EP0015274A1

EP0015274A1 - Improved enkephalin derivatives

Info

Publication number: EP0015274A1
Application number: EP19790900664
Authority: EP
Inventors: Abba J. Kastin; David H. Coy
Original assignee: Individual
Current assignee: Individual
Priority date: 1978-06-05
Filing date: 1980-01-10
Publication date: 1980-09-17
Also published as: WO1980000022A1

Abstract

Polypeptides representes par la formule (R1)5 (H-Tyr-X-Gly-Phe-(R)n)m-Y-Z ou X est la glycine ou un residu a pouvoir d'acide D-amino choisi parmi le groupe constitue D-alanine, D-leucine, D-isoleucine, D-valine, D-norvaline, D-phenylalanine, D-tyrosine, D-tryptophane, D-serine, D-threonine, D-methionine, D-glutamine, acide D-aspartique, D-asparagine, D-lysine, D-proline, D-histidine et D-arginine; R est la methionine lorsque m est leucine et n est 1; n est 0 ou 1; R1 est l'hydrogene ou fluoro; m est 1 ou 2 avec la restriction que lorsque m vaut 1, X ne peut etre la glycine; Y est la lysine lorsque m est 2 et la methionine ou la leucine lorsque m vaut 1 avec la restriction que lorsque m vaut 1, n vaut 0; et Z est choisi parmi le groupe hydroxy, amino, akylamino-inferieur, dialkylamino-inferieur et alkoxy inferieur et leurs sels pharmaceutiquement acceptables.Polypeptides represented by the formula (R1) 5 (H-Tyr-X-Gly-Phe- (R) n) mYZ where X is glycine or a residue with D-amino acid power chosen from the group constitutes D-alanine , D-leucine, D-isoleucine, D-valine, D-norvaline, D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-threonine, D-methionine, D-glutamine, D-aspartic acid, D-asparagine, D-lysine, D-proline, D-histidine and D-arginine; R is methionine when m is leucine and n is 1; n is 0 or 1; R1 is hydrogen or fluoro; m is 1 or 2 with the restriction that when m is 1, X cannot be glycine; Y is lysine when m is 2 and methionine or leucine when m is 1 with the restriction that when m is 1, n is 0; and Z is selected from the group consisting of hydroxy, amino, lower alkyl, lower dialkyl and lower alkoxy and their pharmaceutically acceptable salts.

Description

IMPROVED ENKEPHALIN DERIVATIVES

Background of the Invention

While there are a number of analgesic agents currently utilitzed to relieve mild to severe pain, the search for improved analgesics is a continuing one because of the numerous problems assicated with the presently available agents. Aspirin and related salicylates are considered to be non-narcotic analgesic agents useful for relieving mild to moderate pain in addition to their usefulness as anti-inflammatory and anti-pyretic agents. However, ingestion of salicylic acid or related salicylates may result in epigastric distress, nausea and vomiting. This widely used class of non-narcotic analgesic agents may also cause gastric ulceration and even hemmorhage both in experimaental animals and man. Exacerbation of peptic ulcer symptoms and errosive gastritis have all been reported in patients on high dose therapy, i.e. arthritis patients. Aspirin is also one of the most common causes of drug poisoning in young children and has a potential of serious toxicity if used improperly. Acetominophen is also considered to be a non-narcotic analgesic agent useful in treating mild pain associated with simple headache, common musclar aches, etc. While acetomino-phin is particularly useful for patitents who cannot take aspirin, i.e.,ulcer patients, its use is contraindicated in individuals who have exhibited a sensitivity to it. In addition to their drawbacks in view of their potential side effects, the mild non-narcotic analgesic agents are not sufficiently potent to relieve the severe pain associated with surgery, cancer and the like.

Unfortunately, the potent analgesic agents capable of relieving such severe pain are also narcotic agents and their use entails the risk of producing physical and sometimes psychological dependence. There are as yet no agents effective against severe pain that are entirely free of this risk.

Thus, there is an urgent need for improved analgesic agents for treating mild as well as severe pain. There is also a need to provide improved psychotropic agents or to provide an alternative to current therapy. The compounds of this invention, in addition to their analgesic activity, also exhibit anti-depressant activity and their usefulness as analgesic agents is enhanced since many patitents suffering from pain also exhibit varying states of depression.

