DK171992B1 - Peptides, pharmaceutical preparations which comprise these peptides, the use of the peptides for producing a pharmaceutical preparation for use for stimulating the myelopoiesis system in humans or animals, a process for preparing the peptides, and compounds which can be used as intermediates in the process - Google Patents

Description

DK 171992 B1

The present invention relates to novel peptides having a stimulating effect on the haemopoiesis, a process for their preparation, compounds having use as intermediates therewith, pharmaceutical compositions containing the peptides, and the use of the peptides to prepare a pharmaceutical composition for use in stimulating the myelopoiesis system in humans or animals.

10 Bone marrow cells are derived from multifaceted potato stem cells that mature to form a complex population of morphologically distinct cells, namely megakaryocytes, erythrocytes, granulocytes and lymphocytes. Only approx. 10% of the stem cells are in cell division phase at a given time. In an initial phase of maturation, each stem cell during propagation is "traced" to a particular morphologically distinct form that eventually leads to one of the above four mature cell types. As the cells proliferate, they lose the ability to further proliferate and the mature cells, e.g., erythrocytes or granulocytes, can no longer divide. Since mature cells are still dying, it is crucial that the reproductive capacity of the less mature cells is maintained, especially in the stem cells "intended" for myelopoiesis.

We have now succeeded in finding a group of novel peptides capable of stimulating the proliferation of myelopoietic stem cells.

According to the invention, there are thus provided for-3Q linkers of the general formula A- (NH-CH-CO) - (NH-CH-CO). - (NH-CH-CO) -B I a i b i

(CH) (CH,) CHO I

I 2 ni 2 m I 2 COR COR 'S-- 2 35 2 DK 171992 B1 where all the amino acid units are in L configuration, A represents NH-CH-CO-, NH-CH-CO-, NH ^ -CH-CO - 5 XJ U <{H2 »20 CONH2 or a hydrogen atom, B represents -NH-CH-COR", -NH-CH-CO-R "(CH2) 4 (ch2) 3

NH0 NH

2 I

HN NH 2 or a hydroxy group, n and m independently of each other, represent the number 1 or 2; a and b independently each represent the number 0 or 1; R, R 'and R "independently each represent a hydroxy group or an amino group, 2Q and physiologically acceptable salts thereof, however, so that if A and B are a hydrogen atom and a hydroxy group respectively, then b cannot be the number 0.

Thus, the compounds of formula I are symmetrical dimers.

__ When A represents a glutamine residue or a proline residue, it is preferred that a = b = 1, n = 2, m = 1, R and R 'represent hydroxy groups and B represents a lysine residue.

When B is an arginine residue, it is preferred that A is a pyroglutamate residue, a = b = 1, n = 2 and m = 1, and that R and R 'represent hydroxy groups.

A preferred group of compounds are those in which b = 1, m = 1 and R, R 'and R "are hydroxy groups.

Particularly preferred compounds of the invention are as follows (using the conventional biochemical notation for the amino acids and read from the N-terminal): 3 (Cys - Lys) 2 f (Asp - Cys - Lys) 2 »( Glu - Asp - Cys - Lys) 2, 5 (pGlu - Glu - Asp - Cys) 2, (pGlu - Asp - Cys - Lys) 2, (pGlu - Asp - Asp - Cys - Lys> 2, (pGlu - Glu) - Asp - Cys - Arg> 2, (Gin - Glu - Asp - Cys - Lys) 2 and 10 (Pro - Glu - Asp - Cys - Lys) 2

A further particularly preferred compound is (pGlu-Glu-Asp-Cys-Lys) 2 the compound where a = b = 1, m = 1, n = 2, R and R1 are hydroxy groups, A is a pyroglutamate residue and B is a lysine residue.

Physiologically acceptable salts of the peptides of the invention include acid addition salts such as hydrochlorides, hydrobromides, sulfates, as well as salts with bases such as alkali metal salts, e.g., sodium or potassium salts, alkaline earth metal salts such as calcium salts and amine salts.

Some of the compounds of formula I are dimers of compounds described in EP 0 112 656 A2. This application relates to a small group of haemorrhagic peptides with inhibitory effect on the granulopoiesis.

25 Due to the very small amounts of natural granulocyte factor available, the structure of the natural substance has never been determined. It has never been obtained in crystalline or completely pure form, and it is not known whether this would be possible using material from only natural sources. It has been postulated that the substance has the structure pyroGlu-Asp-Asp-Cys-LysOH, but as stated in the above European patent application, the synthetic compound with this structure was found to be inactive. It has been found that a compound having a stimulatory effect is produced when the natural granulocyte inhibitory factor is subjected to oxidative conditions. However, this product has also never been isolated or identified. In contrast, the peptides of the invention can be recovered in pure form suitable for pharmaceutical use and can be recovered in relatively large quantities.

It has also been reported (Exp. Hematol. J_2 7 (1984)) that when one of the peptides disclosed in the above European patent application, namely 10 pyroGlu-Glu-Asp-Cys-LysOH, freezing and thawing, exhibits stimulatory activity that can be reversed by treatment with mercaptoethanol. However, the stimulatory product was never won in pure form and its structure was never determined.

