The Crotylation Way to Glycosphingolipids: Synthesis of Analogues of KRN7000

Abstract

A synthesis of glycosphingolipids that centers on the reaction of O- and C- glycosyl crotylstannanes and relatively simple lipid aldehydes is described. The modularity of this strategy and versatility of the crotylation products make this an attractive approach to diverse, highly substituted libraries. The methodology is applied to analogues of the potent imunostimulatory glycolipid KRN7000, including O-, methylene- and fluoromethine- linked isosteres with diastereomeric ceramide segments and 2-amido substitutes.

The presentation of glycolipids typified by KRN7000 1 by the CD1 family of antigen presenting molecules to invariant natural killer T (iNKT) cells stimulates the production of cytokines that lead to differentiation of T helper cells into Th1 or Th2 cells (Scheme 1).¹ This polarization results in an inflammatory or immunomodulatory response. Understanding the mechanism of Th1/Th2 balance is a keynote issue that has ramifications on new therapies for a variety of diseases, including cancer and certain autoimmune disorders.² Thus, glycolipids with Th1 or Th2 bias are in demand both for elucidating the molecular basis for cytokine bias and as therapeutic leads. In this context the synthesis and cytokine profiling of novel diverse analogues of KRN7000 has attracted much attention.³ The polar part of the sphingosine residue has come under close scrutiny as subtle alterations in this segment have resulted in very different Th1/Th2 profiles. This behavior may be linked to the intimate interactions of this region within the glycolipid-CD1d-TCR ternary complex.⁴ Among the more well-known analogues of this type are O-and C-glycosides with C2-C3 modifications, including different degrees of hydroxylation, stereochemistry, amide replacements and fluorine substituents.^5-15 Against this backdrop, towards a more rigorous SAR examination of the polar sphingosine segment, we envisaged a synthesis of KRN7000 analogues of the type 2 that centered on the reaction of carbohydrate-derived crotylstannanes 4 with relatively simple aldehydes 5 and elaboration of the crotylation products 3 (Scheme 1).

Scheme 1. The crotylation way to glycosphingolipids.

While crotylations with sugar derived crotylstannanes and aldehydes have been documented the potential of this reaction for glycomimetic synthesis has not been fully tapped.^16,17 Unlike more conventional approaches to glycomimetics, this strategy does not focus on the creation of the glycoside (or pseudoglycoside) bond, but on other linkages, thereby avoiding complications with challenging glycoside isosteres, while opening entry to new glycomimetic space.

For the preliminary test of this methodology, tin-based reagents were chosen because of their ease of preparation and greater stability compared to other classes of crotylating agents (Scheme 2).^18,1,9 Thus 7, 10 and 13 were prepared following the studies by Jarosz, from allylic alcohol precursors 6, 9 and 12 respectively.¹⁷ For 7,6²⁰ was first converted to the xanthate derivative. Thermal rearrangement of the latter and in situ treatment of the resulting secondary dithiocarbonate with Bu₃SnH in the presence of AIBN, gave crotyltin 7 with an E:Z ratio of 3:2, in 57% overall yield from 6. For 10 and 13, allylic alcohols 9 and 12 were prepared from known alkenes 8²¹ and 11²² respectively via standard chain extension-Wittig olefination reaction sequences (Supporting Information). Application of the stannylation protocol to 9 and 12 yielded 10 and 13 as 3:2 E:Z mixtures. The crotyltins were purified using standard extraction and chromatography procedures and could be stored in the refrigerator for several months without decomposition. The E:Z isomers were not easily separable and used as mixtures in the subsequent crotylation reactions.

Scheme 2. Synthesis of crotyltins.

Crotylation reactions were first performed using BF₃.OEt₂ as catalyst (Table 1). Addition of E:Z mixtures of 7, 10 and 13 to a pre-incubated mixture of aldehyde 14¹⁰ and BF₃.OEt₂ in dichloromethane at -78 °C afforded diastereomeric mixtures 15a-c, 16a-c and 17a-c in 86, 70 and 70% yields. These reactions were generally completed within two hours.

Table 1. Reactions of C-glycoside crotyltins and aldehydes.

crotyltin	aldehyde	crotylation productsa
E/Z-7
E/Z-10	14
E/Z-13	14

^aUnless otherwise stated reactions were promoted with BF₃.OEt₂; isolated yields of separated products are indicated in parentheses

^bMgBr₂.OEt₂ promoted reaction.

^c15b/d, 16b/c, 17b/c were isolated as mixtures; diastereomer ratios were determined by NMR

^dStereochemistry of products was not assigned.

