Synthetic Approaches to Zetekitoxin AB, a Potent Voltage-Gated Sodium Channel Inhibitor

Voltage-gated sodium channels (NaVs) are membrane proteins that are involved in the generation and propagation of action potentials in neurons. Recently, the structure of a complex made of a tetrodotoxin-sensitive (TTX-s) NaV subtype with saxitoxin (STX), a shellfish toxin, was determined. STX potently inhibits TTX-s NaV, and is used as a biological tool to investigate the function of NaVs. More than 50 analogs of STX have been isolated from nature. Among them, zetekitoxin AB (ZTX) has a distinctive chemical structure, and is the most potent inhibitor of NaVs, including tetrodotoxin-resistant (TTX-r) NaV. Despite intensive synthetic studies, total synthesis of ZTX has not yet been achieved. Here, we review recent efforts directed toward the total synthesis of ZTX, including syntheses of 11-saxitoxinethanoic acid (SEA), which is considered a useful synthetic model for ZTX, since it contains a key carbon–carbon bond at the C11 position.


Voltage-Gated Sodium Channel Isoforms
Voltage-gated sodium channels (Na V s) are membrane proteins involved in neuronal excitation and transmission [1]. Ten subtypes, Na V 1.1-1.9 and Na V X, have been identified based on sequence determination (Table 1) [2]. These subtypes can be grouped into two types depending upon their sensitivity to the pufferfish toxin, tetrodotoxin (TTX) [3][4][5][6][7]: tetrodotoxin-sensitive Na V s (TTX-s Na V s 1.1-1.4, 1.6, and 1.7) are significantly inhibited by TTX, while tetrodotoxin-resistant Na V s (TTX-r Na V s 1.5, 1.8, 1.9) are not [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. Subtype-selective modulators of Na V s are required for studies to establish the biological functions of these subtypes. Some of the subtypes are also considered to be potential drug targets; for example, Na V 1.7 and 1.8 are potential targets for pain treatment [24][25][26][27][28][29]. Therefore, there is great interest in the development of drugs targeting specific subtypes [30,31].  STX (1) binds to the pore-forming region of the alpha-loop of Na V and blocks the influx of sodium cation in a similar manner to tetrodotoxin (TTX) [42,43]. Recently, Yan's group determined the X-ray structure of the complexes of STX (1) with Na V Pas derived from American cockroach and human-derived Na V 1.7 by using cryoEM (Figure 2) [44,45]. They found that the carbamoyl group at C13 in STX (1) interacts with Gly1407 and Thr1409 in domain III, the two guanidines interact with Glu364 in domain I and Glu930 in domain II, and the geminal diol interacts with Asp1701 in domain IV. Interestingly, residues 1409 and 1410, located in the P2 loop of domain III in Na V 1.7, were mutated to Thr and Ile from Met and Asp, respectively, which may explain why STX (1) has a weaker affinity for Na V 1.7 compared with other subtypes (Met1409 and Asp1410 are conserved in the other subtypes of TTX-s) [46,47]. Mar. Drugs 2020, 18
Among the STX (1) derivatives, zetekitoxin AB (ZTX, 8) has an unusual structure [49]. ZTX (8) was isolated from skin of the Panamanian dart-poison frog Atelopus zeteki in 1969 by Mosher and coworkers [50,51]. It has extremely potent NaV-inhibitory activity (more than 600-fold greater than that of STX (1)), with IC50 values of 6.1 pM, 65 pM, and 280 pM for NaV1.2, NaV1.4, and TTX-r subtype NaV1.5, respectively [49]. Thus, there is great interest in the mode of action of ZTX (8), but studies are hampered by the fact that Atelopus zeteki is designated as an endangered species. Therefore, a chemical synthesis of ZTX (8) is needed. However, ZTX (8) contains a macrocyclic lactam structure in which isoxazolidine is bridged from C6 to C11, and an N-hydroxycarbamate is linked via a methylene group at N7 [49]. These structural features make ZTX (8) synthetically challenging. So far, several synthetic approaches have been reported, but a total synthesis of 8 has not yet been achieved.
Among the STX (1) derivatives, zetekitoxin AB (ZTX, 8) has an unusual structure [49]. ZTX (8) was isolated from skin of the Panamanian dart-poison frog Atelopus zeteki in 1969 by Mosher and coworkers [50,51]. It has extremely potent NaV-inhibitory activity (more than 600-fold greater than that of STX (1)), with IC50 values of 6.1 pM, 65 pM, and 280 pM for NaV1.2, NaV1.4, and TTX-r subtype NaV1.5, respectively [49]. Thus, there is great interest in the mode of action of ZTX (8), but studies are hampered by the fact that Atelopus zeteki is designated as an endangered species. Therefore, a chemical synthesis of ZTX (8) is needed. However, ZTX (8) contains a macrocyclic lactam structure in which isoxazolidine is bridged from C6 to C11, and an N-hydroxycarbamate is linked via a methylene group at N7 [49]. These structural features make ZTX (8) synthetically challenging. So far, several synthetic approaches have been reported, but a total synthesis of 8 has not yet been achieved.  Among the STX (1) derivatives, zetekitoxin AB (ZTX, 8) has an unusual structure [49]. ZTX (8) was isolated from skin of the Panamanian dart-poison frog Atelopus zeteki in 1969 by Mosher and co-workers [50,51]. It has extremely potent Na V -inhibitory activity (more than 600-fold greater than that of STX (1)), with IC 50 values of 6.1 pM, 65 pM, and 280 pM for Na V 1.2, Na V 1.4, and TTX-r subtype Na V 1.5, respectively [49]. Thus, there is great interest in the mode of action of ZTX (8), but studies are hampered by the fact that Atelopus zeteki is designated as an endangered species. Therefore, a chemical synthesis of ZTX (8) is needed. However, ZTX (8) contains a macrocyclic lactam structure in which isoxazolidine is bridged from C6 to C11, and an N-hydroxycarbamate is linked via a methylene group Mar. Drugs 2020, 18, 24 4 of 19 at N7 [49]. These structural features make ZTX (8) synthetically challenging. So far, several synthetic approaches have been reported, but a total synthesis of 8 has not yet been achieved.