In 1975, Hughes et al identified a pentapeptide, methionine enkephalin.which has the following structure, H-Tyr-Gly-Gly-Phe-Met-OH[see Hughs et al, Nature, 258, 577 (1975) ]. The peptide is found in many areas of the brain where it appears to act as a neurotransmitter or neuro- modulator in. a central pain suppressive system. The natura peptide binds stereospecifically to partially purified brain opiate receptor cites [see, for example, Bradbury et al., Nature, 260, 793 (1976)], is very active in Bioassays for opiate activity, but exhibits only weak analgesic activity of short duration when injected directly into the brain of the rat[See, for example, Belluzzi et al.,Nature,260, 625(1976)]. We subsequently found that when methionine enkephalin was substituted in the 2-position with a D-amino acid, potent analgesic agents are obtained. The D-amino acid² analogs ajso exhibit other central nervous system and hormonal activites. We have now found that even greater in vivo analgesia is obtained when the D-amino acid² enkephalins are additionally substituted as disclosed herein. Detailed Description of Preferred Embodiments The polypeptides of this invention are represented by Formula I: (R₁)5

[H-Tyr-X-Gly-Phe-(R)_n]_m-Y-Z wherein X is glycine or a chiral residue of a D-amino acid selected from the group consisting of D-alanine,D-leucine, D-isoleucine, D-tryptophan, D-serine, D-threonine,D-methi-onine,D valine, D-norvaline, D-phenylalanine,D-tryosine, D-glutamic acid,D-glutamine,D-aspartic acid,D-aρaragine, D-lysine,D-proline,D-histidine and D-arginine;R is methionine or leucine when m is 1; n is 0 or 1;R₁_ is hydrogen or fluoro; m is 1 or 2 with the limitation that when m is 1,X cannot be glycine and when m is 2,n is 0; and Z is hydroxy,amino, loweralkylamino,diloweralkylamino or loweralkoxy and the pharmaceutically acceptable salts thereof.

The term"pharmaceutically acceptable salts" refers to the non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium and ammonium salts,prepared by well known methods, as well as the non-toxic acid addition salts which are generally prepared by reacting a compound of this invention with a suitable organic or inorganic acid, or in situ. Representative salts include the hydrochloride, hydrobromide sulfate,bisulfate,acetate, oxalate,valerate, oleate,laurate borate,benzoate, lactate,phosphate,tosylate,napsylate and the like.

The term"loweralkyl"refers to straight or branched chaim alkyl groups having from 1 to 6 carbon atoms such as methyl, ethyl, n-propoyl, iso-propyl,n-butyl, sec-butyl, tert-butyl, n-pentyl and the like.

All chiral amino acid residues are in the natural L configuration unless otherwise specified. In keeping with standard peptide nomenclature,abbreviations for amino acid residues have been used herein as follows: His -L-histidine,D-His, D-histidine, Try-L-tyrosine, D-Tyr D-tyrosine, lie L-isoleucine, D-Ile D-isoleucine, Gly glycine, Phe L-phenylalanine, D-Phe D-phenylalanine, Met L-methionine, D-Met D-methionine, Ala L-alanine, D-Ala D-alanine, Ser L-serine, D-Ser D-serine, Lys L-lysine D-Lys D-lysine, Asn L-asparagine, D-Asn D-asparagine, Asp L-aspartic acid , D-Asp D-aspartic acid, Leu L-leucine, D-Leu D-leucine, Thr threonine D-Thr D-threonine, Val L-valine, D-Val D-valine Pro L-proline, D-Pro D-proline, Gin L-glutamine, D-Gln- D-glutamine, Glu L-glutamic acid, D-Glu D-glutamic acid, Trp L-tryptphan, D-Tryp D-tryptophan.

Also contemplated within the scope of the present invention are intermediates of the formulae II and III:

R₃Tyr(R₂)-X-Gly-Phe-Y-R1 II

N,N-Bis -(R₃-Tyr(R₂)-X-Gly-Phe-Y)-Lys-R₁ III

wherein R₁ is selected from a group consisting of NH₂, 0H, represented

R₂ is a protecting group for the phenolic hydroxyl group of tyrosine selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzoyl, 2,4-dichlorobenzyl, benzyloxcarbonyl and 2-bromobenzyloxycarbonyl(2-BrZ); R₃ is a protecting groups which would be used by one skilled in the art of solid-phase synthesis of the peptides selected from the group consisting of acyl type protecting groups, aromatic urethan-type protecting groups, cycloalkyl urethan protecting groups, thio urethan type protecting groups, trialkylsilance groups or aliphatic urethan protecting groups; and

X is a D-amino acid as defined in formula I or when X is D-tyrosine, D-threonine, D-serine, D-glutamic acid or D-lysine, a protected chiral amino acid residue as defined below.

