.00; Arg, 1.01; 83% peptide.

Thin layer chromatography run (Silica 60 / Analtech): 35 Rf (I) = 0.50 (butanol: acetic acid: water / 3: 1: 1) 96115 15

Rf (II) = 0.59 (butanol: pyridine: acetic acid: water / 4: 1: 1: 2)

Rf (III) = 0.29 (butanol: acetic acid: water ethyl acetate / 1: 1: 1: 1).

5 Comparative Example 2

Synthesis of Tetrapeptide Acetyl-Arg-Pro-Val-Ala-NH2 a. Synthesis

The tetrapeptide was prepared on an Applied Biosystems 430A peptide synthesizer. A 1-hour cycle Melo procedure (manufacturer's form 1.40) was used, which coupled each amino acid once except for BOC-Tos-Arg, which was double coupled. The following prepackaged starting materials were used in the synthesis: 15 Reagent Source mmol Amount p-methyl-BHA resin ABI 0.50 760 mg BOC-Tos-Arg ABI 2.0 857 mg BOC-Pro ABI 2.0 430 mg BOC-Val ABI 2.0 434 mg 20 BOC-Ala ABI 2.0 378 mg

After the BOC group of arginine was removed and the peptide-resin was neutralized and washed, the compound was acetylated using a 10% solution of acetic anhydride in CH 2 Cl 2 · * 25 (15 mL) in the presence of 4-dimethylaminopyridine (20 mg).

After 60 minutes, the peptide was tested in a ninhydrin assay to ensure complete acetylation. The peptide-resin was washed with CH 2 Cl 2 (3 x 15 mL) and dried in vacuo (room temperature) to give 1.0 g of material.

30 b. Cleavage with headset

The peptide was cleaved from the resin using 10 ml of HF in the presence of 1.5 ml of anisole. The mixture was stirred at 0 ° C for 1.5 hours, after which the HF was removed under reduced pressure. The mixture was washed with Et 2 O (3 x 50 mL) and air dried. The peptide / resin mixture was extracted with 25% aqueous HOAc (3 x 50 mL).

96115 16 c. Purifying

The aqueous filtrate was freeze-dried to give 240 mg of crude material. This material was used in Amberlite IRA 68 (acetate form) ion exchange resin in water. Fractions containing peptide 5 were combined and lyophilized (220 mg).

The peptide was purified on a Whatman Partisil 20M 10 ODS3 (22 mm x 50 cm) column using 15% acetonitrile (0.1% trifluoro-10 acetic acid) and 0.1% acetonitrile as eluting solvents (10.0 mL / min). aqueous solution of trifluoroacetic acid. Elution was monitored by HPLC and the appropriate fractions were combined and the solvent removed under reduced pressure. The residue was dissolved in water (H 2 O) and freeze-dried (solution) twice to give 167 mg of product in 50% overall yield.

Amino acid analysis: Arg, 1.04; Pro, 1.02; Vai, 1.00; Ala, 1.01; 72% peptide.

Thin layer chromatography run (Silica Gel GF 250 micrometer plates): 20 Rf (I) = 0.37 (n-BuOH: HOAc: H 2 O / 3: 1: 1)

Rf (II) = 0.15 (CHCl 3: MeOH: HOAc / 60: 35: 5)

Rf (III) = 0.48 (n-BuOH: HOAc: H 2 O: EtOAc / 1: 1: 1: 1).

