DK147918B - METHOD OF ANALOGUE FOR THE PREPARATION OF THYMOSINE ALFA1 (18 - 28) OR PHARMACEUTICAL ACCEPTABLE SALTS THEREOF - Google Patents

Description

147918

Thymosin is a heat stable, highly acidic polypeptide compound of 28 amino acid residues having the following sequence: 5 H-jC-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-15 -Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.

This peptide, which is a potent immunopotensing agent, is isolated by thymosin fraction 5 by Goldstein et al. by a combination of ion exchange chromatography and gel filtration (Proc. Natl. Acad. Sci.

USA 74, 725- 729 (1977)).

147918 2

It has now been found that certain peptides which are sequence fragments of thymosin α1 are effective for the regulation, differentiation and function of T cells, including the undecapeptide of the formula H-Glu-Lys-Lys-Glu-Val-Val-Glu -Glu-Ala-Glu-Asn-OH.

The present invention therefore relates to an analogous method for preparing the novel undecapeptide thymosin α1 (18 - 28) of the formula

H-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH I

in or pharmaceutically acceptable salts thereof, characterized in that the protecting groups are removed from a protected peptide of the formula

Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OB.zl) -Val-Val-Glu (OBzl) -Glu- (OBzl) -Ala-Glu (OBzl) -Asn-OBzl

II

where Boc is tart. -butyloxycarbonyl,

Bzl is benzyl and Z is benzyloxycarbonyl and, if desired, the compound obtained is converted to a pharmaceutically acceptable salt.

The removal of the protecting groups from the protected peptide of formula II is carried out in ways known per se, e.g. by treatment with anhydrous acid such as hydrogen fluoride, preferably in the presence of anisole.

The strategy used in the chemical synthesis of the protected undecapeptide of formula II was as follows: H-Glu (OBzl) -OH is first condensed with Boc-Ala-OSu to form the protected dipeptide fragment Boc-Ala-Glu (OBzl) ) -OH, which is then condensed with HCl.H-Asn-OBzl via the DCC / HOSu process following Wiinsch and Drees, Chem. Ber. £ 9, 110 (1966). The hydrochloride salt of the asparagine benzyl ester is prepared from Boc-Asn-OBzl, which is again synthesized from commercially available Boc-Asn-OH and benzyl bromide using the cesium salt of the amino acid. The Boc protecting group is removed by a 30 minute treatment with 4N HCl in dry THF.

By the reaction of H-Glu (OBzl) ~ OH and Boc-Glu (OBzl) -OSu, Boc-Glu (OBzl) -Glu (OBzl) -OH is obtained as a colorless clear oil. This is then used in the synthesis of the protected pentapeptide Boc-Glu (OBzl) - Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl in a DCC / HOSu mediated fragment condensation using HCl.H-Ala-Glu (OBzl) -Asn-OBzl derived from Boc-Ala-Glu (OBzl) -Asn-OBzl by treatment with 4N HCl / THF. The above protected pentapeptide is obtained in good yield in the form of a crystalline pure material.

For the preparation of the protected octapeptide Boc-Glu (OBzl) -Val - Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, the necessary protected tripeptide Boc-Glu (OBzl) is first prepared. -Val-Val-OH. Boc-Val-OSu is first allowed to react with free valine to form Boc-Val-Val-OH, from which by unblocking with 4N HCl in THF followed by reaction with Boc-Glu (OBzl) -OSu the desired tripeptide is crystallized. as cyclohexylamine salt Boc-Glu (OBzl) -Val-Val-OH.CHA. The cyclohexylamine salt is converted to the free acid and then condensed by DCC in the presence of HOSu with HCl.H-Glu (OBzl) -Glu (OBzl) -Ala-Glu- (OBzl) -Asn-OBzl derived from Boc-Glu ( OBzl) -Glu (OBzl) -Ala-Glu- (OBzl) -Asn-OBzl by treatment with HCl in THF. The protected octapeptide Boc-Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) - Asn-OBzl is obtained in purified form as an amorphous solid.

