Poisonous Piperidine Plants and the Biodiversity of Norditerpenoid Alkaloids for Leads in Drug Discovery: Experimental Aspects

There are famous examples of simple (e.g., hemlock, Conium maculatum L.) and complex (e.g., opium poppy, Papaver somniferum L., Papaveraceae) piperidine-alkaloid-containing plants. Many of these are highly poisonous, whilst pepper is well-known gastronomically, and several substituted piperidine alkaloids are therapeutically beneficial as a function of dose and mode of action. This review covers the taxonomy of the genera Aconitum, Delphinium, and the controversial Consolida. As part of studying the biodiversity of norditerpenoid alkaloids (NDAS), the majority of which possess an N-ethyl group, we also quantified the fragment occurrence count in the SciFinder database for NDA skeletons. The wide range of NDA biodiversity is also captured in a review of over 100 recently reported isolated alkaloids. Ring A substitution at position 1 is important to determine the NDA skeleton conformation. In this overview of naturally occurring highly oxygenated NDAs from traditional Aconitum and Delphinium plants, consideration is given to functional effect and to real functional evidence. Their high potential biological activity makes them useful candidate molecules for further investigation as lead compounds in the development of selective drugs.


Piperidines: Poisonous, Tasty, and Beneficial
Nature is rich with examples of plants that can be described as medicinal or poisonous. The controversy in the description spotlights the fact, well known in pharmacy and the pharmaceutical sciences, that dose and mode of action are critical. Out of thousands of plants with various types of active principles, many examples can be found of natural sources of alkaloids containing a substituted piperidine nucleus.
There are well-known examples of simple piperidine-alkaloid-containing plants, such as the famous poison hemlock (Conium maculatum L.), in the family Apiaceae (formerly Umbelliferae). It is a lethal poison that was given to criminals in ancient Greece and that the Greek philosopher, Socrates, was forced to drink (399 B.C.) [1]. The principal component of poison hemlock is the piperidine alkaloid coniine 1 (Figure 1), which is a nicotinic acetylcholine receptor (nAChR) agonist [1] where the importance of the positive centre was highlighted in the Beers-Reich model [2]. Consumption of C. maculatum leads to various degrees of toxicity in animals, where it has been found that it is more poisonous to cattle than to other animals. Human toxicity signs were described by Socrates' pupil as trembling, staggering, and rapid muscular weakness. Death resulting from hemlock poisoning is mainly due to respiratory failure [1,3]. Another nAChR agonist is anabasine 2, a natural nicotine-3-like isomer compound from Nicotiana spp. (tobacco) of the family Solanaceae. Two important species are N. glauca (wild tree tobacco) and N. tabacum L. Anabasine 2 is the major component in N. glauca, while nicotine 3 is the main constituent in N. tabacum L. [3][4][5]. Another source of piperidine alkaloids is Lobelia spp. (Campanulaceae). An important example is the Indian tobacco, L. inflata L., which is used traditionally in smoking cessation and in the treatment of respiratory conditions [6]. Lobeline 4 is the major and the most  Aconitum L. has been divided into three subgenera (Aconitum, Lycoctonum (DC.) Peterm., and Gymnaconitum (Stapf) Rapes). The Aconitum subgenus Aconitum produces biennial tuberous roots, while subgenus Lycoctonum (DC.) Peterm. species have perennial rhizomes. The only annual species of the Aconitum genus can be found in subgenus Gymnaconitum (Stapf) Rapes. [43]. The Delphinium L. genus is also divided into two subgenera (Delphinastnim (DC.) Wang and Delphinium) [44], and the species within this genus are usually perennial (occasionally annual) [45]. Due to the shape of flowers of the Delphinium Aconitum L. has been divided into three subgenera (Aconitum, Lycoctonum (DC.) Peterm., and Gymnaconitum (Stapf) Rapes). The Aconitum subgenus Aconitum produces biennial tuberous roots, while subgenus Lycoctonum (DC.) Peterm. species have perennial rhizomes. The only annual species of the Aconitum genus can be found in subgenus Gymnaconitum (Stapf) Rapes. [43]. The Delphinium L. genus is also divided into two subgenera (Delphinastnim (DC.) Wang and Delphinium) [44], and the species within this genus are usually perennial (occasionally annual) [45]. Due to the shape of flowers of the Delphinium species which resemble dolphins, the Delphinium genus takes its name from the Greek word delphis [45].

