Markus Laube

Radiolabeled COX-2 Inhibitors for Non-Invasive Visualization of COX-2 Expression and Activity — A Critical Update

Cyclooxygenase-2 (COX-2) is a key player in inflammation. Its overexpression is directly associated with various inflammatory diseases and, additionally, with several processes of carcinogenesis. The development of new selective COX-2 inhibitors (COXIBs) for use in cancer treatment is in the focus of the medicinal chemistry research field. For this purpose, a set of methods is available to determine COX-2 expression and activity in vitro and ex vivo but it is still a problem to functionally characterize COX-2 in vivo. This review focusses on imaging agents targeting COX-2 which have been developed for positron emission tomography (PET) and single photon emission computed tomography (SPECT) since 2005. The literature reveals that different radiochemical methods are available to synthesize COXIBs radiolabeled with fluorine-18, carbon-11, and isotopes of radioiodine. Unfortunately, most of the compounds tested did not show sufficient stability in vivo due to de[18F]fluorination or de[11C]methylation or they failed to bind specifically in the target region. So, suitable stability in vivo, matching lipophilicity for the target compartment and both high affinity and selectivity for COX-2 were identified as prominent criteria for radiotracer development. Up to now, it is not clear what approach and which model is the most suited to evaluate COX-2 targeting imaging agents in vivo. However, for proof of principle it has been shown that some radiolabeled compounds can bind specifically in COX-2 overexpressing tissue which gives hope for future work in this field.

2,3-Diaryl-substituted indole based COX-2 inhibitors as leads for imaging tracer development

A series of 2,3-diaryl-substituted indoles containing a fluorine or methoxy group was synthesized via Fischer indole synthesis, McMurry cyclization, or Bischler–Mohlau reaction to identify potential leads for positron emission tomography (PET) radiotracer development as well as for optical imaging. All 2,3-diaryl-substituted indoles possess autofluorescent properties with an emission maximum in a range of 443–492 nm, which is acceptable for biological studies in vitro and, in part, in vivo. The molecular structure of compounds 3a and 3j was confirmed by X-ray crystal structure analysis. COX inhibitory activity was evaluated by a fluorescence-based and enzyme immunoassay-based assay. Redox activity of all target compounds was also determined. All synthesized 2,3-diaryl-substituted indoles are inhibitors of COX-2 enzyme in the low micromolar range. Compounds 3e, 3f, 3g and 3m displayed a 30–40% inhibition of COX-2 at 0.1 μM concentration while compounds 3f and 3g also exhibited COX-1 inhibitory activity. Various compounds like 3g showed substantial antioxidative potential (RDIENE = 2.85, RHAVA = 1.98), an effect that was most measurable with methoxy-substituted compounds. With respect to PET radiotracer synthesis, OMe-containing compound 3j was selected as a promising candidate for carbon-11 labeling, and F-containing compound 3m as a lead for the development of a fluorine-18 labeled derivative.

2-Carbaborane-3-phenyl-1H-indoles—Synthesis via McMurry Reaction and Cyclooxygenase (COX) Inhibition Activity

Cyclooxygenase‐2 (COX‐2) inhibitors have been the focus of medicinal chemistry efforts for years, and many compounds that exhibit high selectivity and affinity have been developed. As carbaboranes represent interesting pharmacophores as phenyl mimetics in drug development, this paper presents the synthesis of carbaboranyl derivatives of COX‐2‐selective 2,3‐disubstituted indoles. Despite the lability of carbaboranes under reducing conditions, 2‐carbaborane‐3‐phenyl‐1H‐indoles could be synthesized by McMurry cyclization of the corresponding amides. Whereas the meta‐carbaboranyl‐substituted derivatives lacked COX inhibitory activity, an ortho‐carbaboranyl analogue was active, but showed a selectivity shift toward COX‐1.

Diaryl-Substituted (Dihydro)pyrrolo[3,2,1-hi]indoles, a Class of Potent COX-2 Inhibitors with Tricyclic Core Structure

A new compound class of diaryl-substituted heterocycles with tricyclic dihydropyrrolo[3,2,1-hi]indole and pyrrolo[3,2,1-hi]indole core structures has been designed and was synthesized by a modular sequence of Friedel-Crafts acylation, amide formation, and McMurry cyclization. This synthesis route represents a novel and versatile access toward dihydropyrrolo[3,2,1-hi]indoles and is characterized by good chemical yields and high modularity. From a set of 19 derivatives, 11 candidates were selected for determination of their COX inhibition potency and were found to be selective inhibitors with high affinity to COX-2 (IC50 ranging from 20-2500 nM and negligible inhibition of COX-1). The binding mode of the novel inhibitors in the active side of COX-2 was calculated in silico using the protein-ligand docking program GOLD by application of the molecular structures of two compounds derived from X-ray crystallography. Two novel compounds with high affinity to COX-2 (6k = 70 nM, 8e = 60 nM) have a fluoro substituent, making them promising candidates for the development of (18)F-radiolabeled COX-2 inhibitors for imaging purposes with positron emission tomography (PET).