The novel compounds of this invention in chemically pure form have been tested for haemoregulatory activity in vitro and in vivo. In vitro studies of both human and mouse myelopoietic stem cells have shown increases in agar colony formation of up to 20 1500%. The stimulatory effect occurs in the concentration range of 10 to 10 M. In vivo studies in mice, it has been shown that a single injection can increase the number of myelopoietic stem cells by 50% over 49 hours, and with continuous infusion the number of 25 stem cells doubled over 13 days.

The invention is particularly suitable for use in stimulating myelopoiesis in patients suffering from impaired myelopoietic activity, including bone marrow damage, agranulocytosis and aplastic anemia. This includes treating patients with impaired bone marrow function due to immunosuppressive therapy to suppress tissue reactions, for example, by surgical bone marrow transplantation.

The compounds can also be used to promote a faster regeneration of the bone marrow after cytostatic chemotherapy and radiation therapy for neoplastic and drug-induced diseases.

In addition, these new compounds may be of particular value in cases where patients have severe infections due to a lack of immune response due to bone marrow failure.

Another clinical application will be in combination with the corresponding monomers or related myelopoiesis inhibitors as set forth in the above-mentioned European patent to induce alternating peaks of high and low activity in bone marrow cells and thus increase the natural circadian rhythm of the hemopoiesis. In this way, cytostatic treatment can be given during periods of low bone marrow activity, thereby reducing the risk of bone marrow damage, while regeneration will be promoted at the subsequent peak of activity.

It should be noted that there is no stimulatory effect on the cells of other tissues and especially on tumor cells that are related to non-myelopoietic tissue. Thus, the present new compounds selectively stimulate the myelopoietic system.

Thus, the invention also relates to the use of a compound of the invention for the preparation of a pharmaceutical composition for use in stimulating the myelopoietic system in humans or animals.

The peptides have no significant toxicity.

In addition, all hematologic effects observed are reversible and no macroscopic changes were observed in other organs of the animals in which the peptides were injected.

In general, the peptides of the invention for the purpose of exerting stimulatory action may be administered to humans by injection in the dose range of 0.1-10 mg, e.g. 70 kg body weight per day. If administered by infusion or similar technique, the dose may be in the range of 30-300 mg per day. 70 kg body weight, e.g. 100 mg, over 6 days. In principle, it is desirable to induce a concentration of the peptide of approx. 10 M to 10 Mi of the patient's extracellular fluids.

The invention also relates to pharmaceutical compositions containing such active ingredient one or more compounds 5 of the general formula I as defined above or physiologically compatible salts thereof, together with a pharmaceutical carrier or excipient. For example, the compositions of the invention may be worked up into a form suitable for oral, nasal, parenteral or rectal administration.

10 The term "pharmaceutical" also refers in the present description to veterinary applications.

The compounds of the invention may be in conventional pharmacological administration forms such as tablets, coated tablets, nasal spray preparations, solutions, emulsions, powders, capsules or sustained release forms of the active substance. Conventional pharmaceutical excipients as well as the usual production methods can be used to prepare these forms. For example, tablets may be prepared by mixing the active ingredient (s) with known excipients such as with diluents such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as magnesium stearate or talc or to ensure delayed release such as carboxypolymer methylene, carboxymethyl cellulose, cellulose acetate phthalate or polyvinyl acetate.

If desired, the tablets may consist of several layers. Coated tablets can be prepared by coating 30 tablet cores, prepared in a similar manner to tablets, with agents commonly used for tablet coatings, e.g., polyvinylpyrrolidone or shellac, gum arabic, talc, titanium dioxide or sugar. In order to achieve delayed release or to avoid incompatibility problems, the core may also consist of several layers. The tablet coating may also consist of several layers to achieve delayed release, in which case the excipients mentioned above can be used for tablets.

Organ-specific support systems can also be used.

For example, injection solutions may be prepared in a conventional manner such as, for example, by the addition of preservatives such as p-hydroxybenzoate, or stabilizers such as EDTA. The solutions are then filled into vials or ampoules.

20 Nasal spray preparations can be similarly formulated in aqueous solution and packed in spray containers either with an aerosol propellant or provided with manual compression means. Capsules containing one or more active substances may be prepared, for example, by mixing the active ingredients with inert carriers such as lactose or sorbitol and filling the mixture in gelatin capsules.

Suitable suppositories may be prepared, for example, by mixing the active substance or active substance combinations with conventional carriers suitable for this purpose, such as natural fats or polyethylene glycol or derivatives thereof.

Dosage units containing the compounds of the invention preferably contain 0.1-10 mg, eg 1-5 mg of a peptide of formula I or a salt thereof.

The present invention provides a method of stimulating myelopoiesis by administering an effective amount of a pharmaceutical composition as defined above to an individual.

