FLUORINE-SUBSTITUTED

2 ' , 6 ' -DIMETHYL-L-TYROSINE-1 , 2,3, 4-TETRAHYDR0IS0QUIN0LINE-3-CARB0XYLIC ACID

PEPTIDES (DMT-TIC) FOR USE AS MYU- AND DELTA-OPIOID RECEPTOR PROBES IN

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/970,143, filed September 5, 2007, which is incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT scan) provide non-destructive diagnostic examination of tissues or organs. In particular, PET involves the acquisition of physiologic images based on the detection of positron radiation emitted from a radioactive substance administered to the patient. The subsequent images of the human body developed with this technique are used to evaluate a variety of diseases, particularly cancer. Targeted delivery of the radioactive substance to the tissue or organ has advantages such as excellent specificity and high binding affinity, and therefore, has received considerable attention from the industry. Accordingly, there is a desire for substances that target and deliver positron- emitting radionuclides to selected organs or tissues.

BRIEF SUMMARY OF THE INVENTION

[0003] The present invention provides fluorine-substituted derivatives of Dmt-Tic (2',6'- dimethyl-L-tyrosine-l,2,3,4-tetrahydroisoquinoline-3-carboxy lic acid peptide), for example, a compound of formula (I), optical isomer, or a pharmaceutically acceptable salt thereof:

(I) wherein R , R ² , and R ³ are described herein.

[0004] The present invention also provides fluorine-substituted derivatives of methoxytyrosine-Tic and dimethoxytyrosine-Tic, for example, a compound of formula (III), optical isomer, or a pharmaceutically acceptable salt thereof:

(III) wherein R ¹ , R ² , and R ³ are described herein, and wherein R ⁶ is hydrogen and R ⁷ is methoxy or wherein R ⁶ and R ⁷ are both methoxy.

[0005] The invention further provides a compound of formula (II), optical isomer, or pharmaceutically acceptable salt thereof:

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro — R ⁴ — R ⁵

(H) wherein R ⁴ and R ⁵ are described herein.

[0006] Also provided by the present invention is a pharmaceutical composition comprising at least one compound of formula (I), (II) or (III) and a pharmaceutically acceptable carrier.

[0007] The Dmt-Tic pharmacophore binds, advantageously, with affinity to μ-opioid and δ-opioid receptors, which are found in certain cancers (e.g., lung, breast, and/or colorectal cancer). Preferably, the fluorine substituent is a radiolabel, ¹⁸ F. In an embodiment, since the

1 6 radionuclide F emits a positron, the in vitro and in vivo binding characteristics of the radiolabeled compound of formula (I), (II) or (III) can be assessed using diagnostic imaging,

such as positron emission tomography (PET). Accordingly, the present invention further provides a method of locating a tumor comprising a μ- or δ-opioid receptor in a subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0008] Figure 1 illustrates a method of synthesis of the compound BG- 137 in an embodiment of the invention.

[0009] Figure 2 illustrates a method of synthesis of the compound BG-139 in an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides agonists and antagonists of μ-opioid and/or δ- opioid receptors that do not pass the blood-brain barrier (BBB) and can be used to locate tumors that comprise μ-opioid and/or δ-opioid receptors. The compounds of the invention are absorbed systemically.

[0011] The present invention is directed to the Dmt-Tic pharmacophore comprising a fluorine-substituent. More specifically, the present invention is directed to a compound of formula (I):

(I) wherein

R ¹ is selected from the group consisting of hydrogen, amino, alkylamino, dialkylamino, piperidinyl, pyrrolidinyl, pyrrolyl, and pyridinyl; R ² is one or more amino acid residues; and

R is selected from the group consisting of aryl, arylcarbonyl, arylalkylcarbonyl, aralkyloxycarbonyl, arylamido, aralkylamido, and heteroaryl, wherein the aryl, heteroaryl, or the "aryl" part of arylcarbonyl, arylalkylcarbonyl, aralkyloxycarbonyl, arylamido, and aralkylamido is substituted with one or more fluoro or trifluoromethyl groups; an optical isomer thereof; or a pharmaceutically acceptable salt thereof. [0012] In particular, R ¹ is selected from the group consisting of amino, alkylamino, and dialkylamino.

[0013] R ² is one or more amino acid residues, wherein an amino group of the amino acid is bonded to the 3-carboxyl group of the tetrahydroisoquinoline moiety; the amino group that is bonded to the 3-carboxyl group can be an alpha-amino group or another amino such as the side chain amino group of lysine. Preferably, the 3-carboxyl group is bonded to the amino group as an amide group. Preferably, at least one of the amino acid residues has an acidic functionality, such as -COOH or -CONH ₂ . When more than one amino acid residue constitutes R , the residues are covalently bonded through amide bonds. For example, R ² comprises 1-6 amino acid residues, preferably 1-3 amino acid residues, and more preferably 1-2 amino acid residues. Any suitable amino acid, natural or synthetic, can be employed. Typically, the one or more amino acid residues are selected from the group consisting of glycinyl, alaninyl, valinyl, leucinyl, isoleucinyl, phenylalaninyl, asparaginyl, glutaminyl, tryptophanyl, prolinyl, serinyl, threoninyl, tyrosinyl, hydroxyprolinyl, cysteinyl, cystinyl, methioninyl, aspartyl, glutamyl, lysinyl, argininyl, and histidinyl. In an embodiment, R ² comprises one or more amino acid residues selected from the group consisting of phenylalaninyl, asparaginyl, glutaminyl, serinyl, and lysinyl, and more preferably phenylalaninyl and/or lysinyl.

[0014] In an embodiment, for R ³ , the aryl part is substituted with one or more fluoro groups (preferably one) at any suitable ring position. Preferably, the fluoro group is the radiolabel, ¹⁸ F. For example, R ³ is selected from the group consisting of

wherein n is 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6).

