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A promising antitumor xanthone derivative was optimized following a multidimensional approach that involved the synthesis of 17 analogues, the study of their lipophilicity and solubility, and the evaluation of their growth inhibitory activity on four human tumor cell lines. A new synthetic route for the hit xanthone derivative was also developed and applied for the synthesis of its analogues. Among the used cell lines, the HL-60 showed to be in general more sensitive to the compounds tested, with the most potent compound having a GI50 of 5.1 μM, lower than the hit compound. Lipophilicity was evaluated by the partition coefficient (K(p)) of a solute between buffer and two membrane models, namely liposomes and micelles. The compounds showed a logK(p) between 3 and 5 and the two membrane models showed a good correlation (r(2)=0.916) between each other. Studies concerning relationship between solubility and structure were developed for the hit compound and 5 of its analogues.

Our previous work has described a library of thioxanthones designed to have dual activity as P-glycoprotein modulators and antitumor agents. Some of these compounds had shown a significant cell growth inhibitory activity towards leukemia cell lines, without affecting the growth of non-tumor human fibroblasts. However, their effect in cell lines derived from solid tumors has not been previously studied. The present work aimed at: (i) screening this small series of compounds from an in-house library, for their in vitro cell growth inhibitory activity in human tumor cell lines derived from solid tumors; and (ii) initiate a study of the effect of the most potent compound on apoptosis. The tumor cell growth inhibitory effect of 27 compounds was first analysed in different human tumor cell lines, allowing the identification of a hit compound, TXA1. Its hydrochloride salt TXA1 HCl was then synthesized, to improve solubility and bioavailability. Both TXA1 and TXA1 HCl inhibited the growth of MCF-7, NCI-H460, A375-C5, HeLa, 786-O, Caki-2 and AGS cell lines. The effect of TXA1 HCl in MCF-7 cells was found to be irreversible and was associated, at least in part, with an increase in cellular apoptosis. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology, under the projects FEDER COMPETE FCOMP-01-0124-FEDER-015752, FCOMP-01-0124-FEDER-011057, NORTE-07-0162-FEDER-00018—“Contributos para o reforço da capacidade do IPATIMUP enquanto actor do sistema regional de inovação” and NORTE-07-0162-FEDER-000067—“Reforço e consolidação da capacidade infraestrutural do IPATIMUP para o sistema regional de inovação”, both supported by Programa Operacional Regional do Norte (ON.2—O Novo Norte), through FEDER funds under the Quadro de Referência Estratégico Nacional (QREN). This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. The authors also thank FCT for the grant of R.T. Lima (SFRH/BPD/68787/2010) and QREN for the grant of D. Sousa (NORTE-07-0124-FEDER-000023).

The benzopyran and dihydrobenzopyran moieties can be considered as "privileged motifs" in drug discovery being good platforms for the search of new bioactive compounds. These moieties are commonly found fused to the xanthonic scaffold belonging to the biologically important family of the generally designated prenylated xanthones. Several pyranoxanthones have shown promising antitumor activity and since most of them are from natural origin, the biosynthetic pathway only allows a particular pattern of substitution which limits their structural diversity and renders any broad structure-activity study hard to be established. Accordingly, with the aim of rationalizing the importance of the fused ring orientation and oxygenation pattern in pyranoxanthones, this study describes the synthesis of 14 new pyranoxanthones and evaluation of their cell growth inhibitory activity in four human tumor cell lines as well as their lipophilicity in two membrane models. This systematic approach allowed establishing structure-activity and structure-lipophilicity relationships for the obtained compounds in combination with 6 previously described compounds. From this work an angular pyranoxanthone scaffold emerged as particularly promising, presenting a potent cell growth inhibitory activity and suitable drug-like lipophilicity.

(1) Background: Our previous studies unveiled the hit thioxanthone TXA1 as an inhibitor of,P-glycoprotein (drug efflux pump) and of human tumor cells growth, namely of melanoma cells. SinceTXA1 is structurally similar to lucanthone (an autophagy inhibitor and apoptosis inducer) and to N10-substituted phenoxazines (isosteres of thioxanthones, and autophagy inducers), this study aimed at further assessing its cytotoxic mechanism and evaluating its potential as an autophagy modulator in A375-C5 melanoma cells; (2) Methods: Flow cytometry with propidium iodide (PI) for cell cycle profile analysis; Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry with Annexin V/PI labeling and Western blot for apoptosis analysis were conducted. A pharmacophore approach was used for mapping TXA1 onto pharmacophores for autophagy induction. Autophagy analyses included transmission electron microscopy for visualization of autophagic structures, fluorescence microscopy for observation of monodansylcadaverine (MDC) staining, pattern of LC3 expression in the cells and acridine orange staining, and Western blot for autophagic proteins expression; (3) Results: TXA1 induced autophagy of melanoma cells at the GI50 concentration (3.6 μM) and apoptosis at twice that concentration. Following treatment with TXA1, autophagic structures were observed, together with the accumulation of autophagosomes and the formation of autophagolysosomes. An increase in LC3-II levels was also observed, which was reverted by 3-methyladenine (3-MA) (an early stage autophagy-inhibitor) but further increased by E-64d/pepstatin (late-stage autophagy inhibitors). Finally, 3-MA also reverted the effect of TXA1 in cellular viability; (4) Conclusion: TXA1 decreases the viability of melanoma cells by modulation of autophagy and may, therefore, serve as a lead compound for the development of autophagy modulators with antitumor activity. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274)”. The work was also funded by ERDF, COMPETE, and FCT under the projects PTDC/SAU-OSM/101437/2008, PTDC/MAR-BIO/4694/2014, and INNOVMAR—reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR. The authors also thank: FCT for D. Sousa and R.T. Lima grants (PTDC/SAU-FCT/100930/2008 and SFRH/BPD/68787/2010, respectively), QREN for D. Sousa grant (NORTE-07-0124-FEDER-000023).