WO2008016913A1 - Biologically potent analogues of the dmt-tic pharmacophore and methods of use - Google Patents

Abstract

Disclosed are compounds of formula (I) or pharmaceutically acceptable salts thereof: formula (I) in which R1, R2, and R3 are described herein. Also disclosed is a pharmaceutical composition comprising at least one compound of formula (I) and a pharmaceutically acceptable carrier. Also disclosed is a method of agonizing or antagonizing a µ-opioid or δ-opioid receptor in a mammal in need thereof.

Description

BIOLOGICALLY POTENT ANALOGUES OF THE DMT-TIC PHARMACOPHORE AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 60/834,438, filed July 31, 2006, the disclosure of which is incorporated by reference.

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, neuropeptide 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 δ, K and μ. Subtypes of opioid receptors have been suggested, including μl and μ2 (Pasternak et al., Life Sci,. 38: 1889-1898 (1986)) and κl and κ2 (Zukin et al., PNAS USA, S5: 4061-4065 (1988)). Different subtypes of a given type of opioid receptor may co-exist in a single cell (Evans et al., Science, 258 (5090): 1952-1955 (1992); and Kieffer et al., PNAS USA, 89(24): 12048- 12052 (1992)).

[0004] The μ-opioid and δ-opioid receptors have been linked to analgesia and other symptoms. While agonists and antagonists of such receptors have been proposed for treating pain perception and other conditions such as alcoholism and the regulation of food intake, there is a desire for additional agonists and/or antagonists of the μ- and δ-opioid receptors. The present invention provides compounds which are agonists, antagonists, or both.

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention provides lysine derivatives of Dmt-Tic (2',6'-dimethyl-L- tyrosine-l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid peptide), for example, a compound of formula (I) or a pharmaceutically acceptable salt thereof:

(I) wherein R¹, R², and R³ can be hydrogen, functional groups, or protecting groups for the lysine side chain and are described herein.

[0005] Also provided by the present invention is a pharmaceutical composition comprising at least one compound of formula (I) and a pharmaceutically acceptable carrier. [0006] The present invention further provides a method of agonizing or antagonizing a μ- or δ-opioid receptor in a mammal in need thereof. The method comprises administering to the mammal at least one compound of formula (I). The present invention also provides a method of treating a condition in a mammal comprising administering an effective amount of a compound of formula (I) to the mammal in need thereof, wherein the condition is regulation of food intake, treatment of chronic or acute pain, alcoholism, autism, Tourette's syndrome, cocaine addiction, immune system suppression, multiple sclerosis, neurological diseases, neuropeptide or neurotransmitter imbalances, antitussive, or asthma.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0007] Figure 1 illustrates a method of synthesis of compounds 1-6 in an embodiment of the invention.

[0008] Figure 2 illustrates a method of synthesis of compounds 7-10 in an embodiment of the invention. [0009] Figures 3A, 3B, and 3C are graphs illustrating the supraspinal effects of compounds 1, 2, and 3 on morphine- (Figure 3 A, Figure 3B) and deltorphin C-induced antinociception (Figure 3C) in an embodiment of the invention. Figure 3 A is the hot-plate test analysis of time after administration (min) versus response time (sec). The following symbols are used: ♦ represents morphine (1 μg/mouse); Λ represents naltrindole; @ represents compound 1; D represents compound 2; and Δ represents compound 3. Figures 3B and 3C represent the area under the time-response curve (AUC). (***) p < 0.001 denotes significant differences from saline-treated mice by Dunnett's test.

[0010] Figure 4 is a graph comparing intracerebroventricular (icv) injected compound 3 in accordance with an embodiment of the invention (S) with naloxone ( ^A) and D-Phe-c(Cys- Tyr-D-Tφ-Orn-Thr-Pen)-Thr-NH₂ (CTOP) (T). The data are shown as log(g) of the dose versus % MPE (% MPE is the percent maximum possible effect and is calculated after 10 min following icv injection of the compounds). Each value is the mean ± S.E.M. of 5-6 mice. [0011] Figures 5 A and 5B are graphs illustrating the inhibition of antinociception subcutaneously injected compound 3 in an embodiment of the invention. Figure 5 A shows a time course study of time after administration (min) versus % MPE. The following symbols are used: ♦ represents morphine (1 μg/mouse); D represents compound 3 (0.01 mg/kg); O represents compound 3 (0.1 mg/kg); B represents compound 3 (1 mg/kg); and Δ represents compound 3 (3 mg/kg). Figure 5B shows the area under the time-response curve (AUC). Values are the mean ± S.E.M. of 5-6 mice per point. (**) p < 0.01 and (*) p < 0.05 denote significant differences from saline-treated mice by Dunnett's test.

[0012] Figures 6A and 6B are graphs illustrating the AUC of spinal effects of compound 3 on morphine- (Figure 6A) and deltorphin C-induced antinociception (Figure 6B) in an embodiment of the invention. In the tail-flick test results, values are the mean ± S.E.M. of 5- 6 mice per point. (***) p < 0.001 and (*) p < 0.05 denote significant differences from saline- treated mice by Dunnett's test.

[0013] Figures 7A and 7B are graphs illustrating the spinal amelioration of the development of morphine-induced tolerance by compound 3 in an embodiment of the invention. The results are shown as tail-flick test assessment at days 1 and 6 of the treatment regime. Figure 7 A shows a time course study of time after administration (min) versus % MPE. The following symbols are used: ♦ represents morphine (day 1); Δ represents morphine (day 6); O represents morphine plus compound 3 (day 1); and O represents morphine plus compound 3 (day 6). Figure 7B shows the area under the time-response curve (AUC). Values are the mean ± S.E.M. of 8-9 mice per point. (***) p < 0.001, (**) p < 0.01 and (*) p < 0.05 denote significant differences from saline-treated mice by Dunnett's test.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Dipeptides of Dmt-Tic have received considerable attention as agonists of δ- opioid receptors. The present invention seeks to provide agonists and antagonists of μ-opioid and δ-opioid receptors.

[0015] The present invention is directed to the Dmt-Tic pharmacophore comprising a lysine side chain that may be protected or unprotected. The lysine can be either the L or D configuration. More specifically, the present invention provides a compound of formula (I):

wherein

R¹ is selected from the group consisting of hydrogen, alkylcarbonyl, and aralkyloxycarbonyl; and

R² and R³ are the same or different and each is selected from the group consisting of hydrogen, arylamido, aralkylamido, and heteroaryl; an optical isomer thereof; or a pharmaceutically acceptable salt thereof.

[0016] In particular, R¹ is selected from the group consisting of hydrogen, acetyl, and benzyloxycarbonyl, R² is selected from the group consisting of hydrogen, phenylamido, benzylamido, and lH-benzimidazole-2-yl ("Bid"), and R³ is selected from the group consisting of hydrogen, phenylamido, benzylamido, and Bid.

[0017] Referring now to terminology used generically herein, the term alkylcarbonyl refers to the group -C(O)R, in which R is an alkyl group. [0018] The term "alkyl" implies a straight or branched alkyl substituent containing from, for example, 1 to 12 carbon atoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to 6 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecyl, and the like. Examples of an alkylcarbonyl are acetyl, propionyl, and butanoyl. [0019] The term "aralkyloxycarbonyl" refers to an aralkyloxy moiety bound to a carbonyl. The term "aralkyloxy" refers to substituents that have an aralkyl group attached to a divalent oxygen. The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, tolyl, naphthyl, xylyl, anthracenyl and the like. An aryl substituent generally contains from, for example, 6 to 30 carbon atoms, preferably from 6 to 18 carbon atoms, more preferably from 6 to 14 carbon atoms and even more preferably from 6 to 10 carbon atoms. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 π electrons, according to Hϋckel's Rule. The term "alkyl" is as defined herein. An example of an aralkyloxycarbonyl is benzyloxycarbonyl.

[0020] The terms "arylamido" and "aralkylamido" refer to the groups -C(O)NHAr and -C(O)NH(CH₂)_nAr, respectively, in which Ar is an aryl group as described herein. The term "alkyl" is as defined herein. The integer n is 1 to 6.