[0015] In an embodiment, for R ³ , the aryl part is substituted with one or more trifluoromethyl groups (preferably one) at any suitable ring position. Preferably, the

I R 'X trifluoromethyl group contains one, two, or three of the radiolabel, F. For example, R is selected from the group consisting of

[0016] In particular embodiments, R ³ is selected from the group consisting of 2-, 3-, or 4- fluorophenylcarbonyl, 2-, 3-, or 4-fiuorobenzylcarbonyl, 2-, 3-, or 4- fluorobenzyloxycarbonyl, 2-, 3-, or 4-fiuorophenylamido, 2-, 3-, or 4-fluorobenzylamido, and 4-, 5-, 6-, or 7-fluoro-l/i-benzimidazole-2-yl ("F-Bid"). In certain embodiments, R ³ is selected from the group consisting of 4-fluorophenylcarbonyl, 4-fluorobenzylcarbonyl, 4- fluorobenzyloxycarbonyl, 4-fluorophenylamido, 4-fluorobenzylamido, 5-fluoro-lH- benzimidazole-2-yl ("5F-Bid"), and 6-fluoro-lH-benzimidazole-2-yl ("6F-Bid"). Preferably, R ³ is 4-fluorophenylcarbonyl.

[0017] Preferably R is connected to R via any suitable group on R , e.g., a terminal amino or carboxy group on the amino acid residue of R . In embodiments, R is connected via the amino group of the amino acid residue, either an alpha-amino group or a side chain amino group. For example, if R ² is lysinyl, preferably R ³ is bonded to the amino group alpha to the carboxy group. Alternatively, R could be bonded to the side chain amino group on lysinyl.

[0018] The compound of formula (I) can be chiral or achiral. If the compound is chiral, it can be the R enantiomer, the S enantiomer, or a mixture of both (including a racemic mixture). If more than one chiral center is present, the stereoisomers of the compound of formula (I) can be diastereomers of one another and can include a meso compound.

[0019] Specific compounds of the present invention have the formula

(Ia) (Ib) an optical isomer thereof, or a pharmaceutically acceptable salt (e.g., trifluoroacetic acid) thereof. Preferably, the fluoro is the radiolabel, F. Compounds of formula (I) are expec to be δ-opioid selective, for example, compounds of formula (Ia) and (Ib).

[0020] In other embodiments, the present invention is directed to fluorine-substituted derivatives of methoxytyrosine-Tic and dimethoxytyrosine-Tic (2'-methoxy-L-tyrosine- l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid peptide, 6'-methoxy-L-tyrosine-l,2,3,4- tetrahydroisoquinoline-3-carboxylic acid peptide, and 2',6'-dimethoxy-L-tyrosine-l, 2,3,4- tetrahydroisoquinoline-3-carboxylic acid peptide), for example a compound of formula (III):

(III) wherein

R ¹ is selected from the group consisting of hydrogen, amino, alkylamino, dialkylamino, piperidinyl, pyrrolidinyl, pyrrolyl, and pyridinyl;

R ² is one or more amino acid residues; and

R ³ is selected from the group consisting of aryl, arylcarbonyl, arylalkylcarbonyl, aralkyloxycarbonyl, arylamido, aralkylamido, and heteroaryl, wherein the aryl, heteroaryl, or the "aryl" part of arylcarbonyl, arylalkylcarbonyl, aralkyloxycarbonyl, arylamido, and aralkylamido is substituted with one or more fluoro or trifluoromethyl groups; an optical isomer thereof; or a pharmaceutically acceptable salt thereof. [0021] In particular, R ¹ is selected from the group consisting of amino, alkylamino, and dialkylamino.

[0022] R ² is one or more amino acid residues, wherein an amino group of the amino acid is bonded to the 3-carboxyl group of the tetrahydroisoquinoline moiety; the amino group that is bonded to the 3-carboxyl group can be an alpha-amino group or another amino such as the side chain amino group of lysine. Preferably, the 3-carboxyl group is bonded to the amino

group as an amide group. Preferably, at least one of the amino acid residues has an acidic functionality, such as -COOH or -CONH ₂ . When more than one amino acid residue constitutes R ² , the residues are covalently bonded through amide bonds. For example, R ² comprises 1-6 amino acid residues, preferably 1-3 amino acid residues, and more preferably 1-2 amino acid residues. Any suitable amino acid, natural or synthetic, can be employed. Typically, the one or more amino acid residues are selected from the group consisting of glycinyl, alaninyl, valinyl, leucinyl, isoleucinyl, phenylalaninyl, asparaginyl, glutaminyl, tryptophanyl, prolinyl, serinyl, threoninyl, tyrosinyl, hydroxyprolinyl, cysteinyl, cystinyl, methioninyl, aspartyl, glutamyl, lysinyl, argininyl, and histidinyl. In an embodiment, R ² comprises one or more amino acid residues selected from the group consisting of phenylalaninyl, asparaginyl, glutaminyl, serinyl, and lysinyl, and more preferably phenylalaninyl and/or lysinyl.

[0023] In an embodiment, for R ³ , the aryl part is substituted with one or more fluoro groups (preferably one) at any suitable ring position. Preferably, the fluoro group is the radiolabel, F. For example, R is selected from the group consisting of

wherein n is 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6).

[0024] In an embodiment, for R ³ , the aryl part is substituted with one or more trifluoromethyl groups (preferably one) at any suitable ring position. Preferably, the

trifluoromethyl group contains one, two, or three of the radiolabel, ¹⁸ F. For example, R ³ is selected from the group consisting of

[0025] In particular embodiments, R ³ is selected from the group consisting of 2-, 3-, or 4- fluorophenylcarbonyl, 2-, 3-, or 4-fluorobenzylcarbonyl, 2-, 3-, or 4- fluorobenzyloxycarbonyl, 2-, 3-, or 4-fluorophenylamido, 2-, 3-, or 4-fluorobenzylamido, and 4-, 5-, 6-, or 7-fluoro-l//-benzimidazole-2-yl ("F-Bid"). In certain embodiments, R ³ is selected from the group consisting of 4-fluorophenylcarbonyl, 4-fluorobenzylcarbonyl, 4- fluorobenzyloxycarbonyl, 4-fluorophenylarnido, 4-fluorobenzylamido, 5-fluoro-l//- benzimidazole-2-yl ("5F-Bid"), and 6-fluoro-lH-benzimidazole-2-yl ("6F-Bid"). Preferably, R ³ is 4-fluorophenylcarbonyl.

[0026] Preferably R ³ is connected to R ² via any suitable group on R ² , e.g., a terminal amino or carboxy group on the amino acid residue of R ² . In embodiments, R ³ is connected via the amino group of the amino acid residue, either an alpha-amino group or a side chain amino group. For example, if R is lysinyl, preferably R is bonded to the amino group alpha to the carboxy group. Alternatively, R ³ could be bonded to the side chain amino group on lysinyl.

