FI70228B - FRAMEWORK FOR FRAMSTAELLNING AV EN ENKEFALIN ANALOG PEPTID - Google Patents

Description

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c (45) ratct.ttl; ry: '> *. "Ctt7 is co iohg (51) Kv.lk. * / lnt.CI.4 C 07 K 7/12 FINLAND—" FINLAND (21) Patent application - Patentansökning 770254 (22) Application date - Ansökningsdag 26.01.77 (23) Starting date - Giltighetsdag 26.01.77 (41) Become public - Blivit offentllg 27.07.77

National Board of Patents and Registration ^ Date of Publication and Publication— 28 02 86

Patent- och registrstyrelsen Ansökan utlagd och utl.skriften publicerad / (32) (33) (31) Requested privilege — Begärd prloritet 26.01 .76 O3.O3.76, 23.ll.76 England-England (GB) 2900/76, 8481/76, 48821/76 (71) The Wellcome Foundation Limited, 183-193 Euston Road, London NWI,

Englant i-England (GB) (72) Samuel Wilkinson, Beckenham, Kent, Engl anti-Engl and (GB) (74) Oy Koister Ab (54) Method for the preparation of enkephalin analogue peptide -Förfarande för framstälIning av en enkefalin- analog peptide

The invention relates to a process for the preparation of an enkefin analogue-i peptide of the formula H-Tyr-D-Ala-Gly-Phe-D-Leu-R 1 or a pharmaceutically acceptable salt thereof (peptide as carboxylate anion and pharmaceutically acceptable salt). cation), or to prepare a pharmaceutically acceptable acid addition salt thereof, wherein R is NHR or OR, wherein R is an alkyl group having 1 to 4 carbon atoms, and R is hydrogen, an alkyl group having 1 to 4 carbon atoms, or halophenyl .

The peptide or derivatives thereof according to the invention have morphine agonist activity. As is generally accepted and as the term is used herein, a morphine agonist is a compound whose biological activity closely resembles that of a natural alkaloid.

2 70228

The pharmacological properties of morphine and its therapeutic use have been extensively described in the literature (e.g., "The Pharmacological Basis of Therapeutics", Goodman, LS, and Gilman, Δ., Published by The MacMillan Company, New York, 3rd Edition (1965), especially paragraph 15, pp. 246-266, and "Martindale: The Extra Pharmacopoeia", Blacow, NW, published by The Pharmacuetical Press, London, 26th Edition (1972), especially pages 1100-1106).

As is known (Goodman, L. S. et al., Loc. Cit., Paragraph 16), continuous administration of morphine may result in the recipient developing a drug addiction to this drug, tolerance to its effects, and clear withdrawal symptoms when the drug is discontinued. Therefore, many years of research have been carried out with the aim of finding a compound which already has the effect of morphine but which at the same time lacks its disadvantages.

Natural enkephalins are known, such as Met-enkephalin, i.e. L-tyrosylglycylglycyl-L-phenylalanyl-L-methionine, and Leu-enkephalin, i.e. L-tyrosylglycylglycyl-L-phenylalanyl-L-leucine (Nature, 258 (577), s. - 579).

The process according to the invention is characterized in that a) a reagent of formula

H — Y1 — OH II

wherein Y is -Tyr- or a partial radical sequence having -Tyr- at the N-terminus and henceforth identical to said peptide is reacted with the formula 2

H-Y -R III

2, wherein R is as defined above and Y is a partial radical sequence identical to the remainder of the product peptide as defined above, wherein reagents II and III are optionally protected and / or activated, where and when appropriate, and wherein, if appropriate, the protecting groups are removed, or

II

3 70228 b) when it is desired to prepare a pep-2 3 thide of formula I in which R is NHR, a corresponding peptide of formula I in which 2 4 4 R is OR in which R is an alkyl group having 1 to 4 carbon atoms is reacted with 1- With a monoalkylamine containing 4 carbon atoms, or c) when it is desired to prepare such a pep- of formula I. . 2 4 4 a thide in which R is OR, in which R is an alkyl group having 1 to 4 carbon atoms or a halophenyl, the corresponding peptide of the formula I, 2 4 4 in which R is OR in which R is hydrogen, is esterified, followed by the desired or necessary converted to the free base or a pharmaceutically acceptable salt or acid addition salt

Abbreviations used herein for amino acids and their derivatives are commonly used in the art and can be found, for example, in Biochemistry, 11, 1726 (1972). Above and below, all references are to L-amino acids and their derivatives, except in the case of glycs.i.ini and unless otherwise indicated.

