ISOLATION, CHEMICAL MODIFICATION AND CYTOTOXIC EVALUATION OF ATRANORIN, THE MAJOR METABOLITE OF THE FOLIOSE LICHEN PARMOTREMA MELANOTHRIX

The foliose lichen, Parmotrema melanothrix has been chemically screened for the first time and isolated the depside atranorin (1) in significant quantity (2 %) along with methyl 2,4-dihydroxy-3,6-dimethylbenzoate (2) and methyl 3-formyl-2,4-dihydroxy-6methylbenzoate (3). In view of its abundance, interesting structural features and significant biological profile, atranorin was subjected to chemical modification and synthesised five diverse analogues in very good yields (70~93%). The synthesised analogues along with the three isolated compounds were evaluated for their cytotoxic potential against a panel of six human cancer cell lines using MTT assay. Among the tested compounds, 1a showed enhanced activity than the parent compound (1) against almost all the tested cell lines. Significantly, 1a showed highest activity (IC50 = 15.19 μM) against prostate cancer cell line (DU145). The results indicate that complete protection of the phenolic hydroxyls in atranorin as acetates enhances the cytotoxicity, especially against DU 145.


Introduction
Lichens are highly productive natural sources with a conglomerate of two organisms such as fungi and algae or cyanobacteria. 1 They can survive in a wide range of habitats and harsh environmental conditions such as low temperatures, prolonged darkness, drought and continuous light.These extreme conditions encourage the lichens to produce biologically active secondary metabolites such as depsides, depsidones, xanthones, dibenzofurans and chromones. 2Most of these secondary metabolites are unique to lichens and are produced biosynthetically via the polyketide pathway. 3chen substances exhibit a wide range of biological activities such as antimicrobial, anti-inflammatory, HIV-1 integrase inhibitory, antioxidant, antipyretic and antiproliferative or cytotoxic. 4,5The genus Parmotrema, belongs to the Parmeliaceae family, is the largest one in the lichen kingdom.The lichens of this family are characterized by the development of a foliose growth. 6e major secondary metabolites of Parmotrema genus are depsides, which possess two or more aromatic rings joined through ester linkages.8][9] Among the various Parmotrema lichens, P. melanothrix is unexplored both chemically and biologically.In a preliminary study different polar extracts P. melanothrix were tested and reported to exhibit antioxidant and antibacterial activity against S. viridians, S. aureus and Acinetobacter. 10 Virtually, no systematic chemical examination has been done on this species except the preliminary phytochemical screening of Spielmann and Marcelli who identified atranorin and protopraesorediosic acid by performing some spot tests. 11As, there are no systematic chemical and biological studies were carried so far on P.melanothrix, we have now taken up its detailed chemical screening and chemical modification of atranorin, its major metabolite to identify potent cytotoxic leads.

General experimental procedure
Silica gel 60 F254 TLC glass plates (Merck) were used to monitor purity of the isolated compounds and all the reactions.Column chromatography was carried out by using Acme grade silica gel (60-120 mesh).Melting points were determined on Buchi melting point apparatus and are uncorrected.IR spectra were recorded on Nicollet 740 FTIR spectrophotometer using KBr pellets. 1 H and 13 C NMR spectra were recorded on Brucker 300 MHz or Varian 500 MHz in CDCl3 with TMS as internal standard.Chemical shifts were expressed as Hertz (Hz).HRMS spectra were recorded on Agilent-ESI QTOF or JEOL mass spectrometers.

Extraction and isolation
The lichen material was washed under tap water to remove any foreign matter.It was shade dried and powdered in a pulveriser.The powdered lichen (250 g) was extracted with acetone (1 L) at room temperature overnight.Concentration of acetone solubles under vacuum to one quarter volume and keept at room temperature overnight followed by filtration yielded a brown coloured solid (5.5 g, 2.2 %) and an extract (5 g, 2 %).The brown colour solid on recrystallisation using hexane-ethanol solvent system, yielded pure compound 1 (5 g, 2 %).Repetitive Si gel column chromatographic separation of the acetone extract (5 g) afforded compounds 2 (1.0 g, 0.4 %) and 3 (0.5 g, 0.2 %).

Cytotoxicity Assay
The above isolated and synthesized compounds were screened for anticancer activity against five different cell lines including A549 (Lung Cancer), DU145 (Prostate Cancer), MCF-7(Breast Cancer), SiHa (Cervical Cancer), U87MG (Glioblastoma) cell lines.The cells were plated at a density of 5 × 10 3 cells per well in a 96 well plate supplemented with 10 % FBS and after 24 h of incubation at 37 ºC and 5 % CO2, they were treated with respective concentration of the compounds dissolved in the culture media with vehicle controls and known standards for 48 h.
Cell viability was determined by adding 100 µL of 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT reagent (0.5 mg mL -1 ) dissolved in serum free media added to each well and incubated for 4 h.Then the media was aspirated and the formazan crystals were dissolved in 200 µL of DMSO and absorbance was taken at 570 nm in multimode plate reader (BioTek Instruments, Synergy 4, and Winooski, VT)).The percent cell inhibition in treated cells was calculated by normalizing the cells with 0 % inhibition with control group.Then the compounds which exhibited the percentage inhibition of more than 50 % at a concentration of 150 µM in the initial screening were further screened for dose response curve with series of 7 concentrations starting from initial 150 µM.MTT was performed as described previously and IC 50 values were determined from DRC plot by linear regression method.Graph was plot between different concentration and percentage Inhibition.All the values were expressed as Mean ± SEM in three different experiments in which each treatment was performed in triplicates.