[0027] The compound of formula (III) can be chiral or achiral. If the compound is chiral, it can be the R enantiomer, the S enantiomer, or a mixture of both (including a racemic

mixture). If more than one chiral center is present, the stereoisomers of the compound of formula (III) can be diastereomers of one another and can include a meso compound. [0028] In another embodiment, the invention is directed to fluorine-substituted derivatives of dermorphin, a μ-selective opioid agonist, of the formula (II):

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro R ⁴ — R ⁵

(H) wherein

R ⁴ is one or more amino acid residues; and

R ⁵ is selected from the group consisting of aryl, arylcarbonyl, arylalkylcarbonyl, aralkyloxycarbonyl, arylamido, aralkylamido, and heteroaryl, wherein the aryl or heteroaryl group or the "aryl" part of arylcarbonyl, arylalkylcarbonyl, aralkyloxycarbonyl, arylamide, and aralkylamino is substituted with one or more fluoro or trifluoromethyl groups; an optical isomer thereof; or a pharmaceutically acceptable salt thereof. [0029] R ⁴ comprises one or more amino acid residues, wherein an amino group of the amino acid is bonded to the carboxyl group of the prolinyl moiety; the amino group that is bonded to the carboxyl group of proline can be an alpha-amino group or another amino group such as the side chain amino group of lysine. Preferably, the carboxyl group is bonded to the amino group as an amide group. Preferably, at least one of the amino acid residues has an acidic functionality, such as -COOH or -CONH ₂ . When more than one amino acid residue constitutes R ⁴ , the residues are covalently bonded through amide bonds. For example, R ⁴ comprises 1-6 amino acid residues, preferably 1-3 amino acid residues, and more preferably 1-2 amino acid residues. Any suitable amino acid, natural or synthetic, can be employed. Typically, the one or more amino acid residues are selected from the group consisting of glycinyl, alaninyl, valinyl, leucinyl, isoleucinyl, phenylalaninyl, asparaginyl, glutaminyl, tryptophanyl, prolinyl, serinyl, threoninyl, tyrosinyl, hydroxyprolinyl, cysteinyl, cystinyl, methioninyl, aspartyl, glutamyl, lysinyl, argininyl, and histidinyl. In an embodiment, R ⁴ comprises one or more amino acid residues selected from the group consisting of phenylalaninyl, asparaginyl, glutaminyl, serinyl, and lysinyl, and more preferably phenylalaninyl and/or lysinyl.

[0030] In an embodiment, for R ⁵ , the aryl part is substituted with one or more fluoro groups (preferably one) at any suitable ring position. Preferably, the fluoro group is the radiolabel, ¹⁸ F. For example, R ⁵ is selected from the group consisting of

wherein n is 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6).

[0031] In an embodiment, for R ⁵ , the aryl part is substituted with one or more trifluoromethyl groups (preferably one) at any suitable ring position. Preferably, the trifluoromethyl group contains one, two, or three of the radiolabel, F. For example, R is selected from the group consisting of

[0032] In particular embodiments, R is selected from the group consisting of 2-, 3-, or 4- fluorophenylcarbonyl, 2-, 3-, or 4-fluorobenzylcarbonyl, 2-, 3-, or 4- fluorobenzyloxycarbonyl, 2-, 3-, or 4-fluorophenylamido, 2-, 3-, or 4-fluorobenzylamido, and A-, 5-, 6-, or 7-fluoro-l/i-benzimidazole-2-yl ("F-Bid"). In certain embodiments, R ⁵ is selected from the group consisting of 4-fluorophenylcarbonyl, 4-fluorobenzylcarbonyl, 4- fluorobenzyloxycarbonyl, 4-fluorophenylamido, 4-fluorobenzylamido, 5-fluoro-l//- benzimidazole-2-yl ("5F-Bid"), and 6-ftuoro-lH-benzimidazole-2-yl ("6F-Bid"). Preferably, R ⁵ is 4-fluorophenylcarbonyl.

[0033] R ⁵ is connected to R ⁴ via any suitable group on R ⁴ , e.g., a terminal amino or carboxy group on the amino acid residue of R ⁴ . In embodiments, R ⁵ is connected via the amino group of the amino acid residue, either alpha-amino group or side chain amino group. For example, if R ⁴ is lysinyl, preferably R ⁵ is bonded to the amino group. R ⁴ could be bonded to the alpha amino group on lysinyl.

[0034] So as not to pass the BBB, preferably, the compound of formula (II) has a molecular weight greater than or equal to about 1,000 g/mol (e.g., 1,000 g/mol or higher, 1,100 g/mol or higher, 1,200 g/mol or higher, 1,500 g/mol or higher). Also preferably, compounds of formula (II) can include a charged moiety at, for example, an amino (e.g., - NH ₃ ⁺ ) or carboxy (e.g., -CO ₂ ^" ) functionality at a functional pH such as 7.4. The compound

can function as a zwitterion or include a counterion such as a Group I or II cation (e.g., Na ⁺ , K ⁺ , Mg ²⁺ , or Ca ²⁺ ) or a Group VII anion (e.g., Bf, Cl ^" , or F). [0035] In a specific embodiment, the compound of formula (II) is

(H) wherein the fluoro is at the 2, 3, or 4 position of the phenyl group. Preferably, the fluoro is at the 4 position of the phenyl group. Compounds of formula (II) are expected to be μ-opioid selective.

[0036] Referring now to terminology used generically herein, the term "alkyl" implies a straight or branched alkyl moiety 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 moieties include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.

[0037] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic moiety, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, and the like. An aryl moiety 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 most preferably from 6 to 10 carbon atoms. It is understood that the term aryl includes carbocyclic moieties that are planar and comprise 4n+2 π electrons, according to Hϋckel's Rule, wherein n = 1, 2, or 3.

[0038] 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 heteroatoms 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, quinolinyl, isoquinolinyl, thiazolyl, furyl, isoxazolyl, oxadiazolyl, and oxazolyl.

[0039] The term "aralkyl" refers to an alkyl group that is substituted with an aryl group. The aryl and aralkyl groups are as described herein. An example of an aralkyl group is benzyl.

