Design and synthesis of novel prostaglandin E₂ ethanolamide and glycerol ester probes for the putative prostamide receptor(s)

Abstract

Novel prostaglandin-ethanolamide (PGE₂-EA) and glycerol ester (2-PGE₂-G) analogs were designed and synthesized to aid in the characterization of a putative prostamide receptor. Our design incorporates the electrophilic isothiocyanato and the photoactivatable azido groups at the terminal tail position of the prototype. Stereoselective Wittig and Horner–Wadsworth–Emmons reactions install the head and the tail moieties of the PGE₂ skeleton. The synthesis is completed using Mitsunobu azidation and peptide coupling as the key steps. A chemoenzymatic synthesis for the 2-PGE₂-G is described for first time.

Introduction

Prostamides are a class of endogenous eicosanoids resulting from the cyclooxygenase-2 (COX-2) metabolism of the endocannabinoid anandamide (AEA).^1,2 The biosynthesis of prostamides is similar to that of the prostaglandins and involves stimulus-induced phospholipase release of anandamide, oxygenation by COX-2 to a hydroxyl-endoperoxide, and finally enzymatic conversion by one of the prostaglandin synthases.¹ While basal levels of the precursor AEA are not sufficient to produce significant concentrations of prostamides, it is likely that physiological conditions exist, as in inflammatory or infectious conditions, where anandamide release coincides with COX-2 induction.^2,3 The involvement of prostamides in inflammatory settings suggests that they constitute a novel class of lipid mediators, modulation of which could lead to new anti-inflammatory approaches.^4,5

Since their discovery in 1997,⁶ the effects of prostamides have been studied on various systems known to recognize endocannabinoid and prostaglandin classes of compounds. Prostamide E₂ (PGE₂-EA) has shown little to no affinity for cannabinoid receptors CB₁ and CB₂ and little to no activity at the endocannabinoid metabolizing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL).^7,8 While the prostamides have been shown to possess the ability to activate the human prostaglandin receptors (hEP), it is with 100–1000-fold lower affinity than the respective prostaglandins, making prostaglandin receptors an unlikely target for the prostamides.⁹ Additionally, in ex vivo functional assays on isolated tissue preparations from guinea pig known to contain prostaglandin EP₂ and EP₃ receptors, PGE₂-EA activity was found to be approximately 10-fold higher than those determined by the purified hEP₂ and hEP₃ receptor radioligand binding assay and argues for the existence of putative selective receptors within these tissues.⁹ Moreover, the lack of affinity of prostamides at prostaglandin, cannabinoid receptors, and the endocannabinoid enzymes FAAH and MGL suggests that there may be more substantial interactions with other biological targets. A more detailed understanding of the physiological roles of these novel compounds can be greatly enhanced by the availability of unique pharmacological reagents and is the motivating factor for the work presented here.

The current study presents a multiple step synthesis of PGE₂-EA analogs that allows for convergent functionalization of both the head and/or tail groups and provides potentially useful probes for the characterization of the prostamide biochemical system.¹⁰ Because PGE₂-EA is the major product of prostamide biosynthesis, analogs of the E₂ configuration were selected for synthesis.⁶ The initial set of compounds incorporates azido and isothiocyanato functionalities at the terminal tail position as affinity probes for covalent binding to the putative prostamide receptor (Fig. 1). Upon photoirradiation to a nitrene intermediate, the azido moiety can attach to reactive residues in the vicinity of the binding site.^11-13 Similarly, the electrophilic isothiocyanato group is capable of reacting with nucleophilic amino acid residues including thiol, imidazole, or amino groups that are unprotonated under physiological conditions.^14-16 These covalent probes allow for the future isolation and characterization of putative protein targets involved in prostamide function. In earlier work we have shown that introducing steric hindrance and/or chirality adjacent to the amide nitrogen atom in endocannabinoids leads to analogs with enhanced stability toward hydrolyzing enzymes without any reduction in bioactivity.¹⁷ Thus, in our rational design of PGE₂-EAs we have included the R- and S-2-methyl ethanolamide and cyclopropylamide moieties as head group variations (Fig. 1).

Figure 1.

PGE₂-EA and 2-PGE₂-G analogs synthesized.

