PEPTIDES WITH EXTRAORDINARY OPIOID RECEPTOR SELECTIVITY

Abstract: A polypeptide of formula (I) having agonist or antagonist activity to delta opioid receptors. These compounds may be used to induce pharmacological or therapeutic effects, including analgesia, in animals.

Description: PEPTIDES WITH EXTRAORDINARY OPIOID RECEPTOR SELECTIVITY This invention relates to compounds that are rigid analogs of enkephalins having improved delta receptor specificity. This invention also relates to a method of inducing pharmacological anifestions associated with delta receptor agonist and antagonist activity / such as analgesia/ by administering a safe and effective amount of the delta receptor specific compounds. Opioid analgesics are narcotics useful for treating moderate to severe pain and also useful in the treatment of diarrhea and coughing. Morphine/ a plant alkaloid / is one " of the most commonly known opiate drugs. Serious drawbacks associated with the plant opiates include their extreme addictiveness and their inhibition of intestinal transit. Naturally occurring opiates/ known as "enkephalins " are found in the human brain/ as well as in various tissues of lower animals. Naturally occurring enkephalin is a mixture of two pentapeptides and is part of a larger class of opioid peptides known as "endorphins. " A considerab amount of research has been conducted in the hopes of producing a synthetic opiate which does not have the drawbacks associated with morphine. The mechanism of the action of such opiates has onl recently begun to be understood. The key to understanding that mechanism is the opioid receptor. A receptor is that entity, on a cell, which recognizes and binds a chemical substance. An opiate receptor, therefore, recognizes and binds an opiate drug. After binding with the receptor, opioi drugs may act to initiate or block various biochemical and physiological sequences. Such initiation or blockage is oft referred to as transduction. It has been found that there are several types of receptors which are affected by opioids. The major known types of opioid receptors are the mu, delta and kappa receptors. All three receptor types appear to mediate analgesia, but differ considerably in their other pharmacological effects. For examples, mu receptors additionally mediate respiratory depression and inhibit gastrointestinal transit. Kappa receptors mediate sedation. While delta receptors are believed also to produce analgesia as above described, it is believed " that they do not inhibit intestinal transit in the manner associated with mu receptor The biological activity and binding properties of opioids ar directly linked to the opioid structure. Opioid compounds structurally capable of binding a receptor sites may have a variety of biological effects, all of which are useful in attaining a variety of pharmacologica and therapeutic effects. Certain opioids, known as "agonists", inhibit certain electrically stimulated outputs neurotransmitters in tissues containing receptors, and, for example, may inhibit electrically stimulated contractions an other responses. Morphine is an agonist and acts to inhibi transmissions associated with pain and gastrointestinal tra contractions. It is also known that other substances, know as "antagonists", prevent the action of agonists by binding the receptor without inhibiting electrically stimulated outputs in the manner associated with agonists. Naloxone i an antagonist and acts to prevent an agonist from binding a the receptor. Additionally, some substances act as either partial agonists or partial antagonists. Naturally occurring opioid analgesics, known as endorphins, particularly enkephalins, have been extensively studied. The research began with the isolation of naturally occurring enkephalin, which is a mixture of methionine enkephalin (H N-Tyr-Gly-Gly-Phe-Met-OH) and leucine enkephalin (H-N-Tyr-Gly-Gly-Phe-Leu-OH) . Subsequent to the isolation of naturally occurring enkephalin, synthetic enkephalins were produced which displayed the full spectrum of enkephalin like opioid effects. Before proceeding further, it is necessary to explain briefly the terminology used to describe polypeptides. Peptides are identified by amino acid sequence using established abbreviations. For example, as used herein, "Gly" stands for glycine, "Leu" stands for leucine, "Tyr" stands for tyrosine, "Pen" stands for penicilla ine, "Cys" stands for cysteine, "Phe" stands for phenylalanine, "Thr" stands for threonine and "Met" stands for methionine. Polypeptide derivatives in which one or more of the amino acids has been replaced by another amino acid are often described by reference to the basic compound and the position and nature of the substitution. The position of substitution is usually identified by reference to the number of the amino acid in the sequence starting with the amino acid at the amino terminus of the peptide chain. For example, H_N-Tyr-Gly-Gly- 5 ** • Phe-Pen-OH is written as ([Pen ]enkephalin) signifying that penicillamine has been substituted for the leucine or methionine normally forming the fifth amino acid from the amino terminus in enkephalin. Additionally, amino acids may exist as stereoisomers in both L and D configurations. The large scale use of synthetic enkephalins has been impractical due to various difficulties. One of the difficulties associated with enkephalins is that they are extremely unstable and their half-lives in the blood are extremely short. Secondly, enkephalin-like peptides are known not to cross the blood brain barrier easily. They are, however, known to cross the placental barrier and cannot, therefore, be used as analgesics during pregnancy and in childbirth without affecting the unborn child. Attempts at solving these problems focused on altering the structure of the enkephalin molecule. Alterations in the enkephalin structure produce differing pharmacological effects. Each enkephalin analog has fairly selective effects in different systems. Specifically, it has been found that different enkephalin analogs bind to differen opioid receptors. However, it has been difficult to study th role of each receptor type or to induce selectively the pharmacological and therapeutic effects associated with each receptor type because the enkephalin analogs, to date, have not had a high degree of selectivity for a single-receptor . type. Recently, it has been shown that a certain enkephalin analog is highly specific to the mu receptor. See Handa, B. K. , Lane, A. C., Lord, J. A. H., Morgan, B. A., Ranee, M. J. , and Smith, C. F. C. , Eur J. Pharmacol, 70: 531-540(1981); Kosterlitz, H. W., and Paterson, S. J. , Br. J. Pharmacol, 77: 461-468 (1982); and, Gillian, M. G. C. , and Kosterlitz, H. W. , Br. J. Pharmacol. 