Antioxidant , Antimicrobial and Antiproliferative Activities of Synthesized 2 , 2 , 5 , 5-Tetramethyl-9-aryl-3 , 4 , 5 , 6 , 7 , 9-hexahydro-1 H-xanthene-1 , 8 ( 2 H )-dione Derivatives

Ten biologically active 2,2,5,5-tetramethyl-9-aryl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione derivatives were synthesized and their structures were confirmed by IR, 1H and 13C NMR spectroscopy and mass spectrometry. Synthesized compounds were scanned for their antioxidant, antimicrobial and antiproliferative activity. Antibacterial activity was tested by the diffusion and dilution method against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, while antifungal activity was tested against Candida albicans and Saccharomyces cerevisiae. Antiproliferative activity was tested against HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic), hepatocellular carcinoma (HEpG2), lung carcinoma cells (A549) and mouse embryo fibroblast cell line (3T3). The best antioxidant activity showed compound 2 with two hydroxy groups substituted on phenyl ring in positions 2' and 3'. The best antimicrobial activity of all synthesized compounds showed compound 8, while the best antiproliferative activity showed compound 6. Results signify the importance of xanthene-1,8-dione derivatives as potential antioxidant and antiproliferative agents.


INTRODUCTION
ANTHENES and its derivatives are known as an important class of heterocyclic compounds that have been widely used as leuco dyes, pH sensitive fluorescent materials for visualization of biomolecules and in laser technologies due to their spectroscopic properties. [1]Also, xanthenes constitute as an important class of biologically active compounds due to their broad spectrum of pharmacological activities such as antibacterial, [2] antiviral, [3] antiinflammatory activities. [4]Natural and synthetic xanthene derivatives are also well-known for their ability to act as antioxidants and/or enzyme inhibitors. [5]Due to their wide range of applications, these compounds have received a great deal of attention regarding their synthesis.[9][10][11] Antioxidants are necessary to control degenerative reactions produced by reactive oxygen and nitrogen species.These species are involved in several ailments including cancer, heart diseases and Alzheimer's disease. [12]ith the development of new strains of bacteria resistant to many currently available antibiotic treatments, there is increasing interest in the discovery of new antibacterial agents.Antimicrobial resistance refers to microorganism that have developed the ability to inactivate, exclude or block the inhibition or lethal mechanism of the antimicrobial agents. [13]n our previous work we prepared thirteen 2,2,5,5tetramethyl-9-aryl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione derivatives using reliable one-pot synthesis followed by structure elucidating measurements, and performed in vitro antimicrobial potency evaluation against Escherichia coli and Candida albicans strains. [14]Antimicrobial

Instrumentation
Melting points of the compounds were determined with BŰCHI Melting Point B-545 and are presented uncorrected.Infrared (IR) spectra of synthesized compounds were recorded by Shimadzu IR Prestige 21 ID using KBr pellets.The 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at 600 and 150 MHz, respectively, in CDCl3 at 25 °C using NMR spectrometer Bruker AV600, with tetramethylsilane (TMS) as internal reference.Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz.Elemental analyses of synthesized compounds were recorded by Vario EL III C, H, N, S/O Elemental Analyzer, Elementar Analysensysteme GmbH, Hanau-Germany.Electrospray ionization mass spectrometry (ESI-MS) measurements were performed on a high performance liquid chromatography-mass spectrometry (HPLC-MS) triple quadrupole 6420 instrument equipped with an autosampler (Agilent Technologies, Palo Alto, CA, USA).The desolvation gas temperature was 300 °C with flow rate of 6.0 L min -1 .The fragmentor voltage was 135 V and capillary voltage was 4.0 kV.Mobile phase was 0.1 % formic acid in 50 % methanol and a flow rate of mobile phase was 0.2 mL min -1 .

