NO148924B - ANALOGY PROCEDURE FOR THE PREPARATION OF IMMUNPOTENTPENSING PEPTIDES - Google Patents

Description

Thymosin is a heat-stable strong acidic polypeptide compound with 28 amino acid residues with the following sequence:

This peptide which is a strong immunopotentiating agent is isolated from thymosin fraction 5 by Goldstein et al. by a combination of ion-exchange chromatography and gel filtration (Proe. Natl. Acad. Sei. USA _74_, 725-729 (1977)).

It has now been found that certain peptides which are sequence fragments of thymosin also show immunopotential activity, i.e. they influence the regulation, differentiation and function of T cells

Accordingly, the present invention relates to an analogue method for the production of peptides with the general formula in which R<1> is hydrogen, H-Glu-Lys-Lys- or and of pharmaceutically acceptable salts thereof, which method consists in removing the protective groups from a protected peptide of the general formula

wherein R 2 is Boe, Boc-Glu(OBzl)-Lys(Z)-Lys(Z)- or Bon is tert-butyloxycarbonyl,

Bzl is benzyl and

Z is benzyloxycarbonyl,

and, if desired, converting the obtained compound into a pharmaceutically acceptable salt.

Removal of the protective groups from a protective peptide of formula II is easily carried out by known methods such as e.g. by treatment with anhydrous acid such as hydrogen fluoride, preferably in the presence of anisole.

The system used in the chemical synthesis of the peptides of formula I was as follows: H-Glu(OBzl)-OH was first coupled with Boc-Ala-OSu giving the protected dipeptide fragment Boc-Ala-Glu(OBzl)-OH which was then condensed with HC1.H-Asn-OBzl via the DCC/HOSu procedure of Wunsch and Drees, Chem. Pray. 99,110 (1966). The hydrochloride salt of asparagine benzyl ester was prepared from Boc-Asn-OBzl which was in turn synthesized from commercial Boc-Asn-OH and benzyl bromides using the cesium salt of the amino acid. The Boc protecting group was removed by a 30 min. treatment with 4N HCl in dry THF.

The reaction between H-Glu(OBzl)-OH and Boc-Glu(OBzl)-OSu gave Boc-Glu(OBzl)-Glu(OBzl)-OH as a colorless clear oil. It was 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 HC1 " H-Ala-Glu(OBzl)-Asn -OBzl sen derived from Boc-Ala-Glu(OBzl)-Asn-OBzl after 4N HC1/THF treatment, the aforementioned protected pentapeptide was obtained

in good yield as crystalline pure material.

For the preparation of the protected octapeptide Boc-Glu (OBzi )-Val-Val-Glu (OBzl)-G].u (OBz L)-Ala-Glu (OBzl)-Asn-OBzl, the required protected tripeptide Boc-Glu (OBzl)-Val-Val-OH first prepd. Boc-Val-OSu was allowed to react with free valine which gave Boc-Val-Val-OH which after deblocking with 4N.HCl in THF followed by reaction with Boc-Glu(OBzl)-OSu gave the desired tripeptide which was crystallized as cyclohexylarain salt Boc-Glu(OBzl)-Val-Val-OH ' CHA. The cyclohexyamine salt was transferred into the free acid and became

then coupled with DCC in the presence of HOSu to HC1 " H-Glu-(OBzl)-Glu(OBzl)-Ala-Giu(OBzl)-Asn-OBzl which originated from Boc-Glu (OBzl)-Glu(OBzl)-Ala -Glu(OBzl)-Asn-OBzl on treatment with HC1 in THF The protected octapeptide Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl) -Asn-OBzl was obtained in purified form as an amorphous solid. The protecting group was removed hydrogenolytically followed by treatment - with trifluoroacetic acid in the usual manner to give the free octapeptide Glu-Val-Val-Glu-Glu-Ala-Glu-Asn. This product was purified by ion -exchange column chromatography and gave a thin-layer chromatographic paper electrophoresis-

tically homogeneous material.

