SE447262B - ANALOGY PROCEDURE FOR PREPARING AN EMPTY PERIOD WITH IMMUNOPOTENTATIVE PROPERTIES - Google Patents

Description

. Bzl is benzyl and Z is benzyloxycarbonyl, and, if desired, conversion of the obtained compound into a pharmaceutically acceptable salt.

Removal of the protecting groups from the protected peptide of formula II is readily accomplished by methods known per se, for example by treatment with anhydrous acid, such as hydrogen fluoride, preferably in the presence of anisole.

The strategy used in the chemical synthesis of the undecapeptide was as follows: H-Glu (OBzl) -OH was first coupled to Boc-Ala-OSu to give the protected dipeptide fragment Boc-Ala-GlMOBzU-OH which was then condensed. des with HCl. H-Asn-OBzl via the DCC / HOSu method according to Wünsch and Drees, Chem, Ber. 22, ll0 (1966). The hydrochloride salt of the asparagine benzyl ester was prepared from Boc-Asn-OBzl, which in turn was synthesized from commercially available Boc-Asn-OH and benzyl bromide using the cesium salt of the amino acid. The Boc protecting group was removed by 30 minutes treatment with 11-N HCl in dry THF.

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-Oßzl in a DCC / HOSu-mediated fragment condensation using HCl. H-Ala-Glu (OBzl) -Asn-OBzl obtained from Boc-Ala-GIUKOBZD-Asn-OBzl when treated with 11N HCl / THF. The above-mentioned protected pentapeptide time was obtained in good yield as a crystalline pure material.

To prepare the protected octapeptide Boc-Glu (OBzl) -Val- Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-Oßzl, the necessary protected tripeptide Boc-Glu (OBzl) was first prepared ) -Val-Val-OH. Boc-Val-OSu was reacted with free valine to Boc-Val-Val-OH, which upon deblocking with 11N HCl 'in THF followed by reaction with Boc-Glu (OBzl) -OSu gave the desired tripeptide, which crystallized as cyclohexylamine salt BOC -GMOBzU-Val-Val-OH. CHA.

The cyclohexylamine salt was converted to free acid and then coupled with DCC in the presence of Hosu nu Hcl. H-Glr fl oßzn-Gn fl oßzl »Ala-Gmoßzlxasn-oßzl obtained from Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl when treated with HCl in THF. The protected octapeptide ßoc-GluiOlšzll-Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl was obtained in purified form as an amorphous solid.

For synthesis of the protected subcapeptide Boc-Glu (OBzl) -Lys (Z) - Lys (Z) -Glu (OBzl) -Va1-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn -Ol5z1 synthesized the required tripeptide fragment starting from Boc-Lys (Z) -OSu and 11-Lys (Z) -OH. The dipeptide Boc-Lys (Z) -Lys (Z) -OH thus obtained was treated with 4N HCl in THF, and the resulting salt HCl. H-Lys (Z) - Lys (Z) -OH was then reacted with Boc-Glu (OBzl) -OSu to give 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 in Z. Naturforsch. _2_l_b, # 26 (1966), and the solution of the in situ prepared tripeptide ester Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OSu were combined with the trifluoroacetate salt of H-Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzU-Ala-Glu (OBzl) -Asn-OBzl obtained from the corresponding blocked octapeptide by 30 minutes of treatment with TFA, thus adding a small amount of a base gave the desired protected undecapeptide Boc-Glu (OBzD-Lys (Z) -Lys (Z) -Glu (OBzl) -Val- Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl. the protecting groups from the protected subcapeptide Boc ~ Glu (OBzl) -Lys (Z) -I.ys (Z) - Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzD -Asn-Oßzl with anhydrous hydrofluoric acid gave the free undecapeptide Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn, which was uniform in paper electrophoresis after ion exchange column chromatography.

The novel undecapeptide of the present invention, and its pharmaceutically acceptable salts, can be administered to warm-blooded mammals by parenteral application either intravenously, subcutaneously or intramuscularly. These compounds are potent immunopotentiating agents with a daily dose in the range of 1a to 100 mg / kg body weight per day when administered intravenously. obviously the required dose will vary with the particular condition to be 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.

