Advances in the Chemical and Biological Characterization of Amaryllidaceae Alkaloids and Natural Analogues Isolated in the Last Decade

Amaryllidaceae are bulbous wild and cultivated plants well known for their beautiful flowers and pharmaceutical applications, essentially due to the alkaloids and flavonoids content. Hundreds of alkaloids have been isolated until now and several scientific publications reported their sources, chemical structures, and biological activities. During the last decade, some unstudied Amaryllidaceae plants were the object of in-depth investigations to isolate and chemically and biologically characterize new and already known alkaloids as well as some analogues. This review describes the isolation and chemical and biological characterization of the Amaryllidaceae alkaloids, and their analogues obtained in the last decade, focusing the discussion on the new ones.


Introduction
The Amaryllidaceae are wild [1] and cultivated plants in several countries. They are considered ornamental plants for their beautiful flowers and to produce volatile oils. They are dominant plants in Andean South America, the Mediterranean basin, and southern Africa [2,3]. The main metabolites synthesized by Amaryllidaceae are essentially alkaloids which accumulate in their bulbs.
Several transcriptomic and biochemical studies described the molecular features involved in the biosynthesis of Amaryllidaceae alkaloids, including enzymes from the shikimate and phenylpropanoid pathways, were recently reviewed [4]. Amaryllidaceae plants consist of ca. 85 genera and 1100 species, and ca. 600 structurally diverse alkaloids have been isolated from plants and grouped in 12 ring-types [5,6].
The investigation on the Amaryllidaceae alkaloids began in 1877 with the isolation of lycorine from Narcissus pseudonarcissus [7] and then the interest around this group of naturally occurring compounds increased because of the large spectrum of their biological activities. These include antitumor, antibacterial, antifungal, antimalarial, antiviral, analgesic, and cholinesterase (AChE and BuChE) inhibitory activities. The uniqueness of these alkaloid structures provided a viable platform for phytochemical-based drug discovery [6,8,9]. Galanthamine represents the main medicinal application of Amaryllidaceae alkaloids and is commercialized as an Alzheimer's drug [2].
Detailed investigations were carried out on the in vitro antiproliferative, apoptosis-inducing, and antiinvasive activities of Amaryllidaceae alkaloids and their derivatives, aiming to analyze their potential anticancer activity. These studies showed the potency of several Amaryllidaceae alkaloids as well as related isocarbostyryls (pancratistatine and narciclasine) against some solid tumors, reporting the mode of action to explain their cytotoxic activity [8][9][10][11][12][13][14][15][16][17]. Table 1. Amaryllidaceae alkaloids and natural analogues isolated in the last decade.

Alkaloids from Brunsvigia natalensis
Brunsvigia natalensis, also named as Natal Candelabra Flower, occurs in South Africa, Lesotho, and Swaziland, and was used in traditional medicine [35].

Alkaloids from Crinum jagus
Crinum jagus (syn. = Crinum giganteum) grows in Senegal and belongs to a genus which was shown to be very rich in crinine-type alkaloids [36]. The metabolites present in the aqueous and organic extract of C. jagus showed potential for the treatment of inflammatory processes [57], antibacterial [58], sedative [59], inhibition of cholinesterases [60], and antiviral [61] activities. All the alkaloids isolated were tested against parasitic protozoa and for cytotoxicity. Ungeremine, pseudolycorine, and haemanthamine showed good activity in vitro against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Plasmodium falciparum, with IC 50 (half maximal inhibitory concentration) values in the range of 3.66 mM or lower. Ungeremine showed higher toxicity than the other alkaloids but was inactive against Leishmania donovani and showed cytotoxic activity against L6 cells (rat skeletal myoblasts, IC 50 = 65.28 mM). Related compounds with quaternary nitrogen showed strong antiprotozoa activity and the anti-plasmodial activity exhibited by ungeremine, pseudolycorine, and haemanthamine does not depend on interactions with heme [25]. These results increased the knowledge on the structure-antiprotozoal activity relationships in Amaryllidaceae alkaloids, which are scantly investigated. Previously, only the role of a methylenedioxy group and a tertiary non-methylated nitrogen was reported to impart higher activity [38].

