Design, synthesis and bioactions of novel stable mimetics of lipoxins and aspirin-triggered lipoxins

The lipoxins (LX) are a class of potent endogenous oxygenated products that are enzymatically generated from arachidonic acid and have novel anti-inflammatory properties and promote resolution. Elucidation of the biochemical pathways involved in the metabolic inactivation of LX and the discovery of the aspirin-triggered lipoxins (ATL) provided the basis for the design and synthesis of stable analogs of LX and ATL. This special issue review describes the efforts that led to the design and synthesis of stable LX/ATL mimetics, which permitted the detailed elucidation of their novel biological roles, leading to the development of new anti-inflammatory agents that mimic their actions. These synthetic molecules provided the means to uncover the physiologic roles of both the LX and the ATL biosynthetic pathways which led to several unexpected discoveries. Among these findings is the involvement of polyisoprenyl phosphates (PIPP) in intracellular signaling mediated by presqualene diphosphate (PSDP), and the recognition of the novel roles of these lipid mediators in regulating cell trafficking during inflammation as well as in promoting resolution of inflammatory processes. These efforts also provided the basis for examining the potential therapeutic role of LX/ATL stable mimetics and led to the development of new analogs with improved pharmacokinetics that opened the way to potentially new approaches to treating human diseases.     

Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution

Lipoxins (LXs) or the lipoxygenase interaction products are generated from arachidonic acid via sequential actions of lipoxygenases and subsequent reactions to give specific trihydroxytetraene-containing eicosanoids. These unique structures are formed during cell-cell interactions and appear to act at both temporal and spatially distinct sites from other eicosanoids produced during the course of inflammatory responses and to stimulate natural resolution. Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) are positional isomers that each possesses potent cellular and in vivo actions. These LX structures are conserved across species. The results of numerous studies reviewed in this work now confirm that they are the first recognized eicosanoid chemical mediators that display both potent anti-inflammatory and pro-resolving actions in vivo in disease models that include rabbit, rat, and mouse systems. LXs act at specific GPCRs as agonists to regulate cellular responses of interest in inflammation and resolution. Aspirin has a direct impact in the LX circuit by triggering the biosynthesis of endogenous epimers of LX, termed the aspirin-triggered 15-epi-LX, that share the potent anti-inflammatory actions of LX. Stable analogs of LXA4, LXB4, and aspirin-triggered lipoxin were prepared, and several of these display potent actions in vitro and in vivo. The results reviewed herein implicate a role of LX and their analogs in many common human diseases including airway inflammation, asthma, arthritis, cardiovascular disorders, gastrointestinal disease, periodontal disease, kidney diseases and graft-vs.-host disease, as well as others where uncontrolled inflammation plays a key role in disease pathogenesis. Hence, the LX pathways and mechanisms reviewed to date in this work provide a basis for new approaches to treatment of many common human diseases that involve inflammation.     

Anti-inflammatory circuitry: lipoxin, aspirin-triggered lipoxins and their receptor ALX

Endogenous chemical mediators or autacoids play key roles in controlling inflammation and its programmed resolution. Among them, it is known that lipoxins (LX) and aspirin-triggered LX (ATL) evoke bioactions in a range of physiologic and pathophysiologic processes and serve as endogenous lipid/chemical mediators that stop neutrophilic infiltration and initiate resolution. LXA4, ATL and their metabolic stable analogs elicit cellular responses and regulate PMN in vivo via interacting with their specific receptor, namely ALX. ALX is the first cloned and identified lipoxygenase-derived eicosanoid receptor with cell type-specific signaling pathways. Also, ALX could regulate PMN by interacting with each class of ligands (lipid vs. peptide) within specific phases of an inflammatory response. Together LX, ATL and ALX may provide new opportunities to design "resolution-targeted" therapies with high degree of precision in controlling inflammation. In this chapter, we give an overview and update of the current actions for LX and ATL, the identification of ALX and their novel anti-inflammatory and pro-resolving signals.     

