DD296084A5 - PROCESS FOR THE MANUFACTURE OF HUMAN SPLENINE DERIVATIVES - Google Patents

Abstract

The invention relates to human splenin derivatives of the formula I and to processes for their preparation. The novel human splenin derivatives prepared by standard synthetic methods of peptide chemistry have high immunomodulatory activity and high stability, with no neurotropic side effects. The compounds of the present invention can be used to normalize the function of the immune system in primary and secondary immunodeficiencies and to stimulate bone marrow stem cells. {Human splenin; human splenotritins; human splenopentines; acylated splenotritins; acylated splenopentin; acetylation; Immunobiology; immunodeficiencies; Immune system; Autoimmune reactions; Bone marrow stem cells; AIDS; Pharmacy}

Description

6. Process according to claims 1 to 5, characterized in that the splenin derivatives of the formula I in which X is Lys and R ^{1 is} Val-Tyr-OR ³ or Val-Tyr-N (R ³ R ⁴ ) and Arg and / or Lys and / or Val-Tyr are substituted.

Process according to Claims 1 to 6, characterized in that derivatives of formula I selected from the following group are prepared:

(N ^a -acyl-Arg) -X-Al aR ¹

Arg- (N ^e -acyl-Lys) -Ala-R ¹

(N ^a -acyl-Arg) - (N ^C -acyl-Lys) -alka-R \

where acyl is preferably acetyl, X is preferably Lys and R ^{1 is} preferably Val-Tyr-OR ³ or Val-Tyr-N (R ³ R ⁴ ), valine and tyrosine are optionally substituted.

8. A process for the preparation of (N ^ acyl-ArgMN ^ acyl-LysJ-Ala-R ¹ according to claims 1 to 7, characterized in that Arg-Lys-Ala-R ^{1 is} acylated.

9. A process for the preparation of Arg-fNP-acyl-LysHAIa-R ¹ and (N ^ acyl-ArgM-ys-Ala-R ¹ according to claims 1 to 7, characterized in that partially protected Arg-Lys-Ala-R ¹ acylated and then the N ^a - or N ^e protective group is cleaved.

Field of application of the invention

The invention relates to processes for the preparation of human splenin derivatives of the formula I.

These compounds have excellent immunobiological properties. They have an immunomodulating activity and practice u. a. a direct influence on the proliferation and differentiation of bone marrow stem cells. Areas of application of the invention are the medicine and the pharmaceutical industry.

Characteristic of the known state of the art

For the immune defense of the organism, the coordinated interaction of all blood cells is of crucial importance. The proliferation and differentiation (functionalization) of the blood cells is influenced by hormones secreted by primary and secondary lymphatic organs such as thymus and spleen. For example, bovine thymopoietin (bTP), first described in 1981, stimulates T-lymphocyte differentiation and inhibits B-lymphocyte proliferation, as well as neuromuscular transmission, while bovine splenin (bSP) differentiates between T-lymphocyte and T-lymphocyte B-lymphocytes is stimulated and no effect on the neuromuscular transmission occurs (AUDHYA et al .. Biochemistry 1981, 1201, 6195).

In 1987, the hormones hTP and hSP isolated from human thymus and spleen were described (AUDHYA et al., Proc. Nat. Acad. Sci., USA, 1987, [84], 3345).

The biological effects of these 49 or 48 amino acid hormones are reproduced by their so-called "active centers", fragment 32-36.

These pentapeptides, termed thymo- or splenopentines, have the structure Arg Lys X VaI Tyr, in the case of bTP5 and hTP5 X = Asp, while for bSP5 X = GIu and for hSP5 X = Ala.

Currently, hTP5 is used as a "Timunox" for the treatment of secondary immunodeficiencies.

Object of the invention

The object of the invention is to provide the pharmaceutical industry and medicine with new immunobiologically active compounds.

-3- 296 Explanation of the nature of the invention

The invention has for its object to find new peptides, on the one hand have a higher immunomodulating activity and higher stability and on the other hand, no neurotropic side effects and are easy to produce, and to provide methods for their preparation

 The object is achieved by a process for the preparation of human splenin derivatives of the formula I.

Arg-X-Ala-R ¹ I,

Arg possibly substituted arginine,

X is Sar, Pro, Gly, Ala, Leu, He, Thr, Ser, Lys, VaI, Phe or Cys, which are optionally substituted, R '-OR ³ , -N (R ³ R "), -XZO R ³ or -XZN (R ³ R ⁴ ),

R ³ and R ^{4 are} identical or different and are H, C, -C ₁₈ alkyl, Cj-C ₇ alkenyl, C ₂ -C ₇ alkynyl, C ₇ -C ₂₀ alkaryl, Cy-Cj-aralkyl or

