Simple avarone mimetics as selective agents against multidrug resistant cancer cells

In this work, synthesis of alkylamino and aralkylamino derivatives of sesquiterpene quinone avarone and its model compound tert-butylquinone was described. For all obtained derivatives biological activity was studied. Cytotoxic activity of the synthesized derivatives towards multidrug resistant MDR human non-small cell lung carcinoma NCI-H460/R cells, their sensitive counterpart NCI-H460 and human normal keratinocytes (HaCaT) as well as detection of cell death superoxide anion generation were investigated. Antimicrobial activity towards Gram positive and Gram negative bacteria and fungal cultures was determined. The results showed that strong cytotoxic activity toward cancer cells was improved with simple avarone mimetics. Some derivatives were selective towards MDR cancer cells. The most active derivatives induced apoptosis in both cancer cell lines, but not in normal cells. Superoxide production was induced by 2,6-disubstituted compounds in MDR cancer cells and not by less active 2,5-disubstituted compounds and was accompanied by the collapse of the mitochondrial transmembrane potential. Two tert-butylquinone derivatives were particularly selective towards MDR cancer cells. Some tert-butylquinone derivatives exhibited a strong antimicrobial activity.

Biological activity of quinones is dual. It is a combination of Michael addition to enone system and generation of oxygen reactive species (ROS) in redox cycle between quinone and its hydroquinone counterpart via semiquinone anion radical. Studies have shown that TBQ at a low dose induces DNA damage by forming 8-hydroxydeoxyguanosine due to generation of reactive oxygen species (ROS) [22]. TBHQ is O 2 .scavenger with IC 50 of 18.1 μM [23,24].
In this work, a series of of alkylamino and aralkylamino derivatives of avarone and TBQ were synthesized, and their cytotoxic activity was tested against three cell lines, non-small cell lung cancer, both sensitive and MDR, NCI-H460 and NCI-H460/R, respectively, and normal human keratinocytes HaCaT. The type of cell death was discriminated by AV/PI staining, while DHE fluorescent probe was employed to assess superoxide anion generation ability. Mitochondrial transmembrane potential (ΔΨm) was also assessed. Cyclic voltammetry was used to measure half-wave potentials of synthesized compounds in two-stage redox system quinone-semiquinone radical-hydroquinone. In addition, antibacterial and antifungal activities were determined.