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The genus Consolida has proved to be more controversial. A. P. De Candolle separated a group of annual species from the genus Delphinium L. to form an independent section (Consolida DC.). S.F. Gray changed the section Consolida to the rank of a genus in 1821 (Consolida (DC.) S.F. Gray). Boisser gave the rank of genus to the Consolida section of Delphinium L. in 1867 and Huth gave it the rank subgenus in 1895 [46]. Huth was the last worker to include Consolida within Delphinium. Much more recently, Jabbour and Renner (2011) suggested using a DNA phylogenetic study that Consolida should be embedded in Delphinium [47]. Commonly, Delphinium and Consolida species are called larkspur, which is also a name derived from the shape of the flowers [45]. The colourful flowers of the Delphinium species gave rise to many cultivated species (cultivars) that are used as ornamental plants in the garden. These hybrid species come from crossing different parent plants and mainly from the tetraploid D. elatum L. [48,49]. Examples of the hybrid Delphinium varieties are the giant pacific court hybrids which originate from D. elatum and other species such as D. exaltatum and D. formosum [50].

NDA Chemical Toxicity
North Americans have divided the Delphinium (larkspur) plants into three categories depending on habit of growth and environment. First are the tall larkspurs (such as D. barbeyi, D. occidentale), which are 1-2 m tall and generally exist at altitudes above 2400 m in moist habitats. Second are the intermediate larkspurs (such as D. geyeri, plains larkspur), which are 0.6-1 m tall and grow on the short grass prairies of Nebraska, Wyoming, and Colorado. The third category is low larkspurs (such as D. andersonii), which are less than 0.6 m tall and generally grow in the desert/semidesert, foothills, or low mountain ranges [51,52]. Delphinium (larkspur) alkaloids cause economically important livestock toxicity across North American ranges [51,53]. Tall larkspurs contain higher amounts of toxic NDAs and are therefore considered a greater threat [54]. Intoxication happens due to the action of NDAs at the α1-nAChR expressed at neuromuscular junctions (NMJ) [55].
It was found that the livestock intoxication by larkspurs is controlled by different factors, for example, cattle breed and genetics affect the susceptibility to the intoxication. Age is another factor, where young heifers are more susceptible than mature cows. The cattle sex was reported to be an effective factor, where heifers are more prone to the toxicity than steers and bulls. Lastly, the plant factor plays an important role, where the alkaloid concentration and composition of methylsuccinimidoanthranoyl-lycoctonine (MSAL) and non-MSAL ( Figure 2), which depend on the population, species, climate, and the year, affect the toxicity in cattle and the amount needed to develop clinical signs [56,57].
The toxicity of NDAs found in three tall larkspur species (D. barbeyi, D. occidentale, D. glaucescens) was tested in mice. The assay revealed that the 7,8-methylenedioxy-lycoctonine (MDL) alkaloids are the least toxic NDAs. The lycoctonine-type is twice as toxic as MDL, but it is considered to be a low toxic group, where the least toxic alkaloid of this category, brownine 20, has a toxicity which is comparable to the MDL NDA. The MSAL alkaloids MLA 16 and 14-deacetylnudicauline 21 were 10-times more toxic than any other tested NDAs ( Figure 2) [54].
