Design and Synthesis of Antitumor Heck-Coupled Sclareol Analogues: Modulation of BH3 Family Members by SS-12 in Autophagy and Apoptotic Cell Death Shakeel-u-Rehman, †,# Bilal Rah, ‡,§,# Shabir H. Lone, † Reyaz Ur Rasool, ‡,§ Saleem Farooq, ∥ Debasis Nayak, ‡,§ Naveed Anjum Chikan, ⊥ Souneek Chakraborty, § Akanksha Behl, § Dilip Manikaro Mondhe, ‡,§ Anindya Goswami,* ,‡,§ and Khursheed Ahmad Bhat* ,† † Bioorganic Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Srinagar, Jammu & Kashmir-190005, India ‡ Academy of Scientific & Innovative Research, Indian Institute of Integrative Medicine, Jammu, Jammu & Kashmir-180001, India § Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi, Jammu & Kashmir-180001, India ∥ Bioorganic Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Jammu, Jammu & Kashmir-180001, India ⊥ School of Bioscience and Technology, Division of Medical Biotechnology, VIT University, Vellore, Tamilnadu-632014, India * S Supporting Information ABSTRACT: Sclareol, a promising anticancer labdane diterpene, was isolated from Salvia sclarea. Keeping the basic stereochemistry-rich framework of the molecule intact, a method for the synthesis of novel sclareol analogues was designed using palladium(II)-catalyzed oxidative Heck coupling reaction in order to study their structure−activity relationship. Both sclareol and its derivatives showed an interesting cytotoxicity profile, with 15-(4-fluorophenyl)sclareol (SS-12) as the most potent analogue, having IC 50 = 0.082 μM against PC-3 cells. It was found that SS-12 commonly interacts with Bcl-2 and Beclin 1 BH3 domain proteins and enhances autophagic flux by modulating autophagy-related proteins. Moreover, inhibition of autophagy by autophagy inhibitors protected against SS-12-induced apoptosis. Finally, SS-12 effectively suppressed tumor growth in vivo in Ehrlich’s ascitic and solid Sarcoma-180 mouse models. ■ INTRODUCTION Sclareol (Labd-14-ene-8, 13-diol), a well-known fragrance diterpene, isolated mainly from the plant Salvia sclarea, 1 is widely used in the cosmetics industry. 2 The molecule is used as a starting material for the preparation of Ambra odorants (Ambergris) in perfumery. 3 Sclareol has been a subject of a number of studies, being a strongly cytotoxic and cytostatic agent. 4−6 Dimas et al. demonstrated that sclareol induces apoptosis in human leukemic cell lines by down-regulating the expression of proto-oncogene c-myc without affecting the expression of the anti-apoptotic protein, Bcl-2. 4,5 Sclareol induces apoptosis in human colon tumor (HCT116) cells in vitro by activating both the mitochondrial pathway and the death-receptor pathway, and it suppresses HCT116 tumor growth in immunodeficient mice. 7 Sclareol has recently been reported to enhance the activity of known anticancer drugs like doxorubicin, etoposide, and cisplatin against human breast cancer cells. 6 Liposome-incorporated sclareol has been shown to reduce the growth rate of HCT116 developed in severe combined immunodeficiency (SCID) mice, exhibiting superior properties compared to the free sclareol. 8,9 Sclareol modulates the immune response by affecting the cytokine pattern in the splenocytes of intratumorally injected mice. 10 As part of our ongoing research program on bioprospection of natural products, 11 sclareol and its analogues were screened for anticancer activity. The activity profile of sclareol intrigued us to exploit its unique architecture as a basic template to synthesize novel derivatives with improved cytotoxic activity. Structurally, sclareol is a labdane-type ditertiary alcohol that features a stereochemistry-rich framework, including an olefinic moiety (Figure 1). Herein, we report the preparation of semisynthetic analogues of sclareol (Scheme 1) along with cytotoxicity screening in order to study the possible structure− activity relationships (SARs) to explore the detailed biological mechanism of action. Thus, the oxidative Heck reaction was performed to construct a diverse series of different aryl analogues substituted at the C-15 site of sclareol. Until now, there have been no reports wherein any labdane diterpene in general, and sclareol in particular, has been subjected to oxidative Heck coupling. Received: December 3, 2014 Published: March 31, 2015 Article pubs.acs.org/jmc © 2015 American Chemical Society 3432 DOI: 10.1021/jm501942m J. Med. Chem. 2015, 58, 3432−3444