For the preparation of the protected undecapeptide Boc-Glu (OBzl) -Lys- (Z) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) - Asn-4 147918 -OBzl, the necessary tripeptide fragment is prepared from Boc-Lys (Z) - OSu and Η-Lys (Z) -OH. The dipeptide Boc-Lys (Z) -Lys (Z) -OH thus obtained is treated with 4N HCl in THF and the resulting salt HCl.H-Lys (Z) - Lys (Z) -OH is then reacted with Boc Glu (OBzl) -OSu to form the desired tripeptide Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OH. The tripeptide is then activated with DCC and HOSu by the method of Weygand et al., Z. Naturforsch. 21b, 426 (1966), and the solution of the in situ formed tripeptide ester Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OSu is combined with the trifluoroacetate salt of H-Glu (OBzl) -Val-Val-Glu ( OBzl) - Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, which is derived from the corresponding blocked octapeptide by a 30 minute treatment with trifluoroacetic acid. Thus, upon addition of a small amount of base, the desired protected undecapeptide Boc-Glu (OBzl) -Lys (Z) -Lys (Z) - Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) - Ala-Glu (OBzl) -Asn-OBzl.

Upon deprotection of the protected undecapeptide Boc-Glu (OBzl) -Lys (Z) - Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) - Asn - OBzl e.g. with anhydrous hydrogen fluoride, the free undecapeptide H-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH is obtained, which, after ion-exchange column chromatography, is homogeneous by paper electrophoresis.

The novel undecapeptide produced by the method of the present invention, as well as its pharmaceutically acceptable salts, can be administered to warm-blooded mammals by parenteral administration, either intravenously, subcutaneously or intramuscularly. This compound is a potent immunopotensing agent at a daily dose in the range of ca. 1 to 100 mg / kg body weight per day. day by intravenous administration. The dose required will of course vary with the particular condition being treated, the severity of the condition and the duration of treatment. A suitable dosage form for pharmaceutical use is 1 mg of lyophilized peptide, which is reconstituted before use by the addition of sterile water or saline solution.

5 147918

Pharmaceutically acceptable salts of the above peptide include sodium and potassium salts or salts with a strong organic base such as guanidine. In addition, the counterion to these cations, e.g. the chloride, bromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate and the like are included in the composition.

The following example describes in detail the synthesis of the undecapeptide.

The abbreviations used here have the following meanings:

Boc = tert-Butyloxycarbonyl, Bzl = benzyl, DCC = dicyclohexylcarbodiimide, DMF = dimethylformamide, THF = tetrahydrofuran, HOSu = N - hydroxysuccinimide, Triton B = 40% methanolic solution of trimethylbenzylammonium hydroxide , CHA = cyclohexylamine, DCHA = dicyclohexylamine, Z = benzyloxycarbonyl, DMSO = dimethylsulfoxide, TFA = trifluoroacetic acid, TLC = thin layer chromatography, Et ^ N = triethylamine, and HOBT = 1-hydroxybenzotriazole.

Example 15 g (39.5 millimoles) of Boc-Lys (Z) -OH is stirred together with 5.8 g (50.5 millimoles) of HOSu and 8.66 g (42 millimoles) of DCC in 250 ml of THF for 3 hours. An insoluble by-product is filtered off and the filtrate is evaporated to dryness. The syrup obtained as residue (24.2 g) is treated with 150 ml of isopropanol and 150 ml of petroleum ether to give 21 g of an oily product which does not crystallize. Thus, the crude active ester Boc-Lys (Z) -OSu is used to condense 10.6 g (38 millimoles) of H-Lys (Z) -0H in 250 ml of DMF for 72 hours in the presence of 5.5 ml of EtgN. From time to time more Et 1 N is added to maintain the stirred reaction mixture slightly basic. A small amount of undissolved material is then filtered off and the filtrate is evaporated to dryness (45 ° C). The oily residue obtained is treated with 1 liter of 5% acetic acid. The precipitated product is extracted into ethyl acetate and the organic phase is washed with water, dried with sodium sulfate and evaporated to an oil. This is crystallized from 300 ml of ethyl acetate, which contains 10 ml of DCHA in the form of a salt. Recrystallization from methanol and ether gives 22.7 g (72.5%) of Boc-Lys (Z) -Lys (Z) -OH.DCHA, mp 160 - 162 ° C, [α] p 5 = -2.21 ° (c = 1, methanol).