Polyhydroxyalkyl and

Z Tyr, Phe, decarboxy-Tyr, decarboxy-Phe, which are optionally substituted by OH, NO ₂ , halogen and OCH ₃ , or Trp, the amino acids may each have the L or D configuration, as well as by linking the α- or ε-amino group with the a-carboxyl group of the alanine or the amino acid Z directly or with dicarboxylic acids and / or by linking dera-carboxyl group ds alanine or the amino acid Z via polyoxyethylene, Polyoxyethylendiamino- or Polymethylendiamino chains and / or obtained by linking via SS-bonding on the cysteine dimers, as well as their obtained by linking the carboxyl group of the amino acid Z with the amino group of arginine cyclic derivatives, and their salts, complexes, amides, esters and corresponding, optionally partially protected derivatives , The preparation of the splenin derivatives of the formula I is carried out by reacting the corresponding, optionally protected and / or acylated amino acids or their salts, complexes, amides or esters stepwise or by fragment condensation according to known methods of peptide synthesis to the optionally protected splenin derivatives of the formula I or its salts, complexes, amides and esters, which converts the optionally protected free splenin derivatives of the formula I into their salts, complexes, amides or esters and / or from the salts, complexes, amides and esters the free peptide of Formula I releases. The individual amino acids or peptide fragments as well as the splenin derivative of the formula I can be acylated, cyclized or dimerized at the appropriate site of the synthesis in a manner known per se. Likewise, the protective groups can optionally be cleaved selectively in a conventional manner.

The acylation of the optionally protected splenin derivatives of the formula I and the corresponding, optionally protected amino acids or the corresponding, optionally protected di-, tri- and tetrapeptide fragments is preferred with N-hydroxy-norborn-5-ene-2,3-dicarboxymidestern of carboxylic acids, in particular with N-acetoxynorborn-S-ene-S-dicarboximide or with N-acetoxybenzotriazole.

These acylating agents are particularly suitable because of their low propensity for undesirable side reactions and the high yield of splenin derivatives to be achieved thereby.

Due to the high resistance to hydrolysis of N-acetoxynorborn-5-ene-2,3-dicarboximides it has a very good storage stability and can be used in acylation reactions both in organic and in aqueous media. In addition, it has a high reactivity with amino groups even without addition of base.

With regard to their immunomodulating activity and their stability, particular preference is given to compounds of the formula I in which the amino group of the arginine and / or the amino acid X, in particular of the lysine, is substituted by one of the following groups:

l, C ₆ -C ₁₂ aryl, C ₇ -C ₂₀ -alkyl Kary I, CHVAralkyl, _C2-C7 alkenyl, C2-C ₇ alkynyl, C ₃ -C ₇ cycloalkyl, C3 ₇ cycloalkenyl-C , Ci-C ^ alkanoyl, C ₄ -C ₁₂ cycloalkanoyl, C ₇ -C ₁₁ -ArOyI, Cs-C ^ -Polydroxyalkanoyl, C ^ Cw aralkanoyl,

-CO (CH ₂ L-CO-R ⁵ , -CO (CHOH) _n -CO-R ⁵ , where m = 1-8 and η = 1-12,

wherein R ⁵ OH or their ensprechende salts, esters, and amides, N ° -Arg-X-Ala-R ¹ or N ^c -Lys- (Arg) -Ala-R ¹ means. Among the dimers of the splenin derivatives of the formula I, in particular the compounds which are linked via one of the following groupings have a high immunomodulating activity:

-0- (CH ₂ Ip-O-,

-NH- (CH ₂ ) P -NH-,

-O- (CH ₂ -CH ₂ -O -) ,, -,

-NH- (CH ₂ -CH ₂ -O - ^ - CH ₂ -CH ₂ -Nh- where ρ = 1-18 and q = 1-10000.

It has been found that by N ° - and / or N ^e -acylation, in particular acetylation, both the splenotritins and the splenopentines a significant increase in immunomodulating activity is achieved, based on a significant increase in the degradation stability to aminopeptidases and related improved bioavailability is due.

This increase in activity is particularly pronounced for compounds of the formula I in which the N ° position of the arginine and / or the N ^c position of the lysine is acylated, in particular acetylated.

Compounds of the formula I in which XAl, Me, Lys, Sar and Pro mean a particularly high stability.

A particularly high immunomodulating activity and stability without neurotropic side effects show splenin derivatives of the formula I in which X is Lys and R 'is Val-Tyr-OR ³ or VaI is Tyr N (R ³ R ⁴ ) and Arg and / or Lys and / or Val- Tyr are substituted, in particular the following compounds:

(N ^Q acyl-Arg) -X-Ala-R \

Arg- (N ^e -acyl-Lys) -Ala-R ¹ ,

(N ^a -acyl-Arg) - (N ^C -acyl-Lys) -Ala-R ¹ ,

where acyl is preferably acetyl, X is preferably Lys and R ^{1 is} preferably Val-Tyr-OR ³ or Val-Tyr-N (R ³ R ") valine and tyrosine are optionally substituted.

The preparation of (N ^a -acyl-ArgHN ^£ -acyl-Lys) -Ala-R 'can be carried out by acylation of Arg-Lys-Ala-R ¹ .

Arg- (N ^C -acyl-Lys) -Ala-R ¹ and (N ° -acyl-Arg) -Lys-Ala-R 'are prepared by acylation of partially protected Arg-Lys-Ala-R ¹ and subsequent cleavage of the N ° - or N ^c protective group produced.

