Semisynthesis Of Andrographolide Derivatives And Evaluation Of Their Antitumour Properties

Author

JADA SRINIVASA RAO

Date

2004

Keyword

Andrographis paniculata, andrographolide, andrographolide derivatives, Neoandrographolide, in vivo study, antitumour activity, cancer cell lines synthesis, apoptosis

Abstract

Previously, andrographolide, which is the major diterpenoid of Andrographis paniculata, was shown to have in vivo antitumour activity against human breast tumour xenografts. In this study, among the four compounds isolated from A. paniculata, andrographolide was the most potent compound with a mean IC50 value of 8 pM in MCF-7 human breast cancer cells. Neoandrographolide showed a weak cytotoxic effect, whereas 14-deoxy-ll,12-didehydroandrographolide and 14-deoxyandrographolide failed to exhibit growth inhibitory effect at the highest tested concentration of 100µM. Owing to this, andrographolide was considered as the lead compound in the discovery of potent and selective antitumour agents. Using andrographolide isolated from A. paniculata as one of the starting materials, 3,19-benzylidene andrographolide and 3,19-alkylidene andrographolide derivatives were synthesised by coupling of the two -OH groups present at C-3 and C-19 of andrographolide with different benzaldehydes and alkyl aldehydes, respectively. In addition, new derivatives were also synthesised by acetylation, oxidation, Heck and esterolysis reactions. The structures of new derivatives of andrographolide derivatives were confirmed by spectral analysis (1H/13C NMR, MS, FT-FR, UV). Forty seven compounds including andrographolide were tested for antitumour activities in MCF-7 and HCT-116 (colon) cancer cell lines. Using a 72 h MTT cell viability assay, parameters of dose-response effects, GI50, TGI and LC50 were determined. The derivatives had submicromolar GI50 values, except for 3,19-(4-nitrobenzylidene)andrographolide (SRJ58), which showed the most potent activity with a GI50 value of 0.7 µM in MCF-7 cells. Only (Z)-2-[l-benzylamino-2-(5,5,6,8a-tetramethyl-2-methylene-decahydro-naphthalen-l-yl)-ethyl]-4-hydroxy-but-2-enoic acid benzylamide] (SRJ18), displayed a pronounced selectivity (approximately 8-fold) towards HCT-116 cells at the GI50 value compared with MCF-7 cells. Out of the five compounds (3,19-isopropylideneandrographolide (SRJ01), 14-acetylandrographolide (SRJ03), 3,19-(2-bromobenzylidene)-14-deoxy-l 1,12-didehydro andrographolide (SRJ05), 3,19-(2-bromobenzylidene)andrographolide (SRJ09) and 3,19-(3,4-dimethoxybenzylidene)andrographolide (SRJ13)) tested against the 60 National Cancer Institute (NCI) of USA human cancer cell lines, only SRJ09 showed some form of selectivity towards cancers of the colon, central nervous system, renal and melanoma. The mechanism(s) of actions of the compounds were also studied by determining their effect in inducing cell cycle arrest and apoptosis. Andrographolide, SRJ01 and SRJ03 induced G1and G2/M arrest in MCF-7 cells, whereas 3,19-(4-bromobenzylidene)andrographolide (SRJ08), SRJ09, 3,19-(3-bromobenzylidene) andrographolide (SRJ10), 3,19-(3-chloro-4-fluorobenzylidene)andrographolide (SRJ23) and 3,19-(2-fluorobenzylidene)andrographolide (SRJ27) induced only G1-phase arrest in MCF-7 cells. SRJ09 down-regulated CDK4 (a G1 -phase regulator) protein levels in MCF-7 cells, which explains the G1-phase arrest by the compound. NCI's COMPARE mechanistic analysis revealed that the compounds antitumour activities were not similar to that of standard anticancer drugs with known mechanisms of action. Projection of SRJ03 in the Self-Organising Maps (SOMs) analyses of NCI suggested that this compound may be targeting cell cycle related phosphatases or kinases. However, andrographolide, SRJ01, SRJ05, SRJ09 and SRJ13 did not project in the known mechanism categories. The mode(s) of cell death induced by SRJ09 and SRJ23, identified by fluorescence microscopy and flow cytometry, was confirmed to be apoptosis in HCT-116 cells. In conclusion, novel derivatives of andrographolide, especially SRJ09, SRJ18 and SRJ58 are potential lead molecules for future antitumour studies to discover prospective clinical candidates.