DMT-TIC DI-AND TRI-PEPTIDIC DERIVATIVES AND RELATED COMPOSITIONS AND METHODS OF USE TECHNICAL FIELD OF THE INVENTION [0001] The present invention relates to di-and tri-peptidic derivatives comprising the pharmacophore H-Dmt-Tic-X-Y, wherein X is a spacer comprising one or more amino acid residues, and Y comprises an aromatic group, which produce selective bifunctional opioid ligands for the 8 and opioid receptors. The present invention also relates to compositions thereof and methods of use therefor in the antagonism and agonism of 8 and J. opioid receptors.

BACKGROUND OF THE INVENTION [0002] Endogenous opioids are believed to be involved in the modulation of pain perception, in mood and behavior, learning and memory, diverse neuroendocrine functions, immune regulation and cardiovascular and respiratory function. Opioids also have a wide range of therapeutic utilities, such as treatment of opiate and alcohol abuse, neurological diseases, neuropeptidc or neurotransmitter imbalances, neurological and immune system dysfunctions, graft rejections, pain control, shock and brain injuries.

[0003] There are believed to be three types of opiate receptors, namely 8, K and u.

Genes encoding these three main receptor types now have been cloned. Sequencing of the cloned opioid receptor genes has revealed a substantial degree of amino acid homology between different receptor types (Meng et al., PNAS USA 90: 9954-9958 (1993); Thompson et al., Neuron 11: 903-913 (1993); Evans et al., Science 258: 1952- 1955 (1992) ; and Kieffer et al., PNAS USA 89: 12048-12052 (1992) ), which explains the tendency of opioid receptor ligands, even those reported to be selective, to bind to more than one type of opioid receptor. Based on differences in the binding profiles of natural and synthetic ligands, subtypes of opioid receptors have been suggested, including ul and u2 (Pasternak et al., Life Sci. 38: 1889-1898 (1986) ) and Kl and K2 (Zukin et al., PNAS USA 85: 4061-4065 (1988) ). Different subtypes of a given type of opioid receptor may co-exist in a single cell (Evans et al. (1992), supra ; and Kieffer et al. (1992), supra). l0004] The u opioid receptor in the brain appears to mediate analgesia (Kosterlitz et al. , Br. J Pharmacol. 68: 333-342 (1980) ). It is also believed to be involved with other undesirable effects, such as respiratory depression (Ward et al., Soc. Neurosci. Symp. 8: 388 (abstract) (1982) ), suppression of the immune system (Plotnikoff et al. , Enkephalins and Endorphins : Stress and the Immune System, Plenum Press, NY (1986); Yahya et al. , Life Sci. 41: 2503-2510 (1987) ) and addiction (Roemer et al., Life Sci. 27: 971-978 (1981)). Its side effects in the periphery include inhibition of intestinal motility (Ward et al., Eur. J Pharmacol. 85: 163-170 (1982) ) and secretion in the small intestine (Coupar, Br. J. Pharmacol. 80: 371-376 (1983)).

[0005] 8-opioid receptors also mediate analgesic but are not involved in addiction.

They may have an indirect role in immune suppression.

[0006] There appears to be a single binding site for agonists and antagonists in the ligand-binding domain of 8 receptors. Thus, the"message domain"of 8-agonists and 6- antagonists probably presents a similar low energy conformer in order to fit the receptor cavity. The minimum size of that"message domain"constitutes the dimensions of a dipeptide (Temussi et al., Biochem. Biophys. Res. Commun. 198: 933-939 (1994); Mosberg et al., Lett. Pept. Sci. 1: 69-72 (1994); and Salvadori et al., J. Med. Chem. 42: 3100-3108 (1997) ), which has a specific spatial geometry in solution (Bryant et al., Trends Pharmacol. Sci. 18: 42-46 (1998); Bryant et al., Biol. Chem. 378: 107-114 (1997); Crescenzi et al., Eur. J. Biochem. 247: 66-73 (1997); and Guerrini et al., Bioorg.

Med. Chem. 6: 57-62 (1998) ) as seen in the crystallographic evidence for TIPP analogues (Flippen-Anderson et al., J. Pept. Res. 49: 384-393 (1997) ) and N, N (Me) 2- Dmt-Tic-OH.

[0007] The Dmt-Tic pharmacophore represents a distinct class of 8-opioid antagonists (Salvadori et al., Mol. Med. 1: 678-689 (1995); Bryant et al. (1998), supra ; and Lazarus et al., Drug Dev. Today 1998: 284-294). Observations of differences between the 6 opioid receptor binding of Dmt-Tic peptides and their Tyr-Tic cognates (Salvadori et al. (1995), supra ; Lazarus et al. (1998), supra ; and Lazarus et al., Int'l Symp. on Peptide Chem. and Biol., Changchung, PRC (1999) ) indicates that Dmt assumes a predominant role in the alignment or anchoring of the peptide within zut and K opioid receptor binding sites (Bryant et al. (1998), supra ; and Bryant et al. (1997), supra ; Crescenzi et al. (1997), supra ; and Guerrini et al. (1998), supra) or affects the conformation of the dipeptide antagonists in solution (Bryant et al. (1997), supra ; and Crescenzi et al. (1997), supra). Furthermore, observations of differences between the spectra of activity exhibited by the Tyr-Tic cognates of certain Dmt-Tic peptides (Schiller et al., PNAS USA 89: 11871-11875 (1992); Schiller et al., J. Med. Chem. 36: 3182-3187 (1993); Schiller et al., Peptides Hodges and Smith, eds. , ESCOM (1994); pp.

483-486; Temussi et al. (1994), supra ; Mosberg et al. (1994), supra ; Salvadori et al.

(1995), supra ; Lazarus et al. (1998), supra ; and Lazarus et al. (1999), supra) and the corresponding Dmt-Tic peptides suggests that the C-terminal"address"portion of the peptide can influence the"message domain." [0008] Recently, cyclic peptides and di-and tri-peptides comprising the pharmacophore Dmt-Tic have been developed and have been shown to exhibit high selectivity, affinity and potency for the 8-opioid receptor. Such peptides have been shown to function as agonists, partial agonists, antagonists, partial antagonists or mixed antagonists/agonists for opioid receptors (see Lazarus et al. , U. S. Patent No. 5,780, 589, and Schiller, U. S. Patent No. 5,811, 400).

[0009] A variety of modifications to the Tic residue differentially changes receptor selectivity, including alterations in its electronic configuration and chirality, as well as its replacement by heteroaliphatic/heteroaromatic nuclei or D-Phe (Santagada et al., Med.

Chem. Lett., 10, 2745-2748 (2000); Page et al., Bioorg. Med. Chem. Lett., 10, 167-170 (2000); Salvadori et al., Mol. Med, 1 : 678-689 (1995); Balboni et al., Peptides, 21 : 1663-1671 (2000); and Capasso et al., FEBSLett., 417 : 141-144 (1997) ). Changes wrought by altering the distance to a third aromatic center ("Y") at the C-terminus by an interposed sequence, a spacer induces profound changes in the affinity, selectivity and bioactivity of a ligand (Capasso et al., FEBS Lett., 417 : 141-144 (1997); Salvadori et al., J Med Chem., 42 : 5010-5019 (1999); Page et al., J. Med. Chem., 44 : 2387-2390 (2001)).

[0010] The uniqueness of the 8 receptor has led to the use of moderately 8-selective alkaloid antagonists in clinical trials, such as for the amelioration of the effects of alcoholism (Froehlich et al., Alcohol. Clin. Exp. Res. 20: A181-A186 (1996) ), the treatment of autism (Lensing et al., Neuropsychobiol. 31: 16-23 (1995) ), and Tourette's syndrome (Chappell, Lancet 343: 556 (1994) ). The 8-opiate antagonist naltrindole (Portoghese et al., Eur. J. Pharm. 146: 185-186 (1998) ) has been shown to inhibit the reinforcing properties of cocaine (Menkens et al., Eur. J. Pharm. 219: 346-346 (1992)), to moderate the behavioral effects of amphetamines (Jones et al., J. Pharmacol. Exp.

Ther. 262: 638-645 (1992) ), and to suppress the immune system (Jones et al. (1992), supra) for successful organ transplantation (House et al., Neurosci. Lett. 198: 119-122 (1995) ) in animal models (Arakawa et al., TransplantProc. 24: 696-697 (1992); Arakawa et al., Transplant 53: 951-953 (1992); and Arakawa et al., Transplant. Proc.

25: 738-740 (1993) ). The same effects also have been shown for 7- benzylspiroindanylnaltrexone (Lipper et al., Eur. J. Pharmacol. 354: R3-R5 (1998) ).

[0011] The intractable membrane barriers, such as the blood-brain barrier (BBB), must be circumvented in order for peptide antagonists to express activity in vivo (Ermisch et al., Physiol. Rev. 73: 489-527 (1993) ). The requisite physicochemical properties of compounds capable of passing through this barrier include low molecular weight (< 800 Da) and high octanol-water coefficient characteristics.

[0012] In view of the above, the present invention seeks to provide more potent 8- opioid antagonists and 8-opioid antagonists with high dual binding affinity and biological activity toward 6-opioid and u-opioid receptors.

[0013] In addition to the above, one of the major chemical determinants for the inhibition of hMDR-1 involves the presence of strong hydrophobic substituents necessary for lipid solubility (Ford et al., Cancer Res. 50: 1748-1756 (1990); Zamora et al. , Mol. Pharmacol. 33: 454-462 (1988); and Nogae et al., Biochem. Pharmacol. 38: 519-527 (1989) ) as constituted by saturated and aromatic rings, as well as a tertiary nitrogen (Zamora et al. (1988), supra ; Ramu et al., Int. J. Cancer 43: 487-491 (1989); and Pearce et al., PNAS USA 86: 5128-5133 (1989)). In addition to a role in drug resistance, the human Pgp-1 protein is expressed in a variety of normal human tissues and plays an important physiological role in maintenance of the BBB (Schinkel et al., J.

Clin. Invest. 97: 2517-2524 (1996); Schinkel et al., Int. J Clin. Pharmacol. Ther. 36: 9- 13 (1998); and Jonker et al., Br. J : Pharmacol. 127: 43-50 (1999) ). In the BBB, Pgp activity in apical membranes of the capillary endothelial cells functions to at least partially exclude a wide variety of hydrophobic toxicants from the brain (Zamora et al.

(1988), supra ; Schinkel et al. (1996), supra ; and Jonker et al., Br. J Pharmacol. 127: 43-50 (1999) ). These include some drugs with central nervous system activities, such as certain opiate alkaloids and analogues thereof (Callaghan et al., J. Biol. Chem. 268: 16059-16064 (1993) ). Thus, the present invention also seeks to provide inhibitors of hMDR-1.

[0014] These and other objects of the present invention, as well as additional inventive features, will be apparent to the ordinarily skilled artisan from the detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION [0015] The present invention provides a compound of formula: wherein X is a spacer comprising one or more amino acid residues, and Y comprises an aromatic compound.

[0016] More particularly, the present invention provides a compound of formula: wherein R'is selected from the group consisting of wherein, when R'is , R'has a stereocenter (*) having R stereochemistry. [0017] Also provided by the present invention is a composition comprising at least one the above-described compounds and a carrier.

[0018] The present invention further provides methods of treatment. In one embodiment, a method of treating a mammal in need of an antagonist of a 8-opioid receptor is provided. The method comprises administering at least one compound of formula: wherein R'is selected from the group consisting of : in an amount that antagonizes a 8-opioid receptor in said mammal, wherein, when R'is R'has a stereocenter (*) having R stereochemistry.

[0019] The present invention further provides a method of treating a mammal in need of an agonist of a 8-opioid receptor. The method comprises administering at least one compound of formula: wherein R'is selected from the group consisting of : in an amount that agonizes a S-opioid receptor in said mammal.

[0020] In yet another embodiment, the present invention provides a method of treating a mammal in need of an agonist of a p-opioid receptor. The method comprises administering at least one compound of formula: wherein R'is or in an amount that agonizes a u-opioid receptor in the mammal.

[0021] The present invention further provides a method of treating a mammal in need of an antagonist of a 8-opioid receptor and an agonist of a u-opioid receptor. The method comprises administering the compound of formula: wherein R'is in an amount that antagonizes a #-opioid receptor and agonizes a u-opioid receptor in the mammal.

BRIEF DESCRIPTION OF THE FIGURES [0022] Fig. 1A is a graph of fluorescence vs. concentration (uM).

[0023] Fig. 1B is a graph of fluorescence vs. concentration (µM).

[0024] Fig. 1 C is a graph of fluorescence vs. concentration (µM).

[0025] Fig. 1D is a graph of fluorescence vs. concentration (lem).

[0026] Figs. 2A and 2B depicts some of the compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION [0027] The present invention provides a compound of formula: wherein X is a spacer comprising one or more amino acid residues, and Y comprises an aromatic compound.

[0028] The present invention further provides a compound of formula: wherein R'is selected from the group consisting of wherein, when R'is R'has a stereocenter (*) having R stereochemistry.

[0029] With respect to the above-described compounds, when R'is H N CH3 * O \ HN N X, R'desirably has a stereocenter (*) having either R or S , s ab y as s e te () g o S stereochemistry ; when R'is H N X COOH O \ HN N 'R'preferably has a stereocenter (*) having S P Y stereochemistry ; when R'is H N COOH o CONH2 R'desirably has a stereocenter (*) having S stereochemistry ; when R'is H N N COOH HN , R'preferably has a stereocenter (*) having S stereochemistry ; and when R'is H N **'fN NzXCONH2 0 COOH, R'has a stereocenter (*) having S stereochemistry.

[0030] The present inventive compounds can be synthesized by standard methods known to those of ordinary skill in the art. See, for example, Modern Techniques of Peptide and Amino Acid Analysis, John Wiley & Sons (1981) ; Bodansky, Principles of Peptide Synthesis, Springer Verlag (1984) ). Specific examples of the synthesis of the present inventive compounds are set forth in the Examples herein.

[0031] The spacer"X"comprises one or more amino acid residues, which comprise an amino group and a carbonyl, preferably in the form of an amide group. Preferably, the spacer comprises 1-6 amino acid residues, more preferably 1-3 amino acid residues, even more preferably 1-2 amino acid residues, and most preferably 1 amino acid residue. In some embodiments, "X"does not exist.

[0032]"Y"is desirably an aromatic group, such as, for example phenyl, benzyl, or benzoimidazolyl, which are optionally substituted. The aromatic group"Y"can be substituted with 1 to 3 substituents such as, for example, Cl-C6 alkyl, Cl-C6 alkenyl, halo, amino, haloalkyl, hydroxy, Cl-C6 alkoxy, phenyl or nitro. Preferably,"Y"is phenyl, benzyl, or benzoimidazolyl. Fig. 2 provides examples of suitable spacers"X" and aromatic groups"Y".

[0033] Without wishing to be bound to any particular theory, alteration in the C- terminus of the Dmt-Tic pharmacophore appears to mimic changes observed with the amphibian u opioid heptapeptides dermorphin and deltorphin (Melchiorri et al., Gen.

Pharmac., 27 : 1099-1107 (1996); Lazarus et al., Prog. Neurobiol., 57 : 377-420 (1999) ).

Furthermore, a third aromatic nucleus (i. e. ,"Y") determines 8-opioid agonist activity, when it is located at a prescribed distance from the Dmt-Tic pharmacophore by means of a spacer (i. e. ,"X"). This in turn implies that the t receptor binding site not only accommodates, but also requires, a physically larger and bulkier peptide ligand in order to bind and trigger an agonist response.

[0034] Whether an above-described compound functions as an agonist, a partial agonist, an antagonist, a partial antagonist, or a mixed agonist/antagonist is set forth, in part, in the Examples herein. Additionally, conventional techniques known to those of ordinary skill in the art can be used to make such determinations. Examples of such techniques include, but are not limited to, the mouse vas deferens in vitro assay of 8 receptors and the guinea pig ileum in vitro assay of u receptors as described in the Examples. Examples of in vivo studies include, but are not limited to, the tail flick test (Harris et al., J Pharmacol. Meth. 20: 103-108 (1988); and Sing et al. , P. A. Amber (v.

3.0. rev. A), Dept. Pharm. Chem. , University of California, San Francisco (1988)).

[0035] The present invention further provides a composition comprising at least one of the above compounds. Desirably, the composition comprises at least one carrier, which is preferably a pharmaceutically acceptable carrier, diluent or vehicle. Also, desirably, the composition is formulated for human administration. Pharmaceutically acceptable carriers are well-known to those of ordinary skill in the art, as are suitable methods of administration. The choice of carrier will be determined, in part, by the particular method used to administer the composition. One of ordinary skill in the art will also appreciate that various routes of administering a composition are available, and, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, there are a wide variety of suitable formulations of compositions that can be used in the present inventive methods.

[0036] A compound of the present invention can be made into a formulation suitable for parenteral administration, preferably intraperitoneal administration, or dural administration. Such a formulation can include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.

Extemporaneously injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets, as described herein.

[0037] A formulation suitable for oral administration can consist of liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or fruit juice; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solid or granules; solutions or suspensions in an aqueous liquid; and oil-in-water emulsions or water-in-oil emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.

[0038] Similarly, a formulation suitable for oral administration can include lozenge forms, which can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0039] An aerosol formulation suitable for administration via inhalation also can be made. The aerosol formulation can be placed into a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0040] A formulation suitable for topical application can be in the form of creams, ointments, or lotions.

[0041] A formulation for rectal administration can be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. A formulation suitable for vaginal administration can be presented as a pessary, tampon, cream, gel, paste, foam, or spray formula containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0042] Any of the above compositions can further comprise one or more other active agents. Alternatively, any of the above compositions can be administered, by the same or different route, in combination with another composition comprising one or more other active agents, either simultaneously or sequentially in either order sufficiently close in time to realize the benefit of such co-administration. [0043] In view of the above, the present invention provides a method of treating a mammal in need of an antagonist of a 8-opioid receptor. The method comprises administering at least one compound of formula: wherein R'is selected from the group consisting of : in an amount that antagonizes a 8-opioid receptor in said mammal, wherein, when R'is R'has a stereocenter (*) having R stereochemistry.

[0044] The present invention further provides a method of treating a mammal in need of an agonist of a 8-opioid receptor. The method comprises administering at least one compound of formula: wherein R'is selected from the group consisting of : in an amount that agonizes a 8-opioid receptor in said mammal.

[0045] In the context of the above method, if the mammal is also in need of an agonist of a J-opioid receptor, the compound of formula : is the compound wherein R'is and the compound is administered in an amount that agonizes a u-opioid receptor in said mammal. R'is preferably Such a method and compounds are useful to induce analgesia.

[0046] The present invention further provides a method of treating a mammal in need of an antagonist of a 8-opioid receptor and an agonist of a-opioid receptor, which method comprises administering the compound of formula: wherein R'is in an amount that antagonizes a 8-opioid receptor and agonizes a u-opioid receptor in said mammal.

[0047] The present invention also provides a method of treating a mammal in need of an antagonist of a 8-opioid receptor, which method comprises administering the compound of formula: wherein R'is in an amount that antagonizes a 8-opioid receptor in said mammal.

[0048] Moreover, the present invention provides a method of treating a mammal in need of an agonist of a S-opioid receptor, which method comprises administering the compound of formula: wherein R'is in an amount that agonizes a 8-opioid receptor in said mammal.

