Synthesis of tetranor-PGE₁: a urinary metabolite of prostaglandins E₁ and E₂

Abstract

Prostaglandin E₂ is produced in response to inflammation, often associated with human disease. As prostaglandins are rapidly metabolized, quantification of end urinary metabolites depend on chemical synthesis of isotopically labeled standards to support metabolite quantification. A concise synthesis of tetranor-PGE₁ is described including a late stage incorporation of an isotopically labeled side-chain.

Prostaglandins are key lipid mediators produced as an inflammatory response to potentially harmful stimuli and serve as a hallmark of many human diseases including cancer [1]. Due to their rapid metabolism, detection of prostaglandins, such as PGE₂, is not useful as a diagnostic tool but instead detection and quantification of downstream metabolites is useful for prostaglandin analysis (Figure 1) [2]. Metabolic degradation and inactivation of PGE₂ commonly starts with enzymatic oxidation by 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) followed by double bond reduction by 15-keto-prostaglandin Δ₁₃-reductase to afford 13,14-dihydro-15-keto PGE₂ (Figure 1) [3]. While the keto metabolite is detectable in plasma, it has proven unreliable as a biomarker of PGE2 levels as it has a short half-life (t_1/2 ~8 min) and subject to further metabolism leading to tetranor-PGEM, a reliable marker of endogenous PGE₂ production [4]. PGEM is increased in patients with cystic fibrosis [5], correlates with plasma virus load in HIV [6], and is increased in many types of cancer [7]. A recent meta-analysis concluded that PGEM is the most promising urinary biomarker for colorectal cancer risk assessment and screening [7a]. Thus, accurate assessment of endogenous PGE₂ production is essential.

Figure 1.

Two metabolic pathways of prostaglandins E₂ leading to tetranor- PGE₁ and tetranor-PGEM.

Oates and Samuelsson later identified tetranor-PGE₁, a second, albeit minor, urinary metabolite [2b]. In this case the aforementioned oxidation-reduction of the ω-side-chain is by-passed and the α-side-chain cleaved by enzymatic β-oxidation (Figure 1). In support of a proposed clinical study we required a chemical synthesis of tetranor-PGE1 and a deuterium labeled derivative for its quantification in human urine.

The chemical synthesis of prostaglandins and analogues has been extensively studied since the 1960’s [8]. Among the synthetic strategies developed, the two-component (conjugate addition) coupling process suited our needs as the starting materials are easily available and introduction of an isotopically labeled side-chain would be conveniently introduced late in the synthesis (Figure 2). Key to our approach was α- functionalization of iodoenone 1 by a cross-coupling reaction, a strategy popularized by Johnson and co-workers [9].

Figure 2.

Two-component coupling synthetic strategy.

We started our synthetic investigations by examining the cross-coupling of iodoenone 1 with metal homoenolates (Scheme 1). Iodoenone 1 was conveniently prepared in high optical purity following established procedures starting from cyclopentadiene [9,10]. As B-Alkyl Suzuki cross-coupling of iodoenone 1 has been employed en route to prostaglandins and analogues we first examined potassium trifluoroborate 2a as a coupling partner [11]. Unfortunately under reaction conditions used previously to couple with aryl halides we only observed starting material and decomposition. Similar results were obtained when employing zinc homoenolate 2b in a Negishi coupling reaction. Fortunately Heck coupling with acrolein diethyl acetal 2c with iodoenone 1 under Jeffries conditions, previously reported using aryl halide coupling partners [12], afforded ester 3 in 28% yield. Although the yield of this reaction was low, it was reproducible and allowed us to proceed toward the synthesis of tetranor PGE₁.

Scheme 1.

Cross-coupling partners examined and successful Heck coupling with iodoenone 1.

As anticipated, addition of the mixed higher-order cuprate reagent derived from 3S-vinyl iodide 4 to enone 3 afforded cyclopentanone 5 with expected trans, trans diastereoselectivity [9,13]. Vinyl iodide 4 (R = n-C₅H₁₁) was prepared following well-established synthetic methods employing an asymmetric Noyori reduction of an intermediate alkynone derived from alkynylation of hexanoyl chloride [14]. Deuterium labeled d₁₁-4 was prepared from d11-hexanoyl chloride derived from commercial d11-hexanoic acid. Saponification of ester 5 proved problematic due to facile beta-elimination of the TBS ether within the cyclopentanone core. The elimination problem was circumvented using a three-step procedure (5 to 6) reported by Taber as he encountered the same problem in his synthesis of the prostaglandin metabolite PGE₂U_M [15]. Treatment of carboxylic acid 6 with aqueous HF in acetonitrile afforded tetranor-PGE₁.

Upon concentration tetranor-PGE1 tended to undergo decomposition and was therefore kept as a solution in ethyl acetate. The utility d₁₁-tetranor-PGE₁ of the latter in quantifying tetranor-PGE₁ in clinical urine samples will be reported in due course.

Scheme 2.

Synthesis of tetranor-PGE₁ and d₁₁-tetranor-PGE₁.

Highlights.

• Chemical synthesis of prostaglandin E2 metabolite “tetranor PGE1”.

• Heck coupling with acrolein diethyl acetal leads to an ester.

• Cuprate conjugate addition to a 2-substited-5-alkoxycylopentenone.

• Deuterium labeling affords a standard for a prostaglandin urinary metabolite.