Surprisingly, the splenotritin derivatives of the formula I show the same high immunomodulating activity and high stability as the splenopentine derivatives of the formula I.

By means of the dirnerization of the splenin derivatives of the formula I or of the corresponding fragments for the preparation of dimerized splenin derivatives, a further increase in the immunobiological activity can be achieved.

Of particular importance with respect to the immunobiological activity of the peptides of the invention appears to be their effect of increasing the efficiency of the GM-CSF (granulocyte-macrophage colony stimulating factor), thereby increasing the proliferation and differentiation of bone marrow stem cells (Table 1).

Obviously, the splenin derivatives produced according to the invention iniliate simultaneously biological effects such as genetically engineered GM-CSF, which is why the splenin derivatives in such diseases therapy effects in which also the GM-CSF therapy is effective: AIDS in combination with azidothymidine, after chemotherapy and autologous bone marrow transplantation and in HIV-infected patients to prevent the severity of the disease.

Influencing the formation of certain types of blood cells greatly reduces the need for blood transfusions, simplifies and reduces the risk of bone marrow transplantation, and supports the immune system against pathogens (including AIDS) or even cancer.

The splenin derivatives of the formula I according to the invention can be converted into pharmaceutical administration forms by mixing with pharmacologically unavoidable carrier materials and / or diluents.

The following models were used to demonstrate the immunological effects:

"Colony Formation" ("colonies" formation test) by human boiled marrow cells after culturing the cells with human granulocyte-macrophage "colony" stimulating factor (hGM-CSF) and splenin derivatives.

- Antibody formation against sheep erythrocytes (plaque-forming spleen cells) by AB / Bln. mice after X-irradiation (Table 2).

The splenin derivatives according to the invention have valuable pharmacological properties, in particular those for normalizing the immune system. Therefore, they are suitable for use in the following medical indications:

- Treatment of viral infections

The splenin derivatives, in particular the acylated splenin derivatives, have proven to be suitable active ingredients against influenza viruses or for pancreatitis treatment after infection with coxsackieviruses. With regard to the extent of the stimulation of the antiviral immune defense, it has been found that administration of amounts of virus which result in a medium severity infection, the disease can be completely prevented and that the necessary for triggering a disease of equal severity necessary dose of virus under treatment is many times higher as in individuals without therapy.

- Treatment after chemotherapy

The pharmaceutical agents based on the splenin derivatives according to the invention are suitable for overcoming immunosuppressive conditions after chemotherapeutic measures, for. As in the treatment of cancer or as a result of overdosed Ciclosporin therapy.

The normalization of the humoral immune response after cyclophosphamide or dexamethasone treatment of mice can be achieved by the administration of the splenin derivatives according to the invention. However, since these have no influence on the formation of the normal antigen-specific humoral immune response, no adverse immunomodulatory side effects are to be expected.

The splenin derivatives cause the accelerated reconstruction of the immune system of patients with secondary immunodeficiency as a result of immunosuppressive therapy.

Stimulating the growth and maturation of bone marrow cells.

The use of the compounds according to the invention leads in vitro to an increase in the formation rate of stem cell colonies from mouse bone marrow cells co-cultured with leukocytes from normal individuals or patients with systemic lupus erythematosus in agar. The increased formation of "colonies" as a result of the presence of the splenin derivatives is an indication that they directly stimulate the proliferation of immature stem cells of the bone marrow and / or leukocytes of humans sezermieren under the influence of splenin derivatives increasingly colony-forming factors.

In sublethally irradiated mice transplanted with syngeneic bone marrow cells, an accelerated reconstitution of the humoral immune response is observed.

The active substances can be used in immunosuppressive or cytostatic therapy, after irradiation and after bone marrow transplantation. They work by possessing specific binding sites (receptors) on human bone marrow cells (especially on stem cells) which can be detected by radioligand binding studies. Such binding sites are also found on thymocytes and can be detected by ³ H-labeled splenin derivatives or by ^1is I-labeled, in particular diacylated splenin derivatives.

- Therapy of HIV infections

The administration of the splenin derivatives according to the invention, in particular of the diacylated compounds, causes an increase in the total number of lymphocytes in the control pond in HIV-infected patients.

A combination with the cytostatic and revertase inhibitor azido-thymidine used in AIDS therapy and effective eren substances of this type (fluoro-thymidine) leads to an accelerated normalization of the adverse effects caused by these substances in the immune system.

- Prevention of immunosuppressive effects of chronic intoxication

The effect of the compounds of the present invention is to suppress the suppression of the antigen-specific humoral immune response induced by alcohol and other chronic noxious agents and to reduce the number or activity of phagocytic cells and to prevent partial atrophy of the thymus and spleen.

