Isolation, Biological Activities, and Synthesis of the Natural Casuarines

Abstract

This chapter describes the isolation, structure elucidation, glycosidase inhibitory activities, and the synthesis of the four naturally occurring casuarines. These are casuarine, casuarine-6-O-α-d-glucoside, 6-epi-casuarine (uniflorine A), and 3-epi-casuarine.

Introduction

Casuarine 1[1], casuarine-6-O-α-glucoside 2[2], 6-epi-casuarine 3 (uniflorine A) [3], [4], [5], and 3-epi-casuarine 4[6] are members of the expanding group of polyhydroxylated 3-hydroxymethylpyrrolizidine natural products (Fig. 1) [7]. This group also includes australine [8], alexine [9] (7a-epi-australine), several other epi-australines (1-epi-australine, 3-epi-australine [10], 2,3-di-epi-australine, 2,3,7-tri-epi-australine) [11], 1-epi-australine-2-O-α-glucoside, and the more recently isolated hyacinthacine alkaloids of which 19 novel compounds have been identified [12]. This group, along with the polyhydroxylated pyrrolidine, piperidine, indolizidine, and nortropane alkaloids, has glycosidase inhibitory activities and thus has potential utility as antiviral, anticancer, antidiabetic, and antiobesity drugs [7]. Three structurally related synthetic compounds have been marketed as antidiabetic drugs to treat type-II diabetes based on their potent α-glucosidase inhibitory activities, while others have been identified as candidates for therapeutics to treat type-1 Gaucher disease [7]. These potentially useful biological activities, along with the stereochemical richness of these alkaloids (uniflorine A and casuarine have six contiguous stereogenic carbons), have made these compounds attractive and important synthetic targets [13]. This chapter describes the isolation, structure elucidation, glycosidase inhibitory activities, and the synthesis of the four naturally occurring casuarines.

Casuarina equisetifolia L., or commonly called, Australian pine, Filao or beach she oak, is a plant in the family Casuarinaceae, native to South East Asia, islands of the western Pacific Ocean (including French Polynesia, New Caledonia, Vanuatu), Australia (Northern Territory, north and east Queensland, and northeastern New South Wales), and West Africa. It is an evergreen tree that grows to over 6–35 m in height [14].

The first pentahydroxylated pyrrolizidine alkaloid, with six contiguous stereogenic centers and functional groups on all of the eight carbon atoms, was isolated in 1994 from the bark of C. equisetifolia L.[1]. This bark was prescribed as a remedy to treat breast cancer in Western Samoa [2]. Extracts of the wood, bark, and leaves of this plant have also been claimed to be useful for the treatment of diarrhea, dysentery, and colic [1]. This alkaloid was named casuarine 1 ((1R,2R,3R,6S,7R,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine) by Nash et al. [1]. This investigation started with a GC–MS analysis of the per-trimethylsilylated bark extract which revealed a pentahydroxylated pyrrolizidine alkaloid and its glycoside as the major alkaloid components. The 75% aqueous ethanol bark extract was purified by ion-exchange column chromatography with Amberlite CG-120 (NH₄⁺ form) which was eluted with 0.1 M NH₄OH to afford first the glycoside of casuarine 2 and then casuarine 1 itself (Fig. 1). Both alkaloids were isolated in approximately the same amounts with the latter in 0.013% yield based on the weight of the dried ethanol extract [1]. The absolute configuration of casuarine 1 was established by X-ray crystallographic analysis [1].

Eugenia jambolana is a plant in the family Myrtaceae, native to Bangladesh, India, Nepal, Pakistan, and Indonesia. An evergreen tree, it grows to 30 m in height. The extracts of the fruit pulp from E. jambolana have been reported to have antidiabetic properties, although this has been questioned in a more recent study [15]. In 1996, Wormald et al.[2] isolated casuarine 1 and its glucoside 2 from the leaves and the seeds of E. jambolana using Amberlite CG-120 (NH₄⁺ form) ion-exchange chromatography. From 630 g of air-dried leaves they isolated 140 mg of casuarine 1 and 15 mg of the glucoside 2.