Where determined the stereochemistry of crotylation products was assigned in cyclic derivatives and by chemical correlation (vide infra). Unreacted aldehyde and derivatives of the crotylation products showed no indication of epimerization at the α-carbon in the aldehyde during the crotylation reaction. The stereochemical trends of these complex sugar derived crotyltins are similar to the reactions of simple achiral crotyltins.^18,19,23,24 Thus, the preference for 3,4-syn over 3,4-anti diastereomers (ca 7:1 for 7 and 5:1 for 13), is in line with the observation that both E and Z reagents generally show good to high preference for 3,4-syn adducts. This behavior has been reasoned with open transition state models.^18,19,23,24 A preliminary study on the effect Lewis acid on stereochemistry was also performed. Whereas, the BF₃.OEt₂ promoted reaction of 7 and 14 led primarily to the 3,4-syn products 15a and 15b, gave exclusively the Cram (i.e. 4,5-syn) products 15b and 15d, which is expected for such chelating Lewis acids. This erosion in 3,4-syn selectivity in changing from the non-coordinationg to chelating promoter is also precedented.²⁴ Together the data with BF₃.OEt₂ and MgBr₂.OEt₂ illustrate that Lewis acids could be used to control stereoselectivity in these complex crotylations. Stereochemical control through the use of chiral acid promoters and, or other metalating entities is also envisaged.^18,19,25

The stereochemistry of crotylation products 15a-d and 17a-c were determined by ¹H NMR analysis of their derived lactones and chemical correlation. This was achieved via oxidation-reduction sequences on the original crotylation products (Scheme 3, Supporting Information). Thus, transformation of 15a to its benzyl ether and hydroboration-oxidation of the alkene afforded the primary alcohol derivative. Removal of the PMB ether and oxidation of the resulting diol afforded lactone 18a. Similar transformations on 15b, 17a and 17c led to lactones 18b, 19a and 19c respectively. The stereochemistry of these lactones was deduced from analysis of J_H,H values and nOes. The stereochemistry of 15a was confirmed by transformation to C-KRN7000 (vide infra).

Scheme 3. Stereochemical assignment of crotylation products.

The synthesis of C-KRN7000 22, the 2,3-bisepimer of C-KRN7000 23, the fluorinated C-glycoside 24, and the C-KRN7000 analog with an inverse amide 25, provide a glimpse of the diverse sphingolipid analogues that can be prepared via this crotylation methodology (Scheme 4). C-KRN7000 22 is a validated immunostimulant with superior Th1 bias in mice compared to KRN7000.^6,10 The 2,3-bisepimer of C-KRN7000 23 is of interest because another diastereomer, 4-epi-C-KRN7000, exhibits high Th1 cytokine bias and increased potency compared to C-KRN7000.¹² Likewise, other diastereomers of O- and C- KRN7000 have interesting cytokine profiles.^5,9 Fatty acid derivatives of 24 are intriguing because the location and conformational and electronic properties of the fluorine substituent may induce unusual binding in the glycolipid-CD1d-TCR ternary complex, which in turn may elicit unique cytokine activity.^4,15 Similarly the reverse amide 25 is a probe for the binding of the key amide residue.⁴

Scheme 4. Synthesis of C-glycosphingolipids.

For C-KRN7000 22, 15a was processed to carboxylic acid 20 via routine alcohol protecting group and alkene transformations. A Curtius rearrangement protocol using diphenylphosphorylazide (DPPA) on 20 provided carbamate 21.²⁶ Standard conditions for removal of benzyl protecting groups and amine acylation provided 22, which was essentially identical by TLC, NMR and HRMS to an authentic sample.^6,10,11

Using similar reaction sequences 15b and 16a were transformed to 23 and 24 respectively (Supporting Information). This Curtius rearrangement strategy is practical as in situ reaction of the intermediate isocyanate with lipid alcohols, amines and thiols can provide direct entry to carbamates, thiocarbamates and ureas of 22 respectively, which are of interest given the cytokine activity of their O-linked counterparts.¹⁴ Amidation of acid 20 and pentacos-8-en-1-amine, followed by removal of protecting groups, provided the reverse amide 25. Straightforward transformations on alkene 15a to a variety of other novel amide substitutes can be envisaged.

In conclusion, the synthesis of the KRN7000 analogues described herein illustrates the potential of aldehyde crotylation as strategy for accessing diverse glycosphingolipids. The modular plan, rapid build-up of complexity from easily accessible precursors, and versatility of the crotylation products makes this approach appropriate for libraries with different sugar head groups, pseudoanomeric linkages and ceramide segments. Biological evaluation of these new KRN7000 analogues, further investigations on the stereochemistry of these complex crotylation reactions, and applications of this methodology to other classes of glycomimetics are underway.

Supplementary Material

Supplemental Materials

Experimental procedures and analytical data for new compounds (PDF). NMR spectra for selected compounds (PDF)