Biology and Total Synthesis of n-3 Docosapentaenoic Acid-Derived Specialized Pro-Resolving Mediators.

Research over the last 25 years related to structural elucidations and biological investigations of the specialized pro-resolving mediators has spurred great interest in targeting these endogenous products in total synthesis. These lipid mediators govern the resolution of inflammation as potent and stereoselective agonists toward individual G-protein-coupled receptors, resulting in potent anti-inflammatory activities demonstrated in many human disease models. Specialized pro-resolving mediators are oxygenated polyunsaturated products formed in stereoselective and distinct biosynthetic pathways initiated by various lipoxygenase and cyclooxygenase enzymes. In this review, the reported stereoselective total synthesis and biological activities of the specialized pro-resolving mediators biosynthesized from the polyunsaturated fatty acid n-3 docosapentaenoic acid are presented.

The biosynthetic pathways of the protectins.

Evidence for the biosynthetic pathways of the specialized pro-resolving mediator (SPM) protectin D1 (PD1) and its biochemical further local metabolism were presented during the 8th European Workshop on Lipid Mediators, organized June 29th-July 1st, 2022, in Stockholm, Sweden. Herein, we provide an extended and detailed discussion of these topics. PD1, one of 43 SPMs reported so far, exhibits very potent pro-resolution and anti-inflammatory bioactions. Many research groups worldwide have confirmed these and other interesting bioactions. The protectins constitute, together with the lipoxins, resolvins, and maresins, the four individual SPM families, which have received a great interest in basic biomedical research and drug discovery efforts.

The Synthesis of 3-(R)- and 3-(S)-Hydroxyeicosapentaenoic Acid.

Monohydroxylated polyunsaturated fatty acids belonging to the oxylipin class of natural products are present in marine and terrestrial sources as well as in the human body. Due to their biological activities and role in diverse biosynthetic pathways, oxylipins biosynthesized from eicosapentaenoic acid and arachidonic acid have attracted great interest from the scientific community. One example is 3-hydroxyeicosapentaenoic acid where the absolute configuration at C-3 has only been tentatively assigned. In this paper, studies on acetate type aldol reactions that enabled the preparation of 3-(R)-hydroxyeicosapentaenoic acid (3R-HETE, 2) and its enantiomer are presented.

Stereoselective Synthesis and Structural Confirmation of the Specialized Pro-Resolving Mediator Resolvin E4.

Herein, we report the stereoselective and convergent synthesis of resolvin E4, a newly identified specialized pro-resolving mediator. This synthesis proves the absolute configuration and exact olefin geometry. Key elements of the successful strategy include a highly stereoselective MacMillan organocatalytic oxyamination, a Midland Alpine borane reduction, and the use of a 1,4-pentadiyne unit as a linchpin building block. The application of reaction telescoping in several of the synthetic transformations enabled the preparation of the resolvin E4 methyl ester in 10% yield over 10 steps (longest linear sequence). The physical property (UV-Vis and LC-MS/MS) data of synthetic resolvin E4 matched those obtained from biologically produced material.

Stereoselective syntheses and biological activities of E-series resolvins.

Recent research efforts focusing on the many mechanisms participating in the resolution of acute inflammation have uncovered a new genus of pro-resolving lipid mediators. These endogenous molecules include the lipoxins, resolvins, protectins and maresins, collectively coined specialized pro-resolving mediators (SPMs). SPMs are oxygenated polyunsaturated fatty acids biosynthesized by lipoxygenases and cyclooxygenases enzymes. These chemically sensitive molecules are produced in nano- to pico-gram amounts in vivo and exhibit potent anti-inflammatory and pro-resolving bioactions. In addition, SPMs clear bacterial infections, reduce pain and display bioactivities towards host defense, organ protection and tissue remodeling. Altogether, these bioactions and the need for synthetic SPMs for determination of absolute configuration and in vivo experiments have spurred a great interest in the synthetic and biomolecular communities. This review covers reported stereoselective total syntheses and outlines the most significant bioactions of the E-series resolvins.

A new synthetic protectin D1 analog 3-oxa-PD1n-3 DPA reduces neuropathic pain and chronic itch in mice.