Scope of This Review
Synthetic studies of STX (1) and its analogs have been extensive, and several total syntheses have been achieved [52][53][54][55][56][57][58][59][60][61], as recently reviewed by Du Bois [62]. Approaches for developing of subtype-selective modulators based on the STX structure have also been explored [25,47,[63][64][65]. However, in this review, we focus on recent synthetic work related to ZTX (8). As described above, ZTX (8) has a characteristic macrolactam structure though C6 to C11 with an isoxazolidine ring system, and is structurally quite distinct from other STX analogs. To achieve total synthesis of ZTX (8), two key issues must be addressed: (i) carbon-carbon bond formation at the C11 position in the STX skeleton, and (ii) macrolactam formation of the carboxylic acid at C6 with isoxazolidine nitrogen (Figure 4). Regarding the first issue, the STX derivative 11-saxitoxinethanoic acid (SEA, 9) has been used as a synthetic model for 8, since it also has a carbon-carbon bond at the C11 position. As for the second issue, stereoselective synthesis of disubstituted isoxazolidine and oxidation to carboxylic acid at C13, followed by amide formation with the isoxazolidine, have been examined. First, we will consider recent progress in the total synthesis of SEA (9).

Scope of This Review
Synthetic studies of STX (1) and its analogs have been extensive, and several total syntheses have been achieved [52][53][54][55][56][57][58][59][60][61], as recently reviewed by Du Bois [62]. Approaches for developing of subtypeselective modulators based on the STX structure have also been explored [25,47,[63][64][65]. However, in this review, we focus on recent synthetic work related to ZTX (8). As described above, ZTX (8) has a characteristic macrolactam structure though C6 to C11 with an isoxazolidine ring system, and is structurally quite distinct from other STX analogs. To achieve total synthesis of ZTX (8), two key issues must be addressed: (i) carbon-carbon bond formation at the C11 position in the STX skeleton, and (ii) macrolactam formation of the carboxylic acid at C6 with isoxazolidine nitrogen (Figure 4). Regarding the first issue, the STX derivative 11-saxitoxinethanoic acid (SEA, 9) has been used as a synthetic model for 8, since it also has a carbon-carbon bond at the C11 position. As for the second issue, stereoselective synthesis of disubstituted isoxazolidine and oxidation to carboxylic acid at C13, followed by amide formation with the isoxazolidine, have been examined. First, we will consider recent progress in the total synthesis of SEA (9).

