Microwave Irradiation: Alternative Heating Process for the Synthesis of Biologically Applicable Chromones, Quinolones, and Their Precursors.

Microwave irradiation has become a popular heating technique in organic synthesis, mainly due to its short reaction times, solventless reactions, and, sometimes, higher yields. Additionally, microwave irradiation lowers energy consumption and, consequently, is ideal for optimization processes. Moreover, there is evidence that microwave irradiation can improve the regioselectivity and stereoselectivity aspects of vital importance in synthesizing bioactive compounds. These crucial features of microwave irradiation contribute to its inclusion in green chemistry procedures. Since 2003, the use of microwave-assisted organic synthesis has become common in our laboratory, making our group one of the first Portuguese research groups to implement this heating source in organic synthesis. Our achievements in the transformation of heterocyclic compounds, such as (E/Z)-3-styryl-4H-chromen-4-ones, (E)-3-(2-hydroxyphenyl)-4-styryl-1H-pyrazole, (E)-2-(4-arylbut-1-en-3-yn-1-yl)-4H-chromen-4-ones, or (E)-2-[2-(5-aryl-2-methyl-2H-1,2,3-triazol-4-yl)vinyl]-4H-chromen-4-ones, will be discussed in this review, highlighting the benefits of microwave irradiation use in organic synthesis.

Visible-Light-Activated Quinolone Carbon-Monoxide-Releasing Molecule: Prodrug and Albumin-Assisted Delivery Enables Anticancer and Potent Anti-Inflammatory Effects.

The delivery of controlled amounts of carbon monoxide (CO) to biological targets is of significant current interest. Very few CO-releasing compounds are currently known that can be rigorously controlled in terms of the location and amount of CO released. To address this deficiency, we report herein a new metal-free, visible-light-induced CO-releasing molecule (photoCORM) and its prodrug oxidized form, which offer new approaches to controlled, localized CO delivery. The new photoCORM, based on a 3-hydroxybenzo[ g]quinolone framework, releases 1 equiv of CO upon visible-light illumination under a variety of biologically relevant conditions. This nontoxic compound can be tracked prior to CO release using fluorescence microscopy and produces a nontoxic byproduct following CO release. An oxidized prodrug form of the photoCORM is reduced by cellular thiols, providing an approach toward activation in the reducing environment of cancer cells. Strong noncovalent affinity of the nonmetal photoCORM to albumin enables use of an albumin:photoCORM complex for targeted CO delivery to cancer cells. This approach produced cytotoxicity IC50 values among the lowest reported to date for CO delivery to cancer cells by a photoCORM. This albumin:photoCORM complex is also the first CO delivery system to produce significant anti-inflammatory effects when introduced at nanomolar photoCORM concentration.

Response to photo-oxidative stress of Pseudomonas aeruginosa PAO1 mutants impaired in different functions.

Clinicians often have to deal with infections that are difficult to control because they are caused by superbugs resistant to many antibiotics. Alternatives to antibiotic treatment include antimicrobial photodynamic therapy (aPDT). The photodynamic process causes bacterial death, inducing oxidative stress through the photoactivation of photosensitizer molecules in the presence of oxygen. No PDT-resistant bacteria have been selected to date, thus the response to photo-oxidative stress in non-phototrophic bacteria needs further investigation. The opportunistic pathogen Pseudomonas aeruginosa, in particular, has been shown to be more tolerant to PDT than other micro-organisms. In order to find any genetic determinants involved in PDT-tolerance, a panel of transposon mutants of P. aeruginosa PAO1 involved in the quorum sensing signalling system and membrane cytoplasmic transport were photoinactivated as part of this study. Two pseudomonas quinolone signalling (PQS) knock-out mutants, pqsH- and pqsC-, were as PDT-sensitive as the PAO1 wild-type strains. Two PQS hyperproducer variants, pqsA- and rsaL-, were shown to be more tolerant to photo-oxidative stress than the wild-type strain. In the pqsA- mutant, the hyperpigmentation due to the presence of phenazines could protect cells against PDT stress, while in rsaL- no pigmentation was detectable. Furthermore, a mutant impaired in an ATP-binding cassette transport involved in maintaining the asymmetry of the outer membrane was significantly more tolerant to photo-oxidative stress than the wild-type strain. These observations support the involvement of quorum sensing and the importance of the bacterial cell envelope when dealing with photo-oxidative stress induced by photodynamic treatment.

Exposure to phototoxic NSAIDs and quinolones is associated with an increased risk of melanoma.