Pharmaceutically valuable acid addition salts of the peptide of formula I and its derivatives are those derived from the following acids: a) mineral acids: hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric and sulfuric acids; and b) organic acids: tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, gulonic acid, succinic acid and arylsulphonic acid, for example p-toluenesulphonic acid.

Pharmacological experiments and their results A. In vitro experiments i) The inhibition of nerve-induced contractions of an isolated mouse vas deferens was studied by the method of Hughes et al. (Brain Research 88 (1975) 296) using 0.1 Hz pulses and the known narcotic antagonist naloxone i.e. 1-N-allyl-7,8-dihydro-14-hydroxy-normorphinone.

i.i) The reduction of electrically induced contractions in the isolated ileum of the guinea pig was studied by the method of PatonVin (Brit. J. Pharmacol., 12 (1957) 119-127). Each intestinal block was pierced with an anode and weighed 2-3 g. Stimulus parameters: frequency 0.1 Hz, duration 0.4 ms and voltage up to 30 - 40 V; the contractions were transferred isotonically.

4 70228 B) In vivo experiments i) The analgesic effect of the compounds was determined by a hot plate experiment. Experimental animals (male mouse, CFLP strain, Hacking, and Churchill) were each placed separately in a copper-based plexiglass box floating in a 55 ° C water bath and observed for signs of discomfort such as tremor or paw licking, and reaction time (always max. 30 s) was recorded. Groups of five mice then received either test compounds or saline (0.85%) as a cerebrospinal muscle injection (20 μΐ dose volume), and the experiment was repeated every 15 min after treatment. The ED50 values of the test compounds were calculated from the number of animals having a post-treatment reaction time that was twice the pretreatment number.

ii) The antidiarrheal effect of the compounds was determined as follows: Female Cops Wistar rats (Charles River) were fasted for 24 hours, after which an aqueous solution of the test compound (10 ml / kg) was administered subcutaneously. After 15 minutes, each rat received 1 ml of castor oil orally and the animals were observed for one hour for the presence of diarrhea. The ED 2 Q number of each compound was the mg / kg of free peptide required to prevent diarrhea in 50% of the animals.

The results of the experiments performed are shown in the following table.

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The peptides of the formula I and the abovementioned derivatives thereof can be prepared by methods known per se. Thus, they can be formed by sequential coupling of appropriate amino acids using either classical procedures of peptide synthesis or solidase methods, or by first preparing peptide subunits and then joining them together.

Such reactions can be effected, for example, by activating the carboxyl group of the associated amino acid and protecting the amino and carboxylic acid groups not involved in the reaction. Such methods are normal in the peptide field. Details of suitable activating and protecting (covering) groups, as well as suitable reaction conditions (for both coupling reactions and deprotection) which provide less racemization, can be found in the literature, for example: a) GB Patents No. 1,042,487, 1,048,086 and 1,281,383> b) Schröder and Lubke, The Peptides (Academic Press) (1,965); c) Bellean and Malek, J. Am. Chem. Soc. 90 (1968) 165; d) Tilak, Tetrahedron Letters (1970) 849; e) Beyerman, Helv. Chim. Acta 56 (1973), 1729; f) Stewart and Young, "Solid Phase Peptide Synthesis" (W. H.

Freeman et al.) (1969).

