Cyclic Imines: Chemistry and Mechanism of Action: A Review

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Cyclic Imines: Chemistry and Mechanism of Action: A Review Alberto Otero, María-Jose Chapela, Miroslava Atanassova, Juan M. Vieites, and Ana G. Cabado* Microbiology and Biotoxins Area, ANFACO-CECOPESCA, Col. Univ. 16, 36310 Vigo (PO), Spain ABSTRACT: In recent years, there has been an increase in the production of shellfish and in global demand for seafood as nutritious and healthy food. Unfortunately, a significant number of incidences of shellfish poisoning occur worldwide, and microalgae that produce phycotoxins are responsible for most of these. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Cyclic imines (CIs) are a recently discovered group of marine biotoxins characterized by their fast acting toxicity, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. These toxins are macrocyclic compounds with imine (carbonnitrogen double bond) and spiro-linked ether moieties. They are grouped together due to the imino group functioning as their common pharmacore and due to the similarities in their intraperitoneal toxicity in mice. Spirolides (SPXs) are the largest group of CIs cyclic imines that together with gymnodimines (GYMs) are best characterized. Although the amount of cyclic imines in shellfish is not regulated and these substances have not been categorically linked to human intoxication, they trigger high intraperitoneal toxicity in rodents. In this review, the corresponding chemical structures of each member of the CIs and their derivatives are reviewed as well as all the data accumulated on their mechanism of action at cellular level.

’ CONTENTS Introduction Chemical Structures of the Known Cyclic Imines As Part of Fast-Acting Toxins Gymnodimines Spirolides Pinnatoxins and Pteriatoxins Prorocentrolides and Spiroprorocentrimines Mechanism of Action Antagonism on Nicotinic Acetylcholine Receptors Antagonism on Muscarinic Acetylcholine Receptors Acknowledgment Abbreviations References

1817 1818 1818 1819 1820 1824 1825 1826 1826 1827 1827 1827

’ INTRODUCTION Shellfish are an important and traditional food source for many people, in particular those that live within coastal regions. Nowadays, there is an increase in production of shellfish and a global demand of seafood as nutritionally healthy food. Unfortunately, each year a significant number of shellfish poisoning incidents occur worldwide, and microalgae that produce phycotoxins are responsible for most of these incidences. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Each phycotoxin group typically consists of several main compounds, r 2011 American Chemical Society

based on the same or similar chemical structure, and also of several analogues, produced by microalgae or obtained through biotransfromations by marine organisms. These toxins accumulate not only in shellfish but also in crustacean and fish and may pose a direct threat to human beings. In order to enhance the safety of consumers, the study of chemical structure, identity, and mechanism of action of these toxins is required. Spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs), pteriatoxins (PtTXs), prorocentrolides, and spiro-prorocentrimine are marine biotoxins produced by dinoflagellates that belong to the group of cyclic imines (CIs). These toxins are macrocyclic compounds with imine (carbonnitrogen double bond) and spiro-linked ether moieties. They are grouped together due to first the imino group functioning as their common supposed pharmacophore1 and second the similarities in their intraperitoneal toxicity in mice.2 The key that allowed the identification of this recently discovered group of marine biotoxins was their “fast acting toxicity”, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. Furthermore, these polar fast acting toxins were reported to display an “all or nothing” effect.3 The largest group of CIs are the SPXs that together with GYMs are the best characterized. Presently, 14 SPX analogues have been isolated of which 13-desmethyl SPX C is the most commonly found in shellfish. The chemical structures of three GYM (GYM A-G) analogues have been characterized, GYM A being the first one isolated. PnTX and PtTXs are the CIs most closely related to the chemical structure of SPXs. Seven PnTXs Received: May 2, 2011 Published: July 08, 2011 1817

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Figure 1. 2D Structures of known members of the gymnodimine family.20

analogues (PnTXs A-G) have been chemically characterized. PtTXs and PnTXs are almost structurally identical, and the latest studies suggest that PnTXs analogues F and G are progenitors of all the known PnTXs and PtTXs via metabolic and hydrolytic transformation in shellfish.2,4 Finally, two structures of prorocentrolide (A and B) and a spiro-prorocentrimine were elucidated so far. They are potent neurotoxins, and there is evidence that their neurotoxic action is based on the inhibition of both the nicotinic and muscarinic acetylcholine receptors in the central and peripheral nervous system5 and at the neuromuscular junction.69 The toxicological database for SPXs, GYMs, PnTXs, and PtTXs is limited, comprising mostly acute toxicity studies. In view of the acute toxicity and the lack of chronic toxicity data, the EFSA Panel on Contaminants in the Food Chain could not establish an acute reference dose. Thus, nowadays the amount of cyclic imines in shellfish is not regulated in Europe or in other parts of the world. However, at least regarding SPXs and GYMs, even though these CIs have not been categorically linked to any human intoxication, they trigger high intraperitoneal toxicity in rodents. Many scientists agree about the regulation of the allowed levels of these toxins by setting the limits with the oral toxicity of laboratory animals.