PURPOSE: Ultraviolet radiation exposure is the most important exogenous risk factor for cutaneous malignancies. It is possible that phototoxic drugs promote the development of cutaneous melanoma (CM) by intensifying the effect of ultraviolet light on the skin. We investigated the association between the use of common systemic phototoxic drugs and development of CM. METHODS: This study was a case-control study in a Dutch population-based cohort. The drug dispensing data was obtained from PHARMO, a Dutch drug dispensing and hospital admissions registry, and linked to PALGA, the nationwide pathology network of the Netherlands. The cases were patients diagnosed with pathologically confirmed primary CM between 1991 and 2004. Controls were sampled from the PHARMO population. Exposure to systemic phototoxic drugs was measured and included antimicrobial agents, diuretics, antipsychotic drugs, antidiabetic drugs, cardiac drugs, antimalarials and nonsteroidal anti-inflammatory drugs (NSAIDs). A multivariate conditional logistic regression analysis was performed to study the association between exposure to phototoxic drugs and CM. RESULTS: The study population included 1,318 cases and 6,786 controls. Any phototoxic drug during the study period was dispensed for 46 % of the cases and 43 % of the controls (p = 0.012). The use of quinolones [odds ratio (OR) 1.33, 95 % confidence interval (CI) 1.01-1.76] and propionic acid derivative NSAIDs (OR 1.33, 95 % CI 1.14-1.54) had a positive association with CM. CONCLUSIONS: Our study shows that the use of phototoxic drugs is associated with an increased risk of developing CM. Even a short-term use of phototoxic quinolones and propionic acid derivative NSAIDs may increase the risk for CM. Patient education to promote sun-protective behaviour is essential to avoid immediate adverse effects and possible long-term effects of phototoxic drugs.

UV photolysis for accelerated quinoline biodegradation and mineralization.

Sequentially and intimately coupled photolysis with biodegradation were evaluated for their ability to accelerate quinoline-removal and quinoline-mineralization kinetics. UV photolysis sequentially coupled to biodegradation significantly improved biomass-growth kinetics, which could be represented well by the Aiba self-inhibition model: UV photolysis increased the maximum specific growth rate (µ max) by 15 %, and the inhibition constant (K SI) doubled. An internal loop photo-biodegradation reactor (ILPBR) was used to realize intimately coupled photolysis with biodegradation. The ILPBR was operated with batch experiments following three protocols: photolysis alone (P), biodegradation alone (B), and intimately coupled photolysis and biodegradation (P&B). For P&B, the maximum quinoline removal rate (r max) increased by 9 %, K SI increased by 17 %, and the half-maximum-rate concentration (K S) decreased by 55 %, compared to B; the composite result was a doubling of the quinoline-biodegradation rate for most of the concentration range tested. The degree of mineralization was increased by both forms of photolysis coupled to biodegradation, and the impact was greater for intimate coupling (18 % increase) than sequential coupling (5 %). The benefits of UV photolysis were greater with intimate coupling than with sequential coupling due to parallel transformation by biodegradation and photolysis.

Retained Functionality of Atherosclerotic Human Arteries Following Photoactivated Linking of the Extracellular Matrix by Natural Vascular Scaffolding Treatment.

In this study, we investigated natural vascular scaffolding (NVS) treatment on vascular functionality using freshly isolated human popliteal arteries in vitro. Arteries were exposed to intraluminal NVS treatment consisting of a compound (4 amino-1,8-naphthalimide) photoactivated by a 450-nm light-emitting light fiber placed inside the artery. This procedure results in covalent linking between the extracellular matrix proteins to achieve a larger vessel diameter post-angioplasty and minimizing elastic recoil. Immediately following NVS treatment, rings were cut from the treated arteries and mounted in organ baths for contractility testing in response to U46619 and sodium nitroprusside. We also investigated the effect of NVS treatment on IL-6 cytokine release from vascular rings following a 4-h organoculture post-NVS treatment. Based on our results, we conclude that exposure of the vessels to NVS treatment does not adversely affect the contractile responsiveness of the vascular smooth muscle and exerts no pro-inflammatory effect. Graphical abstract.

Androgen receptor-mediated apoptosis is regulated by photoactivatable androgen receptor ligands.