However, another major use of the new peptides consists in the production of material for immunological testing techniques. The peptide can thereby be covalently bound to a suitable high molecular weight carrier such as albumin, polylysine or polyproline to be injected into antibody producing animals (e.g. rabbits, guinea pigs or goats). In vitro immunization techniques can also be used. High specificity application is obtained using well known absorption techniques using the high molecular weight

Λ A

learn carrier. By introducing radioactivity ("h, 4C, 35 S) into the peptide molecule, a radioimmunoassay sample can easily be designed and used for assay in various biological fluids such as serum (plasma), urine and cerebrospinal fluid.

The peptides of the invention can be synthesized in any convenient manner. Generally, the reactive side chain groups present (amino, thiol and / or carboxyl) will be protected during the coupling reactions included in the total synthesis and the final step will then consist of removing protecting groups on a protected derivative of formula I. Normally, all COOH groups will 15 N1 groups and the NH group in a pyroglutamyl or proline residue are protected.

Thus, a further feature of the invention is a method for producing a peptide according to the invention, which is advantageous in removing protecting groups from a compound of the general formula

A- (NH-CH-CO) - (NH-CH-CO). - (NH-CH-CO) -B I and I o I

(CH-) (CH2) CH0

25 i 2 η I 2 m | 2 II

COR1 COR2 S - 2 where A represents R3N-CH-CO-, R4N-CH-CO-, R5NH-CH-CO- 30 JJ U <Ίν2

O CONHR

a hydrogen atom or an amine protecting group; B represents -NH-CH-COR8, -NH-CH-COR10 (ch2) 4 <(h2) 3 NHR7

5 C

r \ 9

HN NHR

a hydroxy group or a carboxy1 blocking group; n and m independently represent one of the numbers 1 and 2; a and b independently represent one of the numbers 0 and 1; 12 8 10 R, R, R and R are amino groups, hydroxy groups, protected amino groups or carboxyl protecting groups; R 1, R 2, R 8, R 8, R 7 and R 2 are hydrogen atoms or amine protecting groups; however, such that when A is a hydrogen atom or an amine protecting group and B is a hydroxy group or a carboxyl protecting group, then b cannot be the number 0.

In general, the protected derivatives of formula II can be prepared by techniques suitable for peptide synthesis.

A possible pathway utilizes cystine and links it to the remaining amino acid residues such that two identical chains of the dimer are joined which are then pooled and bonded to each other via the disulfide bond already present in cystine. Another method consists of synthesizing the corresponding monomeric compound in S-protected form using S-protected cysteine, removing the S-protecting group and performing dimerization. It is possible to use S-protecting groups which can be removed by oxidizing agents providing a disulfide bond directly in a protective removal step; thus, for example, trityl groups can be selectively removed by iodine, preferably in a suitable solvent such as dimethylformamide; such dimerization may be carried out on the C- and N-protected monomer, followed by removal of the C- and N-protecting groups, or may, according to the invention, be carried out on a monomer of Formula III A- (NH-CH-CO) - (NH -CH-CO), - (NH-CH-CO) -BI aibi

(CH ~) (CH0) (CH0) III

In 2 n T 2 m I 2 COR COR 'SR "10 where A, B, n, m, a, b, R and R' have the above meanings and R" is a hydrogen atom or thiol protecting group which can be removed under oxidizing conditions.

In the construction of the peptide chains, one can in principle start either with the C-terminal or with the N-terminal, but the procedure of starting with the C-terminal is in general use.

Thus, one can begin at the C-end by reacting with a suitably protected derivative of, for example, lysine with a suitably protected derivative of cysteine or cystine. The light derivative will contain a free α-amino group, while the second reactant will have either a free or an activated carboxyl group and a protected amino group. After the coupling, the intermediate can be purified, for example by chromatography, and then the N-protecting group can be selectively removed to allow the addition of an additional amino acid residue. This procedure is continued until the desired amino acid sequence is complete.

Carboxylic acid activating substituents which may be used, for example, include symmetrical and mixed anhydrides and activated esters such as the p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, N-hydroxybenzotriazole ester (OBt) or N-hydroxysuccinimidyl ester (OSu).

For example, the coupling of free amino and carboxyl groups 35 can be carried out by means of dicyclohexylcarbodiimide (DCC). Another coupling agent which may be used, for example, is N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

In general, it is convenient to carry out the coupling reaction at low temperatures, for example from -20 ° C to ambient temperature, preferably in a suitable solvent system such as tetrahydrofuran, dioxane, dimethylformamide, methylene chloride or a mixture of these solvents.

It may be more convenient to perform the synthesis on a solid phase resin support. A useful type of carrier is chloromethylated polystyrene (crosslinked with 1% divinylbenzene); in this case, the synthesis will begin with the C-terminal, for example, by coupling N-protected lysine to the carrier.

A number of suitable solid phase techniques are described by Eric Atherton, Christopher J. Logah and Robert C. Sheppard, J. Chem.Soc. Perkin I, 538-46 (1981); James P. Tam, Foe S.

15

Tjoeng and R.B. Merrifield, J. Am. Chem. Soc. 102, 6117-6127 (1980); James P. Tam, Richard D. Dimarchi and R.B. Merrifield, Int. J. Peptide Protein Res. J 4, 412-425 (1980); Manfred Mutter and Dieter Bellof, Helvetica Chimica Acta, 67, 2009-2016 (1984).