Aspirin-triggered lipoxin A4 and B4 analogs block extracellular signal-regulated kinase-dependent TNF-alpha secretion from human T cells

Lipoxins (LX) and their aspirin-triggered 15-epimer endogenous isoforms are endogenous anti-inflammatory and pro-resolution eicosanoids. In this study, we examined the impact of LX and aspirin-triggered LXA(4)-stable analogs (ATLa) on human T cell functions. 15-epi-16-(p-fluoro)phenoxy-LXA(4) (ATLa(1)) blocked the secretion of TNF-alpha from human PBMC after stimulation by anti-CD3 Abs, with the IC(50) value of approximately 0.05 nM. A similar action was also exerted by the native aspirin-triggered 15-epi-LXA(4), a new 15-epi-16-(p-trifluoro)phenoxy-LXA(4) analog (ATLa(2)), as well as LXB(4), and its analog 5-(R/S)-methyl-LXB(4). The LXA(4) receptor (ALX) is expressed in peripheral blood T cells and mediates the inhibition of TNF-alpha secretion from activated T cells by ATLa(1). This action was accomplished by inhibition of the anti-CD3-induced activation of extracellular signal-regulated kinase, which is essential for TNF-alpha secretion from anti-CD3-activated T cells. These results demonstrate novel roles for LX and aspirin-triggered LX in the regulation of T cell-mediated responses relevant in inflammation and its resolution. Moreover, they provide potential counterregulatory signals in communication(s) between the innate and acquired immune systems.     

Induction of angiogenic response by chemically stable prostacyclin analogs

Angiogenic activities of several chemically stable prostacyclin analogs (isocarbacyclins and 7-fluoro prostacyclin) were evaluated by the chick embryo chorioallantoic membrane assay. These compounds showed potent angiogenic activity at very low concentration (0.1 micrograms/egg 1.0 micrograms/egg), whereas naturally occurring prostaglandins such as prostacyclin and PGE1 were almost ineffective up to 1 microgram/egg. Pretreatment of chorioallantoic membranes with dexamethasone or indomethacin inhibited the angiogenic response induced by these chemically stable prostacyclin analogs. These results indicate that these prostacyclin analogs induce the angiogenic response of chick chorioallantoic membranes via a mechanism involving activation of inflammatory cells, as well as through their direct angiogenic activity.     

Discovery of a new class of potent, selective, and orally active prostaglandin D2 receptor antagonists

The process of discovering a series of N-(p-alkoxy)benzoyl-2-methylindole-4-acetic acid analogs is presented since these compounds represent a new class of potent, selective, and orally active prostaglandin D2 (PGD2) receptor antagonists. Most of these compounds exhibit strong PGD2 receptor binding and PGD2 receptor antagonism in cAMP formation assays. When given orally, these new antagonists dramatically suppress allergic inflammatory responses, such as the PGD2-induced or OVA-induced increase of vascular permeability. Structure-activity relationship (SAR) data are also discussed.     

Second-generation beta-oxidation resistant 3-oxa-lipoxin A4 analogs

Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 are structurally and functionally distinct eicosanoids, with potent anti-inflammatory and immunomodulatory actions. Therapeutic use of LXA4 is greatly limited by its rapid metabolism in vivo and chemical instability. First-generation synthetic LXA4 analogs such as methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), were designed to minimize metabolism from the omega-end of the molecule. Pharmacokinetic analysis of ATLa revealed beta-oxidation as a novel route for LXA4 metabolism, prompting the development of second-generation 3-oxa-LXA4 analogs with improved pharmacokinetic disposition. Second-generation 3-oxa-LXA4 analogs such as (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-3-oxa-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoic acid (3), have shown potency and efficacy comparable to ATLa in diverse animal models after topical, intravenous or oral delivery. These include several acute (2-24 h) inflammatory reactions: calcium ionophore-induced skin edema and inflammation (topical), LTB4/PGE2-induced skin inflammation and vascular leak (topical), zymosan A-induced peritonitis (i.v. and oral) and ischemia-reperfusion-induced secondary organ injury (i.v.). Remarkably, 3-oxa-LXA4 analogs have potent once daily oral efficacy in preventing and promoting the resolution of established colitis induced by the hapten trinitrobenzene sulphonic acid (TNBS), an acute/chronic 7-14-day model of Crohn's disease. The second-generation 3-oxa-LXA4 analogs thus provide new stable pharmacophores with which to explore the emerging role of lipoxins as a new therapeutic principle for regulating inflammation, allergy and immune dysfunction in preclinical and clinical research.     

Physical exercise increases urinary excretion of lipoxin A4 and related compounds.