For the synthesis of the protected undecapeptide Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu (OBzl)-Asn -OBzl, the required tripeptide fragment was synthesized from Boc-Lys(Z)-OSu and H-Lys(Z)-OH. Dipep-

the time Boe-Lys(Z)-Lys(Z)-OH thus obtained was treated with 4N HC1 in THF and the formed salt HC1. H-Lys(Z)-Lys(Z)-OH was then allowed to react with Boc-Glu(OBzl)-OSu which

gave the desired tripeptide Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH. The tripeptide was then activated with DCC and HOSu according to Weygand et al., Z. Naturforsch. 21b, 426 (1966), and the solution of the active tripeptide ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu prepared in situ combined with the trifluoroacetate salt H-Glu(OBzl)-Val-Val-Glu( OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl which originated from the corresponding blocked octapeptide at a 30 min. treatment with TFA. After addition of a small amount of a 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 with anhydrous hydrofluoric acid gave the free undecapeptide Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn which was homogeneous by papix electrophoresis after ion-exchange column-chromatography.fi.

The synthesis of the protected tetradecapeptide followed a similar scheme. Boc-Leu-OSu was coupled with H-Lys(Z)-OH giving Boc-Leu-Lys(Z)-OH. After removal of the KK-Boc group with 4N HC1 in THF and reaction with Boc-Asp (OBzl)-OSu, the protected tripeptide Boc-Asp(OBzl)-Leu-Lys(Z)-OH was obtained as a crystalline solid

fabric. It was transferred in the active ester Boc-Asp(OBzl)-Leu-Lys(Z)-OSu

and condensed with the trifluoroacetate salt of H-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu (OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn- OBzl obtained from TFA treatment of the corresponding blocked undecapeptide. The desired product Boc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu (OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl) -Ala-Glu(OBzl)-Asn-OBzl was obtained in

good yield. Thin layer chromatography indicated that the product was homogeneous.

The free tetradecapeptide Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn was obtained from the protected compound by treatment with anhydrous hydrofluoric acid and purification on an ion-exchange column.

The new octa-, undeca- or tetradecapeptides and their pharmaceutically acceptable salts can be administered to warm-blooded mammals

animals by parenteral application either intravenously, subcutaneously or intramuscularly. These compounds are strong immunopotentiating agents at a daily dosage in the range from approx. 1-100 mg/kg body weight per day by intravenous administration.

Obviously, the required dose will vary with the particular condition being treated, the degree of the condition and the duration of the treatment. A suitable dosage form for pharmaceutical use is a 1 mg freeze-dried peptide which must be reconstituted before use by adding sterile water or saline.

The following table shows the results of an experiment demonstrating the activity of peptides I (called octa-, undeca-

and tetradecapeptides) in eliciting a delayed-type hypersensitivity (DTH) reaction that was partially suppressed by the injection of 5-fluoro-uracil (5-Fu) in mice.

The pharmaceutically acceptable salts of the aforementioned pep-

times include the sodium and potassium salts or salts with a strong organic base such as guandine. In addition, the anions of these cations such as chlorine, bromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate etc.,

included in the preparations.

The abbreviations used herein have the following meanings: Boc=t-butyloxycarbonyl; Bzl=benzyl; DCC=dicyclohexylcarbodiimide; DMF=dimethylformamide; THF=tetrahydrofuran; HOSu= N-hydroxysuccinimide; Triton B=4 0% methanol solution of trimethylbenzylammonium hydroxide; NMM=N-methylmorpholine; CHA=cyclohexylamine; DCHA= dicyclohexylamine; Z=benzyloxycarbonyl; DMSO=dimethylsulfoc-

south; TFA=trifluoroacetic acid; TLC=thin layer chromatography; ET^N= triethylamine; HOBT=1-hydroxybenzotriazole.

The following examples describe in detail the synthesis of the octa-, undeca- and tetradecapeptides of formula I.