2-Glycerol ester and 15R isomers were also incorporated in this set of compounds to explore their effects on metabolic stability and bioactivity. 2-Arachidonoylglycerol (2-AG) is an additional key representative of another class of endocannabinoids and was found to be subject to the same biosynthetic pathway via COX-2 to the prostaglandin glycerol esters.¹⁸ For this reason the respective 2-PGE₂-G analog was included in this series of targeted analogs. Also, while the 15S isomer is the native prostanoid configuration, the 15R isomer mimics the stereochemistry of the aspirin-triggered lipoxins (ATL), shown to have a leading role in mediating inflammation resolution.¹⁹ This COX-2 mediated metabolite of AA not only exhibits anti-inflammatory and pro-resolving properties, but in its 15R configuration also increases potency and metabolic stability over that of the native 15S compounds.²⁰ Thus, representative analogs with the 15R configuration were included in the targeted set of compounds synthesized in this study.

Results and discussion

Our overall synthetic strategy incorporates Corey’s prostaglandin synthesis²¹ along with an additional protecting group at the tail terminal carbon. It is essential that the protective scheme allows for complete selectivity in removal of the terminal protecting group to avail further functionalization at this site. In the initial synthesis, triisopropylsilyl (TIPS) groups at the 11 and 15 secondary alcohol positions and a tert-butyldimethylsilyl (TBS) group at the primary terminal position were chosen for this purpose.

The synthesis begins with the commercial starting material, benzoyl protected Corey’s lactone aldehyde 1 (Scheme 1), undergoing a Horner–Wadsworth–Emmons reaction to give the E-enone 3 exclusively as confirmed by ¹H NMR analysis (³J_CH=CH = 15.8 Hz) and in near quantitative yield. The phosphonate reagent 2 used here is prepared by reaction of n-butyllithium with dimethyl methyl phosphonate followed by ring opening of ε-caprolactone and protection of the terminal hydroxyl with TBS chloride (details are given under Supporting information).²² Asymmetric reduction of 3 to enol 4 using Corey’s oxazaborolidine catalyst²³ was attempted, however enantiomeric excess never exceeded 30%. Alternatively, we elected to perform a non-stereoselective Luche reduction,^24,25 isolate both 15S and 15R isomers (86% overall yield), and carry on the remainder of the synthesis in parallel. It should be noted that determination of the exact stereochemistry at C15 in the two isomers 15S (less polar, R_f = 0.55, 60% AcOEt in hexanes) and 15R (more polar, R_f = 0.45, 60% AcOEt in hexanes) was based on the synthesis of the respective Mosher esters (see Supporting information for details) followed by comparative analysis of their ¹H NMR spectral data.^26,27 Our ¹H NMR configurational correlations are in agreement with previous assignments done on prostaglandin intermediates with a straight chain where the less polar compound was determined to be the 15S secondary alcohol and the more polar compound was found to be the 15R.²⁸ Hydrolysis of the benzoyl group (89–91% yields) followed by TIPS protection of the secondary free hydroxyls (90–93% yields) establishes the last of our strategic protecting groups for intermediate 6. Reduction of the lactone to the lactol using DIBAL-H (93% yield) is followed by a Wittig reaction to introduce the head carboxylic acid chain with the desired Z-stereochemistry at the newly formed double bond (73–78% yields). Key intermediate 8 avails the choice of establishing either the head or tail moieties first. In our case, we decided to first incorporate the tail moiety and were thus able to synthesize several head group analogs.

Modifications at the tail aimed at incorporating azido and isothiocyanato functionalities are shown in Schemes 2 and 3. Upon exposure to TMSCHN₂ in MeOH, the acid 8 is converted to its methyl ester 9 (92–93% yields) and the 9S-hydroxy on the ring is oxidized to the ketone 10 (96% yield) with Dess–Martin periodinane buffered with pyridine to avoid potential elimination of the 11-hydroxy substituent to form the α,β-unsaturated ketone. The terminal hydroxy is then selectively deprotected using AcOH/THF/H₂O (3:1:1) to give 11 in high yields (91–93%). At this point we have established a path to synthesize the key primary alcohol 11 which allows the further functionalization of the terminal tail carbon. In the current study we focused on putative affinity probes incorporating azido and isothiocyanato groups. A modified Mitsunobu reaction provides the subsequent conversion to azide 12 in 91% yield (IR: s, 2093 cm⁻¹). Throughout the synthesis certain conditions, specifically those encountered during the amide coupling, led to the formation of the undesirable α,β-unsaturated ketone by dehydration of the 11-hydroxyl group. To prevent this, we reduced the 9-keto back to its hydroxyl counterpart and reintroduced it again after the amide coupling step. This strategy is essentially utilized as a protection/deprotection approach during the incorporation of the azido moiety and was found to be advantageous over the use of additional 9-OH protection groups that would require selective deprotection. Thus, Luche reduction of the ring ketone (83–89% yields) is followed by hydrolysis of the methyl ester 13 (NaOH, THF/H₂O) to give the precursor acid 14 in 84–85% yields.