Depending on the reaction conditions, the peptides of the formula I and their derivatives are obtained in the form of the free base or its acid addition salt or, in the case of the peptides themselves, in the form of their salt. Acid addition salts can be converted to the free bases or salts of other acids, and the bases can be converted to their acid addition salts by methods known in the art. Similarly, peptides can be converted to their salts, and salts can be converted to peptides or other salts by well-established methods.

li 7 70228

The following examples illustrate the invention. The following abbreviations are used in the examples: HOBT 1-hydroxybenzodiazole DCCI dicyclohexylcarbodiimide DCU dicyclohexylurea NMM N-methylimorpholine Olin DMF dimethyl.formamide THF tetrahydrofuran i-Pr isopropanol DIPE diisopropyl ether PE

EtOAc ethyl acetate i-BuOCOCl isobutyl chloroformate Z benzyloxycarbonyl

Bu tert-butyl BOC tert-butyloxycarbonyl

Bzl benzyl CP 4-chlorophenyl

Peptides were analyzed by thin layer chromatography using Merck silica gel plates and the following solvent systems: 1) Methyl ethyl ketone 2) n-butanol / acetic acid / water (3: 1: 1) 3) chloroform / methanol / 32% acetic acid (12: 9: 4) 4 ) chloroform / methanol / 0.88-ammonia (12: 9: 4) 5) ethyl acetate / n-butanol / acetic acid / water (1: 1: 1: 1) 6) chloroform / methanol (8: 1 ) 7) chloroform.i / methanol / 32-% acetic acid (1 20: 90: 5) 8) chloroform.i / methanol / 32-% ammonia (120: 90: 5)

All amino acids had the L-configuration unless otherwise indicated.

Optical rotation angles were determined with a Bendix NPL automatic polarimeter.

The amino acid composition of the peptide hydrolysates (prepared by treatment with 6N hydrochloric acid at 110 ° C for 24 hours in vacuo) was determined on a Beckman-Spingo Model 120C or Rank Chromostak amino acid analyzer.

8 70228

The following general procedures were used in all peptide syntheses: a) Coupling was performed in DMF and mediated with DCCl.

b) Amino acid ester hydrochlorides were converted to the free esters by the addition of a tertiary base, either trethylamine or N-methylmorpholine.

c) HOBT was added in the coupling step when the fragment condensation comprised a peptide having an optically active carboxy-terminal amino acid, e.g. by coupling with BOC-Tyr-D-Ala-Gly-Phe-OH.

d) Connections were allowed to continue for 24 hours in the cold. , .o mode at +4 C.

e) After coupling, purification was performed by washing with acid and base to remove unchanged reactants.

f) Alkaline saponifications were performed in aqueous methanol on an autotitrator at pH 11.5 to 12.0 with 1 N NaOH.

g) Benzyloxycarbonyl protecting groups were removed by hydrogenolysis in methanol / acetic acid with 10% palladium / carbon.

h) The acetate salts obtained from the above hydrogenolysis were converted to the corresponding hydrochlorides by the addition of methanolic hydrogen chloride.

i) Benzyl protection was removed by hydrogenolysis in methanol with 10% palladium / carbon.

j) The tert-butyl and tert-butyloxycarbonyl protecting groups were removed with N-hydrogen chloride in acetic acid in the presence of anisole as a detergent. Separation was allowed to continue for 60-90 minutes.

k) The OBu protecting groups in the alcohol groups of threonine and serine were removed with trifluoroacetic acid containing 10% water, allowing the separation to continue for 90 minutes.

l) The final peptides were isolated as their hydrochloride and lyophilized from aqueous solution.

Il 9 70228

Example 1

H.Tyr - D-Ala - Gly - Phe - D-Leu.OH

Bzl BOC.Tyr.OH HC1.D-Ala.OMe, BZl © BOC.Tyr - D-Ala.OMe

Bzl ^ I (i) 30C.Tyr ——- D-Ala.OH Gly. QMe BOC.PheOH D-Leu.CMe

Bzl I (3) (J) BOC.Tyr - D-Ala -—- Gly. QMe BOC.Phe ——— D-Leu.CMe

Bzl r-N

I (4) (ö) BOC.Tyr - D-Ala - Gly.OH MCI.Phe - ^ - D-Leu.CVe

Bzl L © BOC.Tyr - D-Ala - Gly - Phe D-Leu.CMe

Bzl I @

BOC.Tyr - D-Ala - Gly - Phe D-Leu.CH

(Γ)