[0021] The term "heteroaryl" refers to aromatic 5 or 6 membered monocyclic groups, 9 or 10 membered bicyclic groups, and 11 to 14 membered tricyclic groups which have at least one heteroatom (O, S or N) in at least one of the rings. Each ring of the heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatorns in each ring is four or less and each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms and may be aromatic, saturated, partially saturated, or unsaturated. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. Heteroaryl groups which are bicyclic or tricyclic must include at least one fully aromatic ring but the other fused ring or rings may be aromatic or non-aromatic. The heteroaryl group may be attached at any available nitrogen or carbon atom of any ring. Illustrative examples of heteroaryl groups are pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl (e.g., lH-benzimidazole-2-yl), triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (l,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, thienyl, isothiazolyl, thiazolyl, furyl, isoxazolyl, oxadiazolyl, and oxazolyl. [0022] The term "pharmaceutically acceptable salt" is meant to include salts of the compound of formula (I) which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of formula (I) contain relatively acidic functionalities, base addition salts can be obtained by contacting the free acid form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include alkali or alkaline earth metal salts, such as sodium, potassium, calcium, magnesium salts, or ammonium, organic amino, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the free base form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, trifluroacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Trifluoroacetic acid salts are preferred. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1-19 (1977)). In accordance with embodiments of the invention, compounds of the present invention can contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0023] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0024] The term "antagonist," as used herein, refers to a compound that competes with an endogenous δ-opioid or μ-opioid ligand and inhibits δ- or μ-opioid signaling. In contrast, the term "agonist," as used herein, refers to a compound that competes with the endogenous δ- opioid or μ-opioid ligand and activates or enhances δ- or μ-opioid signaling. [0025] The present inventive compounds can be synthesized by any suitable method. See, for example, Modern Techniques of Peptide and Amino Acid Analysis, John Wiley & Sons, 1981; Bodansky, Principles of Peptide Synthesis, Springer Verlag, 1984). In general, compounds of formula (I) can be made according to an embodiment of the invention, starting from protected Lys and further protecting it, then selectively deprotecting the first protecting group, adding protected Tic, selectively deprotecting Tic, adding protected Dmt, selectively deprotecting Dmt, and then optionally further deprotecting Lys. Specific examples of a method of synthesis of the present inventive compounds are set forth in the Examples herein. See, also, Figures 1 and 2. [0026] Specific compounds of the present invention have the formula

thereof, or a pharmaceutically acceptable salt (e.g., trifluoroacetic acid) thereof.

[0027] The present invention further provides a pharmaceutical composition comprising at least one compound of formula (I) and a pharmaceutically acceptable carrier. 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.

[0028] A compound of the present invention can be made into a formulation suitable for parenteral administration, preferably oral, subcutaneous, intraperitoneal, 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. [0029] 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.

[0030] 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.

[0031] 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.

[0032] A formulation suitable for topical application can be in the form of creams, ointments, or lotions.

[0033] 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.

[0034] 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. For example, time release or controlled release encapsulation based on state of the art methodology would permit the distribution of the active ingredient over a substantial time period in order to maintain adequate levels of the compound throughout the body. Additional active agents include, for example, pain relievers, including non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., acetaminophen, aspirin, methyl salicylate, diflunisal, indomethacin, sulindac, diclofenac, ibuprofen, ketoprofen, naproxen, ketorolac, meloxicam, piroxicam, celecoxib, valdecoxib, parecoxib, etoricoxib), and corticosteroids (e.g., cortisone, hydrocortisone, prednisone, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone). [0035] Whether an above-described compound functions as an agonist, a partial agonist, an antagonist, a partial antagonist, an inverse agonist, 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 δ-receptors and the guinea pig ileum in vitro assay of μ-receptors as described in the Examples. The specificity and affinity of the inventive compounds for μ- and δ-opioid receptors can be determined using any suitable method, such as a non-radiolabelled competitive binding assay (see, e.g., Balboni et al, J. Med. Chem., 45: 5556-5563 (2002), Lazarus et al., J. Med. Chem., 34: 1350-1359 (1991), Salvadori et al., J. Med. Chem., 42: 5010-5019 (1999), and Balboni et al., Bioorg. Med. Chem., 11: 5435-5441 (2003)). Examples of in vivo studies include, but are not limited to, the hot-plate test or tail-flick test, as described in the Example and in the literature (Harris et al., J. Pharmacol. Metk, 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). [0036] The present invention is directed to a method of agonizing or antagonizing a μ- or δ-opioid receptor in a mammal in need thereof, which method comprises administering an effective amount of at least one compound of formula (I). More specifically, an embodiment of the present invention is a method of agonizing a μ-opioid receptor in a mammal in need thereof comprising administering a compound selected from the group consisting of

pharmaceutically acceptable salt thereof. [0037] In another embodiment, the present invention provides a method of antagonizing a μ-opioid and δ-opioid receptor in a mammal in need thereof comprising administering a compound selected from the group consisting of a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0038] In addition, the present invention provides a method of antagonizing a μ-opioid receptor in a mammal in need thereof comprising administering a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0039] The present invention also provides a method of selectively antagonizing a δ- opioid receptor in a mammal in need thereof comprising administering a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0040] It is contemplated that compounds of formula (I) exhibiting pharmacological properties of μ agonism/δ agonism can be useful as analgesics which could have a low dependence for chronic use for the amelioration of pain. Compounds of formula (I) with a mixed μ agonist/δ antagonist activity profile can have diminished propensity to induce tolerance and therefore it is contemplated that they can have therapeutic advantages over μ agonist analgesics for long term treatment of pain. δ-Opioid receptor agonists in accordance with an embodiment of the invention can be used as analgesics with relatively few side effects. It is also contemplated that δ-opioid receptor agonists of the invention can have antidepressant-like and anxiolytic-like effects and can be used to regulate BDNF mRNA expression in rodents, such that the regulation of BDNF mRNA expression could be useful in the treatment of multiple sclerosis and related diseases. Further, in accordance with the invention, it is expected that ^-opioid receptor activation protects cortical neurons, producing hibernation and neuroprotection. Activation of δ- and κ-opioid receptors affords cardioprotection.

[0041] In a specific embodiment, the invention provides a method of ameliorating morphine tolerance in a mammal in need thereof comprising administering a compound of the formula:

, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0042] It is further contemplated that δ-selective compounds of formula (I) can be used for the amelioration of the effects of alcoholism, the treatment of autism, and/or Tourette's syndrome. Compounds of formula (I) that are δ-opiate antagonists are contemplated for inhibiting the reinforcing properties of cocaine, moderating the behavioral effects of amphetamines, suppressing the immune system (e.g., for successful organ transplantation), used as an antitussive agent, and/or in the treatment of asthma.

[0043] In embodiments of the invention, it is contemplated that a compound of formula (I) can be useful in medicinal applications. Such applications include the regulation of food intake, treatment of chronic or acute pain (e.g., nociception), alcoholism, autism, Tourette's syndrome, cocaine addiction, immune system suppression, multiple sclerosis, neurological diseases, and neuropeptide or neurotransmitter imbalances. Accordingly, the present invention provides a method of treating a condition in a mammal comprising administering an effective amount of a compound of formula (I) to the mammal in need thereof, wherein the condition is regulation of food intake, treatment of chronic or acute pain (e.g., nociception), alcoholism, autism, Tourette's syndrome, cocaine addiction, immune system suppression, multiple sclerosis, neurological diseases, neuropeptide or neurotransmitter imbalances, antitussive, or asthma.

[0044] In another specific embodiment, the invention provides a method of reducing pain sensitivity produced within neurons when an opioid combines with a receptor, known as antinociception. The method is a method of antagonizing centrally mediated morphine- and/or deltorphin C-induced antinociception in a mammal in need thereof comprising administering a compound selected from the group consisting of

isomer thereof, or a pharmaceutically acceptable salt thereof.

[0045] The dose administered to a mammal, particularly a human, in the context of the methods of the present invention should be sufficient to affect a response, including 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 agonizing/antagonizing the receptor, 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. [0046] 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 δ-opioid receptor or a μ-opioid receptor as desired.

[0047] 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.

[0048] 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. Various general considerations taken into account in determining the "effective amount" are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

[0049] 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 hot plate and tail-flick test (see, e.g., U.S. Patent 5,780,589). In vitro models are also available, examples of which are set forth in the Examples herein.

[0050] The dose of the compound of formula (I) desirably comprises about 0.1 mg per kilogram (kg) of the body weight of the mammal (mg/kg) to about 400 mg/kg (e.g., about 0.75 mg/kg, about 5 mg/kg, about 30 mg/kg, about 75 mg/kg, about 100 mg/kg, about 200 mg/kg, or about 300 mg/kg). In another embodiment, the dose of the compound of formula (I) comprises about 0.5 mg/kg to about 300 mg/kg (e.g., about 0.75 mg/kg, about 5 mg/kg, about 50 mg/kg, about 100 mg/kg, or about 200 mg/kg), about 10 mg/kg to about 200 mg/kg (e.g., about 25 mg/kg, about 75 mg/kg, or about 150 mg/kg), or about 50 mg/kg to about 100 mg/kg (e.g., about 60 mg/kg, about 70 mg/kg, or about 90 mg/kg).

