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.

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.

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2’,6’-dimethyl-l-tyrosine) analogues were synthesized containing alkylated Phe3 derivatives, 2’-monomethyl (2, 2’), 3’,5’- and 2’,6’-dimethyl (3, 3’, and 4’, respectively), 2’,4’,6’-trimethyl (6, 6’), 2’-ethyl-6’-methyl (7, 7’) and 2’-isopropyl-6’-methyl (8, 8’) groups or Dmt (5, 5’). They had the following characteristics: (i) [Xaa3]EM-2 analogues improved μ- and δ-opioid receptor affinities, the latter were inconsequential (Kiδ= 491–3,451 nM); (ii) [Dmt1,Xaa3]EM-2 analogues enhanced μ- and δ-opioid receptor affinities (Kiμ = 0.069–0.32 nM; Kiδ = 1.83–99.8 nM) and lacked interaction with κ-opioid receptors, and (iii) elevated μ-bioactivity (IC50 = 0.12–14.4 nM) and abolished δ-agonism (IC50 > 10 µM; 2’, 3’, 4’, 5’, 6’); however, 4’ and 6’ exhibited mixed μ-agonism/δ-antagonism (4’: IC50μ = 0.12, pA2 = 8.15; 6’: IC50μ = 0.21 nM, pA2 = 9.05), and 7’ was a dual μ-/δ -agonist (IC50μ = 0.17 nM; IC50δ = 0.51 nM). Alteration of EM-2 activity by Dmt1 and alkylated Phe3 residues retained μ-receptor bioactivity and formed dual μ-/δ -agonists and mixed μ-agonists/δ-antagonists.

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.

H-Dmt-Tic-NH-CH2-Bid (UFP-502) was the first δ opioid agonist prepared from the Dmt-Tic pharmacophore. It showed interesting pharmacological properties, such as stimulation of mRNA BDNF expression, and antidepression. To evaluate the importance of 1H-benzimidazol-2-yl (Bid) in the induction of δ agonism, it was substituted by similar heterocycles: The substitution of NH(1) by O or S, transforms the reference δ agonist into δ antagonists. Phenyl ring of benzimidazole is not important for δ agonism; in fact 1H-imidazole-2-yl retains δ agonist activity.

Dimeric opioid analogues linked to a pyrazinone platform, 3-[Tyr-NH(CH2)m]-6-[Tyr-NH(CH2)n]-2(1H)-pyrazinone (n, m = 3 or 4) and their corresponding Dmt derivatives were synthesized. Whereas the Try-containing compounds were essentially inactive, the Dmt derivatives exhibited high affinity for the µ-opioid receptor (Kiµ; 0.021–0.051 nM) with corresponding agonism (IC50 = 1.79–4.93 nM). Interestingly, while one compound (m = 4, n = 3) revealed modest δ-agonism, the converse analogue (m = 3, n = 4) was inactive. The data suggest that the spatial conformation, linker length and covalent bonding position on the pyrazinone ring are important for opioid activity.

Substitution of Gly with side-chain protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH2-Ph, μ agonist / δ antagonist; H-Dmt-Tic-Gly-NH-Ph, μ agonist / δ agonist and H-Dmt-Tic-NH-CH2-Bid, δ agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high δ- (Kiδ = 0.068-0.64 nM) and μ-opioid affinities (Kiδ = 0.13-5.50 nM) with a bioactivity that ranged from μ-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective μ-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2μ = 7.96)] and a selective δ-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2δ = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and / or antagonist properties.

A wide range of bioactivities are induced by Lys when introduced at the C-terminus of the δ-opioid Dmt-Tic pharmacophore through the α-amine group, such as improved δ-antagonism, and presence of μ-agonism and μ-antagonism. We report the synthesis of a new series of compounds with the general formula H-Dmt-Tic-NH-(CH2)4-CH(R)-R’ (R = -NH2, -NH-Ac, -NH-Z; R’ = CO-NH-Ph, -CO-NH-CH2-Ph, -Bid) in which Lys is linked to Dmt-Tic through its amine group side chain. The compounds (1-9) displayed a potent and selective δ-antagonism (pA2 = 7.81-8.27) independent of the functionalized α-amine and carboxylic groups of the C-terminal Lys. This suggests direct application as a prototype intermediate, such as Boc-Dmt-Tic-ε-Lys(Z)-OMe, which could be applied in the synthesis (after Z or methyl ester removal) of unique “designed multiple ligands” containing the pharmacophore of the quintessential δ-antagonist Dmt-Tic and another opioid or biologically active non-opioid ligand.

Twelve 2′,6′-dimethyl-l-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH2)n]-3- or 6-R-2(1H)-pyrazinone (n = 1 - 4). 3-[H-Dmt-NH-(CH2)4]-6-β-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to μ-opioid receptors with high affinity (Kiμ = 0.13 nM; Kiδ/Kiμ = 447) with μ-agonism (GPI IC50 = 15.9 nM) and weak δ-antagonism (MVD pA2 = 6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.