Development of Carbon-Carbon Linkage at C11 of STX, And Application to The Synthesis of 11-Saxitoxinethanoic Acid (SEA, 9)
The STX analog 11-saxitoxinethanoic acid (SEA, 9) was isolated from Atergatis floridus, an Indo-Pacific crab from the family Xanthidae, by Onoue and co-workers ( Figure 5) [66]. SEA (9) has an acetic acid moiety linked to C11 through a carbon-carbon bond, as seen in ZTX (8), and is regarded as a promising synthetic model compound for 8 in terms of construction of the carbon-carbon connection at C11.
Recently, three total synthesis of SEA (9) were independently reported, including one by our group [67][68][69]. When 9 was first isolated, its toxicity to mice was reported to be 830 μmol/MU, which is similar to that of gonyautoxin II (GTX II, 6) and one-third of that of STX (1), but no information about the NaV-inhibitory activity was provided. After the synthesis of 9, Du Bois and our group

Development of Carbon-Carbon Linkage at C11 of STX, And Application to The Synthesis of 11-Saxitoxinethanoic Acid (SEA, 9)
The STX analog 11-saxitoxinethanoic acid (SEA, 9) was isolated from Atergatis floridus, an Indo-Pacific crab from the family Xanthidae, by Onoue and co-workers ( Figure 5) [66]. SEA (9) has an acetic acid moiety linked to C11 through a carbon-carbon bond, as seen in ZTX (8), and is regarded as a promising synthetic model compound for 8 in terms of construction of the carbon-carbon connection at C11. an IC50 value of 47.0 ± 1.2 nM ( Figure 6B) [67]. Du Bois and co-workers evaluated the inhibitory activity of 9 against NaV1.4, and found that SEA (9) showed similar inhibitory activity to gonyautoxin III (GTX III, 7) (9: IC50 = 17 ± 1.9 nM; GTX III (7): IC50 = 14.9 ± 2.1 nM), even though it was a diastereomeric mixture of α:β = 3:1 at C11 ( Figure 6A) [68]. They suggested that the β-form of 9 binds to NaV preferentially, and then the α-form of 9 isomerizes to the β-form, which shows a similar level of inhibitory activity to GTX III (7) ( Figure 6C).

Carbon-Carbon Bond Formation at C11 by Mukaiyama Aldol Condensation Reaction, as Applied for The Synthesis of (+)-SEA by Nagasawa's Group
For the construction of a carbon-carbon bond at C11, Nagasawa and co-workers utilized ketone 10, which was previously developed by their group [67], to install an acetic acid equivalent at C11. They firstly investigated the alkylation reaction of the enolate of ketone 10a with alpha-halo-ethyl acetate. With various bases and halogens, the alkylation did not take place at all, and the starting ketone 10a was recovered. Next, they investigated the Mukaiyama aldol reaction [71][72][73]. Thus, silyl enol ethers 11a and 11b were synthesized from the ketone by reaction with tert-butyldimethylsilyl chloride in the presence of NaHMDS as a base. Then, the Mukaiyama aldol reaction was examined with ethyl glyoxylate under various conditions. Lewis acids, such as TiCl4 or BF3 Et2O [74,75], removed the tert-butoxycarbonyl (Boc) protecting group of guanidine, and no coupling products with ethyl glyoxylate were obtained. In the case of the fluoride anion agent Bu4NF [76], the reaction did Recently, three total synthesis of SEA (9) were independently reported, including one by our group [67][68][69]. When 9 was first isolated, its toxicity to mice was reported to be 830 µmol/MU, which is similar to that of gonyautoxin II (GTX II, 6) and one-third of that of STX (1), but no information about the Na V -inhibitory activity was provided. After the synthesis of 9, Du Bois and our group independently evaluated the Na V -inhibitory activity of 9. Nagasawa, Yotsu-Yamashita, and co-workers evaluated the Na V -inhibitory activity of SEA (9) by utilizing neuroblastoma Neuro 2A cells, which is known to express Na V 1.2, 1.3, 1.4, and 1.7 [70], and found moderate inhibitory activity with an IC 50 value of 47.0 ± 1.2 nM ( Figure 6B) [67]. Du Bois and co-workers evaluated the inhibitory activity of 9 against Na V 1.4, and found that SEA (9) showed similar inhibitory activity to gonyautoxin III (GTX III, 7) (9: IC 50 = 17 ± 1.9 nM; GTX III (7): IC 50 = 14.9 ± 2.1 nM), even though it was a diastereomeric mixture of α:β = 3:1 at C11 ( Figure 6A) [68]. They suggested that the β-form of 9 binds to Na V preferentially, and then the α-form of 9 isomerizes to the β-form, which shows a similar level of inhibitory activity to GTX III (7) ( Figure 6C).