MSAL is much more toxic than MDL, and the MSAL level in the tall larkspurs mainly contributes to livestock poisoning. A report investigated the importance of the MDL alkaloids and found that MDL alkaloids exacerbate the toxicity of the MSAL alkaloids; as the ratio of MDL to MSAL increases, the amount of MSAL that is needed to develop clinical signs decreases. The exact mechanism of this MDL action is not known, but it was suggested that MDL may act as a co-agonist in an allosteric manner or at the orthosteric ligand binding site of the receptor to exacerbate the toxicity of MSAL-type alkaloids on nAChR and therefore increase their toxicity [58]. The observed action could also be due to an effect of MDL alkaloids on metabolic enzymes which results in prolonged exposure to the MSAL alkaloids, but further investigation is needed. MSAL is much more toxic than MDL, and the MSAL level in the tall larkspurs mainly contributes to livestock poisoning. A report investigated the importance of the MDL alkaloids and found that MDL alkaloids exacerbate the toxicity of the MSAL alkaloids; as the ratio of MDL to MSAL increases, the amount of MSAL that is needed to develop clinical signs decreases. The exact mechanism of this MDL action is not known, but it was suggested that MDL may act as a co-agonist in an allosteric manner or at the orthosteric ligand binding site of the receptor to exacerbate the toxicity of MSAL-type alkaloids on nAChR and therefore increase their toxicity [58]. The observed action could also be due to an effect of MDL alkaloids on metabolic enzymes which results in prolonged exposure to the MSAL alkaloids, but further investigation is needed.

Norditerpenoid Alkaloid (NDA) Biodiversity
NDAs have complex highly oxygenated hexacyclic systems, and as many of them are of pharmacological importance, their structures and 3D configuration are significant factors in their actions at various biological targets [59]. The majority of NDAs possess an N-Et group, as shown in Table 1, which shows various NDA skeletons and their abundance in the SciFinder database. Substitution at position 1 is important to determine the NDA skeleton conformation, as ring A in 1-OMe NDA free bases exists in a twisted-chair conformation, and in 1-OH NDA ring A adopts a twisted-boat conformation [60,61]. Table 1 shows that 1-OMe NDA abundancy is 10-times higher than 1-OH NDA. The biological activity of NDAs attracts natural product chemists to investigate their sources, and that has resulted in the discovery of interesting NDA skeletons, some of them with pharmacological importance.

Norditerpenoid Alkaloid (NDA) Biodiversity
NDAs have complex highly oxygenated hexacyclic systems, and as many of them are of pharmacological importance, their structures and 3D configuration are significant factors in their actions at various biological targets [59]. The majority of NDAs possess an N-Et group, as shown in Table 1, which shows various NDA skeletons and their abundance in the SciFinder database. Substitution at position 1 is important to determine the NDA skeleton conformation, as ring A in 1-OMe NDA free bases exists in a twisted-chair conformation, and in 1-OH NDA ring A adopts a twisted-boat conformation [60,61]. Table 1 shows that 1-OMe NDA abundancy is 10-times higher than 1-OH NDA. The biological activity of NDAs attracts natural product chemists to investigate their sources, and that has resulted in the discovery of interesting NDA skeletons, some of them with pharmacological importance.           . Vilmorine D 26 exhibited moderate to weak antioxidant activity (Fe 2+ chelation activity) with IC 50 = 33.6 ± 0.2 µg/mL, and it showed antibacterial activity against Staphylococcus aureus and Bacillus subtilis with MICs of 64 and 32 µg/mL, respectively. Vilmorine A 23 has an unusual spiro junction. Only three such compounds were isolated with that characteristic skeleton (Table 1). Vilmorine A 23 also has the really unusual 1-β-OMe group, whereas the vast majority of the position 1 substituents have an α-configuration. Vilmorines B-C 24-25 have an unusual cyclopropyl moiety, and they are rare examples containing an imine (piperideine) ( Table 1).      [70]. Ding and coworkers also isolated three new NDAs, vilmotenitines A-C 46-48 from A. vilmorinianum var. patentipilum, ( Figure 5) where vilmotenitines A and B 46-47 had an unusual (spiro) rearranged six-membered B ring [71], as they had found in vilmorine A 23 with the inverted substituent stereochemistry at position 1 [63].  Wang and co-workers discovered three new C19 NDAs, szechenyianine A, B, and C 55-57, from A. szechenyianum [74]. All three compounds were tested against nitric oxide (NO) release inhibition, as they were considered potential anti-inflammatory agents. Szechenyianine A 55 showed activity with IC 50 36.6 ± 7 µM, while szechenyianine B 56 had IC 50 3.3 ± 0.1 µM, and that highlights the importance of the N-O moiety. Szechenyianine C 57 ( Figure 6) which is a 7,17-secoaconitine-type NDA, also showed potent activity, with IC 50 7.5 ± 0.9 µM.