Although specific protective groups are used in these

examples, it will be obvious to one skilled in the art to employ equivalent protecting groups in such syntheses.

Example 1

A.a) Boc-Asn-OH (11.0 g, 47.5 mmol) was dissolved in 200 mL of MeOH

and 20 ml of water was added. The solution was titrated to pH 7.0

with a 20% aqueous solution of Cs 2 CO 3 (ca. 55 ml). The mixture was evaporated to dryness and the residue was evaporated twice from DMF (120 mL each time, 45°C). The white solid obtained was then stirred with 8.9 g of benzyl bromide (52 mmol) in 120 ml of DMF for 6 hours. By evaporation to dryness and treatment with

a large volume of water, the product immediately solidified. It was collected by filtration, dissolved in ethyl acetate, washed with water, dried over Na 2 SO 4 , evaporated to a solid and crystallized from ethyl acetate with petroleum ether. Yield 13.8 g (90.3%) of Boc-Asn-OBzl; m.p. 120-122°C; 00 δ = -17.29° (c=1, DMF).

Boc-Asn-OBzl (13.7 g, 42.4 mmol) was dissolved in 80 mL of THF

and treated with 500 mL of 4N HCl in THF. The mixture was allowed to stand

45 min. below which some product began to precipitate. On treatment with 1000 ml of ether, a white solid formed immediately. The product was filtered, washed with ether and dried over NaOH pellets in vacuo. Yield: 10.3 g (94%) of HCl'H-Asn-OBzl; m.p. 122-126°C; W^5=+6.8 2°. b) H-Glu(OBzl)-OH (7.0 g, 29.5 mmol)' was finely crushed with a mortar and then stirred with 8.88 g (32.3 mmol) of Boc-Ala-OSu for 48 h in 250 ml DMF in the presence of 6 ml NMM. Some more NMM was added to keep the environment slightly basic during the reaction. The solvent was evaporated and the residue partitioned between 300 ml ethyl acetate and 500 ml H 2 O containing 2 ml of 10% H 2 SO 4 - The organic layer was then washed 3 times with water, dried over Na 2 SO 4 and evaporated to dryness. The product was taken up in a small volume of ether and treated with a large volume of petroleum ether. A white amorphous solid was obtained which was homogeneous in TCL. Yield: 11.0 g (91.5%) of Boc-Ala-Glu(OBzl)-OH; m.p. 84-88°C; £- 1 ^<5> 8.08° (c=1,DMF).

Boc-Ala-Glu(OBzl)-OH (10.4 g, 25.4 mmol), HCl . H-Asn-OBzl

(6.56 g, 25", 4 mmol) and HOSu (5.9 g, 50.8 mmol) were dissolved in DMF (250 mL, 0 °C). DCC (5.7 g, 27.6 mmol) was added immediately followed by Et 3 N (3.5 mL).The mixture was stirred at 0°C for two

7

hours and then at 25°C for 40 hours, during which a little more Et^N was added from time to time to keep the environment slightly basic.

The insoluble by-products formed were filtered off and the filtrate evaporated to dryness. The remaining oily material solidified on treatment with water. The crude product was taken up in CHCl^ and washed with water (3x), dried over Na2SO^ and concentrated

pet to a smaller volume. Some solid formed in this step was filtered off (heavily contaminated with dicyclohexylurea) and the filtrate treated with petroleum ether. A crystalline product was obtained. Yield: 8.0 g (51.4%) of Boc-Ala-Glu(OBzl )-Asn-OBzl ); m.p. 102-105°C; G^3 ^5 = 12.5° (c=1., DMF) .

c) H-Glu(OBzl)-OH (4.74 g, 20 mmol) was crushed in a mortar and stirred with Boc-Glu(OBzl)-OSu (0.7 g, 20 mmol) in DMF in

36 hours in the presence of 3.6 ml of NMM. The resulting solution was evaporated to a syrup and treated with water. The oily precipitate was taken up in ethyl acetate, washed first with 5% HOAc and then water (3x), dried over Na 2 SO 4 and evaporated to dryness, yielding 14.03 g of a clear oil. It was allowed to stand under petrol. The remaining oily Boc-Glu(OBzl)-Glu(OBzl)-