Carbon-Carbon Bond Formation at C11 by Mukaiyama Aldol Condensation Reaction, as Applied for The Synthesis of (+)-SEA by Nagasawa's Group
For the construction of a carbon-carbon bond at C11, Nagasawa and co-workers utilized ketone 10, which was previously developed by their group [67], to install an acetic acid equivalent at C11. They firstly investigated the alkylation reaction of the enolate of ketone 10a with alpha-halo-ethyl acetate. With various bases and halogens, the alkylation did not take place at all, and the starting ketone 10a was recovered. Next, they investigated the Mukaiyama aldol reaction [71][72][73]. Thus, silyl enol ethers 11a and 11b were synthesized from the ketone by reaction with tert-butyldimethylsilyl chloride in the presence of NaHMDS as a base. Then, the Mukaiyama aldol reaction was examined with ethyl glyoxylate under various conditions. Lewis acids, such as TiCl4 or BF3 Et2O [74,75], removed the tert-butoxycarbonyl (Boc) protecting group of guanidine, and no coupling products with

Carbon-Carbon Bond Formation at C11 by Mukaiyama Aldol Condensation Reaction, as Applied for The Synthesis of (+)-SEA by Nagasawa's Group
For the construction of a carbon-carbon bond at C11, Nagasawa and co-workers utilized ketone 10, which was previously developed by their group [67], to install an acetic acid equivalent at C11. They firstly investigated the alkylation reaction of the enolate of ketone 10a with alpha-halo-ethyl acetate. With various bases and halogens, the alkylation did not take place at all, and the starting ketone 10a was recovered. Next, they investigated the Mukaiyama aldol reaction [71][72][73]. Thus, silyl enol ethers 11a and 11b were synthesized from the ketone by reaction with tert-butyldimethylsilyl chloride in the presence of NaHMDS as a base. Then, the Mukaiyama aldol reaction was examined with ethyl glyoxylate under various conditions. Lewis acids, such as TiCl 4 or BF 3 Et 2 O [74,75], removed the tert-butoxycarbonyl (Boc) protecting group of guanidine, and no coupling products with ethyl glyoxylate were obtained. In the case of the fluoride anion agent Bu 4 NF [76], the reaction did not proceed at all. On the other hand, with anhydrous tetrabutyl bisfluorotriphenylphosphine stannate, developed by Raimundo and co-workers [77], the coupling reaction with ethyl glyoxylate proceeded very well to afford the aldol-condensation product 12a a 96% yield (Table 2). Aromatic aldehydes were tolerated, as well as aliphatic aldehydes, and the corresponding aldol condensation products 12a-i were obtained with 42%-80% yield. This reaction afforded mixtures of regioisomers in ratios of 5:1 to >10:1. not proceed at all. On the other hand, with anhydrous tetrabutyl bisfluorotriphenylphosphine stannate, developed by Raimundo and co-workers [77], the coupling reaction with ethyl glyoxylate proceeded very well to afford the aldol-condensation product 12a a 96% yield (Table 2). Aromatic aldehydes were tolerated, as well as aliphatic aldehydes, and the corresponding aldol condensation products 12a-i were obtained with 42%-80% yield. This reaction afforded mixtures of regioisomers in ratios of 5:1 to >10:1. With the aldol condensation product 12b in hand, Nagasawa and co-workers went on to achieve a total synthesis of (+)-SEA (9) for the first time (Scheme 1). Thus, selective reduction of the enone moiety in 12b was carried out with L-selectride, and the protecting group of tert-butyldimethylsilyl (TBS) ether was removed with triethylamine trihydrofluoride (3HF-TEA). The resulting alcohol was reacted with trichloroisocyanate, followed by hydrolysis of the trichloroacetyl group with triethylamine in methanol to give carbamoyl 15. After hydrolysis of ethyl ester in 15 with lithium hydroxide, the Boc group was removed with TFA to give (+)-SEA (9). Scheme 1. Total synthesis of SEA (9) by Nagasawa's group. With the aldol condensation product 12b in hand, Nagasawa and co-workers went on to achieve a total synthesis of (+)-SEA (9) for the first time (Scheme 1). Thus, selective reduction of the enone moiety in 12b was carried out with L-selectride, and the protecting group of tert-butyldimethylsilyl (TBS) ether was removed with triethylamine trihydrofluoride (3HF-TEA). The resulting alcohol was reacted with trichloroisocyanate, followed by hydrolysis of the trichloroacetyl group with triethylamine in methanol to give carbamoyl 15. After hydrolysis of ethyl ester in 15 with lithium hydroxide, the Boc group was removed with TFA to give (+)-SEA (9). a total synthesis of (+)-SEA (9) for the first time (Scheme 1). Thus, selective reduction of the enone moiety in 12b was carried out with L-selectride, and the protecting group of tert-butyldimethylsilyl (TBS) ether was removed with triethylamine trihydrofluoride (3HF-TEA). The resulting alcohol was reacted with trichloroisocyanate, followed by hydrolysis of the trichloroacetyl group with triethylamine in methanol to give carbamoyl 15. After hydrolysis of ethyl ester in 15 with lithium hydroxide, the Boc group was removed with TFA to give (+)-SEA (9). Scheme 1. Total synthesis of SEA (9) by Nagasawa's group. Scheme 1. Total synthesis of SEA (9) by Nagasawa's group.