[0051] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

ABBREVIATIONS

Ac acetyl

Bid lH-benzimidazole-2-yl

Boc fert-butyloxycarbonyl

DAMGO [D-Ala²Λ-Me-Phe⁴,Gly-ol⁵]enkephalin

DEL C deltorphin II (H-TyT-D-AIa-PhC-ASp-VaI-VaI-GIy-NH₂)

DMF N,N-dimethylformamide

DMSO-J₆ hexadeuteriodimethyl sulfoxide

Dmt 2',6'-dimethyl-L-tyrosine

GPI guinea-pig ileum

HOBt 1-hydroxybenzotriazole

HPLC high performance liquid chromatography

MALDI-TOF matrix assisted laser desorption ionization time-of-flight

MPE maximum possible effect

MVD mouse vas deferens

ΝMM 4-methylmorpholine pA₂ negative log of the molar concentration required to double the agonist concentration to achieve the original response

TFA trifluoroacetic acid

Tic l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

TIP(P) H-Tyr-Tic-Phe-(Phe)-OH

TLC thin-layer chromatography

WSC l-ethyl-3-[3'-dimethyl)aminopropyl]-carbodiimide hydrochloride

Z benzyloxycarbonyl [0052] Crude peptides and pseudopeptides are purified by preparative reversed-phase HPLC [Waters Delta Prep 4000 system with Waters Prep LC 40 mm Assembly column Cl 8 (30 cm x 4 cm, 15 μm particle)] and eluted at a flow rate of 25 mL/min with mobile phase solvent A (10% acetonitrile + 0.1% TFA in H₂O, v/v), and a linear gradient from 25 to 75% B (60%, acetonitrile + 0.1% TFA in H₂O, v/v) in 25 min. Analytical HPLC analyses are performed with a Beckman System Gold (Beckman ultrasphere ODS column, 250 mm x 4.6 mm, 5 μm particle). Analytical determinations and capacity factor (K') of the products used HPLC in solvents A and B are programmed at flow rate of 1 mL/min with linear gradients from 0 to 100% B in 25 min. Analogues have less than 1% impurities at 220 and 254 nm. [0053] TLC is performed on precoated plates of silica gel F254 (Merck, Darmstadt, Germany): (A) l-butanol/AcOH/H₂O (3:1:1, v/v/v); (B) CH₂Cl₂/toluene/methanol (17:1:2). Ninhydrin (1% ethanol, Merck), fluorescamine (Hoffman-La Roche) and chlorine spray reagents. Melting points are determined on a Kofler apparatus and are uncorrected. Optical rotations are assessed at 10 mg/mL in methanol with a Perkin-Elmer 241 polarimeter in a 10 cm water-jacketed cell. Molecular weights of the compounds are determined by a MALDI- TOF analysis (Hewlett Packard G2025A LD-TOF system mass spectrometer) and α-cyano-4- hydroxycinnamic acid as a matrix. H NMR (δ) spectra are measured, when not specified, in OMSO-dg solution using a Bruker AC-200 spectrometer, and peak positions are given in parts per million downfield from tetramethylsilane as internal standard. [0054] Morphine sulfate, naloxone hydrochloride, and D~Phe-c(Cys-Tyr-D-Trp-Orn-Thr- Pen)-Thr-NH₂ (CTOP) are obtained from Sigma-Aldrich (St. Louis, MO, USA), and naltrindole hydrochloride from Tocris (Ellisville, MO, USA). [0055] Male Swiss- Webster mice (20-25 g, Taconic, Germantown, NY) are used. Animals are housed in plastic cages and maintained on a 12-h light/dark cycle with free access to food and water. All experiments with animals are carried out according to protocols approved by and on file with the NIEHS Animal Care and Use Committee (ACUC). [0056] Intracerebroventricular (icv) injection is performed with a Hamilton microsyringe fitted with a disposable 26-gauge needle inserted 2.3-3 mm deep as described by Laursen and Belknap (Laursen et al, J. Pharmacol. Meth., 16: 355-357 (1986)). Briefly, the bregma is detected by lightly rubbing the point of the needle over the skull until the suture is felt through the skin (about 1-3 mm rostral to a line drawn through the anterior base of the ears). The needle is inserted about 2 mm lateral to the midline and the total volume injected is 4 μl. Shortly after testing, the animals are sacrificed according ACUC protocols: a slit is made along the midline of the scalp and mice having needle tract 2 mm lateral from the bregma are counted as having been injected correctly.

[0057] Statistical significance of the data is estimated by one-way analysis of variance (ANOVA) followed by Dunnett's test using the computer software program JMP (SAS Institute Inc, Cary, NC, USA). The data are considered significant at P < 0.05. The area under the time-response curve (AUC) is obtained by plotting the response time(s) on the ordinate and time (min) on the abscissa after administration of the compounds. ADs₀ and Hill slope values with their 95% confidence intervals are calculated with a computer associated curve-fitting program (Prism 4™; GraphPad Software Inc., San Diego, CA).

EXAMPLE 1

[0058] This example demonstrates a method of peptide synthesis of Boc-Lys(Z)-NH- CH₂-Ph (Damour et al., Tetrahedron 1999, 55, 10135-10154).

[0059] To a solution of Boc-Lys(Z)-OH (0.23 g, 0.62 mmol) and beri2ylamine (0.07 mL, 0.62 mmol) in DMF (10 mL) at 0 ⁰C, HOBt (0.10 g, 0.68 mmol), and WSC (0.13 g, 0.68 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with citric acid (10% in H₂O), NaHCO₃ (5% in H₂O), and brine. The organic phase is dried (Na₂SO₄) and evaporated to dryness. The residue is precipitated from Et₂O/Pe (1 :9, v/v): yield 0.26 g (89%); RβB) 0.94; HPLC K! 5.55; mp 93-95 ⁰C; [α]²⁰ _D -12.1; w/z 471 (M+H)⁺; ¹H-NMR (DMSO-tf_ff) δ 1.29-1.79 (m, 15H), 2.96 (t, 2H), 4.46-4.53 (m, 3H), 5.34 (s, 2H), 7.06-7.19 (m, 10H).

EXAMPLE 2

[0060] This example demonstrates a method of synthesis of TFAH-Lys(Z)-NH-CH₂-Ph. [0061] Boc-Lys(Z)-NH-CH₂-Ph (0.20 g, 0.43 mmol) are treated with TFA (1 mL) for 0.5 h at room temperature. Et₂O/Pe (1:1, v/v) are added to the solution until the product is precipitated: yield 0.16 g (98%); Rf[A) 0.77; HPLC K' 3.55; mp 112-114 ⁰C; [α]²⁰ _D -13.4; m/z 371 (M+H)⁺.

EXAMPLE 3 [0062] This example demonstrates a method of synthesis of Boc-Tic-Lys(Z)-NH-CH₂-Ph. [0063] To a solution of Boc-Tic-OH (0.13 g, 0.46 mmol) and TFAH-Lys(Z)-NH-CH₂-Ph (0.22 g, 0.46 mmol) in DMF (10 niL) at 0 ⁰C, NMM (0.05 mL, 0.46 mmol), HOBt (0.07 g, 0.51 mmol), and WSC (0.09 g, 0.51 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with citric acid (10% in H₂O), NaHCO₃ (5% in H₂O), and brine. The organic phase is dried (Na₂SO₄) and evaporated to dryness. The residue is precipitated from Et₂O/Pe (1:9, v/v): yield 0.23 g (80%); Rf[B) 0.82; HPLC K' 5.61; mp 105-107 ⁰C; [α]²⁰ _D - 18.2; m/z 630 (M+H)⁺; ¹H-NMR (DMSO-^) δ 1.29-1.79 (m, 15H), 2.92-3.17 (m, 4H), 4.17- 4.53 (m, 5H), 4.92-5.34 (m, 3H), 6.96-7.19 (m, 14H).

EXAMPLE 4

[0064] This example demonstrates a method of synthesis of TFA^'H-Tic-Lys(Z)-NH-CH₂-

Ph.

[0065] Boc-Tic-Lys(Z)-NH-CH₂-Ph (0.17 g, 0.27 mmol) is treated with TFA (1 mL) for

0.5 h at room temperature. Et₂O/Pe (1:1, v/v) are added to the solution until the product is precipitated: yield 0.16 g (96%); Rf[A) 0.49; HPLC K 4.28; mp 118-120 ⁰C; [α]²⁰ _D -20.3; m/z 530 (M+H)⁺.

EXAMPLE 5

[0066] This example demonstrates a method of synthesis of Boc-Dmt-Tic-Lys(Z)-NH- CH₂-Ph.

[0067] To a solution of Boc-Dmt-OH (0.10 g, 0.32 mmol) and TFAH-Tic-Lys(Z)-NH- CH₂-Ph (0.21 g, 0.32 mmol) in DMF (10 mL) at 0 ⁰C, NMM (0.03 mL, 0.32 mmol), HOBt (0.05 g, 0.35 mmol), and WSC (0.07 g, 0.35 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with citric acid (10% in H₂O), NaHCO₃ (5% in H₂O), and brine. The organic phase is dried (Na₂SO₄) and evaporated to dryness. The residue is precipitated from Et₂O/Pe (1:9, v/v): yield 0.25 g (95%); Rf[B) 0.78; HPLC K 5.41; mp 132- 134 ⁰C; [α]²⁰ _D -17.1; m/z 821 (M+H)⁺; ¹H-NMR (DMSO-ck) δ 1.29-1.79 (m, 15H), 2.35 (s, 6H), 2.92-3.17 (m, 6H), 4.41-4.53 (m, 5H), 4.92-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.19 (m, 14H). EXAMPLE 6

[0068] This example demonstrates a method of synthesis of TF AΗ-Dmt-Tic-Lys(Z)-NH- CH₂-Ph (1).