73:299P (1981), all of which are specifically incorporated herein by reference. Receptor specificity has also been achieved by conformationally constraining the enkephalin peptides. Examples of such constraints include alpha or N-methylation o the peptide backbone or cyclization. U.S. Patent No. 4,148,786 to Sarantakis, which is specifically incorporated herein by reference, discloses a cyclic polypeptide having the following formula: in which R is hydrogen, lower alkyl, allyl, 2-isopentenyl, 3-isopentenyl, cyclopropylmethyl, cyclobutylmethyl, phenethyl or arginyl; R_ is hydrogen or lower alkyl; R, is hydrogen or lower alkyl; R 4 is hydrogen, hydroxymethyl, carbo (lower)alkoxy, carbamyl or carboxy; and, X is hydrogen, chloro, fluoro, bromo or iodo, the linear precursors thereof or a pharmaceutically acceptable salt thereof. The compounds disclosed by Sarantakis are said to exert 'an analgesic effect in warm-blooded animals when peripherally administered. However, the Sarantakis compounds are not disclosed as specific to any receptor type. Until now, few enkephalin analogs have been developed which react specifically with the delta receptor. U.S. Patent No. 4,518,711 to Hruby, et al. which is also specifically incorporated herein by reference discloses cyclic polypeptides of the formula: wherein R 1 and R2 which may be the same or different, are hydrogen, methyl or lower alkyl; R 3 and R4 which may be the same or different, are 1 * 3 A hydrogen, methyl or lower alkyl, provided R , R , R and R may not all be hydrogen when both n and m are 0; R is hydrogen, L-tyrosine, D-tyrosine, or L-tyrosine or D-tyrosine substituted on the N- amino with 1 or 2 lower alkyl or alkenyl groups; R is a substituted or unsubstituted aromatic; R is hydrogen or methyl; R is carboxylate, carboxamide or amino acid residue; X and Y are hydrogen or methyl; and n and are each 0 or 1. The compounds therein are said to possess either -^ σ agonist or antagonist activity to opioid receptors, and may be used to induce pharmacological or thearapeutic effects, including analgesia, on humans and other animals. dimethylcysteine. These diastereomeric enkephalin analogs have the structure: .S 20 C(CH 3 ) 2 C ( CH 3 ) 2 Tyr-NH-CH-CO-Gly-Phe-NH-CH-COOH These compounds are said to bind to the/opioid receptor, Mosberg, et al. , in Life Science, 32, 25 2565-2569(1981) disclose that cyclic enkephalin analogs, s s I ) H-Tyr-D-Pen-Gly-Phe-D-Cys-NH_ [D-Pe rn~2 — , D-Cy 1 s 5 j enkephalinamide , 30 H-Tyr-D-Pen-Gly-Phe-L-Cys-NH- S- -S [D-Pe 1 n 2 ,L-Cy * s 5 ] enkephalinamide , 35 H-Tyr-D-Pen-Gly-Phe-L-Cys-OH I I s s ([D-Pen 2, L-Cys5]) enkephalin; and H-Tyr-D-Pen -Gly-Phe-D-Cys-OH s _ s 2 5 [D-Pen -D-Cys ] enkephalin, bind to opioid receptors. The present invention provides novel compounds which are capable of binding with enhanced specificity to the delta receptor (hereinafter sometimes referred to as "delta receptor specificity") . The compounds are a series of cyclic, conformationally restricted analogs of enkephalins which display exceptional delta receptor specificity. The novel compounds include those which function either as agonists or antagonists and may be used to induce pharmacological or therapeutic effects corresponding to agonist or antagonist activity in humans and other animals. A particularly preferred group of compounds are delta receptor agonists and may be used to induce analgesia in humans and lower animals without significant involvement of mu receptors and their associated side effects. Those compounds within the scope of the present invention which function as delta receptor antagonists may be used to block the action of delta receptor agonists prepared in accordance with the present invention when necessary or desirable or may be used to induce other pharmacological and therapeutic effects of opioid antagonists, such as in treatment of Alzheimer's Disease. In accordance with the present invention, there are provided polypeptides of the formula: 1 and pharmaceutically acceptable salts thereof wherein; 1 2 7 8 RR , R , R and R are each independently hydrogen or lower alkyl R 3 and R4 fere each independently hydrogen or lower alkyl; R and R are each independently hydrogen or lower alkyl, provided that at least one of R , R , R and R is other than hydrogen when both n and p are zero; 9 R is lower alkyl or hydrogen; 0 R is hydroxy, lower alkoxy, amino, loweralkyl- amino, lower dialkylamino; R 11, R12 and R13 are each independently hydrogen or lower alkyl; R 14 is hydrogen, lower alkyl or lower alkanoyl; - xz B is Gly or a chemical bond; X is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, carboxy, lower carbalkoxy, carbamoyl, loweralkyl- amino carbonyl, lowerdialkylaminό carbonyl, lower alkoxy, amino, halo, nitro, cyano, lower alkanoyl or formyl; 2 Q Y is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, carboxy, lower carbalkoxy, carbamoyl, lower alkylamino carbonyl, lowerdialkylamino carbonyl, lower alkoxy, chloro bromo, iodo, nitro, cyano/ lower alkanoyl or formyl / provided that X and Y are not both hydrogen, 2 -simultaneously and m, n, p and q are each independently 0/ 1 or 2 As used herein, lower alkyl groups, either singly o in combination with other groups contain up to 6 carbon atoms which may be in the normal or branched configuration and 30 include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, a yl, pen yl, hexyl and the like. The preferred alkyl groups contain 1 to 3 carbon atoms. A specially preferred alkyl group is methyl. 35 -_ Lower alkenyl and alkynyl groups contain from 2 to 6 carbon atoms which may in the normal or branched configuration. Lower alkenyl groups include ethenyl, propenyl, butenyl, isobutenyl, pentenyl and the like. Lower 5 alkynyl groups include ethynyl, propynyl, butynyl, isobutynyl, pentynyl, isopentynyl, and the like. Halo as used herein, is bromo, fluoro, iodo and chloro. Alkanoyl as used herein is a lower alkyl group • J_Q containing a carbonyl group on the main chain. The carbonyl group may be at the end or in the middle of said chain. The preferred alkanoyl group is acetyl. - It is preferred that m, n, p and q are zero. When 1 2 7 8 present, R , R , R and R may be the same or different. The 1 2 7 8 * preferred values of each of R , R , R and R are independently hydrogen or methyl. It is especially preferred 1 2 7 8 that, when present, R , R , R and R are hydrogen. The preferred values of R ., R , R and R are hydrogen or methyl. It is especially preferred that R , R , 7 8 2o R an d R are all methyl. Preferred values of R are hydrogen or methyl. It is preferred that R is hydroxy. Preferred values of R is hydrogen or methyl. Hydrogen is especially preferred. 25 H Hyyddrrooggeenn oorr mmee' 12 thyl is preferred for R . Hydrogen is especially preferred. The preferred value of R is hydrogen or methyl. 14 It is preferred that R is hydrogen or acetyl. It is preferred that B is Gly. Q Preferred values of X include methoxy, iodo, bromo, amino, chloro, nitro and hydrogen. Preferred values of Y are iodo, chloro, bromo, methoxy, nitro and hydrogen. 