Varying the head moieties begins with the free acid (14) coupling to the amine derivatives to give the desired amide analogs (Scheme 3). As we reported earlier for the synthesis of novel anandamide analogs, the carbonyldiimidazole (CDI) activation procedure worked well and provided these compounds in excellent yields (88–92%).^29,30 The Dess–Martin periodinane/pyridine system oxidizes the 9-hydroxy back to the ketone 16 (97–98% yields). The desilylation step leading to azides 17 proved to be challenging because of the enhanced stability of the TIPS group at the 11-hydroxy position. After considerable experimentation we found that the 48% HF/pyridine reagent works effectively and provides the azido analogs 17a–17d in acceptable yields (45–48%) along with unidentified byproducts. Subsequently, the isothiocyanato compounds 18a and 18b were isolated after treatment of the respective azido-ethanolamides 17a and 17b with carbon disulfide in the presence of triphenylphosphine (94–96% yields, IR: s, 2090 cm⁻¹). For the synthesis of the n-pentyl chain tail analogs 23 (Scheme 4) where no selectivity in removal of the silyl protecting groups is required, the full synthesis was carried out using TBS protection for both the 11- and 15-hydroxyls. In the final step, removal of all three silyl groups was carried out smoothly with 1% HCl in 95% MeOH and produced the amides 23 in short reaction time (5 min) and high yields (80–83%). The intermediate ketones 22a–22c were synthesized with high chemical efficiency from the precursor acid 20 which was in turn synthesized in three steps from commercially available 19 following previously reported procedures.³¹ It should be noted that an earlier synthesis of 23a (no yields reported) involved exposure of PGE₂ to ethyl chloroformate and treatment of the in situ generated mixed carbonic anhydride to (S)-(+)-amino-2-propanol.⁷ Carbonyldiimidazole mediated coupling of acid 20 with readily available 1,3-divaleric glycerol³² gave the triglyceride 24 in 82% yield (Scheme 5). Dess–Martin oxidation of the free hydroxy on the cyclopentane ring (97% yield) was followed by the deprotection of both silyl protecting groups (79% yield). Finally, the precursor triacylglycerol 26 was conveniently deprotected in ethanol with Candida rugosa lipase type VII to give the desired 2-PGE₂-G (27) in 46% yield. To the best of our knowledge the chemoenzymatic synthesis of this endogenous substance is reported here for the first time and parallels those we reported earlier for the endocannabinoid lipid 2-AG and its analogs.^33,34 Importantly, our approach provides the 2-PGE₂-G essentially free of 1(3)-acylglycerol rearrangement byproducts as confirmed by ¹H NMR data. In a previous study on the metabolism of prostaglandin glycerol esters the 2-PGE₂-G was the minor product which was synthesized as a mixture with the 1(3) regioisomer in a ratio 1:9.³⁵

The involvement of prostaglandins as pro-inflammatory lipid mediators has long been recognized. However, more recent studies have established that certain prostaglandins, specifically PGE₂ and PGD₂, trigger eicosanoid class switching from pro-inflammatory to local acting pro-resolving lipid mediators.³⁶ However, the role of this new class of prostaglandin related amides and ester is not well understood. In this preliminary study of the actions of a representative analog within the series, the 15S hydroxy, 20-isothiocyanato compound 18a was assessed in vivo using a widely employed model of inflammation, namely zymosan A-stimulated murine peritonitis.³⁷ Prostaglandin PGE₂ was also tested for comparison. The results of administration on cell numbers of both polymorphonuclear neutrophils (PMN) and monocytes are illustrated in Figure 2. We observe that while both PGE₂ and 18a effectively reduce PMN infiltrates in this inflammatory model, 18a was slightly more efficacious.

Scheme 1.