Lipoxins (LX) are lipoxygenase-derived eicosanoids with potent anti-inflammatory activities and vascular bed-dependent vasodilatory actions. LX can be formed in vitro and in vivo in a number of conditions, and we have reported that immunoreactive LXA(4) (iLXA(4)) is physiologically excreted with human urine. Using a recently developed LX extraction method coupled to an ELISA, we examined whether iLXA(4) excretion was modified by strenuous exercise, which is known to trigger potential LX-forming events. Maximal exertion significantly increased iLXA(4) urinary excretion in nine healthy volunteers (0.061 +/- 0.023 vs. 0.113 +/- 0.057 ng/mg creatinine; P = 0.028). iLXA(4) levels returned to baseline after 6 h and increased, although at a smaller extent, after 24 h. A significant correlation (r = 0.988) was denoted between iLXA(4) ELISA measurements and reversed-phase high-performance liquid chromatography quantitation of a previously described urinary tetraene, confirming its LXA(4)-related nature. These findings show for the first time that an increase in excretion of LXA(4)-related compounds can be observed in response to strenuous exercise. This may be the reflection of an enhanced LX biosynthesis, which may represent a safeguard mechanism that keeps the inflammatory reaction triggered by physical stress under control.     

Synthesis and biological evaluation of 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4-ethynylimidazole. A novel and highly potent anti-inflammatory and cytoprotective agent

To explore more potent N-acylimidazole analogues of CDDO than CDDO-Im, which is one of the most potent compounds in several widely used bioassays related to protection against inflammation and carcinogenesis; we have synthesized and evaluated five new N-acyl(acetylenic)imidazole analogues. Among them, 4-ethynylimidazole 4 is nearly equivalent to CDDO-Im in potency in these bioassays. Remarkably, the solid form of 4 is more stable than that of CDDO-Im. These findings suggest that 4 is a very promising anti-inflammatory and cytoprotective agent and its further preclinical evaluation is warranted.     

NO-steroids: potent anti-inflammatory drugs with bronchodilating activity in vitro.

The synthesis of nitric oxide (NO) releasing anti-inflammatory molecules is an innovative strategy to design novel anti-inflammatory drugs. These compounds slowly release NO, via an enzymatic pathway conferring new biological activities. Here we report the potent anti-inflammatory profile and the bronchodilator effect of nitro-derivatives of steroids, prednisolone, especially. The experiments were performed on guinea pig trachea or perfused bronchioles precontracted by methacholine. We demonstrated for the first time that unlike the parent compounds which produced weak bronchodilation at the maximum used dose (10(-4) M), NO-steroids caused a significant bronchodilating activity up to 70% of the maximal relaxation induced by 10(-4) M papaverine. This effect was epithelium- and endogenous-independent but cGMP-dependent. Taken together these data suggest that NO-steroids possessed a more potent anti-inflammatory activity than native compounds coupled with a concentration-dependent bronchodilating activity. Further studies are required to determine if NO-steroids will be effective as anti-inflammatory agents in the clinic.     

Synthesis and antiproliferative activity of imidazole and imidazoline analogs for melanoma

We have previously reported substituted 2-aryl-thiazolidine-4-carboxylic acid amides as potent and selective antiproliferative agents for melanoma. To understand the importance of the thiazolidine ring and to reduce potential complications associated with the two chiral centers, we designed and synthesized sets of new analogs by modifying this ring. These new analogs were tested in two melanoma cell lines and fibroblast cells (negative controls). Compared with the older analogs containing the thiazolidine ring, these new analogs have lower potency in general, but some of these analogs still have very good selectivity. These structure-activity studies indicated that the thiazolidine ring is very critical for the activity for these series of compounds.     

Selective inhibition of glycosyltransferases by bivalent imidazolium salts

Galactosyltransferases (GalTs) extend the glycan chains of mammalian glycoproteins by adding Gal to terminal GlcNAc residues, and thus build the scaffolds for biologically important glycan structures. We have shown that positively charged bivalent imidazolium salts in which the two imidazolium groups are linked by an aliphatic chain of 20 or 22 carbons form potent inhibitors of purified human β3-GalT5, using GlcNAcβ-benzyl as acceptor substrate. The inhibitors are not substrate analogs and also inhibited a selected number of other glycosyltransferases. These bis-imidazolium compounds represent a new class of glycosyltransferase inhibitors with potential as anti-cancer and anti-inflammatory drugs.     

Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages

Lipoxins (LX) are lipoxygenase-derived eicosanoids generated during inflammation. LX inhibit polymorphonuclear neutrophil (PMN) chemotaxis and adhesion and are putative braking signals for PMN-mediated tissue injury. In this study, we report that LXA4 promotes another important step in the resolution phase of inflammation, namely, phagocytosis of apoptotic PMN by monocyte-derived macrophages (Mphi). LXA4 triggered rapid, concentration-dependent uptake of apoptotic PMN. This bioactivity was shared by stable synthetic LXA4 analogues (picomolar concentrations) but not by other eicosanoids tested. LXA4-triggered phagocytosis did not provoke IL-8 or monocyte chemoattractant protein-1 release. LXA4-induced phagocytosis was attenuated by anti-CD36, alphavbeta3, and CD18 mAbs. LXA4-triggered PMN uptake was inhibited by pertussis toxin and by 8-bromo-cAMP and was mimicked by Rp-cAMP, a protein kinase A inhibitor. LXA4 attenuated PGE2-stimulated protein kinase A activation in Mphi. These results suggest that LXA4 is an endogenous stimulus for PMN clearance during inflammation and provide a novel rationale for using stable synthetic analogues as anti-inflammatory compounds in vivo.     

U19052 (ICIAm): a novel leukotriene analog which antagonizes LTC4, LTD4, and LTE4

Chemically stable analogs of peptide leukotrienes (LT) have been developed in our laboratories by replacement of the natural triene backbone with a C7H15 substituted aromatic moiety (1). These analogs are potent agonists of airway smooth muscle. Substitution in the peptide region resulted in U19052, an LT receptor antagonist. U19052 antagonized LT-induced contractions of guinea-pig tracheal spirals in a concentration-related manner. The pA2 values versus LTD4 and LTE4 were 6.0 and 5.7, respectively, with slopes which were not significantly different from unity. LTC4-induced contractions were antagonized by U19052 with a pKB of 5.6 obtained either in the absence or presence of L-serine borate. In contrast, carbachol and histamine concentration-response curves were not altered by U19052. LTD4 or LTE4 contractions of isolated guinea-pig ileum were antagonized by U19052 with pKB values of 7.2. The results indicate that potent selective LT antagonists can be developed from stable analogs of leukotrienes. U19052, an example of this series, appears to be as effective in antagonizing LTC4- as well as LD4- and LTE4-induced contractions in guinea-pig tracheal spirals.     

Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.     

Synthesis and anti-inflammatory activity of new arylidene-thiazolidine-2,4-diones as PPARγ ligands

Eight new 5-arylidene-3-benzyl-thiazolidine-2,4-diones with halide groups on their benzyl rings were synthesized and assayed in vivo to investigate their anti-inflammatory activities. These compounds showed considerable biological efficacy when compared to rosiglitazone, a potent and well-known agonist of PPARγ, which was used as a reference drug. This suggests that the substituted 5-arylidene and 3-benzylidene groups play important roles in the anti-inflammatory properties of this class of compounds. Docking studies with these compounds indicated that they exhibit specific interactions with key residues located in the site of the PPARγ structure, which corroborates the hypothesis that these molecules are potential ligands of PPARγ. In addition, competition binding assays showed that four of these compounds bound directly to the ligand-binding domain of PPARγ, with reduced affinity when compared to rosiglitazone. An important trend was observed between the docking scores and the anti-inflammatory activities of this set of molecules. The analysis of the docking results, which takes into account the hydrophilic and hydrophobic interactions between the ligands and the target, explained why the 3-(2-bromo-benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-dione compound had the best activity and the best docking score. Almost all of the stronger hydrophilic interactions occurred between the substituted 5-arylidene group of this compound and the residues of the binding site.     

The 'Ethereal' nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.     

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.     

Anti-inflammatory and antioxidant activity of coumarins designed as potential fluorescent zinc sensors

A series of coumarin analogs, designed and synthesised as potential fluorescent zinc probes were evaluated for their biological activity as anti-inflammatory and antioxidant agents. The effect of the synthesised compounds on inflammation, using the carrageenin-induced rat paw oedema model, was studied. In general, the compounds were found to be potent anti-inflammatory agents (26.5-64%). Compound 5 was found to interact significantly with 1,1-diphenyl-2-picryl-hydrazyl stable free radical (DPPH) whereas the remainder were inactive in this assay. The compounds inhibit in general the soybean lipoxygenase and scavenge superoxide anion radicals. The anti-inflammatory activity seems to be connected with their reducing activity. Their RM values were determined as an expression of their lipophilicity. Theoretical calculations of their lipophilicity as clog P were performed indicating that only a poor relationship exists between their lipophilicity and anti-inflammatory activity.