Organotypical vascular model for characterization of radioprotective compounds: Studies on antioxidant 2,3-diaryl-substituted indole-based cyclooxygenase-2 inhibitors

Radiotherapy of various cancers is closely associated with increased cardiovascular morbidity and mortality. Arachidonic acid metabolites are supposed to play a key role in radiation-induced vascular dysfunction. This study was designed to evaluate the effects of novel, antioxidative 2,3-diaryl-substituted indole-based selective cyclooxygenase-2 (COX-2) inhibitors (2,3-diaryl-indole coxibs) on radiation-induced formation of arachidonic acid metabolites via COX-2 and oxidant stress pathways in an organotypical vascular model of rat aortic rings. Acute and subacute effects of X-ray radiation (4 and 10 Gy; 1 and 3 days post irradiation) with or without the presence of 1 μM of the 2,3-diaryl-indole coxib 2-[4-(aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-indole (C1) or celecoxib as reference compared to sham-irradiated controls were assessed. The following parameters were measured: metabolic activity of the aortic rings; induction and regulation of COX-2 expression; release of prostaglandin E2 and F2α-isoprostane. Irradiation without presence of coxibs resulted in a dose-dependent augmentation of all parameters studied. When aortic rings were exposed to the 2,3-diaryl-indole coxib 1 h before irradiation, metabolic activity was restored and the release of both prostaglandin and isoprostane was inhibited. The latter indicates a direct interaction with oxidant stress pathways. By contrast, celecoxib exhibited only slight effects on the formation of isoprostane. The reduction of radiation-induced vascular dysfunction by antioxidative coxibs may widen the therapeutic window of COX-2 targeted treatment.

Development of Antioxidant COX-2 Inhibitors as Radioprotective Agents for Radiation Therapy-A Hypothesis-Driven Review.

Radiation therapy (RT) evolved to be a primary treatment modality for cancer patients. Unfortunately, the cure or relief of symptoms is still accompanied by radiation-induced side effects with severe acute and late pathophysiological consequences. Inhibitors of cyclooxygenase-2 (COX-2) are potentially useful in this regard because radioprotection of normal tissue and/or radiosensitizing effects on tumor tissue have been described for several compounds of this structurally diverse class. This review aims to substantiate the hypothesis that antioxidant COX-2 inhibitors are promising radioprotectants because of intercepting radiation-induced oxidative stress and inflammation in normal tissue, especially the vascular system. For this, literature reporting on COX inhibitors exerting radioprotective and/or radiosensitizing action as well as on antioxidant COX inhibitors will be reviewed comprehensively with the aim to find cross-points of both and, by that, stimulate further research in the field of radioprotective agents.

Optical imaging of COX-2: Studies on an autofluorescent 2,3-diaryl-substituted indole-based cyclooxygenase-2 inhibitor

This study aimed at in vivo visualization of cyclooxygenase-2 (COX-2) by optical imaging using a representative compound of a class of autofluorescent 2,3-diaryl-substituted indole-based selective COX-2 inhibitors (2,3-diaryl-indole coxibs). COX-2 was successfully visualized in mice models with phorbol myristate ester (TPA)-induced inflammation or bearing xenografted human melanoma cells by 2-[4-(aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-indole (C1). COX-2 protein expression in both TPA-induced inflammatory sites and human melanoma xenografts was confirmed by immunoblotting. Control experiments using surrogate markers, sham injections, and non-COX-2 expressing melanoma cells further confirmed specificity of tissue association of C1. The merging of therapeutic and diagnostic properties of 2,3-diaryl-indole coxibs may widen the range of applications of COX-2-targeted treatment, e.g., for in situ-guided surgery and ex vivo diagnostics.

Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20K in melanoma cells in vitro.

This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors. COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K. COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1. Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.

Protective effects of 2,3-diaryl-substituted indole-based cyclooxygenase-2 inhibitors on oxidative modification of human low density lipoproteins in vitro

It has been suggested that 2,3-diaryl-substituted indole-based cyclooxygenase-2 (COX-2) inhibitors (2,3-diaryl-indole coxibs) do not only appear as potent anti-inflammatory agents but also show the ability to scavenge reactive oxygen species (ROS). This led to the hypothesis that 2,3-diaryl-indole coxibs also may act as potent inhibitors of oxidative modification of low-density lipoprotein (LDL), which is considered a key factor in atherogenesis. The aim of this study was to explore i) the reactivity of a series of new synthesized 2,3-diaryl-indoles with several well characterized LDL oxidation systems and ii) subsequent effects on an inflammatory/atherogenic microenvironment. The results demonstrate that under the present experimental conditions 2,3-diaryl-indoles showed potent ROS scavenging activity and were able to markedly inhibit LDL oxidation. Subsequently, this led to a substantial decrease of modified LDL uptake by scavenger receptors in THP-1 macrophages in vitro and in rats in vivo. Moreover, modified LDL-mediated monocyte/neutrophil adhesion to endothelial cells, macrophage NFκB activation, as well as macrophage and endothelial cell cytokine release was diminished in vitro. The reduction of modified LDL-induced atherogenic effects by antioxidant 2,3-diaryl-indole coxibs may widen the therapeutic window of COX-2 targeted treatment.

Controlled immobilization of His-tagged proteins for protein-ligand interaction experiments using Ni2+-NTA layer on glass surfaces

Gold surfaces functionalized with nickel-nitrilotriacetic acid (Ni²⁺-NTA) as self-assembled monolayers (SAM) to immobilize histidine (His)-tagged biomolecules are broadly reported in the literature. However, the increasing demand of using microfluidic systems and biosensors takes more and more advantage on silicon technology which provides dedicated glass surfaces and substantially allows cost and resource savings. Here we present a novel method for the controlled oriented immobilization of His-tagged proteins on glass surfaces functionalized with a Ni²⁺-NTA layer. Exemplarily, the protein pattern morphology after immobilization on the Ni²⁺-NTA layer of His6-tagged soluble receptor for advanced glycation endproducts (sRAGE) was investigated and compared to non-oriented immobilization of sRAGE on amino SAM by using scanning electron microscopy (SEM). Moreover, we demonstrated interaction of immobilized sRAGE with three structurally different ligands, S100A12, S100A4, and glycated low density lipoproteins (glycLDL), by means of peak-force tapping atomic force microscopy (PF-AFM). We showed a clear discrimination of different protein-ligand orientations by differential height measurements.