GB1596535A - Biologically-active peptides - Google Patents

Description

(54) BIOLOGICALLY-ACTIVE PEPTIDES (71) We, THE WELLCOME FOUNDATION LIMITED, of 183-193 Euston Road, London, N.W.I., a company incorporated in England, do hereby declare that the invention for which we pray that a Patent may be granted to us and the method by which it is performed, to be particularly described in and by the following statement:- This invention relates to peptides and derivatives thereof; to the preparation of such compounds; to formulations containing such compounds and the preparation of such formulations; and to the use of the compounds in human and veterinary medicine.

More particularly the present invention relates to peptides and derivatives thereof which exhibit morphine agonist activity. As generally accepted and as the term is used herein, a morphine agonist is a compound the biological activity of which mimics that of the natural alkaloid.

The pharmacological properties and therapeutic uses of morphine are well documented in the literature, see for example "The Pharmacological Basis of Therapeutics", Goodman, L. S. and Gilman, A. less., published by The MacMillan Company, New York, third edition (1965) especially at Chapter 15, pages 247 to 266, and "Martindale: The Extra Pharmacopoeia", Blacow, N. W. ed., published by The Pharmaceutical Press, London, twenty-sixth edition (1972) especially at pages 1100 to 1106. As is well known however (Goodman, L. S. et al., loc. cit, Chapter 16) repeated administration of morphine can lead to the recipient developing an addiction to the drug and tolerance to its effects and to his manifesting withdrawal symptoms when administration is discontinued. For many years therefore research has been conducted with the aim of obtaining a compound having the activity spectrum of morphine while lacking its disadvantages.

The present invention provides the peptides of formula (I): R-(X1 )rn(X2)nX'X4X5X6R1 (I) together with the salts (as hereinafter defined), esters, amides, N-alkylamides and N,N-dialkylamides thereof, and acid addition salts thereof, (collectively hereinafter referred to as the compounds of formula (I)) which compounds exhibit morphine agonist activity in both in vitro and in vivo tests.

In formula (I): X1 and X2 are the same or different and each is the radical of a basic amino acid (as hereinafter defined) (D or L); X3 is a D or L radical having the formula:

wherein R2 is phenyl or l,4-cyclohexadien-l-yl, a is 0, 1 or 2, b is0 or 1, one of W and W1 is a group -NR3- and the other is hydrogen, provided that W is alaways -NR3- when b is 0 and that when R2 is l,4-cyclohexadien-l-yl a is always 1 and b is always 0, where R3 is hydrogen or a group selected from alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl, and where R2 is optionally substituted by one or more groups each selected from hydroxy, alkoxy, alkanoyloxy, alkyl, nitro, trifluoromethyl, amino, N-alkylamino, halogen, N,Ndialkylamino and benzyloxy (wherein the phenyl ring is optionally substituted by one or more groups each selected from hydroxy, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino and N,N-dialkylamino) X4 is a D radical selected from C-propargylglycyl, alanyl, C-alkyl(2 to 4 carbon atoms)alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl sand glutaminyl, where -each of said radicals is optionally n tr-substituted with an alkyl group; X5 iS glycyl, A-alanyl, C-methylalanyl or a D or L radical selected from Cpropargylglycyl, alanyl, C-alkyl(2 to 4 carbon atoms)alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, where each of said radicals is optionally N-substituted with an alkyl group; Xe is selected from glycyl, a D or L radical selected from methionyl, leucyl, isoleucyl, norleucyl, valyl, norvalyl, prolyl, hydroxyprolyl, alanyl and histidyl, and the meanings recited hereinabove for X3; R is selected from hydrogen, aralkyl, alkyl, akenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl; Rl represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical or a group, replacing said l-carboxyl group, selected from -CH2OR4 (where R4 is hydrogen or alkanoyl) and 5-tetrazolyl optionally substituted in the 1or 2-position with a group selected from alkyl and benzyl; and m and n are each selected from 0 and 1 except for the amides, N-alkylamides and N,N-dialkylamides and acid addition salts thereof wherein X5 is a D or L radical having the formula

wherein R3 is hydrogen or alkyl.

The abbreviations used herein for amino acids and their radicals are those conventional in the art and may be found in, for example, Biochemistrv, II, 1726 (1972). In the above and throughout the following all references are to the Lconfiguration of chiral amino acids and their radicals unless otherwise stated.

By the term "basic amino acid" is herein meant an amino acid having two basic functions and one carboxyl group, and as examples of the radicals X1 and X2 may be mentioned lysyl (D and L), homoarginyl (D and L), ornithyl (D and L), histidyl (D and L), ,y-diaminobutyryl (D and L) and arginyl (D and L).

In formula (I), the optional N" alkyl substituents of the radicals X4 and X5 desirably have 1 to 4 and preferably 1 or 2 carbon atoms. In the phenyl or 1,4cyclohexadien-l-yl ring R2 of the radical X3, and in the optional benzyloxy substituents therein, the optional halogen substituents may be selected from fluorine, chlorine, bromine and iodine and the optional alkyl and alkoxy substituents together with the alkyl moietie, of the optional alkanoyloxy, Nalkylamino and N,N-dialkylamino substituents desirably have 1 to 4 and preferably 1 or 2 carbon atoms.