- Therapy of autoimmune reactions

The substances produced according to the invention are suitable for the therapy of various diseases with an autoimmune component, such as rheumatoid arthritis, SLE and atopic reactions. However, they do not cause the adverse reduction of the antigen-specific antibody titer after immunization.

embodiments

example 1

1. Preparation of (N ^a -acetyl) -arginyl- (N ^c -acetyl) -lysyl-alanyl-valyl-tyrosine (DAc-h-SP-5)

1.1 Synthesis of Boc-Val-Tyr-OBzl

1.2 g (5.5 mmol) Boc-Val-OH in DMF with the addition of 0.65 ml (5.5 mmol) of NMM with 0.68 ml (5.25 mmol) CAIBE at -15 ⁰ C to the mixed anhydride activated. To this is added the cooled solution of 2.22 g (5 mmol) of Tyr-OBzl® Tos in DMF with the addition of 0.55 ml (5 mmol) of NMM. After completion of the reaction, solvent is in Vak. away. The residue is taken up in EA and the organic phase is acid and alkaline, dried over Na ₂ SO ₄ , concentrated in vac. and falls with ether. The solid is filtered off, washed with ether and in vac. dried

 Yield: 2.2 g / 93.5% d. Th.

MG: 470.58

Molecular formula: C ₂₆ H ₃₄ N ₂ O ₆

1.2 Release to H-Val-Tyr-OBzl χ HCl

2g (4.25mMol) Boc-Val-Tyr-OBzl will be 30min with 10.6m! 4n HCI / EE treated. The excess HCI / EE is in Vak. distilled off and the product from EE precipitated, filtered off, washed with EA and in vac. dried over KOH. Yield: 1.37 g / 79.3% of theory.

MG: 406.91

Molecular Formula: C ₂₁ H ₂₇ N ₂ O ₄ Cl

1.3 Synthesis of Boc-Ala-Val-Tyr-OBzl

1.02 g (5.41 mmol) of Boc-Ala-OH are activated in DMF with the addition of 0.59 ml (5.4 mmol) of NMM at -15 ° C. with 0.67 ml (5.16 mmol) of CAIBE and then with the precooled solution of 2.0 g (4.92 mmol) of HVaI Tyr-OBzl x HCl and 0.54 ml (4.92 mmol) of NMM. Workup is analogous to 1.1

 Yield: 2.5 g / 92.4% d. Th.

MG: 541.66

Molecular formula: C ₂₉ H ₃₉ N ₃ O ₇

1.4 Release to H-Ala-Tyr-OBzl x HCI

2.46 g (4.54 mmol) of Boc-Ala-Val-Tyr-OBzl are treated with 12.5 ml (50 mM) 4 N HCI / EA. The excess HCI / EE is in Vak. distilled off and precipitated product with ether, filtered off, washed with ether and in Vak. dried over KOH. Yield: 1.95 g / 89.9% d. Th.

MG: 478.0

Molecular formula: C ₂₄ H ₃₂ N ₃ OsCI

1.5 Synthesis of Boc-Lys (Z) -Ala-Val-Tyr-OBzl

1.9 g (5 mmol) of Boc-Lys (Z) -OH are activated in DMF with addition of 0.55 ml (5 mmol) of NMM at -15 ° C. with 0.62 ml (4.77 mmol) of CAIBE and then with the precooled solution of 2.17 g (4.54 mmol) of H-Ala-Val-Tyr-OBzl x HCl and 0.5 ml (4.54 mmol) of NMM. After completion of the reaction, the reaction solution is stirred into 5% NaHCO ₃ . The product is filtered off and washed neutral, then redissolved in DMF and stirred into 1 η HCl, filtered, washed neutral and dried

 Yield: 3.47 g / 95% d. Th.

MG: 803.92

Molecular formula: C ₄₃ H ₆₇ N ₅ O ₁₀

1.6 Release to H-Lys (Z) -Ala-VaK-Tyr-OBzl χ TFA

3.65 g (4.54 mmol) of Boc-Lys (Z) -Ala-Val-Tyr-OBzl are treated with 17.3 ml (227 mmol) of TFA in 15 ml of dichloroethane for 1 h. After distilling off the excess TFA / dichloroethane, the residue is taken up in a little dichloroethane and stirred into ether. Filtered off product, washed with ether and in Vak. dried over KOH.

Yield: 3.23 g / 87% d. Th.

MG: 817.88

Molecular formula:

1.7 Synthesis of Boc-Arg (NO ₂ Kys (Z) -Ala-Val-Tyr-OBzl

1.39 g (4.35 mmol) Boc-Arg (NO ₂ ) -OH are dissolved in DMF with 0.48 ml (4.35 mmol) at -15 ° C with 0.54 ml (4.15 mmol) CAIBE and reacted with the precooled solution of 3.23 g (3.95 mmol) of H-Lys (Z> -Ala-Val-Tyr-OBzlχTFA and 0.44 ml (3.95 mmol) of NMM analog 1.5.

Yield: 3.48 g / 87.6% d. Th.

MG: 1005.18

Molecular formula: C ₄₉ H ₆₈ N ₁₀ O ₁₃

1.8 Release to H-Arg (NO ₂ M-ys (Z) -Ala-Val-Tyr-OBzlχTFA

3.48 g (3.46 mmol) Boc-Arg (NO ₂ ) -Lys (Z) -Ala-Val-Tyr-OBzl are treated with 13.15 ml (173 mmol) TFA in 12 ml dichloroethane for 1 h. The work-up is analogous 1.6. Yield: 3.4 g / 96.4% d. Th.