Comparative Example 3

Synthesis of the tetrapeptide acetyl-Arg-Pro-Asp-Val-NH2 •: 25 a. Synthesis

The title compound was synthesized by conventional solid phase synthesis using an automated Beckman Model 990 synthesizer. The synthesis was started with 5.0 g of BHA resin (0.67 meg / g). After coupling of BOC-Arg (Tos), the t-bu-30 ethyloxycarbonyl protecting group was removed with a 50% solution of TFA in CH 2 Cl 2 and the resin peptide was acetylated using a 50% solution of acetic anhydride in CH 2 Cl 2 in the presence of a catalytic amount of DMAP. The peptide-resin was washed with ethanol (2 x 50 mL) and dried under reduced pressure overnight before cleavage with HF. The dry peptide-resin weighed 6.5 g · 96115 17 b. Cleavage with HF

The crude tetrapeptide was obtained by cleavage of 6.5 g of resin peptide with HF (25 ml) and anisole (25 ml) at 0 ° C for 4 hours. The HF was evaporated under reduced pressure and diethyl ether (150 ml) was added to the reaction mixture. The crude peptide and resin were transferred to a sintered glass funnel and washed with diethyl ether (3 x 100 mL). The peptide was extracted with 10% HOAc solution (3 x 100 mL) and the aqueous solution was freeze-dried to give 900 mg of a white solid.

10 c. Purifying

The crude product (purity 98% by HPLC) was desalted on a Sephadex LH-20 column (100 g, 2.5 cm x 89 cm) using 10% HOAc as eluent. After freeze-drying, the product was a white fluffy solid weighing 800 mg.

Amino acid analysis: Arg, 0.99; Vai, 1.00; Pro, 1.05; Asp, 1.01.

Thin layer chromatography run (Silica 60 / Merck, 250 F):

Rf (I) = 0.51 (n-BuOH: HCOAc: H 2 O / 3: 1: 1) Rf (II) - 0.60 (n-BuOH: HOAc: H 2 O: EtOAc / 1: 1: 1: 1 )

Rf (III) = 0.59 (n-BuOH: pyridine: HOAc: H 2 O / 4: 1: 1: 2).

Comparative Example 4 Synthesis of N-alpha-formyl-Arg-Pro-Asp-Val a. N-Butyloxycarbonyl-beta-benzyl-aspart ** 25-style-valine benzyl ester In 80 ml of CH 2 Cl 2 was dissolved 3.23 g of Nt- butyloxycarbonyl-beta-benzyl-Asp and 3.97 g of Val-Benzyl ester p-tosylate. Diisopropylethylamine (1.74 ml) was added and the solution was brought to 5 ° C. To mixture 30 was added 2.06 g of DCC. The mixture was stirred at 5 ° C for 30 minutes and then for a further 2 hours at ambient temperature. The precipitate was filtered off and the filtrate was extracted with water, 10% citric acid solution and saturated sodium bicarbonate solution. Removal of solvent 35 gave an oil which was purified by chromatography on silica gel 60 (eluting) with 97: 3 CH 2 Cl 2 -MeOH. The product, (which was) an oil, weighed 3.46 g.

b. Benzyl ester of N-t-butyloxycarbonyl-prolyl-beta-benz-yl-aspartyl-valine Deprotection of 3.41 g of the previous product in 60 ml of 2: 1 CH 2 Cl 2 -TFA for 30 minutes gave the trifluoroacetate salt as an oil. The free amine was obtained by neutralization with saturated sodium carbonate solution and extraction into CH2Cl2. The volume of the extract was reduced by evaporation, after which it was added to 1.05 g of N-t-butyloxycarbonyl-Pro. A solution of 0.73 g of H 2 Bt in 2 ml of DMF was added, followed by 0.99 g of DCC. The mixture was stirred for 2.5 hours, then filtered. The filtrate was extracted with water, 10% citric acid solution and twice with saturated sodium bicarbonate solution. After drying, the solvent was removed to leave an oil. The product was purified by flash chromatography on silica gel 60 (eluting) with 9: 1 CH 2 Cl 2 -CH 3 CN. 2.31 g of a colorless oil were obtained from the fraction containing the main component 20. IR spectra: 1740 cm-1, 1695 cm-1 and 1675 cm-1.