The resolution of inflammation is a biosynthetically active process controlled by the interplay between oxygenated polyunsaturated mediators and G-protein coupled receptor-signaling pathways. These enzymatically oxygenated polyunsaturated fatty acids belong to distinct families of specialized pro-resolving autacoids. The protectin family of mediators has attracted an interest because of their potent pro-resolving and anti-inflammatory actions verified in several in vivo disease models. Herein, we present the stereoselective synthesis and biological evaluations of 3-oxa-PD1n-3 DPA, a protectin D1 analog. Results from mouse models indicate that the mediators protectin D1, PD1n-3 DPA and the new analog 3-oxa-PD1n-3 DPA all relieved streptozotocin-induced diabetic neuropathic pain at doses of 90 and 300 pmol, equivalent to 30 and 100 ng, respectively, following intrathecal (I.T.) injection. Of interest, at a low dose of only 30 pmol (10 ng; I.T.) only 3-oxa PD1n-3 DPA was able to alleviate neuropathic pain, directly compared to vehicle controls. Moreover, using a chronic itch model of cutaneous T-cell lymphoma (CTCL), all three compounds at 300 pmol (100 ng) showed a significant reduction in itching for several hours. The biomolecular information on the structure-functions of the protectins and the new synthetic analog 3-oxa-PD1n-3 DPA is of interest towards developing new immunoresolvents.

New CDK8 inhibitors as potential anti-leukemic agents - Design, synthesis and biological evaluation.

Cyclin-dependent kinase 8 (CDK8) plays a vital role in regulating cell transcription either through its association with the mediator complex or by the phosphorylation of transcription factors. CDK8-mediated activation of oncogenes has proved to be important in a variety of cancer types including hematological malignancies. We have designed and synthesized a series of new synthetic steroids. The compounds were evaluated as CDK8 inhibitors in vitro. The three most potent compounds exhibit Kd-values towards CDK8 in the low nanomolar range (3.5-18 nM). Furthermore, the compounds displayed selectivity for CDK8 in a panel of 465 different kinases. The cell studies indicated a selectivity to kill AML-cancer cell lines compared to normal cell lines.

The First Total Synthesis of the Lipid Mediator PD2n-3 DPA.

The resolution of inflammation is governed by the active biosynthesis of specialized pro-resolving mediators using ω-6 and ω-3 polyunsaturated fatty acids as substrates. These mediators act as resolution agonists and display several interesting bioactivities. PD2n-3 DPA is an oxygenated polyunsaturated fatty acid biosynthesized from n-3 docosapentaenoic acid belonging to the specialized pro-resolving lipid mediator family named protectins. The protectins exhibit anti-inflammatory properties and pro-resolving bioactivities. These endogenously produced compounds are of interest as leads in resolution pharmacology and drug development. Herein, together with its NMR, MS, and UV data, a stereoselective total synthesis of PD2n-3 DPA is presented.

Enzymatic studies with 3-oxa n-3 DPA.

Cyclooxygenase-2 and several lipoxygenases convert polyunsaturated fatty acids into a large variety of products. During inflammatory processes, these enzymes form several distinct families of specialized pro-resolving lipid mediators possessing potent anti-inflammatory and pro-resolving effects. These mediators have attracted a great interest as leads in drug discovery and have recently been the subject of biosynthetic pathway studies using docosahexaenoic and n-3 docosapentaenoic acid as substrates. Herein we present enzymatic studies with cyclooxygenase-2 and 5-, 12- and 15-lipoxygenase enzymes using 3-oxa n-3 DPA as a synthetic mimic of n-3 docosapentaenoic acid. Structural elucidation based on data from RP-HPLC UV and LC/MS-MS experiments enabled the identification of novel enzymatically formed products. These findings constitute the basis for further biosynthetic studies towards understanding the mechanisms regulating substrate utilization in the biosynthesis of specialized pro-resolving lipid mediators.

Stereoselective synthesis of MaR2n-3 DPA.