[0069] Boc-Dmt-Tic-Lys(Z)-NH-CH₂-Ph (0.19 g, 0.23 mmol) is treated with TFA (1 mL) for 0.5 h at room temperature. Et₂CVPe (1 :1, v/v) are added to the solution until the product is precipitated: yield 0.16 g (96%); Rf(A) 0.45; HPLC K' 4.81; mp 128-130 ⁰C; [α]²⁰ _D -15.1; m/z 721 (M+H)⁺; ¹H-NMR (DMSO-J₁₅) δ 1.29-1.79 (m, 6H), 2.35 (s, 6H), 2.92-3.17 (m, 6H), 3.95-4.53 (m, 4H), 4.92-5.34 (m, 3H), 6.29 (s, 2H), 6.96-7.19 (m, 14H).

EXAMPLE 7

[0070] This example demonstrates a method of synthesis of Boc-Lys(Ac)-NH-CH₂-Ph. [0071] This compound is obtained by condensation of Boc-Lys(Ac)-OH with benzylamine via WSC/HOBt as reported for Boc-Lys(Z)-NH-CH₂-Ph: yield 0.32 g (82%); Rf[B) 0.88; HPLC K' 3.76; mp 101-103 ⁰C; [α]²⁰ _D -13.0; m/z 379 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.79 (m, 15H), 2.02 (s, 3H), 3.20-4.53 (m, 5H), 7.06-7.14 (m, 5H).

EXAMPLE 8

[0072] This example demonstrates a method of synthesis of TFA^'H-Lys(Ac)-NH-CH₂-

Ph.

[0073] Boc-Lys(Ac)-NH-CH₂-Ph is treated with TFA as reported for TFAH-Lys(Z)-NH-

CH₂-Ph: yield 0.19 g (98%); Rf[A) 0.74; HPLC K 2.14; mp 120-122 ⁰C; [α]²⁰ _D -14.3; m/z

279 (M+H)⁺.

EXAMPLE 9

[0074] This example demonstrates a method of synthesis of BoC-TiC-LyS(Ac)-NH-CH₂-

Ph.

[0075] This compound is obtained by condensation of Boc-Tic-OH with TFAH-Lys(Ac)-

NH-CH₂-Ph via WSC/HOBt as reported for Boc-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.46 g

(83%); Rf[B) 0.76; HPLC K' 5.02; mp 111-113 ⁰C; [α]²⁰ _D -19.1; m/z 537 (M+H)⁺; ¹H-NMR

(DMSO-4) δ 1.29-1.79 (m, 15H), 2.02 (s, 3H), 3.05-3.20 (m, 4H), 4.22-4.92 (m, 7H), 4.92-

5.34 (m, 3H), 6.96-7.14 (m, 9H). EXAMPLE 10

[0076] This example demonstrates a method of synthesis of TFAH-Tic-Lys(Ac)-NH-

CH₂-Ph.

[0077] Boc-Tic-Lys(Ac)-NH-CH₂-Ph is treated with TFA as reported for TFAH-Tic-

LyS(Z)-NH-CH₂-Ph: yield 0.32 g (98%); Rf[A) 0.46; HPLC K' 3.02; mp 124-126 ⁰C; [α]²⁰ _D -

21.2; m/z 437 (M+H)⁺.

EXAMPLE 11

[0078] This example demonstrates a method of synthesis of Boc-Dmt-Tic-Lys(Ac)-NH-

CH₂-Ph.

[0079] This compound is obtained by condensation of Boc-Dmt-OH with TFAΗ-Tic-

Lys(Ac)-NH-CH₂-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.15 g (85%); Rf[B) 0.72; HPLC K! 4.94; mp 138-140 ⁰C; [α]²⁰ _D -18.0; m/z 729

(M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.79 (m, 15H), 2.02 (s, 3H), 2.35 (s, 6H), 3.05-3.20 (m,

6H), 4.46-4.53 (m, 5H), 4.92 (m, 3H), 6.29 (s, 2H), 6.96-7.14 (m, 9H).

EXAMPLE 12

[0080] This example demonstrates a method of synthesis of TFA^'H-Dmt-Tic-Lys(Ac)-

NH-CH₂-Ph (2).

[0081] Boc-Dmt-Tic-Lys(Ac)-NH-CH₂-Ph is treated with TFA as reported for TFAH-

Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.07 g (95%); Rf[A) 0.42; HPLC K' 3.62; mp 134-136

⁰C; [Ct]²⁰ _D -16.0; m/z 629 (M+H)⁺; ¹H-NMR [OMSO-d₆) δ 1.29-1.79 (m, 6H), 2.02 (s, 3H),

2.35 (s, 6H), 3.05-3.20 (m, 6H), 3.95-4.53 (m, 6H), 4.92 (m, IH), 6.29 (s, 2H), 6.96-7.14 (m,

9H).

EXAMPLE 13

[0082] This example demonstrates a method of synthesis of Boc-Dmt-Tic-Lys-NH-CH₂-

Ph.

[0083] To a solution of Boc-Dmt-Tic-Lys(Z)-NH-CH₂-Ph (0.10 g, 0.12 mmol) in methanol (30 mL) is added C/Pd (10%, 0.07 g), and H₂ is bubbled for 1 h at room temperature. After filtration, the solution is evaporated to dryness. The residue is crystallized from Et₂O/Pe (1:9, v/v): yield 0.08 g (96%); Rf(B) 0.65; HPLC K 4.99; mp 141-143 ⁰C; [α]²⁰ _D -18.3; m/z 687 (MH-H)⁺.

EXAMPLE 14

[0084] This example demonstrates a method of synthesis of 2TFA^'H-Dmt-Tic-Lys~NH- CH₂-Ph (3).

[0085] Boc-Dmt-Tic-Lys-NH-CHrPh is treated with TFA as reported for TFAH-Dmt- Tic-Lys(Z)-NH-CH₂-Ph: yield 0.07 g (95%); RJ[A) 0.39; HPLC K' 3.32; mp 147-149 ⁰C; [α]²⁰ _D -16.2; m/z 587 (M+H)⁺; ¹H-NMR (OMSO-d₆) δ 1.29-1.79 (m, 6H), 2.35 (s, 6H), 2.65- 3.17 (m, 6H), 3.95-4.53 (m, 6H), 4.92 (m, IH), 6.29 (s, 2H), 6.96-7.14 (m, 9H).

EXAMPLE 15

[0086] This example demonstrates a method of synthesis of Boc-Lys(Z)-NH-Ph. [0087] This compound is obtained by condensation of Boc-Lys(Z)-OH with aniline via WSC/HOBt as reported for Boc-Lys(Z)-NH-CH₂-Ph: yield 0.23 g (82%); Bf(B) 0.89; HPLC K 5.15; mp 90-92 ⁰C; [α]²⁰ _D -15.2; m/z 456 (M+H)⁺; ¹H-NMR (DMSO-rf₆) δ 1.29-1.89 (m, 15H), 2.96 (t, 2H), 4.53-5.34 (m, 3H), 7.00-7.64 (m, 10H).

EXAMPLE 16

[0088] This example demonstrates a method of synthesis of TFAH-Lys(Z)-NH-Ph. [0089] Boc-Lys(Z)-NH-Ph is treated with TFA as reported for TFAH-Lys(Z)-NH-CH₂- Ph: yield 0.13 g (97%); Rf[A) 0.65; HPLC K! 3.63; mp 110-120 ⁰C; [α]²⁰ _D -15.9; m/z 356 (M+H)⁺.

EXAMPLE 17

[0090] This example demonstrates a method of synthesis of Boc-Tic-Lys(Z)-NH-Ph. [0091] This compound is obtained by condensation of Boc-Tic-OH with TFAH-Lys(Z)- NH-Ph via WSC/HOBt as reported for Boc-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.26 g (88%); Rf[B) 0.77; HPLC K' 5.47; mp 97-99 ⁰C; [α]²⁰ _D -19.5; m/z 616 (MH-H)⁺; ¹H-NMR (DMSO- d₆) δ 1.29-1.79 (m, 15H), 2.92-3.17 (m, 4H), 4.17-4.53 (m, 2H), 4.92-5.34 (m, 3H), 6.96-7.19 (m, 14H). EXAMPLE 18

[0092] This example demonstrates a method of synthesis of TFAΗ-Tic-Lys(Z)-NH-Ph. [0093] Boc-Tic-Lys(Z)-NH-Ph is treated with TFA as reported for TFAH-Tic-Lys(Z)- NH-CH₂-Ph: yield 0.17 g (98%); Rf[A) 0.44; HPLC K 431; mp 114-116 ⁰C; [α]²⁰ _D -20.4; m/z 516 (M+H)⁺.