The pharmaceutically acceptable salts of the above peptide include the sodium and potassium salts or salts with a strong organic base such as guanidine. In addition, the counter ions of these cations such as chloride, bromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate and the like may be included in the formulation.

The following example describes in detail the synthesis of the undeca peptide. While specific protecting groups have been used in this example, it is within the skill of the art to use equivalent protecting groups in such synthesis.

The abbreviations used herein have the following meanings: Boc = t-butyloxycarbonyl; Bzl = benzyl; DCC = dicyclohexylcarbodiimide; DME '= dimethylformamide; THF = tetrahydrofuran; HOSu = N-hydroxysuccinimide; Triton B = 40% methanol solution of trimethylbenzylammonium hydroxide; NMM = N-methylphospholine; CHA = cyclohexylamine; DCHA = dicyclohexylamine; Z = benzyloxycarbonyl; DMSO = dimethyl sulfoxide; TFA = trifluoroacetic acid; TLC = thin layer chromatography; Et 3 N = triethylarnin; HOBT = 1-hydroxybenzotriazole.

Example Boc-Lysβ11-OH (15 g, 39.5 mmol) was stirred with HOSu (5.8 g, 50.5 mmol) and DCC (8.66 g, 42 mmol) in THF (250 mL) for Bh. An insoluble by-product was filtered off and the filtrate was evaporated to dryness. The remaining syrup (2452 g) was treated with isopropanol (150 ml) and petroleum ether (150 ml) to give an oily product (21 g) which could not crystallize. Thus, the crude active ester Boc-Lys (Z) -OSu was used for condensation with H-Lys (Z) -OH (1.0.6 g, 38 mmol) in DMF (250 mL) for 72 hours in the presence of 5 5 ml EtBN.

More Et 3 N was added from time to time to keep the stirred reaction mixture slightly basic. A small amount of undissolved material was then filtered off and the filtrate was evaporated to dryness (HO). The residual oily residue was treated 447,262 with 1 liter of 596-g HOAc. The precipitated product was extracted into ethyl acetate and the organic phase was washed with water, dried over Na 2 SO 4 and evaporated to an oil. It was crystallized from ethyl acetate (300 ml) containing DCHA (10 ml) as a salt. Recrystallization from MeO1-1 and ether gave 22.7 g <7z, s sa) ßOc-Lys (z) -Lys (z) -oii. Dei-IA; m.p. iso-isf; [α] 25 D = -2.21 ° (e = 1, iAeoH).

Boc-Lys (Z) -Lys (Z) -OH. DCHA (10 g, 12.14 mmol) was partitioned between EtOAc (1 L) and 0.1 N 112504 (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 thus obtained, Boc-Lys (Z) -Lys (Z) -OH, was treated with freshly prepared 11N HCl in THF for 30 minutes. The solvent and excess acid were evaporated (300) and the residue was evaporated twice with THF. The remaining residue solidified on treatment with ether. Salted HCl. H-Lys (Z) -1.ys (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 EtBN (1.63 mL) followed by Boc-Glu (OBZ1) -OSu (256 g, 12.76 mmol). The mixture was stirred at 0 DEG for one hour and then at 250 DEG for 20 hours. More EtBN was added during this time to keep the reaction mixture at about pH 7.5. A few milliliters of acetic acid were added to make the reaction mixture acidic (pH 3.5) and the solvent was evaporated by evaporation. The resulting residue was taken up in EtOAc, washed with water (3x), dried over Na 2 SO 4 and evaporated to dryness when 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. 153-1550; [Α] D = -2.71 ° (e = i, THF).