c. Benzyl ester of N-alpha-formyl-N * -nitro-arginyl-prolyl-beta-benzyl-aspartyl-valine To 2.23 g of the product of the previous step was added 60 "25 ml of 1: 2 TFA-CH2Cl2. The solution was stirred for 30 minutes, after which the solvent was removed under reduced pressure to leave a waxy solid which was washed with Petro ether. 0.72 g of N-alpha-formyl-N * -nitroarginine and 0.33 ml of N-methylmorpholine-30 were dissolved in 15 ml of DMF. The solution was cooled to -20 ° C and 0.40 g of isobutyl chloroformate was added dropwise. The mixture was stirred at -20 to 10 ° C for 20 minutes, after which a cooled solution of benzyl ester of prolyl-beta-benzyl-aspartyl-valine trifluoroacetate-35 in 20 ml of DMF and 0.33 ml of: in N-methylmorpholine 96115 19 nia. The mixture was stirred at -15 ° C for 20 minutes, after which the cooling bath was removed and stirring was continued at ambient temperature for 2 hours. Most of the solvent was removed under reduced pressure. The residue was dissolved in 50 mL of CH 2 Cl 2 and extracted with water, 10% citric acid solution and saturated sodium bicarbonate solution. Drying of the organic phase and evaporation of the solvent gave 2.08 g of product.

TLC (silica gel-GF): 10 Rf (I) = 0.56 (CH 2 Cl 2: MeOH / 9: 1)

Rf (II) = 0.72 (CHCl 3: MeOH: HOAc / 8: 1: 1).

d. N-alpha-formyl-arginyl-prolyl-aspartylvaline

The protecting groups were removed by transfer hydrogenation using 30 ml of 5% formic acid-ammonium formate on palladium black. The reaction mixture was stirred in a ball sealed flask for 18 hours. The mixture was filtered through Celite, after which the solvent was removed under reduced pressure. The residue was dissolved in water and (solution) lyophilized. The product was purified on a 1.6 x 60 cm DEAE-Sephadex column. Elution was started with 0.02 M ammonium bicarbonate solution, pH 8.5, collecting 150 drop fractions. After collecting 52 fractions, a gradient of 0.02 to 0.20 M ammonium bicarbonate was introduced using a 2 liter liquid volume. The largest peak in the Kro-25 matogram eluted in the middle of fraction 88. Fractions 75-100 were combined and lyophilized to give a fluffy amorphous solid. HPLC: rt = 9.4 min, 10% MeOH - 0.01 N NH 4 OAc, pH 5, 1.5 mL / min, Bondapak

CI8.

30 Thin layer chromatography run (silica gel 60):

Rf (I) = 0.16 (n-BuOH: HCOAc: H 2 O / 3: 1: 1)

Rf (II) - 0.27 (n-BuOH: HCOAc: H2O: pyr / 15: 3: 12:10)

Rf (III) 0.42 (EtOAc: pyr: HOAc: H 2 O / 5: 5: 1: 3). Amino acid analysis: Asp, 0.99; Pro, 0.97; Vai, 35 1.05; Arg, 1.00; 38% peptide.

96115 20

Example 1

Synthesis of the pentapeptide Arg-Pro-Ala-Val-Tyr The peptide was synthesized by the solid phase method starting from BOC- (BZLCl2) -Tyr resin ester (4.4 g, 0.40 meg / 5 g). Three (3) equivalents each of BOC-Val, BOC-Ala, BOC-Pro and CBZjArg were coupled to the resin sequentially. DCC and HOB in 4: 1 CH 2 Cl 2: DMF were used as coupling agents. The resin was digested with HF (40 mL) in anisole (5 mL) for 45 minutes at 0 ° C. The solid residue was extracted with 10 mL of HOAc and the (solution) was filtered, followed by lyophilization of the aqueous solution to give 1.56 g of crude peptide.