Chao Zhan et al. reported caerudelphinine A 58, a new 1-OH C-19 lycoctonine-type NDA from D. caeruleum Jacq. ex Camp [75]. Grandiflorine B 59 was also reported as a new C-19 lycoctonine-type NDA from D. grandiflorum [76]. The unusual skeleton of grandiflorine B 59 shows cleavage of the 7-17 bond and N-C19 bond and the formation of an unusual N-C7 bond. Zhao et al. reported three new NDAs, nagaconitine A-C 60-62 from A. nagarum var. heterotrichum [77]. Nagaconitine A 60 has a unique acyl group which was reported in only three NDAs. 8,14-Diacetate diester 62 showed antitumor activity against cancer cell line SK-OV-3. Two more new C-19 NDAs, 14-benzoylliljestrandisine 63 and 14-anisoylliljestrandisine 64, were isolated from A. tsaii (Figure 7) [78]. Wang and co-workers discovered three new C19 NDAs, szechenyianine A, B, and C 55-57, from A. szechenyianum [74]. All three compounds were tested against nitric oxide (NO) release inhibition, as they were considered potential anti-inflammatory agents. Szechenyianine A 55 showed activity with IC50 36.6 ± 7 μM, while szechenyianine B 56 had IC50 3.3 ± 0.1 μM, and that highlights the importance of the N-O moiety. Szechenyianine C 57 ( Figure 6) which is a 7,17-secoaconitine-type NDA, also showed potent activity, with IC50 7.5 ± 0.9 μM. C-19 lycoctonine-type NDA from D. grandiflorum [76]. The unusual skeleton of grandiflorine B 59 shows cleavage of the 7-17 bond and N-C19 bond and the formation of an unusual N-C7 bond. Zhao et al. reported three new NDAs, nagaconitine A-C 60-62 from A. nagarum var. heterotrichum [77]. Nagaconitine A 60 has a unique acyl group which was reported in only three NDAs. 8,14-Diacetate diester 62 showed antitumor activity against cancer cell line SK-OV-3. Two more new C-19 NDAs, 14-benzoylliljestrandisine 63 and 14anisoylliljestrandisine 64, were isolated from A. tsaii (Figure 7)      Guo et al. isolated two new NDAs, 7,8-epoxy-franchetine 71 and N-(19)-en-austroconitine 72, from A. iochanicum [80]. Tested against NO production in macrophages (mouse cell line), they showed a weak anti-inflammatory effect. Liang et al. isolated sinchiangensine A 73 as a new NDA from A. sinchiangense W.T. Wang (Figure 9), and it showed significant antitumour activity against cancer cell lines A-549, SMCC-7721, MCF-7, and SW-480 [81]. The IC50 (μM) values of 73 against these cell lines were 12.8, 9.6, 11.8, and 18.8, respectively, and these values were comparable with cisplatin, the positive control, the IC50 (μM) values of which were 22.3, 18.6, 28.8, and 18.2. Sinchiangensine A 73 also showed potent antibacterial activity against Gram-positive S. aureus ATCC-25923, with an MIC value (μmol/mL) of 0.15 which is comparable to 0.67, the MIC of the positive control berberine HCl.   Fukuyama and co-workers [84] achieved the synthesis of cardiopetaline 83 through Wagner-Meerwein rearrangement of the denudatine skeleton into an aconitine skeleton without the need of pre-activation of the hydroxy group. This means that there is no need to differentiate the hydroxy groups in the poly-oxygenated system as was needed before.  Fukuyama and co-workers [84] achieved the synthesis of cardiopetaline 83 through Wagner-Meerwein rearrangement of the denudatine skeleton into an aconitine skeleton without the need of pre-activation of the hydroxy group. This means that there is no need to differentiate the hydroxy groups in the poly-oxygenated system as was needed before. Fukuyama and co-workers [84] achieved the synthesis of cardiopetaline 83 through Wagner-Meerwein rearrangement of the denudatine skeleton into an aconitine skeleton without the need of pre-activation of the hydroxy group. This means that there is no need to differentiate the hydroxy groups in the poly-oxygenated system as was needed before. Sarpong and co-workers [85]              Ahmad and co-workers reported the isolation of two new C-19 NDAs, jadwarine A-B 119-120 from D. denudatum [95]. They also reported a new lycoctonine-type C-19 NDA, swatinine C 121 ( Figure 18) which showed competitive inhibitory activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [96].     