OH weighed 10.2 g (90%). TLC indicated that the product was homogeneous.M^5 -7.59° (c=1, DMF).

d) Boc-Ala-Glu(OBzl)-Asn-OBzl) (28.2 g; 46 mmol was treated with 1.1 L of 4N HCl in THF for 1 h Evaporation of solvent and excess acid left an oil which was evaporated 2 more times with fresh THF The residual oil passed to a solid

substance when treated with a large volume of ether. The solid HCl H-Ala-Glu(OBzl)-Asn-OBzl was stirred with Boc-Glu(OBzl)-Glu(OBzl)-OH 825.6 g, 46 mmol), HOSu (10.6 g, 92 mmol) and DCC 810.9 g 53 mmol) in DMF (540 mL) at 0° for one hour and then at 25° for 48 hours.

Et^N was added to keep the environment slightly basic throughout the period (approx. 16 ml of Et^N in total). The insoluble byproducts

the solids that formed were filtered off and the filtrate evaporated to dryness. The crude product was dissolved in CHCl 4 , washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness. The product solidified on treatment with petroleum ether. Recrystallization from isopropanol gave 28.9 g (59.8%) of Boc-Glu(OBzl)-Glu (OBzl)-Ala-Glu(OBzD-Asn-OBzl; m.p. 16 9-17 5°C; M ^5 =-11.78°

(c=1, DMF).

Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (3.9 g, 3.48 mmol) was treated with 15 mL of 4N HCl in THF for 30 min. Some crystalline product started and forms. Ether (210 mL) was added and the precipitated solid was collected and washed with ether. The crude material was crystallized from MeOH and ether. Dividend: 2.58

g (75.1%) of HC1'H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl: m.p. 148-151°C: pJ^<5> = -3.65° (c=1, DMF).

e) Boc-Val-OSu (12.6 g, 40 mmol) and H-Val-OH (4.68 g, 40

mmol) was condensed in DMF 8250 ml) for 96 h in the presence of

2 ml Et^N. More Et^N was added as needed to keep the environment slightly basic. The remaining insoluble material was filtered off and the filtrate evaporated to dryness (45°C). The residue was partitioned between ether and dilute ^SO^ (approx. 1%). and the organic layer washed with water (3x), dried over Na 2 SO 4 and evaporated to a frothy glassy mass. The product was crystallized from ether and petroleum ether. Yield: 12.2 g (96.4%) of Boc-Val-Val-OH; m.p. 155-158°C^5 = +1.10° (c = 1, DMF).

Boc-Val-Val-OH (40.5 g, 128 mmol) was treated with 1.8 L of

4N HCl in THF for 60 min. Evaporation to remove excess acid and solvent followed by treatment with ether gave 34.5 g of HC1<*>H-Val-Val-OH as a white amorphous powder. It was treated with Boc-Glu(OBzl)-OSu (55.6 g, 128 mmol) in 1 L of DMF for 24 h in the presence of 54 mL of Et₂N. The reaction mixture was filtered to remove any insoluble material and the filtrate was evaporated to dryness. The remaining oily residue was taken up in EtOAc (1.5 L) and washed with 5% HOAc (2x) followed by water (3x).

The organic layer was dried (Na 2 SO 4 ) and evaporated to dryness to give a colorless clear oil which did not crystallize.

It was then dissolved in 3.2 L of ether and treated with CHA (17 mL) until the pH of the mixture was 7.5. The solid salt obtained was collected and recrystallized from MeOH and ether. Yield: 58.9 g 72.7%) of Boc-Glu(OBzl)-Val-Val-OH'CHA; m.p. 158 - 160°C;

^] 25 = 33.41<0> (c = 1f MeOH) .