’ CHEMICAL STRUCTURES OF THE KNOWN CYCLIC IMINES AS PART OF FAST-ACTING TOXINS In this review, the corresponding chemical structures of each member of the CIs toxins and their derivatives will be reviewed separately. Gymnodimines. The gymnodimine marine toxins group, produced by the dinoflagellate Karenia selliformis, possesses a macrocyclic structure (spiro center) with an ether or polyether subunit and exercises imine functions.10 The gross structure was initially reported by Yasumoto and co-workers in 1995, and further on, Munro and Blunt and colleagues reported the relative and absolute stereochemistry by X-ray crystallographic analysis of a reduced N-acylated derivative in 2000.11,12 The chemical structures of the known gymnodimine analogues have been gathered for this review in Figure 1. The biotoxin gymnodimine 1 (also called gymnodimine A (GYM-A)), was first isolated from oysters (Tiostrea chilensis), collected at the Foveaux Strait, South Island of New Zealand and was found to exhibit neurotoxic effects. Gymnodimine was then described as a complex pentacyclic derivative incorporating a C24 carboxylic acid and a fused azine and was obtained in the form of a colorless amorphous solid substance.11 Its molecular mass is of 504.704 g/mol, and the simple molecular formula is C32H45NO4. Besides in the New Zealand coastal area, outbreaks of intoxication by gymnodimine A have also been observed in Tunisia.13

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The next two analogues from the group, GYM-B and GYM-C, were discovered shortly afterward, in cell cultures of the toxic phytoplankton producer from the coast of New Zealand.14,15 The structure of gymnodimine B is similar to that of GYM-A but contains an exocyclic methylene at position C-17 and allylic hydroxyl group at position C-18, while GYM-C is an oxidized isomer of GYM-B at position C-18.16 Since gymnodimines A and B are known to cause a characteristic rapid death in the intraperitoneal mouse bioassay, being called “fast-acting toxins”, their interest for the pharmacological industry has brought about very detailed chemical studies and the development of different synthetic methods for their enhanced production. The main pharmacological interest of that group and of the other related spiro-cyclic imine toxins resides in the spirocyclic imine ring.17 Any further exploration of the toxicological effects of these molecules and their pharmacology requires reliable and active supply of the pure compounds. Thus, Mountfort and colleagues have developed a biosynthetic method for enhanced gymnodimine production from axenic K. selliformis cultures through the addition of organic acids, amino acids, glycolate, and acetate with an optimized lighting regime. Some chemically modified analogues of GYM-A as gymnodimine acetate, gymnodimine methyl carbonate, and gymnodamine were synthesized as pro-drug forms of the actual gymnodimine by Dragunow and co-workers from a purified GYM-A fraction from a laboratory culture of K. selliformis for the study of the toxin’s receptor binding mechanisms (Figure 2). All of the four molecules (gymnodimine A and its three structural analogues, used in this study, which were selected to be chemically quite distinct) produced a similar effect on neuroblastoma cell lines, at similar concentrations, causing a strong and consistent sensitization of neurons to the toxic effects of another marine toxin, okadaic acid. This fact suggested that certain conserved regions from the gymnodimine structure could be responsible for its pharmacologically important activity.18 Although gymnodimine has a relatively simple architecture when compared to that of other members of the cyclic imines family, it conceals subtle, challenging structural elements such as, for example, the labile butenolide part, representing difficulties for the total synthesis of this toxin. One of the first research groups to attempt artificial chemical synthesis, Yang and colleagues, has developed a partial synthetic process for the gymnodimine molecule starting with a tetrahydrofuran fragment and developing a strategy toward the addition of the spirocyclic imine fragment.10 Certain steps of the process developed suffered from low stereoselectivity. In another work, of White and colleagues, published in 2006, two principal subunits from the gymnodimine molecule were synthesized.19 The first subunit, trisubstituted tetrahydrofuran representing carbon atoms from C10 to C18 of the toxin was prepared by high stereoselective cyclization. Then a cyclohexene subunit corresponding to the C1C8 and C19C24 positions of gymnodimine were synthesized via another approach (catalytic asymmetric DielsAlder cycloaddition), and the two subunits were linked at position C18C19 through B-alkyl coupling. Several subsequent transformations were applied to position all functional groups in the product thus coupled in order to prepare it for a future macrocyclization event that would close the 15-membered ring of gymnodimine. In this way, the developed convergent approach of advanced gymnodimine intermediate section (C3 C32) synthesis completed the previous studies on the subject and laid the groundwork upon which the final stages of a complete chemical synthesis of gymnodimine could be based. 1818

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Figure 2. Chemically modified pro-drug analogues of GYM-A (1), gymnodimine acetate (2), gymnodimine methyl carbonate (3), and gymnodamine (4) according to Dragunow et al.,18 synthesized in the following conditions: (i) Ac2O, Et3N, CH2Cl2, DMAP, 0 °C, 1 h; (ii) methyl chloroformate, CH2Cl2, DMAP, 0 °C, 6 h; and (iii) NaCNBH3, MeOH, AcOH, 0 °C, 5 h.