BOC-Asn-OH (01.0 g, 47.5 mmol) was dissolved in 1 ml of 1 MeoH and 20 ml of water were added. The solution was titrated to pH 7.0 with a 2096 g aqueous solution of CszCO 2 (about 55 ml). The mixture was evaporated to dryness and the residue was re-evaporated twice from DMF (120 ml each, 1450). The resulting white solid was then stirred with 8.9 g of benzyl bromide (52 mmol) in 120 mL of DMF for 6 hours. Upon evaporation to dryness and treatment with a large volume of water, the product solidifies immediately. It was collected by filtration, dissolved in ethyl acetate, washed with water, dried over Na 2 SO 4, evaporated to a solid mass and crystallized from ethyl acetate with petroleum ether.

Yield 13.8 g 90.396) Boc-Asn-OBzl; m.p. 120-1220; [α] 25 D = -17.29 ° (C = 1, DMF). 447,262 Boc-Asn-OBzl (1 3.7 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 for 45 minutes, during which time some product began to precipitate. Upon treatment with 1,000 ml of ether, a white solid formed immediately. The product was filtered, washed with ether and dried over NaOH pellets in vacuo. exchange; 10.3 g (94%) HCl. H-Asn-oßzi; m.p. 122-12e °; [št-jš fi = 5.32% H-Glu (OBz1) -OH (7.0 g 29.5 mmol) was finely ground in a mortar with a pestle and then stirred with 8.88 g (32.3 mmol) of Boc-Ala -OSU for 48 hours in 250 ml of DMF in the presence of 6 ml of NMM. A little more NMM was added to pour the reaction mixture slightly basic during the reaction. The solvent was evaporated, and the residue was partitioned between 300 ml of ethyl acetate and 500 ml of H 2 O, which contained 2 ml of 1096 μg 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 uniform by TLC. yield: 11.0 g (91, va) ßoc-Aia-cxu-oszn-one; m.p. s4-ss °; [wjü = s, os ° (C = 1, DMF).

Boc-Ala-GlMOBzU-OH (1.0 + g, 25.4 mmol), HCl. -1-Asn-OBzl (6.56 g, 25.11 mmol) and HOSu (5.9 g, 50.8 mmol) were dissolved in DMF (250 mL, 0 O). DCC (5.7 g, 27.6 mmol) was added immediately followed by EtaN (3.5 mL). The mixture was stirred at 0 ° for 2 hours and then at 25 ° for # 0 hours, during which time slightly more Et 3 N was added from time to time to keep the reaction mixture slightly basic. The insoluble by-products formed were filtered off and the filtrate was evaporated to dryness.

The remaining oily material solidified on treatment with water.

The crude product was taken up in CHCl 3, washed with water (Bx), dried over Na 2 SO 4 and evaporated to a smaller volume. A small amount of solid formed in this step was filtered off (heavily contaminated with dicyclohexylurea) and the filtrate was treated with petroleum ether. A crystalline product was obtained.

Yield: 8.0 g 61.496) Boc-Ala-G1u (OBzl) -Asn-OBzl; m.p. l02-105 °; [TYJZS = 12, s ° (c = 1, DMF).

H-Glu (OBzl) -OH 04.74 g, 20 mmol) was ground with mortar and pestle and stirred with Boc-Glu (OBzl) -OSu (0.7 g, 20 mmol) in DMF for 36 hours in the presence of 3 , 6 ml NMM. The resulting solution was evaporated to a syrup and treated with water. The oily precipitate was taken up in ethyl acetate, washed successively with 596 g of HOAc and water (Bx), dried over Na 2 SO 4 and evaporated to dryness to give 14.03 g of a clear oil. It was allowed to stand immersed under petroleum ether. The residual oily product Boc-Glu- (OBzl) -G1u (O1§§l) -OH weighed 10.2 g (90.096). TLC showed that the product was 447,262 uniform. [α] 25 D = -7.59 ° (C = 1, DMF), Boc-Ala-Glu (OBzl) -Asn-OBzl (28.2 g; 46 mmol) was treated with 1.1 liters of 4N HCl in THF for 1 hour. Evaporation of the solvent and excess acid gave an oil, which was evaporated twice more with fresh THF. The residual oil turned to a solid on treatment with a large volume of ether. The solid HCl. H-Ala-Glu (OBzl) -Asn-OBzl was stirred with Boc-Glu (OBzl) -Glu (OBzl) -OH (25.6 g, 146 mmol), HOSu (1.0.6 g, 92 mmol) and ncc ( 1o, 9 g, 53 mmei) 1 DMF (5110 m1) at o ° 1 1 hour an seden at 25 ° 1 # 8 hours. An 3N was added to keep the reaction mixture slightly basic throughout the time period (about 16 ml of EtBN in total). The insoluble by-products formed were filtered off, and the filtrate was evaporated to dryness. The crude product was dissolved in CHCl 3, 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 (59.896) of Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -exox-oxy; m.p. 169-179; [mp -11.7 ° (e e 1, DMF).

Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl (3.9 g, 3.148 mmol) was treated with 15 mL of 1N HCl in THF for 30 minutes. Some crystalline product began to form. Ether (210 ml) was added and the precipitated solid was collected and washed with ether. The crude material was crystallized from MeOH and ether.

Yield: 2.53 g (75.1%) of Hci. H-Giu (oßzU-Gu fl oßzl) -A1e-Git fl oßzll-Asn-oßzi; m.p. 14s-151 °; Ms fi -s, e5 ° (e = 1, DMF).

Boc-Val-OSu (12.6 g, 40 mmol) and H-Val-OH 04.68 g, 140 mmol) were condensed in DMF (250 mL) for 96 hours in the presence of 2 mL of EtBN. More Et 3 N was added as needed to keep the reaction mixture slightly basic. The remaining insoluble material was filtered off and the filtrate was evaporated to dryness (UO). The residue was partitioned between ether and dilute H 2 SO 4 (ca. 196) and the organic layer was washed with water (3x), dried over Na 2 SO 4 and evaporated to a foam-like glass mass. The product was crystallized from ether and petroleum ether.

Yield: 12.2 g 06.496) ßee-vei-veu-on; m.p. 155-15s °; [α] D = + 1.1 ° (c = 1, DMF).

Boc-Val-Val-OH (40.5 g, 128 mmol) was treated with 1.8 liters of 1N HCl in THF for 60 minutes. Evaporation to eliminate excess acid and solvent followed by treatment with ether gave 34.5 g of HCl. H-Val-Val-OH as a white amorphous powder. It was treated with Boc-Glu (OBzl) -OSu (55.6 g, 128 mmol) in 1 liter of DMF for 24 hours in the presence of 54 ml of EtBN. The reaction mixture was filtered to eliminate a small amount of insoluble matter and the filtrate was evaporated to dryness. The residual oil residue was taken up in EtOAc (1.5 L) and washed with 596 HOAc (ZX) 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 thus dissolved in 3.2 liters of ether and treated with CHA (17 ml) until the pH of the mixture was 7.5. The resulting solid salt was collected and recrystallized from MeOH and ether.

Yield: 58.9 g (72.7%) of Boc-Glu (OBzl) -Val-Val-OH. CHA; m.p. l88-160 °; [ejšj = 33, a1 ° (c = 1, theory).

