Endomorphin analogues with mixed µ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking ß-arrestin2 recruitment activity.

Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH2) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH2) modified at position 3, with various phenylalanine analogues (Xaa=Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high µ-opioid (MOP) receptor binding affinity (KiMOP=0.13-0.81nM), modest MOP-selectivity (Kiδ-opioid (DOP)/KiMOP=3.5-316), and potent functional MOP agonism (GPI, IC50=0.274-249nM) without DOP and κ-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH2) and 9 (Dmt-Pro-1-Nal-NH2) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of ß-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds.

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 delta/mu antagonists, as determined by the functional [S(35)]GTPgammaS binding assay, the dimerization of potent Aia-based 'parent' ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds.

Role of 2',6'-dimethyl-l-tyrosine (Dmt) in some opioid lead compounds.

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 pK(a) 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 delta agonist (MVD, IC(50)=0.12nM); H-Dft-Tic-Asp *-Bid and H-Tyr-Tic-Asp *-Bid are potent and selective delta antagonists (pA(2)=8.95 and 8.85, respectively). When these amino acids are employed in the synthesis of deltorphin B and its Dmt(1) and Dft(1) analogues, the three compounds maintain a very similar delta agonism (MVD, IC(50) 0.32-0.53 nM) with a decrease in selectivity relative to the Dmt(1) analogue. In the less selective H-Dmt-Tic-Gly *-Bid the replacement of Dmt with Dft and Tyr retains the delta agonism but with a decrease in potency. Antagonists containing the Dmt-Tic pharmacophore do not support the exchange of Dmt with Dft or Tyr.

delta-Opioid receptors protect from anoxic disruption of Na+ homeostasis via Na+ channel regulation.

Hypoxic/ischemic disruption of ionic homeostasis is a critical trigger of neuronal injury/death in the brain. There is, however, no promising strategy against such pathophysiologic change to protect the brain from hypoxic/ischemic injury. Here, we present a novel finding that activation of delta-opioid receptors (DOR) reduced anoxic Na+ influx in the mouse cortex, which was completely blocked by DOR antagonism with naltrindole. Furthermore, we co-expressed DOR and Na+ channels in Xenopus oocytes and showed that DOR expression and activation indeed play an inhibitory role in Na+ channel regulation by decreasing the amplitude of sodium currents and increasing activation threshold of Na+ channels. Our results suggest that DOR protects from anoxic disruption of Na+ homeostasis via Na+ channel regulation. These data may potentially have significant impacts on understanding the intrinsic mechanism of neuronal responses to stress and provide clues for better solutions of hypoxic/ischemic encephalopathy, and for the exploration of acupuncture mechanism since acupuncture activates opioid system.

Conformationally constrained opioid ligands: the Dmt-Aba and Dmt-Aia versus Dmt-Tic scaffold.

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 mu-selective agonists (Structures 5 and 12) as well as potent and selective delta-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.

The novel micro-opioid receptor antagonist, [N-allyl-Dmt(1)]endomorphin-2, attenuates the enhancement of GABAergic neurotransmission by ethanol.

AIMS: We investigated the effects of [N-allyl-Dmt(1)]endomorphin-2 (TL-319), a novel and highly potent micro-opioid receptor antagonist, on ethanol (EtOH)-induced enhancement of GABA(A) receptor-mediated synaptic activity in the hippocampus. METHODS: Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were isolated from CA1 pyramidal cells from brain slices of male rats using whole-cell patch-clamp techniques. RESULTS: TL-319 had no effect on the baseline amplitude of eIPSCs or the frequency of sIPSCs. However, it induced a dose-dependent suppression of an ethanol-induced increase of sIPSC frequency with full reversal at concentrations of 500 nM and higher. The non-specific competitive opioid receptor antagonist naltrexone also suppressed EtOH-induced increases in sIPSC frequency but only at a concentration of 60 microM. CONCLUSION: These data indicate that blockade of micro-opioid receptors by low concentrations of [N-allyl-Dmt(1)]endomorphin-2 can reverse ethanol-induced increases in GABAergic neurotransmission and possibly alter its anxiolytic or sedative effects. This suggests the possibility that high potency opioid antagonists may emerge as possible candidate compounds for the treatment of ethanol addiction.

Synthesis of a potent and selective (18)F-labeled delta-opioid receptor antagonist derived from the Dmt-Tic pharmacophore for positron emission tomography imaging.

Identification and pharmacological characterization of two new selective delta-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high delta selectivity, H-Dmt-Tic--Lys(Z)-OH (reference compound 1) is a useful starting point for the synthesis of (18)F-labeled compounds prepared by the coupling of N-succinimidyl 4-[ (18)F]fluorobenzoate ([(18)F]SFB) with Boc-Dmt-Tic--Lys(Z)-OH under slightly basic conditions at 37 degrees C for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the decay-corrected radiochemical yield of [(18)F]- 1 was about 25-30% ( n = 5) starting from [(18)F]SFB ( n = 5) with an effective specific activity about 46 GBq/micromol. In vitro autoradiography studies showed prominent uptake of [ (18)F]- 1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective delta-opioid receptor antagonist), suggesting high specific binding of [(18)F]- 1 to delta-opioid receptors. Noninvasive microPET imaging studies revealed the absence of [(18)F]- 1 in rat brain, since it fails to cross the blood-brain barrier. This study demonstrates the suitability of [ (18)F]- 1 for imaging peripheral delta-opioid receptors.

Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist.

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.20) and selectivity (>150-fold) for DOP sites. In functional studies ([35S]GTP gamma S binding in CHOhDOP membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective full agonist showing potency values more than 100-fold higher than DPDPE. In vivo, in the mouse forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration. Similar effects were recorded in rats. Moreover, UFP-512 evoked anxiolytic-like effects in the mouse elevated plus maze and light-dark aversion assays. All these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole (3 mg/kg, s.c.). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly potent and selective agonist at DOP receptors and corroborate the proposal that the selective activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.

Synthesis and in vitro evaluation of a library of modified endomorphin 1 peptides.

Endomorphin 1 (Endo-1=Tyr-Pro-Trp-Phe-NH(2)), an endogenous opioid with high affinity and selectivity for mu-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2',6'-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for mu-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers. C-terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt[1]]-Endo-1 is nine times more stable (t(1/2)=43.5min), >8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater mu-opioid receptor affinity (K(imu)=0.08nM).

Role of benzimidazole (Bid) in the delta-opioid agonist pseudopeptide H-Dmt-Tic-NH-CH(2)-Bid (UFP-502).

H-Dmt-Tic-NH-CH(2)-Bid (UFP-502) was the first delta-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 delta-agonism, it was substituted by similar heterocycles: The substitution of NH(1) by O or S transforms the reference delta-agonist into delta-antagonists. Phenyl ring of benzimidazole is not important for delta-agonism; in fact 1H-imidazole-2-yl retains delta-agonist activity.

Inhibition of the development of morphine tolerance by a potent dual mu-delta-opioid antagonist, H-Dmt-Tic-Lys-NH-CH2-Ph.

Three analogues of the dual mu-/delta-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 (delta-antagonist), but 93% as effective as naloxone (nonspecific opioid receptor antagonist) and 4% as active as CTOP, a mu 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.

Bifunctional [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed mu-agonist/delta-antagonist and dual mu-agonist/delta-agonist opioid ligands.

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2',6'-dimethyl-l-tyrosine) analogues, 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'), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved mu- and delta-opioid receptor affinities. The latter, however, were inconsequential (Kidelta = 491-3451 nM). (ii) [Dmt1,Xaa3]EM-2 analogues enhanced mu- and delta-opioid receptor affinities (Kimu = 0.069-0.32 nM; Kidelta = 1.83-99.8 nM) without kappa-opioid receptor interaction. (iii) There were elevated mu-bioactivity (IC50 = 0.12-14.4 nM) and abolished delta-agonism (IC50 > 10 muM in 2', 3', 4', 5', 6'), although 4' and 6' demonstrated a potent mixed mu-agonism/delta-antagonism (for 4', IC50mu = 0.12 and pA2 = 8.15; for 6', IC50mu = 0.21 nM and pA2 = 9.05) and 7' was a dual mu-agonist/delta-agonist (IC50mu = 0.17 nM; IC50delta = 0.51 nM).

Synthesis of opioidmimetics, 3-[H-Dmt-NH(CH(2))(m)]-6-[H-Dmt-NH(CH(2))(n)]-2(1H)-pyrazinones, and studies on structure-activity relationships.

Opioidmimetics containing 3-[H-Dmt-NH-(CH(2))(m)]-6-[H-Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinone symmetric (m = n, 1-4) (1 - 4) and asymmetric (m, n = 1 - 4) aliphatic chains (5 - 16) were synthesized using dipeptidyl chloromethylketone intermediates. They had high mu-affinity (K(i)mu = 0.021 - 2.94 nM), delta-affinity (K(i)delta = 1.06 - 152.6 nM), and mu selectivity (K(i)delta/K(i)mu = 14 - 3,126). The opioidmimetics (1 - 16) exhibited mu agonism in proportion to their mu-receptor affinity. delta-Agonism was essentially lacking in the compounds except (4) and (16), and (1) and (2) indicated weak delta antagonism (pA(2) = 6.47 and 6.56, respectively). The data verify that a specific length of aliphatic linker is required between the Dmt pharmacophore and the pyrazinone ring to produce unique mu-opioid receptor ligands.

Effect of lysine at C-terminus of the Dmt-Tic opioid 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-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 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.

New opioid designed multiple ligand from Dmt-Tic and morphinan pharmacophores.

Here, we report the 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 those 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.