Boc-Glu(OBzl)-Val-Val-OH"CHA (1.69 g, 2.66 mmol) was prepared

slurry in water (40 ml) and ethyl acetate (40 ml) in a separatory

funnel while adding 4 ml of 1 M H2S04. After heavy pipe-

ing, the solid was dissolved, and the organic layer was washed several times with water, dried over Na^SO^ and in-

evaporated to an oil (81.45 g). The free tripeptide thus obtained was then condensed with 2.58 g of HCl' H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (2.61 mmol) in 15 ml of DMF in the presence of

HOSu (0.612 g, 5.32 mmol), NMM (0.3 mL, 2.66 mmol) and DCC (0.63 g, 3.0 6 mmol) during one hour at 0°C and 60 h at 25 C.

More NMM was added when needed to -keep the environment slightly alkaline. An insoluble by-product formed was filtered off and the filtrate evaporated to dryness (45°C). The remaining oil asset

the rest solidified by treatment with water. The solid raw product

tet was dissolved in DMF (50 mL) and quenched with MeOH (300 mL).

Yield: 2.25 g (58.7%) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu (OBzl)-Ala-Glu(OBzl)-Asn-OBzl; m.p. 277-280°C C^J^5 = -12.43°

(c = 1, DMF).

This product (0.72 g, 0.49 mmol) was hydrogenated over 15% Pd/BaSO 4 (0.5 g) for 3 h at 3.4 atm. in a mixture of 4 0

ml DMF/30 ml MeOH/2 ml H 2 O. The mixture was then filtered and the filtrate evaporated to dryness. It was then treated with 5 ml of TFA for 30 min. and the residue obtained after evaporation of the acid was triturated several times with ether. The solid formed was taken up in water (20 mL) and lyophilized to give

0.47 g crude product. The compound was loaded onto a 3 x 32 cm column of a strongly basic polystyrene resin (Bio-Rad AG1-X 2) equilibrated with pH 8.1 ammonium acetate buffer (2% HOAc adjusted to pH 8.1 with NH 2 ). The column was eluted successively with

200 ml of 0.025 M pH 5.5 NH4OAc, 0.025 M HOAc, 0.05 M HOAc,

0.25 M HOAc, 0.5 M HOAc, 0.75 M HOAc, 1M HOAc. Fractions .on

12 ml was collected and the eluate from each tube examined by

TLC. The fractions containing the desired material (tubes 225-229) were pooled and lyophilized twice to give 0.223

g (48.1%) of pure Glu-Val-Val-Glu-Glu-Ala-Glu-Asn which was homogeneous in TLC and paper electrophoresis.

Example 2

Boc-Lys(Z)-OH (15 g, 39.5 mmol) was stirred with HOSu (5.3 g,

50.5 mmol) and 8.66 g, 42 mmol) in THF (250 mL) for three hours.

An insoluble by-product was filtered off and the filtrate in-

steamed to dryness. The remaining syrup (24.2 g) was treated with isopropanol (150 ml) and petroleum ether (150 ml) to give an oily product (21 g) which did not crystallize. The crude active ester Boc-Lys(Z)-OSu was thus used for condensation with H-Lys(Z)-OH (10.6 g, 38 mmol) in DMF (250 mL)

for 72 hours in the presence of 5.5 ml Et^N. More Et^N was added occasionally to keep the stirred reaction mixture slightly basic.

A small amount of undissolved material was then filtered off and the filtrate evaporated to dryness (45°C). The remaining oil residue was treated with 1.5 ml of 5% HOAc. The precipitated product was extracted into ethyl acetate and the organic phase washed with water, dried over Na2S©4 and evaporated to an oil.

It was crystallized from ethyl acetate (300 mL) containing DCHA (10 mL) as a salt. The recrystallization from MeOH and

ether gave 22.7 g (72.5%) of Boc-Lys(Z)-Lys(Z)-OH'DCHA; m.p. 160-16 2°C; E<:]^5 -2.21° (c = 1.M.eOH).