The first total synthesis of the n-3 docosapentaenoic derived oxygenated product MaR2n-3 DPA has been achieved. The 13R and 14S stereogenic centers were introduced using 2-deoxy-d-ribose in a chiral pool strategy. The geometry of the Z,E,E-triene moiety was prepared using highly E-selective Wittig- and Takai-olefination reactions as well as the Z-stereoselective Lindlar reduction. LC/MS-MS data of synthetic MaR2n-3 DPA matched data for the biosynthetic formed product that enabled the configurational assignment of this oxygenated natural product to be (7Z,9E,11E,13R,14S,16Z,19Z)-13,14-dihydroxydocosa-7,9,11,16,19-pentaenoic acid.

Synthesis and Biological Evaluation of Analogs of Didehydroepiandrosterone as Potential New Anticancer Agents.

The synthesis, cytotoxicity and inhibition of CDK8 by thirteen analogs of cortistatin A are reported. These efforts revealed that the analogs with either a 6- or 7-isoquinoline or 5-indole side chain in the 17-position are the most promising anti-proliferative agents. These compounds showed potent cytotoxic effects in CEM, HeLa and HMEC-1 cells. All three compounds exhibited IC50 values < 10µM. The most interesting 10l analog exhibited an IC50 value of 0.59 µM towards the human dermal microvascular endothelial cell line (HMEC-1), significantly lower than the reference standard 2-methoxyestradiol. At a concentration at 50 nM the most potent 10h compound reduced the activity of CDK8 to 35%.

Resolving Inflammation: Synthesis, Configurational Assignment, and Biological Evaluations of RvD1n-3 DPA.

New drugs that can resolve inflammation without immunosuppressive effects are at the medicinal chemistry frontier. Pro-resolving endogenously formed small molecules, that is, the resolvins, are excellent candidates displaying such bioactions. The first total synthesis of the specialized pro-resolving mediator RvD1n-3 DPA has been achieved using the underutilized sp3 -sp3 Negishi cross coupling reaction and an alkyne hydrosilylation-protodesilylation protocol. Biological evaluations revealed that this novel mediator displays low nanomolar pro-resolving properties and potently activates the human DRV1/GPR32 receptor. As such, this endogenous natural product is a lead compound for the development of novel immunoresolvents.

Catalytic enantioselective iodolactonization reactions.

The halolactonization reaction is a useful chemical transformation for the construction of lactones from γ- or δ-substituted alkenoic carboxylic acids or carboxylic esters. Traditionally, the stereoselectivity of these reactions has been controlled by the substrates or the reagents. The substrate-controlled method has been extensively studied and applied in the synthesis of many natural products. However, catalytic, enantioselective iodolactonizations of γ- or δ-substituted alkenoic carboxylic acids have only recently been developed. This review article highlights the advances that have emerged over the last decade.

Molecular modelling, synthesis, and biological evaluations of a 3,5-disubstituted isoxazole fatty acid analogue as a PPARα-selective agonist.

The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC50 = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.

Atomic determinants of BK channel activation by polyunsaturated fatty acids.

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery.

Synthesis, Structural Confirmation, and Biosynthesis of 22-OH-PD1n-3 DPA.

PD1n-3 DPA belongs to the protectin family of specialized pro-resolving lipid mediators. The protectins are endogenously formed mediators that display potent anti-inflammatory properties and pro-resolving bioactivities and have attracted interest in drug discovery. However, few studies have been reported of the secondary metabolism of the protectins. To investigate the metabolic formation of the putative C22 mono-hydroxylated product, coined 22-OH-PD1n-3 DPA, a stereoselective synthesis was performed. LC/MS-MS data of synthetic 22-OH-PD1n-3 DPA matched the data for the biosynthetic formed product. Cellular studies revealed that 22-OH-PD1n-3 DPA is formed from n-3 docosapentaenoic acid in human serum, and we confirmed that 22-OH-PD1n-3 DPA is a secondary metabolite produced by ω-oxidation of PD1n-3 DPA in human neutrophils and in human monocytes. The results reported are of interest for enabling future structure-activity relationship studies and provide useful molecular insight of the metabolism of the protectin class of specialized pro-resolving mediators.