20

Many different amino acid protecting groups are known and are exemplified in E. Schroder and K. Lvibke, The Peptides, Volumes 1 and 2, Academic Press, New York and London, 1965 and 1966; GR Pettit, Synthetic Peptides, Vols 1-4, Van Nostrand, Reinhold, New York 1970, 1971, 1975 and 1976; Houben-Weyl, Methods of Organisms

Chemistry, Synthesis of Peptides, Volume 15, Georg Thieme Verlag, Stuttgart 1974; Amino Acids, Peptides and Proteins, Vols 4-8, The Chemical Society, London 1972, 1974, 1975 and 1976; Peptides, Synthesis-Physical Data 1-6, Wolfgang 30

Voelter, Eric Schmidt-Siegman, Georg Thieme Verlage Stuttgart, NY, 1983; The Peptides, Analysis, Synthesis, Biology 1-7, ed .; Erhard Gross, Johannes Meienhofer, Academic Press, NY, San Francisco, London; Solid Phase Peptide Synthesis, 2nd Edition, John M. Stewart, Janis D. Young,

Pierce Chemical Company.

Thus, for example, amine protecting groups which may be used include protecting groups such as carbobenzoxy (hereinafter also referred to as Z) t-butoxycarbonyl (hereinafter also referred to as Boc), 4-methoxy-2,3,6-trimethylbenzene-5 sulfonyl (Mtr) and 9-fluorenylmethoxycarbonyl (hereinafter also referred to as Fmoc). It will be appreciated that when the peptide is constructed from the C-terminal end, there will be an amine protecting group present on the α-amino group in any newly added residue, and it will be necessary to remove it selectively prior to the next coupling step. A particularly useful group for such temporary amine protection is the Fmoc group, which can be selectively removed by treatment with piperidine in an organic solvent.

Carboxyl protecting groups which may be used, for example, include readily cleavable ester groups such as benzyl (Bzl), p-nitrobenzyl (ONb), pentachlorophenyl (OPC1P), pentafluorophenyl (OPFP) or t-butyl (OtBu) as well as the coupling groups on solid carriers, e.g. bonded to polystyrene.

20 Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) and acetamidomethyl (Acm).

It will be understood that a wide variety of other such groups exist as, for example, discussed in detail in the aforementioned literature references, and the use of all such groups in the processes described above is within the scope of the invention.

There are a wide variety of methods for removing amine and carboxyl protecting groups. The methods used must agree with the synthesis strategy used. The side chain protecting groups must be stable under the conditions used to remove the temporary α-amino protecting groups prior to the next coupling step.

Such amine protecting groups as Boc and carboxyl protecting groups such as tBu can be removed simultaneously by acid treatment, for example with trifluoroacetic acid. The thiol protecting groups such as Trt can be selectively removed by an oxidizing agent such as iodine.

The invention also relates to the protected forms of the peptides of general formula II 'as defined in claim 8.

The following examples are for illustrative purposes only.

Solvents were redistilled from commercial material and stored as follows: dimethylformamide (DMF) over molecular sieve material 4A, dichloromethane (DCM) over CaCl 2 triethylamine (TEA) over a Na / Pb alloy (Baker) and trifluoroacetic acid ( TFA) over molecular sieve material 4A.

TLC systems were as follows: S,: Silica / CHCl3: MeOH (98: 2) S2: Silica / CHCl3 *. MeOH (95: 5) S3: Silica RP 8 / 0.1% TFA in 5% EtOH (aqueous).

The purified final products were analyzed by reverse phase high performance liquid chromatography (HPLC). The HPLC system consisted of an HP 1090M chromatograph with a built-in self-acting sampler and an HP 1040 diode array (Hewlett-Packard, Waldbronn, FRG), and a supelcosil LC-18 column 25 (250 x 4.6 mm, 5µ large particles).

The samples were dissolved in 0.1% by volume aqueous TFA and eluted with a linear gradient from 0 to 30% acetonitrile in 0.1% s TFA (aqueous). The flow rate was 2 ml / min. The eluent was monitored at 214 nm with a bandwidth of 30 4nm. The solvent chromatogram was electronically subtracted and the results expressed as area percentages.

Amino acid analysis:

The cystine-containing peptides were oxidized with permic acid to convert the acid-labile cystine residue to the acid-stable cysteic acid before acid hydrolysis in 6M HCl at 110 ° C for 16 hours. The dry hydrolysates were then derivatized by phenyl isothiocyanate and analyzed as described by Heinrikson (Anal. Bioch. 136, 65-74, 1984).

5

Example 1 (pGlu-Glu-Asp-Cys-Lys) → 1) (Fmoc-Cys-Lys (Νε-Boc) -OtBu) 2 g (2.275 mM) (Fmoc-Cys-OSu) g (5.0 mM)

Light (Νε-Boc) -OtBu.HCl and 0.58 g (5 mM) of N-ethylmorpholine were dissolved in 7.5 ml of DMF and stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in 50 mL of CHCl 3 and washed with 3 x 12 mL of 0.1N HCl and 3 x 12 mL of 1M NaCl. The organic layer was filtered through a phase separation filter paper (Whatman) and evaporated to dryness under reduced pressure.