The first complete synthetic work on gymnodimine was recently published by Kong and co-workers, providing suitable intermediates for the eventual production of an enzyme-linked immunosorbent assay (ELISA) for gymnodimine detection and also for further mode-of-action studies.20 The method developed used an unusual Barbier-type macrocyclization with tBuLi at ambient temperature and then the appendage of a chiral butenolide element to the synthesized part through vinylogous Mukaiyama aldol addition. In this context, a way for the production of pure standard gymnodimine was provided, which would greatly aid all toxicological and pharmaceutical studies. Spirolides. Spirolids (SPXs) are macrocyclic compounds classified as “fast acting toxins” because they cause rapid death when administered intraperitoneally in mice. Six major spirolides (AF) are known in addition to two derivatives, 13-desmethyl C (SPX-1) and 13-desmethyl D. However, to date, 14 members of the spirolide family have been isolated all around the world.4,2126 The structure of the spirolides can be divided into two parts: the upper part containing a 6,7 bicyclic spiroimine unit, believed to be the pharmacophore, and the lower unit containing a spiroacetal or bis spiroacetal moiety27 (Figure 3). These groups of toxins were discovered in 1995 during routine monitoring of a polar lipophilic toxic compound in mollusks extracts; an unusual toxicity was found during a regular mouse bioassay testing of scallop (Placopecten magellanicus) and mussel (Mytilus edulis) viscera harvested along the southeastern coast of Nova Scotia in Canada. These toxins are metabolites of the dinoflagellates Alexandrium ostenfeldii and Alexandrium peruvianum and are sometimes found in the presence of other toxins such as PSP toxins. After chromatographic purification of methanolic extracts of frozen digestive glands of shellfish, four pure novel compounds, spirolides A, B, C, and D, were isolated. Spirolides B and D were isolated in sufficient quantity to determine their molecular formulas as C42H63NO7 and C43H65NO7, respectively.28 IR spectra and 13C analysis established that these molecules contained a γ lactone, a vinyl group, and a double bond between a carbon and either carbonyl or imine functionality. Also, spectra were obtained for spirolide D allowing a partial structure elucidation of this new toxin.

In 1996, the same research group reported the isolation of two further spirolides E and F from shellfish extracts.29 NMR data of spirolide F revealed a close resemblance to spirolide B, but a difference appeared around C28. The IR data also confirmed the presence of an amine and a ketone, establishing spirolide F as a keto amine derivative of spirolide B. For spirolide E, the molecular formula was determined to be C42H64NO8, which suggested this new compound to be a keto amine derivative of spirolide A. In 2001, the structural elucidation of spirolides A and C was reported, and the molecular formula of spirolide A was established as C42H61NO7, which contains two protons less than the known spirolide B. The molecular formula of spirolide C was established to be C43H63NO7 with the extra methyl group being assigned to position 31 on the cyclic imine system and the extra double bond positioned between C2 and C3 in the γ lactone unit. In addition, the structure of another new congener, 13-desmethyl spirolide C, was reported. The molecular formula of this compound was attributed to be C42H61NO7 showing that this new derivative was an isomer of spirolide A. NMR data demonstrated that part of the structure is identical to spirolide C with the only difference occurring at C13. Moreover, LC-MS and MS/MS analysis of phytoplankton extracts also identified 13-desmethyl spirolide D, another desmethyl derivative of spirolide D, but no structural elucidation was carried out for this new congener. Ten years after the first detection of the spirolides, two novel spirolide derivatives were isolated and characterized from the culture of two clonal strains of A. ostenfeldii isolated from Limfjorden in Denmark. The LC MS profile of these extracts revealed the presence of the known 13-desmethyl spirolide C and two unknown components suspected to be spirolide C/D derivatives. The molecular formula was established to be C41H59NO7 and C42H61NO7 for the two new compounds, called 13,19 didesmethyl spirolide C and spirolide G. Subsequently, analysis of blue mussel (Mytilus edulis) digestive glands and algal biomass samples collected during a bloom at Sognefjord, Skjer in Norway allowed the structure elucidation of a new analogue of spirolide G, which contained an additional methyl group at C20. The accurate mass of this compound was determined to be C43H64NO7. 1819



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Figure 3. Structural variants and molecular weights (Mw) of known spirolides isolated from shellfish and plankton.35

In 2007, investigation of a large culture of an Adriatic A. ostenfeldii strain allowed the isolation and structure elucidation of another congener of the spirolide family, 27-hydroxy-13,19-desmethylspirolide C with a molecular formula of C41H59NO8. To date, spirolides H and I are the two most recent reported compounds of the spirolide family. The molecular formulas were established as C40H60NO6 and C40H62NO6 for spirolides H and I, respectively. The spiroimine unit of the spirolides has not yet been synthesized and appears to be a synthetic challenge. To date, the total synthesis of the spirolides has not yet been reported, but the syntheses of the 5,5,6-bispiroacetal moiety of spirolides B and D have been reported by Ishihara et al.30 and the Brimble group.31,32 Studies on the unusual 6,7-spiroimine moiety of the spirolides are in progress in the Zakarian33 and Brimble groups.34 In conclusion, the spirolide family of marine biotoxins discovered 15 years ago still represents an active area of research in

both the chemical and biological sciences. To date, the total synthesis of spirolides remains a challenge, and even the putative pharmacophore of the spirolides has not yet been synthesized. Moreover, the low natural abundance of spirolides currently hampers efforts toward pharmacological evaluation, detection assay development, and possible medicinal applications. Pinnatoxins and Pteriatoxins. Pinnatoxins and pteriaroxins are another group of marine toxins belonging to the cyclic imine family. These are structurally and synthetically related to the better studied group of spirolides. Pinnatoxins have been the first to be discovered in extracts from the digestive glands of the pen shell Pinna attenuata after an outbreak of shellfish poisoning in China and Japan.36 These extracts were referred to as pinnatoxin. The adductor muscle of the bivalve Pinna attenuata is a very popular sushi ingredient in Japan and China, and food poisoning resulting from its ingestion occurs often. The two-dimensional structure and relative stereochemistry by NMR analysis of the 1820



