Click Modification in the N⁶ Region of A₃ Adenosine Receptor-Selective Carbocyclic Nucleosides for Dendrimeric Tethering that Preserves Pharmacophore Recognition

Abstract

Adenosine derivatives were modified with alkynyl groups on N6 substituents for linkage to carriers using Cu(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose, behaved as A3 adenosine receptor (AR) agonists: (5′-methyluronamides) or partial agonists (4′-truncated). Terminal alkynyl groups on a chain at the 3 position of a N6-benzyl group or simply through a N6–propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A3AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and 18F radiolabeling. N6-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative 32 displayed a Ki of 9.1 nM at A3AR with ~1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A3AR in the 5′-methyluronamide, but not 4′ truncated series. N6-Benzyl nucleoside conjugate 43 (apparent Ki 24 nM) maintained binding affinity of the monomer better than a N6-triazolylmethyl derivative. Thus, the N6 region of 5′-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A3AR agonist affinity and enhanced selectivity.

Characterization by flow cytometry of fluorescent, selective agonist probes of the A3 adenosine receptor

“…Both MRS5218 and MRS5704 [19,20] contained a fluorophore linked through the C2 position of the adenine ring. Furthermore, N 6 -(3-chlorobenzyl) and rigid (N)-methanocarba ring modifications that enhanced A 3 AR selectivity were present.…”

“…IR dye 700 DX derivative 6 (C2-tethered) was prepared by amide formation, and Alexa Fluor 488 derivatives 7 and 8 , both of which are N 6 -tethered, were synthesized by the general methods of click coupling of azido (fluorophore) and terminal alkyne (pharmacophore) components used to prepare a series of 1,2,3-triazole derivatives as A 3 AR agonists [20]. The Cy5 N -hydroxysuccinimide ester (variation containing an N -ethyl group) for preparation of 1 was purchased from GE Healthcare Life Sciences (Piscataway, NJ) or from Selleckchem.com (Houston, TX).…”

“…The Cy5 N -hydroxysuccinimide ester (variation containing an N -ethyl group) for preparation of 1 was purchased from GE Healthcare Life Sciences (Piscataway, NJ) or from Selleckchem.com (Houston, TX). The probes were purified using HPLC as described [20] to demonstrate >95% purity. Characterization by NMR using a 400MHz Advance™ III HD-NanoBay (Bruker BioSpin Corp., Billerica, MA, USA) and by high resolution mass spectrometry (proteomics optimized Micromass Q-TOF-2 (Waters, Milford, MA) using external calibration with polyalanine) confirmed the assigned structure.…”

“…All contained a fluorophore linked through either the C2 or N 6 position of the adenine ring. Furthermore, the rigid (N)-methanocarba ([3.1.0-bicyclohexane]) ring modification that enhances A 3 AR selectivity is present in all of the derivatives [19,20]. …”

Characterization of polyamidoamino (PAMAM) dendrimers using in-line reversed phase LC electrospray ionization mass spectrometry

“…Therapeutic and experimental drugs 1–4 , dyes 5, 6 , solubilizing chains 7 or a mixture of all three 8 can subsequently be added to these functional groups creating powerful multi-functional biological agents. There exist a large number of scientific reports regarding the therapeutic potential of dendrimers and especially polyamidoamino (PAMAM) dendrimers 9–12 .…”