Intra-tumoural administration of tigilanol tiglate has been shown effective in local treatment of cutaneous equine neoplasias. The drug causes rapid oncolysis and tumour slough, but may also be associated with comorbidities in sensitive and/or intricate anatomical sites due to its initial pro-inflammatory mode of action. Here we describe two approaches to optimise and further de-risk treatment of equine sarcoids with tigilanol tiglate, especially in difficult locations. Firstly, we provide preliminary clinical and observational data on the efficacy of low dose administration of tigilanol tiglate (metronomic or single intra-tumoural administration). Secondly, we provide proof of therapeutic concept for tigilanol tiglate eluting calcium sulphate beads by evaluating elution characteristics and assessing clinical efficacy treating equine sarcoids. Clinical efficacy, at substantially lower than previously reported tigilanol tiglate treatment doses, was demonstrated by adopting either a single low-dose or metronomic dosing schedule or an eluting implant. Treatments resulted in complete tumour slough in all instances while evoking a perceived reduction in inflammatory responses compared to the use of previously established higher doses for equine sarcoids. While implanted calcium sulphate beads loaded with cytostatic drugs have been used previously for local treatment of equine neoplasia, their utility for local delivery of tigilanol tiglate has not been explored before. Elution analysis of tigilanol tiglate from 5 and 2.5 mm loaded calcium sulphate beads showed beads eluted ~40% and ~24% of their tigilanol tiglate content in horse serum (HS) with continued release observed from intact beads for at least 4 days. The ease of implanting absorbable tigilanol tiglate loaded calcium sulphate beads, and its clinical efficacy in sarcoid treatment, showed the potential of this approach to eliminate common challenges associated with intra-tumoural injection such as inadvertent diffusion from injection sites or the difficulty injecting fibrous tumours. This contributed to reducing the apparent extent of local inflammatory responses and limiting possible associated morbidities. Apart from mild leukotrichia that developed at some treatment sites, healing and cosmetic outcome was deemed excellent. Ongoing studies evaluating the clinical efficacy and safety of lowered tigilanol tiglate doses in a statistically relevant, representative equine population are required to further develop the reported treatment regimens.

Epoxytiglianes are a novel class of diterpene esters. The prototype epoxytigliane, EBC-46 (tigilanol tiglate), is a potent anti-cancer agent in clinical development for local treatment of a range of human and animal tumors. EBC-46 also consistently promotes wound re-epithelialization at the treatment sites, mediated via activation of classical protein kinase C (PKC) isoforms. We have previously shown that epoxytiglianes stimulate proliferative and wound repopulation responses in immortalized human skin keratinocytes (HaCaTs) in vitro, abrogated by pan-PKC inhibitor, bisindolylmaleimide-1. In this study, we further investigate the specific PKC isoforms responsible for inducing such wound healing responses, following HaCaT treatment with 1.51 nM-15.1 mu M EBC46 or analogue, EBC-211. Classical PKC inhibition by GO6976 (1 mu M), significantly attenuated epoxytigliane induced, HaCaT proliferation and wound repopulation at all epoxytigliane concentrations. PKC-(3I/-(3II isoform inhibition by enzastaurin (1 mu M), significantly inhibited HaCaT proliferation and wound repopulation responses induced by both epoxytiglianes, especially at 1.51-151 nM. PKC-alpha inhibitor, Ro 31-8220 mesylate (10 nM), exerted lesser inhibitory effects on HaCaT responses. Epoxytigliane changes in key keratin (KRT17) and cell cycle (cyclin B1, CDKN1A) protein levels were partly attenuated by GO6976 and enzastaurin. GO6976 also inhibited increases in matrix metalloproteinase (MMP-1, MMP-7, MMP-10) activities. Phospho-PKC (p-PKC) studies confirmed that epoxytiglianes transiently activated classical PKC isoforms (p-PKC alpha, p-PKC-(3I/-(3II, p-PKC gamma) in a dose- and time-dependent manner. By identifying how epoxytiglianes stimulate classical PKCs to facilitate keratinocyte healing responses and re-epithelialization, these findings support further epoxytigliane development as topical therapeutics for clinical situations involving impaired re-epithelialization, such as non-healing wounds in skin.