When the phenyl or cyclohexadien-l-yl ring of the radical X3 has I or 2 substituent groups thereon said groups are desirably located at the 3- and/or 4position(s) of the said ring.

The alkyl identity for R and for the group R3 of the radical X3 together with said moiety in the carboxyalkyl identity therefor may in particular have 1 to 4 carbon atoms, for example 1 or 2, but such groups having for example I to 10 or I to 20 carbon atoms are to be understood as also included. The alkenyl and alkynyl identities for R and R3 together with said moieties in respectively the carboxyalkenyl and carboxyalkynyl identities therefor may in particular have 2 to 4 carbon atoms but such groups having for example 2 to 10 or 2 to 20 carbon atoms are to be understood as within the scope of formula (I). As particular values for said alkenyl and alkynyl groups respectively may be mentioned allyl (cow, P or y) and propargyl.

When R is aralkyl this may be for example benzyl optionally substituted in the phenyl ring by one or more groups each selected from those recited hereinbefore in respect of the optional benzyloxy substituent of the group R2 in the radical X3.

When R' is a group -CH2OR4 wherein R4 is alkanoyl the alkyl moiety of said alkanoyl group desirably has 1 to 4 and preferably 1 or 2 carbon atoms. When R' is 5-tetrazolyl the optional alkyl substituent thereon desirably has I to 5 carbon atoms.

Among the esters of the peptides of formula (I) may be mentioned the alkyl esters and the aryl esters. The alkyl esters include in particular those wherein the alkyl group has I to 4 carbon atoms, for example the methyl, ethyl and t-butyl esters, but esters wherein the alkyl group has for example 1 to 10 or 1 to 20 carbon atoms are to be understood as also included. The aryl esters include phenyl esters, for example halophenyl esters where the halo is for example chloro as in pchlorophenyl.

In the N-alkylamides and N,N-dialkylamides of the peptides of formula (I) the alkyl groups may in particular have 1 to 5 carbon atoms but alkyl groups having for example 1 to 10 or 1 to 20 carbon atoms are to be understood as also included and in the N,N-dialkylamides the alkyl groups may be the same or different. The amides of the peptides should be understood to include those notionally derived not only from ammonia but also from heterocyclic bases such as pyrrolidine, piperidine and morpholine, that is to say, the pyrrolidineamides, piperidineamides and morpholineamides respectively.

Thus a sub-class of the amides, N-alkylamides and N,N-dialkylamides of the peptides of formula (I) may be represented by the formula:

wherein X', X2, X3, X4, X, X65 m, n and R have the meanings as hereinabove recited in formula (I) and R5, R6 and~the nitrogen atom to which they are attached together comprise a group selected from amino, pyrrolidino, piperidino, morpholino, N-alkylamino and N,N-dialkylaminb where the alkyl in each instance has 1 to 20 carbon atoms.

In the acid addition salt compounds of formula (I) the activity resides in the base and the acid is of less importance although for therapeutic purposes it is preferably pharmacologically and pharmaceutically acceptable to the recipient.

Examples of such suitable acids include (a) mineral acids: hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; (b) organic acids: tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, gulonic, succininc and arylsulphonic, for example p-toluenesulphonic acids. The pharmaceutically and pharmacologically acceptable acid addition salts together with those salts which are not thus acceptable (for example salts of hydrofluoric and perchloric acids) have utility in isolation and purification of the bases, and of course the unacceptable salts are also valuable in the preparation of the acceptable salts by techniques well known in the art. Those bases containing a plurality of free amino groups may be obtained in the form of mono- or poly-acid addition salts, or as mixed salts of a plurality of acids.

Likewise in the salts of the peptides (comprising the peptide as the carboxylate anion together with a cation) the identity of the cation is of less importance although for therapeutic purposes it is preferably pharmacologically and pharmaceutically acceptable to the recipient. Examples of such suitable cations include sodium and potassium.

The morphine agonist properties of the compounds of formula (I) include the following, which are given solely by way of illustration and should be understood to be non-limiting.

(A) In vitro: (i) Inhibition of neurally evoked contractions of the isolated mouse vas deferens when tested by the method of Hughes et al (Brain Research, 88 (1975) 296) (using pulses at 0.1 Hz), the inhibition being abolished by the known narcotic antagonist naloxone (1 -N-allyl-7,8-dihydro- 1 4-hydroxy normorphinone).

(ii) Reduction of electrically-induced contractions of the isolated guinea-pig ileum when prepared for stimulation after the manner of Paton (Brit. J. Pharmacol., 12 (1957) 119-127). (Each intestinal segment was impaled by the anode and suspended with a 2-3 g load. Stimulus parameters: frequency: 0.1 Hz; duration: 0.4 ms; voltage (supramaximal) 30-40 V; the contractions were transduced isotonically).

(B) In vivo: (i) The compounds exhibit analgesic activity, for example they are effective in mice in the "hot plate" procedure standard in the art when tested by a modification of the method of Eddy, N. B. et al. (J. Pharm. Exp. Therap. 107. 385 (1953)), the compounds being administered by intracerebroventricular injection, and this activity is abolished by naloxone.

(ii) The compounds exhibit antitussive activity, for example when tested in guinea-pigs according to the method of Boura et al. Brit. J. Pharmacol, 39 (1970) 225.