EXAMPLE 19

[0094] This example demonstrates a method of synthesis of Boc-Dmt-Tic-Lys(Z)-NH-

Ph.

[0095] This compound is obtained by condensation of Boc-Dmt-OH with TFAΗ-Tic-

Lys(Z)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.22 g (84%); Rf[B) 0.73; HPLC K' 5.44; mp 127-129 ⁰C; [α]²⁰ _D -16.4; m/z 807 (M+H)⁺; ¹H-

NMR (DMSO-J₆) δ 1.29-1.89 (m, 15H), 2.35 (s, 6H), 2.92-3.17 (m, 6H), 4.41-4.53 (m, 3H),

4.92-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.64 (m, 14H).

EXAMPLE 20

[0096] This example demonstrates a method of synthesis of TFAΗ-Dmt-Tic-Lys(Z)-NH- Ph (4).

[0097] Boc-Dmt-Tic-Lys(Z)-NH-Ph is treated with TFA as reported for TFAH-Dmt-Tic- LyS(Z)-NH-CH₂-Ph: yield 0.13 g (93%); Rf[A) 0.38; HPLC K' 4.70; mp 124-126 ⁰C; [α]²⁰ _D - 14.4; m/z 707 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.89 (m, 6H), 2.35 (s, 6H), 2.96-3.05 (m, 4H), 3.95-4.53 (m, 4H), 4.92-5.34 (m, 3H), 6.29 (s, 2H), 6.96-7.64 (m, 14H).

EXAMPLE 21

[0098] This example demonstrates a method of synthesis of Boc-Lys(Ac)-NH-Ph. [0099] This compound is obtained by condensation of Boc-Lys(Ac)-OH with aniline via WSC/HOBt as reported for Boc-Lys(Z)-NH-CH₂-Ph: yield 0.35 g (92%); Rf[B) 0.83; HPLC K 3.85; mp 96-98 ⁰C; [α]²⁰ _D -16.1; m/z 365 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.89 (m, 15H), 2.02 (s, 3H), 3.20-4.53 (m, 3H), 7.00-7.64 (m, 5H).

EXAMPLE 22 [00100] This example demonstrates a method of synthesis of TFA^'H-Lys(Ac)-NH-Ph. [0100] Boc-Lys(Ac)-NH-Ph is treated with TFA as reported for TFAH-Lys(Z)-NH-CH₂-

PPhh:: yyiieelldd 0.21 g [9S%); Rf[A) 0.62; HPLC K' 2.31; mp 116-118 ⁰C; [α]²⁰ _D -16.8; m/z 265

(M+H)⁺.

EXAMPLE 23

[0101] This example demonstrates a method of synthesis of Boc-Tic-Lys(Ac)-NH-Ph. [0102] This compound is obtained by condensation of Boc-Tic-OH with TFAΗ-Lys(Ac)- NH-Ph via WSC/HOBt as reported for Boc-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.45 g (83%); Rf[B) 0.71; HPLC K' 4.86; mp 103-105 ⁰C; [α]²⁰ _D -20.4; m/z 523 (M+H)⁺; ¹H-NMR (DMSO-rf₆) δ 1.29-1.89 (m, 15H), 2.02 (s, 3H), 3.05-3.20 (m, 4H), 4.22-4.92 (m, 5H), 6.96- 7.64 (m, 9H).

EXAMPLE 24

[0103] This example demonstrates a method of synthesis of TFAΗ-Tic-Lys(Ac)-NH-Ph. [0104] Boc-Tic-Lys(Ac)-NH-Ph is treated with TFA as reported for TFAH-Tic-Lys(Z)- NH-CH₂-Ph: yield 0.30 g (96%); Rj[A) 0.41; HPLC K' 3.28; mp 120-122 ⁰C; [α]²⁰ _D -21.3; m/z 423 (M+H)⁺.

EXAMPLE 25

[0105] This example demonstrates a method of synthesis of Boc-Dmt-Tic-Lys(Ac)-NH-

Ph.

[0106] This compound is obtained by condensation of Boc-Dmt-OH with TFA.H-Tic-

Lys(Ac)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield

0.14 g (80%); Rββ) 0.67; HPLC K' 4.25; mp 133-135 ⁰C; [α]²⁰ _D -17.3; m/z 715 (M+H)⁺; ¹H-

NMR [OMSO-d₆) δ 1.29-1.89 (m, 15H), 2.02 (s, 3H), 2.35 (s, 6H), 3.05-3.20 (m, 6H), 4.46-

4.53 (m, 3H), 4.92 (m, 3H), 6.29 (s, 2H), 6.96-7.64 (m, 9H).

EXAMPLE 26

[0107] This example demonstrates a method of synthesis of TFA^'H-Dmt-Tic-Lys(Ac)- NH-Ph (5).

[0108] Boc-Dmt-Tic-Lys(Ac)-NH-Ph is treated with TFA as reported for TFAH-Dmt- Tic-Lys(Z)-NH-CH₂-Ph: yield 0.07 g (98%); Rf[A) 0.35; HPLC K! 3.73; mp 130-132 ⁰C; [α]²⁰ _D -145.3; m/z 615 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.89 (m, 6H), 2.02 (s, 3H), 2.35 (s, 6H), 3.05-3.20 (m, 6H), 3.95-4.53 (m, 4H), 4.92 (m, 3H), 6.29 (s, 2H), 6.96-7.64 (m, 9H).

EXAMPLE 27

[0109] This example demonstrates a method of synthesis of Boc-Dmt-Tic-Lys-NH-Ph. [0110] Boc-Dmt-Tic-Lys(Z)-NH-Ph is treated with H₂ in presence of C/Pd 10% as reported for Boc-Dmt-Tic-Lys-NH-CH₂-Ph: yield 0.18 g (94%); Rf[B) 0.64; HPLC K 4.71; mp 138-140 ⁰C; [α]²⁰ _D -17.8; m/z 673 (M+H)⁺.

EXAMPLE 28

[0111] This example demonstrates a method of synthesis of 2TFAH-Dmt-Tic-Lys-NH- Ph (6).

[0112] Boc-Dmt-Tic-Lys-NH-Ph is treated with TFA as reported for TFAH-Dmt-Tic- LyS(Z)-NH-CH₂-Ph: yield 0.05 g (92%); Rf[A) 0.34; HPLC K' 3.15; mp 149-151 ⁰C; [α]²⁰ _D - 15.8; m/z 573 (M+H)⁺; ¹H-NMR (DMSO-J₁₅) δ 1.29-1.89 (m, 6H), 2.35 (s, 6H), 2.65-3.05 (m, 4H), 3.95-4.53 (m, 4H), 4.92 (m, IH), 6.29 (s, 2H), 6.96-7.64 (m, 9H).

EXAMPLE 29

[0113] This example demonstrates a method of synthesis of benzyl 5-(tert-butyl 3- carbamoyl-3,4-dihydroisoquinoline-2(lH)-carboxyloyl)-5-(lH-benzo[J]imidazol-2- yl)pentylcarbamate {Boc-Tic-NH-CH[(CH₂)₄-NH-Z]-Bid} .

[0114] To a solution of Boc-Tic-OH (0.33 g, 1.20 mmol) and 2TFAH₂N-CH[(CH₂)₄-NH- Z]-Bid [benzyl 5-amino-5-(lH-benzo[<i]imidazol-2-yl)pentylcarbamate]⁴³ (0.70 g, 1.20 mmol) in DMF (10 mL) at 0 ⁰C, NMM (0.26 mL, 2.40 mmol), ΗOBt (0.20 g, 1.32 mmol), and WSC (0.25 g, 1.32 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with NaHCO₃ (5% in H₂O), and brine. The organic phase is dried (Na₂SO₄) and evaporated to dryness. The residue is precipitated from Et₂OZPe (1:9, v/v): yield 0.59 g (80%); RJ[B) 0.63; HPLC K 4.94; mp 137-139 ⁰C; [α]²⁰ _D -13.3; m/z 613 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.84 (m, 15H), 2.92-3.17 (m, 4H), 4.17-4.87 (m, 3H), 4.92-5.34 (m, 3H), 6.96-7.70 (m, 13H). EXAMPLE 30

[0115] This example demonstrates a method of synthesis of 2TFAH-Tic-NH-CH[(CH₂)₄-

NH-Z]~Bid.

[0116] Boc-Tic-NH-CH[(CH₂)₄-NH-Z]-Bid is treated with TFA as reported for TFAH-

Tic-Lys(Z)-NH-CH₂-Ph: yield 0.43 g (97%); Rf[A) 0.45; HPLC K' 3.72; mp 140-142 ⁰C;

[α]²⁰ _D -14.6; m/z 513 (M+H)⁺.