When X is D-tyrosine, the protected chiral residue is X(R₂) wherein Ro is as described above.

When X is D-serine or D-threonine, the protected residue is X(R₂) wherein R₂ is a protecting group for the alcoholic hydroxy function and is as defined above.

When X is D-glutamic acid, D-aspartic acid, the protected residue is X(RA) wherein R^ is benzyl or tert-butyl. In the case of lysine, the protected residue is X(R₅) wherein R₅ is a protecting group for the epsilon amino function selected from the group consisting of benzyloxycarbonyl or 2-chlorobenzyloxycarbonyl.

When X is D-arginine, the protected residue is X(R₆) wherein R₆ is a protecting group for the guanidino function such as tosyl or nitro.

The term "acyl type protecting groups" refers to groups illustrated but not restricted to foπnyl, trifluoro-acetyl, tosyl, nitrosulfonyl , and the like.

The term "aromatic urethan-type protecting groups" is represented by groups such as benzyloxcarbonyl (Cbz), ρ_-methoxybenzyloxycarbonyl , p_-biphenyl, isopropyloxycarbonyl, 2,5-dimethoxycarbonyl, cyclohexylcarbonyl, isobornyloxy- carbonyl, etc.

"Urethan type protecting groups" include compounds such as trimethylsilane, triethylsilane, tributylsilane and the like.

The preferred protecting groups, the "aliphatic urethan protecting groups" include tert-butyloxycarbonyl, diisopropyloxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl and the like .

The polystyrene support resin is preferably a copoly of styrene with about 1-2% divinyl benzene as a cross-linking agent which causes the polystyrene polymer, to be completely insoluble in most organic solvents. In formula IV, ∅ is phenyl.

In selecting a particular side-chain protecting group to be used in the synthesis of the peptides of formula I, several conditions must be met: (a) the protecting group must stable to the reagent and under reaction conditions selected for removing the alpha-amino protecting group at each step of the synthesis; (b) the protecting group must retain its protect ing properties and not be chemically modified; and (c) the si chaim protecting group must be removable at the end of the solid-phase synthesis under reaction conditions that will not alter the peptide chain.

The peptides of this invention are prepared using standard solid-phase techniques. The synthesis is commenced from the C-terminal end of the peptide using an alpha- amino protected resin. A suitable starting material can be prepared, for instance, by attaching an alpha-amino protected methionine to a chloromethylated resin, a hydroxymethyl resin or a benzhydrylamine resin. One such chloromethylated resin is sold under the tradename Bio-beads SX-1 by Bio Rad • Laboratories , Richmond, California and the preparation of the hydroxymenthyl resin is described by Bodanszky et al., Chem. Ind. (London) 38, 1957(1966). The benzyhydryl resin has been described by Pietta and Marshall, Chem. Commun. , 650 (1970) and is commercially available from Beckman Instrument, Palo Alto , California.

In the preparation of the compounds of this invention, an α-amino protected methionine or leucine or phenylalanine or pentafluorophenylalanine is coupled to the chloromethylated resin with the aid of, for example, cesium bicarbonate catalyst, according to the method described by Gisin, Helv. Chim Acta,56, 1476(1973). After the initial coupling, the α-amino protecting group is removed by a choice of reagents including trifluoroacetic acid or hydrochloric acid solutions in organic solvents at room temperature. After removal of the α-amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order to obtain the compounds of formula II. Each protected amino acid is generally reacted in a 3-fold excess using an appropriate carboxyl group activator such as dicyclohexylcarbodiimide in solution in, for example, methylene chloride-dimethylformamide mixtures.

In the case of the bis-derivatives of this invention, the bis-t-Boc derivative of lysine is coupled to the chloromethylated resin with the aid of, for example, cesium bicarbonate catalyst according to the method of Gisin, supra. After the intial coupling, the amino protecting groups are removed as described above.

