Total Synthesis and Biological Activity of the Arachidonic Acid Metabolite Hemiketal E₂

Abstract

The total synthesis of hemiketal E₂ (HKE₂) has been accomplished using a gold(I)-mediated cycloisomerization followed by oxidation of the enol ether product to introduce a unique keto-hemiketal, the core structure of HKE₂. Synthetic hemiketal E₂ reproduced biosynthetically derived HKE₂ in the inhibition of human platelet aggregation.

Graphical Abstract

Eicosanoids are produced by enzymatic oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1, COX-2), lipoxygenases (LOX), and cytochromes P450 in response to a variety of cellular stimuli such as hormones, stress, and cytokines.¹ These lipid mediators regulate a variety of biological responses and pathological processes such as inflammation, cancer, asthma, and autoimmune diseases.² Studies on eicosanoid biosynthesis, metabolism, and function have been extensive. The biosynthetic pathways leading to prostaglandins and leukotrienes are generally believed to diverge at the point of the initial enzymatic oxygenation of arachidonic acid by cyclooxygenases and lipoxygenases, respectively.³ However, in 2011, Schneider and co-workers described the biosynthesis of eicosanoids HKD₂ (3) and HKE₂ (4) as products of the consecutive oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2 (AA → 1 → 2 → 3/4, Figure 1).⁴

Figure 1.

Structures of 5S-HETE (1), diendoperoxide (2), hemiketal E₂ (3), and hemiketal D₂ (4).\.

The discovery of HKD₂ and HKE₂ resulted from the hypothesis that 5S-HETE (1), an oxidation product of 5-LOX and arachidonic acid, may serve as a substrate of the COX enzyme. In vitro experiments demonstrated 5S-HETE to be converted by COX-2 (but not COX-1), affording diendoperoxide 2 as the enzymatic product.⁵ The latter underwent rearrangement to HKD₂ and HKE₂ in a process reminiscent of the well-known transformation of intermediate endoperoxide PGH₂ to PGD₂ and PGE₂; thus, it is the designated nomenclature for these unusual hemiketals.⁴ The possible relevance of the crossover pathway to living systems was further demonstrated by their ability to induce tubulogenesis of endothelial cells⁴ and their biosynthesis following stimulation of 5-LOX and COX-2 in human leukocytes ex vivo using lipopolysaccharide (LPS).⁶ Further study of HKD₂ and HKE₂ requires their total synthesis, as currently hemiketals D₂/E₂ are produced enzymatically in small quantities starting from 5S-HETE and recombinant COX-2.⁷ Described herein is the first total synthesis of hemiketal E₂ (3).

Hemiketals D₂ and E₂ share common side chains that incorporate secondary alcohols of (S)-configuration of reversed attachment to the heterocyclic core (cf. Figure 2, Rα/Rω). A second difference between the two structures is the configuration of C9 (9S-HKD₂) and C11 (11R-HKE₂) as reflected in the cis and trans relationships between C8–C9 (HKD₂) and C11–C12 (HKE₂), respectively (3/4, Figure 1). Based on this structural analysis, we proceeded with a synthetic strategy whereby the absolute stereochemistry of 9S (HKD₂) and 11R (HKE₂) would be introduced in 2-alkoxy(siloxy)-aldehydes (cf. 5, Figure 2) and relative stereochemistry (C8–C9 and C11–C12) would be established in a vinyl metal addition to 5 directed by the adjacent stereocenter (OP) by way of either a Felkin–Anh (2-siloxyaldehyde) or chelation-controlled (2-alkoxyaldehyde) addition.⁸ The cyclic hemiketal would be introduced by a two-step process starting with a metal-mediated cycloisomerization followed by oxidation of the intermediate enol ether (Figure 2).⁹ We chose as our first objective the total synthesis of hemiketal E₂ (Scheme 1).

Figure 2.

Synthetic strategy to access hemiketals D₂ and E₂.

Scheme 1.

Total Synthesis of HKE₂

Our synthesis started with racemic glycidol 7, as derivatives of glycidol are readily resolved into single enantiomeric products using the Jacobsen hydrolytic–kinetic resolution.¹⁰ For example, PMB ether 8 was readily resolved into (R)-8 using the (S,S)-Co(salen) catalyst.¹¹ After epoxide opening with trimethylsilyl acetylide, the resulting secondary alcohol (9)¹² tected as a TBS ether in anticipation of a Felkin–Anh directed addition to aldehyde 13. Aldehyde 13 was obtained from 10 by way of the three-step deprotection-oxidation sequence as shown in Scheme 1. Next, treatment of known vinyl iodide 14,¹³ with t-butyllithium afforded an intermediate vinyllithium reagent that was reacted with dimethylzinc leading to a nonbasic vinyl zincate which was added to aldehyde 13 to give allylic alcohol 15 in 61% yield (>95:5 diastereoselectivity). Sonogashira coupling of acetylene 15 with vinyl iodide 16¹⁴ gave enyne 17 in 68% yield.

Either a 6-endo or 5-exo-dig mode of cyclization of alkynol 17 was projected to afford the desired hemiketal product following oxidation of either the pyran or furan product, respectively.⁹ After various conditions were examined, it was determined that gold(I) chloride in tetrahydrofuran proved most efficient to afford the 5-exo product, furan 18, in 75% yield.¹⁵ While furan 18 could be purified using buffered silica gel and characterized by NMR analysis, it had a finite lifetime in benzene-d₆ of less than 24 h. Typically, 18 was immediately epoxidized using dimethyl dioxirane following isolation. The epoxide product was not isolated, but instead, diol 19, a product of hydrolysis, was obtained and subsequently oxidized using IBX in DMSO to give hemiketal 20 in 62% yield from enol ether 18. Removal of TBS protecting groups was effected using HF-pyridine in acetonitrile to provide methyl ester 21. Saponification of ester 21 provided hemiketal E₂ (3), identical by ¹H NMR to HKE₂ derived enzymatically.⁷

Hemiketal E₂ inhibited human platelet aggregation induced by the thromboxane receptor agonist U46,619 in a dose-dependent manner (IC₅₀ < 500 nM). Inhibition was more potent in platelets isolated from individuals in whom PGE₂ at 100 nM concentration was unable to inhibit aggregation.¹⁶ Chemically synthesized and enzymatically prepared HKE₂ (1 μM) were equally effective at inhibiting platelet aggregation (Figure 3).

Figure 3.

HKE₂ inhibits platelet aggregation. Human platelet-rich plasma was preincubated with vehicle (v) or HKE₂ (1 μM) obtained from chemical synthesis (A) or enzymatic synthesis (B) for 10 min at 37 °C before stimulation of aggregation by the thromboxane receptor agonist U46,619.¹⁷

Unfortunately, efforts directed toward the total synthesis of hemiketal D₂ starting from 9S-5 (P = Bn) proved unproductive. Problems encountered in effecting the chelation-controlled carbonyl addition included low stereoselectivity and narrow scope of tolerated functionality in the vinyl metal reagent (5 to 6, Figure 2). Access to both hemiketals will provide opportunity for further biological study of these interesting and unusual eicosanoids.