MG: 1019.07

Molecular formula: C ₄₆ H ₆₁ Ni ₀ O ₁₃ F ₃

1.9 Hydrogenation to H-Arg-Lys-Ala-Val-Tyr-OH χ 3AcOH

3.0 g (2.94 mmol) H-Arg (NO ₂ ) -Lys (Z) -Ala-Val-Tyr-OBzl χ TFA are dissolved in 90% AcOH, 0.3 g Pd / A charcoal (10% ) and introduced at RT and atmospheric pressure H ₂ -GaS. After complete deprotection catalyst is filtered off, the solution was concentrated and stirred into ether. The product is filtered off, washed with ether and dried. Yield: 2.28 g / 95% of theory. Th.

MG: 815.78

Sum of photos: C ₃₅ H ₆₁ N ₃ O ₁₃

1.10 Acetylation to Ac-Arg-LysfAct-Ala-Val-Tyr-OH AcOH

0.816 g (1 mmol) of Arg-Lys-Ala-Val-Tyr x 3AcOH, dissolved in 10 ml of water, with a solution of 0.66 g OmMoI) AcONB (N-acetoxy-norborn-ö-en ^^ - dicarboximide) 10ml of DMF reacted. After completion of the reaction, the Lösungsmitlelgemisch in vac. concentrated and the residue was added dropwise to ether. The product is filtered off, washed with ether and in Vak. dried. Yield: 0.7 g / 90% of theory.

MG: 779.91

Molecular formula: C ₃₅ H ₅₇ N ₉ O ₁₁

Example 2

1. Preparation of N-acetyl-) arginyllysyl-alanyl-valyl-tyrosine (a-MAc-SP-5 χ 2AcOH)

1.1 Synthesis of Ac-Arg (NO ₂ ) -Lys (Z) -Ala-Val-Tyr-OBzl

1.09 g (1 mmol) of H-Arg (NO ₂ ) -Lys (Z) -Ala-Val-Tyr-OBzl χ TFA are dissolved in 5 ml of DMF and after addition of 1801 NMM (or another tertiary amine) with 0 , 27g (1.2 mmol) AcONB reacted. After 30 min, the reaction mixture is added with stirring in 1 η HCl, the precipitate is filtered off with suction and washed with water. The product is dissolved in about 10 ml of DMF and precipitated with 5% NaHCO ₃ solution, filtered, washed with water and dried. Yield: 0.92 g / 96.6% d. Th.

MG: 947.01

Molecular formula: C ₄₆ H69N ₁₀ O ₁₂

1.2 Hydrogenation to Ac-Arg-Lys-Ala-Val-Tyr-OH χ 2AcOH

0.9 g (0.95 mmol) Ac-Arg (NO ₂ ) -Lys (Z) -Ala-Val-Tyr-OBzl are hydrogenated analogously to working example 3 pt. 1.9. Yield: 0.7 g / 92% d. Th.

MG: 797.93

Molecular formula: C ₃ SH ₆₉ N ₉ Oi ₂

Example 3

1. Preparation of H-Arg-Lys (Ac) -Ala-Val-Tyr-OH χ 2AcOH

1.1 Hydrogenation to Boc-Arg-Lys-Ala-Val-Tyr-OH x 2AcOH

in the same way as in 3 pt. 1.9, 5.02 g (5 mmol) of Boc-Arg (NO ₂ ) -Lys (Z) -Ala-Val-Tyr-OBzl are hydrogenated. Yield: 3.98 g / 93% d. Th.

MG: 856

Molecular formula: C ₃₈ H ₆ SN ₉ O ₁₃

1.2 acetylation to Boc-Arg-Lys (Ac) -Ala-Val-Tyr-OH χ AcOH

3.42 g (4 mmol) Boc-Arg-Lys-Ala-Val-Tyr-OH χ 2AcOH are acetylated analogously to Example 3 pt. 1.10. Yield: 2.98 g / 89% of theory.

MG: 837.98

Sum formula: C ₃ SH ₆₃ N ₉ O ₁₂

1.3 Deblocking to H-Arg-Lys (Ac) -Ala-Val-Tyr-OH x 2AcOH

Analogously to Example 3 pt. 1.8, 2.51 g (3 mmol) of Boc-Arg-LysfAct-Ala-Val-Tyr-OH χ AcOH are deblocked. After complete deblocking reaction mixture in vac. concentrated, taken up residue in glacial acetic acid and dripped in ether, filtered, washed with ether and in vac. dried. Yield: 2.03 g / 85% d. Th.