After the desired amino acid sequence has been completed, the desired peptide is removed from the resin support by treatment with a reagent such as hydrogen fluoride which not only cleaves the peptide from the resin, but also cleaves all remaining side-chain protecting groups. When the chloromethylated resin is used, hydrogen fluoride treatment results in the formation of, the free peptide acids (Z=0H). When the benzyhydrylamine resin is used, hydrogen fluoride treatment resuts directly in the free peptide amides (Z=NH₂) • Alternatively, when the chloromethylated resin is employed, the side-chain protected peptide can be cleaved by treatment of the peptide-resin with ammonia to give the desired side chain protected amide or with an alkylamine or dialkylamine to give a side-chain protected alkylamide or dialkylamide. Side-chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the amides of this invention.

In preparing the esters. of this invention(Z=lower-alkoxy), the resin used to prepare the acids (Z=0H) is employed and the peptide is removed from the resin by treatment with base and the appropriate alcohol,, i. e. ,methanol. Side chain protection is then removed in the usual fashion by treatment with hydrogen chloride to obtain the desired ester. The solid phase procedure discussed above is well kno in the art and has been essentially described by J.M. Stewart "Solid Phase Peptide Synthesis",Freeman and Co., San Francisc 1969. The compounds of this invention are useful as analge agents when administered to mammalian hosts at dosages of from 0.1 to 100 mg/kg of body weight daily, preferrably in divided dosages. The compounds are administered by parenteral routes of administration, i.e. intravenous, intraperitoneal, intramuscluar or subcutaneous routes of administration, but can also be administerd by a variety of other routes including sublingual, nasal, vaginal, rectal, or, at higher dosages, oral. Accordingly, one aspect of the present inventi inlcudes pharmaceutical compositions suitable for such routes of administration.

The analgesic activity of the compounds of this invention was established in the rat tail flick test, D'Amour and Smith, J. Pharmac. Exp. Ther., 72, 74(1941). The following examples further illustrate the present invention. Examples 1-8 are drawn to the preparation of compounds wherein n is 1 and Y is methionine or leucine. The remaining examples are drawn to the N,N-bis-penta and tetra peptides derivatives wherein n is 2 and Y is lysine. The bis-peptides of this invention are represented by the formulae VI and VII:

EXAMPLE 1 Preparation of 0-2-bromobenzyloxycarbonyl-L-tyrosyl- D-alanyl-glycyl-L-pentafluorophenylalanyl-L-methionyl- benzhydrylamine resin Benzhydrylamine resin(0.94 g, 0.50 mmole).purchased from Beckman Instruments, Palo Alto, California, was placed in the reaction vessel of a Beckman Model 990 automatic peptide synthesizer, programmed to carry out the following cycle of washes and reastions: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride( 2 times for 2.5 and 25 minutes each); (c) methylene chloride; (d) ethanol; (e) chloroform; (f) 10% triethylamine in chloroform(2 times for f minutes each); (g) chloroform; and (h) methylene chloride. The free amino resin was then stirred with tert-butyloxycarbonyl (t-Boc) methionine(347 mg, 1.5 mmoles) in methylene chloride and dicyclohexylcarbodiimide(1.5 mmoles) was added thereot. The mixture was stirred at room temperature for 2 hours and the peptide resin was then washed successively with methylene chloride(3 times), ethanol (3 times) and methylene chloride(3 times). The attached amino acid was deportected with 33 % trifluoroacetic acid in methylene chloride( 2 times for 2.5 and 25 minutes each) and then steps (c) through (h) as described in the above wash were performed. The following amino acids (1.5 mmoles) were then couples successively by the same cycle of events; t-Boc- pentafluorophenylalanine; t-Boc-glycyine, t-Boc D-Ala; and t-Boc-Tyr(2BrZ). The completed pentapeptide resin was washed with methanol(3 times) and dried under reduced pressure whereupon 1.26 g of material was obtained. EXAMPLE 2

Preparation of L-tyrosyl-D-alanyl-L-glycyl-L-pentafluorophenyl- alanyl-L-methionine enkephalin amide

Removal of the protecting group and cleavage of the pentapeptide from the resin obtained according to the method of Example 1 was carried out by treating 1.26 g of the peptide-resin with hydrogen fluroide(20 ml) and anisole(2 ml) at 0°C for 45 minutes. The hydrogen fluoride was removed at reduced pressure and the anisole removed by washing with ethyl acetate. The crude peptide was purified by gel filtration on column(2.5 x 95 cm) of Sephadex G-15 gel by elution with 0.2 molar acetic acid and fractions shown to contain a major peak by uv absorption at 280 nm were pooled and evaporated to dryness.