[0040] The terms "arylcarbonyl" and "arylalkylcarbonyl" refers to the group -C(O)R, in which R is an aryl or aralkyl group, respectively. The aryl and aralkyl groups are as described herein. An example of an arylcarbonyl group is phenylcarbonyl. An example of arylalkylcarbonyl group is benzylcarbonyl.

[0041] The term "aralkyloxycarbonyl" refers to an aralkyloxy moiety bound to a carbonyl. An example of an aralkyloxycarbonyl is benzyloxycarbonyl. [0042] The term "alkylamino" refers to a group with one hydrogen and one alkyl group directly attached to a trivalent nitrogen atom. The term "dialkylamino" refers to a group with two of the same or different alkyl groups directly attached to a trivalent nitrogen atom. The terms "arylamido" and "aralkylamido" refer to the groups -C(O)NHAr and -C(O)NH(CH ₂ ) _n Ar, 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.

[0043] 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, /7-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. 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.

[0044] 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.

[0045] Preferably, the radiolabel is ¹⁸ F. ¹⁸ F has a half-life (t*0 of 110 minutes, emits β+ particles at an energy of 635 keV, and is 97% abundant. F can be obtained from cyclotrons after bombardment of ¹⁸ O-enriched water with protons. The enriched water containing H- ¹⁸ F can be neutralized with a base having a counter-ion that is any alkali metal (M), such as potassium or another monovalent ion, and the water can be evaporated off to give a residue of M- ¹⁸ F, which can be taken up in an organic solvent for further use. In general, the counter- ion is selected to enable the fluoride ion to react rapidly in an organic phase with a halogen. Potassium is generally used as a counter-ion because it is cheaper than cesium. However,

with carrier-free F, trivial amounts of counter-ion are required, and the counter-ion cost is minimal.

[0046] Cesium is useful as a counter ion since it is a larger ion with a more diffuse charge. Accordingly, cesium has looser ionic interactions with the small fluoride atom, and therefore does not interfere with the nucleophilic properties of the fluoride ion. For similar reasons, potassium is preferred to sodium, and, in general, the suitability of a Group Ia metal as a counter-ion in accordance with the present invention increases down the periodic table. Group Ib reagents, such as silver, also are useful as counter-ions. Further, organic phase transfer-type ions, such as tetraalkylammonium salts, also can be used as counter-ions. [0047] Fluoride salts can have a tendency to become hydrated and lose their nucleophilic character. To minimize this, the labeling reaction is preferably performed under anhydrous conditions. For example, fluoride (as potassium fluoride or as a complex with any of the other counter-ions discussed above) can be placed in an organic solvent, such as acetonitrile or tetrahydrofuran. With the help of agents which bind to the counter-ion, such as Kryptofix 2.2.2 (4,7,13, 16,21,24-hexaoxa-l,10-diazabicyclo[8.8.8]-hexacosane), the fluoride ion is very nucleophilic in these solvents.

[0048] The present inventive compounds can be synthesized by any suitable method. See, for example, U.S. Patents 6,916,905 and 6,753,317, 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) or (II) in accordance with embodiments of the invention, can be made by starting from a protected amino acid residue (e.g., Lys) and further protecting it. Substituent R ³ is added and then the first protecting group on the amino acid residue can be selectively deprotected. Next additional amino acid residues (R ² ) are optionally added, then protected Tic is added, and then selectively deprotected. Protected Dmt is added next and then selectively deprotected. The first amino acid residue (R ² ) can be further deprotected if necessary. Compounds of formula (II) can be made from dermorphin. Once R ⁴ and R ⁵ have been prepared and coupled, as described above, the remaining peptide sequence can be added using known techniques. For the fluoro substituent on R ³ , the radiolabel ¹⁸ F can be used. Specific examples of synthesis methods of the present inventive compounds are set forth in the Examples herein. [0049] The present invention further provides a pharmaceutical composition comprising at least one compound of formula (I), (II), or (III), optical isomer, or salt thereof 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.

[0050] A compound of the present invention can be made into a formulation suitable for parenteral administration, e.g., intravenal, 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. [0051] 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.

[0052] 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.

[0053] 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.

[0054] A formulation suitable for topical application can be in the form of creams, ointments, or lotions.

[0055] 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.

[0056] The dose administered to a subject, particularly a mammal (e.g., human), in the context of the methods of the present invention should be sufficient to produce and image and/or affect a 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, 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] The dose of the compound of formula (I), (II), or (III), optical isomer or salt thereof 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) or (II) 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).

[0061] 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. [0062] 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. 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). [0063] Certain tumors and carcinomas have been shown to contain high levels of μ- and/or δ-opioid receptors, such as pulmonary carcinomas, including lung carcinoma, colorectal carcinoma, neuroblastomas, and breast cancer (e.g., Madar et al., J Nucl. Med., 48(2): 207-213 (2007); Campa et al., Cancer Research, 56: 1695-1701 (1996); and Rigaudy et al., Cancer Research, 49: 1846-1832 (1989)). Thus, the present invention further provides a method of locating a μ- and/or δ-opioid receptor that is contained in a tissue or organ of a subject. The method comprises the steps of: a) administering a radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof to the subject,

b) obtaining a diagnostic image of the tissue or organ,

c) determining the location of radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof bound to the tissue or organ; and

d) correlating the location of the bound radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof with the location of the μ- and/or δ-opioid receptor in the subject. The method correlates to tumor detection once the μ- and/or δ-opioid receptor has been located in the subject.

[0064] In some embodiments, the receptor is a μ-opioid receptor. In such embodiments, a compound of formula (I) or (III), optical isomer or salt thereof preferably is administered. In other embodiments, the receptor is a δ-opioid receptor. In such embodiments, a compound of formula (II), optical isomer or salt thereof preferably is administered. Preferably, the

radiolabeled compound of formula (I), (I), or (III), optical isomer or salt thereof is administered in an amount effective to provide an image.

[0065] The term "subject" used herein includes animals such as humans, sheep, horses, cattle, pigs, monkeys, dogs, cats, rats, and mice.

[0066] The tissue or organ in the subject to be tested is any tissue or organ that is known to contain a μ- and/or δ-opioid receptor. Preferably the tissue is peripheral to the brain. Such tissues and organs include the entire organ or a tissue sample of a breast, an ovary, a salivary gland, a stomach, a kidney, a colon, a rectum, a cervix, a bladder, a head, a neck, including an esophagus, or the pulmonary system (e.g., a lung, trachea, nasal cavity, mouth, larynx, pharynx, and epiglottis). In preferred embodiments, the tissue is from the lung, colon, rectum, or breast. More preferably, the tissue is from the lung.