BOC.Tyr - D-Ala - Gly ——— Phe - D-Leu.CH

(T)

H.Tyr - D-Ala - Gly - ^ - Phe - D-Leu.CH

Step 1 Bz 1 BOC.Tyr.D-Ala.QMe

12.25 g of BOC.Tyr.OH were dissolved in 60 ml of THF, cooled to -25 ° C, 3.33 g of N-methylimorpholine in THF (10 ml) and 4.30 g of iso -butyl chloroformate in THF (10 mL). After stirring at -25 ° C for 2 minutes, a mixture of 4.18 g of D-Ala.OMe.HCl and 3.03 g of N-methylmorpholine in dimethylformamide (80 ml) was added pre-cooled to -25 ° C. ).

10 70228

The mixture was stirred at -15 ° C for 2.5 h, the vessel was transferred to an ice bath and stirred at 0 ° C with 2 M KHCO 3 (36 mL) for an additional 30 min. The THF was removed in vacuo and the residue partitioned between ethyl acetate (30 mL) and 50% saturated brine. The organic phase was first washed with 2 x 75 ml of 5% → 200: 3; 1 x 50 mL of 50% saturated NaCl, dried over magnesium sulfate and concentrated in vacuo.

The residue was crystallized from ethyl acetate / naphtha to give colorless prisms; mp. 117-119 ° C (12.05 g, 80% 1) yield); 0.70.

C 25 H 32 N 2 O 6 calculated: c 65.79 H 7/02 N 6.14 found: C 65.99 H 7.24 N 5.93.

Step 2 Bzl

BOC.Tyr.D-Ala.OH

Bzl

12.05 g of BOC.Tyr.D-Ala.OMe were dissolved in methanol (250 ml) and 2N NaOH (14 ml) was added and stirred at room temperature for 2 hours. The methanol was removed in vacuo and water (150 mL) was added. After filtration, the solution was acidified with solid citric acid and extracted twice with EtOAc (250 mL). Washed with 2 x 100 mL of water, and the organic phase was separated and dried over magnesium sulfate and concentrated in vacuo.

The residue was crystallized from EtOAc / naphtha to give prismatic crystals; mp. 158-159 ° C (10.72 g, 92%).

Rf 0.677); 0.608); 0.802 '.

C_.H_.N-0 ,. calculated: C 65.16 H 6.79 N 6.33 24 30 2 6 found: C 65.01 H 6.47 N 6.08.

Valhe 3 Bzl BOC.Tyr.D-Ala.Gly.OMe

The procedure was as described in Step 1, but starting from the mixed anhydride prepared from BOC.Tyr.D-Ala.OH (2.43 g), iBuOCOCl (716.6 mg) and NMM (555.5 mg) in THF. (20 mL), and a mixture of Gly.OMe.HCl (627 mg) and NMM (505 g) in DMF (20 mL) to give the product which crystallized as prisms; mp. 125-127 ° C from EtOAc / naphtha (2.30 g, 89%).

Rf 0.857); 0.828); 0.652) 'C 27 H 35 N 3 O 7 calculated: C 63.16 H 6.82 N 8.19 found: C 63.33 H 6.76 N 7.92.

II

1 1

Step 4 - 70228

Bzl

BOC.Tyr.D-Ala.Gly.OH

Bzl 1 BOC.Tyr.D-Ala.Gly.OMe (2.30 g) was saponified in methanol (50 mL) in the presence of 2N NaOH (2.5 mL) as in Step 2 to give the acid which crystallized as needles in EtOAc. / light gasoline; mp. 99-100 ° C (1.85 g, 83%).

Rf 0.72?); 0.648); 0.602).

C_, H__N_0_ calculated: C 62.53 H 6.61 N 8.42 2b jj 3 / found: C 61.96 H 6.80 N 7.99.

Step 5 BOC.Phe.D-Leu.OMe

19.85 g of BOC.Phe in DMF (150 ml) were dissolved, the solution was cooled to -5 ° C, and 20.25 g of HOBT and 15.45 g of DCl were added.