Evaluation of Antioxidant Activity by DPPH Method
The free radical scavenging activity of synthesized 2,2,5,5tetramethyl-9-aryl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-diones was evaluated using a stable radical, 1,1diphenyl-2-picrylhydrazyl (DPPH • ) in methanol solution described in literature. [19]hen determining the antioxidant activity by using DPPH method, the absorbance of the 0.2 mM methanol solution of the test substance was mixed with the same ratio of 0.2 mM DPPH solution, representing the absorbance of the sample (AA (t)).The control solution is a mixture of methanol and DPPH.The measurement was done 30 minutes after the addition of DPPH at 517 nm.In order to determine the concentration of a compound that inhibits DPPH activity by 50 % (EC50), a dilution series was made for each sample individually, and from the calibrated curves the concentration ratio and percent inhibition were determined by EC50.The inhibition percentage (%) of radical scavenging activity was calculated by using the equation 1: where Ac (0) is the absorbance of control at t = 0 and AA (t) is the absorbance of antioxidant at t = 30 min.All measurements were done in triplicate. [20,21]

Antimicrobial Activity
Antibacterial activity was tested by the diffusion method against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 9027 and Pseudomonas aeruginosa ATCC 15442, while antifungal activity was tested against Candida albicans ATCC 1023 and Saccharomyces cerevisiae ATCC 9763.Test samples were dissolved in 99.5% dimethyl sulfoxide (DMSO) to obtain a 1 mg/mL stock solutions which were then applied to Müller-Hinton and Sabouraud nutritious bases.The inhibition zones for bacteria were measured in millimetres at the end of an incubation period of 18 h at 37 °C, and for fungal strains after 48 h at 25 °C.Compounds that showed good antimicrobial activity tested by the diffusion method were further tested by dilution method.For this, Casein soya bean digest broth (Triptic soya bujon) was used.As referent compounds chloramphenicol and fluconazole are used in concentration of 500 µg mL -1 .Test solution of the compound was prepared, followed by formation of a series of 12 dilutions with liquid nutritious base.After the incubation for 24 h, the last tube with no growth of microorganisms was taken to represent minimum inhibitory concentration (MIC) expressed in mg mL -1 .The concentrations of the prepared solutions were in range 0.5-0.00024mg mL -1 .

Proliferation Assays
A panel of adherent tumour cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at 5000 cells per well.Test agents were then added in five, 10fold dilutions (0.01 µM to 100 µM) and incubated for further 72 h.Working dilutions were freshly prepared on the day of testing in the growth medium.The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in the working concentrations (DMSO concentration never exceeded 0.1 %).After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay.End-point absorbance was measured at 570 nm.Each test point was performed in quadruplicate in three individual experiments.Experimentally determined absorbance values were transformed into a cell percentage growth (PG) using the formulas proposed by NIH and described previously. [22]This method directly relies on control cells at the day of assay because it compares the growth of treated cells with the growth of untreated cells in control wells on the same plate.The results are therefore a percentile difference from the calculated expected value.The IC50 and LC50 values for each compound were calculated from dose-response curves using linear regression analysis by fitting the mean test concentrations that give PG values above and below the reference value.If, however, all of the tested concentrations produce PGs exceeding the respective reference level of effect (e.g.PG DOI: 10.5562/cca3225 Croat.Chem.Acta 2018, 91 (1)   value of 50) for a given cell line, the highest tested concentration is assigned as the default value (in the screening data report that default value is preceded by a ">" sign).