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Figure 4. Chemical and structural data of all known pinnatoxin analogues to date and related 13-desmethylspirolide C and pteriatoxin structures, shown for comparison.43

first member of the group, pinnatoxin A, have been described shortly after.37,38 It was reported to be a water-soluble, amphoteric compound. Pinnatoxins B and C (in a 1:1 mixture) and pinnatoxin D were isolated from the viscera of the prickly pen shell Pinna muricata.37,3941 The stereochemistry of all these toxins was uncovered by NMR profiling and different synthetic approaches.38,39,42 Very recently, three novel analogues of pinnatoxin A, namely, pinnatoxins E, F, and G were isolated and structurally characterized by Sellwood and colleagues from the digestive glands of two bivalve species: Pacific oysters (Crassostrea gigas) and razor fish (Pinna bicolor) from the South Australian coast.43 The two bivalve species studied showed very different pinnatoxin profiles, both in terms of quantities and types of analogues. In 2010, pinnatoxin presence (pinnatoxins

G and A) has also been reported in shellfish from Norwegian waters.9 The genus Pinna is distributed worldwide, inhabiting shallow and warm tropical waters, with certain species restricted to the Indo-Pacific region.44 Extracts from the digestive glands of several Pinna sp. including P. muricata, P. attenuata, P. atropurpurea, and commonly eaten shellfish Atrina pectinata all have been shown to produce the same toxicity symptoms. These data suggest that Pinna shellfish become toxic as the result of feeding on toxic organisms such as dinoflagellates.45 The dinoflagellate producer of pinnatoxins (pinnatoxin G) is not yet known, although, recently, a peridinoid benthic dinoflagellate producer of pinnatoxins E and F has been isolated in a pure culture, and its molecular identification is being studied.46 In this same study, its 1821



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Figure 5. Proposed metabolic pathway for the conversion of pinnatoxins F (8) and G (5) as precursors of the known pinnatoxins and pteriatoxins (7 and 8 depicted with C-36 arbitrary stereochemistry).43

genetic relationship to Scrippsiella trochoidea and Pfiesteria sp. has been shown. Pinnatoxins are amphoteric macrocyclic compounds possessing a common 27-membered carbocyclic backbone composed of a unique 6,7-azaspiro-linked imine fragment (AG ring), a bridged 5,6-bicycloketal (EF ring), and a 6,5,6-dispiroketal (BCD ring), as well as varying functional group substitutions at positions C21, C22, C28, and C33.44 The chemical structures with the corresponding formula and molecular masses of the known pinnatoxin analogues have been summarized in Figure 4. Pinnatoxin D, isolated as a colorless solid, unlike AC, bears substituents on the D ring (cis-C22 hydroxyl and C21 methyl groups) and possesses a more elaborate side chain at C33, but lacks a C28 hydroxyl in the E-ring.42 From all pinnatoxins, the most toxic are B and C, which have an amino acid structure with two epimers at the R-position.47 It was shown that most of the pinnatoxin analogues were chemically more stable than the related spirolides B and D. Both pinnatoxin G and E were stable under base hydrolysis with no release of fatty acid esters. In water and methanol, the lactone ring of pinnatoxin F was opened to form pinnatoxin E or 7-methyl ester depending on the initial conditions. Under weakly acidic conditions, the reverse reaction could also take place with partial cyclization to the lactone. Slow isomerization was detected in pinnatoxins E and F, but not in pinnatoxin G, to a lateeluting epimer (by HPLC) supposing involvement of the 22-OH group in the spiroketal rearrangement taking place. These epimers were present as 520% of the parent compound in natural samples containing the parent toxins.

Certain analogues found in Pacific oysters and razor fish appeared to occur due to metabolism by the shellfish.43 LC-MS analysis of extracts from both species revealed the presence of multiple analogues of pinnatoxins A, G, D, E, and F (epoxides and oxidized or hydrolyzed forms), total concentrations of which were higher in razor fish than in Pacific oysters. Some putative pinnatoxins were metabolites of pinnatoxin G and intermediates to the formation of pinnatoxins A, B, C, and pteriatoxins A, B, and C. In razor fish, pinnatoxin G appeared to be readily metabolized to pinnatoxin A and pinnatoxins E and F to pinnatoxin D. The same metabolic processes seemed to occur in Pacific oysters but at a much slower rate. A brief biogenetic pathway for the synthesis of pinnatoxins and pteriatoxins via shellfish metabolism has been proposed by the research group of Selwood (Figure 5). Regarding the artificial synthesis of pinnatoxins, there are several groups that have contributed to the accumulation of the current knowledge in this respect.44 An extension of the synthetic method developed for the complete chemical synthesis of pinnatoxin A has provided a complete stereochemical assignment for pinnatoxins B and C. The first synthetic study on pinnatoxin A was carried out by Kishi and co-workers in 1998 and consisted of various steps, applied for the construction of the three different ring parts of the toxin molecule. The synthesis started from pent-4-yn-1-ol and included a dithiane coupling, a DielsAlder reaction, and, finally, a NozakiHiyamaKishi reaction.48 In the period 19972001, Murai and colleagues developed a convergent approach to the BCDEF ring polyether fragment.49 Under optimized conditions, the spirocyclic adduct 1822