Background Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear.Methods A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed.Results Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade.Conclusions These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Background Tigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator that disrupts tumour vasculature and causes direct oncolysis of tumour cells. Together, these activities lead to haemorrhagic necrosis of injected tumours with enduring ablation of >70% of target tumours in both pre-clinical xenograft/syngeneic mouse models and cutaneous tumours presenting in the veterinary clinic.1–3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.4 However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood. Methods A combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (HMGB1, calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10 tumor bearing mice. Analysis of DAMP release and immune cell infiltration into TT treated human head and neck tumours (ACTRN12619001407189) was performed by immunohistochemistry. Results Our data reveal that therapeutic concentrations of TT induce the death of cancer and endothelial cell lines via a pathway involving caspase activation and cleavage of the pore forming protein gasdermin E. TT promotes this mechanism of cell death by interacting with ER membranes, causing an ER stress response that results in loss of mitochondrial membrane potential, ATP depletion, organelle swelling and oncosis/pyroptosis. Treatment of cells with TT also led to the release of damage associated molecular patterns (DAMPs), indicative of an immunogenic cell death (ICD) pathway that also resulted in the generation of tumour-specific T cells in CT-26 tumor bearing mice. Whilst the induction of ICD is largely PKC-independent in vitro, PKC/C1 domain signaling appears necessary for efficacious tumour ablation in vivo. Consistent with our pre-clinical data, immunohistochemical analysis of TT-treated head and neck tumors found that drug stimulated DAMP release/externalisation and the recruitment of immune cells, principally CD8+ T cells, into remnant tumour mass. Conclusions These data indicate that TT is an oncolytic small molecule with the potential to ablate target tumours and enhance immunotherapy combinations through promoting immune cell infiltration. TT is currently undergoing Phase II trials in head and neck cancer (NCT05234437) and soft tissue sarcoma (NCT05755113). References Boyle GM, D'Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887. Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci. Rep. 2021;11:207. De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J. Vet. Intern. Med. 2021;35:415–429. Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EBioMedicine. 2019;50:433–441. Ethics Approval The study obtained ethics approval from the following commitees and boards: Metro South Human Research Ethics Committee, 199 Ipswich Road, Woolloongabba, QLD, 4102, Australia. Ethics approval number: HREC/2019/QMS/54004. Bellberry Limited, 123 Glen Osmond Road, Eastwood, SA 5063, Australia. Ethics approval number: 2019–10-846 (REGIS 2019/ETH13063). Tata Memorial Hospital - Institutional Review Board, IRB Office, Dr. E. Borges Marg, Parel, Mumbai - 400 012, India. Ethics approval number: IEC/1219/3370/001. Tata Medical Center - Institutional Review Board, 14 Major Arterial Road (EW), New Town, Rajarhat, Kolkata - 700 160, India. Ethics approval number: 2019/PHARMA/57/IRB39.

The kernels of the Australian blushwood tree (Fontainea picrosperma) are the source of the veterinary anticancer drug tigilanol tiglate (2a, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by 1H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, 2a), EBC-47 (4a), EBC-59 (5a), EBC-83 (6a), and EBC-177 (7a) were identified. By chemoselective acylation of EBC-46 (2a) and EBC-177 (7a) the natural triesters 2b and 7b and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester 2b in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate (2a) under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in F. picrosperma, suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.

Background Tigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator that disrupts tumour vasculature, leading to haemorrhagic necrosis of the lesion.1 Strikingly, in both preclinical syngeneic mouse models and cutaneous/subcutaneous tumours presenting in the veterinary clinic, I.T. injection of TT results in complete and enduring ablation of target tumours in >70% of patients.1,2,3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.4 However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood. Methods A combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10-OVA tumor bearing mice, with or without anti-PD1/anti-CTLA4 treatment. Results Our data demonstrates that therapeutic concentrations of TT induce death of cancer and endothelial cell lines, both in vitro and in vivo, via oncosis. Whilst largely PKC-independent, PKC/C1 domain signaling appears necessary for timely oncolysis in vitro and efficacious tumor ablation in vivo. Our results also show that TT binds to ER membranes, causing ER stress with subsequent activation of the integrated stress response. This is followed by mitochondrial membrane potential loss, ATP depletion, organelle swelling, oncosis and terminal necrosis. We also found that TT treatment promoted the release/externalization of DAMPs (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through rechallenge experiments using CT-26 tumour bearing mice, which also demonstrated that TT promoted the development of tumour-specific T cells. In addition to stimulating immune cell infiltration into tumours, TT significantly improved treatment response in the B16-F10-OVA mouse melanoma model when combined with immune checkpoint blockade. Conclusions These data indicate that TT is an oncolytic small molecule with the potential to enhance responses to immunotherapy. TT is currently undergoing Phase I/II trials in head and neck cancers (ACTRN12619001407189), soft tissue sarcomas, Stage III melanoma in-transit (NCT05234437) and non-resectable Stage IIIB to IV M1c melanoma (TT/pembrolizumab combination: NCT04834973).5 References Boyle GM, D'Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887. Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci Rep 2021;11:207. De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J Vet Intern Med 2021;35:415–429. Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EbioMedicine 2019;50:433–441. QBiotics Group Ltd. website. https://qbiotics.com/ Ethics Approval All animal procedures were approved in accordance with NHMRC guidelines (Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition, 2013; National Health and Medical Research Council of Australia) by the QIMR Berghofer Animal Ethics Committee: A0106-042M, A0404-606M and A01047M.