(iii) The compounds exhibit antidiarrhoeal activity, for example they are effective in reducing castor oil-induced diarrhoea in rats.

As subclasses of the compounds of formula (I) may be mentioned those compounds wherein: (i) m and n are both 0; (ii) R is hydrogen; (iii) X3 is L-tyrosyl; (iv) X4 is D-alanyl; (v) X5 iS glycyl; (vi) Xe is selected from L-phenylalanyl, L-4-chlorophenylalanyl and L-4nitrophenylalanyl, preferably from L-4-chlorophenylalanyl and L-4nitrophenylalanyl, and is most preferably L-4-nitrophenylalanyl; and (vii) R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical.

As a subclass of the peptides of formula (I) may be mentioned those of the formula: H . Tyr . D-Ala . Gly . X6. R1 together with the salts (as hereinbefore defined), esters, amides, N-alkylamides and N,N-dialkylamides thereof, and acid addition salts thereof, wherein Xe is selected from L-phenylalanyl, L-4-chlorophenylalanyl and L-4nitrophenylalanyl; and R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical.

Preferred esters of this subclass are alkyl esters wherein the alkyl group has 1 to 10, more preferably 1 to 4 and most preferably 1 or 2, carbon atoms and the preferred N-alkylamides and N,N-dialkylamides are those wherein the alkyl group(s) has 1 to 5, more preferably 1 or 2, carbon atoms.

The compounds of formula (I) as hereinabove recited may be prepared by any of the methods known in the art for the preparation of compounds of analogous structure. Thus they may be formed by the sequential coupling of appropriate amino acids using either classical methods of peptide synthesis or solid phase procedures, or by the initial preparation and subsequent coupling of peptide subunits.

Such reactions may be affected by, for example, activating the carboxylic acid group of the ingoing amino acid and protecting the non-reacting amino and carboxylic acid groups. Such techniques are standard in the peptide art. Details of suitable activating and protecting (masking) groups and of suitable reaction conditions (both for the coupling reactions and for the removal of protecting groups) giving the minimum of racemisation may be found in the following literature which is given purely by way of exemplification and which is intended to be neither exhaustive nor limiting.

(a) Published United Kingdom patent specifications Nos. 1,042,487; 1,048,086: and 1,281,383.

(b) Schrder and Lebke, "The Peptides" (Academic Press) (1965).

(c) Bellean and Malek, J. Am. Chem. Soc., 90, 165 (1968).

(d) Tilak, Tetrahedron Letters, 849 (1970).

(e) Beyerman, Helv. Chim. Acta., 56. 1729 (1973).

(f) Stewart and Young, "Solid Phase Peptide Svnthesis" W. H. Freeman and Co.) (1969).

Depending upon the reaction conditions the compounds of formula (I) are obtained in the form of the free base or as an acid addition salt thereof or (in the case of the peptides themselves) as a salt as above defined. The acid addition salts may be converted into the free bases or salts of other acids, and the bases may be converted into acid addition salts thereof, by techniques well known in the art.

Likewise the peptides may be converted to salts thereof, and the salts converted to the peptides or to other salts, by well established techniques.

The compounds of formula (I) as hereinabove recited may thus be prepared by reacting a reagent (II) R-Y1-OH (Il) wherein Y1 is selected from the radical (X1)m as defined hereinbefore and a partial radical sequence having the radical (X1)m at its N-terminal end and from thereon corresponding to formula (1), with a reagent (III) H-Y2 (III) wherein Y2 corresponds to the balance of the above defined product compound, the reagents (II) and (III) being optionally protected and/or activated where, and as, appropriate; followed, as appropriate, by deprotection of the product and conversion of the product into the base or a salt or an acid addition salt thereof.

It will be appreciated by those skilled in the peptide art that the arginyl (D or L) and homoarginyl (Har) (D or L) radicals may. not only be incorporated into the peptide chain in the fashion described above but may also be formed in situ in the assembled chain, or in a subunit thereof, by guanidation of an ornithyl (D or L) or lysyl (D or L) radical respectively, using a reagent such as: I-guanyl-3,5dimethylpyrazole.

It will also be appreciated that other in situ conirersions of the compounds of formula (I) are possible. Thus the amides, N-alkylamides and N,N-dialkylamides may be prepared by for example reaction of a peptide alkyl ester such as the methyl ester with ammonia, a heterocyclic base or a monoalkylamine or dialkylamine, as appropriate. The peptide esters may be prepared from the peptides by standard esterification procedures and the esters may be converted to the peptides by saponification. Substituent hydroxy group(s) in the group R2 of the radical X3 may be converted to alkoxy or benzyloxy groups by the use of the appropriate diazoalkane, for example diazomethane to provide methoxy groups. Substituent benzyloxy and alkanoyloxy group(s) in the group R2 of the radical X3 may be removed to leave hydroxy groups by hydrogenolysis in methanol using 10% (w/w) palladium on charcoal catalyst and by alkaline hydrolysis respectively, and the hydroxy group(s) may be converted to alkanoyloxy groups by standard alkanoylation procedures. All these are conventional techniques in the peptide art and reference may be made to the literature referred to hereinabove for details of reaction conditions and of appropriate protection/deprotection procedures.