The crude product, 3.32 g, was recrystallized from 5 ml of 2q hot methanol (15 hours), filtered, washed with methanol and ether and air dried. Yield 1.75 g (61%). The white solid exhibited one major spot UV UV254 + CC ^ / dicarboxidine +, ninhydrin *) on TLC (S ^: Rf = 0.667, S2: Rf = 0.829).

2) (Cys-Lys (Νε-Boc) -OtBu) 2 1.5 g (1.19 mM) of compound (1) was dissolved in 10 ml of 20% piperidine in DMF and stirred at room temperature for 30 minutes. TLC (S2) showed that all the starting material was consumed and that only one new product was formed (UV9t .. +, 30 C2 / dicarboxidine, ninhydrin). The solvent is evaporated under reduced pressure. The residue was dissolved in 20 ml of ether and washed with 2 x 3 ml of 0.1N HCl and 3 ml of 1M NaHCO 3. The organic layer was filtered through a phase separation filter paper and evaporated to dryness. Yield 1.2 g. TLC 35 showed only a ninhydrin positive component (s Rf = 0.216).

DK 171992 B1 3) (Fmoc-Asp (O-OtBu) -Cys-Lys (Νε-Boc) -OtBu) 2

Ca. 1.19 mM of compound (2) and 1.32 g (2.6 mM) of Fmoc-Asp (O-OtBu) -OSu were dissolved in 6 ml of CH2 Cl2 and shaken. at room temperature for 2 hours. TLC (S 2) showed that all ninhydrin positive material had been consumed and that one new UV ^^ positive major product had been formed. The residue after evaporation was dissolved in 30 ml of ethyl acetate, washed with 4 x 10 ml of 1M NaCl, filtered and evaporated as 1 g written for (1). Crude product was dissolved in 2.5 ml of ether and precipitated as a semi-crystalline solid by the addition of 4 ml of n-hexane (refrigerator for 16 hours). The solvent was decanted and the product washed with 5 ml of n-hexane and dried under reduced pressure. Yield 1.272 g (67%). TLC showed one high-1 g bead (UV 254 + Cl 2 / dicarboxidine, ninhydrin *; Rf = 0.333, S2: Rf = 0.658).

4) (Asp (O-OtBu) -Cys-Lys (Νε-Boc) -OtBu) 2.0 g (0.62 mM) of compound (3) was dissolved in 20 ml of 20% s piperidine in CH 2 Cl 2 and treated as described for (2). Yield 0.469 g (65%). TLC showed only one ninhydrin positive spot (S2: Rf = 0.22).

5) (Fmoc-Glu (γ-OtBu) -Asp (O-OtBu) -Cys-Lys (Νε-Boc) -OtBu) 0 ------------------ --------------------------------- é.

0.469 g (0.407 mM) of compound (4) and 0.47 g (0.90 mM)

Fmoc-Glu (γ-OtBu) -Su was dissolved in 5 ml of CH 2 Cl 2 and stirred at room temperature for 15 hours. The reprocessing process was as for (3). Yield 0.62 g (77%). TLC showed only one major spot (UV 254 + 'cl2 // <^: - carboxidine +, ninhydrin *,: Rf = 0.312, S2: Rf = 0.536).

6) (Glu (γ-OtBu) -Asp (O-OtBu) -Cys-Lys (Νε-Boc) -OtBu) 0.35 g (0.25 mM) of compound (5) was dissolved in 2.5 20 ml of piperidine in CH 2 Cl 2 and treated as described for (2).

16 DK 171992 B1 TLC showed only one ninhydrin positive spot (S2:

R f = 0.154).

7) (pGlu-Glu (γ-OtBu) -Asp (β-OtBu) -Cys-Lys (Νε-Boc) -OtBu)

Ca. 0.25 mM of compound (6) and 0.206 g (0.546 mM) of pGlu-OPCIP were dissolved in 2.5 ml of DMF and stirred at room temperature for 15 hours. The work-up was done as described under (3). Yield: 0.259 g (59%). TLC showed only one 1q spot, UV254 + 'ninhydrin * and Cl2 / dicarboxidine + (S2: Rf = 0.117) and only traces of impurities.

8) (p-Glu-Glu-Asp-Cys-Lys) 2 0.110 g (0.063 mM) of compound (7) was dissolved in 5 ml of 80% s TFA (CH 2 Cl 2) and stirred at room temperature for 30 minutes. The solvent was evaporated and the residue was dissolved in 4 ml of H 2 O + 2 ml of CHCl 3. The aqueous phase was washed with 2 x 3 ml of CHCl 3 and evaporated to dryness under reduced pressure (g). The crude product, 0.050 g, was purified on a Lobar (A) 20

Rp 8 column (Merck) and eluted with 0.1% TFA in 7.5% aqueous ethanol. After freeze-drying the product, it was re-chromatographed on the same column and eluted with 0.1% TFA in 5% aqueous ethanol. The product was freeze-dried. Yield 0.016 g. The product containing ninhydrine, Cl- / dicarboxy-25 ^ din + and UV254 * 'was homogeneously evaluated by TLC, S ^: Rf = 0.436, and only traces of impurities were detected by HPLC.