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/ antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree (Fontainea picrosperma), and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)-dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing-associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.

The long-standing perception of Protein Kinase C (PKC) as a family of oncoproteins has increasingly been challenged by evidence that some PKC isoforms may act as tumor suppressors. To explore the hypothesis that activation, rather than inhibition, of these isoforms is critical for anticancer activity, we isolated and characterized a family of 16 novel phorboids closely-related to tigilanol tiglate (EBC-46), a PKC-activating epoxytigliane showing promising clinical safety and efficacy for intratumoral treatment of cancers. While alkyl branching features of the C12-ester influenced potency, the 6,7-epoxide structural motif and position was critical to PKC activation in vitro. A subset of the 6,7-epoxytiglianes were efficacious against established tumors in mice; which generally correlated with in vitro activation of PKC. Importantly, epoxytiglianes without evidence of PKC activation showed limited antitumor efficacy. Taken together, these findings provide a strong rationale to reassess the role of PKC isoforms in cancer, and suggest in some situations their activation can be a promising strategy for anticancer drug discovery.

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound reepithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM-151 mu M concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM-15.1 mu M) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51-151 nM) and EBC-211 (1.51 nM-15.1 mu M) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51-151 nM) with proliferation inhibitor, mitomycin C (1 mu M), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 mu M). By identifying how epoxytiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds.

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

A detailed examination of [4+2] cycloaddition reactions between 1,8-disubstituted cyclooctatetraenes and diazo compounds revealed that 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) reacts to form either 2,3- or 3,4-disubstituted adducts. The product distribution can be controlled by modulating the electron density of the cyclooctatetraene. Unprecedented [4+2] cycloadditions between diisopropyl azodicarboxylate (DIAD) and 1,8-disubstituted cyclooctatetraenes are also described and further manipulation of a resulting cycloadduct uncovered a new pathway to the synthetically challenging bicyclo[4.2.0]octa-2,4-diene family. Variation of the substituents resulted in a range of compounds displaying selective action against different human tumour cell types.

Four plant species, Hordeum vulgare, Oryza sativa, Panax quinquefolium and Nicotiana tabacum, grown as cell suspension cultures, were used for the biotransformation of an anticancer compound, ingenol-3-angelate (1). Three compounds (1a-1c) were detected predominantly in the cultured medium and their structures were determined as 16-hydroxy-ingenol-3-angelate (1a), ingenol (1b) and ingenol-5-angelate (1c), based on MS and NMR spectroscopic evidence. 16-Hydroxy-ingenol-3angelate (1a) was the only compound produced by H. vulgare cell cultures except that, at high substrate concentration (266 mg L-1), 1c was produced with a low yield both in the medium and within cells. In contrast, compounds 1a and 1b were produced in different yields and proportions in the other three cell cultures. The effect of substrate concentration, addition and incubation time on the production of 1a by H. vulgare cell cultures was investigated, and compounds 1a and 1b were assayed to be active in inhibiting the growth and inducing a bipolar morphology of MM96L melanoma cells.

EBC-23, 24, 25, 72, 73, 75 and 76 were isolated from the fruit of Cinnamomum laubatii (family Lauraceae) in the Australian tropical rainforests. EBC-23 (1) was synthesized stereoselectively, in nine linear steps in 8 % overall yield, to confirm the reported relative stereochemistry and determine the absolute stereochemistry. Key to the total synthesis was a series of Tietze-Smith linchpin reactions. The novel spiroacetal structural motif, exemplified by EBC-23 (1), was found to inhibit the growth of the androgen-independent prostate tumor cell line DU145 in the mouse model, indicating potential for the treatment of refractory solid tumors in adults.

EBC-23 (2), a prostate anticancer agent, was isolated from the fruit of Cinnamomum laubatii (family Louraceae) in the Australian tropical rainforest. Extensive NOE experiments enabled the relative stereochemistry of the proposed EBC-23 (2) structure to be determined. Total synthesis of both enantiopodes over nine linear steps, involving challenging RCM and spiroacetal cyclizations, confirmed the gross structure and relative and absolute stereochemistry.