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Table 1. Binding Sites for Spirolides toxin

receptor

effect

reference

13-desmethyl spirolide C

muscle and neuronal-type nAChRs

potent antagonism


soluble ACh binding proteins

bind

61


rat mAChRs and nAChRs

upregulation of gene expression

60


human mAChRs

competitive irreversible antagonism

62 and 6

spirolides

L type calcium channels

weak activator

28

61 and 60

Table 2. Binding Sites for Gymnodimines toxin

receptor

effect

reference

gymnodimine A

muscle-type and neuronal nAChRs

potent antagonism

61 and 8

gymnodimine A gymnodimine and gymnodimine acetato

soluble ACh binding proteins

bind sensitization to OA in neuro2a cells

61 18

gymnodimine

human mAChRs

competitive reversible antagonism

62

Table 3. Information on the Occurrence and Toxicity of Pinnatoxins9 toxin pinnatoxin A

pinnatoxin Ga

toxicity (i.p. mouse, μg/kg)

where found Japan

Pinna og Pteria spp.

LD99 180

New Zealand Australia

green mussels, oysters oysters, Pinna sp., mussels, sediment

LD99 135

Norway

mussels, seawater

pinnatoxin B/C

pinnatoxin Ga

Japan

Pinna sp.

LD99 22

pinnatoxin D

pinnatoxin Fa

Japan

Pinna sp.

LD99 400

New Zealand

Pinna sp., oysters

Australia

oysters

New Zealand

oysters, green mussels, seawater, algae

Australia Cook islands

oysters, Pinna sp., seawater, sediment seawater

New Zealand

oysters, algae, green mussels

pinnatoxin E

pinnatoxin F

pinnatoxin G

pteriatoxin A pteriatoxin B/C a

presumed source

pinnatoxin Fa

new alga

unknown alga

pinnatoxin Ga pinnatoxin Ga

Australia

oysters, Pinna sp., seawater, sediment

Cook islands

seawater

New Zealand

Grenn mussels, oysters, cockels

Australia

oysters, Pinna sp., mussles, seawater, sediment

Norway

seawater, mussels

Japan Japan

Pteria sp. Pteria sp.

Via metabolism or hydrolysis.

originating as a result of the developed methodology was generated in 82% yield and had very high enantio- and exoselectivity, making the method applicable to the synthesis of other marine spiroimine toxins. The total synthesis of pinnatoxins A was recently reported by the NakamuraHashimoto research group.50 The synthesis strategy applied consisted in an exo-selective intermolecular DielsAlder cyclization and Ru-catalyzed cycloisomerization methodology according to the protocol of Trost and Toste.5153 The total synthesis of pinnatoxin A included 53 synthetic steps and obtained a final overall yield of 0.21%. Finally, the research group of Zakarian described also in 2008 a series of synthetic studies culminating in the total synthesis of the natural enantiomer of pinnatoxin A.54 The plan of this synthetic pathway was based on a convergent strategy and generally consisted

of the preparation of two large fragments and their joining by alkyllium addition at the C6C7 site, followed by macrocycle formation upon ring-closing metathesis (at the C26C27 site) with subsequent elaboration of the EF ketal. As demonstrated, pinnatoxin A, being a prototype of this family of spiroimine natural molecules and a very complex compound, has inspired the development of a lot of synthetic strategies. These could be further used for thorough studies and the development of specific synthetic schemes for the other known pinnatoxin analogues. It is to be outlined that chemical synthesis is still the only realistic source in the case of pinnatoxin A for the production of this pharmaceutically important natural product since the producing organism remains elusive. Miles et al.9 compiled the existing knowledge about occurrence, toxicity, and location of the several pinnatoxin analogues (Table 3). 1823



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Figure 6. 1H NMR data for pteriatoxins A (1), B (2), and C (3) from Takada et al.41

Figure 7. Three building blocks of PtTXs AC from Matsuura et al.55 Reprinted from ref 55. Copyright 2006 American Chemical Society.