After stirring for 30 minutes, D-Leu.OMe.HCl (13.61 g) and NEt (10.38 ml) were added and stirring was continued for 24 hours at + 4 ° C.

The DCU was filtered, washed with cold DFM and the filtrate was concentrated in vacuo. The residue was dissolved in diethyl ether (900 mL), first washed with 2 x 100 mL of 10% citric acid; then 100 ml of 50% NaCl; 3 x 100 mL of 10% NaHCO 3 and finally 100 mL of 50% saturated NaCl, dried over magnesium sulfate and concentrated in vacuo. Crystallized from EtOAc / naphtha to give needle-like crystals of m.p. 141-142 ° C (27.8 g, 88%).

Rf 0.641; 0.756).

C 21 H 32 N 2 O 5 calculated: c 64.29 H 8.16 N 7.14 found: C 64.10 H 8.44 N 7.29.

Step 6

Phe.D. Leu.OMe.HCl BOC.Phe.D.Leu.OMe was stirred for 1 hour with 1N HCl in glacial acetic acid. Concentrated in vacuo and triturated with light gasoline to give an amorphous solid (22.57 g, 97%) which was dried in vacuo and not further purified.

Rf 0.65?); 0.778); 0.552).

12 ", h 7 70228

Step 7

Bzl BOC.Tyr.D.Ala.Gly.Phe.D. Leu.OMe

Bzl

30.1 g of BOC.Tyr.D.Ala.Gly.OH were dissolved in DMF (125 ml) cooled to -5 ° C and HOBT (16.29 g) and DCI ( 12.43 g). After stirring for 1 hour, Phe.D.Leu.OMe.HCl (19.82 g) and NEt (8.35 mL) were added and kept at + 4 ° C for 24 hours. The DCU was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in EtOAc (900 mL) and acid / base washed as usual. After drying over magnesium sulfate and concentration in vacuo, the residue was crystallized from EtOAc / ether; to give prism-like crystals, m.p. 164-166 ° C (40.36 g, 87%).

Rf 0.927); 0.908); 0.872); 0.531).

C. ~ H NcO- Calculated: C 65.20 H 7.12 N 9.06 42 55 5 9 Found: C 65.08 H 7.09 N 9.51.

Step 9

BOC.Tyr.D.Ala.Gly.Phe.D-Leu.OH

Bzl i

34.7 g of BOC were dissolved. Tyr. D. Ala. Gly. Phe .D-Leu. OH in MeOH (300 mL) and hydrogenated in the presence of 10% palladium / carbon until the theoretical amount of hydrogen was consumed. The catalyst was filtered off, and washed with methanol; the filtrate was concentrated in vacuo. The residue was triturated with ether and the amorphous solid was filtered off and crystallized from EtOAc / DIPE; needle-like crystals were obtained, m.p.

138-140 ° C (dec.) (39.28 g, 99%).

Rf 0.657); 0.608).

C 34 H 47 N 5 O 7 Calculated: C 60.62 H 7.58 N 10.40 Found: C 60.70 H 7.35 N 9.98.

Il 70228 1 3

Step 10 H.Tyr.D.Ala.Gly.Phe.D-Leu.OH.HCl

The material from Step 9 (30.28 g) was stirred for 90 minutes in a mixture of anisole (75 mL) and 1N HCl / HOAc (225 mL). The solution was concentrated in vacuo and the residue was triturated in dry ether; the resulting material was filtered off and dried in vacuo. The product was dissolved in water (500 mL), filtered and extracted with 2 x 150 mL of ether, and the aqueous phase was separated and lyophilized to give a colorless amorphous powder (25.12 g, 92%).

Rf 0.93?); 0.808); 0.572).

I + 25 + 31.5 ° (c = 0.53, in methanol).

C 29 H 39 N 5 O • 1.5 H 2 O requires: C 54.93 H 6.79 N 11.00 found: C 54.96 H 6.80 N 10.95.

The peptide can be purified from small impurities either by chromatography on CM52 using a gradient elution of 0.005-0.1 M NH 2 OAc (pH 5.1) or by HPLC using Zorbax C8 isocratically eluted with 0.1 M NH 2 OAc (pH 4.0) with 30% acetonitrile, both lyophilized and isolated as the free base.