[0049] With respect to the above-described methods, when R'is H N CH3 my O \ HN N H, R'desirably has a stereocenter having either R or S , y st o () g S stereochemistry ; when R'is H N X COOH O \ HN N 4, R'preferably has a stereocenter (*) having S Y stereochemistry ; when R'is H N COOL o CONH2, R ! desirably has a stereocenter (*) having S stereochemistry ; when R'is H y N N H COOH HN , R'preferably has a stereocenter (*) having S stereochemistry ; and when R'is H N y CONH2 0 COOH, R'has a stereocenter (*) having S stereochemistry.

[0050] The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the individual over a reasonable time frame. The dose will be determined by the potency of the particular compound employed for treatment, the severity of any condition to be treated, as well as the body weight and age of the individual. The size of the dose also will be determined by the existence of any adverse side effects that may accompany the use of the particular compound employed. It is always desirable, whenever possible, to keep adverse side effects to a minimum.

[0051] The dosage can be in unit dosage form, such as a tablet or capsule. The term "unit dosage form"as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular embodiment employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an effective amount, i. e. , an amount effective to antagonize or agonize a 8-opioid receptor or a u-opioid receptor as desired.

[0052] Since the"effective amount"is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on interindividual differences in pharmacokinetics, drug distribution, and metabolism. The"effective amount"can be defined, for example, as the blood or tissue level desired in the patient that corresponds to a concentration of one or more compounds according to the invention. The"effective amount"for a given compound of the present invention also can vary when the composition of the present invention comprises another active agent or is used in combination with another composition comprising another active agent.

[0053] One of ordinary skill in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired"effective amount"in the individual patient.

One skilled in the art also can readily determine and use an appropriate indicator of the "effective amount"of the compound of the present invention by pharmacological end- point analysis.

[0054] Further, with respect to determining the effective amount in a patient, suitable animal models are available and have been widely implemented for evaluating the in vivo efficacy of such compounds. These models include the tail flick test (see, e. g. , U. S.

Patent No. 5,780, 589). In vitro models are also available, examples of which are set forth in the Examples herein.

[0055] Generally, an amount of a present inventive compound up to about 50 mg/kg body weight, preferably from about 10 mg/kg body weight to about 50 mg/kg body weight is preferred, especially from about 10 mg/kg body weight to about 20 mg/kg body weight. In certain applications, multiple daily doses are preferred. Moreover, the number of doses will vary depending on the means of delivery and the particular compound administered.

EXAMPLES [0056] The following examples serve to illustrate further the present invention and are not intended to limit its scope in any way.

[0057] Nomenclature as established by the IUPAC-IUB Commission on Biochemical Nomenclature (J. Biol. Chem. 260: 1442 (1985) ) will be used herein. In addition, the following symbols and abbreviations will be used: AD: 1-adamantyl amide BBB: blood-brain barrier Bid: 1H benzimidazole-2-yl Boc: tert-butyloxycarbonyl DAGO: H-Tyr-D-Ala-Gly-N-MePhe-Gly-ol (, u agonist) DAMME : H-Tyr-D-Ala-Gly-N-MePhe-Met (O)-ol (p agonist) DCC: N, N'-dicyclohexylcarbodiimide Deltorphin A: H-Tyr-D-Met-Phe-His-Leu-Met-NH2 (81 agonist) Deltorphin B: H-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (82 agonist) DER: dermorphin or H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (u. agonist) DMF : N, N dimethylformamide DMSO: dimethylsulfoxide Dmt or D: 2', 6'-dimethyl-L-tyrosine DPD: H-Dmt-D-Phe-Gly-Val-Val-NH2 (p antagonist) DPDPE: H-Tyr- [D-Pen-Gly-Phe-D-Pen] enkephalin (81 agonist) DSB: H-Dmt-Sar-Bid (8/L agonist) DTA: H-Dmt-Tic-Ala-NH2 (8 antagonist) DtMe: H-Dmt-Tic-NHCH3 (8 antagonist) Et: ethyl Et2O : diethyl ether EtPt: petroleum ether GPI: guinea pig ileum (p receptor bioassay) H-aDmt-OH: 2-methylamino-3- (2', 6'-dimethyl-4-hydroxyphenyl) -propionic acid H-ßTic-OH : 1, 2,3, 4-tetrahydroisoquinoline-3-yl acetic acid hMDR-1 : human multidrug resistance glycoprotein (Pgp)-1 HOBt : 1-hydroxybenzotriazole HPLC : high performance liquid chromatography Ke : the antilog of pA2 in molar concentration LiAlH4 : lithium aluminum hydride Me: methyl MeDTOH : N,N@ (Me) 2-Dmt-Tic-OH (8 antagonist) MeOH : methanol MVD : mouse vas deferens (8 specific bioassay) NaBH3CN: sodium cyanoborohydride NAL: naltrindole NH-tBut : tert-butyl amine NMM: N-methyl morpholine OMe: methyl ester PA2 : negative log of the molar concentration required to double the agonist concentration to achieve the original response PipDTOH: [des-NH2-a-piperidine-1-yl] Dmt-Tic-OH (8 antagonist) tBu : tert-butyl TEA: triethylamine TFA: trifluoroacetic acid Tic or T: 1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid TIP (P): H-Tyr-Tic-Phe- (Phe)-OH TLC: thin layer chromatography [Trp4, Tyr5] DER: H-Tyr-D-Ala-Phe-Trp-Tyr-NH2 (L agonist) [Trp4, Lys-OH7] DER : H-Tyr-D-Ala-Phe-Trp-Tyr-Pro-Lys-OH (u agonist) WSC: 1-ethyl-3- [3'-dimethyl-aminopropyl] carbodiimide hydrochloride Z: benzyloxycarbonyl [0058] Verapamil, colchicine and sodium pyruvate were obtained from Sigma Chemical Co. (St. Louis, MO). DPDPE and DAGO were obtained from Bachem (Torrance, CA). Boc-Tic-OH was obtained from Bachem Feinchemikalien AG (Bubendorf, Switzerland). Naloxone, naltrindole and BNTX were obtained from Research Biochemicals International (Natick, MA). Vybrant MDR assay is a product of Molecular Probes (Eugene, OR). [3H] DPDPE was from NEN-DuPont (Billerica, MA) and [3H] DAGO (60 Ci/mmol) was from Amersham (Arlington Heights, IL).

[00591 H-L-Dmt-OH was synthesized as previously described (Dygos et al., Synthesis 8: 741-743 (1992) ). Boc-Tic-OH was obtained from Bachem (Heidelberg, Germany). All peptides were prepared by standard solution methods (Lazarus et al., Biochem. Biophys. Res. Commun. 178: 110-115 (1991) ; Sagan et al., Biochem. Biophys.

Res. Commun. 187: 1203-1210 (1992) ). Dipeptides were obtained by condensation of Boc-L-Dmt-OH with Tic derivatives (H-Tic-OtBu, H-Tic-NHMe, H-Tic-NH-1- adamantane and H- (3Tic-OMe) or by condensation of Boc-Dmt-Tic-OH with tert-butyl amine or 5-aminotetrazolyl via DCC/HOBt. Tripeptides were obtained by condensation of Boc-L-Dmt-Tic-OH with Ala derivatives (H-Ala-NHMe, H-Ala-NH-1-adamantane, H-Ala-NH-tBu and H-Ala-OMe) via DCC/HOBt. Final products were obtained, when necessary, by ester function hydrolysis with 1 N NaOH and removal of the Boc protecting group in TFA. N-alkylated di-and tripeptide derivatives were obtained by reductive alkylation of the corresponding deprotected linear peptides with aldehydes (formaldehyde, glutaraldehyde, succinaldehyde) and NaBH3CN in acetonitrile (Borch et al., J. Org. Chem. 37: 1673-1674 (1972)). HCl'H-pH-Tic-OMe was prepared by reduction of H-Tic-OMe with LiAlH4 to the corresponding 3-hydroxymethyl-Tic that was transformed first in 3-bromomethyl-Tic and then in 3-cyano-Tic. Treatment of the cyano group with HCl-methanol gave the final product (Crabb et al., J. Chem. Soc.

Perkins Trans. 11 : 370-378 (1977)). a-H (R, S) -Dmt-Tic-OH was obtained by condensation of (R, S)-2-cyano-3- (4-hydroxy-2, 6-dimethylphenyl) -propanoic acid with H-Tic-OtBu via DCC/HOBt. In turn, (R, S)-2-cyano-3- (4-hydroxy-2, 6-dimethylphenyl) - propanoic acid was prepared from ethyl cyanoacetate and O-carbethoxy-3, 5-dimethyl-4- chloromethylphenol (Abrash et al., Biochemistry 2: 947-952 (1972)).

[0060] Preparative reversed-phase HPLC was conducted with either a Waters Delta Prep 3000 A (30 x 3 cm; 15 Am) column or a Waters Delta Prep 4000 system with Waters Prep LC 40 mm Assembly column C18 (30 x 4 cm, 300 A, 15 pm particle size column). Peptides were eluted with a gradient of either 0-60% or 0-50% B in 25 min at a flow rate of 50 ml/min using the following mobile phases: solvent A (10% acetonitrile in 0. 1% TFA, v/v) and solvent B (60% acetonitrile in 0. 1% TFA, v/v). Analytical HPLC analyses were carried out with a Bruker Liquid Chromatographer LC21-C instrument using a Vydac 218 TP 5415 C18 column (250 x 4.6 mm, 5 um particle size) and equipped with a Bruker LC 313 UV variable wavelength detector. Recording and quantification were accomplished with a chromatographic data processor coupled to an Epson computer system (QX-10). Analytical determinations and capacity factor (K') of the products were determined using HPLC conditions in the above solvent systems (solvents A and B) programmed at a flow rate of 1 ml/min using the following linear gradients: (a) from 0 to 100% B in 25 min, and (b) from 10 to 70% B in 25 min. All analogues showed less than 1% impurities when monitored at 220 and 254 nm.

[0061] TLC was performed on precoated plates of silica gel F254 (Merck, Darmstadt, Germany) using the following solvent systems: (a) 1-butanol/AcOH/H2O (3: 1: 1, v/v/v) and (B) CH2Cl2/toluene/methanol (17: 1: 2, v/v/v). Ninhydrin (1 %, Merck), fluorescamine (Hoffman-LaRoche), and chlorine reagents were used as sprays. Open column chromatography (2 x 70 cm, 0.7-1 g material) was used on silica gel 60 (70-230 mesh, Merck) using the same eluent systems.

[0062] Melting points were determined on a Kofler apparatus and are uncorrected.

Optical rotations were determined at 10 mg/ml in methanol with a Perkin-Elmer 241 polarimeter with a 10 cm water-jacketed cell. All 1H-NMR spectra were recorded on a Bruker 200 MHZ spectrometer. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analyses of peptides were conducted using a Hewlett Packard G 2025 A LD-TOF system. The samples were analyzed in the linear mode with 28 kV accelerating voltage, mixing them with a saturated solution of a- cyano-4-hydroycinnamic acid matrix.

Example 1 [0063] This example describes the synthesis of Boc-Dmt-Tic-OMe.

[0064] HOBt (0.22 g, 1.42 mmol) and DCC (0.29 g, 1.42 mmol) were added to a solution of Boc-Dmt-OH (0.4 g, 1.29 mmol) and H-Tic-OMe (0.25 g, 1.29 mmol, Hayashi et al., Chem. Pharm. Bull. 31: 312-314 (1983) ). The reaction was stirred for 3 hr at 0°C and for 24 hr at room temperature (RT). After evaporation of DMF, the residue was solubilized in EtOAc and washed with citric acid (10%), NaHC03 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et20/PtEt (1: 1, v/v): yield 0.52 g (83%); Rf (B) 0.81 ; HPLC K'= 7.91 ; mp 135-137 °C ; [a] 20D-13. 2; MHF 483 ;'H-NMR (DMSO) 8 = 1. 37-1.46 (d, 9H), 2.16 (s, 6H), 2.96-3. 01 (m, 2H), 3.08-3. 13 (m, 2H), 3.46-3. 56 (m, 1H), 3.72 (s, 3H), 4.34-4. 88 (m, 3H), 6.46 (s, 2H), 7.18-7. 21 (m, 4H), 8.32 (bs, 1H).

Example 2 [0065] This example describes the synthesis of Boc-Dmt-Tic-NHNH2.

[0066] NH2NH2H2O (1 ml) was added to a solution of Boc-Dmt-Tic-OMe (0.26 g, 0.54 mmol) in MeOH (10 ml). The reaction mixture was stirred for 24 hr at RT. After evaporation of the solvent, the residue was crystallized from Et2O/PtEt (1 : 1, v/v): yield 0.25 g (94%); Rf (B) 0.75 ; HPLC K'= 6.83 ; mp 154-156 °C ; [a] 20D-14. 5; MHF 483; 'H-NMR (DMSO) 6 = 1. 35-1.44 (d, 9H), 2.16 (s, 6H), 3.07-3. 40 (s, 4H), 3.79 (m, 1H), 4.29-4. 78 (m, 5H), 6.34 (m, 2H), 6.95 (bs, 1H), 7.14 (3,4H), 8.22 (bs, 1H).

Example 3 [0067] This example describes the synthesis of TFA-H-Dmt-Tic-NHNH2 (compound 6 in Table II).

[0068] Boc-Dmt-Tic-NHNH2 (0.2 g, 0.41 mmol) was treated with TFA (1 ml) for 0.5 hr at RT. ET20/PtEt (1: 1, v/v) was added to the solution until the product precipitated: yield 0.18 g (91%) ; Rf (A) 0.69 ; HPLC K'= 2. 52; mp 143-145 °C ; [afD +13.5 ; MH+ 382. Anal. (C12H26N4O3#TFA) C, H, N (C 65.95, H 6.88, N 14.65).

Example 4 [0069] This example describes the synthesis of Z-Tic-NHMe.

[0070] NMM (0.35 ml, 3.21 mmol), HOBt (0.54 g, 3.53 mmol) and DCC (0.73 g, 3.53 mmol) were added to a solution of Z-Tic-OH (1 g, 3.21 mmol, Abrash (1972), supra) and HClH2NMe (0.22 g, 3.21 mmol) in DMF (10 ml) at 0 °C. The reaction mixture was stirred for 3 hr at 0 °C and 24 hr at RT. After evaporation of DMF, the residue was solubilized in EtOAc and washed with citric acid (10%), NaHC03 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et20/PtEt (1: 1, v/v): yield 0.90 g (87%); Rf (B) 0.97 ; HPLC K'= 8. 56; mp 137-139 °C ; [a] 20D +13. 5; MHf 325 ; lH-NMR (DMSO) 8 = 2.44-2. 46 (d, 3H), 3.12-3. 24 (m, 2H), 4.36-4. 46 (m, 3H), 5.17 (s, 2H), 7.18-7. 21 (m, 5H), 7.36-7. 39 (m, 4H), 7.74-7. 77 (m, 1H).

Example 5 [0071] This example describes the synthesis of H-Tic-NHMe.

[0072] C/Pd (5%, 0.05 g) was added to a solution of Z-Tic-NHMe (0.90 g, 2.78 mmol) in MeOH (30 ml) and H2 was bubbled for 1 hr at RT. After filtration, the solution was evaporated to dryness. The residue was crystallized from Et20/PtEt (1: 1, v/v): yield 0.49 g (92%); Rf (B) 0.38 ; HPLC K'= 5. 03; mp 123-125 °C ; [a] 20D +18. 7; MH+ 191.

Example 6 [0073] This example describes the synthesis of Boc-Dmt-Tic-NHMe.

[0074] Boc-Dmt-OH was condensed with H-Tic-NHMe via DCC/HOBt as described in Example 1 in order to obtain Boc-Dmt-Tic-NHMe: yield 0.13 g (85%); Rf (B) 0.73 ; HPLC K'= 6. 34; mp 142-146 °C ; [a] 20D - 15. 3; NET 482 ;'H-NMR (DMSO) # = 1. 35- 1.44 (d, 9H), 2.16 (s, 6H), 2.44-2. 46 (d, 3H), 3.05-3. 41 (m, 4H), 3.79 (m, 1H), 4.29-4. 78 (m, 3H), 6.34 (s, 2H), 6.95 (bs, 1H), 7.14 (s, 4H), 8.22 (bs, 1H).

Example 7 [0075] This example describes the synthesis of TFA-H-Dmt-Tic-NHMe (compound 7 in Table II).

[0076] Boc-Dmt-Tic-NHMe was treated with TFA as reported in Example 3: yield 0.12 g (93%); Rf (A) 0.69 ; HPLC K'= 3. 10; mp 152-154 °C ; [a] 20D = 22.0 ; MH+ 382.

Anal. (C22H27N303*TFA) C, H, N (C 69.27, H 7.13, N 11.02).

Example 8 [0077] This example describes the synthesis of TFA-N, N (Me) 2-Dmt-Tic-NHMe (compound 13 in Table II).

[0078] This compound was obtained by exhaustive methylation of TFA*H-Dmt-Tic- NHMe as described in Example 30: yield 0.12 g (96%); Rf (A) 0.71 ; HPLC K'= 3.19 ; mp 156-158 °C ; [α]20D-19. 3; MH+ 410. Anal. (C24H3lN303aTFA) C, H, N (C 70.39, H 7.63, N 10. 26).

Example 9 [0079] This example describes the synthesis of TFA-H-Dmt-Tic-OMe (compound 8 in Table II).

[0080] Boc-Dmt-Tic-OMe was treated with TFA as described in Example 3: yield 0.25 g (92%); Rf (A) 0. 81 ; HPLC K'= 3. 83; mp 118-120 °C; [α]20D-24. 0; MH+ 382.

Anal. (C22H26N204TFA) C, H, N (C 69.09, H 6.85, N 7.32).

Example 10 [0081] This example describes the synthesis of Boc-Dmt-ßHTic-OMe.

[0082] This compound was obtained by condensation of Boc-Dmt-OH with HCl#ßHTic-OMe (Crabb et al., J. Chem. Soc. Perkins Trans. II: 370-378 (1977) ) [Rf (B) 0.38, HPLC K'= 2. 31, mp 153-155 °C, [afD-37. 5; MH 206] via DCC/HOBt as reported for Boc-Dmt-Tic-OMe: yield 0.42 g (97%); Rf (B) 0.93 ; HPLC K'= 9.27 ; mp 94-96 °C ; [a] 20D +33.9 ; MH+ 497 ;'H-NMR (DMSO) 8 = 1.82 (s, 9H), 2.35 (s, 6H), 2.80-3. 50 (m, 6H), 3.70 (s, 3H), 3.90 (m, 1H), 4.30 (m, 2H), 4.40 (dd, 1H), 6.43 (s, 2H), 7.20 (m, 6H), 9.50 (bs, 1H).

Example 11 [0083] This example describes the synthesis of Boc-Dmt-ßHTic-OH.

[0084] Sodium hydroxide (1N, 1.34 ml) was added to a solution of Boc-Dmt- (3Tic- OMe (0.42 g, 0.90 mmol) in MeOH (10 ml). The reaction mixture was stirred for 24 hr at RT. After evaporation of the solvent, the residue was solubilized in EtOAc and washed with citric acid (10%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et20 : yield 0.42 g (98%); Rf (B) 0.35 ; HPLC K'= 7.54 ; mp 117-119 °C ; [a] 20D +36. 2; MHf 483.

Example 12 [0085] This example describes the synthesis of TFA-H-Dmt-HTic-OH (compound 1 in Table II).