Results and Discussion.
Hydroquinones (tert-butylhydroquinone and avarol) were oxidized by silver(I) oxide to corresponding quinones 1 and 2. Quinones were treated with amines to yield two regioisomers of aminoquinones 3a-i and 4a-i. The reaction steps are addition of an amine to form the substituted hydroquinone intermediate, which is immediately oxidized by the excess of the starting quinone.
With tert-butylquinone 2,6-disubstituted quinone product by far dominated 2,5-disubstituted product in the mixture, while with avarone the excess of 2,6-product was much less pronounced, so that with phenethyl amine the major product was 2,5-product. The exception in the synthetic route is preparation of ethylamino derivatives 3b and 4b, which were obtained in the reaction of the quinone with diethylamine, in which the initial addition product undergoes Hoffman-like elimination [25] which results in low yields of products.
All 3 derivatives display long-range W coupling between quinone protons with 4 J around 2 Hz, resulting with signals of these hydrogens to be dublets. On the other hand, 4 derivatives' quinone protons are five bonds away and their long-range coupling is too small to be detected, resulting in their signals to be singlets. Additionally, experimentally measured 13  For all the synthesized compounds, half-wave potentials were recorded at glassy carbon disk (3mm diameter) in dimethyl sulfoxide against silver wire immersed in electrolyte solution containing 0.01 M silver ions as the reference electrode, and ferrocene as reference compound.
Results are given in Table 1 and Fig. 1.
Cyclic voltammograms of avarone amino derivatives show typical behavior of quinone/hydroquinone redox pair. Two waves, corresponding to reversible or quasireversible one electron processes were observed. The reversibility of the first wave attributed to quinone/semiquinone anion radical redox system, was not perturbed by derivatization. Similarly, second wave assigned to semiquinone anion radical/hydroquinone dianion redox system behaves as quasireversible.
As expected, amino derivatization of avarone moiety led to negative shift of first reduction peak potentials with respect to avarone. tert-Butylquinone derivatives were reduced at slightly more negative potential, due to a greater electron donating ability of tert-butyl group. Aralkyl derivatives were reduced at a slightly less negative potential probably due to a slight electron withdrawing effect of phenyl group. Changes in length of the side chain did not produce significant effect on the redox properties, as expected. Finally, 2,5-disubstituted derivatives were always, except for the small methylamino substituent, reduced at a more negative potential.
Consequently, similar behavior of avarone and its tert-butyl analogue justifies application of the latter as model system of avarone.
When comparing activities of tert-butylquinone derivatives with avarone counterparts, it can be noticed that the former ones are generally more selective to cancer cell lines. Among TBQ derivatives aralkyl ones showed the best activity and selectivity to both sensitive and MDR cancer cells. Within the avarone series, large alkylamino derivatives were less active, probably due to excessively high log P. According to cytotoxicity evaluation, the most promising derivatives are: 2-tert-butyl-6-(phenethylamino)-1,4-benzoquinone (3f), 3'-(butylamino)avarone (3g) and 3'-(phenethylamino)avarone (3i) due to their high activity and good selectivity to the MDR cancer cell line ( Table 2). For comparison, parent compounds TBQ (1) and avarone (2) were less potent than their derivatives, while cisplatin (CDDP), an FDA-approved drug for nonsmall cells lung carcinoma treatment [26], showed strong cytotoxicity towards MDR cancer cells, but even stronger cytotoxicity towards normal human keratinocytes (Table 2).
Then, the type of cell death induced by 25 M of tert-butyl compounds 3d-4d, 3f-4f and by their sesquiterpene counterparts 3h-4h, 3i-4i was compared to parent compounds 1, 2 and control compound CDDP (Table 3, Fig. S2). The results analyzed after 72 h showed the highest increase in necrotic cells (AV-PI+). tert-Butyl compounds, particularly 3f, significantly increased a portion of apoptotic cells (AV+PI-and AV+PI+). Therefore, it is reasonable to assume that increase in fraction of dead cells after 72 h is a consequence of apoptotic cell death in case of tert-butyl compounds. On the contrary, necrosis seems to be the type of death induced by avarone derivatives 3h and 3i (Figs. S2 and S4). Also, with tert-butyl compounds, 2,6-derivatives (3d, 3f and 3i) induced a significantly higher fraction of apoptotic cells than 2,5-derivatives (4d, 4f and 4i). Selectivity towards cancer cells was additionally confirmed with 3d and 3f, while 3f was considerably more active against MDR cancer cells ( showed pro-apoptotic activity against all three cell lines showing no selectivity to cancer cells (Table 3, Fig. S3).
In addition to genomic DNA damage, many anticancer drugs act on multiple cellular levels affecting different organelles and signaling pathways. One of the proposed mechanisms behind their toxicity is the generation of ROS, which mostly affect the mitochondrial function [27].
Quinones are generally thought to exhibit their toxicity through oxygen activation by redox cycling and alkylation of essential macromolecules [28]. However, their biological effect can be cell type-dependent as it was reported that p-benzoquinone cytotoxicity correlated with ROS formation in primary rat hepatocytes, but not in PC12 cells. Furthermore, tert-butylhydroquinone was shown to exhibit anticancer effect through the ability to induce phase II xenobiotic metabolizing enzymes via an Nrf2-dependent pathway [29].
Therefore, in this work the potential of quinone derivatives to generate superoxide anions in resistant NCI-H460/R cells was examined by dihydroethidium (DHE) staining (Fig. 2).
Mean DHE fluorescence intensity showed significant potential of 2,6-disubstituted derivatives (3d, 3h, 3f and 3i) to generate superoxide anions in MDR cancer, while treatment with 2,5disubstituted derivatives (4d, 4h, 4f and 4i) was not prooxidative compared to untreated control ( Fig. 2A). This effect was more pronounced with avarone derivatives (3h and 3i). The higher activity of 2,6-disubstituted compounds cannot be directly ascribed to differences of redox properties, since there is no significant difference in their electrochemical parameters (E 0 1 is more negative for 2,5-disubstituted compounds for only 0.01-0.02 V). Therefore, the elevation of ROS levels could be the consequence of the antioxidant enzymes activity modulation. As described previously [30], MDR cancer cells (NCI-H460/R) have lower antioxidant capacity than their corresponding sensitive counterpart (NCI-H460). Particularly, the expression of MnSOD enzyme (responsible for superoxide anion metabolism) is significantly decreased in MDR cancer cells.
Consequently, 3d, 3f and 3h showed different pattern of action in sensitive NCI-H460 by lowering the superoxide anion content ( Fig. 2A) which is in accordance with previous findings that TBHQ acts as O 2 .scavenger [23]. A putative target responsible for different mode of action in MDR cancer cells and assumed as a mechanism of selectivity towards MDR cancer cells remains to be identified, which will be a subject of further studies. In normal cells, TBQ derivatives 3d and 3f did not change superoxide anion content, while corresponding avarone derivatives 3h and 3i increased its production although showing weaker pro-oxidative effect than that observed in MDR cancer cells ( Fig. 2A). Control compound 2 was pro-oxidative in MDR cancer cells, while CDDP induced increase in O 2 .production in all tested cell lines ( Fig. 2A).
The representative flow-cytometric profile of 3d and 3f-induced DHE fluorescence is shown in Besides regulation of fundamental cellular processes such as respiration and oxidative phosphorylation, mitochondria also provide signals that trigger apoptosis [31]. Cytochrome c release from mitochondria is a central event in apoptosis initiation, which induces the assembly of the apoptosome required for activating downstream caspases [32,33]. Mitochondrial membrane potential (∆Ψm) is vital for ATP generation and major loss of ∆Ψm is generally followed by substantial cell death. ∆Ψm and ROS levels give valuable insight into the physiological condition of the cell and are good indicators of mitochondrial dysfunction and oxidative stress. Several p-benzoquinones were reported to disrupt mitochondrial membrane potential in rat hepatocytes and ROS formation in PC12 cells [28]. tert-Butylhydroquinone and 2tert-butyl-1,4-benzoquinone were shown to display a strong anticancer potential [29]. These compounds decreased the mitochondrial membrane potential, disturbed the mitochondrial structure, caused the mitochondrial release of cytochrome c, activated caspases and induced poly ADP ribose polymerase (PARP) cleavage in human monocytic leukemia U937 cells [20]. Our study has shown that superoxide production induced by 3d was accompanied by the collapse of the mitochondrial transmembrane potential (ΔΨm). The results were assessed by JC-1 assay in cancer (NCI-H460 and NCI-H460/R ) and normal (HaCaT) cells (Fig. 3 Tables 4 and 5.
Antibacterial activity evaluation showed that the most sensitive bacterial strains were E. coli, S. The reported compounds were also tested for activity against three fungal strains: C. albicans, S.
cerevisae and A. brasiliensis. Of all the compounds 3c and 3f showed the most potent and the broadest activity versus all three strains, significantly higher than nystatin. Interesting behaviour was noted for regioisomeric pairs 3c-4c and 3f-4f. Pair 3c-4c displayed the same activity toward C. albicans and S. cerevisae but 3c was much more active against A. brasiliensis. On the other hand, 3f-4f pair showed the same activity toward A. brasiliensis, with 3f being much more active against the other two strains. 3-Substituted compounds generally showed a stronger activity than their 4-substituted counterparts. Avarone derivatives again showed a low activity, with their MIC values falling beyond the concentration range used in the assay. Besides that, the structure requirements were not as clear-cut as for the antibacterial activity.

