Rapid Identification of Cyclic Tetrapyrrolic Photosensitisers for Photodynamic Therapy Using On‐line Hyphenated LC‐PDA‐MS Coupled with Photo‐cytotoxicity Assay P. J. Tan, a D. R. Appleton, b,c M. R. Mustafa b and H. B. Lee a * ABSTRACT: Introduction – Photodynamic therapy is a treatment modality that involves site‐directed generation of cytotoxic reactive oxygen species by light‐activated photosensitisers. Objective – In order to rapidly identify new photosensitisers from natural extracts, we developed a liquid chromatography– photodiode array–mass spectrometry (LC‐PDA‐MS) method to rapidly identify plant extracts that contain photosensitisers, particularly those possessing a cyclic tetrapyrrole structure. Method – Six previously isolated compounds (1–6) were identified in bioactive fractions derived from 15 plant extracts on the basis of their chromatographic retention times, UV–visible profiles, accurate mass and fragmentation patterns. Results – Samples containing uncommon photosensitisers were rapidly identified using this method, and subsequent scale‐up isolation efforts led to two new compounds (7 and 8) which were confirmed to be active photosensitisers in a photo‐cytotoxicity assay. Conclusion – This method serves as a useful tool in prioritising samples that may contain new photosensitisers out of a larger group of photo‐cytotoxic natural products extracts. Copyright © 2011 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: photosensitiser; dereplication; photodynamic therapy; LC‐PDA‐MS; cyclic tetrapyrrole Introduction Photodynamic therapy (PDT) is a treatment regime for certain forms of cancer and some vascular diseases of the eye. It involves site‐directed light activation of photosensitisers and subsequently cytotoxic reactive oxygen species (ROS) are locally generated (Verma et al., 2007). Compared with conventional chemotherapy and radiotherapy, PDT is more selective towards cancer tissues, has fewer side effects and has lower rates of treatment‐induced resistance. There are only three clinically approved photosensitisers approved for cancer treatment; Photofrin®, Foscan® and Levulan® (Allison and Sibata, 2010). In addition, these photosensitisers suffer from limitations such as low therapeutic effect in deep tissues due to poor penetration of the required wavelength of activation light and prolonged skin photosensitivity in patients (Allison and Sibata, 2010). This has prompted research efforts into new synthetic and naturally derived photosensitisers that possess more desirable characteristics, which are currently in various stages of preclinical and clinical development. Nature has an impressive track record of providing useful chemotypes for the pharmaceutical industry. For example, nearly 80% of all small molecules entering clinical development for cancer treatment between January 1981 and October 2008 were either natural products or natural product‐derived (Cragg et al., 2009). Malaysia is one of the 12 mega‐diverse centres of the world and therefore its flora and fauna are an attractive target for the discovery of novel pharmaceutical leads. Hence, our research group is actively investigating Malaysian plants (Chee et al., 2005; Ong et al., 2009), microbes (Kamal et al., 2009) and marine organisms for novel potential PDT agents. We recently reported the first isolation of the cyclic tetrapyrrole photosensitiser, 15 1 ‐hydroxypurpurin‐7‐lactone methyl ethyl diester from the Araceae plant family from a sample of Aglaonema simplex. 15 1 ‐Hydroxypurpurin‐7‐lactone methyl ethyl diester exhibited strong photocytotoxicity against leukaemia (HL60) and oral cancer cells (HSC2 and HSC3) (Chee et al., 2005). A semi‐synthesised analogue, 15 1 ‐hydroxypurpurin‐7‐lactone dimethyl ester, was observed to effectively occlude the blood vessels in the chick * Correspondence to: H. B. Lee, Cancer Research Initiatives Foundation, Second floor, Outpatient Centre, Sime Darby Medical Centre, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia. E‐mail: hongboon.lee@carif.com.my a Cancer Research Initiatives Foundation, Second floor, Outpatient Centre, Sime Darby Medical Centre, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia b Centre for Natural Product Research and Drug Discovery, Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia c Sime Darby Technology Centre Sdn Bhd, First Floor, Block B, UPM‐MTDC Technology Centre III, Lebuh Silikon, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia Phytochem. Anal. 2012, 23, 52–59 Copyright © 2011 John Wiley & Sons, Ltd. Research Article Received: 7 December 2010; Revised: 31 January 2011; Accepted: 10 February 2011 Published online in Wiley Online Library: 21 June 2011 (wileyonlinelibrary.com) DOI 10.1002/pca.1324 52