[0067] The tissue can also include a human cancer cell line known to contain a μ- and/or δ-opioid receptor. Such cell lines include NCI-H 146 (small cell lung cancer, δ-opioid receptor), NCI-Hl 87 (non-small cell lung cancer, δ-opioid receptor), SHS Y5 Y (neuroblastoma, μ- and δ-opioid receptors), NS20Y (neuroblastoma, δ-opioid receptor), SK- N-SH (neuroblastoma; μ-opioid receptor), NG108-15 (neuroblastoma-glioma hybrid, δ- opioid receptor), and T47D (breast cancer, δ-opioid receptor).

[0068] Obtaining a diagnostic image of the tissue or organ in step b) typically comprises exposing the tissue or organ in the subject to an energy source, whereupon a diagnostic image of the tissue or organ is obtained. The diagnostic image can be, for example, positron emission tomography (PET) image, a magnetic resonance image (MRI), a computerized tomography (CT) scan, single photon emission computed spectroscopy (SPECT) image, or the like.

[0069] The diagnostic image can be an MRI. When administered to a subject, the radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof distributes in various concentrations to different tissues, and catalyzes the relaxation of protons in the tissues that have been excited by the absorption of radiofrequency energy from a magnetic resonance imager. This acceleration of the rate of relaxation of the excited protons provides for an image of different contrast when the subject is scanned with a magnetic resonance imager. The magnetic resonance imager is used to record images at various times, generally either before and after administration of the ¹⁸ F labeled compound of formula (I), (II), or (III),

or after administration only, and the differences in the images created by the presence of the radiolabeled compound of formula (I) or (II) in tissues are used in diagnosis. Guidelines for performing imaging techniques can be found in Stark et al., Magnetic Resonance Imaging, Mosbey Year Book: St. Louis, 1992, hereby incorporated by reference. [0070] Single Positron Emission Computed Tomography (SPECT) is a non-invasive imaging method to localize the position of a target such as a cancer metastasis, based on radioactive substances that emit gamma radiation when decaying.

[0071] A CT scan provides anatomical detail, such as size and location of the tumor or mass. Digital geometry processing is used to generate a three-dimensional image of the internals of an object from a large series of two-dimensional X-ray images taken around a single axis of rotation. CT produces a volume of data which can be manipulated, through a process known as windowing, in order to demonstrate various structures based on their ability to block the X-ray beam. Combined techniques such as PET/CT and PET/MRI are suitable for use in the invention.

[0072] Preferably, step b) comprises obtaining a positron emission tomography (PET) image of the tissue or organ. PET is a non-invasive imaging method to localize the position of a target such as a cancer metastasis. In PET, 511 keV gamma photons produced during positron annihilation decay are detected. A positron-emitting radionuclide, such as ¹ F, is introduced, usually by injection, and accumulates in the target tissue or organ. As it decays it emits a positron, which promptly combines with a nearby electron resulting in the simultaneous emission of two identifiable gamma rays in opposite directions. These are detected by a PET camera and give very precise indication of their origin. A PET scan can provide in vivo physiology such as metabolic detail (e.g., cellular activity) of the tumor or mass. The diagnosis is at a molecular level thereby providing detection of a tumor or mass at an early stage.

[0073] Since PET is a quantitative tool, the present invention provides a method of measuring the quantity of a μ- and/or δ-opioid receptor that is contained in a tissue or organ of a subject. The method comprises the steps of: a) administering a radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof to the subject,

b) obtaining a positron emission tomography (PET) image of the tissue or organ.

c) determining the amount of radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof bound to the tissue or organ; and

d) correlating the amount of the bound radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof with the quantity of μ- and/or δ-opioid receptor in the subject. In some embodiments, the receptor is a μ-opioid receptor. In other embodiments, the receptor is a δ-opioid receptor. Preferably, the radiolabeled compound of formula (I), (II), or (III), optical isomer or salt thereof is administered in an amount effective to provide an image.

[0074] In a specific embodiment, the invention provides a method of locating a tumor comprising a δ-opioid receptor in a mammal in need thereof comprising administering a compound of the formula selected from the group consisting of:

(Ia) (Ib) an optical isomer thereof, or a pharmaceutically acceptable salt (e.g., trifluoroacetic acid) thereof.

[0075] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

ABBREVIATIONS

Bid lif-benzimidazole-2-yl

Boc tert-butyloxycarbonyl

DAMGO [D-Ala ² ,λ/-Me-Phe ⁴ ,Gly-ol ⁵ ]enkephalin

DEL C deltorphin II (H-Tyr-D-Ala-Phe-Asp-Val- VaI-GIy-NH ₂ )

DMF iV,jV-dimethylformamide

DMSOd ₆ 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

NMM 4-methylmorpholine pA ₂ negative log of the molar concentration required to double the agonist concentration to achieve the original response

TFA trifluoroacetic acid

Tic 1 ,2,3 ,4-tetrahydroisoquinoline-3 -carboxylic acid

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

TLC thin-layer chromatography

WSC 1 -ethyl-3-[3'-dimethyl)aminopropyl]-carbodiimide hydrochloride

Z benzyloxycarbonyl

[0076] Crude compounds are purified by preparative reversed-phase HPLC (Waters Delta Prep 4000 system with Waters Prep LC 40 mm Assembly column Cl 8 (30 x 4 cm, 15 μm particle size)) and eluted at a flow rate of 20 mL/min with mobile phase solvent A (10% acetonitrile + 0.1% TFA in H ₂ O, v/v), and a linear gradient from 10 to 60% solvent B (60%, acetonitrile + 0.1% TFA in H ₂ O, v/v) in 30 min. Analytical HPLC analyses are performed with a Beckman System Gold (Beckman ultrasphere ODS column, 250 x 4.6 mm, 5 μm particle size). Analytical determinations and capacity factor (K') of the products that are used in HPLC in solvents A and B are programmed at flow rate of 1 mL/min with a linear gradient from 0 to 100% B in 25 min. Analogues have less than 1% impurities when monitored at 220 and 254 nm.