Conclusion.
Some general conclusions can be made regarding the optimization of scaffold structure, position and nature of substituents. Most importantly, tert-butyl derivatives showed at least the same or better cytotoxic activity and selectivity as avarone counterparts. This is if great importance in view of the availability of the precursor compound.
2,6-Disubstituted quinones were shown to be more active than the corresponding 2,5disubstituted quinones in both series of compounds, due to differences in redox status. Selectivity to MDR cell line occurred with medium sized or longer alkylamino as well as with aralkylamino substituents. The most promising derivatives are 2-tert-butyl-6-(octylamino)-1,4-benzoquinone and 2-tert-butyl-6-(phenethylamino)-1,4-benzoquinone, because of their selectivity towards both sensitive and MDR cancer cells in comparison with normal cells confirmed on several different levels: apoptosis, changes in redox balance and loss of mitochondrial membrane potential.
Since several compounds showed a good selectivity to MDR cells, the results of this study might contribute to selecting candidates for overcoming the problem of multidrug resistance in cancer cells.

EXPERIMENTAL SECTION
General procedure. Reagents and solvents were obtained from commercial sources (Fluka, Autopol IV polarimeter. UV-Vis spectra were recorded using Cintra 40 UV-Visible spectrometer. (1). tert-Butylhydroquinone (2 g; 12 mmol) was dissolved in diethyl ether (100 mL). Silver(I) oxide (3.74 g; 16.14 mmol) was added in portions to the reaction mixture, and the mixture was stirred for 2 hours at room temperature. After this period stirring was stopped, sodium sulphate was added and the mixture was left overnight. Silver, excess of silver(I) oxide and sodium sulphate were removed by filtration over Kieselgur. Diethyl ether was removed by evaporation and tert-butyl-1,4-benzoquinone used without further purification. The reaction yield was quantitative.

tert-Butyl-1,4-benzoquinone
Avarone (2). Avarol (1.1 g; 3.5 mmol) was dissolved in diethyl ether (100 mL). Silver(I) oxide (1.1 g; 4.7 mmol) was added in portions to the reaction mixture, and the mixture was stirred for 2 h at room temperature. The reaction mixture work-up was the same as above. The reaction yield was quantitative. Organic phase was separated, dried with anhydrous calcium chloride, and the solvent removed by evaporation under vacuum. Crude products were separated by column chromatography and purified by preparative thin-layer chromatography, with the indicated solvents.
Statistical analysis. Statistical analysis was performed by GraphPad Prism 6 Software. In MTT assay analysis, the differences between groups were tested by Student's t-test and were considered statistically significant if p < 0.05. Flow cytometry results were examined by Twoway ANOVA and analyzed by Tukey's multiple comparisons test.
Antimicrobial Activity Determination. Antimicrobial activity was evaluated using a broth microdilution method according to NCCLS [35].