New 2',6'-dimethyl-L-tyrosine (Dmt) opioid peptidomimetics based on the Aba-Gly scaffold. Development of unique mu-opioid receptor ligands.

The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed micro/delta or delta opioid receptor activities with micro agonism. Substitution of Tic by Aba-Gly coupled to -NH-CH2-Ph (1), -NH-Ph (2), or -Bid (Bid=1H-benzimidazole-2-yl) (3) shifted affinity (Ki(micro)=0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to micro-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.

6-N,N-dimethylamino-2,3-naphthalimide: a new environment-sensitive fluorescent probe in delta- and mu-selective opioid peptides.

A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3-naphthalimide (6DMN) was introduced in the delta-selective opioid peptide agonist H-Dmt-Tic-Glu-NH(2) and in the mu-selective opioid peptide agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)). 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 delta-selective irreversible antagonists (H-Dmt-Tic-Glu-NH-(CH(2))(5)-CO-Dap(6DMN)-NH(2) (1) and H-Dmt-Tic-Glu-Dap(6DMN)-NH(2) (2)) were identified as potential fluorescent probes showing good properties for use in studies of distribution and internalization of delta receptors by confocal laser scanning microscopy.

Potent in vivo antinociception and opioid receptor preference of the novel analogue [Dmt1]endomorphin-1.

[Dmt1]Endomorphin-1 is a novel analogue of the potent mu-opioid agonist endomorphin-1. Given the physiological role of endomorphin-1 in vivo, this compound was investigated to determine if the antinociception occurred through systemic, supraspinal or in a combination of both neuronal pathways. This compound exhibited a potent dose-dependent effect intracerebroventricularly in both spinal and supraspinal regions, and was blocked by opioid antagonist naloxone, which verified the involvement of opioid receptors. Specific opioid antagonists characterized the apparent receptor type: beta-funaltrexamine (mu1/mu2-irreversible antagonist) equally inhibited spinal- and central-mediated antinociception; on the other hand, naloxonazine (mu1-subtype) was ineffective in both neural pathways and naltrindole (delta-selective antagonist) partially (26%), though not significantly, blocked only the spinal-mediated antinociception. Therefore, spinal antinociception was primarily triggered by mu2-subtypes without involvement of mu1-opioid receptors; however, although a slight enhancement of antinociception by delta-receptors cannot be completely ruled out since functional bioactivity indicated mixed mu-agonism/delta-antagonism. In terms of the CNS action, [Dmt1]endomorphin-1 appears to act through mu2-opioid receptor subtypes.

Enantioselective synthesis of a phenylalanine library containing alkyl groups on the aromatic moiety: confirmation of stereostructure by X-ray analysis.

Six phenylalanine analogues containing 2'-methyl-, 2',6'-dimethyl-, 2'-ethyl-6'-methyl-, 2'-isopropyl-6'-methyl-, 2',4',6'-trimethyl-, and 3',5'-dimethyl-L-phenylalanine were synthesized enantioselectively through asymmetric hydrogenation of acetamidoacrylate derivatives. Enzymatic digestion and X-ray analysis supported the L-configuration of the phenylalanine derivatives obtained.

Structural function of C-terminal amidation of endomorphin. Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy, molecular calculation, and X-ray crystallography.

To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)), an endogenous micro-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D (1)H-NMR measurement and molecular modelling calculation. Both peptides were in equilibrium between the cis and trans rotamers around the Tyr--Pro w bond with population ratios of 1 : 1 to 1 : 2 in dimethyl sulfoxide, TFE and water, whereas they predominantly took the trans rotamer in DPC micelle, except in EM2OH at pH 5.2, which had a trans/cis rotamer ratio of 2 : 1. Fifty possible 3D conformers were generated for each peptide, taking different electronic states depending on the type of solvent and pH (neutral and monocationic forms for EM2, and zwitterionic and monocation forms for EM2OH) by the dynamical simulated annealing method, under the proton-proton distance constraints derived from the ROE cross-peak intensities. These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical 7 (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the backbone structure. Most EM2 conformers in neutral (in TFE) and monocationic (in water and DPC micelles) forms adopted the open structure (mixture of major rS-type and minor n7-type conformers) despite the trans/cis rotamer form. On the other hand, the zwitterionic EM2OH in TFE, water and DPC micelles showed an increased population of F1- and F2-type folded conformers, the population of which varied depending on their electronic state and pH. Most of these folded conformers took an F1-type structure similar to that stabilized by an intramolecular hydrogen bond of (Tyr1)NH(3) (+)...COO(-)(Phe4), observed in its crystal structure. These results show that the substitution of a carboxyl group for the C-terminal amide group makes the peptide structure more flexible and leads to the ensemble of folded and open conformers. The conformational requirement of EM2 for binding to the micro-opioid receptor and the structural function of the C-terminal amide group are discussed on the basis of the present conformational features of EM2 and EM2OH and a possible model for binding to the micro-opioid receptor, constructed from the template structure of rhodopsin.