[0077] TLC is performed on precoated plates of silica gel F254 (Merck, Darmstadt, Germany): (A) l-butanol/AcOH/H ₂ O (3:1:1, v/v/v); (B) CH ₂ Cl ₂ /toluene/methanol (17:1:2).

Ninhydrin (1% ethanol, Merck), fluorescamine (Hoffman-La Roche) and chlorine spray reagents are used. 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 G2025 A LD-TOF system mass spectrometer) and α-cyano-4-hydroxycinnamic acid as a matrix. ¹ H NMR (δ) spectra are measured, when not specified, in DMSO-cfc solution using a Bruker AC-200 spectrometer, and peak positions are given in parts per million downfield from tetramethylsilane as internal standard. [0078] All experiments with animals are carried out according to protocols approved by and on file with the NIEHS Animal Care and Use Committee (ACUC).

EXAMPLE 1

[0079] This example demonstrates a synthesis of TFAH-Tic-ε-Lys(Z)-OMe useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 1. [0080] Boc-Tic-£-Lys(Z)-OMe (Balboni et al., Bioorg. Med. Chem. 2007, 15, 3143-3151) (1.67 g, 3.02 mmol) is treated with TFA (2 mL) for 0.5 h at room temperature. Et ₂ O/Pe (1:1, v/v) are added to the solution until the product precipitates out of solution: yield 1.58 g (92%); Rf(A) 0.49; HPLC K' 4.96; mp 118-120 ⁰ C; [α] ²⁰ _D -19.6; m/z 454 (M+H) ⁺ .

EXAMPLE 2

[0081] This example demonstrates a synthesis of Boc-Dmt-Tic-ε-Ly S(Z)-OMe useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 1.

[0082] To a solution of Boc-Dmt-OH (0.22 g, 0.71 mmol) and TFA H-Tic-e-Lys(Z)-OMe (0.4 g, 0.71 mmol) in DMF (10 mL) at 0 °C, NMM (0.08 mL, 0.71 mmol), HOBt (0.12 g, 0.78 mmol), and WSC (0.15 g, 0.78 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰ C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with citric acid (10% in H ₂ O), NaHCO ₃ (5% in H ₂ O), and brine. The organic phase is dried (Na ₂ SO ₄ ) and evaporated to dryness. The residue is precipitated from Et ₂ O/Pe (1:9, v/v): yield 0.46 g (87%); Rf[B) 0.91; HPLC K 7.30; mp 133-135 ⁰ C; [α] ²⁰ _D -

17.5; m/z 746 (M+H) ⁺ ; ¹ H-NMR (DMSO-(I ₆ ) δ 1.29-1.90 (m, 15H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.67 (s, 3H), 4.41-5.34 (m, 7H), 6.29 (s, 2H), 6.96-7.19 (m, 9H).

EXAMPLE 3

[0083] This example demonstrates a synthesis of Boc-Dmt-Tic-s-Lys-OMe useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 1. [0084] To a solution of Boc-Dmt-Tic-£-Lys(Z)-OMe (0.37 g, 0.5 mmol) in methanol (30 mL) is added Pd/C (10%, 0.1 g). H ₂ is bubbled for 1 h at room temperature. After filtration, the solution is evaporated to dryness. The residue is precipitated from Et ₂ OZPe (1 :9, v/v): yield 0.27 g (90%); Rf[B) 0.74; HPLC K 5.06; mp 139-141 °C; [α] ²⁰ _D -18.1; m/z 612 (M+H) ⁺ .

EXAMPLE 4

[0085] This example demonstrates a synthesis of Boc-Dmt-Tic-£ ⁱ -Lys(4- fluorobenzoyl)-OMe useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 1.

[0086] To a solution of Boc-Dmt-Tic-£-Lys-OMe (0.43 g, 0.7 mmol) and 4-fluorobenzoic acid (0.1 g, 0.7 mmol) in DMF (10 mL) at 0 °C, HOBt (0.12 g, 0.77 mmol), and WSC (0.15 g, 0.77 mmol) are added. The reaction mixture is stirred for 3 h at 0 ⁰ C and 24 h at room temperature. After DMF is evaporated, the residue is dissolved in EtOAc and washed with citric acid (10% in H ₂ O), NaHCO ₃ (5% in H ₂ O), and brine. The organic phase is dried (Na ₂ SO ₄ ) and evaporated to dryness. The residue is precipitated from Et ₂ OZPe (1 :9, v/v): yield 0.45 g (88%); Rf[B) 0.79; HPLC K 5.25; mp 134-136 °C; [α] ²⁰ _D -17.3; m/z 734 (M+H) ⁺ ; ¹ H-NMR (DMSO-^) δ 1.29-1.94 (m, 15H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.67 (s, 3H), 4.41-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.93 (m, 8H).

EXAMPLE 5

[0087] This example demonstrates a synthesis of Boc-Dmt-Tic-£-Lys(4-fluorobenzoyi)- OH useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 1.

[0088] To a solution of Boc-Dmt-Tic-£-Lys(4-fluorobenzoyl)-OMe (0.51 g, 0.7 mmol) in ethanol (10 niL) at room temperature, IN NaOH (1.1 mL, 1.1 mmol) is added. The reaction mixture is stirred for 4 h at room temperature. After ethanol is evaporated, the residue is dissolved in EtOAc and washed with citric acid (10% in H ₂ O) and brine. The organic phase is dried (Na ₂ SO ₄ ) and evaporated to dryness. The residue is precipitated from Et ₂ O/Pe (1 :9, v/v): yield 0.45 g (90%); RJ[B) 0.75; HPLC K 5.14; mp 141-143 °C; [α] ²⁰ _D -17.9; m/z 720 (M+H) ⁺ .

EXAMPLE 6

[0089] This example demonstrates a synthesis of TFAH-Dmt-Tic-£-Lys(4- fluorobenzoyl)-OH (BG-137) in accordance with an embodiment of the invention. See Figure 1.

[0090] Boc-Dmt-Tic-£-Lys(4-fluorobenzoyi)-OH is treated with TFA as reported for TFAH-Tic-£--Lys(Z)-OMe: yield 0.09 g (94%); Rf[A) 0.42; HPLC K 4.86; mp 149-151 °C; [Oc] ²⁰ _D -18.5; m/z 620 (M+H) ⁺ ; ¹ H-NMR (DMSO-J ₆ ) δ 1.29-1.82 (m, 6H), 2.35 (s, 6H), 2.92- 3.20 (m, 6H), 3.95-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.93 (m, 8H).