10 g (12.14 millimoles) of Boc-Lys (Z) -Lys (Z) -OH.DCHA is partitioned between 1 liter of ethyl acetate and 1 liter of 0.1N sulfuric acid. The organic phase is then washed three times with water, dried over sodium sulfate and evaporated to dryness to give 7.9 g. The thus obtained free acid Boc-Lys (Z) -Lys (Z) -OH is treated with freshly prepared 4N HCl in THF for 30 minutes. The solvent and excess acid are evaporated (30 ° C) and the residue is evaporated twice with THF. The residue obtained solidifies by ether treatment. The salted HCl.H-Lys- (Z) -Lys (Z) -OH is collected by filtration and washed several times with ether to give 6.7 g of white powder. It is dissolved in 70 ml DMF, cooled in an ice bath and treated with 1.63 ml Et 2 N followed by 5.54 g (12.76 millimoles) of Boc-Glu (OBzl) -OSu. The mixture is stirred at 0 ° C for 1 hour and then at 25 ° C for 24 hours. More Et 2 N is added during this period to maintain the reaction environment at a pH of about 7.5. Add a few ml of acetic acid to make the reaction mixture acidic (pH 3.5) and evaporate the solvent. The residue obtained is taken up in ethyl acetate, washed three times with water, dried over sodium sulfate and evaporated to dryness as the product begins to solidify. It is triturated in ether and recrystallized from ethyl acetate. Yield: 7.26 g (69.5%) of Boc-Glu- (OBzl) -Lys (Z) rLys (Z) -OH, m.p. 153 - 155 ° C, [α] D = -2.71 ° (c = 1 , THF).

Dissolve 11.0 g (47.5 millimoles) of Boc-Asn-OH in 200 ml of methanol and add 20 ml of water. The solution is titrated to pH 7.0 with a 20% aqueous solution of cesium carbonate (about 55 ml). The mixture is evaporated to dryness and the residue is twice evaporated from dimethylformamide (120 ml each time, 45 ° C). The white solid obtained is then stirred together with 8.9 g (52 millimoles) of benzyl bromide in 120 ml of dimethylformamide for 6 hours. After evaporation to dryness and treatment with a large volume of water, the product immediately solidifies. It is filtered off and dissolved in ethyl acetate, washed with water, dried over sodium sulfate and evaporated to give a solid mass which is crystallized from ethyl acetate with petroleum ether. There is thus obtained 13.8 g (90.3%) of Boc-Asn-OBzl, mp 120 - 122 ° C, [α] p 5 = -17.29 ° (c = 1, DMF).

Dissolve 13.7 g (42.4 millimoles) of Boc-Asn-OBzl in 80 ml of tetrahydrofuran and treat with 500 ml of 4N HCl in THF. The mixture is allowed to stand for 45 minutes, during which time some product begins to precipitate. When treated with 1000 ml of ether, a white solid forms immediately. The product is filtered off, washed with ether and dried over sodium hydroxide beads in vacuo. Yield: 10.3 g (94%) of HCl.H-Asn-OBzl, m.p. 122 - 126 ° C, [α] 25 D = + 6.82 °.

7.0 g (29.5 millimoles) of H-Glu (OBzl) -OH are finely ground in a mortar and then stirred with 8.88 g (32.3 millimoles) of Boc-Ala-OSu for 48 hours in 250 ml of DMF in the presence of 6 ml of NMM. An additional NMM is added to maintain the reaction environment slightly alkaline during the reaction. The solvent is evaporated and the residue is partitioned between 300 ml of ethyl acetate and 500 ml of water containing 2 ml of 10% sulfuric acid.

The organic phase is then washed three times with water, dried over sodium sulfate and evaporated to dryness. The product is taken up in a small volume of ether and treated with a large volume of petroleum ether. Thereby a white amorphous solid is obtained which is homogeneous on TLC. Yield: 11.0 g (91.5%) of Boc-Ala-Glu (OBzl) -OH, mp 84-88 ° C, [ct] J = 8.08 ° (c = 1, DMF).

10.4 g (25.4 millimoles) of Boc-Ala-Glu (OBzl) -OH, 6.56 g (25.4 millimoles) of HCl.H-Asn-OBzl and 5.9 g (50.8 millimoles) of HOSu dissolve in 250 ml of dimethylformamide at 0 ° C. DCC (5.7 g, 27.6 millimoles) is added immediately followed by 3.5 mL of Et 2 N. The mixture is stirred at 0 ° C for 2 hours and then at 25 ° C for 40 hours, occasionally adding some more EtgN to maintain a weak basic reaction environment. The insoluble by-products formed are filtered off and the filtrate is evaporated to dryness. The oily material obtained from the residue solidifies by treatment with water. The crude product is taken up in chloroform, washed three times with water and dried over sodium sulfate and evaporated to a smaller volume. Some solid formed in this step is filtered off (heavily contaminated with dicyclohexylurine) and the filtrate is treated with petroleum ether. A crystalline product is obtained. Yield: 8.0 g (51.4%) of Boc-Ala-Glu (OBzl) -Asn - OBzl, mp 102 - 105 ° C, [α] D = 12.5 ° (c = 1, DMF).