Pteriatoxins were first described by Takada et al.41 that observed that a moray eel vomiting the viscera of the Okinawan oyster Pteria penguin. These authors successfully isolated pteriatoxins A, B, and C as extremely toxic and minor components from P. penguin and reported the structural determination of these three compounds. The molecular formulas of pteriatoxins A, B, and C were determined to be C45H70N2O10S (A) and C45H70N2O10S (B and C). The position of duplicate signals in the 1H NMR spectrum suggested that pteriatoxins B (2) and C (3) are C-34 epimers of each other (Figure 6). Therefore, pteriatoxins A, B, and C have the same polyether macrocycles as in pinnatoxin A, and they are considering pinnatoxins analogues containing a cysteine moiety. On the basis of the presence of pinnatoxin analogues in both Pinna sp. and Pteria sp., Takada et al.41 suggested that the pinnatoxin series may be synthesized by common symbionts. They proposed that these three compounds and PnTX A share the same stereochemistry at the macrocyclic core; however, the C34 and C20 stereochemistry of PtTXs AC were unassigned. The naturally occurring PtTXs B/C were isolated as a mixture and shown to be chromatographically inseparable. However, Matsuura et al.55 reported a total synthesis of all members of the PtTX class of natural compounds. In general, PtTXs are envisioned to be assembled from three building blocks: dithiane (1), vinyl iodide 2, and alkyl iodide 3. Building blocks 1 and 2 are also used for PnTx A synthesis.55 PtTXs are thought to be biosynthesized in a way similar to that of an epoxide, followed by nucleophilic addition of the cysteine moiety. Recently, all eight possible stereoisomers were synthesized by Matsuura et al.55 (Figure 7). Also, the stereochemistries of PtTxs AC, have been established, and two natural C34 sterisomers (PtTXs B and C) were isolated from the PtTX B/C series, whereas only one steroisomer (PtTX A) was in the PtTx A series. It was concluded that natural PtTx A is stereochemically homogeneous.56 Prorocentrolides and Spiroprorocentrimines. Prorocentrolide A has been isolated from Prorocentrum lima for the first

Figure 8. Prorocentrolide A structure from Torigoe et al.57 Reprinted from ref 57. Copyright 1988 American Chemical Society.

time in 1988.57 The dinoflagellate was isolated at Sesoko Island, Okinawa, in 1985 and cultured in seawater enriched with ES-1 nutrient 3 at 25 °C for 5 weeks. The planar structure of prorocentrolide A was also elucidated by these authors (Figure 8). Hu et al.58 isolated prorocentrolide B, a new toxin from P. maculosum, which causes the characteristic “fast acting” symptoms. As the structural details began to emerge, it became apparent that the new compound was related to prorocentrolide A, previously isolated from a strain of P. lima. No stereochemical details have been reported for prorocentrolide A, and details of the relative stereochemistry of B are shown in Figure 9. However, the absolute stereochemistry of the rings and the relative configuration of one ring with respect to the others remain undetermined. The full distribution of this family of toxins is not well known, but it is reasonable to conclude from the structural data that prorocentrolide B from P. maculosum shares a common biosynthetic pathway with prorocentrolide A from P. lima.58 Spiro-prorocentrimine, C42H69NO13S, is a polar lipid-soluble toxin that was isolated from a laboratory-cultured benthic 1824



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Figure 9. Relative stereochemistry of the 5- and 6-membered rings of prorocentrolide B from Hu et al.58 Reprinted from ref 58. Copyright 1996 American Chemical Society.

Figure 10. Structure of spiro-prorocentrimine from Lu et al.59 Reprinted with permission from ref 59. Copyright 2001 Elsevier.

Prorocentrum species of Taiwan by Lu et al.59 X-ray diffraction analysis of the compound revealed a spiro-linked cyclic imine with the ortho, para-disubstituted 3%-cyclohexene in addition to its macrolide skeleton (Figure 10).

’ MECHANISM OF ACTION Since its first discovery in 1990,36 the biological activity of different toxin groups of cyclic imines has been investigated. The lack of reference or toxic material has increased the difficulty of these investigations; thus, only few works on pharmacological effects of gymnodimines and spirolides exist until now (Tables 1 and 2). From a toxicological point of view, the toxic effects of cyclic imines have two main characteristics: a complex cascade of neurological symptoms appearing in few minutes after intraperitoneal administration and an all or nothing action, meaning that injected mice either die in the first 20 min after injection or that they completely recover without any apparent sequels. This neurological symptomatology suggests that the mechanism of action may be interfering in the action of certain neurotransmitters. Hu and co-workers studied in 199528 the in vitro effect of spirolides, observing that the molecules had no effect on NMDA, AMPA, or kainate receptors, that they did not inhibit PP1 nor PP2A phosphatases, and that they did not activate or block voltage dependent sodium channels. However, they showed a

weak activation effect on L type calcium channels. These channels are high voltage activated channels that are present in excitable cells of several tissues: skeletal, smooth and cardiac muscle, cortical neurones, and endocrine system cells. Its activation can cause muscle contraction, upregulation of gene expression, or liberation of hormones or neurotransmitters depending on the type of cell. However, no further investigations on the spirolides activity upon these receptors have been published. In order to elucidate the possible biological receptors of the spirolides, Richard and co-workers6 studied the effect of preadministration of therapeutic doses of several well-known drugs followed by a lethal dose of an Alexandrium ostenfeldii extract containing spirolides. The hypothesis was that decreases or increases in dead time could be used to infer the possible mode of action of the toxin. In an initial screening, four drugs were investigated: epinephrine, propanolol, physostigmine, and atropine. Both epinephrine and propanolol showed little effect at the doses studied. However, the physostigmine treatment, a cholinesterase inhibitor, resulted in an increase in the dead time, while the mice treated with atropine died more quickly. This suggested that spirolides and atropine had similar effects: anticholinergic activity by blocking the muscarinic acetylcholine receptors. Consequently, four additional drugs, tropicamide, gallamine, pirenzepine, and methacoline, were studied. The results agreed with those previously obtained, resulting in a reduction of the dead time when mice were treated with acetylcholine antagonists, while agonists showed a protection effect against spirolide toxicity. It was concluded that spirolides produced an antagonistic effect on muscarinic acetylcholine receptors, although the possibility that they might produce effects on other biological receptors also exists.6 Gill and co-workers investigated the histological injuries and the transcriptional analysis of neural injury markers in the brain and other internal organs of rats and mice treated with different doses of 13-desmethyl-spirolide.60 The only tissue affected was the brain in both species, but histological and transcriptional analyses were different in the two species. In rats, the upregulation in the transcriptional biomarkers affected the cerebellum and the brain stem but not the cerebrum. Among others, the upregulation of several types of nicotinic and muscarinic receptors was remarkable. In contrast, no histological changes were 1825


