Trigonal scaffolds for multivalent targeting of melanocortin receptors

Abstract

Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (∼2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed.

Figure 1

Saturation binding curves for probe 19 generated using the six-well plate assay method. Top: Total binding (●) and non-specific binding (■). Bottom: Specific binding (▲). The calculated K_d = 21 ± 3 nM (n = 5).

Figure 2

Competitive binding curves for the compounds 14a–c (top), 16a–c (middle), and 18 (bottom) against probe 19 (20 nM) generated using the six-well plate assay method. Control compounds 12a and 15a were not competitive inhibitors of 19 over the concentration ranges tested.

Figure 3

Views of representative conformations observed for compounds 6, 14c, 16c, and 18. The distances from the N-terminal nitrogen atoms of the histidine residues are given in Angstroms as a measure of the three inter-ligand distances. The average distances between ligands for 6, 14c, 16c, and 18 are 22, 22, 19, and 21 Å, respectively.

Scheme 1

Synthesis of phloroglucinol-based compounds 5a and 14a–c.

Scheme 2

Synthesis of tripropargylamine-based compounds 15a and 16a–c.^{a a}Conditions A: TACP, sodium ascorbate, DMF, microwave irradiation to maintain 100 °C, 4 h; used in the synthesis of 16a and 16b. Conditions B: TACP, 2,6-lutidine, H₂O/MeCN (1:1), rt, 4d; used in the synthesis of 16c.

Scheme 3

Synthesis of 1,4,7-triazacyclononane-based compound 18.