Synthetic manipulations of polyunsaturated fatty acids as a convenient strategy for the synthesis of bioactive compounds.

Stereoselective synthesis of Z-configured double bonds is central in organic synthesis due to the presence of such motifs in polyunsaturated fatty acids and many natural products. Traditionally, reductions of internal alkynes or Wittig, Ando or Still-Gennari reactions, are often used for preparing such compounds. The substrate scope is limited for both the Ando and the Still-Gennari reactions, while the Wittig reaction often gives low Z-selectivity for the synthesis of polyunsaturated Z-configured methylene interrupted (skipped) double bonds. Reductions of internal alkynes are challenging due to diminished Z-selectivity, poor catalyst reproducibility and over-reductions. An alternative and highly attractive approach is to employ naturally occurring and commercially available polyunsaturated fatty acids as starting materials. The main advantage of this strategy is the conservation of the multiple Z-configured double bonds present in the starting material, allowing a precise incorporation of the desired double bonds into the final product. In particular, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid have been used for the stereoselective synthesis of polyunsaturated fatty acids, their derivatives and other polyunsaturated natural products. Herein, such efforts are reviewed.

Synthesis, molecular modelling studies and biological evaluation of new oxoeicosanoid receptor 1 agonists.

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC50) and efficacy (Emax) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles.

Structural review of PPARγ in complex with ligands: Cartesian- and dihedral angle principal component analyses of X-ray crystallographic data.

Two decades of research into the ligand-dependent modulation of the activity of the peroxisome proliferator-activated receptor γ (PPARγ) have demonstrated the heterogeneous modes of action of PPARγ ligands, in terms of their interaction surfaces in the ligand-binding pocket, binding stoichiometry and ability to interact with functionally important parts of the receptor, through both direct and allosteric mechanisms. These findings signal the complex mechanistic bases of the distinct biological effects of different classes of PPARγ ligands. Today, the development of PPARγ ligands focuses on partial- and non-agonists as opposed to classical agonists, due to the severe side effects observed with PPARγ classical agonists as therapeutic agents. To aid this development, we performed principal component analyses of the atomic (Cartesian) coordinates (cPCA) and dihedral angles (dPCA) of the structures of human PPARγ from X-ray crystallography, available in the public domain, seeking to reveal ligand-induced trends. In the cPCA, projections of the structures along the principal components (PCs) demonstrated a moderate correlation between cPC1 and structural parameters related to the stabilization of helix 12, which is central to the transcriptional activation by PPARγ classical agonists. Consequently, the presented cPCA mapping of the PPARγ-ligand complexes may guide in silico drug discovery programs seeking to avoid stabilization of helix 12 in their development of partial- and non-agonistic PPARγ ligands. Notably, while the dPCA could identify key regions of dihedral fluctuation in the structural ensemble, the distributions along dPC1 - 2 could not be classified according to the same parameters as the distribution along cPC1. Proteins 2017; 85:1684-1698. © 2017 Wiley Periodicals, Inc.

The novel lipid mediator PD1n-3 DPA: An overview of the structural elucidation, synthesis, biosynthesis and bioactions.

Resolvins, protectins and maresins are individual families of specialized pro-resolving mediators biosynthesized from the dietary n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid. These enzymatically oxygenated polyunsaturated lipid mediators were first elucidated during the resolution phase of acute inflammation in animal models of self-limited inflammation. Specialized pro-resolving mediators display potent bioactions when administrated in vivo. Biosynthetic pathway studies have revealed that individual lipoxygenases and cyclooxygenase-2 converts eicosapentaenoic acid and docosahexaenoic acid into distinct families of the resolvins, protectins and maresins. Recently n-3 docosapentaenoic acid was found to be a substrate for the biosynthesis of several novel families of specialized pro-resolving mediators. One example is PD1n-3 DPA. During the 6th European Workshop on Lipid Mediators, Frankfurt, Germany, the structural elucidation, total organic synthesis, studies on the biosynthetic pathway, as well as the potent anti-inflammatory and pro-resolving properties of PD1n-3 DPA were presented. Herein, we provide an overview of these topics for the new member PD1n-3 DPA of the super-family of pro-resolving mediators.