Because of their morphine agonist activity already alluded to the peptides of formula (I) together with their pharmacologically and pharmaceutically acceptable salts (as hereinbefore defined), esters, amides, N-alkylamides and N,Ndialkylamides and pharmacologically and pharmaceutically acceptable acid addition salts thereof may be used in the treatment of mammals in the fields of both human and veterinary medicine in any condition where an agent with a morphinelike effect is indicated. Specific utilities that may be mentioned, by way of example, include the following: (I) The relief of pain (analgesia), for example pain arising from spasm of smooth muscle as in renal or biliary colic, pain due to terminal illness such as cancer, pain in the post-operative period, and obstetrical pain.

(2) Sedation, for example in pre-anaesthetic medication; tranquillization; the induction of sleep, especially where sleeplessness is due to pain or cough; and the relief of anxiety in general.

(3) The suppression of cough.

(4) The relief of dyspnoea, for example that of acute left ventricular failure of pulmonary oedema.

(5) The induction of constipation, for example after ileostomy or colostomy, and the treatment of diarrhoea and dysentery.

(6) The induction of euphoria and the treatment of depression, for example when allied to the relief of pain in terminal illness such as cancer.

For each of these utilities the amount required of a compound (hereinafter referred to as the active ingredient) will vary with the route of administration and with the severity of the condition to be treated, and will ultimately be at the discretion of the physician or veterinarian. In general however for each of these utilities the dosage will be in the range 0.0025 pg. to 40 mg. per kilogram bodyweight of mammal, preferably OP25 ,ug. to 4.0 mg./kg., and optimally 0.25 to 400 ,ug./kg. (all dosages calculated with reference to the peptide base).

The active ingredients may be administered by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (buccal), vaginal and parenteral (including subcutaneous, intramuscular and intravenous).

While it is possible for the active ingredients to be administered as the raw chemical it is preferable to present them as a pharmaceutical formulation preparation.

The formulations, both veterinary and for human use, of the present invention comprise an active ingredient, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof Desirably the formulations should not include oxidising agents and other substances with which peptides are known to be incompatible.

The formulations include those suitable for oral, rectal, nasal, topical (buccal), vaginal or parenteral (including subcutaneous, intramuscular and intravenous) administration, although the most suitable route in any given case will depend upon for example the active ingredient and the condition to be treated. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. The formulations may thus be prepared by admixture of the active ingredient with liquid carriers or finely divided solid carriers or both and, if necessary, shaping of the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; or as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oilin-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as a powder or-gr nules, optionally mixed with a binder, lubricant, diluent, lubricating, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, while a suitable formulation for nasal administration is nasal drops comprising the active ingredient in aqueous or oily solution.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.

Formulation suitable for vaginal administration may be presented as pessaries, creams, pastes or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration conveniently comprise sterile aqueous solutions of the active ingredient, which solutions are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving solid active ingredient in water to produce an aqueous solution, and rendering said solution sterile and isotonic with the blood of the recipient. The formulations may be presented in unit-or in multi-dose containers, for example sealed ampoules or vials.

Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size preferably in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.

It should be understood that in addition to the aforementioned ingredients the formulations of this invention may include one or more additional ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants and preservatives (including anti-oxidants).

Where the formulation, for human or for veterinary use, is presented in unit dosage form, for example those unit dosage forms specifically mentioned above, each unit thereof conveniently contains the active ingredient (as above defined) in an amount in the range 0.125 ,ug. to 2 g., preferably 1.25 ,ug. to 200 mg. And optimally 12.5 ,ug. to 20 mg. (all weights calculated with reference to the peptide base).

It will be appreciated from the foregoing that the present invention in particular provides the following: (a) The peptides of formula (I) as hereinbefore defined, together with the salts (as hereinbefore defined), esters, amides, N-alkylamides and N,N-dialkylamides thereof, and acid addition salts thereof.

(b) Methods as described hereinbefore for the preparation of the compounds recited in (a) above.

(c) Pharmaceutical formulations comprising a compound recited in (a) above together with an acceptable carrier therefor, provided that where said compound is a salt (as hereinbefore defined) or an acid addition salt then the said compound is pharmacologically and pharmaceutically acceptable.

(d) Methods for the preparation of the pharmaceutical formulations defined in (c) above.

(e) A method for the treatment of a mammal other than man for a condition wherein an agent with a morphine-like effect is indicated, comprising the administration 'to the mammal of a treatment-effective, non-toxic amount of a compound. recited in (a) above, provided that where said compound is a salt (as hereinbefore defined) or an acid addition salt then the said compound is pharmacologically and pharmaceutically acceptable.

(f) A method according to (e) above for the treatment of a condition selected from those specifically identified hereinabove finder (1), (2), (3), (4), (5) or (6).

The following Examples serve to illustrate the present invention but should not be construed as in any way providing a limitation thereof. All temperatures are in degrees Celcius.