Ahmad and co-workers reported the isolation of two new C-19 NDAs, jadwarine A-B 119-120 from D. denudatum [95]. They also reported a new lycoctonine-type C-19 NDA, swatinine C 121 ( Figure 18) which showed competitive inhibitory activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [96]. Ahmad and co-workers reported the isolation of two new C-19 NDAs, jadwarine A-B 119-120 from D. denudatum [95]. They also reported a new lycoctonine-type C-19 NDA, swatinine C 121 ( Figure 18) which showed competitive inhibitory activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [96].  Extraction of the roots of A. brevicalcaratum led to the isolation of three new C-19 NDAs, brochyponines A-C 122-124 ( Figure 18) [97]. Abjalan et al. discovered a new lycoctonine-type C-19 NDA, aemulansine 125 from D. aemulans Navaski, which showed in vitro cytotoxicity [98]. Two novel 8,15-seco C-19 NDAs, nagarine A 126 and B 127 (Figure 19), were isolated from A. nagarum [99]. The variation in pharmacological activities of the NDAs, despite their structural similarities, is an attractive aspect for synthetic chemists to work on to obtain a better understanding of the structure-activity relationships (SAR). Liu et al. reported a total synthesis of the ABCDE system of the C-19 NDAs [100]. Another study reported the construction of the AEF ring system attached to a phenyl group as an analogue to ring D [101]. The construction of the fused CD-bicycle of aconitine was also achieved [102]. Lv et al. built the hexacyclic ring system of franchetine 128, a 7,17-seco NDA [103]. The importance of such NDAs continues to attract chemists to attempt to make a total synthesis of them. Progress has been made in the total synthesis of aconitine 12, but the construction of a pentacyclic system of the aconitine skeleton failed [104]. On the other hand, a total synthesis of talatisamine 129 (Figure 20) has been completed in 33 steps [105]. In addition, the synthesis of a [6-6-6] ABE-tricyclic analogue of MLA 16 has been achieved [106]. A syn- Extraction of the roots of A. brevicalcaratum led to the isolation of three new C-19 NDAs, brochyponines A-C 122-124 ( Figure 18) [97]. Abjalan et al. discovered a new lycoctonine-type C-19 NDA, aemulansine 125 from D. aemulans Navaski, which showed in vitro cytotoxicity [98]. Two novel 8,15-seco C-19 NDAs, nagarine A 126 and B 127 (Figure 19), were isolated from A. nagarum [99].  Extraction of the roots of A. brevicalcaratum led to the isolation of three new C-19 NDAs, brochyponines A-C 122-124 ( Figure 18) [97]. Abjalan et al. discovered a new lycoctonine-type C-19 NDA, aemulansine 125 from D. aemulans Navaski, which showed in vitro cytotoxicity [98]. Two novel 8,15-seco C-19 NDAs, nagarine A 126 and B 127 (Figure 19), were isolated from A. nagarum [99]. The variation in pharmacological activities of the NDAs, despite their structural similarities, is an attractive aspect for synthetic chemists to work on to obtain a better understanding of the structure-activity relationships (SAR). Liu et al. reported a total synthesis of the ABCDE system of the C-19 NDAs [100]. Another study reported the construction of the AEF ring system attached to a phenyl group as an analogue to ring D [101]. The construction of the fused CD-bicycle of aconitine was also achieved [102]. Lv et al. built the hexacyclic ring system of franchetine 128, a 7,17-seco NDA [103]. The importance of such NDAs continues to attract chemists to attempt to make a total synthesis of them. Progress has been made in the total synthesis of aconitine 12, but the construction of a pentacyclic system of the aconitine skeleton failed [104]. On the other hand, a total synthesis of talatisamine 129 (Figure 20) has been completed in 33 steps [105]. In addition, the synthesis of a [6-6-6] ABE-tricyclic analogue of MLA 16 has been achieved [106]. A syn- The variation in pharmacological activities of the NDAs, despite their structural similarities, is an attractive aspect for synthetic chemists to work on to obtain a better understanding of the structure-activity relationships (SAR). Liu et al. reported a total synthesis