The residual oil was applied to a column (2.5 x 95 cm) of Sephadex G-25 gel, previously equilibrated with the lower phase followed by the upper phase of n-butanol: acetic aci water(4:l:5) solvent system. Elution with the upper phase yielded a major symmetrical peak and material from, this was evaporated to dryness and lyophilized from dilute acetic acid solution to yield a white, fluffy powder(45 mg) .

The product was homogeneous by thin layer chromatogra-phy in four separate solvent systems on silica gel when loads of 20 μg were applied and visualized by exposure to ninhy reagent followed by chlorine/starch-potassium iodide reagent The following Rf values were obtained: (A) 1-butanol:acetic acid:water(4:1:5 upper phase), 0.58; (B) ethyl acetate: pyridine"acetic acid:water(5:5:l:3)0.84; (C) 2-proρanol: 1M acetic acid(2:1),0.70; (D) 1-butanol: acetic acid:water: ethyl acetate(1:1:1:1), 0.68. Amino acid analysis gave: Gly:1.00; Ala, 0.98; Met+pentafluorophenylalanine, 2.02; Tyr,0.99.

EXAMPLE 3 Preparation of 0-bromobenzyloxycarbonyl-L-tyrosyl-D-leucyl- L-glycyl-L-pentafluorophenylalanyl-L-methionyl-benzhydryl- amine resin

Using the conditions described in Esample 1, the t-Boc derivatives of pentafluorophenylalanine, glycine, D-leucine and tyrosine(2BrZ) are coupled successively to a methionine benzydryl resin to produce the desired resin.

EXAMPLE 4 Preparation of L-tyrosyl-D-leucyl-L-glycyl-L-pentafluoro- phenylalanyl-L-methionine enkephalin amide The above-named compound is prepared by deprotecti and cleaving the desired peptide from the resin support of Example 3 under the conditions described in Example 2. The crude peptide is purified by gel filtration on a column (2.5 x 95 cm) of Sephadex G-15 by elution with 2.0 molar acetic acid followed by partition chromatography as described in Example 2.

EXAMPLE 5 Preparation of 0-2-bromobenzyloxycarbonyl-L-tyrosyl-D-phenyl- alanyl-L-pentafluoroalanyl-L-methionyl-benhydrylamine resin Using the conditions described in Example 1, the t-Boc derivatives of pentafluorophenylalanine, glycine, D-phenylalanine and tyrosine[F₅Phe,Gly, D-Phe and Tyr(2BrZ) are coupled successively to a methionine-benzhydryl resin(0.63 g, 0.50 mmole) to yield the desired peptide resin. EXAMPLE 6

Preparation of L-tyrosyl-D-phenylalanyl-L-glycyl-L-pentafluoro- phenylalanyl-L-methionine enkephalin amide

The pentapeptide of Example 5 is deprotected and cleaved from the resin support of Example 5 under the conditions described in Example 2. The crude peptide is purified by gel filtration on a column (2.5 x 95 cm) of Sephadex G-15 gel by elution with 50% acetic acid followed by partition chromatography as described in Example 2.

EXAMPLE 7 Preparation of O-2-bromobenzyloxycarbonyl-L-tyrosyl-D-alanyl-

L-pentafludrophenylalanyl-L-methionyl-O-CH?-resin

Using the conditions described under Example 1, the t-Boc derivatives of pentaflurophenylalanine, glycine, D-alanine and tyrosine(2BrZ) were couples to tert-butyloxycarbonyl methionine-0-CH₂-resin(0.5 mmole) prepared by the method of Gisin, supra to obtain the desired resin.

EXAMPLE 8 Preparation of L-tyrosyl-D-alanyl-glycyl-L-pentafluorophenyl- alanyl-L-methionine enkephalin The above pentapeptide was deprotected and cleaved from the resin support of Example 7 under the conditions described in Example 2. The crude material was purified by gel filtration and partition chromatography as described in Example 2 to yield the desired product. EXAMPLE 9

Preparation of N N -bis(0-2-bromobenzyloxycarbonyl-L- tryosyl-D-alanyl-glycyl-L-phenylalanyl-L-methionyl)-2- chlorobenzyloxycarbonyl-L-lysyl-benzhydrylamine resin

Benzhydrylamine resin(1.88 g, 1.0 mmole), purchased from Beckman instruments, Palo Alto, California, was placed in the reaction vessel of a Beckman MDdel 990 automatic pepticie synthesizer, programmed as described in Example 1.