C 29 H 39 N 5 O 7 · 4 H 2 O requires: C 54.29 H 7.33 N 10.92 found: C 54.47 H 6.92 N 11.13.

70228 14

Example 2

H. Tyr- D.Ala - Gly - Phe - D-Leu.OCP

BOC.D-Leu 0 DCI / HOBT / p-chlorophenone BOC.D-Leu.OCP 01-11 HCl / HOAc

Bzl w

BOC.Tyr - D.Ala - Gly - Phe HC1 .D-Leu.OCP

f3) DCI / H0BT (90.5%)

Bzl

BOC.Tyr - D.Ala Gly Phe - D-Leu.OCP

@ H2 / PdC (90-%)

BOC.Tyr - D.Ala Gly Phe - D-Leu.OCP

01-n HCl / HOAc / anisole (87-%)

HC1 H.Tyr - D.Ala Gly Phe - D-Leu-OCP

H.Tyr.D.Ala.Gly.Phe.D-Leu.OCP

Prepared according to the scheme and crystallized from ether; prism-like crystals, m.p. 182 ° C.

Rf 0.752); 0.695); / TaJO = + 38.9 ° (c = 0.5).

Found: C, 62.81; H, 6.18; N, 10.3. Found: C, 62.00; H, 6.45; N, 10.51.

li 15 70228

Example 3 H-Tyr - D-Ala -Gly - Phe ----- D-Leu.OMe BOC.Gly Phe.CMe Θ H0BT / DC1 BOC.Gly - Phe.CMe © N.HCl / HOAc ZD-Ala HCl.Gly - Phe.CMe © H0BT / DC1 (77-%) ZD-Ala - Gly ---_ Pne.OMe (T) H? / PdC (92-%)

Bzl BOC.Tyr HCl.D-Ala Gly - Phe.CMe

Bzl ©) DC 1 (53-%) BOC.Tyr - D-Ala Gly - Pne.OMe © H0 / PdC (90-%)

'<L

BOC.Tyr - D-Ala Gly - Phe.CMe (7) MaOH / MeOH (93-%) BOC.Tyr - D-Ala Gly _ Phe.CM D-Leu.OMe @ HOBT / DCl (82-%) BOC.Tyr - D-Ala Gly - Phe - D-Leu.OMe © 1-n HCl / HOAc (86-%) H.Tyr --- D-Ala - Gly - Phe - D-Leu.OMe H .Tyr.D-Ala.Gly.Phe.D-Leu.OMe.HC1

Prepared according to the above scheme and isolated as an amorphous powder by lyophilization.

Rf O, 928); 0.632).

Δ + 39.6 ° (c = 0.51, in methanol).

C30H41N5O7'HCl, H2O calculated: c 56.47 H 6.90 N 10.98 found: C 56.17 H 6.97 N 10.72.

16 70228

Example 4

50 mg of H.Tyr.D-Ala.Gly.Phe.D.Leu.OH in 1N HCl in methanol (4 ml) was dissolved and allowed to stand at room temperature for 48 hours. Concentrated in vacuo and triturated in ether to give amorphous methyl ester hydrochloride (quantitative yield) which was the same as in the known sample.

Example 5 H.Tyr - D.Ala- Gly - Phe - D.Leu.NHEt BZ1 BOC.Tyr - D.Ala - Gly - Phe - D.Leu.OMe Br 1 / - \ (ONH ~ Et BOC.Tyr - - D.Ala - Gly —-- Phe. D.Leu.NHEt

nft „(2) H2 / PdC

BOC.Tyr D.Ala —- Gly —-- Phe - D.Leu.NHEt (l) 1-n.HCl / HQAc / anisole (87-%) H · 'ΥΓ - D.Ala --- Gly - - Phe _ D.Leu.NHEt

Step 1 Bzl - 1

2.68 g of BOC.Tyr.D.Ala.Gly.Phe.D-Leu.OMera were dissolved in a mixture of methanol (25 ml) and ethylamine (100 ml) and stirred in a sealed tube at room temperature for 24 hours. The solution was concentrated in vacuo and the residue was crystallized from isopropanol to give prismatic crystals, m.p. 218-220 ° C (2.06 g).