We have studied nonsteroidal ligands of the human androgen receptor (hAR) and have shown elsewhere that when photoactivated by visible light they collide with O2 to yield singlet oxygens (1O2) in vitro. Here we report cell killing after brief light activation (405 nm) of 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ) in human prostate tumor cells. TDPQ/AR complexes were required for the death response because AR-positive LNCaP cells were killed, whereas AR-negative PC-3 cells were resistant. Excess dihydrotestosterone (DHT) blocked the TDPQ effect when the two were added together; irradiation of cells containing DHT alone had no effect. When LNCaP AR expression was suppressed using small interfering oligonucleotides targeting AR, photocytotoxicity was diminished. Conversely, stable transfection of hAR into PC-3 cells made the cells photosensitive to TDPQ. Similar results were obtained using a structural isomer of TDPQ, and also the synthetic steroidal AR ligand R1881. Cell death occurred via apoptosis as demonstrated by annexin V immunostaining, nuclear condensation, and caspase inhibition. Death involved oxidative stress, because it was prevented by addition of the antioxidant ascorbic acid during photoactivation. Detection of elevated levels of 8-hydroxy-2'-deoxyguanosine in nuclei of irradiated cells indicated oxidative DNA damage. Apoptosis spread into adjacent nonirradiated cells by direct cell-cell contacts, indicative of a bystander effect. Other photoactivatable ligands are described, implying a general method for ablation of cells bearing specific nuclear hormone receptors.

Lewis-acid-catalyzed photodimerization of coumarins and N-methyl-2-quinolone.

The BF(3)-catalyzed photodimerization of parent coumarin (1), three 6-alkylcoumarins (2-4) and N-methyl-2-quinolone (5) in dichloromethane was studied by time-resolved UV-vis spectroscopy. The lowest triplet state properties in the absence and presence of BF(3) were outlined, in particular the effect of self-quenching which initiates dimerization. The quantum yield of intersystem crossing (Phi(isc)) of 1-4 increases with BF(3) concentration, approaching Phi(isc) = 0.3. Phi(isc) and the relative quantum yield of dimerization go along, thereby favoring an overall triplet mechanism in both the direct and BF(3)-catalyzed photodimerization. The product ratio of 5 changes strongly with the BF(3) concentration from 100%anti-hh for the free quinolone to 100%syn-ht for the 1:1 complex.

Cell- and Tissue-Based Proteome Profiling and Bioimaging with Probes Derived from a Potent AXL Kinase Inhibitor.

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Recently, our group has developed a lead compound, 9im, capable of displaying potent and specific inhibition of AXL. To further identify the cellular on/off targets, in this study, competitive affinity-based proteome profiling was carried out, leading to the discovery of several unknown cellular targets such as BCAP31, LPCAT3, POR, TM9SF3, SCCPDH and CANX. In addition, trans-cyclooctene (TCO) and acedan-containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of AXL in various biosystems.

Structure and medium effects on the photochemical behavior of nonfluorinated quinolone antibiotics.

The photophysical behavior of the quinolone antibiotics, oxolinic (OX), cinoxacin (CNX) and pipemidic (PM) acids was studied as a function of pH and solvent properties. The ground state of these compounds exhibits different protonated forms, which also exist in the first excited states. Theoretical calculations of the Fukui indexes allowed to assigning the different protonation equilibria. The pK values indicate that the acidity of the 3-carboxylic and 4-carbonyl groups increases with the N-atom at position 2 in CNX. It has been found that fluorescence properties are strongly affected by pH, the more fluorescent species is that with protonated carboxylic acid, protonated species at the carbonyl group and the totally deprotonated form present very low fluorescence. The fluorescence behavior also depends on the chemical structure of the quinolone and on the solvent properties. The analysis of the solvent effect on the maximum and the width of the fluorescence band of OX, using the linear solvent-energy relation solvatochromic equation, indicates that the polarizability and hydrogen bond donor ability are the parameters that condition the spectral changes. The hydrogen bond acceptor ability of the solvents also contributes to the spectral shifts of CNX. The compound bearing the piperazinyl group at the position 7, PM only is fluorescent in high protic solvents. These results are discussed in terms of the competition between the intra- and intermolecular hydrogen bonds. The irradiation of OX, CNX and PM using 300 nm UV light led to a very low photodecomposition rate. Under the same conditions the nalidixic acid (NA), a structurally related quinolone, photodecomposes two orders of magnitude faster.

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine.

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC-MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5-400 ng/mL on PE Sciex API 3000 LC-MS/MS system. The assay intraday accuracy and precision were 92.1-104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299-307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739-752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.

Structure and medium-dependent photodecomposition of fluoroquinolone antibiotics.