of the ABCDE system of the C-19 NDAs [100]. Another study reported the construction of the AEF ring system attached to a phenyl group as an analogue to ring D [101]. The construction of the fused CD-bicycle of aconitine was also achieved [102]. Lv et al. built the hexacyclic ring system of franchetine 128, a 7,17-seco NDA [103]. The importance of such NDAs continues to attract chemists to attempt to make a total synthesis of them. Progress has been made in the total synthesis of aconitine 12, but the construction of a pentacyclic system of the aconitine skeleton failed [104]. On the other hand, a total synthesis of talatisamine 129 ( Figure 20) has been completed in 33 steps [105]. In addition, the synthesis of a [6-6-6] ABE-tricyclic analogue of MLA 16 has been achieved [106]. A synthetic approach has also been established for the BCD-tricyclic system [107].

Conclusions
It is clear that nature is rich with many examples of plants that can be described as medicinal or poisonous. The poisonous piperidine plants show that dose and mode of pharmacological action are critical. The biodiversity of natural sources of NDAs, based upon a substituted piperidine nucleus, are important for continuing to provide leads in drug discovery. NDAs from Aconitum and Delphinium have complex, highly oxygenated hexacyclic systems, and as many of them are of pharmacological importance, their structures and 3D configuration are significant factors in their actions at various protein targets with respect to medicine and toxicology.
The majority of NDAs possess an N-Et group. We investigated the occurrence count in the SciFinder database for NDA skeletons, including many new NDAs. Substitution at position 1 is important to determine the NDA skeleton conformation, as ring A in 1-OMe NDA free bases exists in a twisted-chair conformation. In 1-OH NDA, ring A adopts a twisted-boat conformation. In conclusion, screening NDAs for their biological activity has resulted in the discovery of new sets of ligands. These are promising natural compounds that are pharmacologically active. These hits potentially eventually will become selective leads for the treatment of a wide variety of disease states. These NDAs are new natural products, and if they can be isolated from easy-to-grow Aconitum or Delphinium plants, then the future is bright for further NDA development based on experimental aspects, including phytochemistry leading to SAR studies and hopefully to new, selective, if not specific, drugs.

Conclusions
It is clear that nature is rich with many examples of plants that can be described as medicinal or poisonous. The poisonous piperidine plants show that dose and mode of pharmacological action are critical. The biodiversity of natural sources of NDAs, based upon a substituted piperidine nucleus, are important for continuing to provide leads in drug discovery. NDAs from Aconitum and Delphinium have complex, highly oxygenated hexacyclic systems, and as many of them are of pharmacological importance, their structures and 3D configuration are significant factors in their actions at various protein targets with respect to medicine and toxicology.
The majority of NDAs possess an N-Et group. We investigated the occurrence count in the SciFinder database for NDA skeletons, including many new NDAs. Substitution at position 1 is important to determine the NDA skeleton conformation, as ring A in 1-OMe NDA free bases exists in a twisted-chair conformation. In 1-OH NDA, ring A adopts a twisted-boat conformation. In conclusion, screening NDAs for their biological activity has resulted in the discovery of new sets of ligands. These are promising natural compounds that are pharmacologically active. These hits potentially eventually will become selective leads for the treatment of a wide variety of disease states. These NDAs are new natural products, and if they can be isolated from easy-to-grow Aconitum or Delphinium plants, then the future is bright for further NDA development based on experimental aspects, including phytochemistry leading to SAR studies and hopefully to new, selective, if not specific, drugs.