Experimental Section The following abbreviations are used throughout HOBT l-hydroxybenzotriazole DCCI dicyclohexylcarbodiimide DMF dimethylformamide BOC tertiary butyloxycarbonyl Bzl benzyl Peptides were examined by tIc on Merck (Trade Mark) silica gel plates with the following solvent systems: 1. Methylethylketone 2. n-Butanol:acetic acid:water (3:1:1) (by volume) 3. Chloroform:methanol:32% (v/v) acetic acid (120:90:40) (by volume) 4. Chloroform:methanol:32% (w/v) ammonia (120:90:40) (by volume) 5. n-Butanol:acetic acid:ethylacetate:water (1:1:1:1) (by volume) 6. Chloroform:methanol (8:1) (by volume) 7. Chloroform:methanol:32% (v/v) acetic acid (120:90:5) (by volume) 8. Chloroform:methanol:32% (w/v) ammonia (120:90:5) (by volume) Optical rotations were determined on a Bendix (Trade Mark) NPL automatic polarimeter.

The amino acid compositions of peptide hydrolysates (6N-hydrochloric acid at 110"C. for 24 hours in evacuated sealed tubes) were determined with a Beckman (Trade Mark) Spinco Model 120C amino acid analyser or with a Rank Chromostak amino acid analyser.

The following general procedures were used throughout the syntheses of the peptides.

a) Couplings were carried out in DMF and were mediated by DCCI.

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

c) HOBT was added at the coupling stage when fragment condensation involved a peptide having an optically active carboxy terminal amino acid.

d) couplings were allowed to proceed for 24 hours in the cold room at +4"C.

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

(f) alkaline saponifications were carried out in aqueous methanol with an autotitrator at pH 11.5 to 12.0 with N sodium hydroxide.

g) Benzyloxycarbonyl protecting groups were removed by hydrogenolysis in methanol/acetic acid with 10% (w/w) palladium on charcoal.

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

i) Benzyl protecting groups were removed by hydrogenolysis in methanol with 10% (w/w) palladium on charcoal.

j) Tertiary butyl and tertiary butyloxycarbonyl protecting groups were removed with 1N hydrogen chloride in acetic acid, in the presence of anisole to act as a scavenger. Cleavage was allowed to proceed for 60 to 90 minutes.

The final peptides were isolated as their hydrochlorides and were lyophilised from aqueous solution.

Example I H. Tyr. D-Ala. Gly. Phe. OH This was prepared according to the Scheme set out in Table I (the Pd/C reagent (step 7) was 10% (w/w)). The product, as the hydrochloride addition salt, had the following characterising data after lyophilisation from aqueous solution: Rf: 0.412; 0.664; 0.238 [(u]5: +64.60 (C=0.5 in methanol).

Example 2 H. Tyr. D-Ala. Gly. Phe. OMe This was prepared from the product of Example I by reaction with methanolic hydrogen chloride. The ester, as the hydrochloride addition salt, had the following characterising data after lyophilisation from aqueous solution: Rf: 0.572; 0.883; 0.635 lal25: +57.20 (C=0.5 in methanol).

The same product, with identical characterising data, resulted from treatment of the product of step 5 in Table 1 in the following manner (the Pd/C reagent was 10% (w/w)):

H.Tyr D.Ala Gly a PheOfl BOC.D-Ala. OH H.Gly.OMe O HOBT/DCCI coupling BOC-fl-Ala Gly.OMe Bzl Q 1N HC1/HOAc BOC-Tyr.OH 11.0-Ala Uty Gly .OM e Q3 HOBT/OCCI coupling Bzl : , BOC-Tyr D-Ala Gly. OMe (E) 11N NaOH/MeOH Uzi BOC-Tyr O-Ala Gly.OH llPhe. ------ Gly . O H H. Phe. OMe Q5 HOBT/DCCI coupling Bzl BOC.Tyr D-Ala 0All ---------- Gly -------- Phe.OMe INNaOH/MeOH Bzl 1 UDETyr ----------- D-Ala Gly Phe. OH ( 10% Pd /C ~~~~~~~~~~ H2 2 DOC.Tyr D-Ala --------- Gly Phe. OH (D 1N HCllHOAc 11. F ----------- Tyr U-Ala Gly Phe.Oll TABLE 1 The following peptides were prepared, with the characterising data respectively shown therefor, according to standard procedures in peptide chemistry analogous to those set out in the foregoing Examples. C-Terminal derivatives are indicated according to convention, that is to say: -OMe: methyl ester -O(n-heptyl): n-heptyl ester -NHEt: ethylamide Ex. No. Comnound Rf [α]D25 (in methanols 3 H. Tyr. D-Ala. Gly. D-Phe. OH 0.605; 0.447 4 H. Tyr. D-Ala. Gly. Phe(4C1). OH 0.417 +55.9 (c=0.3) 5 H. Tyr. D-Ala. D-Ala. Phe. OH HCI 0.723; 0.675 6 H. Tyr. D-Ala. Gly. Phe. O(n-heptyl) HCI 0.652 +46.1" (c=0.2) 7 H. Tyr. D-Ala. Gly. Phe(4NO2). OMe HCI 0.607; 0.548 +59 4O (c=0.5) 8 H, Tyr. D-Ala. Gly. Phe(4NO2). NHEt HCI 0.532; 0.687; 0.618 +36.9 (c=0.5) 9 H. Tyr. D-Ala. Gly. Phe(4NO2). OH HCI 0.432; 0.497; 0.558 +66.3 (c=0.5) Example 10 Pharmacological Activity Peptides of the foregoing Examples were tested for the following activities according to standard pharmacological procedures.