35 A preferred embodiment of the present invention has the formula: where n R 3 , R 4 , R 6 , R" R 10 ,11 ,12 R 13 R 14 B, X and Y are as defined heretofore. Especially, preferred compounds of the present invention have the formula: wherein; X is methoxy, iodo, bromo, chloro, nitro, amino or hydrogen ; Y is iodo, chloro, bromo, methoxy, nitro or hydrogen ; I R 13 is hydrogen or methyl; R 14 is hydrogen, lower alkanoyl or alkyl and B is Gly or a chemical bond. In the above formulae, all of the amino acid residues, except Gly, are in the L-configuration except for the penicillamine at position 2 and 5 and the amino acid in position 4 (which may be phenylalanine or derivatives thereof or homophenylalanine or derivatives thereof, etc.) . The amino acid in position 4 may be in either the D or L configuration. The penicillamine in position 2 is in the D-configuration while the penicillamine in position 5 can be D or L. It is preferred that the pencillamine in position 5 be in the D-configuration. Preferred species of the present invention are: p-Cl-Phe 4 ]DPDPE p-I-Phe 4 ]DPDPE 3 , -N0 2 -Tyr 1 ]DPDPE 3'-_I-_ Tyr 1 ]D p D p E wherein , i.e., The superscript adjacent to the amino acid refers to its position in the polypeptide relative to the amino terminus. For example, Tyr is" the first amino acid, pencillamine is second, glycine is third, phenylalanine is fourth and pencillamine is fifth. The compounds of the present invention include both agonists and antagonists. The present invention is also directed to a process for inducing analgesia in human and lower animals by administering a safe and effective amount of an opioid receptor agonist with delta receptor specificity. The compounds which are agonists may be useful as analgesics without producing the undesirable side effects associated with previously known opioids. It is believed tha the agonists of the present invention are useful in pregnancy and child birth because they will not cross the placental barrier, and therefore, they will not harm the unborn child. The antagonist compounds may also be useful in the treatment of schizophrenia, Alzheimer's disease, as well as in the treatment of respiratory and cardiovascular functions. Specifically, the polypeptides of the present invention are cyclic opioid compounds having greatly improved specificity for the delta opioid receptor. The increased specificity is believed to be due to the increased structural rigidity of the claimed compounds. The structural rigidity appears to stabilize the three-dimensional conformation required for activity at the delta opioid receptor, but appears to exclude the optimal conformation required for activity at the mu receptor. In a preferred group it is believed that particularly increased specificity is attributed to the presence of geminal dialkyl substituents on the peptide ring and, in a particularly preferred ή A embodiment, to the presence of β, -dialkyl substituents in the sulfur-containing residues at positions 2 and/or 5 of th peptide. Such geminal dialkyl groups, combined with the S-S bridge, produce enkephalin analogs which are conformationall stable. Thus, these compounds possess the three dimensional conformation required for activity at the delta receptor and at the same time exclude the optimal conformation required for activity at the mu receptor. For example, increased rigidity and thus increased delta receptor specificity can be conferred on the peptides disclosed by Sarantakis if the half-cysteine in the 2 position is replaced by half-penicillamine ( β ,p -dimethyl half-cysteine) , preferably the D-isomer. The half-penicillamine enkephalin analog can have as much as a six hundred fold increased selectivity in delta receptor specific assays, compared to activity in mu receptor specific assays. The geminal dialkyl substituted compounds of the present invention also display increased delta receptor specificity in rat brain binding assays. A unique feature of some of the preferred enkephalin analogs of the present invention is the incorporation of a half-penicillamine amino acid residue into the two and/or five position of the enkephalin. Penicillamine is beta,beta-dimethylcysteine. These compounds share the common feature of having at least one pair of geminal dialkyl groups in the ring. These dialkyl groups impose a particularly high degree of conformational restriction and steric hindrance resulting in particularly high delta receptor specificity. It is also believed that unfavorable steric and/or polar-non-polar interactions between the fi r β -dialkly groups and the mu receptor binding site contributes to the observed delta receptor specificity. By altering the various amino acids, differing pharmacological properties are obtained. An agonist will inhibit an electrically stimulated contraction in smooth muscle and an antagonist will reverse the inhibition caused by an agonist. Whether a particular compound of the present invention is an agonist, partial agonist, antagonist or partial antagonist can be determined by routine experimentation by those skilled in the art in light of the teachings contained herein. However, the compounds of the present invention are believed to share the property of having enhanced specificity for the delta receptor. Modifications in ring size will also produce varying pharmacological and biological effects. For example, it is possible to substitute D- or L-homocysteine for the amino acids in either the two or five position of the polypeptides of the present invention. Similarly, L-homocysteine may be substituted for pencillamine, or cysteine in the two or five position of the polypeptides of the present invention. All of these modifications are within the scope of the invention. The compounds of the present invention can be prepared by art recognized techniques. An examplary procedure is as follows. The polypeptide of Formula VI depicted hereinbelow: 1 14 wherein X,Y, B, R -R , , n, p and q are as defined hereinabove, is reacted with an oxidizing agent, such as K 3 [Fe(CN)g] under disulfide bond forming conditions to form a polypeptide of Formula I. More specifically, the disulfide bond can be formed by dissolving the linear peptide in previously degassed water at a basic pH, e.g., pH 8.4 (which can be achieved by adjusting the pH with aqueous ammonia) . The solution is titrated with the oxidizing reagent, such as K 3 [Fe(CN) g ] . Next the pH is adjusted to 5 with acid, e.g., glacial acetic acid. The crude product is then purified by techniques known to one skilled in the art. For example, an anion exchange resin e.g., 15 L of Amberlite IRA-45, is added to remove the excess ferri- and the ferrocyanide and the mixture is filtered and lyophilized. The crude product is then desalted on a Sephadex G-10 column with 15% acetic acid. Further purification can be made on RP-HPLC [Perkin Elmer, Vydac 218TP1010 C18 reverse phase column (25 cm x 1 cm) using a gradient of 20-40% acetonitrile in 0.1% trifluoroacetic acid 1%/min, at a flow rate of 4 ' mL/min with UV detection at 214 or 280nm. Compounds of Formula VI can be prepared by art recognized techniques. The polypeptide of Formula VI can be prepared under peptide forming conditions from the amino acid moieties depicted hereinbelow: O II H IV R " V wherein B, X, Y, R1-R14 , m, n, p and q are as defined hereinabove. In the following scheme, the various amino acid moieties will be indicated by I, II, in, iv or V. c SCHEME V-A + Z-IV -Z IV-V-A ;-III -z III-IV-V-A +Z-II -z II-III-IV-V-A + Z-I 0 -Z -A I-II-III-IV-V In the scheme hereinabove, Z is an amino protecting 5 group and A is a carboxy protecting group. The various protecting groups that can be employed may be found in "Protective Groups in Organic Synthesis" by T. W. Green, John Wiley and Sons 1981. It is preferred that Z is benzyloxycarbσnyl or N-t-butyloxycarbonyl and that A is : ^ benzyloxy carbonyl. As indicated in the