Boc-Lys(Z)-OH DCHA (10 g, 12.14 mmol) was partitioned between

EtOAc (1 L) and 0.1 N H 2 SO 4 (1 L). The organic layer was then washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness (7.9 g). The free acid, Boc-Lys(Z)-Lys(Z)-OH thus obtained, was treated with freshly prepared 4N HCl

in THF for 30 minutes. Solvent and excess acid were evaporated (30°C) and the residue evaporated again 2 times with THF.

The remaining residue solidified by treatment with ether. HC1<*>H.Lys(Z)-Lys(Z)-OH was collected by filtration and washed several times with ether to give 6.7 g of white powder. It was dissolved in DMF (70 mL), cooled in an ice bath and treated with Et-3N (1.63 mL) followed by Boe-Glu(OBzl)-OSu (5.54 g, 12.76 mmol). The mixture was stirred at 0°C for one hour and then at 25°C for 24 hours. More Et-3N was added during this time to keep the reaction close to pH 7.5. A few ml of acetic acid were added to acidify the environment (pH 3.5) and the solvent removed by evaporation. The resulting residue was taken up in EtOAc, washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness, whereupon the product began to solidify. It was triturated in ether and recrystallized from ethyl acetate. Yield: 7.26 g (69.5%) of Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH; m.p. 153-155°C; G-O^5 = -2.71° (c = 1, THF).

g) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (1.7 g, 1.16 mmol was treated with TFA (24 ml ) in 30

minutes. After evaporation of excess acid (30°C), the residue was triturated with ether. The powder obtained was washed thoroughly with ether and petroleum ether and dried over NaOH in vacuo to give the trifluoroacetate salt of the octapeptide (1.71 g). The active ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu was then prepared in situ by

to stirring Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH (0.998 g, 1.16 mmol), HOSu (0.16 g, 1.4 mmol) and DCC (0.274 g, 1, 33 mmol) in 15 ml

DMF at 0°C for 3 hours. To this solution containing the active tripeptide ester was added the octapeptide salt CF3COOH'H-Glu (OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl

(1.71 g) along with 0.2 ml of Et-^N. A few more drops of Et₂N and DMF (15 mL) were added and the mixture was stirred for 3 days at 25°C. A gel-like semi-solid mass was formed. It was acidified with acetic acid and treated with water. The white solid precipitate was collected and washed (H 2 O, MeOH, ether) to give 2.25 g of crude product which melted at 310-313°C. It was dissolved in DMF and precipitated with MeOH. 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;

m.p. 314-316°C; 1?<<i>3^<5> = 13.68° (c = 1, DMSO); homogeneous in TLC.

Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu (OBzl)-Ala-Glu(OBzl)-Asn-OBzl (0.5 g, 0.226 mmol) was dissolved

in 2 ml TFA and stirred with 15 ml HF at 0°C for 15 min. After evaporation of excess acid (0°C), the residue was dissolved in 5% . aqueous HOAc, washed with ether (3x), evaporated to a small volume and lyophilized to give 0.34 g of crude product. It was chromatographed on the ion exchange column described above for octapeptide and gave 0.13 g (42.1%) of pure^Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn;f< *] ^5 = -86.65° (c = 1, H 2 O) .

Example 3

Boc-Leu-OSu (4.0 g, 12.2 mmol) and H-Lys(Z)-OH (3.42 g, 12.2 mmol) were condensed in DMF (75 mL) over 48 h in presence of Et 3 N (1.7 mL). The pH of the reaction mixture was maintained at 7.5 by adding Et^N periodically as usual. The remaining insoluble material was precipitated and filtered and the filtrate evaporated to dryness. The foamy glassy mass formed was dissolved in ether (200 mL) and the mixture treated with 3 mL of DCHA to give crystalline material which was collected, washed with ether and recrystallized from MeOH and ether. Yield: 5.7 g (69.5%) of Boc-Leu-Lys(Z)-OH.DCHA; m.p. 140-142°C; <f6>! Q <5> = -7.20° (c = 1.MeOH).Boc-Leu-Lys(Z)-OH.DCHA (2.97 g,

