Synthesis of C-glycoside Analogues of α-GalactosylCeramide via Linear Allylic C-H Oxidation and Allyl Cyanate to Isocyanate Rearrangement

Abstract

C-Glycoside analogues of α-galactosylceramide were synthesized in which several significant modifications known to promote Th-1 cytokine production were included. The key transformations include C-H oxidation, Sharpless asymmetric epoxidation, olefin cross metathesis, and an allyl cyanate to isocyanate rearrangement.

α-Galactosylceramide (1, also known as α-GalCer or KRN7000), an optimized synthetic material originating from a marine sponge, is the most widely studied glycolipid antigen for activating invariant natural killer T (iNKT) cells.¹ These cells are a subset of T lymphocytes that interact with glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d.² Immunoregulatory cytokines, such as IFN-γ (Th-1 type) and IL-4 (Th-2 type), produced by stimulated iNKT cells hold substantial promise in immunotherapy and for development of vaccine adjuvants.³ However, phase I clinical trials of 1 in the treatment of solid tumors have been ineffective, perhaps as a consequence of counteraction of the Th-1 and Th-2 cytokines induced by 1.⁴

A variety of glycolipid antigens that can differentially elicit distinct effector functions in iNKT cells have been identified. For example, installing a OMe group at the 6’-position of the galactosyl moiety gave rise to the strong Th-1 biasing ligand RCAI-61 (2).⁵ Introduction of a p-fluorophenyl group at the terminus of the fatty amide chain led to 7DW8-5 (3), which induced selective production of IFN-γ.⁶

α-C-GalCer (4), a C-glycoside analogue of KRN7000,⁷ binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo.⁸ In fact, comparisons of 1 and 4 in mouse models of disease revealed that the C-glycoside 4 displayed higher activity.^7b Interestingly, 4 was found to be a weak agonist of human iNKT cells in vitro, but C-glycoside analogues that feature an E-alkene as a spacer between the galactose moiety and the ceramide, such as GCK152 (5), activate human iNKT cells and induce the maturation and activation of human dendritic cells through iNKT-cell activation.⁹

As part of our ongoing investigations of C-glycoside analogues of KRN7000,¹⁰ we have designed analogues 6a,b, which combine several significant structural modifications for selective Th-1 cytokine production. In addition to the installation of the 6’-OMe and p-fluorophenyl substitutions stated above, the report that 4-deoxy-KRN7000 initiates cytokine production similarly to that induced by 1 in human iNKT cells in vitro, and in its murine counterpart in vivo,¹¹ prompted the synthesis of the corresponding 4-deoxyphytosphingosine moiety in C-glycoside 6. Our retrosynthetic plan is illustrated in Scheme 1. It was reported that cross-metathesis¹² (CM) of vinyl-C-galactosides requires high loading of catalysts, and that the yields of coupling product are low and sensitive to the protecting groups on the vinyl-C-galactoside.^13,14 Therefore, a CM disconnection of 8 was deemed more practical than for 7, leading to the simple synthons 9 and 13. The required amide at C-3 could potentially be delivered with stereocontrol from C-1 via an allyl cyanate to isocyanate rearrangement¹⁵ (8 to 7). The concerted nature of the sigmatropic rearrangement would secure a high level of [1,3]-chirality transfer during the C-O to C-N bond reorganization. The stereocontrolled construction of the hydroxyl group at the allylic position in 9 could be fulfilled via zinc-mediated reductive elimination of the asymmetric epoxide halide 10, which might be accessible from terminal alkene 11 via linear C-H oxidation¹⁶ followed by Sharpless asymmetric epoxidation (SAE).¹⁷ α-Allyl galactoside 11 could possibly be prepared from β-galactoside 12 through straightforward transformations according to known protocols.¹⁸ Similarly, allylic ester 13, the lipid olefin of CM, could be obtained through a sequence of SAE and reductive elimination from known epoxy alcohol 14,^19,20 which is accessible from palmitaldehyde 15.

Scheme 1.

