Insertion of an Amphipathic Linker in a Tetrapodal Tryptophan Derivative Leads to a Novel and Highly Potent Entry Inhibitor of Enterovirus A71 Clinical Isolates

Figures

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  Figure 1. Structure of dendrimer AL-385 and reduced-sized prototypes AL-470 and AL-471.

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  Figure 2. Structural modifications explored in this work. A. d-Trp instead of l-Trp. B. Alkyl urea linkers of different length between the isophthalic acid moiety and the C2 position of Trp. C. Trp-less analogues.

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  Scheme 1. Synthesis of the d-Trp counterpart of AL-471. Reagents and conditions: (a) dimethyl 5-iodoisophthalate, Pd(OAc)2, AgBF4, TFA, anh. DMF, microwave, 120 °C, 30 min; (b) HCO2NH4, Pd/C (10%), DMF, rt, 3 h; (c) HATU, DIPEA, anh. DMF, 30 °C, 40 h; (d) LiOH·H2O, THF/H2O, rt, overnight, then 1 M HCl.

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  Scheme 2. Synthesis of C2-alkylated tetrapodal derivatives 23–25. Reagents and conditions: (a) PdCl2, norbornene, K2CO3, anh. DMF, water (0.2 M), 60 °C, 24 h; (b) H2NNH2·H2O, EtOH, 30 °C, 5 h; (c) dimethyl 5-isocyanatoisophthalate, DIPEA, anh. DMF, 30 °C, 4 h; (d) TFA, CH2Cl2, rt, 6 h; (e) HATU, DIPEA, anh. DMF, 30 °C, 40 h; (f) LiOH·H2O, THF/H2O, rt, overnight, then 1 M HCl.

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  Scheme 3. Synthesis of C2-alkylated tetrapodal derivatives 32 and 33. Reagents and conditions: (a) PdCl2, norbornene, K2CO3, anh. DMF, water (0.2 M), 60 °C, 24 h; (b) TFA, CH2Cl2, rt, 6 h; (c) HATU, DIPEA, anh. DMF, 30 °C, 40 h; (d) LiOH·H2O, THF/H2O, rt, overnight, then 1 M HCl.

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  Scheme 4. Synthesis of the truncated analogue 35. Reagents and conditions: (a) dimethyl 5-aminobenzene-1,3-dicarboxylate, HATU, DIPEA, anh. DMF, 30 °C, 40 h; (b) LiOH·H2O, THF/H2O, rt, overnight, then 1 M HCl.

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  Scheme 5. Synthesis of the tetrapodal Trp-less derivative 40. Reagents and conditions: (a) dimethyl 5-isocyanatoisophthalate, DIPEA, anh. DMF, 30 °C, 4 h; (b) HCO2NH4, Pd/C (10%), DMF, rt, 3 h; (c) HATU, DIPEA, anh. DMF, 30 °C, 40 h; (d) LiOH·H2O, THF/H2O, rt, overnight, then 1 M HCl.

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  Figure 3. Proposed high-affinity binding site in EV-A71 of the l-Trp unit, alkylurea linker, and isophthalic acid moiety of 23 (AL-534). (A) Simplified spacefill model of the EV-A71 capsid (PDB entry 3VBS) showing VP1, VP2, and VP3 subunits colored in green, cyan, and pink, respectively (VP4 is hidden from view). (B) Cartoon representation of VP1 (C atoms in green), as found in the fivefold vertex of the EV-A71 capsid, and consensus binding location of five separate legs of 23 (AL-534) (all shown as CPK models with C atoms colored in violet), each one at a subunit interface. For reference, the side chains of amino acids at positions 98 (B–C loop) and 145 (D–E loop) lining the pore are displayed as sticks; the positively charged Lys242 and Lys244 are also displayed as sticks with C atoms colored in orange; Pro96, Pro142, and Pro246 are displayed as spheres, with the C atoms of Pro246 in the H–I loop colored in yellow. (C) Zoomed view showing one binding site in greater detail and the side chains of Glu92, Asp94, Tyr116, Arg236, Ser240, and Arg250, all of which are in close proximity to the ligands, shown as sticks and labeled.

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  Figure 4. Molecular model of one EV-A71 VP1 pentamer in complex with a single 23 (AL-534) molecule. Top and side views of ten overlaid ensembles are displayed that were generated from the molecular dynamics simulation lasting 300 ns and subsequent simulated annealing and energy minimization. The protein is shown as a green ribbon enveloped by a semitransparent gray surface to highlight the crevices. For reference, the side chains C atoms of the 241Ser-Pro246 stretch in the H–I loop are colored in yellow, except for Lys242 and Lys244, which are colored in orange. The entry inhibitor is displayed as sticks with C atoms colored in violet. Note how one of the legs of 23 (AL-534) binds permanently at the interface between two VP1 subunits—as shown in Figure 3—while the others extend away from the protruding central pentaerythritol-based scaffold and make additional crucial interactions with residues lining the pore and neighboring subunits. Significantly, in the equilibrated complexes, one of the isophthalic acid rings from another leg stacks on Pro246 (yellow spheres) and its carboxylic groups form ionic bridges with the invariable Lys244 (top view, circled area on the left), whereas the Trp…