EXAMPLE 7

[0091] This example demonstrates a synthesis of Boc-Dmt-Tic-f-Lys-OH useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 1. [0092] To a solution of Boc-Dmt-Tic-ε-Lys-OMe (0.09 g, 0.15 mmol; Example 3) in ethanol (10 mL) at room temperature, IN NaOH (0.23 mL, 0.23 mmol) is added. The reaction mixture is stirred for 4 h at room temperature. After ethanol is evaporated, the residue is dissolved in solvent B and directly purified by preparative HPLC as reported above in general methods: yield 0.1 g (92%); Rf[B) 0.67; HPLC K 4.73; mp 146-148 ⁰ C; [α] ²⁰ _D - 18.4; m/z 598 (M+H) ⁺ ; ¹ H-NMR (DMSO-^) δ 1.29-1.78 (m, 15H), 2.35 (s, 6H), 2.92-3.49 (m, 7H), 4.41-4.92 (m, 4H), 6.29 (s, 2H), 6.96-7.02 (m, 4H).

EXAMPLE 8 [0093] This example demonstrates a synthesis of the compound [ F]fluoro-BG-137

in accordance with an embodiment of the invention.

[0094] Boc-Dmt-Tic-ε-Lys-OH (Example 7) is coupled with N-succinimidyl-4- [ ^l FJfluorobenzoate under slightly basic conditions at 60 ⁰ C for 40 min. The resulting product is deprotected with TFA, and then purified by HPLC to yield [ ¹⁸ F]fluoro-BG-137. Yield: 3.2 mg.

EXAMPLE 9

[0095] This example demonstrates a synthesis of Boc-Dmt-Tic-Phe-Lys-OMe useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure

2.

[0096] To a solution of Boc-Dmt-Tic-Phe-Lys(Z)-OMe (0.71 g, 0.8 mmol) in methanol

(30 mL) Pd/C (10%, 0.2 g) is added, and H ₂ is bubbled for 1 h at room temperature. After filtration, the solution is evaporated to dryness. The residue is precipitated from Et ₂ O/Pe

(1:9, v/v): yield 0.58 g (88%); Rf[B) 0.81; HPLC K 5.24; mp 144-146 °C; [α] ²⁰ _D +35.2; m/z

759 (M+H) ⁴

EXAMPLE 10

[0097] This example demonstrates a synthesis of Boc-Dmt-Tic-Phe-Lys(4- fluorobenzoyl)-OMe useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 2.

[0098] This compound is obtained by condensation of Boc-Dmt-Tic-Phe-Lys-OMe with 4-fluorobenzoic acid via WSC/HOBt as reported for Boc-Dmt-Tic-ε-Lys(4-fluorobenzoyl)- OMe: yield 0.23 g (85%); RflB) 0.78; HPLC K' 5.10; mp 135-137 ⁰ C; [α] ²⁰ _D +30.6; m/z 881 (M+H) ⁺ ; ¹ H-NMR (OMSO-d ₆ ) δ 1.29-1.90 (m, 15H), 2.35 (s, 6H), 2.92-3.20 (m, 8H), 3.67 (s, 3H), 4.41-4..92 (m, 6H), 6.29 (s, 2H), 6.96-7.93 (m, 13H).

EXAMPLE I l

[0099] This example demonstrates a synthesis of Boc-Dmt-Tic-Phe-Lys(4- fluorobenzoyl)-OH useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 2.

[00100] This compound is obtained by hydrolysis of Boc-Dmt-Tic-Phe-Lys(4- fluorobenzoyl)-OMe with IN NaOH as reported for Boc-Dmt-Tic-£-Lys(4-fluorobenzoyl)-

OH: yield 0.15 g (91%); Rf[B) 0.75; HPLC K 5.0; mp 139-141 °C; [α] ²⁰ _D +31.4; m/z 867

(M+H) ⁺ .

EXAMPLE 12

[00101] This example demonstrates a synthesis of TF A H-Dmt-Tic-Phe-Lys(4- fluorobenzoyl)-OH (BG-139) in accordance with an embodiment of the invention. See Figure 2.

[00102] Boc-Dmt-Tic-Phe-Lys(4-fluorobenzoyl)-OH is treated with TFA as reported for TFAH-Tic-^-Lys(Z)-OMe: yield 0.08 g (96%); Rf[A) 0.40; HPLC K' 2.87; mp 146-149 °C; [Ot] ²⁰ _D +31.1; m/z 161 (M+H) ⁺ ; ¹ H-NMR (DMSO-Cf ₆ ) δ 1.29-1.78 (m, 6H), 2.35 (s, 6H), 2.92- 3.20 (m, 8H), 3.95-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.93 (m, 13H).

EXAMPLE 13

[00103] This example demonstrates a synthesis of Boc-Dmt-Tic-Phe-Lys-OH useful in the synthesis of a compound in accordance with an embodiment of the invention. See Figure 2. [00104] The title compound is obtained by hydrolysis of Boc-Dmt-Tic-Phe-Lys-OMe (Example 9) with IN NaOH as reported for Boc-Dmt-Tic-^-Lys-OH: yield 0.06 g (86%); Rf[B) 0.65; HPLC K! 4.75; mp 140-142 °C; [α] ²⁰ _D +36.1; m/z 745 (M+H) ⁺ ; ¹ H-NMR (DMSO-^) δ 1.29-1.78 (m, 15H), 2.35 (s, 6H), 2.92-3.17 (m, 8H), 4.41-4.92 (m, 6H), 6.29 (s, 2H), 6.96-7.21 (m, 9H).

EXAMPLE 14 [00105] This example demonstrates a synthesis of the compound [ ¹⁸ F]fluoro-BG-139

in accordance with an embodiment of the invention.

[00106] Boc-Dmt-Tic-Phe-Lys-OH (Example 13) is coupled with N-succinimidyl-4- [ ¹⁸ F]fluorobenzoate under slightly basic conditions at 60 ⁰ C for 40 min. The resulting product is deprotected with TFA, and then purified by HPLC to yield [ ¹⁸ F]ftuoro-BG-139.