Amino acid analysis: Asp 1.02, Glu 1.91, Cys 1.08,

Lys 0.93.

30

Example 2 9) (Cys-Lys) 2

The compound (2) of Example 1 was dissolved in TFA and stirred at room temperature for 40 minutes. Solvent-35.0 was easily evaporated under reduced pressure (30 ° C) and the water-soluble (S) -like portion of the residue was chromatographed on a "Lobar, Wy 17 DK 171992 B1 (B) RP8 column (Merck) using 0.1 % TFA (aqueous) in 5% aqueous ethanol as mobile phase The column was monitored at UV22Q · Fractions containing the pure product (evaluated by TLC) were combined and lyophilized The product 5 was homogeneous according to TLC (S 57) and showed positive reaction with ninhydrin and no absorption by UV 254. HPLC analysis: 93.5% pure by area determination Amino acid analysis: Cys 1.01, Lys 0.99.

Example 3 10) (Asp-Cys-Lys) 2

The compound (4) treated as described in Example 2 (2% ethanol in mobile phase) led to the compound (10). The product was homogeneously evaluated by TLC (S +: Rf = 0.64) and showed positive reaction with ninhydrin and no absorption by UV 254 * HPLC analysis: 97.9% pure by area. Amino acid analysis: Asp 0.99, Cys 1.03, Lys 0.98.

Example 4 11) (Glu-Asp-Cys-Lys) 2

The compound (6) treated as described in Example 2 led to compound (11). The product was homogeneous according to 25 TLC (S 2: R f = 0.83) and showed positive reaction with ninhydrin and no absorption by UV 254 * HPLC analysis: 100% pure by area. Amino acid analysis: Asp 1.08, Cys 0.97, Glu 0.98, Lys 0.98.

Example 5 12) (pGlu-Glu-Asp-Cys) 2

Compound (12) was synthesized as monomer by continuous phase solid phase peptide synthesis using a "Biolynx" 4170 Automated Peptide Synthesizer (LKB) and DMF as the solvent.

18 DK 171992 B1

Reagents: FMOC-Cys (Trt) -SASRIN Resin (Bachem); Fmoc-Asp (0-OtBu) -OPFP; FMOC-Glu (y-OtBu) -OPFP; pGlu-OPCLP; HOBT as a catalyst. FMOC was removed after each coupling step using 20% piperidine in DMF. The disulfide bridge and thus the dimer were established by treating the fully protected peptide on the solid support with 0.1M iodine (2-3 equivalents) in DMF for 15 minutes. After washing with DMF, the peptide separated from the solid support and residual protecting groups was removed in one step by treatment with 50% TFA in CHCl3 for 40 minutes. The resin was filtered off and the filtrate was evaporated to dryness under reduced pressure (30 ° C). The residue was dissolved in 0.1% aqueous TFA and chromatographed on a "Lobar" ® (B) RP8 column using 0.1% aqueous TFA in 4% s propan-2-ol as mobile phase. The column was monitored at UV22Q · Fractions containing the pure product (as determined by HPLC) were combined and lyophilized. The product showed no reaction with ninhydride and no absorption by UV254 HPLC analysis: 96.9% pure by area. Amino acid analysis: 1.15, Cys 0.99, Glu 1.85.

20

Example 6 13) (pGlu-Glu-Cys-Lys) 2

Compound (13) was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. Reagents not previously described: FMOC-Lys (Ne-BOC) -SASRIN resin; FMOC-Cys (S-Trt) -OPFP. The product was homogeneous by TLC (S +: Rf = 0.49) and showed positive reaction with ninhydrin and no absorption by UV254 * 30 HPLC analysis: 100% pure by area. Amino acid analysis: Cys 1.01, Glu 1.98, Lys 1.01.

Example 7 14) (pGlu-Asp-Cys-Lys) 2

Compound 14 was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. The product was homogeneous by TLC (S +: Rf = 0.55) and gave positive reaction with ninhydrin and no absorption at UV254.

HPLC analysis: 97.5% pure by area. Amino acid analysis: Asp 1.04, Cys 0.96, Glu 0.97, Lys 1.02.

Example 8 15) (pGlu-Asp-Cys-Lys) 21 The compound (15) was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. The product was homogeneous by TLC (S +: Rf = 0.42) and gave positive reaction with ninhydride and no absorption at UV254.

1b HPLC analysis: 99.3% pure, according to area. Amino acid analysis: Asp 1.03, Cys 0.96, Glu 2.09, Lys 0.94.