[0086] Boc-Dmt-ßHTic-OH was treated with TFA as described in Example 3: yield (0.26 g (68%); Rf (A) 0.82 ; HPLC K'= 4. 9; mp 118-120 °C ; [a] 20D +95.0 ; MH 383; 'H-NMR (DMSO) 6 = 2. 2 (s, 6H), 2.80-3. 0 (m, 4H), 3.4 (s, 2H), 4.1 (m, 1H), 4.3 (m, 2H), 4.5 (m, 1H), 6.43 (s, 2H), 7.20 (m, 4H), 9.2 (bs, 3H). (C22H26N204-TFA) C, H, N (C 69.09, H 6.85, N 7.32).

Example 13 [0087] This example describes the synthesis of Boc-Dmt-Tic-OH.

[0088] Sodium hydroxide (1 N, 1.3 ml) was added to a solution of Boc-Dmt-Tic- OMe (0. 52 g, 1. 08 mmol) in MeOH (10 ml). The reaction mixture was stirred for 24 hr at RT and treated as described in Example 11: yield 0.45 g (89%); Rf (B) 0.36 ; HPLC K'= 6. 71; mp 147-149 °C ; [α]20D -15. 6; MH+ 469.

Example 14 [0089] This example describes the synthesis of Boc-Dmt-Tic-NH-tetrazole-5-yl.

[0090] HOBt (0.14 g, 0.94 mmol) and DCC (0.19 g, 0.94 mmol) were added to a solution of Boc-Dmt-Tic-OH (0.4 g, 0.85 mmol) and 5 aminotetrazole monohydrate (0.1 g, 0.85 mmol) in DMF (10 ml) at 0 °C. The reaction mixture was stirred for 3 hr art 0 C and 24 hr at RT. After evaporation of the DMF, it was treated as described in Example 11; however, the residue was crystallized from Et20/PtEt (1 : 1, v/v): yield 0.19 g (42%); Rf (B) 0.75 ; HPLC K'= 7. 18; mp 146-148 °C ; [a] 20D-23. 1; MH+ 537 ;'H-NMR (DMSO) 8 = 1.36-1. 45 (d, 9H), 2.16 (s, 6H), 2.96-3. 0 (m, 2H), 3.08-3. 13 (m, 2H), 3.46- 3.56 (m, 1H), 4.34-4. 88 (m, 3H), 6.46 (s, 2H), 7.18-7. 21 (m, 4H), 8.32 (bs, 1H), 8.45 (bs, 1H), 14.39 (bs, 1H).

Example 15 [0091] This example describes the synthesis of TFAH-Dmt-Tic-NH-tetrazole-5-yl (compound 10 in Table II).

[0092] Boc-Dmt-Tic-NH-tetrazole-5-yl was treated with TFA as described in Example 3: yield 0.17 g (87%); Rf (A) 0.79 ; HPLC K'= 3.75 ; mp 131-133 °C ; [a] 20D- 18.3 ; MH+ 436 ;'H-NMR (DMSO) 8 = 2.18 (s, 6H), 2.81-3. 03 (m, 2H), 3.12-3. 18 (m, 2H), 3.66-3. 76 (m, 1H), 4.38-4. 52 (m, 3H), 6.45 (s, 2H), 7.21-7. 25 (m, 4H), 8.27 (bs, 3H), 8.9 (bs, 3H), 14.39 (bs, 1H). Anal. (C22H2sN703*TFA) C, H, N (C 69.68, H 5.79, N22. 51).

Example 16 [0093] This example describes the synthesis of Boc-Dmt-Tic-Ala-OMe.

[0094] NMM (0.05 ml, 0.42 mmol), HOBt (0.07 g, 0.46 mmol) and DCC (0.09 g, 0.46 mmol) were added to a solution of Boc-Dmt-Tic-OH (0.2 g, 0.42 mmol) and HClH-Ala-OMe (0.06 g, 0.42 mmol) in DMF (10 ml) at 0 °C. the reaction was stirred for 3 hr at 0 °C and 24 hr at RT. After evaporation of DMF, the residue was treated as described in Example 14: yield 0.2 g (85%); Rf (B) 0.77 ; HPLC K'= 7.68 ; mp 124-126 °C ; [a] 20D +40.1 ; MH 555 ;'H-NMR (DMSO) 6 = 1. 37-1.46 (m, 12H), 2.16 (s, 6H), 2.96-3. 01 (m, 2H), 3.08-3. 13 (m, 2H), 3.46-3. 56 (m, 1H), 3.73 (s, 3H), 4.03-4. 07 (q, 1H), 4.34-4. 88 (m, 3H), 6.46 (s, 2H), 7.18-7. 21 (m, 4H), 8.32 (bs, 1H), 8.51 (bs, 1H).

Example 17 [0095] This example describes the synthesis of TFA*H-Dmt-Tic-Ala-OMe (compound 19 in Table II). Boc-Dmt-Tic-Ala-OMe was treated with TFA as described in Example 3: yield 0.19 g (92%); Rf (A) 0.64 ; HPLC K'= 3.72 ; mp 130-132 °C ; [a] 20D +90.1 ; MH+ 454. Anal. (C2sH3, N303aTFA) C, H, N (C 66.21, H 6.89, N 9.26).

Example 18 [0096] This example describes the synthesis of Boc-Tic-NH-1-adamantane.

[0097] NMM (0.20 ml, 1.8 mmol), HOBt (0.30 g, 1.98 mmol) and DCC (0.41 g, 1.98 mmol) were added to a solution of Boc-Tic-OH (0.5 g, 1.8 mmol) and 1- aminoadamantane hydrochloride (0.34 g, 1.8 mmol) in DMF (10 ml) at 0 °C. The reaction was stirred for 3 hr at 0 °C and 24 hr at RT. After evaporation of DMF, the residue was treated as described in Example 14: yield 0.6 g (81%) ; Rf (B) 0. 89 ; HPLC K'= 9.68 ; mp 127-129 °C ; [a] 20D +15.3 ; MH"412 ;'H-NMR (DMSO) 8 = 1. 39.-1. 46 (d, 9H), 1.65 (s, 6H), 1.93-2. 07 (m, 9H), 3.08-3. 13 (m, 2H), 4.34-4. 88 (m, 3H), 7.17- 7.20 (m, 4H), 8.08 (bs, 1H).

Example 19 [0098] This example describes the synthesis of TFAcH-Tic-NH-1-adamantane.

[0099] Boc-Tic-NH-1-adamantane (0.6 g; 1.46 mmol) was treated with TFA (2 ml) for 0.5 hr at RT. Et2O/EtPt (1: 1, v/v) was added to the solution until the product precipitated: yield 0.57 g (92%); Rf (A) 0.73 ; HPLC K'= 5.80 ; mp 157-159 °C ; [a] 20D +18. 4; MH+ 311.

Example 20 [00100] This example describes the synthesis of Boc-Dmt-Tic-NH-1-adamantane.

[00101] This compound was obtained by condensation of Boc-Dmt-OH with TFA-H- Tic-NH-1-adamantane via DCC/HOBt as described in Example 1: yield 0.16 g (85%); Rf (B) 0.93 ; HPLC K'= 9. 28; mp 142-144 °C ; [a] 20D +28.1 ; MH+ 603 ;'H-NMR (DMSO) # = 1. 38-1.45 (d, 9H), 1.64 (s, 6H), 1.93-2. 08 (m, 9H), 2.17 (s, 6H), 2.96-3. 01 (m, 2H), 3.08-3. 13 (m, 2H), 3.47-3. 54 (m, 1H), 4.34-4. 88 (m, 3H), 6.46 (s, 2H), 7.17- 7.20 (m, 4H), 8.27 (bs, 1H), 8.47 (bs, 1H).

Example 21 [00102] This example describes the synthesis of TFA*H-Dmt-Tic-NH-1-adamantane (compound 11 in Table II).

[00103] Boc-Dmt-Tic-NH-1-adamantane was treated with TFA as described in Example 3: yield 0.15 g (94%; Rf (A) 0.64 ; HPLC K'= 6.98 ; mp 180-182 °C ; [a] 20D- 2.7 ; Mu+ 502. Anal. (C3, H39N303-TFA) C, H, N (C 74.22, H 7.84, N 8. 38).

Example 22 [0100] This example describes the synthesis of Boc-D-Tic-NH-1-adamantane.

[0101] This compound was obtained by condensation of Boc-D-Tic-OH with 1- amino adamantane hydrochloride as described in Example 18: yield 0.6 g (81%); Rf (B) 0.89 ; HPLC K'= 9.68 ; mp 127-129 °C ; [a] 20D-15. 3; MH+ 412 ;'H-NMR (DMSO) 8 1.39-1. 46 (d, 9H), 1.65 (s, 6H), 1.93-2. 07 (m, 9H), 3.08-3. 13 (m, 2H), 4.34-4. 88 (m, 3H), 7.17-7. 20 (m, 4H), 8.08 (bs, 1H).

Example 23 [0102] This example describes the synthesis of TFA-H-D-Tic-NH-1-adamantane.

[0103] Boc-D-Tic-NH-1-adamantane was treated with TFA as reported for TFA'H- Tic-NH-1-adamantane : yield 0.57 g (92%); Rf (A) 0.73 ; HPLC K'= 5.80 ; mp 157-159 °C ; [a] 20D-18. 4; MH+ 311.

Example 24 [0104] This example describes the synthesis of Boc-Dmt-D-Tic-NH-l-adamantane.

[0105] This compound was obtained by condensation of Boc-Dmt-OH with TFA-H- D-Tic-NH-1-adamantane via DCC/HOBt as described in Example 1: yield 0.16 g (85%); Rf (B) 0.87 ; HPLC K'= 9. 54; mp 135-137 °C ; [a] 20D +14.2 ; MH'-603 ;'H-NMR (DMSO) 8 = 1.39-1. 46 (d, 9H), 1.63 (s, 6H), 1.94-2. 09 (m, 9H), 2.16 (s, 6H), 2.97-3. 03 (m, 2H), 3.05-3. 14 (m, 2H), 3.49-3. 56 (m, 1H), 4.41-4. 91 (m, 3H), 6.41 (s, 2H), 7.18- 7.20 (m, 4H), 8.31 (bs, 1H), 8.43 (bs, 1H).

Example 25 [0106] This example describes the synthesis of TFA#H-Dmt-D-Tic-NH-1- adamantane (compound 12 in Table II).

[0107] Boc-Dmt-D-Tic-NH-1-adamantane was treated with TFA as described in Example 3: yield 0.15 g (94%); Rf (A) 0.58 ; HPLC K'= 7.24 ; mp 164-166 °C ; [a] 20D +27.5 ; Mu"502. Anal. (C3lH39N303aTFA) C, H, N (C 74.22, H 7.84, N 8.38).

Example 26 [0108] This example describes the synthesis of Boc-Ala-NH-1-adamantane.

[0109] NMM (0.20 ml, 1.8 mmol), HOBt (0.30 g, 1.98 mmol) and DCC (0.41 g, 1.98 mmol) were added to a solution of Boc-Ala-OH (0.34 g, 1.8 mmol) and 1-amino adamantane hydrochloride (0.34 g, 1.8 mmol) in DMF (10 ml) at 0 °C. The reaction was stirred for 3 hr at 0 °C and 24 hr at RT. After evaporation of DMF, the residue was treated as described in Example 14: yield 0.52 g (89%); Rf (B) 0.85 ; HPLC K'= 7.73 ; mp 107-109 °C ; [a] 20D +5.9 ; MH+ 324 ; IH-NMR (DMSO) # = 1. 38-1.45 (m, 12H), 1.65 (s, 6H), 1.93-2. 07 (m, 9H), 4.04-4. 07 (q, 1H), 8.08 (bs, 1H), 8.42 (bs, 1H).

Example 27 [0110] This example describes the synthesis of TFAH-Ala-NH-1-adamantane.

[0111] Boc-Ala-NH-1-adamantane (0.52 g, 1.6 mmol) was treated with TFA (2 ml) for 0.5 hr at RT. Et20/EtPt (1: 1, v/v) was added to the solution until the product precipitated: yield 0.5 g (92%); Rf (A) 0.82 ; HPLC K'= 4.95 ; mp 131-133 °C ; [a] 20D +6.8 ; MH+ 224.

Example 28 [0112] This example describes the synthesis of Boc-Dmt-Tic-Ala-NH-1- adamantane.

[0113] This compound was obtained by condensation of Boc-Dmt-Tic-OH with TFA*H-Ala-NH-1-adamantane via DCC/HOBt as described in Example 16: yield 0.12 g (87%); Rf (B) 0.91 ; HPLC K'= 9.32 ; mp 137-139 °C ; [afD +15.5 ; MH+ 674 ; lH- NMR (DMSO) 6 = 1. 38-1.45 (d, 12H), 1.64 (s, 6H), 1.93-2. 08 (m, 9H), 2.17 (s, 6H), 2.96-3. 01 (m, 2H), 3.08-3. 13 (m, 2H), 3.47-3. 54 (m, 1H), 4.03-4. 07 (m, 1H), 4.34-4. 88 (m, 3H), 6.46 (s, 2H), 7.17-7. 20 (m, 4H), 8.27 (bs, 1H), 8.47 (bs, 1H), 8.53 (bs, 1H).

Example 29 [0114] This example describes the synthesis of TFAH-Dmt-Tic-Ala-NH-1- adamantane (compound 21 in Table II).

[0115] Boc-Dmt-Tic-Ala-NH-1-adamantane was treated with TFA as described in Example 3: yield 0.11 g (92%); Rf (A) 0.65 ; HPLC K'= 6. 91; mp 163-165°C ; [a] 20D +20.1 ; MHF 574. Anal. (C34H44N404*TFA) C, H, N (C 71.3, H 7.74, N 9.78).

Example 30 [0116] This example describes the synthesis of TFA*NN- (Me) 2-Dmt-Tic-NH-1- adamantane (compound 15 in Table II).

[0117] NMM (0. 01 ml, 0.11 mmol), 37% aqueous formaldehyde (0.07 ml, 0.83 mmol) and sodium cyanoborohydride (0.016 g, 0.25 mmol) were added to a stirred solution of TFA#H-Dmt-Tic-NH-1-adasmantane (0.7 g, 0.11 mmol) in acetonitrile (10 ml). Glacial acetic acid (0.02 ml) was added over 10 min and the reaction was stirred at RT for 2 hr. The reaction mixture was evaporated in vacuo to give a crude product that was purified by preparative HPLC : yield 0.06 g (90%); Rf (A) 0.64 ; HPLC K'= 7.44 ; mp 118-120 °C ; [a] 20D-58. 01; MH+ 530. Anal. (C33H43N3O3#TFA) C, H, N (C 74.82, H 8.18, N 7. 93).

Example 31 [0118] This example describes the synthesis of TFA-N, N-(Me) 2-Dmt-D-Tic-Ala- NH-1-adamantane (compound 16 in Table II).

[0119] This compound was obtained by exhaustive methylation of TFAH-Dmt-D- Tic-NH-1-adamantane as described in Example 30: yield 0.06 g (90%); Rf (A) 0.61 ; HPLC K'= 7.53 ; mp 134-136 °C ; [ (X] 2'D +8.2 ; MH+ 530. Anal. (C33H43N3O3#TFA) C, H, N (C 74.82, H 8.18, N 7.93).

Example 32 [0120] This example describes the synthesis of Boc-Dmt-Tic-Ala-NHMe.

[0121] This compound was obtained by condensation of Boc-Dmt-Tic-OH with HCl*H-Ala-NHMe via DCC/HOBt as described in Example 16: yield 0.25 g (84%); Rf (B) 0.78 ; HPLC K'= 8.90 ; mp 131-133 °C ; [a] 20D +42.7 ; MH+ 553; 1H-NMR (DMSO) 8 = 137-1. 46 (m, 12H), 2.16 (s, 6H), 2.44-2. 46 (d, 3H), 2.96-3. 01 (m, 2H), 3.08-3. 13 (m, 2H), 3.46-3. 56 (m, 1H), 4.03-4. 07 (q, 1H), 4.34-4. 88 (m, 3H), 6.46 (s, 2H), 7.18- 7.35 (m, 4H), 8.32 (bs, 1H), 8.43 (bs, 1H), 8.51 (bs, 1H).

Example 33 [0122] This example describes the synthesis of TFA*Dmt-Tic-Ala-NHMe (compound 18 in Table II).

[0123] Boc-Dmt-Tic-Ala-NHMe was treated with TFA as described in Example 3: yield 0.24 g (91%); Rf (A) 0.58 ; HPLC K'= 4. 29; mp 142-144 °C ; [a] 20D +28.5 ; Mi 453. Anal. (C25H32N4O4#TFA) C, H, N (C 66.35, H 7.13, N 12.38).

Example 34 [0124] This example describes the synthesis of TFA-[des-NH2-a-piperidine-1-yl- Dmt] -Tic-OH (compound 3 in Table II).

[0125] NMM (0.07 ml, 0.62 mmol), 50% aqueous glutaraldehyde (0.38 ml, 2.36 mmol) and sodium cyanoborohydride (0.45 g, 0.73 mmol) were added to a stirred solution of TFAH-Dmt-Tic-OH (0.15 g, 0. 31 mmol, Matthes et al., Nature 383: 819- 823 (1996) ) in acetonitrile (10 ml). Glacial acetic acid (0.06 ml) was added over 10 min and the reaction was stirred at RT for 2 hr. The reaction mixture was evaporated in vacuo to give a crude product that was purified by preparative HPLC : yield 0.16 g (90%); Rf (A) 0.74 ; HPLC K'= 3.66 ; mp 205-207 °C ; [a] 20D-14. 2; MHF 437. Anal.

(C26H32N204*TFA) C, H, N (C 71.53, H 7.39, N 6.42).

Example 35 [0126] This example describes the synthesis of TFA [des-NH2-a-pyrrolidine-1-yl- Dmt] -Tic-OH (compound 4 in Table II) and TFA* [des-NH2-a-pyrrole-1-yl-Dmt]-Tic- OH (compound 5 in Table II).

[0127] These two compounds were obtained by reductive alkylation of TFA-H-Dmt- Tic-OH with succinaldehyde as described in Example 34. Analytical data for compound 4: yield 0. 016 g (20%); Rf (A) 0. 81 ; HPLC K'= 2.68 ; mp 168-170 °C ; [a] 20D +45.2 ; MH+ 423. Anal. (C25H3oN2O4-TFA) C, H, N (C 71.07, H 7.16, N 6.63). Analytical data for compound 5: yield 0.1 g (80%); Rf (A) 0.67 ; HPLC K'= 5.85 ; mp 185-187 °C ; [a] 20D-56. 4; MH+ 421. Anal. (C25h26N2O4#TFA) C, H, N (C 71.75, H 6.26, N 6.29).

Example 36 [0128] This example describes the synthesis of Boc-Dmt-Tic-NH-tBu.

[0129] This intermediate was obtained by condensation of Boc-Dmt-Tic-OH with tert-butyl amine via DCC/NMM as described in Example 14: yield 0.26 g (87%); Rf (B) 0.88 ; HPLC K'= 7.02 ; mp 153-155 °C ; [a] 20D-8. 2; MH"524 ;'H-NMR (DMSO) 8 = 1.23-1. 44 (m, 18H), 2.16 (s, 6H), 3.05-3. 41 (m, 4H), 3.79 (m, 1H), 4.29-4. 78 (m, 3H), 6.34 (s, 2H), 6.95 (bs, 1H), 7.14 (s, 4H), 8.22 (bs, 1H).

Example 37 [0130] This example describes the synthesis of TFA#H-Dmt-Tic-NH-tBu (compound 9 in Table II).