Results and discussion
Chemistry P. melanothrix was collected from the lower temperate region of Tirumala Hills (13.65000N 79.420 E) located in Andhra Pradesh, India.This region is having more saxicolous kind of P. melanothrix.Before carrying out extraction, it was washed with fresh water to remove foreign matter.The lichen material was shade dried, powdered in a pulveriser and extracted with acetone at room temperature overnight.The extract was concentrated to one quarter volume and left overnight at room temperature.Filtration of the extract yielded a brown coloured solid (2.2 %) and filtrate (2 %).The solid on recrystallisation from hexaneethanol solvent system, yielded a single and pure compound 1 (Scheme 1).The residue obtained on evaporation of the filtrate, was subjected to silica gel flash column chromatography with n-hexane and ethyl acetate solvents as eluents to yield two pure compounds 2 and 3 (Scheme 1).The structures of the isolated compounds were elucidated by their spectral data ( 1 H & 13 C NMR, IR and HRMS).Compound 1 was obtained as colourless crystals and its molecular formula was deduced as C19H18O8 from its HRMS, which showed the molecular ion peak at m/z 373.0917 [M-H] + .
The IR spectrum of the compound showed the absorption bands at 3450 and 1730 cm -1 corresponding to hydroxyl and ester functionalities respectively. 1H and 13 C NMR spectra of the compound revealed its identity as atranorin.Its structure was further confirmed by comparing its physical and spectral data with reported values. 135][16] However, its present isolation assumes significance as it is now isolated in high yield (2 %).Compound 2 was obtained as colourless crystals (0.4 %).Its molecular formula was deduced as C10H12O4 by ESI-HRMS, which showed the protonated molecular ion at m/z 197.0810 [M+H] + .Its IR spectrum showed the absorption bands at 3404 and 1627 cm - 1 corresponding to hydroxyl and ester functionalities respectively.Its 1 H and 13 C NMR spectra identifies its structure as methyl 2, 4-dihydroxy-3, 6-dimethylbenzoate.Its identity was further confirmed by comparing its spectral data with the reported values. 17Compound 3 was obtained as colourless crystals (0.2 %) and its molecular formula was deduced as C10H10O5 from ESI-HRMS, which showed the molecular ion peak at m/z 211.0608 [M+H] + .Its IR spectrum showed absorption bands for hydroxyl (3404 cm -1 ) and aldehydic carbonyl (1644 cm -1 ) functionalities.Its 1 H and 13 C NMR spectra reveals its identity as methyl 3formyl-2,4-dihydroxy-6-methylbenzoate. 18mong the isolated metabolites, atranorin (1) was obtained in significant quantity (2%).It is to mention here that atranorin was reported to exhibit potent biological activities such as anticancer, 19 anti-inflammatory (Inhibition of LTB4 biosynthesis). 20Further atranorin was reported to be non toxic to normal cells and has an added advantage as a promising scaffold for making diverse analogues to identify potent bio-active lead molecules.Hence, compound 1 was fine tuned by chemical modification of its active functional groups (2 phenolic hydroxyls and 1 aldehydic group) and synthesised five diverse analogues as shown in scheme 1 (1a-1e).The structures of the synthesised analogues were confirmed by their spectral data ( 1 H and 13 C NMR, IR, HRMS).Treatment of 1 with acetic anhydride in presence of pyridine afforded compound 1a in 93% yield.Its structure was confirmed by spectral data.The IR spectrum of compound 1a showed sharp peaks at 1776 cm -1 and 1728 cm -1 corresponding to the carbonyl group in acetoxyl and ester functionalities.The 1 H NMR displayed peaks between δ 2.01 ~ 2.39, corresponding to the methyl protons of acetyl group.The 13 C NMR spectrum of compound 1a showed

Conclusion
In conclusion, chemical screening of P. melanothrix resulted in the isolation three orcinol based metabolites (1-3) with the depside, atranorin (2 %) as the major metabolite.In view of its abundance and interesting skeletal features, compound (1) was subjected to chemical modification and synthesised five diverse analogues (1a-1e).Among the synthesised compounds, 1a showed enhanced cytotoxicity against almost all the cancer cell lines screened with highest activity against the prostate cancer cell line with an IC50 of 15.19μM.

Table 1 .
Biological profile of atranorin derivatives over cancer cells.
13e 1 H and13C NMR spectra of compound 1c confirmed the absence of aldehydic group and presence of a hydroxy methylene group in the compound.The HRMS spectra of compound 1c further confirmed its structure by exhibiting the sodiated molecular ion [M+Na] + at m/z 441.1554.The analogue 1e was obtained, when compound 1 was treated with phenyl hydrazine in dichloromethane and sodium acetate to yield the product 1e in 82 % yield.The structure of compound 1e was identified by its spectral data, which exhibited absorption band at 1645 cm -1 corresponding to the imine functionality in IR spectrum and showed diagnostic signals at δ 8.37 and between δ = 7.29-7.32 in 1 H NMR spectrum corresponding to imine and aryl ring protons respectively.This further supported by the13C NMR spectrum, which showed characteristic signals δ = 143.38 corresponding to the imine carbons.The HRMS of compound 1e showed [M+H] + molecular ion peak at m/z 465.1653 confirming its structure.