The photochemical reactivity of four fluoroquinolone antibiotics is examined. For norfloxacin (NOR), enoxacin (ENX) and lomefloxacin (LOM), the only process occurring is defluorination (from position 6 for the first two drugs, from position 8 for the last one). The quantum efficiency is both structure and medium dependent (phi close to 0.5 both in water and in 0.1 M phosphate buffer for LOM; 0.01 for ENX and 0.004 for NOR in buffer, but more than an order of magnitude higher in neat water). Ofloxacin (OFL) is less light sensitive (phi 0.001) and undergoes, in part, reactions different from defluorination. The photoreaction involves heterolytic C-F bond fragmentation and its efficiency is determined by the internal charge-transfer character of the excited state (increasing in the series OFL < NOR < ENX < LOM according to the electronegativity of the substituent in position 8) and by the stabilization of the resulting aryl cation (larger for the 8-cation than for the 6-cation). The relevance of these data for the rationalization of the known phototoxicity of these drugs is discussed.

Photochemical decomposition of lomefloxacin in vitro and in vivo.

To obtain an idea of the photostability of Lomefloxacin (Lom) under in vivo conditions the compound was exposed to UV-A (310-360 nm) in PBS buffer pH 7.4. Exposure of 10 microg/ml of Lom in PBS pH 7.4 led to more than 50% decomposition within 10 min. Loss of the fluorine atom at C-8 and partial breakdown of the piperazine ring occurred. The only two photoproducts formed under these conditions were AEA, 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminoethyl-amino)-4-oxo-3-quinolinecarboxylic acid, and APA, 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminopropyl-amino)-4-oxo-3-quinolinecarboxylic acid. When Lom was exposed in whole blood in vitro, the same photochemical decomposition was observed in the plasma as in PBS buffer: APA and AEA were the only products. During UV-A exposure, Lom was shown to be taken up by the leukocytes. This process appeared to be less rapid during UV-A exposure than in the dark. As soon as UV-A exposure commenced, AEA and APA were found. As in the plasma, the total amount of Lom and the two photoproducts in the leukocytes was not significantly different from the amount of Lom found in unexposed cells at the same time point. The erythrocytes did not take up Lom, but exposure of whole blood to Lom and UV-A under the above conditions led to more than 7% haemolysis. Treatment of rats with a combination of Lom and UV-A demonstrated photodecomposition of Lom in vivo. In urine produced during exposure and by the irradiated rats during the twilight period after exposure, a considerable amount of AEA and APA was found. The blood plasma from rats exposed simultaneously to UV-A and Lom proved to contain AEA and APA and, in the leukocytes, APA. This was not the case with animals kept in twilight.

Enantioselective, intermolecular [2+2] photocycloaddition reactions of 3-acetoxyquinolone: total synthesis of (-)-pinolinone.

The natural product (-)-pinolinone was synthesised via a concise route (six steps, 17% overall yield) from 3-acetoxyquinolone, employing an enantioselective intermolecular [2+2] photocycloaddition as the key step.

Optical control of antibacterial activity.

Bacterial resistance is a major problem in the modern world, stemming in part from the build-up of antibiotics in the environment. Novel molecular approaches that enable an externally triggered increase in antibiotic activity with high spatiotemporal resolution and auto-inactivation are highly desirable. Here we report a responsive, broad-spectrum, antibacterial agent that can be temporally activated with light, whereupon it auto-inactivates on the scale of hours. The use of such a 'smart' antibiotic might prevent the build-up of active antimicrobial material in the environment. Reversible optical control over active drug concentration enables us to obtain pharmacodynamic information. Precisely localized control of activity is achieved, allowing the growth of bacteria to be confined to defined patterns, which has potential for the development of treatments that avoid interference with the endogenous microbial population in other parts of the organism. 

ESIPT-mediated photocycloadditions of 3-hydroxyquinolinones: development of a fluorescence quenching assay for reaction screening.

Irradiation of 1,2-dimethyl-3-hydroxyquinolinone (DMQ) leads to excited state intramolecular proton transfer (ESIPT) generating a 3-oxidoquinolinium species which undergoes [3 + 2] photocycloaddition with dipolarophiles. A parallel, fluorescence quenching assay using a microplate format has been developed to evaluate fluorescence quenching of this species with a range of dipolarophiles.

Characterization of intermediate compounds formed upon photoinduced degradation of quinolones by high-performance liquid chromatography/high-resolution multiple-stage mass spectrometry.