(A) Analgesia in mice in the hot plate test (modification of the method of Eddy, N.B. et al., J. Pharm. Exp. Therap. (1953) 107, 385, the peptide being administered by intracerebroventricular injection).

(B) Antidiarrhoeal activity in the rat. In this procedure rats were starved for 24 hours, the peptide then administered subcutaneously followed after 15 minutes by I ml. Castor oil per rat given orally.

From the data obtained the respective ED80 figures were calculated (i.e. the dose required to elicit the appropriate effect in 50% of the animals), expressed as weight of peptide base per mouse/rat.

Analgesia Antidiarrhoeal (mouse: hot plate) (rat) Peptide ED50 i.c.v. ED50 s.c.

of Example (ug/mouse) (mg/kg) 1 0.7 5.0 2 0.2 0.7 4 5.0 1.0 6 3.0 3.0 7 0.5 0.3 8 0.07 0.3 9 1.0 0.5 Pharmaceutical Formulations A) Tablet Formulation (20 mg/tablet) Compound of formula (I) 20 mg Lactose 76 mg Maize starch 10 mg Gelatin 2 mg Magnesium stearate 2 mg Mix together the compound of formula (I), lactose and maize starch. Granulate with a solution of the gelatin dissolved in water. Dry the granules, add the magnesium stearate and compress to produce tablets, 110 mg per tablet.

B) Suppository (5 mg/product) Compound of formula (I) 250 mg Suppository base (Massa Esterinum C) to 100 g Melt the suppository base at 400 C. Gradually incorporate the compound of formula (I) in fine powder form and mix until homogeneous. Pour into suitable moulds, 2 g per mould, and allow to set.

Massa Esterinum C is a commercially available suppository base consisting of a mixture of mono, di, and tri-glycerides of saturated vegetable fatty acids. It is marketed by Henkel International, Dusseldorf.

C) Pessary (5 mg/product) Compound of formula (I) 5 mg Lactose 400 mg Polyvinylpyrrolidone 5 mg Magnesium stearate 5 mg Mix together the compound of formula (I) and lactose. Granulate with a solution of polyvinylpyrrolidone in 50% (v/v) aqueous ethanol. Dry the granules, add the magnesium stearate and compress on suitably shaped punches, 415 mg per pessary.

D) Freeze-dried Injection (100 mg/vial) Compound of formula (I) 100 mg Water for injections to 2.0 ml Dissolve the compound of formula (I) in the water. Sterilise the solution by passage through a membrane filter, 0.2 ,tlm pore size, collecting the filtrate in a sterile receiver. Fill into sterile glass vials, 2 ml/vial under aseptic conditions and freeze-dry. Close the vials with sterile rubber closures secured with an aluminium seal.

The injection is reconstituted prior to administration by the addition of a convenient volume of water for injections or sterile saline solution.

In the foregoing, the weight of the compound of formula (I) is in each instance calculated with reference to the peptide base.

WHAT WE CLAIM IS: 1. A peptide of formula (I): R--(X' ),~(X2),~X3~X4~X 5~X6~R1 (I) or a salt (as hereinbefore defined), ester, amide, N-alkylamide or N,N-dialkylamide thereof, or an acid addition salt thereof, wherein X' and X2 are the same or different and each is the radical of a basic amino acid (as hereinbefore defined) (D or L) X3 is a D or L radical having the formula:

wherein R2 is phenyl or 1,4-cyclohexadien-1-yl, a is0, 1 or 2, b is0 or I, one of W and W1 is a group -NR3- and the other is hydrogen, provided that W is always -NR3- when b is 0 and that when R2 is 1,4-cyclohexadiene-l-yl a is always 1 and B is always 0, where R3 is hydrogen or a group selected from alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl, and where R2 is optionally substituted by one or more groups each selected from hydroxy, alkoxy, alkanoyloxy, alkyl, nitro, trifluoromethyl, amino, N-alkylamino, halogen, N,Ndialkylamino and benzyloxy (wherein the phenyl ring is optionally substituted by one or more groups each selected from hydroxy, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino and N,N-dialkylamino); X4 iS a D radical selected from C-propargylglycyl, alanyl, C-alkyl(2 to 4 carbon atoms)alanyl, valyl, norgalyl, leucyl, isoleueyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, where each of said radicals is optionally N"-substituted with an alkyl group; X5 is glycyl, 13-alanyl, C-methylalanyl or a D or L radical selected from C propargyiglycyl, aJanyl, C-alkyl(2 to 4 carbon atoms)alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, where each of said radicals is optionally N"-substltuted with an alkyl group; Xs is selected from glycyl, a D or L radical selected from methionyl, leucyl, isoleucyl, norleucyl, valyl, norvalyl, prolyl, hydroxyprolyl, alanyl and histidyl, and the meanings recited hereinabove for X3; R is selected from hydrogen, aralkyl, alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl; R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical or a group, replacing said carboxyl group, selected from -CH2OR4 (where R4 is hydrogen or alkanoyl) and 5-tetrazolyl optionally substituted in the 1or 2-position with a group selected from alkyl and benzyl; and m and n are each selected from 0 and 1, except for the amides, N-alkylamides and N,N-dialkylamides and acid addition salts thereof wherein X8 is a D or L radical having the formula

wherein R3 is hydrogen or alkyl.