MG: 797.93

Molecular formula: C ₃₅ H ₅₉ N ₉ O ₁₂

Example 4

1. Preparation of Ac-Arg-Lys (Ac) -Ala-OH AcOH

1.1 Synthesis of Boc-Lys (Z) -Ala-OBzl

MA Synthesis Analogous to Exemplary Embodiment 3 pt. 1.1 of Boc-Lys (Z) OH with H-Ala-OBzI χ HCl Yield: about 75% of theory

MG: 541.62

Molecular formula: C ₂₉ H ₃₉ N ₃ O ₇

1.2 Release to H-Lys (Z) -Ala-OBzl χ HCl Deblocking Analogous to Example 3 Point 1.2 of Boc-Lys (Z) -Ala-OBzl Yield: Oil about 90%

MG: 478.01

Molecular formula: C ₂₄ H ₃₂ N ₃ O ₅ CL

1.3 Synthesis of Boc-Arg (NO ₂ ) -Lys (Z) -Ala-0Bzl

MA synthesis analogous to Example 3 pt. 1.1 of Boc-Arg (NO ₂ ) -OH with H-Lys (Z) -Ala-OBzl χ HCl. Yield: 80% of theory.

MG: 742.85

Molecular formula: C ₃₅ Hs ₀ N ₈ OiO

1.4 Release to H-Arg (NO ₂ ) -Lys (Z) -Ala-OBzl × HCl Deblocking Analogous to Example 3 Point 1.4 of Boc-Arg (NO ₂ ) -Lys (Z) -Ala-OBzl Yield: 80% of theory .th.

MG: 679.23

Molecular formula: C ₃₀ H ₄₃ N ₈ O ₈ CI

1.5 Hydrogenation to H-Arg-Lys-Ala-OH χ 3AcOH

analogously to Example 3, Section 1 H-Arg (NO ₂ ) -Lys (Z) -Ala-O8zl χ HCl is hydrogenated. Yield: 91% of theory.

MG: 553.62

Molecular formula: C ₂₁ H ₄₃ N ₇ O ₁₀

1.6 Acetylation to Ac-Arg-Lys (Ac) -Ala-OH x AcOH

Analogously to Example 3, Section 1.10, H-Arg-Lys-Ala-OH is acetylated x 3 AcOH. Yield: 88% of theory.

MG: 517.59

Molecular formula: C ₂₁ H ₃₉ N ₇ Oe

Example 5

1. Preparation of Ac-Arg-Lys-Ala-OH x 2AcOH

1.1 Acetylation to Ac-Arg (NO ₂ ) -Lys (Z) -Ala-OBzl

Analogously to Example 4, Section 1.1, H-Arg (NO ₂ H-ys (Z) -la-OBzl x HCl (Example 6.1.4) is acetylated.) Yield: 87% of theory.

MG: 684.77

Molecular formula: C ₃₂ H ₄₄ N ₈ O ₉

1.2 Hydrogenation to Ac-Arg-Lys-Ala-OH x 2AcOH

Analogously to Example 3, Section 1.9, Ac-Arg (NO ₂ ) -Lys (Z) -Ala-OBzl is hydrogenated. Yield: 92% of theory.

MG: 535.59

Molecular formula: C ₂₁ H ₄₁ N ₇ O ₉

Example 6

1. Preparation of H-Arg-Lys (Ac) -Ala-OH x 2AcOH

1.1 Hydrogenation to Boc-Arg-Lys-Ala-OH x 2AcOH

Boc-Arg (NO ₂ H-ys (Z) -Ala-OBzl χ 2AcOH (Ex.6.1.3) is hydrogenated analogously to Example 3, Section 1.9.) Yield: 90% of theory.

MG: 593.69

Molecular formula: C ₂₄ H ₄₇ N ₇ OiO

1.2 Acetylation to Boc-Arg-Lys (Ac) -Ala-OH AcOH

analogously to Example 3 pt. 1.10 Boc-Arg-Lys-Ala-OH χ 2AcOH is acetylated. Yield: 87% of theory.

MG: 575.68

Molecular formula: C ₂₄ H ₄₅ N ₇ O ₉

1.3 Deblocking to Arg-Lys (Ac) -Ala-OH x 2AcOH After deblocking Boc-Arg-Lys (Ac) -Ala-OH x 2 AcOH with TFA / dichloroethane (1: 1), the reaction mixture is concentrated in vacuo.

, dissolves residue in glacial acetic acid and drips into ether.

Yield: 86% of theory.

MG: 535.59

Molecular formula: C ₂ IH ₄ IN ₇ O ₉

Example 7

Preparation of Peptide Hydrochlorides By dissolving the peptides described in Examples 3.1.9, 3.1.10.4.1.2.5.1.3, 6.1.5.6.1.6.7.1.2 and 8.1.3 in 1.1 equivalents 0 , 1 η HCl and subsequent lyophilization, the corresponding peptide hydrochlorides are obtained.

Example 8

Preparation of the Free Peptides By dissolving the peptides in water and described in Examples 3.1.9, 3.1.10.4.1.2.5.1.3.6.1.5, 6.1.6, 7.1.2 and 8.1.3 or in Example 9 Titration with 0.1 η NaOH, the corresponding free peptides are obtained by column chromatography desalting and lyophilization.