Synthesis of the C-8 and C-12 chain appendages. Reagents and conditions: (a) 2, NaH, THF, 0 °C, 1 h, then 1, 0 °C to rt, 2.5 h, 100%; (b) CeCl₃, NaBH₄, MeOH, 0 °C, 30 min, then chromatographic separation of 4a and 4b, 86% overall yield; (c) K₂CO₃, THF/MeOH, 50 °C, 2.5 h, 91% for 5a and 89% for 5b; (d) TIPSOTf, 2,6-lutidine, CH₂Cl₂, 0 °C, 1 h, 90% for 6a and 93% for 6b; (e) DIBAL-H, CH₂Cl₂, −78°C, 30 min, 93% for 7a and 7b; (f) Br Ph₃P–(CH₂)₄–COOH, t-BuOK, THF, 0 °C, 30 min, then 7a or 7b, 1 h, 73% for 8a and 78% for 8b.

In summary, a successful synthetic strategy has been established for the construction of a focused family of prostamide analogs containing head and tail modifications to aid in the characterization of a putative prostamide receptor. This synthesis incorporates the strategic use of silyl protecting groups for selective deprotection and functionalization of the ω terminus. Azide and isothiocyanate functionalities are introduced at this tail position to serve as affinity probes designed to covalently attach to target proteins associated with this prostamide thus aiding in their identification. In a preliminary biological study we found that the isothiocyanato compound 18a effectively reduces PMN infiltrates in the zymosan A-stimulated murine peritonitis model of inflammation and is more efficacious than PGE₂. A more detailed biochemical characterization of the compounds reported here is currently being explored.

Figure 2.

Comparisons for PGE2 and 18a in reducing leukocytes in murine peritonitis. Polymorphonuclear neutrophils (PMN) and monocytes were enumerated following injection with 1μg/mouse compound iv, after 5 min initiation of peritonitis with 1 mg zymosan A. After 4 h, leukocytes were obtained via lavages and leukocytes were identified and enumerated using light microscopy as well as fluorescence-activated cell sorting.

Scheme 2.

Synthesis of the functionalized azido tail. Reagents and conditions: (a) TMSCHN₂, Et₂O, MeOH, rt, 15 min, 93% for 9a and 92% for 9b; (b) Dess–Martin periodinane, pyridine, CH₂Cl₂, 0 °C to rt, 1 h, 96% for 10a and 10b; (c) AcOH:THF:H₂O (3:1:1), rt, overnight, 93% for 11a and 91% for 11b; (d) Zn(N₃)₂-Py, DIAD, PPh₃, toluene, rt, 7 h, 91% for 12a and 12b; (e) CeCl₃, NaBH₄, THF/MeOH, 0 °C, 1 h, 83% for 13a and 89% for 13b; (f) NaOH, THF/H₂O, rt, 24 h, 85% for 14a and 84% for 14b.

Scheme 3.

Final steps to the azido and isothiocyanato substituted prostamide probes. Reagents and conditions: (a) carbonyldiimidazole, CH₂Cl₂, rt, 30 min, then R-NH₂, 3h, 88–92%; (b) Dess–Martin periodinane, pyridine, CH₂Cl₂, 0 °C to rt, 1 h, 97–98%; (c) 48% HF, pyridine, THF/MeCN, 0 °C to rt, overnight, 45–48%; (d) CS₂, PPh₃, THF, rt, 48 h, 94– 96%.

Scheme 4.

Synthesis of the n-pentyl chain tail analogs. Reagents and conditions: (a) carbonyldiimidazole, CH₂Cl₂, rt, 30 min, then R-NH₂, 3 h, 87–90%; (b) Dess–Martin periodinane, pyridine, CH₂Cl₂, 0 °C to rt, 1 h, 89–91%; (c) 1% HCl, aqueous MeOH, 5 min, 80–83%.

Scheme 5.

Chemoenzymatic synthesis of the glycerol ester analog. Reagents and conditions: (a) HO–CH[CH₂–O–C(O)–(CH₂)₃CH₃]₂, carbonyldiimidazole, CH₂Cl₂, 0 °C to rt, 24 h, 82%; (b) Dess–Martin periodinane, pyridine, CH₂Cl₂, 0 °C to rt, 1 h, 97%; (c) 1% HCl, aqueous MeOH, rt, 5 min, 79%; (d) Candida rugosa lipase, EtOH, 2 h, 46%.