2. A peptide according to claim 1 of formula: H. Tyr. D-Ala. Gly. X6. R1 or a salt (as hereinbefore defined), ester, amide, N-alkylamide or N,N-dialkylamide thereof, or an acid addition salt thereof, wherein X8 is selected from L-phenylalanyl, L-4-chlorophenylalanyl and L-4nitrophenylalanyl: and

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (48)

**WARNING** start of CLMS field may overlap end of DESC **. In the foregoing, the weight of the compound of formula (I) is in each instance calculated with reference to the peptide base. WHAT WE CLAIM IS:

1. A peptide of formula (I): R--(X' ),~(X2),~X3~X4~X 5~X6~R1 (I) or a salt (as hereinbefore defined), ester, amide, N-alkylamide or N,N-dialkylamide thereof, or an acid addition salt thereof, wherein X' and X2 are the same or different and each is the radical of a basic amino acid (as hereinbefore defined) (D or L) X3 is a D or L radical having the formula:

wherein R2 is phenyl or 1,4-cyclohexadien-1-yl, a is0, 1 or 2, b is0 or I, one of W and W1 is a group -NR3- and the other is hydrogen, provided that W is always -NR3- when b is 0 and that when R2 is 1,4-cyclohexadiene-l-yl a is always 1 and B is always 0, where R3 is hydrogen or a group selected from alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl, and where R2 is optionally substituted by one or more groups each selected from hydroxy, alkoxy, alkanoyloxy, alkyl, nitro, trifluoromethyl, amino, N-alkylamino, halogen, N,Ndialkylamino and benzyloxy (wherein the phenyl ring is optionally substituted by one or more groups each selected from hydroxy, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino and N,N-dialkylamino); X4 iS a D radical selected from C-propargylglycyl, alanyl, C-alkyl(2 to 4 carbon atoms)alanyl, valyl, norgalyl, leucyl, isoleueyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, where each of said radicals is optionally N"-substituted with an alkyl group; X5 is glycyl, 13-alanyl, C-methylalanyl or a D or L radical selected from C propargyiglycyl, aJanyl, C-alkyl(2 to 4 carbon atoms)alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, where each of said radicals is optionally N"-substltuted with an alkyl group; Xs is selected from glycyl, a D or L radical selected from methionyl, leucyl, isoleucyl, norleucyl, valyl, norvalyl, prolyl, hydroxyprolyl, alanyl and histidyl, and the meanings recited hereinabove for X3; R is selected from hydrogen, aralkyl, alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl; R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical or a group, replacing said carboxyl group, selected from -CH2OR4 (where R4 is hydrogen or alkanoyl) and 5-tetrazolyl optionally substituted in the 1or 2-position with a group selected from alkyl and benzyl; and m and n are each selected from 0 and 1, except for the amides, N-alkylamides and N,N-dialkylamides and acid addition salts thereof wherein X8 is a D or L radical having the formula

wherein R3 is hydrogen or alkyl.

2. A peptide according to claim 1 of formula: H. Tyr. D-Ala. Gly. X6. R1 or a salt (as hereinbefore defined), ester, amide, N-alkylamide or N,N-dialkylamide thereof, or an acid addition salt thereof, wherein X8 is selected from L-phenylalanyl, L-4-chlorophenylalanyl and L-4nitrophenylalanyl: and

R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical.

3. A compound according to claim I of formula:

or an acid addition salt thereof, wherein X', X2, X3, X4, X5, X6 m, n and R are as defined in claim 1 and R5, R8 and the nitrogen atom to which they are attached together comprise a group selected from amino, pyrrolidino, piperidino, morpholino, N-alkylamino and N,N-dialkylamino where the alkyl in each instance has 1 to 20 carbon atoms.

4. A compound according to claim 3 wherein R5, R6 and the nitrogen atom to which they are attached together comprise an N-alkylamino or N.N-dialkylamino group where the alkyl in each instance has 1 to 5 carbon atoms.

5. A compound according to either of claims 1 and 2 which is an alkyl ester or an acid addition salt thereof, wherein the alkyl group has 1 to 4 carbon atoms.

6. A compound according to either of claims I and 2 wherein R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical.

7. A compound according to any of claims 1 and 3 to 6 wherein R is hydrogen.

8. A compound according to any of claims 1 and 3 to 7 wherein m and n are both 0.

9. A compound according to any of claims 1 and 3 to 8 wherein X3 iS L-tyrosyl.

10. A compound according to any of claims 1 and 3 to 9 wherein X4 iS D-alanyl.

11. A compound according to any of claims 1 and 3 to 10 wherein X5 is glycyl.

12. A compound according to any of claims 1 and 3 to 11 wherein X is selected from L-phenylalanyl, L-4-chlorophenylalanyl and L-4-nitrophenylalanyl.