The paper deals with the photocatalytic transformation of two antibacterial agents, ofloxacin and ciprofloxacin, under simulated solar irradiation using titanium dioxide as photocatalyst. The investigation involved monitoring decomposition of the drugs, identifying intermediate compounds, assessing mineralization, and evaluating the toxicity of drug derivatives. High-resolution mass spectrometry was employed to assess evolution of the photocatalyzed process over time. Respectively 15 and 8 main species were identified after transformation of ofloxacin and ciprofloxacin. Through the full analysis of MS and MSn spectra and a comparison with parent drug fragmentation pathways, the different isomers were characterized. In the ofloxacin molecule, the initial transformation attacks are confined to the piperazine moiety and to the methyl groups, while the fluoroquinolone core is unmodified. Conversely, ciprofloxacin degradation involves two parts of the molecule: the piperazinic moiety and the quinolone moiety. All these intermediates are easily degraded and by 4 h mineralization is complete. Toxicity assays using Vibrio fischeri prove that neither ciprofloxacin nor its intermediates exhibit acute toxicity. In ofloxacin the secondary degradation products exhibit toxicity; a correlation exists between the evolution of the intermediate compounds and the toxicity connected to them.

Rufloxacin-induced photosensitization in yeast.

The fluoroquinolone Rufloxacin (RFX) is active as specific inhibitor of bacterial gyrase. The adverse effects of the photosensitization induced by fluoroquinolones are well known. A predominant type II photosensitizing activity of Rufloxacin has already been demonstrated on simpler models (free nucleosides, calf thymus DNA), whereas a cooperative mechanism was corroborated on more complex ones (plasmid and fibroblast). The purpose of this study is to examine the drug photocytoxicity in another complex cellular model, a wild-type eukaryotic fast-growing microorganism whose cultivation is cheap and easily managed, Saccharomyces cerevisiae. This work represents the first report of the potential photogenotoxicity of Rufloxacin. Particular emphasis was given to DNA modifications caused in yeast by the formation of Rufloxacin photomediated toxic species, such as hydrogen peroxide and formaldehyde. Drug phototoxicity on yeast was evaluated by measuring DNA fragmentation (single/double strand breaks) using single cell gel electrophoresis assay and 8-OH-dGuo, a DNA photooxidation biomarker, by HPLC-ECD. Cellular sensitivity was also assessed by cell viability test. The extra- and intracellular RFX concentration (as well as its main photoproduct) was verified by HPLC-MS, whereas the cytotoxic species were evaluated by colorimetric assays. The results confirm the phototoxicity of Rufloxacin on yeast cell and are in agreement with those previously obtained with human fibroblast and with simpler models used recently, and provide a clear link between DNA photosensitization and overall phototoxicity.

Photochemistry of 4-(2'-aminoethyl)quinolones: enantioselective synthesis of tetracyclic tetrahydro-1aH-pyrido[4',3':2,3]- cyclobuta[1,2-c] quinoline-2,11(3H,8H)-diones by intra- and intermolecular [2 + 2]-photocycloaddition reactions in solution.

Enantioselective [2 + 2]-photocycloaddition reactions on 4-(2'-aminoethyl)quinolones in solution were studied using the enantiomerically pure complexing agent 1 as source of chirality. The intermolecular reactions of fully N-protected substrates 5a-5c with different 2-alkyl-substituted acrylates 12-15 represent the first systematic study on the diastereoselectivity of their intermolecular [2 + 2]-photocycloadditions to unsymmetrically 1,1-disubstituted olefins (75-91% yield, d.r. = 58/42-95/5). N-Benzylic-protected photoproducts exo-16a/b-19a/b could easily be converted into lactams 20a/b-23a/b by a sequence of Boc deprotection and thermal lactamization (74-98% yield). Identical products 20a-22a were directly accessible by the intramolecular [2 + 2]-photocycloaddition of acrylic acid amides 2-4 (41-61% yield). The suitability of both pathways for an enantioselective reaction variant was proven (70-92% ee). Thus, tetracyclic lactams possessing the carbon framework C were obtained with good yields and enantioselectivities of up to 92% ee in intramolecular reactions. Comparative investigation of both routes showed that quinolone dimerization was the single most decisive factor preventing a complete chirality transfer. Functional group manipulations were successfully conducted with the primary photoproduct exo-17a. Finally, a new and unexpected type of benzylic hydrogen abstraction-radical cyclization reaction was discovered for substrate 5a, which explains the photochemical instability of substrates 2-5 under short wavelength irradiation (lambda = 300 nm).