The free amino acid resin was then stirred with N^α,N^ε-bis-tert-butyloxycarbonyl(t-boc)lysine(1.04 g, 3. Ommoles) in methylene chloride and dicyclohexylcarbodiimide(3. Ommoles) was added thereto. The mixture was stirred at room temperature for 2 hours and the peptide resin was then washed successively with methylene chloride(3 times), ethanol(3 times) and methylene chloride(3 times). The attached amino acid was deprtected with 33 % trifluoroacetic acid in methylene chloride( 2 times for 2.5 and 25 minutes each) and then steps (c) through (h), as described in Example 1, were performed.

The following amino acids (6.0 mmoles) were then coupl successively by the same cycle of events; t-Boc-phenylalanine,glycine,t-Boc-D-alanine and t-boc tyrosine(2BrZ). The completed dried peptide resin weighed 3.43 g.

EXAMPLE 10 Preparation of N^α,N^ε-bis(L-tyrosyl -D-alanyl-glycyl-L-phenylalanyl-L-methionyl)-L-lysine amide The branched-chain peptide amide was deprotected and cleaved from the resin support of Example 1 by treating the resin with hydrogen fluoride(20 ml) and anisole(2 ml) at 0°C for 45 minutes. The hydrogen fluroide was removed at reduced pressure and the anisole removed by washing with ethylacetate.

The crude peptide was purufied by gel filtration on a column of Sephadex G-25(2.5 x 95 cm) gel by elution with 0.2 molar acetic acid followed by partition chromatography on Sephadex G-25 gel using 1-butanol:acetic acid:water(4:1:5) as the eluant. The peptide was obtained as a fluffy white powder (290 mg).

The product was homogeneous by thin layer chromatography using the spray reagents and solvency systems described in Example 2: (A), 0.32; (B), 0.88; (C), 0.59; (D), 0.64. Amino acidanalysis gave: Gly,1.96;Ala, 2.00;Met,1.52;Tyr, 2.00;Phe, 2.06; Lys, 1.00.

EXAMPLE 11 Preparation of N^α,N^ε-bis(0-2-bromobenzyloxycarbonyl-L-tyrosyl-D-alanyl-glycyl-L-phenylalanyl)-2-chlorobenzyloxycarbonyl-L-lysyl-benzyhydrylamine resin Preparation of N^α, N^ε -bis(O-bromobenzyloxycarbonyl-L-tyrosyl- D-alanyl-glycyl-L-phenylalahyl)-2-chlorobenzyloxycarbonyl-L- lysyl-benzyhydrylamine resin

Using the conditions described in Examples 1 , 2 and 9, the t-Boc derivatives (6.0 mmoles) of phenylalanine, glycine, D-alanine and tyrosine were coupled successivley to Lys-benzhydrylamine resin(1.0 mmole). The completed, dried peptide resin weighed 3.28 g.

EXAMPLE 12 Preparation of N^α,N^ε'-bis(L-tyrosyl-D-alanyl-glycyl-L-phenylalany)-L-lysine amide

The branched chaim peptide amide was deprotected and cleaved from the resin support of Example 3 under the conditions described in Example 10 and Example 2. The crude material was purified by gel filtration and partition chromatography as described in Example 10 to yield a fluffy white powder (440 mg) . The product was homogeneous by thin layer chromatography using the spray reacgents and solvent system described in Example 10: (A), 0.27; (B) 0.84; (C), 0.56; (D), 0.63. Amino acid analysis gave: Gly, l.97;Ala, 2.04; Tyr, 2.00; Phe,2.04;Lys, 1.01.

The present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient at least one of the compounds of formula I in association with a pharmaceutical carrier or diluent. The compounds of this invention can be administered by oral, parenteral,nasal, vaginal, rectal or sublingual routes of administration and can be formulated in dosage forms appropriate for each route of administration. Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one intert diluent such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration includ pharmaceutically acceptable emulsions, solutions, suspensio syrups and elixirs containing inert diluents commonly used in the art such as water. Besides inert diluents, such compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.

Preparations according to this invention for paren eral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving wetting, emulsifying and dispersing agents. They may be sterilized by, for example, filtration through a bacteria- retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Compostions for rectal or vaginal administration are preferably suppoitories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.