Purification was performed on Sephadex SPC 25 resin by chromatography (2.6 x 90 cm column) eluting with a gradual NH 4 OAc gradient: 0.05 M, pH 5 (2 liters), 0.15 M, pH 5 (1 liter), 0.15 M, pH 6.7 (2 liters); flow rate 100 ml / hour, 12.5 ml fractions, detection 280 nm. Isolation of fractions 198-207 gave the title peptide as a colorless solid, 1.13 g.

TLC run (silica gel G, 250): 20 Rf (I) = 0.24 (n-PrOH: NH 4 OH / 84: 37)

Rf (II) = 0.64 (trifluoroethanol: NH 4 OH / 78: 22)

Rf (III) = 0.58 (n-BuOH: HCOAc: H2O: pyr / 15: 3: 12:10). Amino acid analysis: Arg, 1.00; Ala, 0.96; Pro, 0.97; Vai, 1.03; Tyr, 1.01; 67% peptide.

25 Example 2

Biological Activity: Cyclic GMP Assay This assay measures the ability of a peptide prepared in accordance with this invention to bind to a cell membrane receptor in an intact MOLT-4 cell and selectively stimulate the production of cyclic GMP as well as humanized splenin and human thymopentin.

The MOLT-4 cell line was obtained from the American Type Culture Collection, Rockville, Md. Freshly inoculated MOLT-4 cells were grown for 3 days 35, then harvested as described in T. Audhya 96115 21 et al. Arch. Biochem. Biophys. 234 (1984) 167-177. The cells were washed 3 times in PBS and resuspended in RPMI 1640 medium at a concentration of 1.0 x 10 7 cells / ml, then allowed to equilibrate at 37 ° C for 30 minutes 5 before the addition of test pentapeptides (25 microliters) and control peptides. The incubation was allowed to proceed with shaking in a water bath for 4-5 minutes, after which it was stopped by adding 1 ml of ice-cold TCA (10% solution).

Cells in TCA were homogenized and sonicated to release the cyclic nucleotide. The slurry was centrifuged at 3000 x g for 20 minutes at 4 ° C. The resulting precipitate was dissolved in 0.1 N NaOH solution to determine the protein content. TCA was removed from the supernatant fraction by extraction 4 times with 5 ml of water-saturated diethyl ether. After the last extraction, the remaining ether residue was removed by heating in a water bath at 50 ° C for 10 minutes. After lyophilization, the sample was reconstituted in 50 mM acetate buffer (pH 6.2) for 20 cyclic GMP radioimmunoassay.

Figure 1 shows typical dose-response profiles determined for concentrations of 1 to 1000 micrograms / ml for the active peptides acetyl-Arg-Pro-beta-D-Asp-Val-NH2, Arg-Pro-Ala-Val-Tyr and acetyl-Arg-Pro-Ala-25 Val-NH2, compared to thymopentin and inactive peptides acetyl-Arg-Pro-Asp-Pro-NH2, Ala-Lys-Asp-Val and Lys-Lys-Asp-Val-NH2 MOLT -4-cells.

Threshold activity was determined for each peptide tested. This is defined as the lowest concentration of test peptide 30 that induces a higher intracellular level of cyclic GMP than two (2) standard deviations above the control. The intracellular cyclic GMP readings of the controls were less than 0.5 picomol / ml (mean +/- standard deviation). Experimental results were considered positive if the level of cyclic GMP was higher than 2 times (2,916,112 standard deviations +) the level determined for the parallel negative control.

The results of the cyclic GMP assays are shown in Figure 1 and the corresponding Table I, in which pep-5 tides have been analyzed compared to thymopentin and control peptides. These results were compared to thymopentin (Arg-Lys-Asp-Val-Tyr) in MLTLT-4 cells because the humanization-plenin pentapeptide "SP-5" (Arg-Lys-Ala-Val-Tyr) is inactive in MLTLT-4 cells. . These results demonstrate the biological activity of pep-10 tides in stimulating T cell helper activity in MLTLT-4 cells.

