Synthesis and Study of Building Blocks with Dibenzo[b,f]oxepine: Potential Microtubule Inhibitors.

The synthesis of biphenylmethoxydibenzo[b,f]oxepine or photoswitchable fluorinated dibenzo[b,f]oxepine derivatives with one or three azo bonds, potential microtubule inhibitors, is described. Our studies provide a concise method for constructing derivatives containing the dibenzo[b,f]oxepine skeleton. An analysis of products was run using experimental and theoretical methods. Next, we evaluated the E/Z isomerization of azo-dibenzo[b,f]oxepine derivatives, which could be photochemically controlled using visible-wavelength light.

Development of 5-fluorouracil-dichloroacetate mutual prodrugs as anticancer agents.

5-Fluorouracil (5-FU) is one of the most widely applied chemotherapeutic agents with a broad spectrum of activity. However, despite this versatile activity, its use poses many limitations. Herein, novel derivatives of 5-FU and dichloroacetic acid have been designed and synthesized as a new type of codrugs, also known as mutual prodrugs, to overcome the drawbacks of 5-FU and enhance its therapeutic efficiency. The stability of the obtained compounds has been tested at various pH values using different analytical techniques, namely HPLC and potentiometry. The antiproliferative activity of the new 5-FU derivatives was assessed in vitro on SK-MEL-28 and WM793 human melanoma cell lines in 2D culture as well as on A549 human lung carcinoma, MDA-MB-231 breast adenocarcinoma, LL24 normal lung tissue, and HMF normal breast tissue as a multicellular 3D spheroid model cultured in standard (static) conditions and with the use of microfluidic systems, which to a great extent resembles the in vivo environment. In all cases, new mutual prodrugs showed a higher cytotoxic activity toward cancer models and lower to normal cell models than the parent 5-FU itself.

Systematic Studies on Anti-Cancer Evaluation of Stilbene and Dibenzo[b,f]oxepine Derivatives.

Cancer is one of the most common causes of human death worldwide; thus, numerous therapies, including chemotherapy, have been and are being continuously developed. In cancer cells, an aberrant mitotic spindle-a microtubule-based structure necessary for the equal splitting of genetic material between daughter cells-leads to genetic instability, one of the hallmarks of cancer. Thus, the building block of microtubules, tubulin, which is a heterodimer formed from α- and ß-tubulin proteins, is a useful target in anti-cancer research. The surface of tubulin forms several pockets, i.e., sites that can bind factors that affect microtubules' stability. Colchicine pockets accommodate agents that induce microtubule depolymerization and, in contrast to factors that bind to other tubulin pockets, overcome multi-drug resistance. Therefore, colchicine-pocket-binding agents are of interest as anti-cancer drugs. Among the various colchicine-site-binding compounds, stilbenoids and their derivatives have been extensively studied. Herein, we report systematic studies on the antiproliferative activity of selected stilbenes and oxepine derivatives against two cancer cell lines-HCT116 and MCF-7-and two normal cell lines-HEK293 and HDF-A. The results of molecular modeling, antiproliferative activity, and immunofluorescence analyses revealed that compounds 1a, 1c, 1d, 1i, 2i, 2j, and 3h were the most cytotoxic and acted by interacting with tubulin heterodimers, leading to the disruption of the microtubular cytoskeleton.

Synthesis and Study of Dibenzo[b, f]oxepine Combined with Fluoroazobenzenes-New Photoswitches for Application in Biological Systems.

Dibenzo[b, f]oxepine derivatives are an important scaffold in natural, medicinal chemistry, and these derivatives occur in several medicinally relevant plants. Two dibenzo[b, f]oxepines were selected and connected with appropriate fluorine azobenzenes. In the next step, the geometry of E/Z isomers was analyzed using density functional theory (DFT) calculations. Then the energies of the HOMO and LUMO orbitals were calculated for the E/Z isomers to determine the HOMO-LUMO gap. Next, modeling of the interaction between the obtained isomers of the compounds and the colchicine α and ß-tubulin binding site was performed. The investigated isomers interact with the colchicine binding site in tubulin with a part of the dibenzo[b, f]oxepine or in a part of the azo switch, or both at the same time. Based on the UV-VIS spectra, it was found that in the case of compounds with an azo bond in the meta position, the absorption bands n→π* for both geometric isomers and their separation from π→π* are visible. These derivatives therefore have the potential to be used in photopharmacology.

Synthesis and Investigations of Building Blocks with Dibenzo[b,f] Oxepine for Use in Photopharmacology.

The synthesis of photoswitchable azo-dibenzo[b,f]oxepine derivatives and microtubule inhibitors were described. Subsequently, we examined the reaction of methoxy derivative 3-nitrodibenzo[b,f]oxepine with different aldehydes and in the presence of BF3·OEt2 as a catalyst. Our study provided a very concise method for the construction of the azo-dibenzo[b,f]oxepine skeleton. The analysis of products was run using experimental and theoretical methods. Next, we evaluated the E/Z isomerization of azo-dibenzo[b,f]oxepine derivatives, which could be photochemically controlled using visible-wavelength light.

Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells.

Microtubules (MTs), highly dynamic structures composed of α- and ß-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule-MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the consequences of such a phenomenon, including drug resistance development.

Studies on the Synthesis of Endocyclic Enol Lactones via a RCM of Selected Vinyl Esters.

The novel and efficient approach toward the synthesis of endocyclic enol lactones was devised based on ring-closing metathesis of unsaturated carboxylic acids vinyl esters. Systematic studies revealed that vinyl esters are suitable substrates for RCM reaction. The developed methodology offers an easy route for synthetically challenging target molecules with different functional groups and substitution. We have also demonstrated that vinyl esters of cyclic carboxylic acids can be successfully applied for domino ring-opening ring-closing metathesis sequences.

Evaluation of Pseudoenantiomeric Mixed Carbonates as Efficient Fluorogenic Probes for Enantioselectivity Screening.

We report mixed carbonates as enzyme substrates and demonstrate their application as fluorogenic probes for lipase and esterase enantiopreference screening. By the application of pseudoenantiomers with distinct fluorescence behaviors, it is possible to evaluate the activity and enantiopreference of hydrolytic enzymes. In order to validate our method, enantioselectivities calculated from fluorometric measurements were compared with the results obtained from larger-scale kinetic resolution.

Mixed carbonates as useful substrates for a fluorogenic assay for lipases and esterases.

Mixed carbonates of 7-hydroxy-4-methylcoumarin were shown to be a new class of probe for fluorogenic assays. They are promising substrates for fingerprinting enzyme hydrolytic activity, and proved particularly useful because of the low level of nonspecific degradation and ease of synthesis. They are highly relevant for screening lipase and esterase libraries. These advantages make umbelliferyl carbonates highly suitable substrates for high-throughput screening. Moreover, we report the use of chiral fluorogenic carbonates as enantiopreference probes.

First-in-Class Colchicine-Based Visible Light Photoswitchable Microtubule Dynamics Disrupting Agent.

Compounds that disrupt microtubule dynamics, such as colchicine, paclitaxel, or Vinca alkaloids, have been broadly used in biological studies and have found application in clinical anticancer medications. However, their main disadvantage is the lack of specificity towards cancerous cells, leading to severe side effects. In this paper, we report the first synthesis of 12 new visible light photoswitchable colchicine-based microtubule inhibitors AzoCols. Among the obtained compounds, two photoswitches showed light-dependent cytotoxicity in cancerous cell lines (HCT116 and MCF-7). The most promising compound displayed a nearly twofold increase in potency. Moreover, dissimilar inhibition of purified tubulin polymerisation in cell-free assay and light-dependent disruption of microtubule organisation visualised by immunofluorescence imaging sheds light on the mechanism of action as microtubule photoswitchable destabilisers. The presented results provide a foundation towards the synthesis and development of a novel class of photoswitchable colchicine-based microtubule polymerisation inhibitors.

Development of novel derivatives of stilbene and macrocyclic compounds as potent of anti-microtubule factors.

Microtubules (composed of α- and ß-tubulin heterodimers) ubiquitous cellular polymers are important components of the cytoskeleton and play diverse roles within the cell, such as maintenance of cell structure, protein trafficking or chromosomal segregation during cell division. The polymers of tubulin play a pivotal role in mitosis and are regarded as an excellent target for chemotherapeutic agents to treat cancer. This review presents a brief overview of the synthesis and mechanism of action of new compounds targeting the dynamic of microtubule - tubulin polymerization/depolymerization. It is divided into the following parts: section I concerns targeting microtubules- tubulin-binding drugs derivatives of stilbene. In section II there are presented photoswitchable inhibitors of microtubule dynamics. Section III concerns using macrocyclic compounds as tubulin inhibitors. In this review, the authors focused primarily on reports produced inthe last five years and the latest strategies in this field.

The stilbene and dibenzo[b,f]oxepine derivatives as anticancer compounds.

In the present study, the synthesis and cytotoxic effect of six stilbenes and three oxepine derivatives against two cancerous - HeLa and U87, and two normal - EUFA30 and HEK293 cell lines has been reported. The results of cytotoxic assay and flow cytometry analysis revealed that compounds 9-nitrobenzo[b]naphtho[1,2-f]oxepine (4), (E)-3,3',4,4',5,5'-hexamethoxystilbene (6) and 4-hydroxy-2',4'-dinitrostilbene (8) were the most active and their interaction with tubulin (crystal structure from PDB) has been analyzed by computer molecular modeling. Molecular docking of these compounds on colchicine binding site of the tubulin indicates the interaction of (4), (6) and (8) with tubulin. The compound (4) could interact stronger with tubulin, relative to colchicine, however, with no selectivity of action against cancer and normal cells. Conversely, compounds (6) and (8) interact more weakly with tubulin, relative to colchicine but they act more selectively towards cancerous versus normal cell lines. Obtained results proved that the compounds that are the most active against cancerous cells operate through tubulin binding.