The synthesis of 3-azabicyclo[4.3.0]nonane scaffolds from brefeldin A

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Highlights

  • Large scale fermentations of Penicillium camberti generated multi-kg quantities of brefeldin.

  • A triazoline derived from brefeldin serves as a useful building block for di-and tri-cyclic scaffolds.

  • New brefeldin derivatives were generated facilitated by microwave induced N2 expulsion and ring expansion.

Abstract

Brefeldin A (BFA) is a fungal metabolite that displays a wide range of biological activities making it an attractive target for drug discovery. To explore the biological potential of the BFA scaffold an amine-macrocycle target 3 was proposed. To access, we dissembled BFA into smaller building blocks in preparation for regenerating the macro-lactone. These entities can then be used to synthesise new brefeldin analogues with various new structural features for biological testing. To overcome supply issues a scaled fermentation process of Penicillium camemberti was employed generating 4.5 kg of BFA. Novel N-BFA type derivatives were generated and herein we describe the synthesis of two new scaffolds 12 and 14 from BFA over six steps in 24% and 17% yield, respectively. A trans-fused bicyclic cyclopentanoid piperidine scaffold 16 can be generated from 12 that may serve as a valuable new scaffold for the synthesis of novel natural product-like compounds.

Introduction

Brefeldin A (BFA, 1; Fig. 1) is a naturally occurring macrolide produced by various fungal species [1], [2]. “The name “brefeldin A” was assigned to a metabolite from the fermentation of Penicillium brefeldianum Dodge [3] and shown to be identical to decumbin from Penicillium decumbens [4], [5]. Other names were reported [6], [7] until X-ray crystallographic data cemented the name “brefeldin A” [7].

The first total synthesis of racemic BFA was reported in 1976 and the synthesis of natural (+)-BFA was completed just a few years later [8], [9]. BFA is a natural product of considerable interest demonstrating antibiotic [10], antifungal [11], antiviral [12], antimitotic [13] and anti-inflammatory through to anti-tumor biological activity [3], [6]. Its use as a chemotherapeutic agent [6], [14] was sparked by promising data from the National Cancer Institute’s (NCI) human tumor cell line panel [15]. The compound was found to induce differentiation and apoptosis of tumor cells [16], [17]; and trigger disassembly of the Golgi apparatus by inhibition of ADP-ribosylation factor 1 (ARF1) activation impeding the wider function of the trans-Golgi network (TGN) [18], [19], [20], [21], [22], [23], [24]. Because ARF1 is also implicated in tumor progression and invasion it represents a potential target for the development of new cancer therapeutics [25]. The progress of BFA in clinical trials is hampered by adverse pharmacokinetic properties, such as poor aqueous solubility and bioavailability, rapid clearance and off-target effects [24](a), [26]. Improved water solubility and stability was achieved with the 7-N,N-dimethylglycinate prodrug, breflate (2), but undesirable side-effects remained an issue [27], [28], [29]. Therefore the preparation of new BFA analogues for biological assessment is warranted. One way to achieve this is through the application of a “top-down-approach” [30]. Thus, BFA was disassembled into intermediates to serve as starting points for the synthesis of new analogs of the natural product. The original intention was the reassembly of the BFA macrocycle with an amine linkage to probe the biochemical properties of 3 and analogues (variation of n and R; Fig 1). Initially, to provide sufficient BFA starting material, despite many advances in the total synthesis of BFA [31], we relied on microbial fermentation [32]. The manufacture of 4.5 kg of Brefeldin A with 99% purity by fermenting Penicillium camemberti in fermentation vessels of up to 1500 L was completed [33]. Herein, we will outline this process (full fermentation details to be reported at a later date) and describe our results of a feasibility study into the synthesis of novel N-BFA type derivatives (3).

Section snippets

Fermentation

Penicillium camemberti was recovered onto potato dextrose agar before inoculation into shake flasks. After 48 h the 1.7 L of monoculture was charged to 225 L of media in a 300 L fermenter, the culture grew for a further 8 days before 20 L was removed and charged to 1100 L of media in a 1500 L fermenter. A further 11 days of fermentation in the 1500 L fermenter resulted in brefeldin expression recorded at 4.5 g/kg. The 300 L fermenter was permitted to progress to day-10, where the brefeldin

Conclusion

In summary, a microbial manufacturing process has produced kilogram quantities of BFA of high purity. This process relies on the fermentation with Penicillium camemberti in 300 and 1500 L fermentation vessels and enabled the production 4.5 Kg of BFA in 99% purity. We have also outlined our first results from a feasibility study into the synthesis of novel N-BFA type derivatives via a top-down-approach using BFA as a starting material. Although this study has not yet provided a direct route to

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank GlycoSyn (Callaghan Innovation, Lower Hutt, New Zealand) and IRLBioPharm (Industrial Research Limited 1999-2008, Lower Hutt, New Zealand) for permission to reproduce the manufacturing process of brefeldin A and for access to the final product.33 BS gratefully acknowledges the University of Otago for the provision of a PhD scholarship.

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