EXAMPLE 31

[0117] This example demonstrates a method of synthesis of Boc-Dmt-Tic-NH- CH[(CH₂)₄-NH-Z]-Bid.

[0118] To a solution of Boc-Dmt-OH (0.10 g, 0.32 mmol) and 2TFAH-Tic-NH- CH[(CH₂)₄-NH-Z]-Bid (0.24 g, 0.32 mmol) in DMF (10 mL) at 0 ⁰C, NMM (0.07 mL, 0.64 mmol), HOBt (0.05 g, 0.35 mmol), and WSC (0.07 g, 0.35 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with NaHCO₃ (5% in H₂O), and brine. The organic phase is dried (Na₂SO₄) and evaporated to dryness. The residue is precipitated from Et₂O/Pe (1:9, v/v): yield 0.23 g (91%); Rf[B) 0.63; HPLC K' 4.88; mp 140-142 ⁰C; [α]²⁰ _D -14.8; m/z 804 (M+H)⁺; ¹H-NMR (DMSO-4) δ 1.29-1.84 (m, 15H), 2.35 (s, 6H), 2.92-3.17 (m, 6H), 4.41-4.87 (m, 3H), 4.92-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.70 (m, 13H).

EXAMPLE 32

[0119] This example demonstrates a method of synthesis of 2TFAΗ-Dmt-Tic-NH-

CH[(CH₂)₄-NH-Z]-Bid (7).

[0120] Boc-Dmt-Tic-NH-CH[(CH₂)₄-NH-Z]-Bid is treated with TFA as reported for

TFAH-Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.04 g (96%); Rf[A) 0.31; HPLC K' 3.90; mp

146-148 ⁰C; [α]²⁰ _D -18.3; m/z 704 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.84 (m, 6H), 2.35

(s, 6H)₅ 2.96-3.05 (m, 6H), 3.95-4.46 (m, 3H), 4.87-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.70 (m,

13H).

EXAMPLE 33

[0121] This example demonstrates a method of synthesis of Boc-Tic-NH-(Φ>CH[(CH₂)₄- NH-Z]-Bid. [0122] This compound is obtained by condensation of Boc-Tic-OH with 2TFA' H₂N-(D,)- CH[(CH₂)₄-NH-Z]-Bid via WSC/HOBt as reported for Boc-Tic-NH-CH[(CH₂)₄-NH-Z]-Bid: yield 0.56 g (83%); Rj(B) 0.64; HPLC K' 4.87; mp 139-141 ⁰C; [α]²⁰ _D +6.8; m/z 613 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.84 (m, 15H), 2.92-3.17 (m, 4H), 4.17-4.87 (m, 3H), 4.92-5.34 (m, 3H), 6.96-7.70 (m, 13H).

EXAMPLE 34

[0123] This example demonstrates a method of synthesis of 2TFAH-Tic-NH-(D>

CH[(CH₂)₄-NH-Z]-Bid.

[0124] Boc-Tic-NH-(2⁾j-CH[(CH₂)₄-NH-Z] -Bid is treated with TFA as reported for

TFAH-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.39 g (92%); Rf(A) 0.47; HPLC K' 3.71; mp 143-145

⁰C; [Ct]²⁰ _D +7.5; m/z 513 (M+H)⁺.

EXAMPLE 35

[0125] This example demonstrates a method of synthesis of Boc-Dmt-Tic-NH-(2)j-

CH[(CH₂)₄-NH-Z]-Bid.

[0126] This compound is obtained by condensation of Boc-Dmt-OH with 2TFAH-Tic-

NH-(D>CH[(CH₂)₄-NH-Z]-Bid via WSC/HOBt as reported for Boc-Dmt-Tic-NH-

CH[(CH₂)₄-NH-Z]-Bid: yield 0.17 g (87%); Rf[B) 0.65; HPLC K' 5.17; mp 142-144 ⁰C;

[α]²⁰ _D +4.9; m/z 804 (M+H)⁺; ¹H-NMR (DMSO-4) δ 1.29-1.84 (m, 15H), 2.35 (s, 6H), 2.92-

3.17 (m, 6H), 4.41-4.87 (m, 3H), 4.92-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.70 (m, 13H).

EXAMPLE 36

[0127] This example demonstrates a method of synthesis of 2TFAΗ-Dmt-Tic-NH-(Z)>

CH[(CH₂)₄-NH-Z]-Bid (8).

[0128] Boc-Dmt-Tic-NH-(7J>>CH[(CH₂)₄-NH-Z]-Bid is treated with TFA as reported for

TFAH-Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.06 g (91%); Rf[A) 0.33; HPLC K! 3.90; mp

149-151 ⁰C; [α]²⁰ _D +5.6; m/z 704 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.84 (m, 6H), 2.35

(s, 6H), 2.96-3.05 (m, 6H), 3.95-4.46 (m, 3H), 4.87-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.70 (m,

13H). EXAMPLE 37

[0129] This example demonstrates a method of synthesis of Boc-NH-CH[(CH₂)₄-NH- Ac]-Bid.

[0130] A solution of Boc-Lys(Ac)-OH (0.3 g, 1.04 mmol) and NMM (0.11 niL, 1.04 mmol) in DMF (10 mL) is treated at -20 ⁰C with IBCF, 0.14 mL, 1.04 mmol). After 10 min at -20 ⁰C, σ-phenylendiamine (0.11 g, 1.04 mmol) is added. The reaction mixture is allowed to stir while slowly warming to room temperature (1 h) and is then stirred for an additional 3 h. The solvent is evaporated, and the residue is partitioned between EtOAc and H₂O. The EtOAc layer is washed with 5% NaHCO₃ and brine and dried over Na₂SO₄. The solution is filtered, the solvent is evaporated, and the residual solid is dissolved in glacial AcOH (10 mL). The solution is heated at 65 ⁰C for 1 h. After the solvent is evaporated, the residue is precipitated from Et₂O/Pe (1:9, v/v): yield 0.3 g (80%); RβB) 0.71; HPLC K 3.03; mp 141- 143 ⁰C; [(X]²⁰ _D -7.6; m/z 362 (M+H)⁺; ¹H-NMR (DMSO-^₆) δ 1.29-1.84 (m, 15H), 2.02 (s, 3H), 3.20 (m, 2H), 4.87 (m, IH), 7.26-7.70 (m, 4H).

EXAMPLE 38

[0131] This example demonstrates a method of synthesis of 2TFA-H₂N-CH[(CH₂)₄-NH-

Ac]-Bid.

[0132] Boc-NH-CH[(CH₂)₄-NH-Ac]-Bid is treated with TFA as reported for TFAH-

LyS(Z)-NH-CH₂-Ph: yield 0.16 g (90%); Rf[A) 0.48; HPLC K' 2.08; mp 149-151 ⁰C; [α]²⁰ _D -

9.8; m/z 262 (M+H)⁺.

EXAMPLE 39

[0133] This example demonstrates a method of synthesis of Boc-Tic-NH-CH[(CH₂)₄-

NH-Ac]-Bid.

[0134] This compound is obtained by condensation of Boc-Tic-OH with 2TFAH₂N-

CH[(CH₂)₄-NH-Ac]-Bid via WSC/HOBt as reported for Boc-Tic-NH-CH[(CH₂)₄-NH-Z]-

Bid: yield 0.45 g (85%); RβB) 0.57; HPLC K 4.09; mp 143-145 ⁰C; [α]²⁰ _D -14.2; m/z 521

(M+H)⁺; ¹H-NMR (DMSO-ck) δ 1.29-1.84 (m, 15H), 2.02 (s, 3H), 3.05-3.20 (m, 4H), 4.22-

4.92 (m, 5H), 6.96-7.70 (m, 8H). EXAMPLE 40

[0135] This example demonstrates a method of synthesis of 2TFA-H-Tic-NH-

CH[(CH₂)₄-NH-Ac]-Bid.

[0136] Boc-Tic-NH-CH[(CH₂)₄-NH-Ac]-Bid is treated with TFA as reported for TFAH-

Tic-Lys(Z)-NH-CH₂-Ph: yield 0.31 g (97%); RJ[A) 0.42; HPLC K' 3.38; mp 146-148 ⁰C;

[α]²⁰ _D -15.5; m/z 421 (M+H)⁺.

EXAMPLE 41

[0137] This example demonstrates a method of synthesis of Boc-Dmt-Tic-NH-

CH[(CH₂)₄-NH-Ac]-Bid.

[0138] This compound is obtained by condensation of Boc-Dmt-OH with 2TFAH-Tk-

NH-CH[(CH₂)₄-NH-Ac]-Bid via WSC/HOBt as reported for Boc-Dmt-Tic-NH-CH[(CH₂)₄-

NH-Z]-Bid: yield 0.13 g (78%); RβB) 0.57; HPLC K' 4.31; mp 150-152 ⁰C; [α]²⁰ _D -15.7; m/z 712 (M+H)⁺; ¹H-NMR (DMSO-^) δ 1.29-1.84 (m, 15H), 2.02 (s, 3H), 2.35 (s, 6H),

3.05-3.20 (m, 6H), 4.46 (s, 2H), 4.87-4.92 (m, 3H), 6.29 (s, 2H), 6.96-7.70 (m, 8H).

