Stereocontrolled Total Synthesis of Resolvin D4 and 17(R)-Resolvin D4

Abstract

The total synthesis of Resolvin D4 and its 17(R)-hydroxy-epimer is reported. These lipid-based natural products are biosynthesized from docosahexaenoic acid (DHA, C22:6) during the body’s rapid cellular and chemical response to injurious stimuli and are part of a large class of bioactive molecules that resolve inflammation. Our convergent synthesis employed a chiral pool strategy starting from glycidol derivatives and D-erythrose to introduce stereogenic centers. A copper(I)-mediated cross coupling between propargyl bromide and terminal acetylenic precursors yielded core structures of late-stage key intermediates. A simultaneous Lindlar reduction of the skipped diynyl moiety followed by silyl group cleavage securely completed the synthesis. The synthetic availability of these molecules helped further elucidate their stereoselective biofunctions.

Graphical Abstract

Introduction

Omega-3 fatty acids such as docosahexaenoic acid (DHA) have well-known benefits against a wide range of inflammatory conditions.¹ Experimental studies involving the enzymatic oxygenation of DHA and other polyunsaturated fatty acids during the physiological response to injuries and infections led to the identification of several lipid mediators that play key roles in inflammatory resolution.² These biologically active chemical signals include the DHA-derived (D-series) Resolvins, which are a subclass of signaling molecules also termed specialized pro-resolving mediators (SPMs).^2–4 The discovery and investigation of SPMs offered new insights into the mechanistic biological roles of essential fatty acids.

The D-series Resolvins were originally identified in resolving murine exudates, and this work resulted in the isolation of six distinct biologically active metabolites denoted Resolvin D1 (RvD1) through Resolvin D6 (RvD6).^2,3 In addition, generally referred to as the aspirin-triggered Resolvins are the mediators carrying a 17(R)-hydroxyl residue, owing to their biosynthetic routes involving acetylsalicylic acid³ and other enzymatic pathways such as CYPs. These autacoids are produced in miniscule in vivo quantities, requiring the use of synthetic versions in order to elucidate their physical and biological properties.

Herein we disclose the total synthesis of Resolvin D4 (RvD4) and its epimeric isomer 17(R)-Resolvin D4 (AT-RvD4). This work is an elaborate follow-up and expansion of our previous report on the synthesis of RvD4.⁵ Our chiral pool-based synthesis is complimentary to the reported strategies by us and collaborators^5,6 and by Kobayashi.⁷ The key features of Kobayashi’s approach encompass a Wittig olefination between the C₁-C₁₀ dienal and C₁₁-C₂₂ phosphonium salt to forge the docosahexaenoate carbon backbone, and Sharpless kinetic resolution to secure the stereocenters.

As part of our continuing interest and contribution in the field of lipid mediators with versatile anti-inflammatory and protective properties, we therefore deemed it essential to expand on our previous work on the synthesis of Resolvin D4 in order to offer a broader array of practical synthetic options of these biologically important, yet synthetically challenging natural substances.

Similar to other Resolvins, Resolvin D4 possesses anti-inflammatory and pro-resolution activities such as governing neutrophilic movement as well as clearing of apoptotic cells and microbial particles.³ Resolvin D4 boosts the ability of human macrophages and dermal fibroblasts to phagocytize apoptotic neutrophils at doses as low as 1 nM.⁵

A proposed biosynthetic pathway of Resolvin D4 is illustrated in Scheme 1.^2,3 The biosynthesis is initiated by the conversion of DHA to 17S-hydroperoxy-DHA (17S-HpDHA) catalyzed by 15-lipoxygenase (15-LOX). The hydroperoxyl intermediate can also be further reduced to the hydroxyl product leading to 17S-HDHA. Both 17S-HpDHA and 17S-HDHA undergo a second lipoxygenation via 5-LOX yielding a new hydroperoxide at the C-4 position. This peroxol is then transposed to a transient epoxide-containing intermediate, 4S,5S-epoxy-17S-HDHA,⁸ followed by enzymatic hydrolysis to produce Resolvin D4 (1A).

Scheme 1.

Biosynthetic pathway of Resolvin D4 (1A) and AT-Resolvin D4 (1B).

Aspirin-triggered 17(R)-Resolvin D4 has a related biosynthetic pathway (Scheme 1).^2,3 Notably, the oxygenation of DHA is initiated by acetylated cyclooxygenase-2 (COX-2), in the presence of aspirin, or via the P450 pathway, leading to the insertion of molecular oxygen with opposite stereochemistry at C-17 to form 17R-hydroperoxy-DHA (17R-HpDHA). This intermediate is further reduced to a hydroxyl residue establishing the 17R-HDHA intermediate. Subsequent enzymatic processes lead to AT-Resolvin D4 (1B).

Results and Discussion

The structures of Resolvin D4 (1A) and AT-Resolvin D4 (1B) were originally inferred using mass spectrometric analyses of resolving murine exudates.³ The detailed double bond geometries and alcohol group stereochemistries were speculated based on hypothesized biosyntheses. Stereoselective total syntheses of several lipid mediators,^9,10 including ours outlined in the present report in Scheme 2, allowed the structural confirmations as well as biological and pharmacological studies of these vital molecules.

Scheme 2.

Retrosynthetic analysis of Resolvin D4 (1A) and AT-Resolvin D4 (1B).