[0131] Boc-Dmt-Tic-NH-tBu was treated with TFA as described in Example 3: yield 0.12 g (93%); Rf (A) 0.74 ; HPLC K'= 5. 35; mp 150-152 °C ; [a] 20D-15. 6; MH+ 424.

Anal. (C2gH33N303TFA) C, H, N (C 70.89, H 7.85, N 9.92).

Example 38 [0132] This example describes the synthesis of TFAN, N-(Me) 2-Dmt-Tic-NH-tBu (compound 14 in Table II).

[0133] This N,N-alkylated peptide was obtained by exhaustive methylation of TFA*H-Dmt-Tic-NH-tBu (compound 9 in Table II) as reported in Example 21 : yield 0.12 g (88%); Rf (A) 0.78 ; HPLC K'= 6.01 ; mp 157-159 °C ; [a] Z°D-18. 7; MHF 452.

Anal. (C27H37N303aTFA) C, H, N (C 71.81, H 8.26, N 9.30).

Example 39 [0134] This example describes the synthesis of Boc-Dmt-Tic-Ala-NH-tBu.

[0135] This compound was obtained by condensation of Boc-Dmt-Tic-OH with HCl#H-Ala-OtBu via DCC/HOBt as reported for Boc-Dmt-Tic-Ala-OMe: yield 0.2 g (87%); Rf (B) 0.85 ; HPLC K'= 8. 09; mp 150-152 °C ; [a] 20D +28.2 ; E+ 595 ;'H-NMR (DMSO) 6 = 1. 23-1.48 (m, 21H), 2.16 (s, 6H), 3.05-3. 41 (m, 4H), 3.79 (m, 1H), 4.29- 4.78 (m, 4H), 6.34 (s, 2H), 6.95 (bs, 1H), 7.14 (s, 4H), 8.22 (bs, 1H), 8.36 (bs, 1H).

Example 40 [0136] This example describes the synthesis of TFAnDmt-Tic-Ala-NH-tBu (compound 20 in Table II).

[0137] Boc-Dmt-Tic-Ala-NH-tBu was treated with TFA as described in Example 3: yield 0.18 g (91%); Rf (A) 0.64 ; HPLC K'= 5. 35; mp 150-152 °C ; [a] 20D +25.7 ; MHF 495. Anal. (C28H38N404*TFA) C, H, N (C 67.99, H 7.74, N 11.33).

Example 41 [0138] This example describes the synthesis of (R, S)-2-cyano-3- (4-hydroxy-2', 6'- dimethylphenyl) -propanoic acid ethyl ester.

[0139] Ethyl cyanoacetate (0.62 ml, 7.08 mmol) and, after ten minutes, O-carbethoxy-3, 5-dimethyl-4-chloromethylphenol (1.8 g, 7.42 mmol) were added to a solution of sodium ethoxide (Na, 0.17 g, 7.2 mmol; anhydrous EtOH, 12 ml). The reaction mixture was refluxed for 2 hr, cooled and filtered. The solution was evaporated in vacuo.

The residue was crystallized from H20/acetone (5: 1, v/v). The product was purified by column chromatography [Si02 ; Et20/AcOEt (1: 1 v/v) ]: yield 1.05 g (60%); Rf (B) 0.74 ; HPLC K'= 5. 38 ; mp 132-134 °C ; MH+ 248 ;'H-NMR (DMSO) 8 = 1.25-1. 81 (t, 3H), 2.23 (s, 6H), 3.08-3. 14 (m, 2H), 4.14-4. 27 (m, 3H), 6.44 (s, 2H), 9.17 (s, 1H).

Example 42 [0140] This example describes the synthesis of (R, S)-2-cyano-3- (4, hydroxy-2', 6'- dimethylphenyl) -propanoic acid.

[0141] Sodium hydroxide (1N, 4.68 ml, 4.68 mmol) was added to a solution of (R, S)-2-cyano-3- (4-hydroxy-2', 6'-dimethylphenyl) -propanoic acid ethyl ester (1.05 g, 4.24 mmol) in ethanol (10 ml). The reaction mixture was stirred for 24 hr at RT. After evaporation of the solvent, the residue was dissolved in EtOAc and washed with citric acid (10%) and brine. The organic phase was dired and evaporated to dryness. The residue was crystallized from Et2O/PtEt (1: 2, v/v): yield 0.79 g (85%); Rf (B) 0. 31 ; HPLC K'= 3. 54; mp 154-156 °C ; MHF 220.

Example 43 [0142] This example describes the synthesis of [des-NH2-a-cyano- (R, S) -Dmt]-Tic- OtBu.

[0143] HOBt (0.09 g, 0.58mmol) and DCC (0.12 g, 0. 58 mmol) were added to a solution of (R, S)-2-cyano-3- (4-hydroxy-2', 6'-dimethylphenyl) -propanoic acid (0.12 g, 0.53 mmol) and H-Tic-OtBu (0.12 g, 0.53 mmol) in DMF (10 ml) at 0 °C. The reaction mixture was stirred for 3 hr at 0 °C and 24 hr at RT. After evaporation of DMF, the residue was treated as reported for Boc-Dmt-Tic-NH-tetrazole-5-yl: yield 0.18 g (80%) ; Rf (B) 0.82 ; HPLC K'= 8.40 ; mp 140-142 °C ; [α] 20D-13. 85; MH+ 435.

Example 44 [0144] This example describes the synthesis of [des-NH2-a-cyano- (R, S) -Dmt]-Tic- OH.

[0145] [des-NH2-a-cyano- (R, S) -Dmt] -Tic-OtBu (0.18, 0.42 mmol) was treated with TFA (1 ml) for 0.5 hr at RT. Et20/EtPt (1: 1, v/v) was added until the product precipitated: yield 0.15 g (96%); Rf (A) 0.68 ; HPLC K'= 5.55 ; mp 142-144 °C ; [a] 20D - 15. 31 ; MH+ 379; IR (KBr) 3420 (OH), 2360 (nitrile), 1636 (C=O, amide), 1734 (C=O, acid), 1142 cm-1 (C-0, carboxylate anion).

Example 45 [0146] This example describes the synthesis of aH (R, S) -Dmt-Tic-OH (compound 2 in Table II).

[0147] HC1 (1 N, 4.5 ml) and Pt02 (0.05 g) were added to a solution of [des-NH2-a- cyano- (R, S) -Dmt] -Tic-OH (0.15 g, 0.4 mmol) in EtOH (30 ml) and H2 bubbled for 8 hr at RT. After filtration, the solution was evaporated to dryness. The residue was crystallized from Et2O/PtEt (1: 1, v/v): yield 0.15 g (98%); Rf (A) 0.39 ; HPLC K' 2. 51 ; mp 157-159 °C ; [a] 20D-18. 7; MH+ 383 ; IR (KBr) 3432 (NH3+), 1683 (C=O, amide), 1616 (C=O, carboxylate anion), 1203 (C-O, carboxylate anion) cm-'. Anal.

(C22H26N204*TFA) C, H, N (C 69.09, H 6.85, N 7.32).

Example 46 [0148] This example describes the synthesis of N, N (Et) 2-Dmt-Tic-OH (compound 17 in Table II).

[0149] To a stirred solution of TFA. H-Dmt-Tic-OH (0.27 g, 0.56 mM) in CH3CN (10 ml) was added CH3CHO (0.147 ml, 4.26 mM) followed by sodium cyanoborohydride (0.829 g, 1.32 mM). Glacial acetic acid (0.11 ml) was added over 10 min and the reaction was stirred at room temperature for 2hr. The reaction mixture was poured into 100 ml of ethyl acetate and then washed with brine. The organic solvent was dried (Na2S04) and evaporated in vacuo to give the crude product that was purified by preparative HPLC : yield 0.26 g (90%); Rf 0.73 ; HPLC K'= 4. 18; mp 117-119; [a] 20D- 120; MH+ 425. Reference and analytical determinations as in Salvadori et al., J Med.

Chem. 40: 3100-3108 (1997).

Example 47 [0150] This example describes the binding of the compounds of the present invention to 8-opioid receptors and u-opioid receptors.

[0151] The present inventive compounds were assayed with a rat brain synaptosomal preparation (P2) that had been preincubated in 0.1 M NaCl, 0.4 mM GDP, 50 mM HEPES, pH 7.5, and 50 g/ml soybean trypsin inhibitor for 60 min at RT to remove endogenous opioids (Lazarus et al., J. Biol. Chem. 264: 354-362 (1989) ). The assays were conducted as previously described (Salvadori et al. (1995), supra ; Salavadori et al.

(1997), supra ; and Lazarus et al., Eur. J. Med. Chem. 27: 791-797 (1992) ). Briefly, the agonists [3H] DPDPE (30-60 Ci/mmol, NEN-DuPont) and [3H] DAGO (30-60 Ci/mmol, Amersham) were used to label 8 and 11 sites, respectively, under saturation binding conditions (2 hrs at 22 °C). Excess unlabeled peptide (2 uM) established non-specific binding levels and the labeled membranes were rapidly filtered on Whatman GF/C glass fibre filters, thoroughly washed, dried and measured for radioactivity using CytoScint (ICN, Irvine, CA). The 8 antagonist [3H] N, N-(CH3) 2-Dmt-Tic-OH was catalytically dehalogenated from a diiodo-intermediate to a specific activity of 59.88 Ci/mol and binds to 8 receptors with a Kd = 0.39 nM (Kertesz et al., J Label. Compds.

Radiopharmac. 41: 1083-1091 (1998) ). The receptor binding and biological properties of the unlabeled peptide were previously described (Salvadori et al. (1997), supra). All analogues were analyzed in duplicate using 5-9 dosages and at least three independent repetitions using different synaptosomal preparations were conducted for each peptide (actual n values are listed in Table II in parentheses) with results given as mean + SEM.

The affinity constants (Kj) were calculated according to Cheng and Prusoff (Biochem.

Pharmacol. 22: 3099-3108 (1973)).

[0152] As shown in Table II, the elimination of the carboxylate function of H-Dmt- Tic-OH substantially increased the pL affinity of di-and tripeptide derivatives (see compounds 6-16 and 18-21). The net effect was the loss of 6-opioid selectivity and the appearance of compounds that were either essentially nonselective (compounds 6,9, 10 and 15), weakly selective (compounds 8,11 and 16), or moderately p selective (compound 12). The 8 affinity generally remained high for most C-terminally modified analogues with K ; values ranging from 0.07 to 1 nM. Nonetheless, the binding data revealed that several analogues lost high 8 affinity, in particular the methyl ester derivative (compound 8) and those containing the D-Tic enantiomer (compounds 12 and 16).

[0153] Interposing a methylene spacer (see compound 1) between the Ca of the Tic residue and the carboxylate function (see Table I) to prevent diketopiperazine formation had minimal effect on 8 affinity, but enhanced u affinity. The same chemical approach employing a methylene group between the amino group and the Ca of Dmt (compound 2) was more detrimental as compared to H- (R, S) Dmt-Tic-OH. The dipeptide analogues containing hydrazide (compound 6), methyl amide (compound 7), and tetrazole-5-yl (compound 10) exhibited high 8 affinities, but each with a marked gain in u affinity, which was also observed with the Ala-containing tripeptide methyl ester (compound 19) relative to its title compound (H-Dmt-Tic-Ala-NH2).

[0154] The largest increase in p affinity occurred in the Dmt-Tic analogues C- terminally substituted with either tert-butyl amide (compounds 9 and 14) or 1- adamantyl amide (compounds 11,12, 15,16 and 21). In comparison to H-Dmt-Tic- OH, the 11 affinity of compound 15 rose nearly 2, 200-fold relative to N, N (Me) 2-Dmt- Tic-OH. Comparisons between the amidated parental peptides to their C-terminal derivatives, however, indicated smaller changes in u affinities.

[0155] N-alkylation of Dmt-Tic with piperidine-1-yl (compound 3), pyrrolidine-1-yl (compound 4) or pyrrole-1-yl groups (compound 5) (see Table II) decreased 8 affinity, particularly the latter compound whose receptor binding was comparable to H-aDmt- Tic-OH (compound 2), H-Dmt-Tic-OMe (compound 8), and H-Dmt-D-Tic-NH-1- adamantane (compound 12). In spite of the bulky N-terminal substituents, the 6 selectivities of compounds 3 and 4 were analogous to other modified peptides.

[0156] Receptor binding with the 8 antagonist [3H] N, N (Me) 2-Dmt-Tic-OH yielded similar Ki values to those obtained with the agonist [3H] DPDPE in over 80% of the peptides listed in Table II. Exceptions (i. e. , peptides whose Ki values differed by at least an order of magnitude) were the title peptide H-Dmt-Tic-OH, which is relatively unstable and forms a diketopiperazine (Marsden et al., Int. J. Pept. Prot. Res. 41: 313- 316 (1993); Carpenter et al., J. Am. Chem. Soc. 116: 8450-8458 (1994); Caspasso et al., Int. J. Pept. Prot. Res. 45: 567-573 (1995); and Balboni et al., Biol. Chem. 378: 19-29 (1997) ) and analogues (compounds 2,6, 8 and 10).

TABLE I HO 3 Q < X CH3 o ho hic han 'I 4 Compound R'Compound R" No. No. 2 H2NH2C-1 CH2COOH 3/\ 2-5, 17 COOH Zu Compound R' Compound R"<BR> No. No.

Compound R' Compound R"<BR> No. No.

Compound R' Compound R"<BR> No. No.

With respect to Table I, the bold numbers refer to the analogues in Table II. Asterisks denote hiral centers.

TABLE II<BR> MEMBRANE RECEPTOR BINDING O THE MODIFIED DMT-TIC PHARMACOPHORE Ki#(nM) Compound Peptide [3H]DPDPE [3H]N,N(CH3)2- Kiµ(nM) Agonist Antagonist No. Dmt-Tic-OH [3H]DAGO Kiµ/Ki# Kiµ/Ki# Parental Compounds H-Dmt-Tic-OH 0.022 ~ 0.002(6) 0.34 ~ 0.05 (4) 3320 ~ 435 (7) 150800a 9852 H-Dmt-Tic-NH2 1.22 ~ 0.09 (6) 2.13 ~ 0.12 (3) 277 ~ 26 (3) 227a 130 H-Dmt-Tic-Ala-OH 0.29 ~0.03 (6) 0.29 ~ 0.05 (3) 813 ~ 68 (4) 2852a 2784 H-Dmt-Tic-Ala-NH2 0.24 ~ 0.02 (5) 0.69 ~ 0.13 (3) 47 ~ 3.4 (4) 195a 68 N,N(Me)2-Dmt-Tic-OH 0.12 ~ 0.02 (3) 0.07 ~ 0.01 (4) 2435 ~ 462 (3) 20636b 34785 H-(R,S)Dmt-Tic-OH 0.46 ~ 0.001 (3) 0.80 ~ 0.19 94) 1158 ~ 327 (3) 2517 1449 cyclo (DMT-Tic) 9.58 ~ 1.98 (3-5) no activity 635 ~ 69 (3-5) no activity Dipeptide Derivatives 1 H-Dmt-ßTic-OH 0.85 ~ 0.20 (5) 0.71 ~ 0.04 (3) 418 ~ 86 (3) 498 590 2 H-αDmt-Tic-OH 11.2 ~ 3.5 (3) 475 ~ 37 (4) 1740 ~ 16 (3) 155 4 Ki#(nM) Compound Peptide [3H]DPDPE [3H]N,N(CH3)2- Kiµ(nM) Agonist Antagonist No. Dmt-T$ic-OH [3H]DAGO Kiµ/Ki# Kiµ/Ki# 3 [des-NH2-α-piperidine-1-yl]-Dmt-Tic-OH 1.18 ~ 0.10 (3) 0.74 ~ 0.30 (3) 2039 ~ 264 (3) 1728 2755 4 [des-NH2-α-pyrrolidine-1-yl]-Dmt-Tic-OH 1.62 ~ 0.19 93) 1.42 ~ 0.14 (3) 814 ~ 65 (3) 502 573 5 [des-NH2-α-pyrrole-1-yl]-Dmt-Tic-OH 16.6 ~ 2.5 (5) 9.94 ~ 2.5 (3) 5591 ~ 41 (3) 338 562 6 H-Dmt-Tic-NHNH2 0.99 ~ 0.04 (3) 42.0 ~ 7.9 (5) 85.1 ~ 7.3 (3) 86 2 7 H-Dmt-Tic-NHMe 0.47 ~ 0.09 (3) 1.24 ~ 0.15 (4 ) 85.5 ~ 7.7 (3) 182 69 8 H-Dmt-Tic-OMe 9.64 ~ 2.2 (3) 500 ~ 90 (5 ) 423 ~ 25 (3) 44 0.8 9 H-Dmt-Tic-NH-tBu 0.43 ~ 0.07 (5) 0.93 ~ 0.15 (4) 5.96 ~ 0.82 (4) 14 6 10 H-dmt-Tic-NH-tetrazole-5-yl 0.70 ~ 0.03 (3) 9.75 ~ 1.73 (5) 37.0 ~ 4.5 (3) 53 4 11 H-Dmt-Tic-NH-1-adamantane 0.26 ~ 0.05 (4) 1.01 ~ 0.26 (3) 0.76 ~ 0.05 (4) 3 0.8 12 H-Dmt-D-Tic-NH-1-adamantane 24.5 ~ 4.9 (6) 70.3 ~ 7.25 (3) 0.26 ~ 0.08 (4) 0.01 0.004 13 N,N(Me)2-Dmt-Tic-NHMe 0.54 ~ 0.07 (3) 0.28 ~ 0.02 (3) 359 ~ 62 (3) 669 1268 14 N,N(Me)2-Dmt-Tic-NH-tBu 0.61 ~ 0.02 (3) 0.11 ~ 0.04 (3) 226 ~ 21 (3) 369 2132 15 N,N(Me)2-Dmt-Tic-NH-1-adamantane 0.16 ~ 0.17 (3) 0.12 ~ 0.02 (3) 1.12 ~ 0.10 (3) 7 9 16 N,N(Me)2-Dmt-D-Tic-NH-1-adamantane 140 ~ 30 (6) 120 ~ 25.9 (3) 50.5 ~ 2.6 (3) 0.36 0.42 17 N,N(Et)2-DMT-Tic-OH 0.982~ 0.19 (3-5) not tested 35.3 ~ 1.96 (3-5 ) not tested Ki#(nM) Compound Peptide [3H]DPDPE [3H]N,N(CH302. Kiµ(nM) Agonist Antagonist No. Dmt-Tic-OH [3H]DAGO Kiµ/Ki# Kiµ/Ki# Tripeptide Derivatives 18 H-Dmt-Tic-Ala-NHMe 0.058 ~ 0.01 (3) 0.12 ~ 0.04(3) 5.75 ~ 0.72 (3) 100 47 19 H-Dmt-Tic-Ala-OMe 0.23 ~ 0.09 (3) 0.14 ~ 0.04 (3) 11.3 ~ 1.87 (3) 48 83 20 H-Dmt-Tic-Ala-NH-tBu 0.066 ~ 0.01 (3) 0.08 ~ 0.02 (3) 4.03 ~ 0.21 (3) 61 48 21 H-Dmt-Tic-Ala-NH-1-adamantane 0.073 ~ 0.02 (3) 0.04 ~ 0.01 (3) 2.52 ~ 0.56 (4) 35 72 [0157] With respect to Table II, the numeric value in the parentheses indicates the number (n) of repetitions of independent binding assays using different synaptosomal preparations. aSalvadori et al. (1995), supra. bSalvadori et al. (1997), supra.

Example 48 [0158] This example describes the functional bioactivity of the present inventive compounds.