Rf 0.501).

Ο, οΗγοΝ, Ο. calculated: C 65.65 H 7.38 N 10.69 4 J Do O δ found: C 65.93 H 7.63 N 10.58.

Step 2

The material from Step 1 was hydrogenated in methanol (100 mL) in the presence of 10% palladium (1 g). Filtered, evaporated to dryness and recrystallized from isopropanol; prism-like crystals were obtained, m.p. 212-213 ° C (dec.) (1.9 g).

Rf 0.451).

C, HC 2 N 0 O calculated: C 62.07 H 7.47 N 12.07 36 bz 6 o found: C 61.88 H 7.78 N 11.74.

1] 70228

Step 3

The protecting group was removed from the material from step 2 by stirring for 1 hour with 1N HCl / HOAc (100 mL) in the presence of anisole (10 mL). Concentrated, triturated in ether, and lyophilized to give an amorphous solid (1.56 g, 87%).

21 5) 4)

Rf 0.60; 0.613 '; 0.93 * / + 46.5 ° (c = 0.5, in methanol) C., H. , Nr0r.HCl.1.5 Ho0 calculated: C 56.40 H 7.30 N 12.70 I 3 4 4 6 6 2 found: C 55.97 H 7.29 N 12.31.

Claims (3)

A process for preparing a therapeutically useful enkephalin analogue peptide of the formula H-Tyr-D-Ala-Gly-Phe-D-Leu-R 2 wherein R is NHR or OR, wherein R is an alkyl group of 1-4 carbon-4 atoms and R is hydrogen, an alkyl group of 1-4 carbon atoms or halogenophenyl, or a pharmaceutically acceptable salt (the peptide in the form of a carboxylate anion and a pharmaceutically acceptable cation ) or a pharmaceutically acceptable acid addition salt thereof, may be -subscribed therefrom, that a) a reagent of the formula H-Y1-OH II Ί where Y is -Tyr- or a partial radical sequence with -Tyr- at its N-terminal end and hence preferably identical with said peptide, is reacted with a reagent of the formula HY 2 -R III 2. wherein R is the same as before and Y is a partially partial radical sequence identical to the final portion of the product peptide, wherein reagents II and III are possibly protected and or activated, whenever and where appropriate, and where appropriate, protection the groups are removed, or b) when such a peptide of formula I is desired, where R is NHR, a 2 4.4 .. corresponding peptide of formula I, where R is OR, where R is an alkyl group of 1-4 carbon atoms, is reacted with a monoalkylamine having 1-4 carbon atoms, or 2 c) when such a peptide of formula I is desired, where R is OR är, where R ^ is an alkyl group of 1-4 carbon atoms, or halogen-4,4-phenyl, the corresponding peptide of formula I wherein R is OR, where R is hydrogen is esterified, and the product obtained, if desired or if required, is converted to a free base or a pharmaceutically acceptable salt or acid addition salt thereof. 2. A process according to claim 1 for the preparation of a peptide of formula H-Tyr-D-Ala-Gly-Phe-D-Leu-OH or a pharmaceutically acceptable salt or acid addition salt thereof, characterized in that a reagent of formula Η -Υ1-OH II where Y is -Tyr- or a partial radical sequence of -Tyr- at its N-terminal end and where from f is measured identically to said peptide, is reacted with a reagent of the formula h-y2-oh III 2 where Y is a partial radical sequence identical to the end portion of the previously defined product peptide, wherein reagents II and III are possibly protected and / or activated as and when appropriate, and where appropriate, the protecting groups are removed, whereupon the product obtained if desired or if required is converted to a free base or a pharmaceutically acceptable salt or acid addition salt thereof. 3. A process according to claim 1 for the preparation of a peptide of the formula H-Tyr-D-Ala-Gly-Phe-D-Leu-NCl or a pharmaceutically acceptable acid addition salt thereof, characterized in that a peptide of the formula