13. H. Tyr. D-Ala. Gly. Phe. OH or a salt (as hereinbefore defined) or acid addition salt thereof.

14. H. Tyr. D-Ala. Gly. Phe. OMe or an acid addition salt thereof.

15. H. Tyr. D-Ala. Gly. D-Phe. OH or a salt (as hereinbefore defined) or acid addition salt thereof.

16. H. Tyr. D-Ala. Gly. Phe(4C1). OH or a salt (as hereinbefore defined) or acid addition salt thereof.

17. H. Tyr. D-Ala. D-Ala. Phe. OH or a salt (as hereinbefore defined) or acid addition salt thereof.

18. H. Tyr. D-Ala. Gly. Phe. O(n-heptyl) or an acid addition salt thereof.

19. H. Tyr. D-Ala. Gly. Phe(4NO2). OMe or an acid addition salt thereof.

20. H. Tyr. D-Ala. Gly. Phe(4NO2). NHEt or an acid addition salt thereof.

21. H. Tyr. D-Ala. Gly. Phe(4NO2). OH or a salt (as hereinbefore defined) or acid addition salt thereof.

22. A compound according to any of claims 1,2,6 to 13, 15 to 17 and 21 which is a salt (as hereinbefore defined), the said salt being pharmacologically and pharmaceutically acceptable.

23. A compound according to any of claims 1 to 21 which is a pharmacologically and pharmaceutically acceptable acid addition salt.

24. The addition salt with hydrochloric acid of a peptide according to any of claims 13 to 21.

25. A pharmaceutical formulation comprising a compound as defined in any of claims 1 to 21 together with an acceptable carrier therefore, provided that where said compound is a salt (as hereinbefore defined) or an acid addition salt then the said compound is pharmacologically and pharmaceutically acceptable.

26. A formulation according to claim 25, suitable for administration by a route selected from oral, rectal, nasal, buccal, vaginal and parenteral.

27. A formulation according to claim 25, suitable for administration by a route selected from oral, rectal, vaginal and parenteral.

28. A formulation according to claim 25 comprising the compound in solution in an aqueous medium.

29. A formulation according to any of claims 25 to 28 in unit dosage form.

30. A formulation according to claim 29 in the form of a tablet suitable for oral administration.

31. A formulation according to claim 29 in the form of a suppository suitable for rectal administration.

32. A formulation according to claim 29 in the form of a pessary suitable for vaginal administration.

33. A formulation according to claim 29 in the form of a sterile injection solution suitable for parenteral administration.

34. A freeze-dried formulation according to claim 33.

35. A formulation according to any of claims 29 to 34 wherein each unit dosage contains the compound in an amount in the range 0.125 Mg to 2 g, calculated with reference to the peptide base.

36. A formulation according to any of claims 25 to 35 wherein the compound is as defined in any of claims 13 to 21.

37. A method for preparing a formulation as defined in any of claims 25 to 36 comprising admixture of the ingredients thereof and, if necessary, shaping of the product.

38. A method for the treatment of a mammal other than man for a condition wherein an agent with a morphine-like effect is indicated, comprising the administration to the mammal of a treatment-effective, non-toxic amount of a compound as defined in any of claims 1 to 21, provided that where said compound is a salt (as hereinbefore defined) or an acid addition salt then the said compound is pharmacologically and pharmaceutically acceptable.

39. A method according to claim 38 for the relief of pain.

40. A method according to claim 38 for the induction of constipation or for the treatment of diarrhoea or dysentry.

41. A method according to any of claims 38 to 40 wherein the compound is administered at a dosage in the range 0.0025 yg to 40 mg per kilogram body weight of the mammal, calculated with reference to the peptide base.

42. A compound according to any of claims 1 to 21, provided that where said compound is a salt (as hereinbefore defined) or an acid addition salt then the said compound is pharmacologically and pharmaceutically acceptable whenever used in a method according to any of claims 38 to 41.

43. A method for the preparation of a compound of formula (I) as defined in any of claims 1 to 24 comprising (a) the reaction of a reagent (II) R-Y1-OH (Il) wherein R is as defined in claim 1 and Y' is selected from the radical (X1)m as defined in claim 1 and a partial radical sequence having the radical (X1)m at its Nterminal end and from thereon corresponding to formula (I), with a reagent (III) H-Y2 (III) wherein Y2 corresponds to the balance of the above defined product compound, the reagents (II) and (III) being optionally protected and/or activated where, and as, appropriate; followed, as appropriate, by deprotection of the product; or (b) where the compound of formula (I) is an amide, N-alkylamide or N,Ndialkylamide, the reaction of a corresponding alkyl ester compound of formula (I) with ammonia, a heterocyclic base or a monoalkylamine or dialkylamine, as appropriate; or (c) where the compound of formula (I) is an ester, the esterification of the corresponding compound of formula (I) wherein R' represents the hydroxyl of the carboxyl group of the C-terminal amino acid radical; or (d) where in the compound of formula (I) R' represents the hydroxyl of the 1 carboxyl group of the C-terminal amino acid radical, the saponification of a corresponding ester compound of formula (I); followed as appropriate by conversion of the product into the base or a salt (as hereinbefore defined) or an acid addition salt thereof.

44. A compound of formula (I) as defined in any of claims 1 to 24, when prepared by a method according to claim 43.

45. A compound of formula (I) as claimed in claim 1 substantially as hereinbefore described.

46. A pharmaceutical formulation substantially as hereinbefore described with particular reference to Examples (A) to (D).

47. A method for the preparation of a compound of formula (I) as defined in claim 1, substantially as hereinbefore described with particular reference to Examples I to 9.

48. A method for the treatment of a mammal other than man for a condition wherein an agent with a morphine-like effect is indicated, substantially as hereinbefore described with particular reference to Example 10.