Cellular Localization of Dieldrin and Structure–Activity Relationship of Dieldrin Analogues in Dopaminergic Cells

The incidence of Parkinson’s disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure–activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and <i>cis</i>-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the <i>cis</i>-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity

Conversion of the Potent δ-Opioid Agonist H-Dmt-Tic-NH-CH₂-Bid into δ-Opioid Antagonists by N-Benzimidazole Alkylation¹

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

From the Potent and Selective μ Opioid Receptor Agonist H-Dmt-d-Arg-Phe-Lys-NH₂ to the Potent δ Antagonist H-Dmt-Tic-Phe-Lys(Z)-OH

H-Dmt-d-Arg-Phe-Lys-NH2 ([Dmt]DALDA) binds with high affinity and selectivity to the μ opioid receptor and is a potent and long-acting analgesic. Substitution of d-Arg in position 2 with Tic and masking of the lysine amine side chain by Z protection and of the C-terminal carboxylic function instead of the amide function transform a potent and selective μ agonist into a potent and selective δ antagonist H-Dmt-Tic-Phe-Lys(Z)-OH. Such a δ antagonist could be used as a pharmacological tool

Development of Potent μ-Opioid Receptor Ligands Using Unique Tyrosine Analogues of Endomorphin-2

Six analogues of tyrosine, which contained alkyl groups at positions 2‘, 3‘, and 6‘, either singly or in combination on the tyramine ring, were investigated for their effect on the opioid activity of [Xaa]endomorphin-2 (EM-2). The opioid analogues displayed the following characteristics:  (i) high μ-opioid receptor affinity [Ki(μ) = 0.063−2.29 nM] with selectivity [Ki(δ)/Ki(μ)] ranging from 46 to 5347; (ii) potent functional μ-opioid agonism [GPI assay (IC50 = 0.623−0.924 nM)] and with a correlation between δ-opioid receptor affinities and functional bioactivity using MVD; (iii) intracerebroventricular administration of [Dmt1]- (14) and [Det1]EM-2 (10) produced a dose−response antinociception in mice, with the former analogue more active than the latter; and (iv) a marked shift occurred from the trans-orientation at the Tyr1-Pro bond to a cis-conformer compared to that observed previously with [Dmt1]EM-2 (14) (Okada et al. Bioorg. Med. Chem. 2003, 11, 1983−1984) except [Mmt1]EM-2 (7). The active profile of the [Xaa1]EM-2 analogues indicated that significant modifications on the tyramine ring are possible while high biological activity is maintained

Bifunctional [2‘,6‘-Dimethyl-l-tyrosine]endomorphin-2 Analogues Substituted at Position 3 with Alkylated Phenylalanine Derivatives Yield Potent Mixed μ-Agonist/δ-Antagonist and Dual μ-Agonist/δ-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 μ- and δ-opioid receptor affinities. The latter, however, were inconsequential (Kiδ = 491−3451 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) without κ-opioid receptor interaction. (iii) There were elevated μ-bioactivity (IC50 = 0.12−14.4 nM) and abolished δ-agonism (IC50 > 10 μM in 2‘, 3‘, 4‘, 5‘, 6‘), although 4‘ and 6‘ demonstrated a potent mixed μ-agonism/δ-antagonism (for 4‘, IC50μ = 0.12 and pA2 = 8.15; for 6‘, IC50μ = 0.21 nM and pA2 = 9.05) and 7‘ was a dual μ-agonist/δ-agonist (IC50μ = 0.17 nM; IC50δ = 0.51 nM)

New 2‘,6‘-Dimethyl-l-tyrosine (Dmt) Opioid Peptidomimetics Based on the Aba-Gly Scaffold. Development of Unique μ-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 μ/δ or δ opioid receptor activities with μ 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(μ) = 0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to μ-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief

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-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 (Kiμ = 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

6-<i>N,N</i>-Dimethylamino-2,3-naphthalimide: A New Environment-Sensitive Fluorescent Probe in δ- and μ-Selective Opioid Peptides

A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3-naphthalimide (6DMN) was introduced in the δ-selective opioid peptide agonist H-Dmt-Tic-Glu-NH2 and in the μ-selective opioid peptide 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 good properties for use in studies of distribution and internalization of δ receptors by confocal laser scanning microscopy

Oral Bioavailability of a New Class of μ-Opioid Receptor Agonists Containing 3,6-Bis[Dmt-NH(CH₂)<i>_n</i>]-2(1<i>H</i>)-pyrazinone with Central-Mediated Analgesia

The inability of opioid peptides to be transported through epithelial membranes in the gastrointestinal tract and pass the blood−brain barrier limits their effectiveness for oral application in an antinociceptive treatment regime. To overcome this limitation, we enhanced the hydrophobicity while maintaining the aqueous solubility properties in a class of opioid-mimetic substances by inclusion of two identical N-termini consisting of Dmt (2‘,6‘-dimethyl-l-tyrosine) coupled to a pyrazinone ring platform by means of alkyl chains to yield the class of 3,6-bis[Dmt-NH−(CH2)n]-2(1H)-pyrazinones. These compounds displayed high μ-opioid receptor affinity (Kiμ = 0.042−0.115 nM) and selectivity (Kiδ/Kiμ = 204−307) and functional μ-opioid receptor agonism (guinea-pig ileum, IC50 = 1.3−1.9 nM) with little or undetectable bioactivity toward δ-opioid receptors (mouse vas deferens) and produced analgesia in mice in a naloxone reversible manner when administered centrally (intracerebroventricular, icv) or systemically (subcutaneously and orally). Furthermore, the most potent compound, 3,6-bis(3'-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone (7‘), lacked functional δ-opioid receptor bioactivity and was 50−63-fold and 18−21-fold more active than morphine by icv administration as measured analgesia using tail-flick (spinal involvement) and hot-plate (supraspinal effect) tests, respectively; the compound ranged from 16 to 63% as potent upon systemic injection. These analgesic effects are many times greater than unmodified opioid peptides. The data open new possibilities for the rational design of potential opioid-mimetic drugs that pass through the epithelium of the gastrointestinal tract and the blood−brain barrier to target brain receptors

Potent Dmt-Tic Pharmacophoric δ- and μ-Opioid Receptor Antagonists

A series of dimeric Dmt-Tic (2‘,6‘-dimethyl-l-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) analogues (8−14, 18−22) were covalently linked through diaminoalkane and symmetric or asymmetric 3,6-diaminoalkyl-2(1H)-pyrazinone moieties. All the compounds exhibited high affinity for both δ-opioid receptors [Ki(δ) = 0.06−1.53 nM] and μ-opioid receptors [Ki(μ) = 1.37−5.72 nM], resulting in moderate δ-receptor selectivity [Ki(μ)/Ki(δ) = 3−46]. Regardless of the type of linker between the Dmt-Tic pharmacophores, δ-opioid-mediated antagonism was extraordinarily high in all analogues (pA2 = 10.42−11.28), while in vitro agonism (MVD and GPI bioassays) was essentially absent (ca. 3 to >10 μM). While an unmodified N-terminus (9, 13, 18) revealed weak μ-opioid antagonism (pA2 = 6.78−6.99), N,N‘-dimethylation (21, 22), which negatively impacts on μ-opioid-associated agonism (Balboni et al., Bioorg. Med. Chem. 2003, 11, 5435−5441), markedly enhanced μ-opioid antagonism (pA2 = 8.34 and 7.71 for 21 and 22, respectively) without affecting δ-opioid activity. These data are the first evidence that a single dimeric opioid ligand containing the Dmt-Tic pharmacophore exhibits highly potent δ- and μ-opioid antagonist activities