Accomplishments

Medicinal Chemistry Group: Drug Development

• Synthesis of receptor selective opioid ligands containing the Dmt-Tic pharmacophore

  The two most important contributions in our lengthy studies on opioid ligands entailed the introduction of Dmt into δ-opioid receptor antagonists1 and conversion of a potent δ-opioid receptor antagonist into a strong δ-opioid receptor agonist. The replacement of the critical Tyr at the first position led to the verification and expansion of several pertinent aspects of opioid ligand chemistry, while subtle modification of the C-terminus of dipeptides completely change the bioactivity spectrum of the peptide. Dmt, being more hydrophobic residue than Tyr, interacts with comparable regions in the ligand-binding site(s) and provides clues on formulating a molecule that can pass through gastrointestinal epithelium and the tight junctions that comprise the blood-brain barrier.

• Formation of a high affinity and selective δ-opioid receptor ligand: [3H]N,N(CH₃)₂-Dmt-Tic OH

  Catalytic tritiation of the potent δ-opioid antagonist N,N(CH₃)₂-Dmt-Tic OH,5 yielded a peptide with high specificity and a K_d of 0.42 nM and a B_max of 63.1 fmol/mg protein. The radiolabeled compound inhibited the binding of [³⁵S]GTP_γS in cell membranes and had equivalent receptor binding as the unlabeled compound6 and other tritiated δ-agonists.

• Modification of Tic in the Dmt-Tic pharmacophore

  In an attempt to understand the influence, Tic was either modified or substituted by other residues. In terms of activity toward δ-opioid receptors, an intact and unmodified Tic residue was considered essential. Halogenation, presence of nitrate, amine, hydroxyl or methoxyl groups at positions 6 or 7 of Tic were deleterious. However, the C-terminal CH₂OCH₂-Ph group yielded a compound with antagonist activity, that provided clues for additional substitutions at this position in the pharmacophore.

• Formation of opioid substances containing heteroaromatic or heteroaliphatic nuclei

  A large number of analogues were prepared into order to test the hypothesis that other aromatic might replace Tic in the Dmt-Tic pharmacophore, including benzimidazole, pyridoindole, and spiroindole groups. Of these opioids, only a benzimidazole derivative demonstrated high δ-opioid receptor affinity and antagonism with modest µ-opioid agonism, indicative of the formation of multifunctioning opioids and led to the formation of dual or bifunctional ligands.

• Inverse agonism by Dmt-Tic pharmacophore analogues

  Dmt was responsible for the full inverse agonism observed with N,N-(CH₃)-Dmt-Tic-NH₂, which was greater than any previously described opioid substance. H-Dmt-Tic-OH exhibited partial inverse agonism and H-Dmt-Tic-NH2 was a neutral antagonist comparable to naltrindole.

• N-alkylation and C-terminus modification to increase hydrophobicity of the Dmt-Tic pharmacophore

  To ensure greater penetrability of the opioids through membrane barriers, two distinct schemes were instituted: (i) N-alkylation of the free amine and (ii) C-terminus modification with bulky hydrophobic groups with or without N-alkylation. In essence, mono- or dimethylation, which form secondary and tertiary amines, respectively, had no deleterious effect on δ-opioid receptor affinity, although µ-opioid receptor affinity was markedly affected.

• Formation of multifunctional opioidmimetic opioid ligands. H-Dmt-Tic-NH-CH(R)-R and differentiation of multifunctional compounds

  Another series of compounds, H-Dmt-Tic-NH-CH(R)-R, where R = an alkyl chain or an amino acid residue linker (spacer) and R = 1H-benzimidazole-2-yl (Bid) or phenyl (Ph), which represented a third aromatic nucleus, attempted to increase µ-opioid receptor affinity and µ-opioid agonism while maintaining δ-opioid receptor properties inherent in the Dmt-Tic moiety. Three critical factors permitted the differentiation between ligands with agonism and antagonism: (i) the distance between the third aromatic center and Tic determined whether the ligand exhibited δ-opioid antagonism or δ-opioid agonism; (ii) the importance of the composition of the third aromatic nucleus; and (iii) N-methylation enhanced δ-opioid receptor antagonism, while decreasing these factors toward µ-opioid receptors.

• Hydrophobic endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) analogues

  In order to develop more hydrophobic endomorphin-2 analogues with the potential to pass through the blood-brain barrier, Dmt replaced Tyr in the first position in a series of analogues. Other analogues contained aromatic, heteroaromatic or hydrophobic substituents at the C-terminal in lieu of Phe4-NH2. Although [Dmt¹,5-quinolyl⁴]endomorphin-2 exhibited a ten-fold gain in µ-opioid receptor affinity, the extraordinary selectivity was lost due to increased δ-opioid receptor affinity, verifying the observations that Dmt augments binding to both δ- and µ-opioid receptors, indicative of a common or structurally similar binding domain in different opioid receptor types.

• Unique opioidmimetic ligands for the µ-opioid receptor. 1. Bis-[Dmt-NH-R]-pyrazinone

  The chemistry of pyrazinone ring formation permitted synthesis of symmetric and asymmetric analogues containing Dmt N-termini. Incorporation of Dmt in lieu of Tyr increased µ-opioid affinities by two to three orders of magnitude and exhibited very high affinity, with Ki values consistently < 1 nM, and potent agonism; Tyr derivatives were biologically inactive.

• Unique opioidmimetic ligands for the µ-opioid receptor. 2. Bis-[Dmt-NH]-alkyl opioidmimetics

  Elimination of the intervening pyrazinone ring yielded two N-termini separated by a simple non-branched alkyl chain that had high µ-opioid receptor affinity and agonism. Optimum length of the carbon chain fell between four and six methylene units. These compounds represent the simplest form of an opioid peptide known to date.

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