EXAMPLE 42

[0139] This example demonstrates a method of synthesis of 2TFA-H-Dmt-Tic-NH- CH[(CH₂)₄-NH-Ac]-Bid (9).

[0140] Boc-Dmt-Tic-NH-CH[(CH₂)₄-NH-Ac]-Bid is treated with TFA as reported for TFAH-Dmt-Tic-Lys(Z)-NH-CH₂-Ph: yield 0.06 g (93%); Rf[A) 0.28; HPLC K' 3.16; mp 153-155 ⁰C; [α]²⁰ _D -19.2; m/z 612 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.84 (m, 6H), 2.02 (s, 3H), 2.35 (s, 6H), 3.05-3.20 (m, 6H), 3.95-4.46 (m, 3H), 4.87-4.92 (m, 3H), 6.29 (s, 2H), 6.96-7.70 (m, 8H).

EXAMPLE 43

[0141] This example demonstrates a method of synthesis of Boc-Dmt-Tic-NH-

CH[(CH₂)₄-NH₂]-Bid.

[0142] Boc-Dmt-Tic-NH-CH[(CH₂)₄-NH-Z]-Bid is treated with H₂ in presence of C/Pd

10% as reported for Boc-Dmt-Tic-Lys-NH-CHz-Ph: yield 0.17 g (92%); Rj[B) 0.52; HPLC

K 4.02; mp 147-149 ⁰C; [α]²⁰ _D -16.3; m/z 670 (M+H)⁺. EXAMPLE 44

[0143] This example demonstrates a method of synthesis of 3TFAΗ-Dmt-Tic-NH-

CH[(CH₂)₄-NH₂]-Bid (10).

[0144] Boc-Dmt-Tic-NH-CH[(CH₂)₄-NH₂]-Bid is treated with TFA as reported for

TFAH-DnIt-TiC-LyS(Z)-NH-CH₂-Ph: yield 0.03 g (93%); Rf[A) 0.29; HPLC K' 2.98; mp

156-158 ⁰C; [α]²⁰ _D -20.4; m/z 570 (M+H)⁺; ¹H-NMR (DMSO-J₆) δ 1.29-1.84 (m, 6H), 2.35

(s, 6H), 2.65-3.05 (m, 6H), 3.95-4.46 (m, 3H), 4.87-4.92 (m, 2H), 6.29 (s, 2H), 6.96-7.70 (m,

8H).

EXAMPLE 45

[0145] This example demonstrates a method to determine the equilibrium receptor binding.

[0146] The assay is conducted as described in detail elsewhere using rat brain synaptosomes (P2 fraction) (Salvadori et al, MoI. Med. 1995, 1, 678-689; Salvadori et al., J. Med. Chem. 1997, 40, 3100-3108; Balboni et al., Peptides 2000, 21, 1663-1671; Balboni et al., J. Med. Chem. 2002, 45, 713-720; Balboni et al., Bioorg. Med. Chem. 2003, 11, 5435- 5441; Balboni et al., J. Med. Chem. 2004, 47, 6541-6546; and Balboni et al., J. Med. Chem. 2005, 48, 8112-8114). Membrane preparations are preincubated to eliminate endogenous opioid peptides and after extensive washing with ice cold buffer, stored at -80 ⁰C in buffered 20% glycerol containing 50 μg/mL soybean trypsin inhibitor. Each analogue is analyzed in duplicate using five to eight different doses of peptide and independent repetitions with different synaptosomal preparations ranging from 400-600 μg total protein (n values are listed in Table 1 in parentheses and the results are the mean ± SE). Unlabeled peptide (2 /M) determined non-specific binding in the presence of 1.9 nM [³H]deltorphin C (45.0 Ci/mmol, Perkin-Elmer, Boston, MA; Kn - IA nM) for ^-opioid receptors; for //-opioid receptors, 3.5 nM [³H]DAMGO (50.0 Ci/mmol, Amersham Biosciences, Buckinghamshire, U.K.; Ku = 1.5 nM). After an incubation of 2.5 hr room temperature (ca. 22 ⁰C), the synaptome-radioligand complex is filtered through glass fibre filters (Whatman GFC), presoaked in 0.1% polyethylenimine to enhance the signal/noise ratio of the bound radiolabeled ligand- synaptosome complex, and the filters are washed thrice in ice-cold buffered BSA. The affinity constants (Ki) are calculated according to Cheng et al. (Biochem. Pharmacol. 1973, 22, 3099-3108). [0147] Receptor binding and functional bioactivities are reported in Table 1. In Table 1 , the notation "^α" indicates that the K_\ values (nM) are determined according to Chang and Prusoff (Biochem. Pharmacol. 1973, 22, 3099-3108). The mean ± SE with n repetitions in parenthesis is based on independent duplicate binding assays with five to eight peptide doses using several different synaptosomal preparations. In addition, the notation "^δ" indicates that agonist activity is expressed as IC₅₀ obtained from dose-response curves. These values represent the mean ± SE for at least five to six fresh tissue samples. Deltorphin II and endomorphin-2 are the internal standards for MVD (<5-opioid receptor bioactivity) and GPI (//-opioid receptor bioactivity) tissue preparation, respectively. The notation "^c" indicates that the pA₂ values of opioid antagonists against the agonists (deltorphin II and endomorphin- 2) are determined by the method of Kosterlitz and Watt (Sasaki et al., Bioorg. Med. Chem. 2003, 11, 675-678). Finally, the notation "n.a." indicates the compound had no antagonism.

EXAMPLE 46

[0148] This example demonstrates the functional bioactivity in isolated organ preparations.

[0149] Preparations of myenteric plexus-longitudinal muscle are obtained from male guinea pig ileum (GPI, enriched in //-opioid receptors) and preparations of MVD (containing ^-opioid receptors) are used for field stimulation with bipolar rectangular pulses of supramaximal voltage (Balboni et al., J. Med. Chem. 2004, 47, 4066-4071 and Balboni et al., J. Med. Chem. 2005, 48, 5608-5611). Agonists are evaluated for their ability to inhibit the electrically evoked twitch. The biological potency of the compounds is compared against the activity of the //-opioid receptor agonist dermorphin in GPI and with MVD for the <£-opioid receptor measured agonist deltorphin C. The results are expressed as the IC₅₀ obtained from dose-response curves. To evaluate antagonism, compounds are added to the bath and allowed to interact with tissue receptor sites 5 min before adding the standard peptide. Competitive antagonist activities are evaluated for their ability to shift the deitorphin C (MVD) and endomorphin-2 (GPI) log concentration-response curve to the right; pyi₂ values are determined using the Schild Plot. IC₅₀ (nM, mean ± SE) as well as the py4₂ are obtained from five to six experiments conducted with fresh tissues.

[0150] Compounds (1-10) are tested in the electrically stimulated MVD and GPI assays for intrinsic functional bioactivity (Table 1). LVM 701619

DHHS E-103-2000/3-PCT-02

37

Table 1. Receptor Binding and Functional Bioactivity. receptor affinity⁰ selectivity functional bioactivity

MVD GPI MVD GPI

Comp. Structure Kiδ (nM) Kiμ/Kiδ

IC₅₀ (nM)^δ IC₅₀ (nM)^έ pA₂ ^c V^2^C

Endomorphin-2 13.7

Deltorphin~π 0.371

1 H-Dmt-Tic-Lys(Z)-NH-CH₂-Ph 0.31±0.02 (3) 4.41±0.52 (5) 15 >10000 >10000 6.21 5.82

2 H-Dmt-Tic-Lys(Ac)-NH-CH₂-Ph 0.068±0.009 (3) 5.50±0.18 (3) 81 >10000 >10000 10.4 8.16

3 H-Dmt-Tic-Lys-NH-CH₂-Ph 0.50±0.07 (4) 4.05±0.54 (5) 8 >10000 >10000 6.01 7.96

4 H-Dmt-Tic-Lys(Z)-NH-Ph 0.57±0.06 (3) 1.47±0.20 (4) 3 >10000 438±65 5.77 -

5 H-Dmt-Tic-Lys(Ae)-NH-Ph 0.13±0.003 (4) 0.63±0.065 (3) 5 >10000 1248±194 12.0 -

6 H-Dmt-Tic-Lys-NH-Ph 0.42±0.02 (4) 0.93±0.05 (4) 2 >10000 1254±205 5.38 -

7 H-Dmt-Tic-NH-CH[(CH₂)₄NH-Z]-Bid 0.64±0.02 (3) 0.37±0.040 (3) 1.7* 9049±1128 375±33 <5 -

8 H-Dmt-Tic-NH-(D>CH[(CH₂)₄NH-Z]-Bid 0.40±0.08 (4) 0.15±0.018 (3) 2.7* >10000 62.5±13.5 <5 -

9 H-Dmt-Tic-NH-CH[(CH₂)₄NH-Ac]-Bid 0.18±0.03 (4) 0.13±0.02 (4) 1.4* >10000 53.9±7.3 n.a. -

10 H-Dmt-Tic-NH-CH[(CH₂)₄NH₂]-Bid 0.49±0.04 (3) 0.16±0.015 (3) 3.Ϊ* >10000 39.7±6.9 n.a. -

EXAMPLE 47

[0151] This example demonstrates the supraspinal effects of some of the compounds of formula (I) by a hot plate method.