Example 9

Stimulating the growth and maturation of bone marrow stem cells (in vitro)

Table 1

Number of colonies per 10 ⁵ human bone marrow cells (BMC) after culturing ⁵¹ of cells with the human granulocyte macrophage colony stimulating factor (hGM-CSF) ⁶ 'and splenopentin (SP5), splenopentin analogs or thymopentin (TPEi)

No. Peptide" 1 pg χ ml ' Number of colonies P ³¹ 10μg χ ml "' per 10 ⁶ KMZ ² ' 1 nurhGM-CSF 46.7 ± 5.6 2 hGM-CSF 1 μg χ ml " ¹ bSPE: 1C ^ g "ml" ' 46.5 ± 9.4 ns ⁴ ' 69.0 ± 11.9 0.01 3 hGM-CSF 1 μg χ ml "' DAc-EX5: 10μg χ ml "' 92.0 ± 21.6 0.001 56.0 ± 8.7 n.s. 4 hGM-CSF 1 μg χ ml "' HSP5: 10μg x ml "' 70.5 ± 11.4 0.01 77.5 ± 7.5 0.01 5 hGM-CSF 1 μg χ ml "' DAc-HSP5: 10μg χ ml "' 116.2 ± 23.7 0.01 10C ^ gx ml " ¹ 66.7 ± 7.9 n.s. 6 hGM-CSF SP5: 51.2+ 7.3 n.s. 48.3 ± 3.9 n.s. 48.2 ± 4.4 n.s.

1 bSP5 (bovine splenopentin): H-Arg-Lys-Glu-Val-Tyr-OH

DAc-bSP5: (N ^ acetyl-ArginylHN'-acetyl-LysyO-glutamyl-valyl-tyrosine-OH (N'-acetyl-ArginyO-IN'-acetyl-LysyD-gluiamyl-valyl-tyrosine-HCl hSP5 (human splenopentin) : H-Arg-Lys-Ala-Val-Tyr-OH DAc-hSP5 (N-acetyl-Arginyl-HN ^e -acetyl-lysyl) -alanyl-Valyt-tyrosine-OH TP5: (Thymopentin): H-Arg-Lys -Asp-Val-Tyr-OH

2 The values represented the mean + scatter of triplicate determinations of bone marrow cells obtained from three different donors

3 Statistical differences (Mann-Whitney test): Comparison of the values no.2 to no.6 with Nr 1ns. = Not significant difference 5 Methodology- Spooncer et al., Differentation, 1986, (31), j Product of Behringi Works Marburg

The number of "colonies" formed is a measure of the mitotic or differentiating effect of the factors on the bone marrow cells.

5a Thymopention in this test system (Tab. 1, No. 6) has an effect, it is to be assumed that the immunostimulating action of Timunox does not come about via an effect on bone marrow stem cells.

Example 10

Influencing the function of bone marrow cells (in vivo)

Three months old AB / Bln. Mice were X-rayed once with 80OcGy. The animals were then placed in three groups and treated as follows:

Group 1: After irradiation, the animals were transplanted with bone marrow cells from animals of the same inbred strain (injection of 5 times 10 * bone marrow cells per animal). Subsequently, the animals were treated three times a week with DAc-hSP5 at a dose of 10 μg per animal (intraperitoneal application). At various intervals after treatment and during DAc-hSP5 treatment, the JERNE plaque assay was used to examine the ability of the animals to produce antibodies.

Group 2: Same treatment of animals as in group 1, but no treatment of animals with DAc-hSP5.

Group 3: sole X-ray of the animals; no transplantation of syngenic bone marrow cells, no treatment of animals with DAc-hSP5.

Five days before the determination of the pancreatic spleen cells (days indicated in Table 2), the animals were immunized with 5 × 10 ⁸ sheep erythrocytes (intraperitoneal application), method: Eckert et al., Biomed. Biochem. Acta, 1985,

The results show (Table 2) that under in vivo conditions, bone marrow cells are accelerated by DAc-hSP5 to normal antibody production. This implies that bone marrow cells also "mature" in vivo through DAc-hSP5, i.e., differentiate more rapidly into functionally active cells.

The intraperitoneal application of 10μg per animal three times a week had an optimal effect, 1 pg, 2μ and 4pg were not effective under identical conditions, 8pg were suboptimal. After application of 20 pg and 5C ^ g, an effect similar to that of 10pg could be measured. Basically similar results were achieved when using other splenin derivatives of the formula I.

Table 2

Antibody formation against sheep erythrocytes by AB / Bln. Mice after single X-ray irradiation of the animals with 80OcGy, subsequent transplantation of syngenic bone marrow cells (5 χ 10 ⁶ bone marrow cells) and exclusive treatment of the animals with DAc-hSP5 (No 3). Gift of 10pg DAc-hSP5 around the clock three times a week. Each value (mean ± variance) represents the mean of at least five animals.