4.4 mmol) was taken up in the free acid (partitioned between EtOAc and 0.1 N H 2 SO 4 ), and the resulting colorless oil (2.2 g) was treated with 4N HCl in THF (40 mL) for 30 min. The excess acid and solvent were evaporated (30°C) and the residue treated with ether. The remaining oil was dissolved in ether and evaporated 2 more times with new ether. The residue was then stirred with Boc-Asp(OBzl)-OSu (1.85 g, 4.4 mmol) in the presence of Et₂N (1.85 mL) overnight. The reaction mixture was then evaporated to dryness to give an oil residue which was taken up in ethyl acetate, washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness again. The crude product thus obtained was crystallized from ethyl acetate and petroleum ether and gave 1.52 g (49.6%) of pure Boc-Asp(OBzl)-Leu-Lys(Z)-OH; m.p. 109-111°C; [KJ ^<5> -16.14°

(c = 1, DMF).

Boc-Glu (OBzl) -Lys ( Z ) -Lys (Z ) -Glu (OBz 1) -Val-Val-Glu (0 Bzl) - Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (1 .2 g, 0.545 mmol) was treated with 35 ml of TFA for 30 min. The excess acid was quickly evaporated and the residue triturated with ether several times to give 1.2 g of the undecapeptide TFA salt as a white powder. It was dissolved in a mixture of DMF (5 ml) and DMSO (2 ml) and treated with Boc-Asp(OBzl)-Leu-Lys(Z)-OSu prepared in situ by

stir Boc-Asp(OBzl)-Leu-Lys(Z)-OH (0.381 g, 0.545 mmol)

with HOSu (0.126 g, 1.1 mmol) and DCC (0.124 g, 0.599 mmol) in 3 mL DMF at 0°C for three hours. The mixture containing the active tripeptide ester and undecapeptide was stirred at 0°C for 2 hours and then at 25°C for three days, during which Et^N was added from time to time to maintain the pH

weakly basic. A gelatinous substance was formed. It was triturated with 5% HOAc and the resulting white solid was filtered and washed with water, MeOH and ether to give 1.28 g of crude material melting at 325-326°C. Reprecipitate from DMF/DMSO (10 mL/5 mL) and MeOH

(230 mL) gave 1.22 g (80.2%) of pure Boc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl) -Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl); m.p. 326-327°C; [<;>Op<5> = "15.71° (c=

1, DMF/DMSO).

This product (1.128 g, 0.404 mmol) was mixed with 7 mL of anisole and treated with 25 mL of anhydrous HF at 0°C for 15 min. The excess acid was evaporated (0°C) and the remaining residue partitioned between ether and water in the Water layer was washed 2 times with ether, evaporated to half the original volume and lyophilized, yielding 0.69 g of crude material. It was chromatographed in the manner described above for the octapeptide. The material eluted in tubes 101-120 was collected and lyophilized, yielding 0.25 g of product which was shown to be slightly mixed with minor impurities. It was therefore rechromatographed on the same column, which gave 0.155 g (22%) of pure Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn. Paper electrophoresis indicated that it was homogeneous• Ijx^lp"' = "86.27°

(c = 1, 0.1N HCl).

Claims (1)

1. Analogy method for the production of immunopotentiating peptides with the general formula where R''- is hydrogen, H-G.lu-Lys-Lys- or H-Asp-Leu-Lys-Glu-Lys-Lys-, and of pharmaceutically acceptable salts thereof, characterized in that the protective groups are removed from a protected peptide of the general formula where R 2 is Boe, Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-or Boc-Asp-(OBz1)-Leu-Lys(Z)-Glu(OBzl)-Lys (Z)-Lys ( Z)-, Boe is tert-butyloxycarbonyl, Bzl is benzyl and Z is benzyloxycarbonyl, and, if desired, converting this obtained compound into a pharmaceutically acceptable salt.