Compositions for nasal or sublingual administratio are also prepared with standard excipients well known in the art. The dosage of active ingredient in the composition of this invention may be varied, however, it is necessary that the amount of the active ingredient shall be such that a suitable dosage form is obtained. The selected dosages depend upon the desired therapeutic effect, on the route of administration and on the duration of treatment.

The compounds of this invention also exhibit psychotropic activity and may be useful in treating depres and the like states.

The following Examples further illustrate the present invention. EXAMPLE 13 Tablets weighing 200 mg and having the following composition are formulated by standard methods: Ingredient Mg L-Tyrosyl-D-alanyl-glycyl- L-pentafluorophenylalanyl- L-methionine enkephalin amide 25

Starch 145

Colloidal silica 27 Magnesium stearate 3

EXAMPLE 14 Sterile 10 ml ampoules are prepared containing 1 mg per ml of N ,N -bis (L-tyrosyl-D-alanyl-L-glycyl-L-phenylalanyl-L-methionine enkephaline)lysine amide, 0.1 percent sodium bisulfate, 0.7 percent sodium chloride, and 0.5 percent chlorbutanol as a preservative.

EXAMPLE 15 Topical aqueous formulations for administration by nose drops or nasal spray are formulated containing 1 mg of L-tyrosyl-D-leucyl-glycyl-L-pentafluorophenylalanyl-L-leucine enkephalin amide, 3.8 mg glycerine, 40 mg. sorbital, 0.02 mg benzalkonium chloride and purified water q.s. 1 ml.

Claims

1. Polypeptides represented by the formula wherein X is glycine or a chiral residue of a D-amino aci selected from the group consisting of D-alanine, D-leucine, D-isoleucine, D-valine, D-norvaline, D-phenylalanine, D-tyr osine, D-tryptophan, D-serine, D-threonine, D-methionine, D-glutamic acid, D-glutamine, D-aspartic acid, D-asparagine, D-lysine, D-proline, D-histidine and D-arginine; R is methionine when m is leucine and n is 1; n is 0 or 1; R₁ is hydrogen or fluoro; m is 1 or 2 with the limitation that when m is 1, X cannot be glycine; Y is lysine when m is 2 and methionine or leucine when m is 1 with the limitation that when m is 1, n is 0; and Z is selected from the group consisting of hydroxy, amino, loweralkylamino, diloweralkylamino and lower alkoxy and the pharmaceutical acceptable salts thereof.

2. A polypeptide of claim 1 represented by the formula wherein X is as defined in claim 1 except that X cannot be glycine; Y is methionine or leucine and Z is as defined in claim 1.

3. A polypeptide of claim 1 or 2 wherein Y is methionine.

4. A polypeptide of claim 1, wherein Z is hydroxy.

5. A polypeptide of claim 1, wherein Z is amino.

6. A polypeptide of claim 1, wherein Z is lowerealkylamino.

7. A polypeptide of claim 1, wherein Z is diloweralkylamino.

8. A polypeptide of claim 1, wherein Z is loweralkoxy.

9. A compound of claim 1 or methionine enkephalin amide or a pharmaceutically acceptable salt thereof.

10. A compound of claim 1 or 2 wherein Y is leucine.

11. A compound of claim 10 wherein Z is hydroxy.

12. A compound of claim 10 wherein Z is amino.

13. A compound of claim 10 wherein Z is loweralkylamino.

14. A compound of claim 10 wherein Z is diloweralkylammo.

15. A compound of claim 10 wherein A is loweralkoxy.

16 A compound of claim 1 represented by the formula:

17. A compound of claim 16 wherein Z is hydroxy.

18. A compound of claim 16 wherein Z is amino.

19 A compound of claim 16 wherein Z is loweralkylammo.

20. A compound of claim 16 wherein Z is diloweralkylammo.

21. A compound of claim 16 wherein Z is loweralkoxy.

22. A compound of claim 1 represented by the formula:

23. A compound of Claim 22 wherein Z is hydroxy.

24. A compound of Claim 22 wherein Z is amino.

25. A compound of Claim 22 wherein Z is loweralkylammo.

26. A compound of Claim 22 wherein Z is diloweralkylammo.

27. A compound of Claim 22 wherein Z is loweralkoxy.

28. A pharmaceutical composition comprising a compound of Claim 1 and a pharmaceutically acceptable carrier or diluent .