[0159] The functional bioactivity of the present inventive compounds was assessed using standard functional bioassays of 6 and activity in MVD and GPI (Salvadori et al. (1995), supra; and Salvadori et al., R Med. Chem. 36: 3748-3756 (1993) ). Briefly, a 2-3 cm segment of GPI was placed in a 20 ml tissue bath containing Kreb's solution, 70 uM hexamethonium bromide and 0.125 AM mepyramine and aerated with 95% 02/5% C02 at 36 °C. Transmural stimulation of GPI was by means of a square-wave electrical pulse of 0.5 ms duration at a frequency of 0.1 Hz. A single MVD was suspended in 4 ml modified Kreb's solution aerated with 95% 02/5% C02 at 33 °C. An isometric transducer recorded the twitch induced by field stimulation (0.1 Hz for 1 ms at 40 V).

Dose-response curves were obtained (Salvadori et al. (1995), supra) for both tissues.

The u agonist activity was compared against dermorphin (ICo = 1.82 nM) and 8 antagonism was determined through the inhibition of deltorphin C (81 receptor agonist, ICso = 0.54 nM) in comparison to the nonpeptide 8 antagonist naltrindole. Data were derived from at least four independent tissue samples and dose-response curves from which the pA2 values were determined (Salvadori et al. (1997), supra) according to Arunlakshana and Schild (Br. J. Pharmacol. 14: 48-58 (1959) ). In other words, antagonism was expressed as pA2 (negative logarithm of the concentration that causes 50% inhibition) and agonism was expressed as the ICso.

[0160] Dmt-Tic analogues (compounds 1,9, 13,15, 20 and 21) demonstrated the highest 8-antagonist functional bioactivities (Table III) as well as some of the highest 6- receptor affinities (Table II). Of greater interest, however, was the observation that several analogues acquired unusual bioactivity profiles. For example, both of compounds 13 and 15 elicited excellent 8 antagonism, yet the former was a weak pL antagonist and the latter clearly displayed p agonism (Table III). Inclusion of the D-Tic enantiomer (compounds 12 and 16) essentially greatly reduced bioactivity on MVD with uM activity on GPI. Replacement of the N-terminal amine through alkylation by piperidine-1-yl (compound 3), pyrolidine-1-yl (compound 4) or pyrrole-1-yl (compound 5) was detrimental for all bioactivity measurements on MVD (Table III).

N-alkylation by methyl groups to form secondary or tertiary amines was the only N- terminal substitution tolerated (Lazarus et al. (1998), supra ; see Table III).

Interestingly, the non-alkylated, C-terminally modified dipeptides, i. e. , compounds 11 and 12, lacked 8 antagonism; however, compound 11 surprisingly manifested a weak 8 agonism and moderate 11 agonism in spite of its high 8 affinity (see Table II), while other analogues, i. e. , compounds 7,13, 14 and 18, also produced anomalous biological activities on MVD and GPI (see Table III) relative to their receptor binding parameters (see Table II). H-Dmt-Tic-NHMe (compound 7) had extraordinarily weak 8 agonism with low u antagonism, while its Ala-tripeptide derivative (compound 18) gave very weak 8 and p agonism. Despite the high 8-receptor affinity of compound 14, the bioactivity data indicated only modest 8 antagonism and weak u antagonism. The tetrazole-5-yl-amide analogue (compound 10) was a weak 8 antagonist with very minimal activity on GPI (see Table III).

TABLE III<BR> FUNCTIONAL BIOACTIVITY OF THE MODIFIED DMT-TIC PHARMACOPHORE MVD GPI Compound Peptide pA2 (range) Ke(nM) ED50(µM) pA2 (range) Ke(µM) ED50(µM) No. H-Dmt-Tic-OH 8.2 5.7 - - - >10a H-Dmt-Tic-NH2 7.2 42 - - - >10a H-Dmt-Tic-Ala-OH 8.4 4.0 - - - >10a H-Dmt-Tic-Ala-NH2 8.0 9.0 - - - 4.74 ~ 0.9 N,N(Me)2-Dmt-Tic-OH 9.4 0.28 - - - >10b H-(R,S)Dmt-Tic-OH 8.17 (7.8-8.6) 6.76 - - - >10 cyclo (DMT-Tic) 5.43 1 H-Dmt-ßTic-OH 8.8 (8.6-9.1) 1.41 - - - >10 3 [des-NH2-α-piperidine-1-yl]-Dmt-Tic- 7.31 (7.15-7.47) 49 - - - >10 OH MVD GPI Compound Peptide pA2 (range) Ke(nM) ED50(µM) pA2 (range) Ke(µM) ED50(µM) No. 4 [des-NH2-α-pyrrolidine-1-yl]-Dmt- 6.9 (6.9-7.1) 121 - - - >10 Tic-OH 5 [des-NH2-α-pyrrole-1-yl]-Dmt-Tic- 6.39 (6.0-6.7) 408 - - - >10 OH 7 H-Dmt-Tic-NHMe -d >10,000 21.2 (9.0- 5.94 (4.2-7.6) 1.15 >10 45) 9 H-Dmt-Tic-NH_tBu 8.24 (8.2-8.5) 1.74 - - - 1.04 (0.69-1.5) 10 H-Dmt-Tic-NH_tetrazole-5-yl 7.44 (7.1-7.7) 36.3 - - - 8.2 (2.7-24.9) 11 H-Dmt-NH-1-adamantane - >10,000 0.87 (0.8- - - 0.036 (0.019-0.068) 1.2) 12 H-Dmt-D-tic-NH-1-adamantane - >10,000 - - - 1.68 (1.23-2.3) 13 N,N(Me)2-Dmt-Tic 9.39 (8.8-9.9) 0.41 - 6.41 (6.3-6.5) 0.39 >10 14 N,N(Me)2-Dmt-Tic-NH-tBu 7.85 (7.5-8.1) 14.1 - .52 (6.2-6.7) 0.30 >10 15 N,N(Me)2-Dmt-Tic-NH-1-adamantane 9.06 (8.6-9.5) 0.87 - - - 0.016 (0.011-0.023) MVD GPI Compound Peptide pA2(range) Ke(nM) ED50(µM) pA2 (range) Ke(µM) Ed50(µM) No. 16 N,N(Me)2-Dmt-D-Tic-NH-1 6.91 (6.8-7.0) 128 - - - 4.48 (3.17-6.32) adamantane 17 N,N(Et)2-Dmt-Tic-OH not tested not tested 18 H-Dmt-Tic-Ala-NHMe - - 1.29 (1.18- - - 0.29 (0.18-0.45) 1.42) 20 H-Dmt-Tic-Ala-NH-tBu 9.16 (8.7-9.5) 0.69 - 6.76 (6.6-6.9) 0.17 >10 21 H-Dmt-Tic-Ala-NH-1-adamantane 9.29 (9.0-9.5) 0.51 - - - 1.0 (0.7-3.0) With respect to Table III, data were derived from at least four (at least three for compounds 17 and 18) independent tissue samples and dose-<BR> response curves. pA2 is the mean and the range is in parentheses. aSalvadori et al. (1995) , supra. bSalvadori et al. (1997), supra. cTemussi et<BR> al. (1994), supra. dA dash indicates the absence of pharmacological activity.

Example 49 [0161] This example describes the ability of certain compounds of the present invention to inhibit hMDR-1.

[0162] Stably transfected G-185 fibroblasts containing hMDR-1 and NIH 3T3 cell lines were grown to confluent monolayers in DMEM containing 4.5 g glucose/1, 10 % fetal calf serum, L-glutamine, penicillin (50 units/ml), streptomycin (50 pg/ml) and 1 mM sodium pyruvate in 5% CO2 at 37 °C. G-185 cells were maintained in 60 ng/1 colchicine.

[0163] The assay for Pgp is based on the diffusion of non-fluorescent calcein AM across the cell membrane. Calcein AM is then hydrolyzed and accumulates as the fluorescent calcein. In transfected cells expressing excess hMDR-1, Pgp extrudes calcein AM from the cell before hydrolysis occurs; inhibition of hMDR-1 results in the accumulation of intracellular calcein. Transfected G-185 cells and the NIH 3T3 cells were analyzed in triplicate using seven graded dosages of verapamil or the opioid peptides in 96-well plates. Cells (2.5 x 106/well) were incubated in 100 ul culture medium for 15 min at 37 °C and 50 u. of 1 1 uM calcein AM in PBS, plus 50 u. l of verapamil or one of the present inventive compounds in PBS, or 100 u. l of PBS alone (control) were added. Cells were mixed and incubated for 15 min at 37 °C, then centrifuged for 5 min at 2,000 x g, and then washed three times in 200 pl PBS. Cell pellets were resuspended in 200 zip PBS and transferred to white Microfluor Plates (Nunc, Copenhagen, Denmark) in order to measure calcein-specific fluorescence (494 nm absorption; 517 nm emission). Internal controls were carried out with each experiment using verapamil and PBS in order to compare the consistency between cell preparations. Means SE (error bars) were determined and plotted using Prism (GraphPad, San Diego, CA).

[0164] The results are shown in Fig. lA-1D, all of which are graphs of fluorescence vs. concentration (uM). In Fig. 1A, the inhibition of hMDR-1 in G-185 cells (o) and NIH 3T3 cells (9) by verapamil (control) is shown. In Fig. 1B, the inhibition of hMDR- 1 in G-185 cells with NIH 3T3 cells (*) by the 1-adamantyl amide derivatives N, N (Me) 2-Dmt-Tic-NH-l-adamantane (compound 15) (o), H-Dmt-Tic-NH-1- adamantane (compound 11) (A), H-Dmt-Tic-Ala-NH-1-adamantane (compound 12) ('), and N,N@ (Me) 2-Dmt-Tic-OH () is shown. In Fig. 1 C, the inhibition of hMDR-1 in G-185 cells with NIH 3T3 cells (A) by the tert-butyl amide derivatives N, N (Me) 2-Dmt- Tic-NH-tBu (compound 14) (o), H-Dmt-Tic-Ala-NH-tBu (compound 20) (A), and H- Dmt-Tic-NH-tBu (compound 9) (~) is shown. In Fig. 1D, the inhibition of hMDR-1 in NIH 3T3 cells ()) and with naltrindole ('-') or in G-185 cells with naltrindole (A), cyclo (Dmt-Tic) (o) or N, N (Et) 2-Dmt-Tic-OH (compound 17) (~) is shown.

[0165] Inhibition of Pgp in G-185 fibroblasts occurred in a dose-response manner with Dmt-Tic dipeptides containing 1-adamantyl amide and with or without N alkylation (Fig. 1B) comparable to verapamil (Fig. 1A). Peptides lacking N-or C- terminal hydrophobic substituents (Fig. 1A, deltorphin B, deltorphin A, DER, DAGO, DPDPE, DAMME, DtMe, DPD, [Trp4, Tyr'] DER, [Trp4, Lys-OH7] DER, DSB, DTA, PipDTOH and MeDTOH) were inactive. The maximum effective dose of the peptides and verapamil fell within the range of 75-100 pLM. In contrast, NIH-3T3 fibroblasts lacking membrane Pgp-1 were unresponsive. The spatially confined aromatic rings of Dmt-Tic and the saturated rings of 1-adamantane presented an optimal configuration to support the inhibition of hMDR-1 activity, which is diminished by inclusion of the anion function that determines 6-receptor selectivity. The order of inhibition by 1- adamantyl amide derivatives was: N, N (Me) 2-Dmt-Tic-NH-1-adamantane (compound 15) = H-Dmt-Tic-NH-1-adamantane (compound 11) > H-Dmt-Tic-Ala-NH-1- adamantane (compound 21) » N, N (Me) 2-Dmt-Tic-OH (inactive). These results clearly demonstrate that inhibition of hMDR-1 only occurs in the presence of Dmt-Tic modified with hydrophobic groups. Fortuitously, H-Dmt-Tic-NH-1-adamantane (compound 15) only exhibited weak biological activities (Table III) and, thus, may function as a"chemosensitizer"without side effects on the 8-or u-opioid systems.

[0166] Although the 1-adamantyl amide derivative of Dmt-Tic enhanced the inhibition of hMDR-1, incorporation of Ala3-NH-1-adamantyl-amide curtailed activity (see Fig. 1B). These observations suggest that distance between hydrophobic and aromatic centers in the peptide play a definitive role in the interaction with hMDR-1, and that the inhibitor effectiveness of the Dmt-Tic compounds was independent of their receptor binding activities or pharmacological bioactivites (Table III). A reduction in the hydrophobicity at the C-terminus by the graded reduction in hydrophobicity from 1- adamantyl amide to tert-butyl amide and methyl amide (see Fig. 1C) was comparable to the diminution of activity in dipyridamole analogues containing only a single methyl group. The order of activity by tert-butyl amide substances was: N, N (Me) 2-Dmt-Tic- NH-tBu (compound 14) > H-Dmt-Tic-Ala-NH-tBu (compound 20) > H-Dmt-Tic-NH- tBu (compound 9) >> H-Dmt-Tic-NHMe (compound 7) (inactive). In the case of tert- butyl amide derivatives, the additional hydrophobicity afforded by N-alkylation by methyl groups increased the inhibition of hMDR-1 (see Fig. 1C). While N, N (Me) 2- Dmt-Tic-OH was inactive, N-alkylation by ethyl groups increased the inhibition of hMDR-1, but substantially less than that observed with the tert-butyl amide or 1- adamantyl amide conjugates (see Fig. 1D). Interestingly, the elevation in hydrophobicity associated with N-alkylation of the Dmt-Tic pharmacophore enhanced 8 antagonism in vitro by 20-fold without significant changes on 8-opioid binding parameters. On the other hand, 1-adamantyl amide or tert-butyl amide increased pL- receptor binding without interfering with 8-receptor interactions to produce non- selective or u-selective opioid peptides.

[0167] The spontaneous formation of the diketopiperazine, cyclo (Dmt-Tic), was a moderately effective inhibitor of hMDR-1 (see Fig. 1D). These results with cyclo (Dmt- Tic) call into question the requirement for a tertiary amine for Pgp inhibition. On the other hand, the non-peptide 8 antagonist naltrindole exhibited inhibition that dissipated at high concentrations (see Fig. 1D), which suggests possible disruption of membrane integrity. The lack of inhibition with opioid agonists, such as dermorphin, deltorphin and the enkephalin analogues DAGO, DPDPE and DAMME, indicates that the charged function retards passage through the BBB. Thus, the data indicate that enhancing the hydrophobicity of Dmt-Tic analogues in order to accelerate passage through the BBB also makes them better substrates for Pgp. Specifically, the covalent addition of tert- butyl amide or 1-adamantyl amide converts a potent opioid antagonist into an inhibitor of hMDR-1. The fact that H-Dmt-Tic-NH-1-adamantane (compound 15) exhibits minimal bioactivity would make it an erstwhile candidate as a chemosensitizer for chemotherapy of cancers containing hMDR-1.

Example 50 [0168] This example describes the synthesis of (lH-Benzimidazol-2-yl-methyl)- carbamic acid tert-butyl ester (Boc-NH-CH2-Bid).

[0169] A solution of Boc-Gly-OH (1.7 g, 9.6 mmol) and NMM (1 ml, 9.6 mmol) in DMF (10 ml) was treated at-20 °C with isobutyl chloroformate (IBCF) (1.2 ml, 9.6 mmol). After 10 min at-20 °C, o-phenylendiamine (1 g, 9.6 mmol) was added. The reaction mixture was allowed to stir while slowly warming to room temperature (1 h) and then stirred for an additional 3 h.

[0170] The solvent was evaporated and the residue was partitioned between EtOAc and H20. The EtOAc layer was washed with 5% NaHC03 and brine and dried over Na2S04. The solution was filtered, the solvent evaporated and the residual solid dissolved in glacial acetic acid (10 ml). The solution was heated at 65 °C for 1 h. After evaporation of the solvent, the residue was crystallized from Et20/Pe (1: 9, v/v): yield 2.11 g. (89 %); Rf (B) 0.43 ; HPLC K'= 3.95 ; mp 131-133 °C ; MH+ 248 ; 1H-NMR (DMSO) 8 = 1. 40 (s, 9H), 4.22-4. 40 (m, 2H), 7.26-7. 70 (m, 5H), 7.98 (s, 1H).

Example 51 [0171] This example describes the synthesis of Boc-Tic-NH-CH2-Bid.

[0172] To a solution of Boc-Tic-OH (0.3 g, 1.08 mmol) and 2 TFA-H-NH-CH2-Bid (Cescon et al. , J. Org. Chem. , 27: 581-586 (1962) ) (0.41 g, 1.08 mmol) in DMF (10 ml) at 0 °C were added NMM (0.24 ml, 2.16 mmol), HOBt (0.18 g, 1.19 mmol) and WSC (0.23 g, 1.19 mmol). The reaction mixture was stirred for 3 h at 0 °C and 24 h at room temperature. After evaporation of DMF, the residue was solubilized in EtOAc and washed with NaHC03 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et20/Pe (1: 9, v/v): yield 0.36 g. (82%); Rf (B) 0.68 ; HPLC K'= 8. 12; mp 142-144 °C ; [a] 20D-12. 1; MH+ 407; 1H-NMR (DMSO) 6= 1. 38-1.42 (d, 9H), 3.08-3. 15 (m, 2H), 4.22-4. 53 (m, 5H), 7.05-7. 71 (m, 9H), 7.97 (s, 1H).

Example 52 [0173] This example describes the synthesis of 2TFA-H-Tic-NH-CH2-Bid.

[0174] Boc-Tic-NH-CH2-Bid (0.36 g, 0.89 mmol) was treated with TFA (1 ml) for 0.5 h at room temperature. Et20/Pe (1: 1, v/v) were added to the solution until the product precipitated: yield 0.46 g (97%); Rf (A) 0.43 ; HPLC K'= 4,11 ; mp 180-182 °C ; [a] 20D-14. 8; Mu+307.

Example 53 [0175] This example describes the synthesis of Boc-Dmt-Tic-NH-CH2-Bid.

[0176] To a solution of Boc-Dmt-OH (0.25 g, 0.82 mmol) and 2TFA-H-Tic-NH- CH2-Bid (0.44 g, 0.82 mmol) in DMF (10 ml) at 0 °C were added NMM (0.18 ml, 1.64 mmol), HOBt (0.14 g, 0.90 mmol) and WSC (0.17 g, 0.90 mmol). The reaction mixture was stirred for 3 h at 0 °C and 24 h at room temperature. After evaporation of DMF, the residue was solubilized in EtOAc and washed with NaHC03 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et20/Pe (1: 9, v/v): yield 0.39 g (80%); Rf (B) 0.68 ; HPLC K'= 8. 98; mp 142-144 °C ; [α]20D-22. 5; MH"598 ;'H-NMR (DMSO) 8 = 1.32-1. 39 (d, 9H), 2.17 (s, 6H), 2.85-3. 10 (m, 4H), 4.22-4. 65 (m, 6H), 6.37 (s, 2H), 7.05-7. 71 (m, 10H), 7.97 (s, 1H).

Example 54 [0177] This example describes the synthesis of 2TFA-H-Dmt-Tic-NH-CH2-Bid (compound 24).

[0178] Boc-Dmt-Tic-NH-CH2-Bid (0.1 g, 0.17 mmol) was treated with TFA (1 ml) for 0.5 h at room temperature. Et20/Pe (1: 1, v/v) were added to the solution until the product precipitated: yield 0.12 g (97%); Rf (A) 0. 31 ; HPLC K'= 5.40 ; mp 152-154 °C ; [α]20D-35. 2; MHF 499.