Boc ~ Glu (OBzl) -Val-Val-OH. CHA (1.69 g, 2.66 mmol) was suspended in water (40 mL) and ethyl acetate (40 mL) in a separatory funnel to which 11 mL of 1 M H 2 SO 4 was added. After shaking vigorously, the solid was dissolved and the organic layer was washed several times with water, dried over Na 2 SO 4 and evaporated to an oil (135 g). The free tripeptide thus obtained was then condensed with 2.52 g of HCl. H-clu (oß21) -c1u (oszn-Aia-cunoßzn-Asn-oßzi (2, s1 mmol) in 15 ml of DMF in the presence of HOSu (0.612 g, 5.32 YWUOÜ: NMM (013 m ° lf 2.66 mmol) and DCC (0.63 g, 3.06 mmol) for 1 hour at 0 ° and 60 hours at 250. More NMM was added as needed to keep the reaction mixture slightly basic, an insoluble by-product formed was filtered off and the filtrate was evaporated to dryness ( # 50) The residual oil residue solidified on treatment with water, the crude solid was dissolved in DMF (50 ml) and precipitated with MeOH (300 ml) yield: 2.25 g 68,796) B0e-ciu (oßz1) -va1- va1-G1u (oßz1) -ciu (0B2l) -A1a-c1u- (OBzU-Asn-Oßzl; mp 277-280 °; ßdšs = -l2, l¿3 ° (c = 1, DMF) - soc-oiuioßziz -väi-vai-GíuiöB21J-ciuioßzU-Åia-äitíiosäli-Ašhioßzi (1.7 g, 1.6 mmol) was treated with TFA (214 ml) for 30 minutes, after evaporation of the excess acid (30 °) the residue was triturated with ether The resulting powder was washed thoroughly with ether and petroleum ether and dried over NaO1-1 in vacuo to give the trifluoroacetate salt of ok the tapeptide (1.7l g). The active ester Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OSu was then prepared in situ by stirring Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Ol-1 (0.998 g, 1.16 mmol), HOSu (0.6 g, 1.14 mmol) and DCC (0.27l + g, 1.33 mmol) in 15 mL of DMF at 00 for 3 hours. To this solution containing the active tripeptide ester was added v okrapepridsaifet c1 = 3cooH. H-cnoszn-vai-vai-G1u (oBz1) -G1u (OBz1) -A1a- Glu (OBzl) -Asn-OBzl (1.7l g) together with 0.2 ml of Et3N. A few more drops of EtβN and DMF (15 ml) were added and the mixture was stirred for 3 days at 25 °. 447 262 9 A gelatinous semi-solid substance was formed. It was acidified with acetic acid and treated with water. The White. the solid precipitate was collected and washed (H 2 O, MeOH, ether) to give 2.25 g of crude product melting at 30-313 °.

It was dissolved in DMF and precipitated with MeOH.

Yield: 1.75 g (68396) Boc-Glu (OBzl) ~ Lys (Z) -Lys (Z) -Glu (OBzl) -Val-Val-Glu- (OBzU-Glu (OBzl) -Ala-GIMOBZD-Asn -Oßzl; m.p. Bill-HSO; [# 625 = 13.68 ° (c = i, ist-iso »uniform at TLc.

Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzI) - Ala-Glu (OBzl) -Asn-Ol3zl (0.5 g, 0.226 mmol) was dissolved in 2 mL of TFA and stirred with 15 nd iiF at 0 ° for 15 nuns. After evaporation of excess acid (Oo), the residue was dissolved in 596 g of aqueous HOAc, washed with ether (Bx), evaporated to a smaller volume and lyophilized to give 0.34 g of crude product. It was chromatographed on an ion exchange column as described above for the octapeptide to give 0.13 g (42, 1996) of pure Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Aia-ciu-Asn; [wjšj = -ss, e5 ° (c = 1, H20).

The following table shows the results of an experiment which exhibits the immunopotentiating activity of the undecapeptide, namely in restoring the hypersensitivity lbT fl) response of the delayed type which was partially suppressed by injection of S-fluorouracil (S-FU) into mice.

Mice Dos DTH response treated with ug% Iron Transfer Sample - l5.5il, 0 100 5-F0 only - 6.3il, 3 40.8 S-FU plus undecapeptide 0.01 l0.8j¿l, 5 69.9 0, 1 ll, 6_-l¿l, 3 75.3 1.0 l0.7_-l¿2.5 69.3

Claims (1)

A method of analogy for the preparation of a undecapeptide of the formula Q Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH and of pharmaceutically acceptable salts thereof, characterized in that the process comprises removing the protected groups from the protected peptide of the general formula Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) - - G1u (OBzl) -Ala-Glu (OBzl) ~ Asn-OBzl wherein Boo is tert-butyloxycarbonyl, Bzl is benzyl and Z is benzyloxycarbonyl, and, if desired, conversion of the obtained compound into a pharmaceutically acceptable salt. .wo