In accordance with our previous work, the current synthetic strategy established the diene and triene moieties separated by a methylene bridge in the final steps due to the inherent sensitivity of conjugated double bond systems. This required a mild reduction of the acetylenic moieties while preserving the methylene bridge, in a similar approach reported by Inoue on the synthesis of Resolvin E3 stereoisomers.¹¹ The presence of the alcohol residue at the C-4 position promoted the creation of a lactone with the carboxylic acid formed only three carbons away during the silyl cleavage with TBAF. Therefore this observation, also noted in our previous synthesis of a related Resolvin,¹² required reduction of the bis-acetylene to take place prior to desilylation and subsequent hydrolysis of the methyl ester. This was achieved by employing a mild Lindlar hydrogenation to forge the triene and diene units of the target compounds. The bis-acetylene (3) was in turn attained via a Cu(I)-mediated coupling of propargyl bromide intermediate 4 and terminal alkyne 5.

The synthesis of propargyl bromide intermediate 4 is detailed in Scheme 3. This synthesis employed D-erythrose (9) as a chiral feedstock. However, in our latest and separate study⁸ postdating the current synthesis, we found D-erythronolactone as a more suitable chiral synthon than 9 due to its high cost and handling difficulties. A Wittig reaction with commercially available methyl (triphenylphosphoranylidene)acetate furnished intermediate 10 in the precise 4(S), 5(R)-vicinal diol residues present in Resolvin D4 (1A) and AT-Resolvin D4 (1B). Complete reduction with palladium on carbon followed by silylation produced silyl protected alkyl intermediate 11 in 89% yield over two steps. Selective deprotection of the primary alcohol by treatment with camphorsulfonic acid (CSA) with a subsequent Dess-Martin oxidation achieved aldehyde 12 in 80% yield.

Scheme 3.

Synthesis of key intermediate 4.

A homologation step with commercially available Wittig-type reagent (triphenylphosphoranylidene)acetaldehyde constructed intermediate 13, and this extended aldehyde was further converted to the corresponding vinyl halide via Takai olefination,^12,13 affording alkenyl iodide 14 in moderate yields, with an insignificant amount of the Z-isomer. Iodide 14 was then submitted to a copper-free Sonogashira coupling with propargyl alcohol to produce dienyne 15. Using N-bromosuccinimide the alcohol was displaced, generating the target propargyl bromide 4 in good yields over two steps.

Key intermediates 5A and 5B are stereoisomers and were produced via similar synthetic steps from chiral glycidol derivatives 6A and 6B, respectively, as depicted in Scheme 4. The epoxides were opened by addition of lithiated 1-butyne in the presence of boron trifluoride etherate (BF₃•OEt₂) to produce secondary alcohol compounds 16 and 20 in excellent 94–95% yields. The free alcohol residues were then silylated followed by selective mono-deprotection under mild acidic conditions to yield intermediates 17 and 21 in the high 80% yields over 2 steps. Lindlar hydrogenation of the alkynes, followed by Dess-Martin oxidation afforded α-siloxy aldehydes 18 and 22 in the low to mid 80% yields over 2 steps. A homologation with commercially available and stabilized Wittig reagent (triphenylphosphoranylidene)acetaldehyde furnished elongated enals 19 and 23 in moderate 70% yields. These compounds were then homologated to their corresponding terminal alkynes using a Corey-Fuchs reaction¹⁴ to give 5 in good overall yields. In the case of 5B, which was prepared prior to its enantiomer (5A), we proceeded by switching the silyl ether to a less bulky group, envisioning a smooth desilylation at the final step to achieve AT-Resolvin D4 (1B). Interestingly, moving forward with the bulky TBDPS ether (5A) to generate Resolvin D4 (1A) also emerged as favorable.

Scheme 4.

Synthesis of key intermediates 5A and 5B.

Next, the assembly of target molecules AT-Resolvin D4 (1B) and Resolvin D4 (1A) was respectively accomplished in a similar fashion by the union between propargyl bromide 4 and terminal alkyne 5 as outlined in Schemes 5–6. A copper-mediated cross coupling of the two late-stage precursors led to the silyl-protected bis-acetylenic intermediates 3A and 3B in the mid to high 80% yields. Selective reduction of the C-10 and C-13 position triple bonds to cis-alkenes required a mild Lindlar-catalyzed hydrogenation in a mixed solvent system of ethyl acetate/pyridine/1-octene¹⁵ to create silyl ethers (2B) and (2A) in 72% and quantitative yields, respectively. The addition of pyridine controls the activity of the heterogeneous catalyst while the α-olefin plays a sacrificial role to minimize competing over-hydrogenation of the desired product.

Scheme 5.

Convergent synthesis of AT-Resolvin D4 (1B).

Scheme 6.

Convergent synthesis of Resolvin D4 (1A).

Desilylation using TBAF followed by treatment with freshly prepared diazomethane produced a mixture of the methyl ester 24 and lactone 25.¹² Without chromatographic separation, the mixture was immediately subjected to alkaline hydrolysis of the products with lithium hydroxide followed by HPLC purification to afford 1B and 1A, respectively, in comparable mid to high 50% yields over 2–3 steps. Characterization by LC-MS/MS, UV, and NMR confirmed the structures of the target compounds, and these data were in agreement with the published reports.^5–7

In summary, the total synthesis of both Resolvin D4 (1A) and its epimeric isomer 17(R)-Resolvin D4 (1B) is described in 11% yield over 14 steps of the longest linear sequence, expanding the toolbox of practical synthetic strategies and leading to access of these natural products that orchestrate critical physiological responses to injuries and infections. Our convergent synthesis attains the target molecules via the use of nonracemic starting materials and stereocontrolled transforms.