Bifunctional ligands containing an ester linkage between morphine and the δ-selective pharmacophore Dmt-Tic were synthesized, and their binding affinity and functional bioactivity at the μ, δ and κ opioid receptors determined. Bifunctional ligands containing or not a spacer of β-alanine between the two pharmacophores lose the μ agonism deriving from morphine becoming partial μ agonists 4 or μ antagonists 5. Partial κ agonism is evidenced only for compound 4. Finally, both compounds showed potent δ antagonism.

Here we evaluated how the interchange of the amino acids 2′,6′-dimethyl-L-tyrosine (Dmt), 2′,6′-difluoro-L-tyrosine (Dft), and tyrosine in position 1 can affect the pharmacological characterization of some reference opioid peptides and pseudopeptides. Generally, Dft and Tyr provide analogues with a similar pharmacological profile, despite different pKa values. Dmt/Tyr(Dft) replacement gives activity changes depending on the reference opioid in which the modification was made. Whereas, H-Dmt-Tic-Asp*-Bid is a potent and selective δ agonist (MVD, IC50 = 0.12 nM); H-Dft-Tic-Asp*-Bid and H-Tyr-Tic-Asp*-Bid are potent and selective δ antagonists (pA2 = 8.95 and 8.85, respectively). When these amino acids are employed in the synthesis of deltorphin B and its Dmt1 and Dft1 analogues, the three compounds maintain a very similar δ agonism (MVD, IC50 0.32–0.53 nM) with a decrease in selectivity relative to the Dmt1 analogue. In the less selective H-Dmt-Tic-Gly*-Bid the replacement of Dmt with Dft and Tyr retains the δ agonism but with a decrease in potency. Antagonists containing the Dmt-Tic pharmacophore do not support the exchange of Dmt with Dft or Tyr.

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced δ/μ antagonists, as determined by the functional [S35]GTPγS binding assay, the dimerization of potent Aia-based ‘parent’ ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds.

Orally active dual μ-/δ-opioid receptor antagonist, H-Dmt-Tic-Lys-NH-CH2-Ph (MZ-2) was applied to study body weight gain, fat content, bone mineral density, serum insulin, cholesterol and glucose levels in female ob/ob (B6.V-Lep/J homozygous) and lean wild mice with or without voluntary exercise on wheels for three weeks, and during a two week post-treatment period under the same conditions. MZ-2 (10 mg/kg/day, p.o.) exhibited the following actions: (1) reduced body weight gain in sedentary obese mice that persisted beyond the treatment period without effect on lean mice; (2) stimulated voluntary running on exercise wheels of both groups of mice; (3) decreased fat content, enhanced bone mineral density (BMD), and decreased serum insulin and glucose levels in obese mice; and (4) MZ-2 (30 μM) increased BMD in human osteoblast cells (MG-63) comparable to naltrexone, while morphine inhibited mineral nodule formation. Thus, MZ-2 has potential application in the clinical management of obesity, insulin and glucose levels, and the amelioration of osteoporosis.

Opioids containing the Dmt-Tic pharmacophore, especially the δ agonists H-Dmt-Tic-Gly-NH-Ph 1 and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid 4 (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the – NH-Ph and N1H-Bid hydrogens relative to δ agonism. The results provide the following conclusions: (i) Asp increases δ selectivity by lowering μ affinity; (ii) -NH-Ph and N1H-Bid nitrogen methylation transforms δ agonists into δ antagonists; (iii) substitution of Gly with L-Asp/D-Asp in the δ agonist H-Dmt-Tic-Gly-NH-Ph resulted in δ antagonists, while the same substitution in the δ agonist H-Dmt-Tic-NH-CH2-Bid yielded more selective δ agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid; (iv) L-Asp seems important only for functional bioactivity, not receptor affinity; (v) H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) (10) revealed analgesia similar to 4, which was reversed by naltrindole only in the tail-flick test. Compounds 4 and 10 had opposite behaviours in mice: 4 caused agitation, while 10 gave sedation and convulsions.

Replacement of the constrained phenylalanine analogue 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the opioid Dmt-Tic-Gly-NH-Bn scaffold by the 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]azepin-3-one (Aia) and 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffolds has led to the discovery of novel potent μ-selective agonists (Structures 5 and 12) as well as potent and selective δ-opioid receptor antagonists (Structures 9 and 15). Both stereochemistry and N-terminal N,N-dimethylation proved to be crucial factors for opioid receptor selectivity and functional bioactivity in the investigated small peptidomimetic templates. In addition to the in vitro pharmacological evaluation, automated docking models of Dmt-Tic and Dmt-Aba analogues were constructed in order to rationalize the observed structure-activity data.

Three analogues of the dual μ-/δ-antagonist, H-Dmt-Tic-R-NH-CH2-Ph (R = 1, Lys-Z; 2, Lys-Ac; 3, Lys) were examined in vivo: 1 and 2 exhibited weak bioactivity, while 3 injected intracerebroventricularly was a potent dual antagonist for morphine- and deltorphin C-induced antinociception comparable to naltrindole (δ-antagonist), but 93% as effective as naloxone (nonspecific opioid receptor antagonist) and 4% as active as CTOP, a μ antagonist. Subcutaneous or oral administration of 3 antagonized morphine-induced antinociception indicating passage across epithelial and blood-brain barriers. Mice pretreated with 3 before morphine did not develop morphine tolerance indicative of a potential clinical role to inhibit development of drug tolerance.

H-Dmt-Tic-ε-Lys(Z)-OH (1) was used in the synthesis of 18F-labeled opioids for positron emission tomography (PET) imaging by coupling N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) with Boc-Dmt-Tic-ε-Lys(Z)-OH under slightly basic conditions at 37 °C for 15 min, deprotected with TFA and HPLC purification in 120 min with a decay-corrected radiochemical 25–30% yield of [18F]-1 (n = 5) and specific activity ca. 46 GBq/µmol. Autoradiography uptake of [18F]-1 in striatum and cortex was blocked by 1 and UFP-501 demonstrating specific binding to δ-opioid receptors. MicroPET imaging revealed the absence of [18F]-1 in rat brain, suggesting its suitability for imaging peripheral δ-opioid receptors.