Eugenia uniflora, Surinam cherry, Brazilian cherry, or Cayenne cherry, is a plant in the family Myrtaceae, native to tropical America and widely distributed in Paraguay, Uruguay, Argentina, and Brazil [3]. Decoctions of the leaves of this small tree are used as traditional medicines for a number of ailments, including use as an antidiabetic preparation. A number of studies have been made on the biological activities of the leaf extracts [16], [17], [18].

The water-soluble extracts of the leaves of E. uniflora L. have been used as an antidiabetic agent in Paraguayan traditional medicine [3]. In 2000, Arisawa et al.[3] reported the isolation of uniflorine A and B from the leaves of this tree. The water-soluble extract was purified twice on Amberlite ion-exchange resins and then on silica gel and finally HPLC to give samples of uniflorine A, uniflorine B, and (+)(3α,4α,5β)-1-methylpiperidine-3,4,5-triol in undisclosed amounts. The structures of the alkaloids uniflorine A and uniflorine B were deduced from NMR analysis to be that of the pentahydroxyindolizidine structures 3a and 1a, respectively (Fig. 2).

In 2004, Pyne and Davis [19] synthesized the proposed structure of uniflorine A; however, the NMR spectral data for synthetic 3a did not match with those reported for uniflorine A [3]. The structure of their synthetic 3a was unequivocally established by a single-crystal X-ray crystallographic study of its pentaacetate derivative. The Wollongong researchers therefore concluded that the structure originally assigned to uniflorine A was not correct [19]. The initial thoughts of several researchers were that uniflorine A was a diastereoisomer of 3a. In 2006, Dhavale et al. [20], in their paper of partial title, “Attempts To Find the Correct Structure of Uniflorine A,” reported the second synthesis of compound 3a. Their sample of 3a had NMR spectral data identical to those of 3a that was earlier synthesized by Pyne et al. [19]. This paper also reported the synthesis of two diastereomers of 3a, 8a-epi-3a and 1,2,8a-tri-epi-3a. In 2005, Mariano et al.[21] reported the synthesis of 1-epi-3a, while that of 1,2-di-epi-3a had been reported by Fleet et al. in 1996 [22], before uniflorine A was even isolated, and later by Mariano et al.[21] and by Pyne et al. in 2008 [4]. In 2008, Pyne et al. reported the synthesis of 2-epi-3a (Fig. 3) [4]. Despite these synthetic chemistry efforts, these 1,2,6,7,8-pentahydroxyindolizidine molecules also had NMR spectral data significantly different to those of uniflorine A.

From a reexamination of the original NMR data, Pyne, Davis, and Ritthiwigrom reassigned uniflorine B as the known pyrrolizidine alkaloid casuarine 1, while the structure of (−)-uniflorine A was suggested to be that of 6-epi-casusarine 3 (Fig. 2) [4]. The structure of uniflorine A was unequivocally established to be that of 6-epi-casuarine 3 by its total synthesis (see synthesis section) [5], [23], [24].

Myrtus communis L., commonly known as Myrtle or True Myrtle, belongs to the family Myrtaceae. It originates from the Mediterranean, North African, and Western Asia regions. Casuarine 1 and 3-epi-casuarine 4 were isolated from M. communis L. growing in the grounds of the Institute of Grassland and Environmental Research in Aberystwyth, UK. The isolation was conducted using ion-exchange chromatography. Casuarine 1 was the major alkaloid present, which eluted first with water from the anion-exchange resin Dowex 1 (OH form) followed by 3-epi-casuarine 4 ((1R,2R,3S,6S,7R,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine) (Fig. 1). No other epimer of 1 was isolated. Casuarine 1 and 3-epi-casuarine 4 were crystallized from warm 95% aqueous ethanol by layering with acetone. The absolute configuration of 3-epi-casuarine 4 was established by X-ray crystallographic analysis [6].