Example 9 (16) (pGlu-Asp-Asp-Cys-Lys) 2

Compound (16) was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. The product was homogeneous by TLC (S +: Rf = 0.38) and showed positive reaction with ninhydrin and no absorption 25 by UV254 HPLC analysis: 100% pure by area. Amino acid analysis: Asp 2.06, Cys 0.98, Glu 0.98, Lys 0.98.

Example 10 17) (pGlu-Glu-Cys-Lys) 2

Compound 17 was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. The product was homogeneous by TLC (S +: Rf = 0.39) and gave positive reaction with ninhydrin and no absorption at 35 nv uv254 * HPLC analysis: 99.9% pure by area. Amino acid analysis: Cys 0.95, Glu 2.90, Lys 1.15.

20 DK 171992 B1

Example 11 18) (pGlu-Glu-Asn-Cys-Lys) 2

Compound (18) was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. Reagents not previously described: FMOC-Asn-OPFP. The product was homogeneous by TLC (S +: Rf = 0.42) and gave positive reaction with ninhydrin and no absorption at UV254.

HPLC analysis: 98.2% by area. Amino acid analysis: Asx 1.1, Cys 1.03, Glu 1.9, Lys 0.96.

Example 12 (19) (pGlu-Gln-Asp-Cys-Lys) 0 1 5 _ £

Compound (19) was synthesized and purified as described in Example 5 from the corresponding amino acid derivatives. Reagents not previously described: FMOC-Gln-OPFP. The product was homogeneous according to TLC (S +: Rf = 0.45) and gave positive reaction with ninhydrin and no absorption at UV254.

HPLC analysis: 98.3% pure by area. Amino acid analysis: Asp 1.09, Cys 1.06, Glx 1.89, Lys 0.96.

Example 13 20) (pGlu-Glu-Asp-Cys-Arg) 2

Compound (20) was synthesized and purified (5% mobile phase propan-2-ol) as described in Example 5 out of 30 from the relevant amino acid derivatives. The final removal of protecting groups was performed in 10% thioanisole in TFA.

Reagents not previously described: FMOC-Arg (Mtr) -SASRIN resin.

The product gave no reaction with ninhydrin and no absorption by UV254 * DK 171992 B1 21 HPLC analysis: 99.4% purely by area determination. Amino acid analysis: Arg 0.92, Asp 1.12, Cys 1.00, Glu 1.96.

Example 14 (21) (Gln-Glu-Asp-Cys-Lys) 2

Compound (21) was synthesized and purified as described in Example 5 from the relevant amino acid derivatives. The product was homogeneous according to TLC (S +: Rf = 0.57) and gave positive reaction with ninhydrin and no absorption by UV 254 * HPLC analysis: 94.7% purely by area determination. Amino acid analysis: Asp 1.00, Cys 1.01, Glx 2.02, Lys 0.96.

Example 15 22) (Pro-Glu-Asp-Cys-Lys) 2

Compound (22) was synthesized and purified as described in Example 5 using the appropriate amino acid derivatives. The product was pure by TLC (S +: Rf = 0.57) and gave positive reaction with ninhydrin and no absorption by UV 254 * HPLC analysis: 98.2% pure by area determination. Amino acid analysis: Asp 1.06, Cys 1.01, Glu 0.99, Lys 0.99, Pro 0.96.

30 35 22 DK 171992 B1

Determination of stimulatory effect In vitro test

The following peptides according to the invention were dissolved from

5 crystalline material in 0/9% saline and added to C3H

mouse bone marrow cells suspended in Eagle-Dulbecco's medium with 10% serum from newborn calves. After pre-incubation for 1 hour, the cells were plated in agar and the colonies counted after 7 days according to previous standardization of the method. Recombinant GM-CSF (Immunex) was used to give a colony count of the order of 30 to 100 colonies per 35 mm plate. By default, the pre-incubation concentrations of -5-15 peptide were 10 to 10 M, 5 plates per point.

GM-CFS - granulocyte-monocyte colony-stimulating factor

results

The results are listed as "maximum effect" and "wife", meaning that the peptide tested had its maximum effect (in%) at the concentration indicated (in molar units, M).

Peptide: Max. effect: Cone: (p-Glu-Glu-Asp-Cys-Lys) 2 165% 10 25 (Gln-Glu-Asp-Cys-Lys) 2 118% 10 Cys-Arg) 2 124% 10-6 (Asp-Cys-Lys) 2 140% 10 ^ (Cys-Lys) 2 145% 10

The results show that the dipeptide, tripeptide and pentapeptide dimers have stimulatory effect in the in vitro test system.