scheme, subsequent to each coupling thereof the amino protecting group from the growing polypeptide chain is removed by techniques known to one skilled in the art. After the polypeptide is synthesized, the amino and carboxy protecting groups are then removed by techniques known to one skilled in the art. For example, if A is benzyloxy carbonyl, it can be removed by catalytic hydrogenation. In the case when Z is benzyloxy carbonyl, it can b removed by catalytic hydrogenation or by acid reagents, such 3 as HBr/HOAc, HF/Pyr, and the like. Protecting groups can also be used if the 14 substituents X, Y or R are reactive under peptide forming conditions. Again, reference is made to "Protective Groups Organic Synthesis" referred to hereinabove forϊa discussion 35 the various protecting groups that could be employed. As shown by the scheme, the amino acids are added sequentially, one at a time, to form a larger molecule. More specifically, an amino protected amino acid is added to a carboxy protected amino acid to form a peptide consisting of two amino acid moieties. A protected amino acid is then added to the protected peptide, thereby forming a dipeptide. This process is continued until the polypeptide of Formula VI is formed. Coupling of the compound in each step of the Scheme employs established techniques in peptide chemistry. One such technique uses dicyclohexylcarbodimide (DCC) as the coupling agent. The DCC method may be employed with or without additives such as 4-dimethylaminopyridine, HOBt or copper (II) . The DCC coupling reaction generally proceeds at room temperature, however, it may be carried out from about -20° to 50°C. in a variety of solvents inert to the reactants. Thus, suitable solvents include, but are not limited to, N,N-dimethylformamide, methylene chloride, toluene and the like. Preferably the reaction is carried out under an inert atmosphere such as argon or nitrogen." Coupling usually is complete within 2 hours but may take as long as 24 hours depending on reactants. It is preferred that X and Y be present on the amino acids prior to the coupling. X and Y can be added to the appropriate amino acid moieties by techniques known to one skilled in the art, such as electrophilic aromatic addition, e.g, nitration, halogenation, Friedel Crafts alkylation and acylation, and the like. For example, the p-nitro-^- "ethylphenylalanine isomers may be prepared by nitration of erythro and threo S-MePhe, which can be synthesized and separted by Kataoka, et al. , Bulletin Che . Soc. of Japan, 49, 1081-1084 (1976) , which is incorporated herein by reference. It is preferred that the polypeptides be synthesized by solid phase peptide synthesis. Said synthesis takes place on a solid phase matrix, such as chloromethylated polystyrene crosslinked with 1-2% divinylbenzene. The N-protected amino acid at the C-terfninal end, i.e., Z-V can be attached to the resin using the procedure disclosed by Gisin, Helv. Chim, Acta, 56 , 1476 (1973) , which is specifically incorporated herein by reference. The Z-V-resin is placed into a solid phase peptide synthesis reaction vessel and Z-IV, Z-III, Z-II and Z-I are added sequentially under solid phase peptide synthesis conditions. In each step, deprotection can be accomplished by trifluoroacetic acid and anisole in dichloromethane . This is followed by neutralization of the resulting peptide with 10% diisopropylethylamine . This peptide is then coupled with another N-protected amino acid moiety, and the process is continued until the desired polypeptide of Formula VI is synthesized. Upon completion of the solid phase synthesis the polypeptide may be deprotected by techniques known to one skilled in the art. For example, the peptide resin may be washed with dichloromethane, ethanol and DCM and dried. The peptide resins then are cleaved by liquid HF in the presence of anisole. The solvents are evaporated off, the dried product is washed with ethyl ether and the peptide is extracted with glacial acetic acid, 30% acetic acid and 10% acetic acid and filtered and lyophilized, thereby rendering a compound of Formula VI, wherein R is hydroxy, and then converted to the disulfide form as previously discussed. The resulting polypeptide where R is hydroxy can then be transformed to the other polypeptides of Formula VI, wherein R is amino, alkoxy, alkylamino or dialkylamino by techniques known to one skilled in the art. For example, the polypeptides wherein R is alkoxy can be formed under 1 Fischer esterification conditions from the corresponding acid. Alternatively, if R is methoxy or ethoxy, diazomethane or diazoethane, respectively, synthesized in situ can be used. Formation of compounds wherein R 10 is amino, *a ~ ~lkylamino or ~ > dialkylamino can be formed by reacting the ester with ammonia, N-alkylamine or N, N-dialkylamine to form the corresponding unsubstituted, N-substituted or N,N,-disubstituted amide. Purity of the products described hereinabove can be assessed by TLC on silica gel plates (Merck, ieselgel F-254, 5 x 20 cm) and the spots can be detected by UV, ninhydrin or iodine vapor. The following systems may be used; a:) butanol-acetic acid-water (4:1:1) b) butanol-acetic acid-pyridine-water (13:3:12:10) c) isopropanol-ammonium hydroxide-water (3:1:1) d) butanol-acetic _~ ~*. acid-ethyiacetate-water (1:1:1:1). Analytical HPLC can be performed using Vydac 218TP1010 C18 column (25 cm x 1 cm) under isocratic conditions using 25% acetonitrile in 0.1%. trifluoroacetic acid at a flow rate of 4 mL/min with UV detection at 214 or 280 nm. For quantiative amino acid 0 analyses, peptides (ca. 0.5 mg) can be hydrolyzed with constant boiling HCl (0.5 mL) in evacuated and sealed ampoules for 24 h at 110°C. The analyses are performed on a Beckman 20C amino acid analyzer. The present new compounds contain basic nirrogen an *-' can form salts with acids. All such acid salts are contemplated by the invention but especially preferred are salts with pharmaceutically acceptable acids, such as hydrochloric, sulfuric, nitric, toluenesulfonic, acetic, propionic, tarraric, malic and similar such acids well known -- in this art. In addition, quaternary salts can be formed using standard techniques of alkylation employing, for example, hydrocarbyl halides or sulfar.es such as methyl, ethyl, benzyl, propyl or allyl halides or sulfates. 35 The compounds of the present invention can be administered to the host in a variety of forms adapted to the chosen route of administration, i.e., orally, intravenously, intramuscularly or subcutaneous routes. The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains an amount of active compound ranging from about 1 ug/kg of body weight to about 1000 ug/kg of body weight. Preferred dosage of active compound ranges from about 1 ug/kg of body weight to about 20 ug/kg of body weight. The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size, in the case of dispersions, and by the use of surfactants. The prevention of the action of micro-organisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof. The following examples further illustrate the invention. EXAMPLE 1 I 2 ' 5 [D-Pen , D-Pen ] enkephalin (DPDPE. 