Synthesis and Cytotoxic Evaluation of 1 H-1 , 2 , 3-Triazol-1-ylmethyl-2 , 3-dihydronaphtho [ 1 , 2-b ] furan-4 , 5-diones

The 1,2-naphthoquinone compound was previously considered active against solid tumors. Moreover, glycosidase inhibitors such as 1,2,3-1H triazoles has been pointed out as efficient compounds in anticancer activity studies. Thus, a series of eleven 1,2-naphthoquinones tethered in C2 to 1,2,3-1H-triazoles 9a-k were designed, synthesized and their cytotoxic activity evaluated using HCT-116 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma) and RPE (human nontumor cell line from retinal epithelium). The chemical synthesis was performed from C-3 allylation of lawsone followed by iodocyclization with subsequent nucleophilic displacement with sodium azide and, finally, the 1,3-dipolar cycloaddition catalyzed by Cu(I) with terminal alkynes led to the formation of 1H-1,2,3-Triazol-1-ylmethyl-2,3-dihydronaphtho[1,2-b] furan-4,5-diones in good yields. Compounds containing aromatic group linked to 1,2,3-triazole ring (9c, 9d, 9e, 9i) presented superior cytotoxic activity against cancer cell lines with IC50 in the range of 0.74 to 4.4 μM indicating that the presence of aromatic rings substituents in the 1,2,3-1H-triazole moiety is probably responsible for the improved cytotoxic activity.


INTRODUCTION
There are several synthetic and natural low molecular weight naphthoquinones with many applications in various scientific and technological fields.These naphthoquinones also have potential clinical utility in the treatment of various diseases (da Silva and Ferreira 2016, Ferreira et al. 2016).
The quinones can be cytotoxic through several mechanisms of action (Klotz et al. 2014, de Paiva et al. 2015), including redox cycle (dos Santos et al. 2004, Ferreira et al. 2010), arylation of thiol groups of proteins, intercalation, induction of breaks in the DNA chain, generation of free radicals and reactive oxygen species (ROS) (de Castro et al. 2013(de Castro et al. , 1028 INGRID C. CHIPOLINE et al.Kumagai et al. 2012) and bioreductive alkylation via formation of quinone-methoxy (Cao and Peng 2014, Paz et al. 2012).
Although 1,2,3-triazoles are not natural, this class of substances has already been investigated against several important disease targets (Dheer 2017) and the conjugation of these two moieties has shown that it is a molecular combination with potential antitumor synergism.Figure 1 shows some examples of 1,2,3-triazoles and naphthoquinones conjugates such as 1,2-naphthoquinone 1, with activities against MDA-MB-435 melanoma cells (Ferreira et al. 2009), and 2 for MOLT leukemia cells (Cardoso et al. 2014), and also the 1,4-naphthoquinones 3-5 which are active against HL-60 leukemia cells (da Cruz et al. 2014).

MATERIALS AND METHODS
The reagents were purchased from Sigma-Aldrich Brazil and were used without further purification.Column chromatography was performed with silica gel 60 (Merck 70-230 mesh).Analytical thin layer chromatography was performed with silica gel plates (Merck, TLC silica gel 60 F254), and the plates were visualized using UV light or aqueous solutions of ammonium sulfate.The indicated yields refer to chromatographically and spectroscopically homogeneous materials.Melting points were obtained on a Fischer-Johns apparatus and were uncorrected.Infrared spectra were collected using KBr pellets on a Perkin-Elmer model 1420 FT-IR spectrophotometer, and the spectra were calibrated relative to the 1601.8cm -1 absorbance of polystyrene.NMR spectra were recorded on a Varian Unity Plus VXR (500 MHz) instrument in DMSO-d 6 or CDCl 3 solution.The chemical shift data were reported in units of d (ppm) downfield from solvent, and solvent was used as an internal standard; coupling constants (J) are reported in hertz and refer to apparent peak multiplicities.High-resolution mass spectra (HRMS) were recorded on a MICROMASS Q-TOF mass spectrometer (Waters).
The physical and spectroscopic data for 9a-f were previously reported in our studies (Chipoline et al. 2015).
IC 50 values were calculated along with the respective 95% confidence intervals by non-linear regression using GraphPad Prism 5.0 (Intuitive Software for Science).
The structures of the compounds were elucidated by spectroscopic techniques (see Supplementary Material -Figures S1-S34).Taking as an example the compound 9g, the IR spectrum presents stretches at 3478, related to O-H bond, 1686 and 1651 cm -1 to C=O bonds of carbonyls.
The anticancer activity of naphthoquinones 9a-k was assessed against two human cancer and a non-tumor cell lines in comparison to doxorubicin (positive control) using the MTT assay (Mosmann 1983).At 5 µM concentration, seven out of ten tested substances presented growth inhibition superior to 75% to MCF-7 cells, while only three of them were active against HCT-116 cells, as reported in Table I.Most of the substances were also active against the nontumor cells at 5 µM.
The IC 50 values demonstrated that compounds 9c, 9d, 9e, 9i, 9j showed highest activity against all cancer cell lines with IC 50 ranged from 0.74 to 4.4 µM (Table II).The aryl triazolic derivatives 9c and 9d presented selective activity in MCF-7 while 9i seems to be selective to HCT-116 cell line being almost four times more potent than to the other tested cells, appearing as good prototypes for an anti-breast and colon cancer lead molecules, respectively.
In terms of selectivity, the presence of methyl group on aromatic ring in 9d increased toxicity in non-cancer cells compared to 9c, with similar results for 9j which contains the hexyl group.One addition of halogen to propyl group in 9b enhanced anticancer activity, however, decreased the selectivity.
The results indicate that the presence of aryl substituents (9c, 9d, 9e and 9i) in the triazole moiety is relevant for cytotoxic activity and selectivity against cancer cells.