4.74 g (20 millimoles) of H-Glu (OBzl) -OH is ground in a mortar and stirred with 0.7 g (20 millimoles) of Boc-Glu (OBzl) -OSu in DMF for 36 hours in the presence of 3, 6 ml NMM. The resulting solution is evaporated to a syrup and treated with water. The oily precipitate is taken up in ethyl acetate, washed with 5% acetic acid and 3 times with water in the above order, dried over sodium sulfate and evaporated to dryness to give 14.03 g of a clear oil. This is left under petroleum ether. The oily Boc-Glu (OBzl) -Glu (OBzl) -OH obtained residue obtained as the residue weighs 10.2 g (90.0%).

TLC indicates that the product is homogeneous, [α] 25 = -7.59 ° (c = 1, DMF).

28.2 g (46 millimoles) of Boc-Ala-Glu (OBzl) -Asn-OBzl are treated with 1.1 liters of 4N HCl in THF for 1 hour. Evaporation of the solvent and excess acid gives an oil which is evaporated twice more with fresh THF. The oil obtained from the residue solidifies when treated with a large volume of ether. The solid HCl.Η-Ala-Glu (OBzl) - Asn-OBzl is stirred together with 25.6 g (46 millimoles) of Boc-Glu (OBzl) - Glu (OBzl) -0H, 10.6 g (92 millimoles) ) HOSu and 10.9 g (53 millimoles) of DCC in 540 ml of DMF at 0 ° C for 1 hour and then at 25 ° C for 48 hours. Et ^ N is added throughout the period to maintain a weak basic reaction environment (a total of about 16 ml of Et ^ N). The insoluble by-products formed are filtered off and the filtrate is evaporated to dryness. The crude product is dissolved in chloroform, washed three times with water, dried over sodium sulfate and evaporated to dryness. The product solidifies by treatment with petroleum ether. Recrystallization from isopropanol gives 28.9 g (59.8%) of Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, mp 169-175 ° C, [α] -11.78 ° (c = 1, DMF).

3.9 g (3.48 millimoles) of Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn - OBzl are treated with 15 ml of 4N HCl in THF for 30 minutes. Some crystalline product begins to form. 210 ml of ether are added and the precipitated solid is collected and washed with ether. The crude material is crystallized by methanol and ether. Yield: 2.58 g (75.1%) of HCl.Η-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, m.p. 148-151 ° C, [a] p5 = - 3.65 ° (c = 1, DMF).

12.6 g (40 millimoles) of Boc-Val-OSu and 4.68 g (40 millimoles) of H-Val-OH are condensed in 250 ml of DMF for 96 hours in the presence of 2 ml of Et 2 N.

More Et ^ N is added when needed to keep the reaction environment weakly basic. The remaining insoluble material is filtered off and the filtrate is evaporated to dryness (45 ° C). The residue is partitioned between ether and dilute sulfuric acid (about 1%), and the organic phase is washed three times with water, dried over sodium sulfate and evaporated to a foamy glass. The product is crystallized from ether and petroleum ether. Yield: 12.2 g (96.4%) of Boc-Val-Val-OH, mp 155 - 158 ° C, [α] 25 D = + 1.10 ° (c = 1, DMF).

40.5 g (128 millimoles) of Boc-Val-Val-OH are treated with 1.8 liters of 4N HCl in THF for 60 minutes. Evaporation to remove excess acid and solvent followed by ether treatment yields 34.5 g of HCl.H-Val-Val-OH in the form of a white amorphous powder. It is treated with 55.6 g (128 millimoles) of Boc-Glu (OBzl) -OSu in 1 liter of DMF for 24 hours in the presence of 54 ml of Et 2 N. The reaction mixture is filtered to remove some insoluble material and the filtrate is evaporated to dryness. The oily material obtained as the residue is taken up in 1.5 liters of ethyl acetate and washed twice with 5% acetic acid and then three times with water. The organic phase is dried over sodium sulfate and evaporated to dryness to give a colorless clear oil which does not crystallize. It is thus dissolved in 3.2 liters of ether and treated with 17 ml of CHA until the pH of the mixture is 7.5.