Example 55 [0179] This example describes the synthesis of (lH-Benzimidazol-2-ylethyl)- carbamic acid tert-butyl ester (Boc-NH-CH2-CH2-Bid).

[0180] This product was obtained from Boc-pAla-OH and o-phenylenediamine as reported for Boc-NH-CH2-Bid: yield 0.8 g (85%); Rf (B) 0.52 ; HPLC K'= 4.25 ; mp 142-144 °C ; MH+ 262; 1H-NMR (DMSO) 8 = 1.38 (s, 9H), 2.81-3. 29 (m, 4H), 7.25- 7.68 (m, 5H), 7.95 (s, 1H).

Example 56 [0181] This example describes the synthesis of Boc-Tic-NH-CH2-CH2-Bid.

[0182] This substance was obtained by condensation of Boc-Tic-OH with 2TFA- H2N-CH2-CH2-Bid (Cescon et al. , J. Org. Chem., 27 : 581-586 (1962) ) via WSC/HOBt as reported for Boc-Tic-NH-CH2-Bid: yield 0.36 g (88%); Rf (B) 0.72 ; HPLC K'= 8.38 ; mp 147-149 °C ; [α]20D-10. 7; MH+ 421; 1H-NMR (DMSO) # = 1. 39-1.41 (d, 9H), 2.80-3. 31 (m, 6H), 4.21-4. 55 (m, 3H), 7.07-7. 69 (m, 9H), 7.99 (s, 1H).

Example 57 [0183] This example describes the synthesis of 2TFA-H-Tic-NH-CH2-CH2-Bid.

[0184] Boc-Tic-NH-CH2-CH2-Bid was treated with TFA as reported for 2TFA-H- Tic-NH-CH2-Bid : yield 0.35 g (98%); RJ (A) 0. 48 ; HPLC K'= 4. 18 ; 176-178 °C ; [a] 20D-12.3 ; Mu+321.

Example 58 [0185] This example describes the synthesis of Boc-Dmt-Tic-NH-CH2-CH2-Bid.

[0186] This compound was obtained by condensation of Boc-Dmt-OH with 2TFA- H-Tic-NH-CH2-CH2-Bid via WSC/HOBt as reported for Boc-Dmt-Tic-NH-CH2-Bid: yield 0.29 g (83%); Rf (B) 0.72 ; HPLC K'= 9.03 ; mp 151-153 °C ; [a] 20D-19. 9 ; MH+ 612; 1H-NMR (DMSO) 6 = 1. 33-1.41 (d, 9H), 2.16 (s, 6H), 2.81-3. 31 (m, 4H), 4.22- 4.64 (m, 4H), 6.38 (s, 2H), 7.04-7. 69 (m, 10H), 7.98 (s, 1H).

Example 59 [0187] This example describes the synthesis of 2TFA-H-Dmt-Tic-NH-CH2-CH2- Bid (compound 25).

[0188] Boc-Dmt-Tic-NH-CH2-CH2-Bid was treated with TFA as reported for 2TFA -H-Dmt-Tic-NH-CH2-Bid : yield 0.052 g (87%); Rf (A) 0.34 ; HPLC K'= 4. 88; mp 156- 158 °C ; [a] 20D-30.7 ; MH+ 513.

Example 60 [0189] This example describes the synthesis of Boc-Gly-NH-CH2-Bid.

[0190] This compound was obtained from Boc-Gly-Gly-OH and o- phenylenediamine as reported for Boc-NH-CH2-Bid: yield 0.73 g (82%); Rf (B) 0.55 ; HPLC K'= 4. 53; mp 148-150 °C ; MH+ 305; 1H-NMR (DMSO) 8 = 1.40-1. 42 (d, 9H), 3.85-4. 46 (m, 4H), 7.25-7. 71 (m, 6H), 7.94 (s, 1H).

Example 61 [0191] This example describes the synthesis of 2TFA-H-Gly-NH-CH2-Bid.

[0192] Boc-Gly-NH-CH2-Bid was treated with TFA as reported for 2TFA-H2N- CH2-Bid: yield 0.49 g (97%); Rf (A) 0.45 ; HPLC K'= 1. 88; mp 187-189 °C ; MH+ 205.

Example 62 [0193] This example describes the synthesis of Boc-Tic-Gly-NH-CH2-Bid.

[0194] This substance was obtained by condensation of Boc-Tic-OH with 2TFA-H- Gly-NH-CH2-Bid via WSC/HOBt as reported for Boc-Tic-NH-CH2-Bid: yield 0.38 g (84%); Rf (B) 0.73 ; HPLC K'= 8.16 ; mp 146-148 °C ; [a] 20D-10. 4; MH+ 464; 1H-NMR (DMSO) 6= 1. 40-1.42 (d, 9H), 3.08-3. 15 (m, 2H), 3.85-4. 55 (m, 7H), 7.03-7. 70 (m, 10H), 7.95 (s, 1H).

Example 63 [0195] This example describes the synthesis of 2TFA#H-Tic-Gly-NH-CH2-Bid.

[0196] Boc-Tic-Gly-NH-CH2-Bid was treated with TFA as reported for 2TFA-H- Tic-NH-CH2-Bid: yield 0.32 g. (95%); Rf (A) 0.47 ; HPLC K'= 4. 35; mp 163-165 °C ; [α]20D-10.1 ; MH+ 364.

Example 64 [0197] This example describes the synthesis of Boc-Dmt-Tic-Gly-NH-CH2-Bid.

[0198] This compound was obtained by condensation of Boc-Dmt-OH with 2TFA- H-Tic-Gly-NH-CH2-Bid via WSC/HOBt as reported for Boc-Dmt-NH-CH2-Bid: yield 0.28 g (85%); Rf (B) 0.73 ; HPLC K'= 9. 03; mp 146-148 °C ; [a] 20D-19. 3; MH+ 655; 1H-NMR (DMSO) # = 1. 35-1.40 (d, 9H), 2.17 (s, 6H), 2.85-3. 12 (m, 4H), 4.20-4. 72 (m, 8H), 6.37 (s, 2H), 7.05-7. 75 (m, 11H), 7.99 (s, 1H).

Example 65 [0199] This example describes the synthesis of 2TFA-H-Dmt-Tic-Gly-NH-CH2-Bid (compound 26).

[0200] Boc-Dmt-Tic-Gly-NH-CH2-Bid was treated with TFA as reported for 2TFA- H-Dmt-Tic-NH-CH2-Bid: yield 0.068 g (96%); Rf (A) 0.35 ; HPLC K'= 4. 95; mp 155- 157 °C ; [α20D-30. 8; MH+ 557.

Example 66 [0201] This example describes the synthesis of Boc-Tic-Gly-NH-Ph.

[0202] This compound was obtained by condensation of Boc-Tic-OH with TFA-H- Gly-NH-Ph49 as reported for Boc-Tic-NH-CH2-Bid: yield 0.24 g (85%); Rf (B) 0.67 ; HPLC K'= 10.73 ; mp 131-133 °C ; [α]20D-36. 8; MH+ 410; 1H-NMR (DMSO) # = 1. 39- 1.41 (d, 9H), 3.08-3. 15 (m, 2H), 3.61 (d, 2H), 4.22-4. 92 (m, 3H), 6.91-7. 31 (m, 10H), 8.96 (bs, 1H).

Exmaple 67 [0203] This example describes the synthesis of TFA-H-Tic-Gly-NH-Ph.

[0204] Boc-Tic-Gly-NH-Ph was treated with TFA as reported for 2 TFA-H-Tic-NH- CH2-Bid: yield 0.18 g (96%); Rf (A) 0. 35 ; HPLC K'= 6. 07; mp 165-167 °C ; [a] 20D- 32, 5; MH+ 310.

Example 68 [0205] This example describes the synthesis of Boc-Dmt-Tic-Gly-NH-Ph.

[0206] This compound was obtained by condensation of Boc-Dmt-OH with TFA-H- Tic-Gly-NH-Ph as reported for Boc-Dmt-Tic-NH-CH2-Bid: yield 0.14 g (84%); Rf (B) 0.64 ; HPLC K'= 9.9 ; mp 144-146 °C ; [α]20D-19. 7; MH+ 601 ; 1H-NMR (DMSO) 6 = 1.40-1. 42 (d, 9H), 2.17 (s, 6H), 3.08-3. 15 (m, 4H), 3.61 (d, 2H), 4.22-4. 92 (m, 3H), 6.37 (s, 2H), 6.91-7. 31 (m, 11H), 8.94 (bs, 1H).

Example 69 [0207] This example describes the synthesis of TFA-H-Dmt-Tic-Gly-NH-Ph (compound 27).

[0208] Boc-Dmt-Tic-Gly-NH-Ph was treated with TFA as reported for 2 TFA-H- Dmt-Tic-NH-CH2-Bid: yield 0.07 g (97%); Rf (A) 0.41 ; HPLC K'= 7. 18; mp 155-7 °C ; [α]20D-21. 8; MH+ 444.

Example 70 [0209] This example describes the synthesis of Boc-Tic-Gly-NH-Bzl.

[0210] This compound was obtained by condensation of Boc-Tic-OH with TFAXH- Gly-NH-Bzl50 as reported for Boc-Tic-NH-CH2-Bid : yield 0.26 g. (88%) ; Rf (B) 0.72 ; HPLC K'= 11.02 ; mp 123-125 °C ; [a] 20D-35. 2; MH+ 424; 1H-NMR (DMSO) 8 = 1.39- 1.41 (d, 9H), 3.08-3. 15 (m, 2H), 3.61 (d, 2H), 4.22-4. 92 (m, 5H), 6.91-7. 31 (m, 10H), 8.25 (t, 1H).

Example 71 [0211] This example describes the synthesis of TFA-H-Tic-Gly-NH-Bzl.

[0212] Boc-Tic-Gly-NH-Bzl was treated with TFA as reported for 2 TFAXH-Tic- NH-CH2-Bid: yield 0.20 g (97%); Rf (A) 0.38 ; HPLC K'= 6. 14; mp 151-153 °C ; [a] 20D - 30. 8; MH+ 324.

Example 72 [0213] This example describes the synthesis of Boc-Dmt-Tic-Gly-NH-Bzl.

[0214] This compound was obtained by condensation of Boc-Dmt-OH with TFAXH- Tic-Gly-NH-Bzl as reported for Boc-Dmt-Tic-CH2-Bzl: yield 0.15 g (81%) ; Rf (B) 0.68 ; HPLC K'= 10.4 ; mp 138-140 °C ; [a] 20D-18. 9; MH+ 615; 1H-NMR (DMSO) 8 1.40-1. 42 (d, 9H), 2.17 (s, 6H), 3.08-3. 15 (m, 4H), 3.61 (d, 2H), 4.22-4. 92 (m, 5H), 6. 37 (s, 2H), 6.91-7. 31 (m, 11H), 8.25 (t, 1H).

Example 73 [0215] This example describes the synthesis of TFA-H-Dmt-Tic-Gly-NH-Bzl (compound 28).

[0216] Boc-Dmt-Tic-Gly-NH-Bzl was treated with TFA as reported for 2 TFAxH- Dmt-Tic-NH-CH2-Bid : yield 0.07 g (98%); Rf (A) 0.44 ; HPLC K'= 7. 39; 148-150 °C ; [a] 2°D-20. 1; MH+ 516.

Example 74 [0217] This example describes radio-receptor assays.

[0218] The peptides were assayed in a competition assay under equilibrium conditions with a P2 preparation of rat brain synaptosomes (Lazarus et al., J Biol.

Chem., 264: 354-362 (1989) ) using [3H] DPDPE (32.0 Ci/mmol; NEN-DuPont, Bilirica, MA) for 8 receptors and [3H] DAGO (58.0 Ci/mmol; Amersham, Arlington Heights, IL) for p receptors in 50 mM HEPES, pH 7.5, containing MgCl2 and a cocktail of several protease inhibitors and glycerol (Lazarus et al., J. Med. Chem., 34: 1350-1359 (1991)).

After 120 min incubation at room temperature (23-24'C), the samples were rapidly filtered through Whatman GF/C glass fiber filers and washed three times with 2 ml ice- cold 0.05 M Tris, pH 7.4-7. 5, containing 0. 1% BSA. Presoaking filters in 0. 1% polyethylenimine is only required in assays with peptides carrying an overall positive charge, such as dynorphin or nociceptin (Guerrini et al., J. Med. Chem., 40 : 1789-1793 (1997) ). Non-specific binding was determined using 2 uM unlabeled DPDPE or DAGO for 6 or µ assays, respectively. The K ; data, determined according to Cheng and Prusoff (Cheng et al., Biochem. Pharmacol., 22: 3099-3108 (1973) ), represent the means SE from 3 or more independent assays using at least 3 different synaptosome preparations and testing the analogues with 5-8 graded dosages over 2-3 orders of magnitude in concentration.

[0219] The appearance of high 8 affinity (Table IV) depends on the quality of the radioligand, either [3H] DPDPE, [3H] DAGO, [3H] N,N# (CH3) 2-Dmt-Tic-OH (Kertesz et al. , J. Labelled Compd. Radiopharmac., 41 : 1083-1091 (1998); Monory et al., NeuroReport, 11 : 2083-2086 (2000) ) or [3H] deltorphin B, which all yielded consistently comparable K ; values (Salvadori et al., J. Med. Chem., 42 : 5010-5019 (1999); Page et al. , J. Med. Chem., 44 : 2387-2390 (2001) ). As long as the synaptosomal preparations are prepared from fresh rat brains and stored at-80 °C in glycerol (Lazarus et al., J. Biol.

Chem., 264 : 354-362 (1989) ), stability and reliability of the radioligand remained the key factor.

[0220] The Dmt-Tic pharmacophore peptides (23-28) had exceptionally high 6 affinities, in which the K ; values were 0.1 nM or less (Table IV). In Table IV, the Ki values (nM) were determined by Cheng and Prusoff using a rat brain receptor (P2 synaptosome) assay, the mean SE with n repetitions in parentheses, were determined using Prism (GraphPad). Each repetition was an independent bioassay conducted in duplicate using 5-8 graded dosages of peptide ; confidence levels are in parentheses. pA2 is the negative log of the molar concentration required to double the agonist concentration to achieve the original response. pEC50 is the negative log of the molar concentration of an agonist to produce 50% of the maximum effect. The C-terminal substituents containing a spacer and an aromatic ring substituent exerted essentially minimal influence on 8 binding parameters in these analogues; i. e. , no linker (23) or containing NH-CH2 (24), NH-CH2-CH (25), Gly-NH (27), or Gly-NH-CH2 (26,28) were nearly equivalent. The interaction between peptide ligand and, receptors, on the other hand, demonstrated remarkable variation (Table IV). C-Terminal aromatic groups enhanced u-receptor binding by several orders of magnitude with K ; values < 1 nM (24, 27,28), while compounds 23,25 and 26 had Kj, u values of 5-20 nM; however, it should be noted that 25 and 26 were essentially non-selective. Ligand affinity for the 11 receptor increased over 1, 700-fold with 27 and 28 relative to H-Dmt-Tic-NH2, a standard prototypic 6-opioid antagonist (Salvadori et al., Mol. Med., 1 : 678-689 (1995)).

The length of the spacer in the Bid-containing series (23-26) greatly affected the acquisition of high u affinity (Table IV). Ligand interaction with u receptors required a definitive distance between aromatic nuclei of Tic and Bid. The chemical and physical nature of the C-terminal substituent, however, plays a role in receptor recognition.

Example 75 [0221] This example describes functional pharmacological bioassays.

[0222] The in vitro pharmacological assays used a single MVD for S receptors and a 2-to 3-cm segment of GPI for 11 receptors with each suspended in 20 ml organ baths containing balanced salt solutions in a physiological buffer (Salvadori et al., Mol. Med., 1 : 678-689 (1995) ). Peptide analogues were assayed for p agonist activity by the inhibition of electrically stimulated contractions in comparison to dermorphin ; 8 antagonist activity used deltorphin C (81-agonist) (Lazarus et al., Prog. Neurobiol., 57: 377-420 (1999) ). Data were obtained from 4 independent assays and tissue samples.

Agonism is expressed as the IC50 (nM) value and antagonism as pA2 (Labarre et al., Eur. J Pharm., 406 : R1-R3 92000) ).

[0223] The in vitro pharmacological assays are summarized in Tables IV and V. In Table V, the data are the means of at least four independent experiments. pA2 is measured as: log [C-1]/ [antagonist], where C is concentration. H-Dmt-Tic-NH-CH2-Bid (24) and H-Dmt-Tic-Gly-NH-Ph (27) exhibited potent 6-agonist activity, in particular compound 24 (pEC50 = 9.90) was greater than that of deltorphin C; its weak 11 agonism was nearly two orders of magnitude less than dermorphin. In order to determine if the 6 agonist activities of 24 and 27 were mediated by the 8 receptor, the pA2 for naloxone was determined using the 8 selective agonist deltorphin C. The pA2 values were comparable for all 8 agonists confirming that agonist activity in the MVD was mediated by the S receptor (Table V). Similarly, N, N (Me) 2-Dmt-Tic-OH, a highly selective 8 antagonist (Salvadori et al., J. Med. Chem., 40 : 3100-3108 (1997) ), yielded analogous results with deltorphin C and compound 24, while compound 27 had 5-fold lower activity. Thus, the activity of 24 is mediated by 8-opioid receptors, and its 8-agonist activity is more than two orders of magnitude greater than its A-agonist activity.

Interestingly, while compounds 26 and 28 retained potent 8 antagonist activity, only 28 had remarkably high u agonism.

[0224] The functional pharmacological activity of compound 23 had an unusual spectrum of behavior that depended on peptide concentration. At low doses, it was possible to estimate 8 antagonist activity against deltorphin C, while at high concentration (up to 1 pM), electrical stimulation of the tissue was reduced by 83%.

Table IV. In vitro opioid activity of Dmt-Tic analogues. compound Receptor affinity (nM) Functional Bioactivity Ki# Kiµ µ/# MVD MVD GPI (pEC50 ) (pA2) (pEC50) H-Dmt-Tic-OH 0.022 3320 1250,780 -- 8.2 --a H-Dmt-Tic-NH2 1.22 277 227 -- 7.2 --a H-Dmt-Tic-ol 0.44 151 344 -- 7.0 --a H-Dmt-Tic-Ala-NH2 0. 22 47 195--8. 0--a Deltorphin C 0.21387 1840b 9.56 -- -- (0. 30) Dermorphin 82.5 0.28 0.0034c -- -- 9. 15 (0. 10) 23 0.13 7.22 56d 7.28 7.90 -- (0. 45) (0. 30) 24 0. 035 0. 50 14 9. 90--7. 57 (0.32) (0.14) 25 0.067 5.49 82 -- 8.32 6. 97 (0. 25) (0. 18) 26 0.058 20.5 353 -- 9.00 6. 45 (0.29) (0.20) 27 0.042 .16 3.6 8.52 -- 8. 59 (0.99) (0.48) 28 0. 031 0. 16 5. 3 9. 25 8. 57 (0. 3) (0. 14) -, no activity. aSalvadori et al. bLazarus et al. cTomatis et al. dBalboni et al.

Table V. Pharmacological activity of selected 6 agonists/, agonists. compound MVD MVD, antagonist activity (pA2) pEC50 naloxone N, N (CH3) 2-Dmt-Tic-OH Deltorphin C 9. 56 7. 5 9. 76 24 9. 90 7. 45 9. 70 27 8. 52 7. 47 9. 04 [0225] The results presented in Tables IV and V demonstrate that C-terminal modification of the Dmt-Tic pharmacophore converted a 8 antagonist into a pseudopeptide with remarkable new properties. For example, some analogues demonstrated an improvement in u-opioid affinity and u-agonist activity with GPI assay as noted previously (see, e. g. , Balboni et al., Peptides, 21 : 1663-1671 (2000)).