Claims (16)

1. Peptides, characterized in that they have the general formula 5 p A - (NH-CH-CO) - (NH-CH-CO) .- (NH-CH-CO) -BI a I bj (CH,) (CH-) CH, II 2 ni 2 m I 2 COR COR 'S-- _ J 2 10 where all amino acid units are in L configuration, where A represents 15 NH-CH-CO-, NH-CH-CO-, NH, -CH-CO- QXJ U <? H2> 2 0 CONH2 20 or a hydrogen atom; B represents -NH-CH-COR ", -NH-CH-CO-R" 25 (fH2} 4 (^ H2J3 nh9 NH HN NH2 30 or a hydroxy group; n and m independently of each other denote 1 or 2; 35). and b independently represent the number 0 or 1; R, R 'and R "independently represent a hydroxy group or an amino group; or constitute physiologically acceptable salts thereof, however, if A and B represent a hydrogen atom, respectively and a hydroxy group, then b cannot be the number 0. Peptides according to claim 1, characterized in that b represents the number 1, m represents the number 1, and R, R 'and R "represent hydroxy groups. Peptide according to claim 2, characterized in that it is 10 (Cys - Lys) 2, (Asp - Cys - Lys) 2 »(Glu - Asp - Cys - Lys) 2, (pGlu - Glu - Asp - Cys) 2, (pGlu - Asp - Cys - Lys) 2, (pGlu - Asp - Asp - Cys - Lys) 2, (pGlu - Glu - Asp - Cys - Arg) 2, (Gin - Glu - Asp - Cys - Lys) > 2 or (Pro - Glu - Asp - Cys - kiss) 2 20 Peptide according to claim 2, characterized in that it is (pGlu-Glu-Asp-Cys-Lys) 2. Process for the preparation of a peptide according to claim 1, characterized in that 25 protecting groups are removed from a compound of the general formula II A- (NH-CH-CO) .- (NH-CH-CO). - (NH-CH-CO) -B I a 1 b 1 (CH-) (CH,) CH, -_ 30 I 2 n I 2 mj 2 11 COR1 COR2 S - 2 where A represents 35 r3n-ch-co-, rVch-co-, r5rh-ch-co-, IJ IJ <iV2, CONHR6 O DK 171992 B1 a hydrogen atom or an amine protecting group; B represents -NH- <j: H-COR8, -NH-CH-COR10 5 (? H2. * 4 NHR ^ | b 9 HN NHRy 10 represents a hydroxy group or a carboxyl blocking group; n and m independently represent the number 1 or 2; a and b independently represent the number 0 or 1; i 2 8 10 R, R, R and R are amino groups, hydroxy groups, protected amino groups or carboxyl protecting groups; 3. S 6 7 9 R, R, R, R, R and R are hydrogen atoms or amine cooling groups; however, if A is a hydrogen atom or an amine protecting group and B is a hydroxy group or a carboxyl protecting group, then b cannot be the number 0. Process according to claim 5, characterized in that removal of protecting groups is carried out by acid hydrolysis, hydrogenolysis, ammonolysis or enzymatic hydrolysis. Process for the preparation of a peptide according to claim 1, characterized in that a compound of the general formula III is dimerized A- (NH-CH-CO) - (NH-CH-CO) (NH-CH-CO) -B (<fH2> n «rB2> m« K * 111 COR COR1 SR "B1 wherein A, B, n, m, a, b, R and R 'have the meanings specified in claim 1, and R "is a hydrogen atom or a thiol protecting group which can be removed under oxidizing conditions. Compounds of general formula II> A- (NH-CH-CO) (NH-CH-CO) <NH-CH-CO) -B '(2' '<CH2) n 4¾, COR1 COR2 S__ L-J 2 wherein A represents R3N-CH-CO-, R4N-CH-CO-, R5NH-CH-CO-, yes u C) CONHR® a hydrogen atom or an amine protecting group 20 selected from carbobenzoxy (Z) , t-butoxycarbonyl (Boc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and 9-fluorenylmethoxycarbonyl (Fmoc); B denotes -NH-CH-COR8, -NH-CH-COR10 (Ch2) 4 (<fH2> 3 NHR7 / g g HH NHR * 30 a hydroxy group or a carboxyl blocking group selected from benzyl (Bzl), p-nitrobenzyl (ONb) , pentachlorophenyl (OPClP), pentafluorophenyl (OPFP) and t-butyl (OtBu) denote B and n independently of the numbers 1 or 2; a and b independently of each other denote 0 or 1; c 12 8 10 3 R, R, R and R are amino groups, hydroxy groups, protected amino groups wherein the protecting group is selected from carbobenzoxy (Z), t-butoxycarbonyl (Boc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and 9- fluorenylmethoxycarbonyl (Fmoc), or carboscyl protecting groups selected from benzyl (Bzl), p-nitrobenzyl (ONb), pentachlorophenyl (OPC1P), pentafluorophenyl (OPFP) and t-butyl (OtBu) R3, R4, R5, R6, R 9 is hydrogen atoms or amine protecting groups selected from carbobenzoxy 15 (Z), t-butoxycarbonyl (Boc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and 9-fluorenylmethoxycarbonyl (Fmoc), however, so that if A is a hydrogen atom or an amine protecting group and B is a hydroxy group or a carboxyl protecting group, then b cannot be the number 0. 9. Pharmaceutical composition, characterized in that it contains as active ingredient one or more compounds of general formula I as set forth in claim 1 or a physiologically compatible salt thereof f, together with a pharmaceutical carrier or excipient. Use of a compound according to claim 1 for the preparation of a pharmaceutical composition for use in stimulating the myelopoietic system in humans or animals. 30 35