1) was prepared by the methods described by Mosberg, et al. in PNAS _80_, 5871 (1983) . EXAMPLE 2 " f " ~2 ;— 2 " I 5~ [D-Pen , p-F-Phe , D-Pen ] enkephanin (2) . The title compound was prepared by the solid phase method as outlined above starting with 1.17 g of Na,-Boc-D-Pen- (S-pMB) -resin (substitution = 0.86 mmol/g resin, 1.0 mmol) and the following protected amiήo acids were added stepwise to the growing peptide chain: N -Boc-p-F-Phe; ^-Boc-Gly; N a -Boc-D-Pen (S-pMB) ; N^-Boc-Tyr. There was obtained 1.57 g of N^-Boc-Tyr- D-Pen (S-pMB) -Gly-p-F-Phe-D-Pen(S-pMB) -0-resin, which was treated with 15 mL of HF containing 1.5 mL of anisole for 60 min. at 0°C. The HF was rapidly removed by vacuum aspiration at 20°C. The mixture was washed with 3 x 60 mL of ether, and the peptide extracted with 40 mL glacial acetic acid and washed with 3 x 30 mL of 20% aqueous acetic acid. The peptide solution was lyophilized and the residue dissolved in 1,500 mL of deaerated 0.1% aqueous acetic acid. The pH was adjusted to 8.4 with 3N ammonium hydroxide and 150 mL of 0.01 N K,Fe (CN) c 3 6 was added. After 120 min the pH was raised to 4.5 with glacial acetic acid the ferro- and excess ferric-cyanide removed by the anion ion exchange resin Amberlite IRA-45 (Cl~form) . After stirring for 20 min, the resin was filtered off and the resin washed with 3 x 30 mL of 30% aqueous aqueou acetic acid. The solution was evaporated down to 150 mL on a rotary evaporator in vacuo at 25°C and lyophilized. The residue was dissolved in 5 mL of 15% aqueous acetic acid and gel filtered on a 50 x 3.2 cm column containing Sephadex G-10. The major peak was isolated and lyophilized. The powder obtained was dissolved in 2 mL of 20% acetonitrile in 0.1% trifluoroacetic acid and purified on a Vydac. 218TP1010 C18 RP-HPLC column (25 cm x 1 cm) . Conditions: linear gradient elution starting with 20% CH_.CN in 0.1% TFA, 1%/min for 20 min, at a flow rate of 4mL/min. The more lypophilic impurities were washed from the column with 80% CH 3 CH in 0.1% TFA for 5 min., nd after equilibrium (5 min. 20% CH-CN) the column was ready to use. The major peak was isolated as a white powder (yield - 70 mg) . Amino acid analysis: Gly 1.00 (1.00); Tyr, 0.93 (1.00); p-F-Phe 1.01 (1.0) . The analytical data for the purified product 2 is given in Table 1. EXAMPLE 3 • _ ' i [D-Pen , p-Cl-Phe , D-Pen J ] enkephalin (3) . The title compound was prepared as for 2 above except that N* 1 -Boc-p-Cl-Phe was added to the growing peptide chain instead of N a -Boc-p-F-Phe. There was obtained 1.5 g of N^-Boc-Tyr- D-Pen(S-pMB)-Gly-p-Cl-Phe-D-Pen(S-pMB)-0-resin. The peptide resin was treated with HF as before, the peptide isolated and oxidized to the cyclic structure, and then purified as for 2 above. There was obtained 65 mg of the title compound 3 as a white powder. Amino acid analysis: Gly 1.00 (1.00) ; Tyr, 0.94 (1.00) ; p-Cl-Phe, 1.09 (1.00) . The analytical data for the purified product 3 is given in Table 1. EXAMPLE 4 [D-Pen , p-Br-Phe^, D-Pen ] enkephalin (4) . The title compound was prepared as for 2 above except that N* -Boc-p-Br-Phe was used in place of N -Boc-p-F-Phe in synthesizing the growing peptide chain. There was obtained 1.3 g of N*-Boc-Tyr-D-Pen ( S-pMB ) -Gly-p-Br-Phe-D-Pen(s-pMB)-0- Resin. The peptide resin was treated with HF as before, the peptide isolated and oxidized to the cyclic structure, and then purified as for 2 above. There was obtained 43 mg of the title compound 4 as a white powder. Amino acid analysis: Cly, 1.00 (1.00); Tyr, 0.89 (1.00); p-Pr-Phe, 0.95 (1.00) . The analytical data for the product 4 -is given in Table 1. TΛΠΓΛ. l The available purity data are summarized in Table 1. Table 1. Purity of synthetic peptides TLC' i IIPLCb FARMS 'eptides I II III IV V Calc. Found [M+l] + [M+l] + 1 Tyr-D-Pen-Gly-Phe-U-Pcn 0.13 0.66 0.06 0.01 0.91 2 Tyr-D-PeM-Gly-p-F-Phe-O-Pen 0.-15 0.66 0.91 0.03 1.23 664 664 3 T r-jl-Pen-Glγ-p-CI-Phe-lM'en 0.40 0.67 0.92 0.04 2.36 600 600 4 Tyr- -Pen-Gly-p-Gr-Phe-D-Pen 0.54 0.57 °- 92 0.70 2.00 725 724,726 5 Tyr-ID-Pen-Gly-p-I-Phe-D-Pen 0.49 0.60 0.93 0.05 3.00 722 772 a) Merck DC-Fertiplatten iesolgol 60r254 0.25 mm analytical silica gel plates v/ere used. Solvent systems are as follows: I. butanol/acetic acid/water 4 : 1 : 1; II. butanol/acetic acid/piridine/water 13 : 3 : 12 : 10; III. isopropanol/anmionla/water 3 : 1 : 1; IV. butanol/acetic acld/ethylacetate/water 1 : 1 : 1 : 1 b) capacity factor for the following system : Vydac 210TP1011 C\Q reversed phase (RP) column (25 x 1 cm) with 0.1% tri luoroacetic acid/CI^CN (75/25 v/v) and at a flow rate 4 ni /min. All pejxLides were monitored at λ = 214 and 200 run. (S,S) [p-NQ 2 - -MePhe 4 ]DPDPE and (R,R) [p- O - ^- MePhe ]DPDPE. Using 1.75 g/1.5 mraol of N -Boc-S-pMB-D-Pen- resin, two cycles of deprotection, neutralization and coupling were performed with racemic N^-Boc- (S,S) (R,R) -p-NO--/■*•* • '- * MePhe-OH (m : 143°C) , N^-Boc-Gly-OH, N* " -Boc-S-pMB-D-Pen-OH and N -Boc-Tyr-OH using the procedure above yielding 2.43g of protected peptide-resin. After cleavage of peptide from the resin, the linear crude peptide was cyclized by K-.[Fe(CN) g ] as described above. The crude peptide was purified by gel fitration on a Sephadex G10 column with 15% acetic acid. The ma- or peak was pooled, lyophilized to give a white powder. Part of it (100 mg) was purified by semipreparative RP-HPLC using Vydac 218TP1010 C18 column (25 cm x 1 cm) using a combination of isocratic and gradient chromatography. Conditions were: isocratic elution with 25% CH..CN in 01.% trifluσroacetic acid for 20 min at a flow rate of 4 mL/min at 280 nm, followed by linear gradient elution 10%/min from 20 min to 25.5 min, continued isocratic elution with 80% CH-CN i 0.1% TFA for 5 min and linear gradient elution from 80% to 25% CH-CN in 0.1% TFA for 2 min. After 5 min equilibrium time th column is ready to run a new sample. The column was loaded b ca. 5 mg of crude peptide/injection. Two main peaks were collected corresponding to the two diastereoisomeric peptides After lyophilization 18 mg and 23 mg of the pure peptides wer obtained respectively. The determination of optical pure enantiomers were carried out by enzymatic methods. 1 mg peptide corresponding to first or second peak was hydrolyzed with 6 N HCL for 24 hrs at 110°C. The HCL solution was removed in vacuo and the residue was dissolved in 200 jil of ' water. 100 Λil of this solution was used for amino acid analysis, the other 100 μ. of solution was diluted with 100 ι of TRIS buffer, pH=7.2. 