This study reports the synthesis, chromatographic separation and pharmacological evaluation of the two enantiors of the neuropeptide S receptor (NPSR) antagonist (9R/S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68). The (9R)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10) and (9S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10a) were synthesized and their purity assessed by chiral chromatography. The absolute configuration of the enantiomer 10 has been assigned from the crystal structure of the corresponding (S)-phenyl ethyl amine derivative 8. Calcium mobilization studies performed on cells expressing the recombinant NPSR demonstrated that compound 10 is the active enantiomer while the contribution of 10a to the NPSR antagonist properties of the racemic mixture is negligible.

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.

Based on a renewed importance recently attributed to bi- or multifunctional opioids, we report the synthesis and pharmacological evaluation of some analogues derived from our lead μ agonist / δ antagonist, H-Dmt-Tic-Gly-NH-Bzl. Our previous studies focused on the importance of the C-teminal benzyl function in the induction of such bifunctional activity. The introduction of some substituents in the para position of the phenyl ring (-Cl, -CH3, partially −NO2, inactive -NH2) was found to give a more potent μ agonist / antagonist effect associated with a relatively unmodified δ antagonist activity (pA2 = 8.28-9.02). Increasing the steric hindrance of the benzyl group (using diphenylmethyl and tetrahydroisoquinoline functionalities) substantially maintained the μ agonist and δ antagonist activities of the lead compound. Finally and quite unexpectedly D-Tic2, considered as a wrong opioid message now; inserted into the reference compound in lieu of L-Tic, provided a μ agonist / δ agonist better than our reference ligand (H-Dmt-Tic-Gly-NH-Ph) and was endowed with the same pharmacological profile.

Based on a renewed importance recently attributed to bi- or multifunctional opioids, we report the synthesis and pharmacological evaluation of some analogues derived from our lead μ agonist/δ antagonist, H-Dmt-Tic-Gly-NH-Bzl (Dmt = 2′,6′-dimethyl-l-tyrosine, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Bzl = benzyl). Our previous studies focused on the importance of the C-teminal benzyl function in the induction of such bifunctional activity. The introduction of some substituents in the para position of the phenyl ring (−Cl, −CH3, partially −NO2, inactive −NH2) was found to give a more potent μ agonist/antagonist effect associated with a relatively unmodified δ antagonist activity (pA2 = 8.28−9.02). Increasing the steric hindrance of the benzyl group (using diphenylmethyl and tetrahydroisoquinoline functionalities) substantially maintained the μ agonist and δ antagonist activities of the lead compound. Finally and quite unexpectedly d-Tic2, considered as a wrong opioid message now, inserted into the reference compound in lieu of l-Tic provided a μ agonist/δ agonist better than our reference ligand (H-Dmt-Tic-Gly-NH-Ph; Ph = phenyl) and was endowed with the same pharmacological profile.

Peptidomometic analogues, H-Dmt-Tic-NH2-CH2-Ph or -Bid exhibit δ-opioid receptor activities. Substitution of Tic by the Aba-Gly scaffold coupled to the C-termini -CH2-Ph (1), -NH-Ph (2) and Gly*-Bid (3) shifted receptor affinity and selectivity to μ-opioid receptors (Kiμ = 0.46, 1.48 and 19.9 nM, respectively) with μ agonism. These represent templates for a new class of μ-opioid agonists. Further modification with negative or positive charges could yield altered properties suitable for therapeutic application for pain relief.

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.

Neuropeptide S (NPS) regulates various biological functions by activating the NPS receptor (NPSR). Previous studies demonstrated that the substitution of Gly5 with D-amino acids generates NPSR antagonists. Eleven [D-Xaa5]NPS derivatives were synthesized and pharmacologically tested measuring [Ca2+]i in HEK293mNPSR cells. The present results confirmed that the [D-Xaa5] substitution promotes antagonist activity with potency inversely related to the side chain size and allowed to identify the novel potent NPSR peptide antagonist [tBu-D-Gly5]NPS.

Activation of δ-opioid receptors (DOR) attenuates anoxic K+ leakage and protects cortical neurons from anoxic insults by inhibiting Na+ influx. It is unknown, however, which pathway(s) that mediates the Na+ influx is the target of DOR signal. In the present work, we found that in the cortex, 1) DOR protection was largely dependent on the inhibition of anoxic Na+ influxes mediated by voltage-gated Na+ channels; 2) DOR activation inhibited Na+ influx mediated by ionotropic glutamate NMDA receptors, but not that by non-NMDA receptors though both played a role in anoxic K+ derangement; and 3) DOR activation had little effect on Na+/Ca2+ exchanger-based response to anoxia. We conclude that, 1) DOR activation attenuates anoxic K+ derangement by restricting Na+ influx mediated by Na+ channels and NMDA receptors, and 2) non-NMDA receptors and Na+/Ca2+ exchangers, though involved in anoxic K+ derangement in certain degrees, are less likely the targets of DOR signal.