EXAMPLE 15

[00107] This example demonstrates a synthesis of BG-138

BG-138 a compound of formula (II), in accordance with an embodiment of the invention. [00108] Dermorphin is synthesized using Fmoc/t-butyl chemistry with a Syro XP multiple peptide synthesizer (MultiSynTech GmbH, Witten Germany). Fmoc-Rink-amide MBHA resin (0.7 mmol/g, 0.150 g) is treated with 40% piperine/DMF and linked with: Fmoc- Lys(Dde)-OH, Fmoc-Pro-OH, Fmoc-Tyr(φu)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-D- AIa-OH, Boc-Tyr-OH (4 equiv.; 0.5 M in DMF) by using HOBt (5 equiv.; 0.78 M in DMF), DIC (7 equiv.; 1.09 M in DMF) as the coupling reagent. The coupling reaction time is 1.5 h and piperidine (40%)/DMF is used to remove the Fmoc group at each step (20 min). The

peptide resin is washed with CH ₂ Cl ₂ and dried in vacuo to yield the protected Dermorphin resin.

[00109] The protected Dermorphin resin is treated with TFA/H ₂ O/triethylsilane (9 : 0.5 :

0.5 v/v) (10 ml/0.150 g of resin) for 1.5 h at room temperature. After filtration of the exhausted resin, the solvent is concentrated in vacuo, and the residue is triturated with diethyl ether. The crude linear peptide is purified by preparative reverse-phase HPLC to yield a powder after lyophilization.

EXAMPLE 16

[00110] This example demonstrates a method to determine the equilibrium receptor binding of compounds of formula (I) and (II) in accordance with an embodiment of the invention.

[0100] Opioid receptor affinities are determined under equilibrium conditions (2.5 h at room temperature) in a competition assay using brain P ₂ synaptosomal membranes prepared from Sprague-Dawley rats (e.g., Lazarus et al., J. Med. Chem. 1991, 34, 1350-1355; Lazarus et al., Peptides 1993, 14, 21-28). Synaptosomes are preincubated to remove endogenous opioid peptides and stored at -80 °C in buffered 20% glycerol (Lazarus et al., J. Med. Chem. 1991, 34, 1350-1355; Lazarus et al., J Biol. Chem. 1989, 264, 3047-3050). Each compound is analyzed in duplicate assays using five to eight dosages and three to five independent repetitions with different synaptosomal preparations (n values are listed in Table 1 in parenthesis and results are mean ± SE). Unlabeled compound (2 μM) is used to determine non-specific binding in the presence of 1.9 nM [ ³ H]deltorphin II (45.0 Ci/mmol, Perkin Elmer, Boston, MA; K _Ό = 1.4 nM) for ^-opioid receptors and 3.5 nM [ ³ H]DAMGO (50.0 Ci/mmol), Amersham Bioscience, Buckinghamshire, U. K.; K^ = 1.5 nM) for //-opioid receptors. Glass fiber filters (Whatman GFC) are soaked in 0.1% polyethylenimine in order to enhance the signal-to-noise ratio of the bound radiolabeled-synaptosome complex, and the filters are washed thrice in ice-cold buffered BSA (Lazarus et al., J Med. Chem. 1991, 34, 1350-1355). The affinity constants (K _\ ) are calculated according to Cheng and Prusoff (Biochem. Pharmacol. 1973, 22, 3099-3108). Receptor binding and functional bioactivities are reported in Table 1.

Table 1. Receptor binding affinities.

Receptor affinity" Selectivity

a: the K _{ values (nM) are determined according to Chang et al., Biochem. Pharmacol. 1973, 22, 3099-3108

EXAMPLE 17

[0101] This example demonstrates the functional bioactivity of compounds of the invention in isolated organ preparations in accordance with an embodiment of the invention. [0102] The myenteric plexus longitudinal muscle preparations (2-3 cm segments) from the small intestine of male Hartley strain guinea pigs (GPI) measure μ-opioid receptor agonism, and a single mouse vas deferens (MVD) is used to determine <S-opioid receptor agonism as described previously (Sasaki et al., Bioorg. Med. Chem. 2003, 11, 675-678). The isolated tissues are suspended in organ baths containing balanced salt solutions in a physiological buffer, pH 7.5. Agonists are tested for the inhibition of electrically evoked contraction and expressed as IC ₅₀ (nM) obtained from the dose-response curves. The IC ₅₀ values represent the mean ± SE of five or six separate assays. Delta-antagonist potencies in

the MVD assay are determined against the ^-agonist deltorphin-II; //-antagonism in the GPI assay uses the //-agonist endomorphin-2, and both are expressed as pλ ₂ determined using the Schild Plot (Arunlakshana et al., Br. J. Pharmcol. 1959, 14, 48-58). [0103] Compounds from Examples 6 and 12 are tested in the electrically stimulated MVD and GPI assays for intrinsic functional bioactivity (Table 2).

Table 2. Functional bioactivities.

aAgonist activity is expressed as IC ₅₀ obtained from dose-response curves. These values represent the mean ± SE for at least five to six fresh tissue samples. bThe pA ₂ values of opioid antagonists against the agonists are determined by the method of Kosterlitz and Watt. {Br J. Pharmacol 1968, 33, 266-276).

EXAMPLE 18

[0104] This example demonstrates rat brain autoradiography of a compound of formula (I) in accordance with an embodiment of the invention.

[0105] The compound of Example 8 is injected into normal mice for brain imaging. The total synthesis time is 120 min and the decay-corrected radiochemical yield of [ F]fluoro- BG-137 is about 30-32% decay starting from [ ¹⁸ F]SFB. [ ¹⁸ F]Fluoro-BG-137 shows no uptake in mouse brain as it does not cross the blood-brain barrier (BBB). [0106] An in vitro autoradiographic study is also performed by incubating normal rat brain slices with [ ¹⁸ F]fluoro-BG-137 without and with the presence of blocking ligands: BG- 137 and UFP-501 [N(Me) ₂ -Dmt-Tic-OH].

[0107] The [ F]fluoro-BG-137 shows prominent uptake in the striatum, thalamus, and cortex, which are opioid rich regions. Significant blocking of the uptake by BG- 137 and UFP-501 indicates high specific binding of [ ¹⁸ F]fluoro-BG-137 to the δ-opioid receptor.

[0108] 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.

[0109] 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.

[0110] 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.