Benzoimidazole-, pyridoindole-and spiroinden-derivatives of Dmt showed the most interesting properties. Among the benzoimidazole derivatives, 2-amino-l- [3- (lH- <BR> <BR> benzoimidazole-2-yl) -3, 4-dihydro-lH-isoquinoline-2-yl]-3- (4-hydroxyl-2, 6-dimethyl- phenyl)-propane-1-one (Fig. 2, compound 23), showed high 8 binding affinity, being 10-fold higher than H-Dmt-Tic-NH2 and 3-fold greater than H-Dmt-Tic-ol. Compound 23 also increased affinity toward the receptor nearly 40-fold compared to H-Dmt-Tic- NH2 ; published data indicated a 8 antagonism (pA2, 7.67) and u agonist activities (IC50, 30 nM) (Balboni, supra). This observation was substantiated with the functional bioassays of newly synthesized preparations of 23 that revealed 8-antagonist (pA2,7. 90) and 8-agonist activities (pEC50, 7.28) (Table IV). All previously described Dmt-Tic analogues are all 8 antagonists (Lazarus et al., Drug Discovery Today, 3: 284-294 (1998); Bryant et al., Trends Pharmacol. Sci., 19 : 42-46 (1998) ), although hydrophobic and aromatic substituents at the C-terminus provided evidence that they substantially modified the properties of the Dmt-Tic pharmacophore.

[0226] The data clearly demonstrate that a third pharmacophore, namely 1-H- benzimidazol-2-yl (Bid), in lieu of the carboxylic acid function of H-Dmt-Tic-OH or in analogues containing a linker in the general formula H-Dmt-Tic-X-Bid, is generally better than N-phenyl amide to elicit 6 agonist or antagonist activities (compare 25 or 26 to 28). Interestingly, H-Dmt-Tic-Gly-NH-Ph (27) had essentially equivalent activity as a 8 agonist and a u agonist. However, replacing the N-phenyl amide (27) with a N benzyl amide (28) function not only increased the distance between aromatic nuclei, but also converted the analogue into a potent 6 antagonist with excellent u-agonist activity, although about one-fourth of that for dermorphin.

Example 76 [0227] This example describes the synthesis of Boc-Tic-NH-(S)CH (CH3) -Bid.

[0228] To a solution of Boc-Tic-OH (0.55 g, 2 mmol) and 2 TFAoH2N- (S) CH (CH')- Bid [1-(1H-benzimidazol-2-yl)-ethylamine (Cescon et al., supra) (0.8 g, 2 mmol) in DMF (10 mL) at 0°C were added NMM (0.44 mL, 4 mmol), HOBt (0.34 g, 2.2 mmol) and WSC (0.42 g, 2.2 mmol). The reaction mixture was stirred for 3 h at 0°C and 24 h at room temperature. After DMF was evaporated, the residue was solubilized in EtOAc and washed with NaHCO3 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et2O/Pe (1: 9, v/v): yield 0.7 g. (83%); Rf (B) 0.81 ; HPLC K'= 7. 42; mp 215-217 °C ; [a] 20D-18. 5; MH+ 421; 1H-NMR (DMSO) 8 = 1.38-1. 42 (d, 9H), 1.56 (d, 3H), 3.08-3. 15 (m, 2H), 4.22-4. 53 (m, 4H), 7.05-7. 71 (m, 9H), 7.97 (s, 1H).

Example 77 [0229] This example demonstrates the synthesis of 2TFAcH-Tic-NH-(S) (S) CH (CH3) - Bid.

[0230] Boc-Tic-NH- (S) CH (CH3) -Bid (0.4 g, 0.95 mmol) was treated with TFA (1 mL) for 0.5 h at room temperature. Et2O/Pe (1: 1, v/v) was added to the solution until the product precipitated: yield 0.49 g. (95%); Rf (A) 0.36 ; HPLC K'= 5. 32; mp 180-182°C ; [a] 20D-20. 9; Mu+ 321.

Example 78 [0231] This example demonstrates the synthesis of Boc-Dmt-Tic-NH- (S) CH (CH3) - Bid.

[0232] To a solution of Boc-Dmt-OH (0.11 g, 0.37 mmol) and 2TFA#H-Tic-NH- (S) CH (CH3) -Bid (0.2 g, 0.37 mmol) in DMF (10 mL) at 0 °C were added NMM (0.08 mL, 0.74 mmol), HOBt (0.063 g, 0.41 mmol) and WSC (0.079 g, 0.41 mmol). The reaction mixture was stirred for 3 h at 0°C and for 24 h at room temperature. After DMF was evaporated, the residue was solubilized in EtOAc and washed with NaHC03 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et20/Pe (1: 9, v/v): yield 0.18 g. (81%) ; Rf (B) 0.77 ; HPLC K'= 7.8 ; mp 143-144 °C ; [a] 20D-24. 6; MH+ 611 ;'H-NMR (DMSO) d = 1. 32-1.39 (d, 9H), 1.55 (d, 3H), 2.17 (s, 6H), 2.85-3. 10 (m, 4H), 4.22-4. 65 (m, 5H), 6.37 (s, 2H), 7.05-7. 71 (m, 10H), 7.97 (s, 1H).

Example 79 [0233] This example demonstrates the synthesis of 2TFAcH-Dmt-Tic-NH- (S) CH (CH3) -Bid [31] (Table VI).

[0234] Boc-Dmt-Tic-NH-(S(CH (CH3) -Bid (0.061 g, 0.1 mmol) was treated with TFA (1 mL) for 0.5 h at room temperature. Et2O/Pe (1 : 1, v/v) was added to the solution until the product precipitated: yield 0.09 g. (93%); Rf (A) 0.46 ; HPLC K'= 4.34 ; mp 158-160 °C ; [α]20D-26. 1; MH+ 512. Anal. C3oH33Ns032TFA) C, H, N.

Example 80 [0235] This example demonstrates the synthesis of Boc-Tic-NH- (R) CH (CH3)-Bid.

[0236] This intermediate was obtained by condensation of Boc-Tic-OH with 2TFAoH2N- (R) CH (CH3) -Bid via WSC/HOBt as reported for Boc-Tic-NH- (S) CH (CH3) - Bid: yield 0.68 g. (82%); Rf (B) 0.79 ; HPLC K'= 7.38 ; mp 215-217 °C ; [a] 20D +3.5 ; MH+ 421 ; 1H-NMR (DMSO) d = 1. 38-1. 40 (d, 9H), 1.56 (d, 3H), 3.08-3. 17 (m, 2H), 4.20-4. 54 (m, 4H), 7.05-7. 71 (m, 9H), 7.97 (s, 1H).

Example 81 [0237] This example demonstrates the synthesis of 2TFAoH-Tic-NH- (R) CH (CH3)- Bid.

[0238] Boc-Tic-NH- (R) CH (CH3) -Bid was treated with TFA as reported for 2TFA#H-Tic-NH- (S) CH (CH3) -Bid: yield 0.36 g. (94%); RJ (A) 0.41 ; HPLC K'= 5. 75; mpl80-182 °C ; [α]20D +7.9 ; MH+ 321.

Example 82 [0239] This example demonstrates the synthesis of Boc-Dmt-Tic-NH- (R) CH (CH3)- Bid..

[0240] This compound was obtained by condensation of Boc-Dmt-OH with 2TFA#H-Tic-NH-(R) CH (CH3) -Bid via WSC/HOBt as reported for Boc-Dmt-Tic-NH- (S) CH (CH3) -Bid: yield 0.17 g. (79%); Rf (B) 0.85 ; HPLC K'= 7.2 ; mp 142-144 °C ; [a] 20D +12.6 ; MH+ 611 ;'H-NMR (DMSO) d = 1.32-1. 39 (d, 9H), 1.55 (d, 3H), 2.17 (s, 6H), 2.85-3. 15 (m, 4H), 4.20-4. 65 (m, 5H), 6.34 (s, 2H), 7.05-7. 72 (m, 10H), 7.97 (s, 1H).

Example 83 [0241] This example demonstrates the synthesis of 2TFAcH-Dmt-Tic- NH (R) CH (CH3)-Bid [32] (Table VI).

[0242] Boc-Dmt-Tic-NH- (R) CH (CH3) -Bid was treated with TFA as reported for 2TFA#H-Dmt-Tic-NH-(S)CH (CH3) -Bid: yield 0.088 g. (96%); Rf (A) 0.40 ; HPLC K'= 3.78 ; mp 152-154 °C ; [8] 20D +16.1 ; MH+ 512. Anal. C30H33N503*2TFA) C, H, N.

Example 84 [0243] This example demonstrates the synthesis of 3-(1H-Benzimidazol-2-yl)-2-tert- butoxycarbonylamino-propionic acid benzyl ester [Boc-NH- (S) CH (CH2-Bid) - COOBzl].

[0244] A solution of Boc-Asp-OBzl (1 g, 3.1 mmol) and 4-methyl morpholine (NMM, 0.34 mL, 3.1 mmol) in N,N-dimethylformamide (DMF, 10 mL) was treated at - 20 °C with isobutyl chloroformate (IBCF, 0.4 mL, 3.1 mmol). After 10 min at-20 °C, o-phenylendiamine (0. 33 g, 3.1 mmol) was added. The reaction mixture was allowed to stir, while slowly warming to room temperature (1 h), and was then stirred for 3 h. The solvent was evaporated, and the residue was partitioned between ethyl acetate (EtOAc) and H2O. The EtOAc layer was washed with 5% NaHC03 and brine and dried over Na2SO4. The solution was filtered, the solvent was evaporated, and the residual solid was dissolved in glacial AcOH (10 mL). The solution was heated at 65 °C for 1 h. After the solvent was evaporated, the residue was crystallized from diethyl ether (Et20)/petroleum ether (Pe) (1: 9, v/v); yield 0.98 g. (80%); Ff (B) 0.79 ; HPLC K'= 6. 6; mp 141-143 °C ; [α]20D +12.1 MH+ 396 ;'H-NMR (DMSO) d = 1. 40 (s, 9H), 3.09 (d, 2H), 4. 86-5.30 (m, 3H), 7.19-7. 70 (m, 10H), 7.98 (s, 1H).

Example 85 [0245] This example demonstrates the synthesis of 2TFAH2N- (such (CH2-Bid) - COOBzl.

[0246] Boc-NH- CH (CH2-Bid)-COOBzl (0.9 g, 2.28 mmol) was treated with TFA (1 mL) for 0.5 h at room temperature. Et20/Pe (1: 1, v/v) was added to the solution until the product precipitated; yield 1.11 g. (93%); Rf (A) 0.83 ; HPLC K'= 4. 85; mp 152-154 °C ; [a] 20D +15. 3; MH+ 296.

Example 86 [0247] The example demonstrates the synthesis of Boc-Tic-NH- (such (CH2-Bid) - COOBzl.

[0248] This substance was obtained by condensation of Boc-Tic-OH with 2TFAH2N-(S) CH (CH2-Bid)-COOBzl via WSC/HOBt as reported for Boc-Tic-NH- (S) CH (CH3) -Bid: yield 0.74 g (79%); Rf (B) 0.74 ; HPLC K'= 4. 5; mp 166-168 °C ; [a] 20D +8.2 ; MH+ 555 ;'H-NMR (DMSO) d = 1.40 (s, 9H), 3.04-3. 17 (m, 4H), 4.22- 5.32 (m, 6H), 7.01-7. 70 (m, 14H), 7.98 (s, 1H).

Example 87 [0249] This example demonstrates the synthesis of 2TFA#H-Tic-NH- (S) CH (CH2- Bid)-COOBzl.

[0250] Boc-Tic-NH- CH (CH2-Bid)-COOBzl was treated with TFA as reported for 2TFA#H-Tic-NH-(S)CH (CH3) -Bid: yield 0.73 g. (92%); Rf (A) 0. 71 ; HPLC K'= 5. 31 ; mp 158-160 °C ; [a] 20D +10.4 ; MH+ 455.

Example 88 [0251] This example demonstrates the synthesis of Boc-Dmt-Tic-NH-(S)CH (CH2- Bid)-COOBzl.

[0252] This compound was obtained by condensation of Boc-Dmt-OH with 2TFA#H-Tic-NH-(S)CH(CH2-Bid)-COOBzl via WSC/HOBt as reported for Boc-Dmt- Tic-NH-(S) CH (CH3) -Bid: yield 0.44 g. (881%) ; Rf (B) 0.65 ; HPLC K'= 9.7 ; mp 165-167 °C ; [a] 20D +3.5 ; MH+ 746 ;'H-NMR (DMSO) d = 1.40 (s, 9H), 2.35-3. 14 (m, 8H), 4.48- 5.33 (m, 7H), 6.32-7. 70 (m, 16H), 7.98 (s, 1H).

Example 89 [0253] This example demonstrates the synthesis of Boc-Dmt-Tic-NH- (S) CH (CH2- Bid)-COOH.

[0254] To a solution of Boc-Dmt-Tic-NH-(S) CH (CH2-Bid)-COOBzl (0.3 g, 0.4 mmol) in MeOH (30 mL) was added C/Pd (10%, 0.05 g), and H2 was bubbled for 1 h at room temperature. After filtration, the solution was evaporated to dryness. The residue was crystallized from Et2O/Pe (1: 9, v/v): yield 0.24 g. (94%); Rf (B) 0.76 ; HPLC K'= 7.1 ; mp 175-177 °C ; [α]20D +4.8 ; MH+ 656.

Example 90 [0255] This example demonstrates the synthesis of 2TFAH-Dmt-Tic-NH- (S) CH (CH2-Bid)-COOH [35] (Table VI). Boc-Dmt-Tic-NH- CH (CH2-Bid) -COOH was treated with TFA as reported for 2TFA#H-Dmt-Tic-NH-(S)CH (CH3) -Bid: yield 0.17 g. (96%); Rf (A) 0.87 ; HPLC K'= 2. 93; mp 165-167 °C ; [a] 20D +6.2 ; MH+ 556.

Anal. C31H33N5O5#2TFA) C, H, N.

Example 91 [0256] This example demonstrates the synthesis of 3-(1H-Benzimidazol-2-yl)-3-tert- butoxycarbonylamino-propionic acid benzyl ester [Boc-NH- (S) CH (CH2-COOBzl)-Bid].

[0257] This compound was obtained by condensation of Boc-Asp(OBzl)-OH with o- phenylendiamine via IBCF as reported for Boc-NH-(S) CH (CH2-Bid)-COOBzl : yield 1.62 g. (82%); Rf (B) 0.52 ; HPLC K'= 6. 5; mp 136-138 °C [a] 20D +15. 8; MH+ 396 ; tH- NMR (DMSO) d = 1.40 (s, 9H), 2.80 (d, 4H), 5.34-5. 51 (m, 3H), 7.19-7. 70 (m, 10H), 7.99 (s, 1H).

Example 92 [0258] This example demonstrates the synthesis of 2TFAH2N-(S) (S) CH (CH2- COOBzl)-Bid.

[0259] Boc-NH- CH (CH2-COOBzl)-Bid was treated with TFA as reported for 2TFA#H2N-(S)H(CH2-Bid)-COOBzl : yield 1.26 g. (95%); Rf (A) 0.78 ; HPLC K'= 4.2 ; mp 142-144 °C ; [α]20D +17.3 ; MH+ 296.

Example 93 [0260] This example demonstrates the synthesis of Boc-Tic-NH-(S)CH (CH2- COOBzl)-Bid.

[0261] This compound was obtained by condensation of Boc-Tic-OH with 2TFA#H2N-(S)CH(CH2-COOBzl)-Bid via WSC/HOBt as reported for Boc-Tic-NH- (S) CH (CH3) -Bid: yield 0.76 g. (84%); Rf (B) 0.76 ; HPLC K'= 4. 05; mp 159-161 °C [afo 20D +10. 3; MH+ 555 ;'H-NMR (DMSO) d = 1. 40 (s, 9H), 2.80-3. 05 (m, 4H), 4.22- 5.51 (m, 6H), 6.96-7. 70 (m, 14H), 7.98 (s, 1H).

Example 94 [0262] This example demonstrates the synthesis of 2TFA#H-Tic-NH-(S)CH (CH2- COOBzl)-Bid.

[0263] Boc-Tic-NH- CH (CH2-COOBzl)-Bid was treated with TFA as reported for 2TFA#H-Tic-NH-(S)CH (CH3) -Bid: yield 0.62 g. (91%); Rf (A) 0.67 ; HPLC K'= 3. 49; mp 152-154 °C ; [a] 20D +11.8 ; MH+ 455.

Example 95 [0264] This example demonstrates the synthesis of Boc-Dmt-Tic-NH- (S) CH (CH2- COOBzl)-Bid.

[0265] This compound was obtained by condensation of Boc-Dmt-OH with 2TFA#H2N- CH (CH2-COOBzl)-Bid via WSC/HOBt as reported for Boc-Dmt-Tic- NH- (S) CH (CH3) -Bid: yield 0.47 g. (83%); Rf (B) 0.63 ; HPLC K'= 9. 11; mp 171-173 °C ; [a] 20D +4.3 ; MH+ 746 ;'H-NMR (DMSO) d = 1. 40 (s, 9H), 2.35-3. 05 (m, 12H), 4.46-5. 51 (m, 7H), 6.29 (s, 2H), 6.96-7. 70 (m, 13H), 7.99 (s, 1H).

Example 96 [0266] This example demonstrates the synthesis of Boc-Dmt-Tic-NH- CH (CH2- COOH)-Bid.

[0267] Boc-Dmt-Tic-NH- CH (CH2-COOBzl)-Bid was treated with C/Pd and H2 as reported for Boc-Dmt-Tic-NH-(S)CH (CH2-Bid) -COOH: yield 0.28 g. (92%); Rf (B) 0.79 ; HPLC K'= 6. 9; mp 173-175 °C ; [a] 20D +5.2 ; MH+ 656.

Example 97 [0268] This example demonstrates the synthesis of 2TFA*H-Dmt-Tic-NH- (S) CH (CH2-COOH) -Bid [33] (Table VI).

[0269] Boc-Dmt-Tic-NH-(S)CH (CH2-COOH) -Bid was treated with TFA as reported for 2TFA-H-Dmt-Tic-NH-(S) CH (CH3) -Bid: yield 0.17 g. (90%); Rf (A) 0.67 ; HPLC K' = 2.90 ; mp 164-166 °C ; [a] 20D +7.9 ; MH+ 556; Anal. (C31H33 N5O5#2TFA) C, H, N.

Example 98 [0270] This example demonstrates the synthesis of [2-(tert-Butoxycarbonylamino- methyl)-benzimidazol-1-yl]-acetic acid ethyl ester [Boc-NH-CH2-Bid (Nl-CH2-COOEt)].

[0271] To a solution of Boc-NH-CH2-Bid (Balboni et al., J. Med. Chem. 45: 713-720 (2002) ) (0.5 g, 1.63 mmol) in DMF (10 mL) at room temperature K2CO3 (0.79 g, 5.72 mmol), and after 1 h, bromoacetic acid ethyl ester (0.19 mL, 1.73 mmol) were added.