Retrosynthetic Plan For 6a

As shown in Scheme 2, our synthesis commenced with the preparation of α-allyl-C-galactoside 11 from commercially available penta-O-acetyl-β-D-galactose (12) and involved straightforward protecting group manipulations using 16 and 17.¹⁸ Linear C-H oxidation of 11 under conditions developed by Chen and White^16a [Pd(OAc)₂, benzoquinone (BQ), DMSO/AcOH (1:1), 50 °C, 100 h] afforded 18 in low yield (17%) and incomplete conversion (71%). However, treatment of 11 under conditions^16b revised by White et al., followed by acetate deprotection under basic conditions, provided allylic alcohol 19 in 40% yield (two steps) with high E-selectivity (E/Z ratio > 20:1). SAE of 19 smoothly afforded epoxy alcohol 20 in 81% yield. Iodination of 20 afforded 10, which upon zinc-mediated reductive elimination gave rise to 9 (71% yield, two steps). The R configuration of C-1 in 9 was established by the advanced Mosher method (see Supporting Information),²¹ and the dr value was determined on the basis of the ¹H NMR spectra of the corresponding Mosher esters.

Scheme 2.

Synthesis of 9

The synthesis of lipid olefin 21 was accomplished in a similar way, using SAE to introduce the chirality (Scheme 3). Unlike the strategy involving zinc-mediated reductive elimination of an epoxide halide, the transformation of 2,3-epoxy alcohol 14 to allylic alcohol 21 was achieved in one step by using the titanocene-induced regioselective deoxygenation protocol developed by Yadav and coworkers.²² CM with 15 mol % Grubbs 2^nd generation catalyst (G-2) using 3.2 molar equiv of lipid olefin 13 with 9 provided 8 in 70% yield with high E-selectivity (E/Z ratio > 20:1). This reaction required reflux for only 2 h, and 48% of 13 was recovered, along with a small amount of the easily separable homodimers of 13 (11%, based on 13).

Scheme 3.

Synthesis of 6a

Treatment of 8 with trichloroacetyl isocyanate¹⁵ afforded intermediate 22, and hydrolysis with potassium carbonate in aqueous methanol gave carbamate 24, along with 5% of 23. Dehydration of 24 with trifluoroacetic anhydride (TFAA) and triethylamine at 0 °C gave allyl cyanate 25, which immediately underwent the allyl cyanate to isocyanate rearrangement¹⁵ to afford allyl isocyanate 26. It is noteworthy that this rearrangement can occur below room temperature, and thus is milder than the related Overman rearrangement.²³ Isocyanate 26 was further reacted with methanol in the presence of a catalytic amount of tributyltin methoxide²⁴ in situ, providing carbamate 7. The E configuration of the alkene was confirmed by the coupling constants of the vinylic protons (16.0 Hz). Basic hydrolysis of 7 afforded cyclic carbamate 27, which was further treated with 30% aqueous KOH solution at reflux in ethanol to afford amine 28. The absolute configuration at the C-3 position was confirmed by the advanced Mosher method,²¹ which revealed the S configuration at C-3 in 28. The fatty amide chain was then introduced into amine 28 by using cerotyl chloride²⁵ to afford amide 29 in 97% yield over two steps. Finally, global debenzylation using Birch reduction furnished the final analogue 6a in 91% yield.

As shown in Scheme 4, the corresponding carboxylic acid 33 of amide moiety in 3⁶ was prepared in 59% yield (two steps) from commercially available iodide 30 and alkyne 31 via Sonogashira coupling followed by catalytic hydrogenation of alkyne 32. For in situ N-acylation, 33 was converted to acyl chloride 34. In order to avoid defluorination during Birch reduction, debenzylation must be carried out prior to N-acylation. As a result, target 6b was obtained in 29% yield over three steps from 27.

Scheme 4.

Synthesis of 6b

In conclusion, we have developed a highly stereocontrolled total synthesis of C-glycoside analogues of KRN7000 containing an E-alkene linker in 20 steps starting from penta-O-acetyl-β-D-galactose (12) in 2.5% (6a) and 0.8% (6b) overall yield. The synthesis showcases the utility of a linear allylic C-H oxidation in synthetic carbohydrate chemistry and an allyl cyanate to isocyanate rearrangement for stereoselective construction of the stereogenic center in the presence of a sugar moiety. These novel α-GalCer analogues are currently undergoing biological evaluation.

Figure 1.

Structures of glycolipids 1-6