5 mg (2,25U) of L-amino acid oxidase (Sigma) was added and the mixture was incubated for 24 hrs at 37°C. A second part of enzyme (2.5 mg) was added and the incubation was continued for an additionatl 24 hrs. The sample was diluted with citrate buffer, pH=2.2 (Beckman) for 1 mL. Amino acid analyses were performed upon the samples treated or not treated with enzyme. In the sample treated with enzyme from the first peak p-NO--^ -MePhe couldn't be detected by amino acid anaylsis; that means the L-amino acid oxidase enzyme decomposed this amino acid, therefore this peptide consisted of ' the (S ,S) p-NOj ? -MeP the peptide from the second peak was (D isomer) . " Analytical data for (S , : TLC: R.A: 0.46; R-B : 0.64; R^C: 0.S1 ; R-D: 0.82; HPLC: J. t . f K'-1.59; FABMS: MH 705, found 705, H-NMR (500 MHz, DMSO-d,) _ b Tyr 9.4 (H, NH , m) , 4.25 (H, H* , m) , 2.95 (H, H^ , dd) , 2.75 (H, Wβ, dd) ; D-Pen 2 8.53 (H, NH , d) , 4.5 (H, H A , d) , 1.25 (3H, CH 3 , s) , 0.96 (3H, CH 3 , s); Gly 3 ^8.51 (H, NH, t) , 4.13 (K, H a , dd) , 3.10 (H, Ha,, dd) ) ; (S,S) p-N0 2 ~ -MePhe 8.30 (K, NH, d) , 4.31 (H, & , d,dd) , 3.43 (1H, K^, dd) , 1.18 (3H, CH 3 , dd) ; D-Pen 5 $ 7.45 (H, NH, d) , 4.18 (H, H* , d) , 1.22 (3H, CH, , *-*> S) , 1.06 (3H, CH 3 , S) . Analytical data for (R,R) [p-NO_- A-Me-Phe 4 ]DPDPE: TLC: R A; 0.41; R-B: 0.61; R_C: 0.81; R..D: + 1 1 0.82; HPLC: K'=3.66; FABMS: MH 705, found 705; H-NMR (500 MHz, DMS0-d 6 ) ; Tyr 1 cf 9.33 (H, NH, m) , 4.10 (H, H* , ) , 2.84, 2.81 (2H, H , dd) , D-Pen 2 (£8.60 (H, NH, d) , 4.36 (H, H A , d) , 1.24 (3H, CH 3 , ε) , 0.96 (3H, CH 3 , s) ; Gly 3 £ 8.44 (H, NH , d) , 3.65 (H, E ά , dd) 3.51 (H, H , dd) ; (R f R)p-NO,-U -MePhe 4 7.62 (H, HN, d) , 4.76 (H, Hβ , ddd) , 3.46 (H, H/ , dd) , 1.21 (3H, CH.,, s)) : D-Pen 5 £7.78 (H, NH, d) , 4.24 (H, H A , d) 1.24 (3H, CH s) , 0.96 (3K, CH. s)) chain following the protocol outlined above. The peptide wa cleaved from the resin by HF, the cyclization of linear peptide was performed in the same way as described above. T desaltation of crude peptide was carried out by gel filtrati on Sephadex G-10 column. The final purification of the diastereomer peptide was performed by semipreparative RP-HPL using Vydac 218TP1010 C18 column (25 cm x 1 cm) ; combining t isocratic and gradient chromatography. Conditions were: isocratic elution with 23.5% CH 3 C in 0.1% TFA for 20 min. a a flow rate of 4 ml/ in at 280 run, the washing procedure of column was the same as above in the purification of (S,S) an (R,R) DPDPE. Two major peaks were collecte and lyophilized. The determination of configuration of p- O f. -Me-Phe in the optical pure peptide was performed by enzymatic method, similarly to those previously described. The peptide from first peak was the (S,R) [p-N0 2 ~ -MePhe J DPDPE (L isomer) , the peptide from the second peak was the (R,Ξ) [p-N0 -^ Me-Phe 4 ) DPDPE (D isomer). - ** ' 1 Δ Analytical data for (S,R,)p-N0 2 - p -MePhe ]DPDPE: TLC: .R f A: 0.48; R f B: 0.53, R f D: 0.75; HPLC: K'=1.98; FABMS: [M+l] + 705, found 705; I H-NMR (500 MH -£ * , ) Tyr 1 9.32_- (H, NH, m) , 4.23 (H, E f ) , 2.93, 2.74 (2H, E , dd) ; D-Pen $8.64 (H, NH, d) , 4.56 (H, H^, d) , 1.34 (3H, CH 3 , s) 1.02 (3 CH 3 , s), Gly 3 8.63 (H, NH, dd) , 4.47 (H, H , dd) , 3.24 (H, r d d ) ; ( S ,R) -p-N0 2 - -MePhe 4 8.68 (H, NH, d) , 4.42 (H, H , ddd) , 3.57 (H, H , dd) , 1.28 (3H, CH 3 , dd) ; D-Pen 5 ζ 7.3 (H, NH, d) , 4.23 (H, Hβ, d) , 1.34 (3H, CH 3 , s) , 1.02 (3H, CH.- , Analytical data for (R,S) [p-N0 2 ~p -MePhe 4 ] DPDPE: TLC: R f A: 0.48, R f B: 0.53, RfD: 0 .75; HPLC: K > _= 2 . 56 . !_ NMR (500 MHz, DMSO-d 6 ) : Tyr A J 9 .34 (II, NH, m) , 4.09 (H, H_< , m) , 2.88, 2.80 (2H, H β , dd) ; D-Pen 2 ^ 8.10 (H, ^I, d) , 4.39 (H, H., d) , 1.32 (3H, CH 3 , s) , 1.02 (3H, CH, , s) ; Gly 3 ,- 8.81 (H, dd) , 3.80 (H, I , dd) , 3.70 (H, H dd) ; (R, S) p-NO,- -MePhe 4 7.74 (H, NH, d) , 4.72 (H, H , ddd) , 3.56 (H, H έ , dd) , 1 21 (3H, CH 3 dd) ; D-Pen 5 ^7.75 (H, NH , d) , 4.21 (H, H , d) , 1 32 (3H, CH 3 , S) , 1.02 (3H, CH 3 , S) . EXAMPLE 8 r3-NQ 2 Tyr 1 ]DPDPE a) Nitration of DPDPE: Nitration of DPDPE was carried out by the method of Riordah, et al. in JACS, 88, 4104 (1966) which was slightly modified according to Guillemette, et al. in J. Mec. Chem. , 27, 315 (1984). DPDPE (10.4 g, 13.6 u ol) was dissolved in 5mL of a mixture of ethanol 0.01M ammonium acetate, pH=7, (1:1) and stirred at room temperature. Tetranitromethane (Sigma Chemical Corp, 120 mg, 614 umol, 45 times excess) in 5 ml ethanol was added slov/ly over 30 min and stirred for 5 hrs. The reaction was checked by RP-HPLC every 30 min, using a Vydac 218TP1Q10 C18 column (25 cm x 1 cm). Conditions: linear gradient elution, 20-35% CH^CN in 0.1% trifluoroaceric acid, 1%/min, at 280, at a flow rate of 4 mL/ in. During the reaction the removal of DPDPE and the appearance of [3-N0 * __•-Tyr ]DPDPE and the nitroform was seen. After 5 hrs th pH was adjusted to 4 with acetic acid to stop the reaction. The mixture of products was separated by RP-HPLC (see above) . [3*,5' dinitro-Tyr ]DPDPE was not detected in the reaction mix .83, R_D: 0.82; HPL 1. b) 1.17 g (1 m ol) N* * -Boc-S-pMB-D-Pen-resin was used for synthesis which was carried out similarly to those used for [p-FPhe 4 ]DPDPE. TLC: R f A: 0.4, R f B: 0.67, R f C: 0.83, R,D 0.82; HPLC: K'=2.81. FABMS: [M+1J + 691, found 691. EXAMPLE 9 [3'-NH..-Tyr 1 ]DPDPE a) Reduction of [3'-N0 2 Tyr-DPDPE by Na 2 S 2 0 4 was done using t method of Riordan, et al, in Biochimica et Biophvsica Acta, 236, 161-163 (1971) . [3'N0 2 Tyr 1 ]DPDPE (5.0 mg in 2 L of TRIS buffer (pH 8, 0.05 M) was added to sodium dithionite (5.2 mg, 30 umol, four times excess) in 0.5 L TRIS buffer. The yellow color o. 1 solution of [3'-N0 2 Tyr ]DPDPE disappeared immediately. The reaction mixture was purified by HPLC on Vydac 218TP1010 C18 column (25 cm x 1 cm) using the same condition as those used for [3'-N0 2 Tyr]DPDPE. Yield: 80%. TLC: R f A: 0.3, R-B: 0.39, R ■ _-O : 0.62; RP-HPLC: K'=0.63; FABMS: [M+l] + 661, found 661. b) Hvdroσenatxon of [3'-N0-Tvr ]DPDPE. [3'-N0 2 Tyr 1JDPDPE Δ (2.9 g, 3.6 umol) was 10 hydrogenated in the presence of 7 mg of Pd on charcoal (5%)_. for 2 hrs at about 40 p.s.i. of H_ gas. Before filtration of catalyst, 20 uL of dithioeth ane was added to the reaction mixture to protect it from oxidation and to deplace the product from adsorbing the catalyst. The catalyst was **** 5 filtered and the solution was lyopholized. The trace of dithioethane was removed by RP-HPLC using similar conditions as above. Yield: 1 mg, 33%, TLC: R-A: 0.3, R j -D: 0.60; RP-HPLC: K'=0.63. 20 EXAMPLE 10 H'l-Tyr 1 JDPDPE: a) Synthesis of [3 '-ITyr 1 ]DPDPE by SPPS. 1.61 g of N*-Boc-S-pMB-D-Pen-resin (1.5 mmol) was used for the synthesis which was carried out as described for • ^5 [p-FPhe JDPDPE. The cyclized crude peptide was purified by gel filtration on Sephadex G10 column with 30% acetic acid. The main peak was collected, lyophilized and it was purified by partition chromatography on Sephadex G-25 block polymerization column using n-butanol-toluene-pyridine-acetic acid-water (6:3:0.135:0.135:8.55) upper phase as an eluent. The first peak was polymer, the second peak was the [3'-I- 35 Tyr 1 jDPDPE. TLC: R f A: 0.47, R^B: 0.67, R f C: 0.89, R D: 0.84; 1 RP-HPLC: K'=2.74. FABMS: [M+1J 772, found 772. b) Iodination of DPDPE The iodination of DPDPE was carried out by the Hunter-Greenwood method described in Nature, 194 , 495 (1962) 5 as modified by Miller, et al. in Int. J. Peotide Protein Research, 2Λ r 112 (1984) . DPDPE-TFA salt/0.83 mg, 1.1 umol/was dissolved in phosphate buffer (pH 7.2, 0.4 M, 1.5 mL) . 