Structural Characterisation of Compounds 1-10
In all IR spectra of synthesized 2,2,5,5-tetramethyl-9-aryl-3,4,5,6,7,9 -hexahydro-1H-xanthene-1,8(2H)-dione derivatives there are absorption bands characteristic for xanthene structure; absorption bands at 1600-1680 cm -1 come from the C=O group and 1152 cm -1 band from C-O-C vibration.Also, in all IR spectra of synthesized xanthene compounds, there are bands at 1300 cm -1 , characteristic for C-O stretching, and deformation vibrations of the OH group at 1200 cm -1 .The IR spectra of the synthesized xanthene compounds differ in absorption bands that come from the corresponding substituent attached to the phenyl ring.For compounds with trisubstituted benzene ring in the structure there are visible characteristic bands at 860 cm -1 (compounds 8 and 9).For compound with bromine in the structure there is visible characteristic band at 750 cm -1 from C-Br stretching, while for methoxy substituted derivatives (compounds 3, 8, 9 and 10), IR spectra contain bands between 1200 and 1450 cm -1 characteristic for stretching of O-CH3.Compound with dimethylamino group (compound 7) shows band at 1440 cm -1 which originates from N-CH3 stretching.Synthesized compounds with hydroxy groups as substituents (compounds 1, 2, 3, 4) on IR spectra showed bands at 2500-3400 cm -1 .In IR spectra of compound with fluor as substituent (compound 5) there is visible band at 1100 cm -1 from C-F streching.
In order to determine the exact concentration in which the activity of the DPPH reagent was inhibited by % (EC50), dilution series were individually made and for each dilution the absorbances were measured at 517 nm.The results of antioxidant activity are shown in Table 2.
According to the obtained results compound 2 (with two hydroxy groups substituted on phenyl ring in positions 3' and 4') has shown the best antioxidant effect with EC50 of 0.017 mM.The higher scavenging activity of compound 2 could be attributed to the formation of two radicals on hydroxy groups both of which are stabilized by resonance.Similarly compound 1 also has two hydroxy groups on phenyl ring, but in positions 2' and 3' and its EC50 of 0.075 mM indicates antioxidant potency lower than of the compound 2, so we can conclude that position of hydroxy groups has significant effect on the activity.This difference in potency could be due to the difference in the stability of the radicals.Also, in literature good antioxidant activity is described for dibenzoxanthene and xanthene-11-one derivatives against hydroxy radicals which correlates well with our results. [23,24]

Antimicrobial Activity
Microbiological activity was determinated by diffusion and dilution method on four strains of bacteria and two strains of fungi.The results of antimicrobial acivity of synthesized compounds by diffusion method are shown in Table 3 and  Table 4.
Results showed that the best antibacterial activity against Escherichia coli ATCC 9027 and Staphylococcus aureus ATCC 6538P possesses compound 8.The best activity against Pseudomonas aeruginosa ATCC 15442 showed compound 6 and against Bacillus subtilis ATCC 6051 compounds 6 and 7. Also, compound 6 showed the best antifungal activity with zone of inhibition of 18.5 mm against Candida albicans ATCC 1023 and 18 mm against Saccharomyces cerevisiae ATCC 9763.
Described in vitro antibacterial activity of tetrahydrobenzoxanthene-11-one derivative against Pseudomonas strains suggests that antibacterial activity increased with increasing number of hydroxy groups in molecule. [25]In our research the best activity against Pseudomonas strain showed compound with bromine in structure, while compounds with hydroxy groups possess weak antibacterial activity against same bacterium.The results of antimicrobial activity by dilution method are shown in Table 5 and Table 6.
derivatives show characteristic.All spectra possess signals in the range of 26.4-29.9ppm from the carbon atoms of the methyl groups (C-14, C-15, C-16, C-17), also at 30.1-32.02 ppm signals from C-4 and C-10 atoms and at 32.72 ppm signals of the C-13 atom.

Table 2 .
The tests were carried out on gram negative bacteria Escherichia coli ATCC 9027 and Pseudomonas aeruginosa ATCC 15442, and gram positive bacteria Staphylococcus aureus ATCC 6538P and Bacillus subtilis ATCC 6051.The antifungal activity of synthetized xanthene compounds was tested against fungi Candida albicans ATCC Absorbances, percentage of inhibition and EC50 of 0.2 mM solution of synthesized compounds.

Table 3 .
Results of antibacterial activity by diffusion method.

Table 4 .
Results of antifungal activity by diffusion method.

Table 5 .
Results of antibacterial activity by dilution method.

Table 6 .
Results of antifungal activity by dilution method.

Table 7 .
Results of antiproliferative activity presented as IC50.