* 1 ·

Table I

23 9 6115 cGMP concentration (picograms / ml Peptide concentration 5 (micrograms / ml): _1_10_100_1000

Arg-Lys-Asp-Val-Tyr 6.4 18.3 20.8 21.9

Arg-Pro-Asp-Val-NH2 0.1 17.1 19.31 24.8 10 Acetyl-Arg-Pro-Asp- 4.55 8.99 19.62 24.09

Val-NH ^

Acetyl-Arg-Pro-Glu-4j7 9.41 16.76 25.00

Val-NH ^

Acetyl-Arg-Pro-Ala-14.6 19.1 24.5

Val-NH „B 2. 37 and 17.7 24.9

Acetylx-Arg-Aib-Glu- 1 · '»

Val_NH2 18.75 29.68 34.98 40.23

Acetyl-Arg-Pro-Gln

Val-NH 2 4.1 11.9 7.9 30.8

Acetyl-Arg-Pro-Glu-Val

Acetyl-Arg-Aib-Ala-Val- 18.78 30.45 36.17 39.48 nh2 yn Acetyl-Arg-Pro-Ä-D-Asp-6.6 14.3 20.5 25.0

Val-NH2 ^

Acetyl-Arg-Pro - ^ - Asp- 20.56 31.83 36.66 34.95

Gly-NH 2

Lys-Lys-Asp-Val-NH 2 0.33 0.31 0.34 0.33

Lys-Arg-Asp-Val 0.44 0.43 0.44 0.39 25 Arg-Gly-Asp-Ser 0.71 0.50 0.68 0.58

Arg-Pro-Ala-Val-Tyr 0.09 16.41 27.90 37.14

Arg-Pro-Ala-Val-Tyr-NH2 3f7l 14.15 12.83 12.57

Acetyl-Arg-Lys-Ala-Val- 3.00 8.86 12.83 18.43 30 Tyr-NH2 35 0 peptide / control is 0-0.3 picograms / ml

Claims (4)

96115 A process for preparing a therapeutically useful peptide having the amino acid sequence of formula R2-Arg-X'-Al-Y'-Z'-R3 wherein R2 is H, lower alkyl, acetyl, formyl, lower alkanoyl or des amino; X 'is Pro, dehydro-Pro, hydroxy-Pro, D-Lys, Aib or Lys; Y 'is an amino acid in D or L form selected from Val, Ile, Leu, Ala, Asp, Glu, Gln, 15 Lys; Z 'is an amino acid in D or L form selected from Tyr, Val, Leu, His, Ala and Trp; R3 is OH or NR4R5, wherein R4 and R5 are hydrogen or straight-chain or branched alkyls or alkenyls having 1 to 6 carbon atoms, or R4 and R5 together form a cyclic methylene group of 3 to 7 carbon atoms, characterized in that a ) is performed by a solid phase method using a peptide-resin intermediate comprising amino acid groups of the sequence defined above attached to protecting groups which are independently amino-protecting groups, hydroxyl-protecting groups, indole-protecting groups or imidazole-protecting groups, and a resin, resin, arginine can be solidly bonded with a covalent bond; or b) performing solution synthesis of the peptide comprising stepwise or blockwise coupling of the amino acids or peptide fragments of the above formula to form amide bonds therebetween, thereby obtaining a peptide of the formula. 96115 Process according to Claim 1, characterized in that in the peptide X 'is Pro and Y' is Val. Process according to Claim 1, characterized in that the peptide is Arg-Pro-Ala-Val-Tyr Arg-Pro-Ala-Val-Tyr-NH 2 acetyl-Arg-Lys-Ala-Val-Tyr-NH 2. Process according to Claim 1, characterized in that Arg-Pro-Ala-Val-Tyr-NH 2, acyl-Arg-Lys-A1a-Va1-Tyr-NH 2, or Arg-Pro-Ala-Val-Tyr is prepared. . · · »· 115