No. treatment Number of plaque-forming spleen cells per spleen day 14 day 20 day 34 1400 ± 400 9 500 ± 1000 16000 ± 1000 11000 ± 2400 15800 ± 1600 39 000 ± 8 500 Day 48 1 2 3 X-irradiation X-ray and bone marrow transplantation X-ray and bone marrow transplantation 1500 ± 500 1600+ 300 2900 ± 300 41000 + 3000 43000 + 7000 17000+ 2600 28 000 ± 4 300 44000 + 7000 4 without X-ray without any treatment (control) 43100 ± 4600 42,500 ± 8,300

U-Test (Mann-Whitney): Comparison between # 1 and 2: Day 20: p <0.001 Comparison between # 2 and 3: Day 20: ρ <0.001 Comparison between # 3 and # 4: Day 34: No significant difference

List of abbreviations

AcOH - acetic acid

AcONB - N-acetoxy-norborn-5-ene-2,3-dicarboximide

CAIBE - isobutyl chloroformate

DMF - dimethylformamide

EE - ethyl acetate

NMM - N-methylmorpholine

TFA - trifluoroacetic acid

Claims (5)

1. A process for the preparation of human splenin derivatives of the formula I. Arg-X-Ala-R ¹ I, Arg possibly substituted arginine, X is Sar, Pro, Gly, Ala, Leu, He, Thr, Ser, Lys, VaI, Phe or Cys, which are optionally substituted. R ¹ -OR ³ , -N (R ³ R ⁴ ), -XZO R ³ or -XZN (R ³ R ⁴ ), R ³ and R ⁴ are identical or different and are H, C ₁ -C ₈ -AIRyI, C ₂ -C 7 alkenyl, C ₂ -C ₇ alkynyl, C ₇ -C ₂ O-AlClar, C ₇ -C ₂₀ -ArBl ky I or polyhydroxyalkyl and
Z is Tyr, Phe, decarboxy-Tyr, decarboxy-Phe optionally substituted by OH, NO ₂ , halogen and OCH ₃ are substituted or Trp
mean, the amino acids can each have the L- or D-configuration, as well as by linking the α- or ε-amino group with the a-carboxyl group of the alanine or the amino acid Z via polyoxyethylene, polyoxyethylenediamino or polymethylenediamino chains and / or Dimers obtained by linking via SS backbinding to the cysteine, and their cyclic derivatives obtained by linking the carboxyl group of the amino acid Z with the amino group of the arginine, and their salts, complexes, amides, esters and corresponding, optionally partially, protected derivatives, characterized by that the corresponding, optionally protected and / or acylated amino acids or their salts, complexes, amides or esters, in stages or by fragment condensation according to known methods of peptide synthesis to the optionally protected splenin derivatives of the formula I or their salts, complexes, Amides and esters reacted, optionally in a conventional manner, the protecting groups selectively abs acylated, cyclized or dimerized, if appropriate, the optionally protected splenin derivatives of the formula I are converted into their salts, complexes, amides or esters and / or from the salts, complexes, amides and esters free peptide of formula I releases. 2. The method according to claim 1, characterized in that the acylation of the optionally protected splenin derivatives of the formula I and the corresponding, optionally protected amino acids or the corresponding, optionally protected di-, tri- and tetrapeptide fragments with N-hydroxy-norborn 5-en-2,3-dicarboximidestern of carboxylic acids, preferably N-acetoxy-norborn-5-ene-2,3-dicarboximide is performed. 3. The method according to claims 1 and 2, characterized in that the acylation of the optionally protected splenin derivatives of the formula I and the corresponding, optionally protected amino acids or the corresponding, optionally protected di-, tri- and tetrapeptide fragments with N- Acetoxybenzotriazole is performed. 4. Process according to claims 1 to 3, characterized in that splenin derivatives of the formula I in which the amino group of the arginine and / or the amino acid X, in particular of the lysine, is substituted by one of the following groups: C ₁ -C ₇ -alkyl, C ₆ -C ₁₂ -aryl, C ₁ -C ₄ -alkaryl, C ₇ -C ₂₀ -aralkyl, C ₂ -C ₇ -alkenyl, C ₂ -C ₇ -alkynyl, C ₃ -C ₇ -cycloalkyl, C ₃ -C ₇ -cycloalkyl, C 1 -C ₆ -alkanoyl, C ₄ -C ₁₂ -cycloalkanoyl, C ₇ -C 5 -aryl, C ₆ -C ₁₂ -polydroxyalkanoyl, C ₁ -C 4 -alkanoyl, -CO (CH ₂ ) m -CO-R ⁵ , -CO (CHOH) _n -CO-R ⁵ , where m = 1-8 and η = 1-12, wherein R ⁵ OH or their corresponding salts, esters, and amides, fvT-Arg-X-Ala-R ¹ or N ^£ -Lys- (Arg) -Ala-R ¹ means are prepared. 5. Process according to claims 1 to 4, characterized in that such dimers of the splenin derivatives of the formula I are prepared which are obtained by linking the α-carboxyl group of the alanine or the amino acid Z via one of the following groups: -0- (CH ₂ ) PO-, -N H- (CH ₂ Jp -N H-, -CMCH ₂ -CHr-O -) ,, -, -NH- (CH ₂ -CH ₂ -O-Jq-CH ₂ -CH ₂ -NH- where ρ = 1-18 and q == 1-10000.