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as NPSR. In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)5]NPS was identified. [D-Cys(tBu)5]NPS up to 100 μM did not stimulate calcium mobilization in HEK293 cells stably expressing the mouse NPSR (HEK293mNPSR), however, the peptide inhibited in a concentration dependent manner the stimulatory effects elicited by 10 and 100 nM NPS (pK 6.62). In Schild analysis experiments [D-Cys(tBu)5]NPS (0.1 - 100 μM) produced a concentration dependent and parallel rightward shift of the concentration response curve to NPS showing a pA2 value of 6.44. 10 μM [D-Cys(tBu)5]NPS did not affect signalling at seven NPSR unrelated G-protein coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmole intracerebroventricularly reduced the percent of animals losing the RR in response to diazepam 15 mg/kg and their sleeping time. [D-Cys(tBu)5]NPS (1-10 nmoles) was inactive per se but dose dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal promoting action of 1 nmole NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)5]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist, but with moderate potency. Moreover, using this tool together with receptor knockout mice studies we demonstrated that the arousal-promoting action of NPS is due to the selective activation of the NPSR protein.

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.

Neuropeptide S (NPSa), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure-activity study of Gly5 which has been replaced with L and D amino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated that peptide potency is inversely related to the side chain size while peptide efficacy strongly depends on the relative L and D configuration with the L aminoacids favoring agonist while D aminoacids displaying antagonist pharmacological activity. [D-Val5]NPS behaved as NPSR pure antagonist in HEK293mNPSR cells showing the highest potency (pKB 7.56) among this series of peptides. The antagonist action of [D-Val5]NPS was confirmed in vivo in mice where the peptide at a dose of 10 nmoles completely blocked the stimulatory effect of 0.1 nmole NPS on locomotor activity.

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.

We have recently found that in the mouse cortex, activation of δ-opioid receptor (DOR) attenuates the disruption of K+ homeostasis induced by hypoxia or oxygen–glucose deprivation. This novel observation suggests that DOR may protect neurons from hypoxic/ischemic insults via the regulation of K+ homeostasis because the disruption of K+ homeostasis plays a critical role in neuronal injury under hypoxic/ischemic stress. The present study was performed to explore the ionic mechanism underlying the DOR-induced neuroprotection. Because anoxia causes Na+ influx and thus stimulates K+ leakage, we investigated whether DOR protects the cortex from anoxic K+ derangement by targeting the Na+-based K+ leakage. By using K+-sensitive microelectrodes in mouse cortical slices, we showed that 1) lowering Na+ concentration and substituting with impermeable N-methyl-D-glucamine caused a concentration-dependent attenuation of anoxic K+ derangement; 2) lowering Na+ concentration by substituting with permeable Li+ tended to potentiate the anoxic K+ derangement; and 3) the DOR-induced protection against the anoxic K+ responses was largely abolished by low-Na+ perfusion irrespective of the substituted cation. We conclude that external Na+ concentration greatly influences anoxic K+ derangement and that DOR activation likely attenuates anoxic K+ derangement induced by the Na+-activated mechanisms in the cortex.

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.

N1-Alkylation of 1H-benzimidizole of the δ agonist H-Dmt-Tic-NH-CH2-Bid with hydrophobic, aromatic, olefinic, acid, ethyl ester or amide (1–6) became δ antagonists (pA2 = 8.52–10.14). δ- and μ-Opioid receptor affinities were high (Kiδ = 0.12–0.36 nM and Kiμ = 0.44–1.42 nM). Only δ antagonism (pA2 = 8.52–10.14) was observed; μ agonism (IC50 = 30–450 nM) was not correlated with changes in alkylating agent or δ antagonism and some compounds yielded mixed δ antagonism/μ agonism.

Opioid compounds with mixed μ agonist / δ antagonist properties could be used as analgesics with low propensity to induce tolerance and dependence. Here we report the synthesis of a new designed multiple ligand deriving from the μ selective agonist endomorphin-2 and the δ selective antagonist pharmacophore Dmt-Tic. As predicted, the resulting bivalent ligand showed a μ agonist / δ antagonist profile deriving from the corresponding activities of each pharmacophore.

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.

Here we report the facile synthesis of a designed multi-pharmacophore ligand derived from the linkage of a delta selective peptide antagonist (Dmt-Tic) and a mu / kappa morphinan agonist butorphan (MCL 101) through a two methylene spacer. The new compound MCL 450 maintains the same characteristics as the two reference compounds. MCL 450 represents a useful starting point for the synthesis of other multiple opioid ligands endowed with analgesic properties with low tolerance and dependence.

[(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC50 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP−/− mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6 and 3.5 fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1–100 pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t; in both cases, UFP-112 was approx. 100 fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10 pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP−/− mice. Equi-effective high doses of UFP-112 (0.1 nmol) and N/OFQ (10 nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16 h. N/OFQ produced a clear inhibitory effect which lasted for 60 min while UFP-112 elicited longer lasting effects (> 6h). In conscious rats, UFP-112 (0.1 and 10 nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.

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.

A new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN) was introduced in the δ-selective opioid agonist H-Dmt-Tic-Glu-NH2 and in the μ-selective opioid agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2). Environment sensitive fluorophores are a special class of chromophores that generally exhibit a low quantum yield in aqueous solution, but become highly fluorescent in nonpolar solvents or when bound to hydrophobic sites in proteins or membranes. New fluorescent δ-selective irreversible antagonists [H-Dmt-Tic-Glu-NH-(CH2)5-CO-Dap(6DMN)-NH2(1) and H-Dmt-Tic-Glu-Dap(6DMN)-NH2)] (2) were identified as potential fluorescent probes showing properties suitable for studies of distribution and internalization of δ-opioid receptors by confocal laser scanning microscopy.