[0152] The male Swiss-Webster mice are placed on an electrically heated plate (WTC MODEL 39D Hot Plate analgesia meter, IITC Inc, Woodland Hills, CA) at 55° ± 0.1 ⁰C after 10 min following intracerebroventricular (icv) administration of compounds of the invention. Hot-plate latency (HPL) is measured as the interval between placement of mice onto the hot plate and observing movement consisting of either jumping, licking, or shaking their hind paws with a baseline latency of 15 sec and maximal cut off time of 30 sec. Duration time is 10 min, and the test is terminated when HPL is close to the pre-response time. [0153] For studying the icv effect of antagonists, mice are injected with saline containing morphine (control) or morphine mixed with different doses of antagonists. For subcutaneous (sc) and oral (po) administrations, the antagonists are injected 5 and 20 min before morphine (icv), respectively, and the resulting effect is measured 10 min post-morphine injection. Compounds are injected icv with 2.52 μg/mouse and 8 ng/mouse for morphine and deltorphin C-induced antinociception, respectively.

[0154] Test compounds 1 (H-Dmt-Tic-Lys(Z)-NH-CH₂-Ph), 2 (H-Dmt-Tic-Lys(Ac)-NH- CH₂-Ph), and 3 (H-Dmt-Tic-Lys-NH-C^-Ph) exhibit dual μ-/δ-antagonism when injected icv in mice toward morphine (μ) and deltorphin C (δ) antinociception (Figures 3A, 3B, and 3C) based on the hot-plate test (supraspinal activity) (Jinsmaa et al., J. Med. Chem., 47: 2599- 2610 (2004); Jinsmaa et al., J. Pharmacol. Exp. Ther., 309: 432-438 (2004); and Okada et al., J. Med. Chem., 46, 3201-3209 (2003)).

[0155] The efficacy of compound 3 is compared with recognized antagonists, namely naloxone (non-selective), CTOP (D-Phe-c(Cys-Tyr-D-Tφ-Om-Thr-Pen)-Thr-NH₂) (μ-opioid receptor selective) (Braida et al., Peptides, 18, 1189-1195 (1997)), and naltrindole (δ-opioid receptor specific) (Figure 4). While the effective dose of compound 3 to reduce morphine antinociception is 2.52 μg/mouse (AD₅₀ at 10 min = 0.899 μg; CL₉₅o_/o: 0.0116-69.6200), it is nearly half as effective as naloxone (1.08 μg/mouse; AD₅₀ 0.966 μg, CL₉Sy₀: 0.0023-38.9191). On the other hand, compound 3 has about 4% the activity of CTOP (0.106 μg/mouse; ADs₀ 0.0281 μg, CL₉₅O₇₀: 0.000023-0.0338) (Figure 4). However, the inhibition of δ-opioid- mediated activity of compound 3 and naltrindole are similar (Figure 3C). [0156] To assess whether compound 3 is systemically active against centrally-mediated morphine antinociception, subcutaneous (sc) or oral (po) administrations are initiated before the icv injection of morphine. Subcutaneously injected compound 3 antagonizes morphine antinociception in a dose-dependent manner that begins as low as 0.1 mg/kg. (Figure 5A), reaches peak potency within 15 min, and diminishes thereafter depending on the dose. The AUC reveals that 0.1 mg/kg represents the minimum effective dose to exert a significant effect; antinociception is relatively similar between 1 and 3 mg/kg (Figure 5B).

EXAMPLE 48

[0157] This example demonstrates the supraspinal effects of some of the compounds of formula (I) by a tail flick method.

[0158] Spinal effects are measured using a tail-flick instrument (Columbus Instruments,

Columbus, OH). Radiant heat is applied on the dorsal surface of the tail, and the latency for removal of the tail from the onset of the radiant heat is defined as the tail-flick latency (TFL).

The baseline TFL is adjusted between 2 and 3 s (pre-response time) and a cut off time is set at 8 s to avoid external heat-related damage. The measurement of time is the same as described for the hot plate test.

[0159] Spinal effects (tail-flick test) reveal that compound 3 exhibits a potent δ- antagonism at a dose as low as 0.1 mg/kg (sc), but is ineffective at μ-receptor sites even at a

100-fold higher concentration (10 mg/kg; sc) (Figures 6A and 6B), which may be due to the presence of highly reactive δ-opioid receptors in the spinal cord (Jinsmaa et al., Pharmacol.

Biochem. Behav., 84, 252-258 (2006); Jinsmaa et al., Eur. J. Pharmacol, 509, 37-42 (2005); and Gomes et al., Proc. Natl. Acad. ScL U. S. A., 101, 5135-5139 (2004)).

[0160] To develop morphine tolerance, mice are injected sc daily with morphine (10 mg/kg, sc) for 6 consecutive days. Saline (control) or compound 3 (0.1 mg/kg, sc) is injected

30 min before each morphine injection, and the spinal effects (TFL) are determined. The response is measured at day 1 and day 6 following the morphine injections.

[0161] On day one, both groups (morphine, and morphine plus compound 3) exhibit similar antinociception (AUC = 403 and 381, respectively, no significant difference).

However by day 6, the morphine only group reveals a highly significant 62% reduction in the

AUC in contrast to the group that simultaneously receives morphine plus compound 3, and in which the mice remain as sensitive as the saline control (Figure 7). [0162] 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.

[0163] 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. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. 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.

[0164] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may 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.

Claims

CLABlS:

1. A compound of formula (I) :

wherein

R¹ is selected from the group consisting of hydrogen, alkylcarbonyl, and aralkyloxycarbonyl; and

R² and R³ are the same or different and each is selected from the group consisting of hydrogen, arylamido, aralkylamido, and heteroaryl; an optical isomer thereof; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 , wherein R is selected from the group consisting of hydrogen, acetyl, and benzyloxycarbonyl.

3. The compound of claim 1 or 2, wherein R² and R³ are the same or different and each is selected from the group consisting of hydrogen, phenylamido, benzylamido, and lH-benzimidazole-2-yl.

4. The compound of any of claims 1-3, wherein R³ is hydrogen.

5. The compound of any of claims 1-4, wherein R² is benzylamido.

6. The compound of any of claims 1-4, wherein R² is phenylamido.

7. The compound of any of claims 1-4, wherein R² is lH-benzimidazole-2-yl.

The compound of any of claims 1-7, wherein R is hydrogen.

9. The compound of any of claims 1-7, wherein R¹ is benzyloxycarbonyl.

10. The compound of any of claims 1-7, wherein R¹ is acetyl.

11. The compound of any of claims 1 -3 , wherein R² is hydrogen.

12. The compound of any of claims 1 -3 or 11 , wherein R³ is benzylamido.

13. The compound of any of claims 1-3 or 11, wherein R³ is phenylamido.

14. The compound of any of claims 1 -3 or 11 , wherein R³ is lH-benzimidazole-2-yl.

15. The compound of any of claims 11-14, wherein R¹ is hydrogen.

16. The compound of any of claims 11-14, wherein R¹ is benzyloxycarbonyl.

17. The compound of any of claims 11-14, wherein R¹ is acetyl.

18. The compound of any of claims 1-3, wherein the compound is selected from the group consisting of



isomer thereof, or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition comprising a compound of any of claims 1-18 and a pharmaceutically acceptable carrier.

20. A method of agonizing a μ-opioid receptor in a mammal in need thereof comprising administering a compound selected from the group consisting of

pharmaceutically acceptable salt thereof.

21. A method of antagonizing a μ-opioid and δ-opioid receptor in a mammal in need thereof comprising administering a compound selected from the group consisting of a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

22. A method of antagonizing a μ-opioid receptor in a mammal in need thereof comprising administering a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

23. A method of selectively antagonizing a δ-opioid receptor in a mammal in need thereof comprising administering a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

24. A method of ameliorating morphine tolerance in a mammal in need thereof comprising administering a compound of the formula:

an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

25. A method of antagonizing centrally mediated morphine- and/or deltorphin C- induced antinociception in a mammal in need thereof comprising administering a compound selected from the group consisting of

thereof, or a pharmaceutically acceptable salt thereof.