20 uL of Nal solution in water (0.224 mg, 1.5 umol) was added at 0°C. The reaction was started with addition of 20 uL ^ of chloramine (0.824 mg, 2.9 umol) water solution. The mixture was stirred for 1 min. 3 mL of 0.1% TFA was added to stop the reaction. The composition of reaction mixture was determined by HPLC using Vydac 218TP1010 C18 column with linear gradient elution of 20-40% CH 3 C in aqueous 0.1% -~~- trifluoroacetic acid, 1% min at a flow rate of 3 mL/min. Unreacted DPDPE, [mono- iodo-Tyr JDPDPE, trace [3 ' ,5 '-diiodo-Tyr JDPDPE and an unknown product were detected. K 1 value in this conditions: 1.77, 2.6, 3.12, and 1.18 respectively. K 1 value of [monoiodo- 20 Tyr J -DPDPE was identical as that obtained from [3-I-Tyr J — DPDPE prepared by SPPS. EXAMPLE 11 [3 '-OCK_,Tyr JDPDPE: The title compound was prepared as for 2 5 [3 '-NO„Tyr JDPDPE by SPPS except that N^-Boc-3 ' -0CH-.Tyr-0H was d added to the growing peptide chain instead of N -Boc-3 '-NO-Tyr -OH. The work up and purification were the same procedure as for [p-FPhe JDPDPE. TLC: R f A: 0.36, R f B: 0.68, R C: 0.86, R f D: 0.81. HPLC: K'=1.05; FABMS: (M+l) + 676, found 676. 30 The compounds of the present invention were tested for their relative activites in the guinea pig ileum (hereinafter referred to as GPI) and in the mouse vas deferens 35 ^ (hereinafter referred to as MVD) assay systems, as well as for their binding properties in rat-brain receptors in competition with tritiated naloxone (hereinafter sometimes referred to as ! '[ 3 HJNAL" and [ 3 HJ [D-Ala 2 ,D-Leu 5 Jenkephalin (hereinafter 3 sometimes referred to as "[ HjDADLE"). It is generally agreed that the MVD preparation contains largely delta receptors and that the GPI preparation contains largely mu receptors. These assays tested the degree of electrically stimulated contractions in the MVD and GPI tissues. Compounds of the present invention showed a higher activity in the MVD assay than in the GPI assay, thereby confirming their enhanced specificity for the delta receptor. All agonists tested inhibited the contractions. The inhibitions of contractions were reversible by naloxone, the protytype antagonist, and are also believed to be reversible by compounds of the present invention which display antagonist activity. See generally: Kosterlitz, H. ., Lydon, R. J., and Watt, A J., Kosterlitz, H. W. , and Leslie, F. M.., Br. J. Pharmacol. , 53, 371-381 (1975) , which are herein specifically incorporated by More specifically, the following is a protocol for the GPI and MVD bioassays. GPI and MVD Bioassays. Electrically induced smooth muscle contractions of mouse vas deferens and strips of guinea pig ileum longitudinal muscle-myenteric plexus were used as a bioassay. Tissues came from male Hartley guinea pigs weighing 150-400 grams and male ICR mice weighing 25-30 grams. The tissues were tied to gold chains with suture silk, suspended in 20 L baths containing 37°C oxygenated (95% 0, 5% C0 2 ) Krebs-dicarbonate solution (magnesium-free for the MVD) and allowed to equilibrate for 15 min. The tissues were then stretched to 1 g tension (0.5 g for MVD) and allowed to equilibrate for 15 min. The tissues were stimulated transmurally between platinum plate electrodes at 0.1 Hz, 0.4 msec pulses (2.0 msec pulses for MVD) and supramaximal voltage. Drugs were added to the baths in 20-60 uL volumes. The agonists remained in contact with the tissue for 3 min. and the baths were then rinsed several times with fresh Krebs solution. Tissues were given 8 min to re-equilibrate and regain predrug contraction height. Antagonists were a_dded to the bath 2 min prior to the addition of the agonist. Percent inhibition was calculated using the average contraction height for 1 min preceding the addition of the agonist divided by the contraction height 3 min after exposure to the agonist.' ^Q co values represent the mean of 4 tissues. The results are indicated in Table 2. Tasle 2 iKHisiTosY POTENCY A:;D SIL.CTI V. TV G? SELECTED CPDPE AΓ^CG ΞS IK C-PI A.\ ' 3 .".73 AES-.YS The GPI preparation has been shown to contain primarily mu type opiate receptors and the MVD preparation primarily delta type opiate receptors. Thus, comparisons of IC.. Q value in these two assay systems, as shown in Table II provide a measure of the receptor specificity of the tested analogs. The results shown in Table II clearly indicate the high delta receptor selectivity of these analogs. Another bioassay is the Radioreceptor binding assay This assay tests the ability of the enkephalin analogs to inhibit opiate receptor binding and displace tritiated ligands, DPDPE and CTOP, i.e., (D-Phe-Cy≤-Tyr-D-Trp-Orn- Thr-Pen-Thr-NH,) The protocal of this bioassay is as follows: Radioreceptor binding assay Adult male Sprague-Dawley rats (200-250 g) were sacrificed and brains immediately removed and placed on ice. Whole brain minus cerebellum was homogenized using a Polytron homogenizer (Brink an, setting #5, 15 sec). The homogenate was preincubated at 25°C for 30 min to remove endogenous opioids and centrifuged two times at 43,000 g for 10 min before use in the radioreceptor binding assay. [ 3 H]DPDPE (43.0 C i/mmole, 1.59 TBq/mmol, New England Nuclear, Boston, MA) and [ 3 H]CTOP (84,2 Ci/mmole, 3.12 TBq/mmol, New England Nuclear, Boston, MA) bindings were measured by a rapid filtration technique. A 100 juL aliquot of rat brain homogenate (0.5% final) was incubated with either l.OmM [ 3 H]DPDPE or 0.5 nM [ 3 H]CTOP in a total volume of 1 mL of 50 nM Tris-HCl pH 7.4 at 50C° containing 5 nM MgCl„, bovine serum albumin (1 mg/mL) , and phenylmethyl-sulfonylfluoride (100 ΛIL) . All binding measurements were done in duplicate and the binding displaced by 1 uM naltrexon hydrochloride was defined as specific tissue binding. Steady state binding experiments were carried out at 25°C for 120 min. The binding reaction was terminated by rapid filtration of samples through GF/B Whatman glass fiber filter strips pretreated with 0.1 % polyethyleneamine solution with a Brandel cell harvester: this was followed immediately by three rapid washes with 4 mL aliquots of ice cold saline solution. Filters were remove d and allowed to dry before assaying filter bound radioactivity by liquid scintillation spectrophotometry. The data were analyzed by using nonlinear least- square regression analysis on the Apple II computer. Programs were generously provided by SHM Research Corp., Tucson, AZ. The results of this bioassay is shown in Table 3. Tab!e 3 BI ■ NOIKG POTENCIES AND SELECTIVI* S OF SELECTED DPDPE Aft ALGG'JES I:; c ύ :: ? Eτmo: ; WITH [JHJCTOP aπd C-W3?E RECEPTO?. BIK0IK3 In view of the positive results obtained with these " tests, the claimed delta receptor agonist compounds are believed to be useful in the treatment of pain without the undesirable side effects associated with previously known opiates. Compounds according to the present invention having antagonist activity are believed to behave in a manner similar to naloxone and, thereby, are believed to be useful in those areas where narcotic antagonists have been useful in the prior art, including the treatment of Alzheimer's disease. See generally, Reisberg, B., et al. New Eng. J. Med. , Vol. 308: 12, 721-722 (1983) , which is specifically incorporated herein by reference. The above preferred embodiments and examples are given to illustrate the scope and spirit of the present invention. These embodiments and examples will make apparent to those skilled in the art other embodiments and examples. These other embodiments are examples within the contemplatin of the present invention. Therefore the present invention • should be limited only by the appended claims.