The reaction mixture was stirred for 86 h at room temperature. After DMF was evaporated, the residue was solubilized in EtOAc and washed with NaHC03 (5%) and brine. The organic phase was dried and evaporated to dryness. The residue was crystallized from Et2O/Pe (1: 9, v/v): yield 0.46 g. (84%); Rf (B) 0.93 ; HPLC K'= 4.95 ; mp 139-141 °C ; MH+ 334 ;'H-NMR (DMSO) d = 1. 30-1.40 (m, 12H), 4.20-4. 69 (m, 6H), 7.26-7. 70 (m, 4H), 8.00 (bs, 1H).

Example 99 [0272] This example demonstrates the synthesis of 2TFAcH2N-CH2-Bid (N'-CH2- COOEt).

[0273] Boc-NH-CH2-Bid (N'-CH2-COOEt) was treated with TFA as reported for 2TFA#H2N- CH (CH2-Bid)-COOBzl : yield 0.51 g. (98%); Rf (A) 0.88 ; HPLC K'= 2.50 ; mp 222-224 °C ; MH+ 234.

Example 100 [0274] This example demonstrates the synthesis of Boc-Tic-NH-CH2-Bid (Nt-CH2- COOEt).

[0275] This compound was obtained by condensation of Boc-Tic-OH with 2TFA-H2N-CH2-Bid (N'-CH2-COOEt) via WSC/HOBt as reported for Boc-Tic-NH- (S) CH (CH3) -Bid: yield 0.42 g. (86%); Rf (B) 0.92 ; HPLC K'= 9. 12; mp 148-150 °C [a] 20D-11. 6; MH+ 493 ;'H-NMR (DMSO) d = 1.30-1. 40 (m, 12H), 3.05 (m, 2H), 4.12- 4.92 (m, 9H), 6.96-7. 70 (m, 8H), 8.00 (bs, 1H).

Example 101 [0276] This example demonstrates the synthesis of 2TFAH-Tic-NH-CH2-Bid (N1- CH2-COOEt).

[0277] Boc-Tic-NH-CH2-Bid (N'-CH2-COOEt) was treated with TFA as reported for 2TFA#H-Tic-NH-(S)CH (CH3) -Bid: yield 0.36 g. (96%); Rf (A) 0.93 ; HPLC K'= 6. 11 ; mp 187-189 °C ; [α]20D-14. 3; MH+ 393.

Example 102 [0278] This example demonstrates the synthesis of Boc-Dmt-Tic-NH-CH2-Bid (N1- CH2-COOEt).

[0279] This compound was obtained by condensation of Boc-Dmt-OH with 2TFA#H-Tic-NH-CH2-Bid(N1-CH2-COOEt) via WSC/HOBt as reported for Boc-Dmt- Tic-NH- (S) CH (CH3) -Bid: yield 0.33 g. (92%); Rf (B) 0.96 ; HPLC K'= 9.8 ; mp 154-156 °C ; [a] 20D-22. 0; MH+ 684 ;'H-NMR (DMSO) d = 1.30-1. 40 (m, 12H), 2.35 (s, 6H), 3.05 (m, 4H), 4.12-4. 92 (m, 10H), 6.29 (s, 2H), 6.96-7. 70 (m, 8H), 8.00 (m, 2H).

Example 103 [0280] This example demonstrates the synthesis of 2TFAcH-Dmt-Tic-NH-CH2- Bid (N1-CH2-COOEt).

[0281] Boc-Dmt-Tic-NH-CH2-Bid (N'-CH2-COOEt) was treated with TFA as reported for 2TFAoH-Dmt-Tic-NH- (S) CH (CH3) -Bid: yield 0.16 g. (92%); Rf (A) 0. 81 ; HPLC K'= 6. 40; mp 162-164 °C ; [α]20D-34. 7; MH+ 584.

Example 104 [0282] This example demonstrates the synthesis of 2TFAcH-Dmt-Tic-NH-CH2- Bid (N'-CH2-COOH) [37] (Table VI).

[0283] To a solution of 2TFA*H-Dmt-Tic-NH-CH2-Bid (N'-CH2-COOEt) (0.15 g, 0.22 mmol) in EtOH (10 mL) at room temperature, 1 N NaOH (1 mL, 1 mmol) was added. The reaction mixture was stirred for 3 h at room temperature. After EtOH was evaporated, the residue was purified by preparative HPLC without any treatment: yield 0.116 g. (89%); Rf (A) 0.93 ; HPLC K'= 7. 8; mp 179-181 °C; [α]20D-36. 2 MH+ 556; Anal. (C31H33N5O5#2TFA) C, H, N.

Table VI. Elemental analysis of Dmt-Tic pharmacophore compounds<BR> Compound MH+ Analytical<BR> New compounds<BR> TFA#H-Dmt-Tic-D-Ala-NH2 [30] 439 (C24H30N4O4#TFA) C, H, N.<BR> <P>2TFA#H-Dmt-Tic-NH-(S)CH(CH3)-Bid [31] 512 (C30H33N5O3#2TFA) C, H, N.<BR> <P>2TFA#H-Dmt-Tic-NH-(R)CH(CH3)-Bid [32] 512 (C30H33N5O3#2TFA) C, H, N.<BR> <P>2TFA#H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid [33] 556 (C31H33N5O5#2TFA) C, H, N.<BR> <P>TFA#H-Dmt-Tic-Asp-NH2 [34] 483 (C25H30N4 O6#TFA) C, H, N.<BR> <P>2TFA#H-Dmt-Tic-NH-(S)CH(CH2-Bid)-COOH [35] 556 (C31H33N5O5#2TFA) C, H, N.<BR> <P>TFA#H-Dmt-Tic-Asn-OH [36] 483 (C25H30N4O6#TFA) C, H, N.<BR> <P>2TFA#H-Dmt-Tic-NH-CH2-Bid(N1-CH2-COOH) [37] 556 (C31H33N5O5#2TFA) C, H, N.<BR> <P>Published Compounds<BR> TFA#H-Dmt-Tic-Ala-NH2 [29] (Salvadori et al., Mol. Med. 1: 678-689 (1995))<BR> 2TFA#H-Dmt-Tic-NH-CH2-Bid[24] (Balboni et al., J. Med. Chem. 45: 713-720 (2002))<BR> TFA#H-Dmt-Tic-NH-CH2-CH2-Bid [25] (Balboni et al., J. Med. Chem. 45 : 713-720 (2002)) Example 105 [0284] This example demonstrates the opioid receptor affinity of the analogues containing the Dmt-Tic pharmacophore in Table VI.

[0285] The binding affinity of all opioid analogues was determined under equilibrium conditions (2 h at room temperature) in a competition assay system using rat brain P2 synaptosomes, previously preincubated in 0.1 M NaCI, 0.4 mM GDP, 50 mM HEPES, pH 7.5, and 50 mg/mL soybean trypsin inhibitor for 60 min at room temperature to remove endogenous opioids (Lazarus et al., R Biol. Chem. 264: 354-362 (1989) ). After extensive washing of synaptosomes in ice cold buffer containing protease inhibitor, the material was resuspended in buffer, pH 7.5, containing 50 mg/mL soybean trypsin inhibitor and 20% glycerol, aliquoted and stored at-80 °C. b-and p^-Opioid receptors were radiolabeled with [3H] DPDPE (32.0 Ci/mmol; NEN-DuPont, Bilirica, MA) and [3H] DAGO (58.0 Ci/mmol; Amersham, Arlington Heights, IL), respectively, as described in detail elsewhere (Lazarus et al., J. Med. Chem. 34: 1350-1359 (1991)).

Excess unlabeled peptide (2 mM) established non-specific binding. Labeled membranes were rapidly filtered on Whatman GF/C glass fibre filters and thoroughly washed and dried and radioactivity was determined using CytoScint (ICN). All analogues were analyzed in duplicate using 5-8 dosages and 3-5 independent repetitions using different synaptosomal preparations for each analogue (actual n values are listed in Table VII in parenthesis) with results given as mean SEM. The affinity constants (K ;) were calculated according to Cheng and Prusoff (Cheng et al., Biochem. Pharmacol. 22: 3099-3108 (1973)).

[0286] While all of the peptides listed in Table VII exhibited high 8 affinity, several analogues [24,25, 31,32, 35, 37] demonstrated exceptional K ; values (< 0.1 nM). On the other hand, only three Bid-containing peptides had high u affinities (Ki < 1 nM ; [24, 31, 32] ; the 11 affinities of the other compounds ranged from 5 to 80 nM. Alkylation of the carbon atom between the amide and Bid [31] did not substantially modify 8-and u- receptor affinities and pharmacological activities (infra vide) compared to the reference 8 agonist, Dmt-Tic-NH-CH2-Bid [24]. The corresponding diastereomer of [31] ([32]) maintained comparable 8 selectivity and potent agonist activity even though p affinity and agonist potency increased. The control compounds, containing Ala-NH2 instead of Bid [29], [30], exhibited equivalent 8 affinities and selectivities. In addition, the tripeptide analogue controls with C-terminal Asp and Asn ([34] and [36] respectively) had relatively good 8 affinities (0.289 nM and 1.10 nM, respectively) and 8-antagonist bioactivities [Ke (MVD) = 7.58 nM and 5.88 nM, respectively]. The alkylation of the side chain of compound [31] with a carboxylic function [33] improved 8 selectivity relative to [24], [31], and [32] even though 8 affinity fell by about 10-fold (Table VII).

Compound [34] with a very weak K, u. (2,565 nM) had the highest 8 selectivity, comparable to many Dmt-Tic analogues with a free C-terminal carboxyl group [24, 31, 34, 35].

[0287] In the Bid analogues containing carboxylate groups ([33], [35], [37]), µ affinities diminished and 8 selectivities increased, which verified the hypothesis that a negative charge in an opioid ligand retards binding to u receptors without detrimental effects on 8 affinity (Lazarus et al., Prog. Neurobiol. 57: 377-420 (1999); Lazarus et al., J. Med. Chem. 34: 1350-1359 (1991) ). However, the position of the carboxyl group relative to the Bid moiety (compare [35] and [37]) influenced u binding to a greater extent than that for the 8 receptor. Bioactivity data for analogues containing a different chirality at the C-terminal of Dmt-Tic indicated that the change in chirality had essentially no influence on the bioactivity profiles (compare pairs of compounds [29] + [31] and [30] + [32]).

Table VII. Receptor affinitya and functional bioactivityb of analogues containing the Dmt-Tic pharmacophore<BR> Receptor Affinity (nM)<BR> no. peptide Ki# Kiµ µ/# MVD GPI<BR> IC50 (nM) Ke (nM) IC50 (nM)<BR> 29 H-Dmt-Tic-Ala-NH2 0.241 ~0.02 (5) 47.1 ~ 3.4 (4) 195 10.0 + 1.3c 4,740 + 345c<BR> 30 H-Dmt-Tic-D-Ala-NH2 0.301 ~0.021 (4) 47.5 ~ 4.6 (3) 158 25.1 ~ 1.1 >10,000<BR> 31 H-Dmt-Tic-NH-CH(CH3)Bid 0.063 ~ 0.004 (3) 0.411 ~ 0.016 (3) 6.5 0.26 ~ 0.03 - 71.7 ~ 8.2<BR> 32 H-Dmt-Tic-H(R)CH(CH3)-Bid 0.047 ~ 001 (3) 0.233 ~ 0.01 (3) 5 0.026 ~ 0.002 - 6.36 ~ 0.7<BR> 24 H-Dmt-Tic-NH-CH2-Bid 0.035 ~ 0.006 (3) 0.50 ~0.054(3) 14d 0.035 ~ - 40.7 ~ 5<BR> 33 H-Dmt-Tic-NH-CH(CH2-COOH)-Bid 0.443 ~ 0.043 (4) 53.9 ~ 4.4 93) 122 0.12 ~ 0.02 - 1,724 ~ 150<BR> 34 H-Dmt-Tic-Asp-NH2 0.289 ~0.018 (3) 2,565 ~290(3) 8,875 7.58 ~ 0.31 >10,000<BR> 35 H-Dmt-Tic-NH-CH(CH2-Bid)-COOH 0.042 ~ 0.017 (4) 21.68 ~ 5.5 (3) 516 0.015 ~0.003 - 1,558 + 153<BR> 36 H-Dmt-Tic-Asn-OY 1.10 ~0.26(5) 80.3 ~8.2(4) 73 5.88 + 0.21 >10,000<BR> 25 H-Dmt-Tic-NH-CH2-CH2-Bid 0.067 ~0.015 (4) 5.49 ~ 0.03 (3) 82 4.78 ~ 0.37e 107.5 ~ 11.4c<BR> 37 H-Dmt-Tic-NH-CH2-Bid(CH2-COOH) 0.021 ~ 0.0025 (4) 6.92 ~ 0.25 (4) 330 0.27 ~0.015 3,193 ~402<BR> Deltorphin C 0.24 ~ 0.06 (6) 272 ~50(11) 1,135f 0.30 +0.012 - >1,200<BR> Demorphin 178.6 ~18(15) 1.22 ~0.13 (22) 1246g 16.5 ~1.8 - 1.21 ~0.18 aThe Ki values (nM) were determined according the Chang and Prusoff (Chang et al., Life Sci., 18: 1473-1482 (12976)); the mean ~ SE<BR> with n repetitions in parenthesis is based on independent assays with 5-8 peptide dosesd using multiple synaptosome preparations.<BR> bAgonist activity was expressed as IC50 (ke)obtained from concewntration-response curves; these values represent the mean ~ SE of at<BR> least five fresh tissue samples. [D-Ala2]Deltorphin I (deltorphin C) (Lazarus et al., Prog. Neurobiol. 57: 377-420 (12999)) and<BR> dermorphin were the internal standardsd for MVD (# bioactivity) and GPI (µ bioactivity) tissue preparartions, respectively.<BR> cData from Salvadori et al., Mol. Med. 1: 678-689 (1995)<BR> dReceptor binding data from Balboni et al., J. Med. Chem. 45: 713-720 (2002); these bioassay dasta were not reported previously.<BR> eDasta taken from Balboni et al., J. Med. Chem. 45: 713-720 (2002).<BR> fBinding assay data from Salvadori et al. 44 gReceptor data from Lazarus et al., J. Biol. Chem. 264: 354-362 (1989).

[0288] This example demonstrates that compounds 24,25, 31,32, 34, and 37 have high affinity for the 8 opioid receptor, while compounds 24, 31, and 32 also have high affinity for the u opioid receptor.

Example 105 [0289] This example demonstrates the biological activity of the analogues of Table VI.

[0290] Preparations of the myenteric plexus-longitudinal muscle, obtained from the small intestine of guinea pigs (GPI, rich in u-opioid receptors) and preparations of mouse vas deferens (MVD, containing 8-opioid receptors), were used for field stimulation with bipolar rectangular pulses of supramaximal voltage. Agonists were evaluated for their inhibition of the electrically evoked twitch. The results were expressed as the ICSO values obtained from concentration-response curves (Tallarida and Murray, 1986) and converted to pECso. The IC50 values represent the mean SE of not less than five tissue samples. [D-Ala2] Deltorphin I (Lazarus et al., Prog. Neurobiol. 57: 377-420 (1999) ) and dermorphin (Melchiorri et al., Gen. Pharmacol. 27: 1099-1107 (1996) ) were used as internal standards with MVD and GPI tissue preparations, respectively.

[0291] Several peptides (124, 31,32, 33, 35]) acted as potent 8-opioid agonists. The IC50 values ranged nearly 20-fold, from 0. 015 nM to 0.26 nM in isolated organ preparations (Table VII), and they were many times more effective in inhibiting electrically evoked contractions in the mouse vas deferens (MVD) than in the guinea- pig ileum (GPI). Interestingly, the analogues containing carboxylic acids ( [33, 35, 37]) exhibited very weak u agonism relative to [24], [31], and [32]. In addition, compound [37], which displayed the highest 8 affinity, was inactive as an agonist in the MVD preparation at a concentration as high as 137 mM. In fact, compound [37] behaved as a potent antagonist displacing the [D-Ala2] deltorphin I dose response curve to the right (Ke = 0. 27 nM) with a weak bioactive response on GPI (ICso = 3, 193 nM).

[0292] Analogues [29,30, 34, and 36] were pure 8 antagonists that lacked the third aromatic center, Bid. These control compounds exhibited micromolar activity on GPI while, in contrast, compound [25], a potent 8 antagonist containing Bid, displayed weak p agonism (Table VII). None of the peptide analogues (Table VII) exhibited u antagonism.

[0293] This example demonstrated that compounds 24,31, 32,33, and 35 are potent 8 opioid receptor agonists, while compound 37 is a potent antagonist of this receptor.

Example 106 [0294] This example demonstrates the molecular modeling studies of the compounds in Table VI.

[0295] H-Dmt-Tic-NH-CH2-Bid, a 8-opioid receptor agonist [24], was modeled using InsightII Discover software (v. 98.0) manufactured by Accelrys. Coordinates for Dmt and Tic were derived from the X-ray crystal structure of N, N (Me) 2-Dmt-Tic-OH (8 antagonist) (Bryant et al., J. Med. Chem., in press (2002) ). Conformations describing the C-terminal modifications were generated by extensive conformational searching and energy minimization. The Dmt-Tic portion of the molecule was constrained by tethering with a force constant of 1000 kcal/mol-A to preserve the x-ray coordinates and minimized for 1000 steps or until the root mean square (rms) gradient was 0.0002 kcal/mol-A2 using the cff91 force field and the conjugate gradient algorithm.

Minimization parameters included a distant dependent dielectric [e = 1.0 (r)], and all scale terms except non-bonded and coulombic terms were set to 1.0 ; the latter terms were 2.0. Conformational searches were performed via a torsion force with a force constant of 100 kcal/mol-A that was applied to 3 dihedrals labeled Cl (peptide bond between Tic and NH2), C2 (bond between-NH-and-CH2-), and C3 (bond between - CH2-and-Bid). The region evaluated ranged from 0° and 360° ; Cl for 2 intervals (180°), C2 and C3 for 6 intervals (60°). All final conformers were minimized for an additional 200 iterations. The C-terminal region was evaluated using conformational searching techniques, which forces angles of rotation and does not require solvation.

[0296] H-Dmt-Tic-NH-CH2-Bid, a 5-opioid receptor agonist [24], was modeled using coordinates for Dmt and Tic from the X-ray crystal structure of N, N (Me) 2-Dmt- Tic-OH (8-antagonist) (Bryant et al., J Med Chem., in press (2002) ). Features of the Dmt-Tic pharmacophore include cis orientation around the peptide bond, approximately 5 A between the aromatic rings, and near parallel orientation of the aromatic ring structures (Bryant et al., Trends Pharmacol. Sci. 19: 42-46 (1998); Bryant et al., J. Med.

Chem., in press (2002); and Bryant et al., Peptides 2000 407-408 (2000)).

Conformations describing the C-terminal modifications were generated by extensive conformational searching and energy minimization using InsightII. One hundred forty- seven low energy conformers were generated by the search and four of the lowest energy conformations with unique C-terminal orientations were superimposed for comparison and evaluation as potential bioactive forms. The relative energies of four unique low energy structures ranged from 67-97 kcal/mol and centroid distances between the aromatic rings of Tic and Bid ranged from 6.7-9. 9 A. The most notable structural variations were observed around bonds between the amide and methylene linker, as well as the methylene linker and Bid.

[0297] This example demonstrated the accessible confortnational space of the C- terminal portion of these peptides based on the Dmt-Tic pharmacophore generated from the X-ray crystallography data (Bryant et al., J. Med. Chem, in press (2002) ).

[0298] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0299] The use of the terms"a"and"an"and"the"and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e. g. ,"such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0300] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.