Stereospecific Synthesis and Biological Evaluation of KRN7000 Analogues with Thio-modifications at the Acyl Moiety

Abstract

α-Galactosylceramide (KRN7000 or α-GalCer) analogues terminated with phenyl (Ph) groups at the acyl moiety possess more potency than KRN7000 to activate invariant natural killer T (iNKT) cells for inducing a T helper 1 (Th1)-biased immune response. However, biological activities of phenyl glycolipids with thio-modifications at the acyl moiety remain unknown, and facile approaches for highly stereoselective synthesis of KRN7000 and its analogues are rather scarce. Herein, we exploited 4,6-di-O-tert-butylsilylene (DTBS)-directed stereospecific galactosylation to efficiently synthesize various α-GalCer analogues bearing thioamide, terminal thiophenyl and dual modifications at the acyl moiety. Biological evaluations suggest that a new analogue S34 featuring a terminal Ph-S-Ph-F group exhibits a more superior Th1-biased immune response in mice. Molecular docking analysis revealed that the introduction of a sulfur atom influences vital hydrogen bonding interactions between glycolipids and the cluster of differentiation 1d (CDld), thus adjusting the stability of the glycolipid-CDld complex.

Figure 1

Structures of α-GalCer 1 and its analogues 2–8.

Scheme 1. Synthesis of Compounds 1–4

Reagents and conditions: (a) NIS, TfOH, 5 Å MS, CH₂Cl₂, 0 °C → room temperature, 1 h, 64%; (b) i. NiCl₂·6H₂O, NaBH₄, CH₂Cl₂/MeOH, 0 °C, 0.5 h; (c). fatty acids, EDCI, HOBt, DIPEA, THF, room temperature, overnight, two steps 93%–89%; (d) HF-Pyridine, THF, room temperature, 3–4 h; (e) NaOMe, CH₂Cl₂/MeOH, room temperature, overnight, two steps 78%–86%.

Scheme 2. Synthesis of Thioamide-Modified Analogues 5–8

Reagents and conditions: (a) Lawesson’s reagent, toluene, 80 °C → reflux; (b) Ac₂O, DMAP, Pyridine, room temperature, overnight, 62%–90%; (c) Lawesson’s reagent, toluene, 80 °C, 4–5 h, 85–90%; (d) NaOMe, CH₂Cl₂/MeOH, room temperature, 1–2 h, 66%–77%.

Figure 2

Biological evaluations of glycolipids 1–8. (A,B) IL-4 and IFN-γ cytokine secretions induced by glycolipids 1–8 in C57BL/6 mice. Data values (mean ± SEM, n = 3 mice/group) were graphed. (C) Ratio of IFN-γ to IL-4 from compounds 2–8 were compared to that from KRN7000 (1). C57BL/6 mice were injected with indicated glycolipids (200 μg/kg) or vehicle (0.1% DMSO+0.05% Tween-20+PBS). N.D. is below the minimum detection limit of the ELISA kit.

Figure 3

Docking models of compounds to the CD1d protein (PDB code: 3HUJ). (A) The ligand binding site is located at the interface between NKT-TCR and CD1d, formed by CD1d α1 helix and α2 helix. Compounds are shown in sticks. Proteins are shown in a cartoon depiction. (B–E) Binding modes of C34 (B), S-C34 (C), S34 (D), and S-S34 (E) to CD1d in docking models. Compounds and key residues are shown in sticks. Proteins are shown in cartoon depiction.

Figure 4

Comparison of binding modes of different compounds. (A) Binding model of C34. (B) Binding model of S-C34. (C) Binding model of S34. (D) Binding model of S-S34. Proteins are shown in cartoon depiction. Compounds and key residues are shown in sticks. Putative hydrogen bonds are shown as dash lines. Important interactions are indicated with arrows.