Alkaloid from Nerine huttoniae
Nerine huttoniae is a species belonging to the Nerine genus [23], which comprises ca. 23 perennial bulbous species native to southern Africa. This Amaryllidacea is a summer growing, evergreen species, and was essentially found in the western part of the Eastern Cape Province of South Africa [39,40]. It was used in folk medicine and in particular by Sotho and Zulu tribes [41].
From the bulbs of N. huttoniae, a new alkaloid, belonging to the homolycorine-type of Amaryllidaceae alkaloids and named 6-O-methylkrigeine (2, Figure 1, Table 1), was isolated together with the known oxokrigenamine. Compound 2 did not exhibit acetylcholine esterase inhibitory activity when tested at a concentration of 50 µg mL −1 [26].
Subsequently, the new N-methylhemeanthidine chloride (3, Figure 1, Tables 1 and 2) was isolated from Z. candida and exhibited potent cytotoxicity on a spectrum of tumor cells. Table 2. Biological activities of Amaryllidaceae alkaloids and natural analogues isolated in the last decade.

Alkaloids from Narcissus jonquilla quail
Narcissus jonquilla quail, native to Spain and Portugal, has now become naturalized in many regions of Europe and the United States. The extract of bulbs collected in Middlesex county of southeast England, as shown by GC analysis, showed the presence of galanthamine and haemanthamine as the main alkaloids [44].
Further investigation carried out by Masi et al. [29] allowed to isolate abundant amounts of haemanthamine, lycorine and narciclasine, and a new alkaloid, named jonquailine (4, Figure 1, Tables 1  and 2) and belonging to the pretazettine group of Amaryllidaceae alkaloids [29]. An extensive work was carried out by comparing the ECD spectrum of jonquailine and tazettine, and the ECD data reported in the literature for pretazettine showed that compound 4 and pretazettine have the same absolute configuration at the B/C and B/D ring junctions, while they are empimers at C-8. The stereochemistry at C-8 was not previously assigned in pretazettine and thus is not assigned in the alkaloid 4 [44]. Subsequently, the absolute configuration, R, at C-8 of compound 4 was assigned by density functional theory (DFT) calculations of chiroptical properties, namely electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and optical rotatory dispersion (ORD). These results confirmed the absolute configuration of jonquailine and allowed the assignment of an S configuration to C-8 of pretazettine [45].
Jonquailine showed anticancer activity against drug-resistant human tumor models with diverse mechanisms and displayed synergy with paclitaxel. These results and literature data demonstrated that the hydroxylation at C-8 is an important feature to impart the anticancer activity, which is independent from its stereochemistry as both jonquiline and pretazettine showed significant activity, while tazettine, lacking this hydroxyl group, had no activity [29].

Alkaloids from Lycoris longituba
Lycoris longituba is native in Jiangsu province in China [46], and its bulbs were used in folk medicine for different skin diseases [47].

Alkaloids from Nerine sarniensis
Nerine sarniensis belonging to a genus well-known as ornamental plants is an herbaceous bulbous perennial species. Nerine genus is comprised of 24 species in the Amaryllidaceae family and is endemic to South Africa and a few neighboring countries [2]. N. sarniensis is restricted to the Western Cape of South Africa [48].

Alkaloids from Crinum latifolium
Crinum latifolium is widespread in the upper Gangetic Plain. It is also cultivated and used in folk medicine [16]. This Amaryllidacea belongs to Crinum L., which is the only genus mainly distributed in Africa, America, Australia, and southern Asia [49][50][51][52]. This genus contains ca. 110 species [53].

Alkaloids from Zephyranthes grandiflora
Zephyranthes grandiflora, as the above-reported for Z. candida, belong to genus Zephyranthes, and consists of 60 species which are distributed mainly in the warm-temperate regions of the Western hemisphere [54,55]. These plants are well-known for their ornamental use and medicinal properties [56].

Alkaloids from Brunsvigia natalensis
Brunsvigia natalensis, also named as Natal Candelabra Flower, occurs in South Africa, Lesotho, and Swaziland, and was used in traditional medicine [35].

Alkaloids from Crinum jagus
Crinum jagus (syn. = Crinum giganteum) grows in Senegal and belongs to a genus which was shown to be very rich in crinine-type alkaloids [36]. The metabolites present in the aqueous and organic extract of C. jagus showed potential for the treatment of inflammatory processes [58], antibacterial [59], sedative [60], inhibition of cholinesterases [61], and antiviral [62] activities.

Conclusions
This review described the alkaloids and their natural analogues isolated in the last decade from different Amaryllidaceae species, focusing the discussion on the new ones. The world region in which they grow and their diffused use in folk medicine was also reported. Together with the new alkaloids, some new analogues were reported as well as other well-known alkaloids and other metabolites isolated from Amaryllidaceae plants. The description of isolation and chemical and biological characterization of the new alkaloids follows a chronological order and their biological activities in the tests were described and listed in Table 1, together with the plant source and literature. In some cases, results on the structure activity relationships were also described.