Chemical Research in Toxicology observed in this species. In mice, the histological analysis showed widespread neuronal damage throughout the brain, with the hippocampus and brain stem being the most affected areas, but no transcriptional alteration was found. Antagonism on Nicotinic Acetylcholine Receptors. Despite the fact that the first studies on the mechanism of action of cyclic imine marine biotoxins found that muscarinic acetylcholine receptors (mAChRs) were the main target of the toxin, recent works demonstrated that both spirolides and gymnodimines act as a potent antagonists on nicotinic acetylcholine receptors (nAChRs) and that, probably, this antagonism is the main cause of the acute symptomatology observed during the mouse bioassay of samples containing these toxins.8 nAChRs are cholinergic pentameric receptors that form ligandgated ion channels in the plasma membranes of certain neurons and on the postsynaptic side of the neuromuscular junction. The binding with Ach or other agonists causes conformational changes resulting in the opening of the nAChR channel pore. They mediate fast neurotransmission in the central and peripheral nervous system and in neuromuscular transmission. Kharrat and co-workers8 studied the effect of gymnidimine A on neuromuscular transmission and its effect on muscular and neuronal types of nicotinic receptors. They observed that GYM-A interferes in neuromuscular transmission in isolated mouse hemidiafragmatic preparations causing a time and concentration dependent block of twitch responses evoked by nerve stimulation. This effect could be reversed by washing out the toxin from the medium. Interestingly, direct stimulation on muscle fibers elicited twitch and tetanic contraction, suggesting that GYM-A directly interferes in neuromuscular transmission but does not interfere in the contractility of muscular fibers. The study of neuromuscular transmission with intracellular recording confirmed that GYM-A causes a reduction and blockage of the amplitude of miniature-end plate potentials (caused by the liberation of a very small amount of acetylcholine (ACh)) and a reduction of the amplitude of end plate potential so that the threshold necessary to originate an action potential is not reached. These results suggest that GYM-A blocks end plate muscle-type nAChRs. In order to confirm this blockage, the researchers performed patchclamp experiments with Xenopus embryonary myocytes which have been known to express nAChRs on their membrane surface. In these experiments, ACh was applied to myocytes membrane before and after the perfusion of GYM-A. They observed that the toxin had no agonist effect on nAChRs but reduced and blocked nicotinic current elicited by ACh. This effect was reverted after 2030 min of washing with GYM-A free solution. They also investigated whether GYM-A affected neuronal human R7 nAChRs expressed in Xenopus oocytes, observing no agonist effect but a reversible competitive antagonism8,61 Finally, the direct interaction between GYM-A and muscletype nAChRs was demonstrated by the concentration dependent displacement of radio-labeled R-bungarotoxin (a peptidic antagonist) in competition binding experiments performed in HEK293 cells expressing muscle nAChRs. Similar experiments were carried out with labeled epibatidine (an alkaloid agonist) in cells expressing different subtypes of neuronal nAChRs, observing in this case that the displacement of the alkaloid by the toxin depended on the subtype of nicotinic receptor.8 It was concluded that GYM-A broadly interferes in neuromuscular transmission by directly blocking muscle- type nAChRs, and this action could explain some of the acute toxic symptoms observed in mice following GYM-A injection including paralysis