The obtained solid salt is collected and recrystallized from methanol and ether. Yield: 58.9 g (72.7%) of Boc-Glu (OBzl) -Val-Val-OH.CHA, mp 158-160 ° C, [α] D = 33.41 ° (c = 1, methanol) .

1.69 g (2.66 millimoles) of Boc-Glu (OBzl) -Val-Val-OH.CHA is suspended in 40 ml of water and 40 ml of ethyl acetate in a separatory funnel after the addition of 4 ml of 1M sulfuric acid. After vigorous shaking, the solid dissolves and the organic phase is washed several times with water, dried over sodium sulfate and evaporated to 1.45 g of oil. The free tripeptide thus obtained is then condensed with 2.58 g (2.61 millimoles) of HCl.H-Glu- (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl in 15 ml of DMF in the presence of 0.612 g (5.32 millimoles) of HOSu, 0.3 ml (2.66 millimoles) of NMM and 0.63 g (3.06 millimoles) of DCC for 1 hour at 0 ° C and for 60 hours at 25 ° C.

More NMM is added when needed to maintain a weak basic reaction environment. A formed insoluble by-product is filtered off and the filtrate is evaporated to dryness (45 ° C). The oily material obtained from the residue solidifies by treatment with water. The crude solid is dissolved in 50 ml of DMF and precipitated with 300 ml of methanol. Yield: 2.25 g (58.7%) of Boc-Glu (OBzl) -Val-Val-Glu (OBzl) - Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, mp 277-280 ° C = [α] D = -12.43 ° (c = 1, DMF).

1.7 g (1/16 millimole) of Boc-Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) - Ala-Glu (OBzl) -Asn-OBzl is treated with 24 ml of TFA in 30 minutes.

After evaporation of an excess acid (30 ° C), the residue is triturated with ether. The obtained powder is washed thoroughly with ether and petroleum ether and dried over sodium hydroxide in vacuo to give 1.71 g of the trifluoroacetate salt of the octapeptide. The active ester Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OSu is then formed in situ by stirring 0.998 g (1.16 millimoles) of Boc-Glu (OBzl) -Lys (Z) -Lyz ( Z) -OH, 0.16 g (1.4 millimoles) of HOSu and 0.274 g (1.33 millimoles) of DCC in 15 ml of DMF at 0 ° C for 3 hours. To this solution containing the active tripeptide ester is added 1.71 g of the octapeptide salt CF3COOH.H - Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn- OBzl together with 0.2 ml of Et ^ N. A few more drops of Et 2 N and 15 ml of DMF are added and the mixture is stirred for 3 days at 25 ° C. A gelatinous semi-solid is formed. It is acidified with acetic acid and treated with water. The precipitated white solid is collected and washed with water, methanol and ether to give 2.25 g of crude product, which melts at 310 r 313 ° C. It is dissolved in DMF and precipitated with methanol. Yield: 1.75 g (68.3%) of Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val - Val-Glu (OBzl) -Glu (OBzl) -Ala -Glu (OBzl) -Asn-OBzl, mp 314 - 316 ° C, [α] p 5 = 13.68 ° (c = 1, DMSO), homogeneous on TLC.

0.5 g (0.226 millimoles) of Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val - Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) ) -Asn-OBzl is dissolved in 2 ml of TFA and stirred with 15 ml of HF at 0 ° C for 15 minutes. After evaporation of excess acid (0 ° C), the residue is dissolved in 5% aqueous acetic acid, washed three times with ether, evaporated to a smaller volume and lyophilized to give 0.34 g of a crude product. It is chromatographed on a 3 x 32 cm column of highly basic polystyrene resin (Bio-Rad AGl-x 2) equilibrated with ammonium acetate buffer of pH 8.1 (2% acetic acid gives pH 8.1 along with ammonia). The column is eluted successively with 200 ml portions of 0.025 M ammonium acetate of pH 5.5, 0.025 M acetic acid, 0.05 M acetic acid, 0.1 M acetic acid, 0.25 M acetic acid, 0.5 M acetic acid, 0.75 M acetic acid and 1 M acetic acid. Fractions of 12 ml are collected and the eluate from each tube is measured by TLC. The fractions containing the desired material are combined and lyophilized twice to give 0.13 g (42.1%) of pure H-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu Asn-OH, [α] 25 = -85.65 ° (c = 1, H2 <0).