Claims: WHAT IS CLAIMED IS: A polypeptide of the formula: and pharmaceutically acceptable salts thereof wherein; are each independently hydrogen or lower alkyl; R 3 and R4 are each independently hydrogen or lower alkyl; R and R are each independently hydrogen or lower alkyl, provided that at least one of R , R R and R is other than hydrogen when both n and p are zero; 9 R is hydrogen or lower alkyl; R is hydroxy; lower alkoxy, amino, lower alkyla ino, or lower dialkylamino; R 11, R12 and R13 are each independently hydrogen or lower alkyl; R 14 is hydrogen, lower alkyl or lower alkanoyl; B is Gly or a chemical bond; X is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, carboxy, lower carbalkoxy, carbamoyl, lower alkylaminocarbonyl, lower dialkylaminocarbonyl, lower alkoxy, halo, nitro, cyano, lower alkanoyl or formyl; Y is hydrogen, lower alkenyl, lower alkynyl, carboxy, lower carbalkoxy, carbamoyl, lower alkylamino - carbonyl, lower dialkylaminocarbonyl, lower alkoxy, chloro, bromo, iodo, cyano, nitro, lower alkanoyl, or formyl, provided that both X and Y are not both hydrogen, and m, n, p and q are each independently 0, 1 or 2, with the proviso that when m and q are 0, and B is Gly, and x is hydrogen and T is fluoro or nitro, then R,. is lower alkyl. 10 2. The polypeptide according to Claim 1 wherein lower alkyl contains from one to three carbon atoms. 3. The polypeptide according to Claim 1 wherein lower alkyl is methyl. 4. The polypeptide according to Claim 1 wherein B 15 is Gly. The polypeptide according to Claim 1 wherein R is hydroxy. 3 6. The polypeptide according to Claim 1 wherein R , R 4, R5 and R6 are methyl. or) 9 7. The polypeptide according to Claim 6 wherein R , R 11, R12 are hydrogen and R13 is hydrogen or methyl. 8. The polypeptide according to Claim 1 wherein X is methoxy, fluoro, bromo, chloro, nitro or hydrogen and Y is i chloro, bromo, methoxy, nitro or hydrogen. 25 9. The polypeptide according to Claim 1 wherein R 14 is hydrogen or acetyl. 10. The polypeptide according to Claim 1 wherein , n, p and q are zero. 11. The polypeptide according to Claim 10 wherein 30 lower alkyl contains from one to three carbon atoms. 12. The polypeptide according to Claim 10 wherein lower alkyl is methyl. 13. The polypeptide according to Claim 10 wherein B is Gly. 35 14. The polypeptide according to Claim 10 wherein τ R_10 i * s Vh,yd Arrooxxyy.. 15. The polypeptide according to Claim 10 wherein R 3, R4, R5 and R6 are methyl. 16. The polypeptide according to Claim 15 wherein D R9, R11, R12 are hydrogen and R13 is hydrogen or methyl. 17. The polypeptide according to Claim 10 wherein X is methoxy, fluoro, bromo, chloro, amino, nitro or hydrogen and Y is iodo, chloro, bromo, methoxy, nitro or hydrogen. 18. The polypeptide according to Claim 10 wherein R is hydrogen or acetyl. 19. The polypeptide according to Claim 1 having the formula: wherein; 0 X is methoxy, iodo, bromo, chloro, amino, nitro or hydrogen; Y is iodo, chloro, bromo, methoxy, nitro or hydrogen, R ,13 is hydrogen or methyl, 35 14 R is hydrogen, lower alkyl or lower alkanoyl and B is Gly or a chemical bond. 20. The polypeptide according to Claim 19 wherein B is Gly. 21. The polypeptide according to Claim 17 wherein J •**. Pen5 is in the D configuration. 22. The polypeptide according to Claim 19 wherein 14 R is hydrogen or acetyl. 4 23. A polypeptide which is [p-F-Phe ]DPDPE_ 24. The polypeptide according to Claim 1 which is 10 [p-Br-Phe 4 ]DPDPE. 25. The polypeptide according to Claim 1 which is is [p-I-Phe 4 ^DPDPE 26. The polypeptide according to Claim 1 which is [3 '-N0 2 -Tyr 1 ]DPDPE. ^ 27. The polypeptide according to Claim 1 which is [3 '-I-Tyr 1 JDPDPE. 28. The polypeptide according to Claim 1 which is [3 '-CH 3 0-Tyr ~ ~JDPDPE. 29. The polypeptide according to Claim 1 which is 0 [3*-NH 2 -Tyr 1 JDPDPE. 30. The polypeptide according to Claim 1 which is [ (S,S)-p-N0 2 -*£-MePhe 4 ]DPDPE. 31. The polypeptide according to Claim 1 which is [ (R,R) -p-N0 2 - >-MePhe JDPDPE. 25 32. The polypeptide according to Claim 1 which is [ (Ξ,R)-p-N0 2 -A-MePhe 4 JDPDPE. 33. The polypeptide according to Claim 1 which is [ (R,S)-p-N0 2 -p>-MePhe 4 JDPDPE. 34. The polypeptide according to Claim 1 which is ° [p-Cl-Phe 4 JDPDPE. 35. The polypeptide according to Claim 1 wherein said polypeptide has the ability to bind to the delta receptor and, when bound to said delta receptor, has the ability to act as either an agonist or antagonist for said receptor. 35 36. The polypeptide according to Claim 23 wherein said polypeptide has the ability to bind to the delta receptor and, when bound to said delta receptor, has the ability to act as either an agonist or antagonist for said receptor. 37. The polypeptide according to Claim 35 wherein said polypeptide is a delta receptor agonist. 38. The polypeptide according to Claim 35 wherein said polypeptide is a delta receptor antagonist. 39. The polypeptide according to Claim 35 wherein said polypeptide is a delta receptor partial agonist. 40. The polypeptide according to Claim 35 wherein said polypeptide is a delta receptor partial antagonist. 41. A-pharmaceutical composition comprising a pharmaceutically effective amount of the polypeptide according to Claim 1 and a pharmaceutical carrier therefor. - 5 42. A pharmaceutical composition comprising a pharmaceutically effective amount of the polypeptide according to Claim 23 and a pharmaceutical carrier therefor. 43. A process of inducing analgesia in animals comprising administering to said animal an analgesic effectiv 0 amount of a polypeptide according to Claim 1. 44. A process for inducing agonist opioid delta receptor activity in animals which comprises administering to said animal an effective amount of the polypeptide according to Claim 37. -> 45. A process for inducing antagonist opioid delta receptor activity in animals which comprises administering to said animal an effective amount of the polypeptide according to Claim 38. 0 5

Inventors: HRUBY VICTOR JOSEPH, TOTH GEZA, HRUBY, VICTOR, JOSEPH, TOTH, GEZA
Applicants: RES CORP TECHNOLOGIES INC, RESEARCH CORPORATION TECHNOLOGIES, INC.
Application year: 1989
Main classification: C07K7
IPC codes: A61K38, A61P25, C07K14