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of the hind legs and dyspnea. It was also concluded that GYM-A interacts with a broad range of muscular and neuronal types of nAChRs, larger than other well characterized toxins that also act on nicotinic receptors.8 Bourne and co-workers61 confirmed the interaction of SPX and GYM with several muscle and neuronal types of nAChRs by competition binding experiments against labeled R-bungarotoxin and epibatidine. They also performed functional analysis of both toxins using voltage-clamp recordings with oocytes expressing muscular and neuronal types of receptors. No agonist effect was observed with SPX or GYM, but both toxins showed a dosedependent antagonism in muscle and neuronal type receptors. However, while the GYM effect was reverted after washing with free-toxin solution, SPX antagonism was not abolished after 30 to 40 min of washout. It was concluded that SPX and GYM toxins display high affinity and potent antagonism but limited selectivity for the muscle-type versus neuronal subtypes of nAChRs. Moreover, they concluded that 13-desmethyl-SPX is the most potent general nonpeptidic nicotinic antagonist. Their findings during structural studies on interaction of both toxins with acetylcholine binding proteins (AChBP), a soluble receptor surrogates, explained their high affinity and low selectivity to nAChRs. In this interaction, the protonates imine nitrogen shows an important role tethering the toxins to their binding site, while the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclo-hexane-butyrolactone further anchor the toxins in apical and membrane directions. This findings provide a new point of view for the development of more sensitive and versatile spiroimines detection methods based on AChBP binding assays.61 Antagonism on Muscarinic Acetylcholine Receptors. Although initial works on the mechanism of action of cyclic imine toxins suggested the possible implication of muscarinic receptors in the toxicity of these substances, this implication has not been studied in depth and demonstrated until recently. Muscarinic acetylcholine receptors are G protein-coupled acetylcholine receptors found in the plasma membranes of certain neurons and other cells. They can signal through cAMP generation or through an increase of cytosolic calcium concentration, depending on the type of the associated G protein. Wandscheer and co-workers62 studied the effect of spirolides on mAChRs in a human neuronal model known to have an ACh elicited calcium signal dependent on muscarinic AChRs activation. They demonstrated that 13-desmethyl C SPX caused a dose- and time-dependent inhibition on neuroblastoma cell response to ACh extracellular stimulation expressed in the concentration of intracellular calcium ([Ca2+]i response). The addition of 13-desmethyl C SPX did not caused any response in [Ca2+]i. An exposure of 30 min was necessary to achieve a full inhibitory effect at a concentration of 100 nM, while an exposure of 2 min did not cause any effect. Moreover, the inhibitory effect of 13-desmethyl C SPX persisted for 30 min after the removal of the toxin. Simultaneous incubation of neuroblastoma cells with 13-desmethyl C SPX and atropine, a well-characterized competitive reversible muscarinic antagonist, caused a reduction in the inhibitory effect on the ACh-induced calcium signal observed after the removal of the toxins, suggesting a competition of both toxins for the same binding site. Competitive binding assays were carried out with the mAChRs antagonist tritiated quinuclidinyl benzilate ([3H]QNB) in order to elucidate whether the inhibitory effect of 13-desmethyl-SPX occurred at the receptor level. The cells were incubated with 1826


Chemical Research in Toxicology the toxin for 1 h, and then [3H]QNB was added. The spirolide caused a dose-dependent inhibition on the specific binding of [3H]QNB to neuroblastoma cells. This effect was persistent even after the removal of the toxin from the extracellular medium. On the contrary, assays in which cells were incubated simultaneously with atropine and 13-desmethyl-SPX followed by washing showed similar binding levels of [3H]QNB than those without any treatment, demonstrating that the 13-desmethyl SPX is a competitive antagonist on mACHhRs.62 The possible process of internalization and up- and downregulation that stimulation with both agonists and antagonists could cause on mAChRs, similar to other G protein-coupled receptors, was also considered by Wandscheer and co-workers. They investigated by Western Blot the influence of 13-desmethyl SPX on the total and membrane-associated levels of M3 mAChRs on neuroblastoma cells exposed to the toxin for long periods of time. While no difference was found on the total level of receptors between cells exposed and not exposed, a significant reduction in membrane-associated levels of M3 AChrRs was found after 12 h of incubation with 13-desmethyl SPX. These results suggest that 13-desmethyl SPX causes an internalization of the M3 receptor that might be responsible of the inhibitory effect of the toxin on the calcium response to ACh stimulation. However, the probability of this internalization being the only mechanism causing the calcium signal reduction is low given the demonstrated binding of the toxins to the orthosteric binding site of the mAChRs. Viability of the cells was not affected by the presence of the toxin, ruling out the possibility that the inhibitory effect on calcium signal could be caused by dead cells.62 This is in agreement with the results obtained by Dragunow and co-workers, who treated cultures of Neuro2a cells with gymnodimine and gymnodimine acetate observing a very small and inconsistent reduction in the number of cells.18 Funding Sources

This study was financed through research grant AGL2009-13581C02-02 from the Ministerio de Ciencia e Innovacion (Ministry of Science and Innovation, Spanish Government) and through the European research project grant ATLANTOX (2008-1/003) from the ERDF funds within the Atlantic Area Operational Programme.

’ ACKNOWLEDGMENT We thank Dr. Efren Perez Santín for his kind help with the design of the figures. ’ ABBREVIATIONS Ac2O, acetic anhydride; AcOH, acetic acid; ACh, acetylcholine; AChBP, acetylcholine-binding protein; AMPA, R-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid receptor; cAMP, cyclic adenosine monophosphate; CIs, cyclic imines; Da, Daltons; CH2Cl2, dichloromethane; DMAP, 4-dimethylaminopyridine; GYMs, gymnodimines; GYM-A, gymnodimine A; GYM-B, gymnodimine B; GYM-C, gymnodimine C; HEK293, human embryonic kidney 293 cell line; IR, infrared; LC-MS, liquid chromatographymass spectrometry; MeOH, methanol; MW, molecular weight; Neuro 2a, murine neuroblastoma cell line ATCC CCL-131; mAChRs, muscarinic acetylcholine receptors; NMDA, N-methyl D-aspartate; nAChRs, nicotinic acetylcholine receptors; NMR, nuclear magnetic resonance; PSP, paralytic shellfish poisoning; PnTXs, pinnatoxins; PP1, protein phosphatase 1; PtTXs, pteriatoxins; NaBH3CN, sodium cyanoborohydride; SPXs, spirolides;SPX-1, 13-desmethyl

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spirolide C; tBuLi, tert-butyllithium; Et3N, triethylamine; [3H]QNB, tritiated quinuclidinyl benzilate.

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Cyclic Imines: Chemistry and Mechanism of Action: A Review

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