Carbon-Carbon Bond Formation At C11 by Stille Coupling Reaction, As Applied for The Synthesis of (+)-SEA by Du Bois' Group
Another approach for the construction of the carbon-carbon bond at C11 in STX was explored by Du Bois and co-workers, who employed Stille coupling reaction conditions [68]. They firstly examined the coupling reaction of zinc enolate of ethyl acetate or the stannane enolate of ethyl acetate-type agents with vinyl halide 17, which was prepared from 20, developed by their group (Scheme 2), in the presence of palladium catalyst (Table 3, entries 1 and 2) [78][79][80][81][82][83]. Under the conditions examined, decomposition of the starting substrate was observed in the case of zinc agent, and no reaction occurred with the stannane agent. Then they examined the Stille coupling reaction, using vinyl stannane for the construction of the carbon-carbon bond at C11 [84]. A Stille coupling reaction of vinyl iodide 17 with tributyl(vinyl)tin was examined in the presence of a catalytic amount of Pd(PPh 3 ) 4 , with CuI as an additive (a standard condition). Unfortunately, only a trace amount of the corresponding coupling product of 18c was obtained (entry 3). Then, they changed vinyl stannane to cis-tributyl (2-ethoxyvinyl) tin, and included LiCl as an additional additive. Under these conditions, the corresponding coupling product 18d was obtained with 67% yield (entry 4) [85,86]. Interestingly, poor reproducibility or low yield of the coupling reaction was observed when they used a highly oxidized vinyl stannane agent, tributyl(2,2-diethoxyvinyl)stannane (entry 5). This issue was successfully overcome by switching from CuI to copper(I) thiophene-2-carboxylate (CuTC), and 19 was obtained with 60% yield and with good reproducibility (entry 6) [87].
Based upon the Stille coupling strategy, Du Bois and co-workers achieved a total synthesis of SEA (9), as shown in Scheme 2, including the synthesis of vinyl halide 17 as a substrate for the Stille coupling reaction. Firstly, vinyl halide 17 was synthesized from 20 via Mislow-Evans [2,3] rearrangement: bisguanidine 20 was converted to N,S-acetal 21 by reaction with benzenethiol in the presence of BF 3 ·Et 2 O with 84% yield. Upon treatment of 21 with urea-hydrogen peroxide (UHP), the Mislow-Evans [2,3] rearrangement reaction [88,89] took place under heating in the presence of sodium benzenthiolate, and allylic alcohol 23 was obtained with 81% yield in two steps. After oxidation of the alcohol with Dess-Martin periodinate, the resulting enone 24 was reacted with iodine in the presence of pyridine to give vinyl iodide 17 [90,91], which was further elaborated to 19 by Stille coupling reaction with 25 with 60% yield. Then, the double bond in enone 19 was hydrogenated under high pressure in the presence of Crabtree catalyst 26. Deprotection of the tert-butyldiphenylchlorosilane (TBDPS) ether in 27 with tetrabutylammonium (TBAF) was followed by installation of a carbamoyl group on the resulting hydroxyl group. Finally, deprotection of Tces and Troc and hydrolysis of the ester group were carried out to give (+)-SEA (9). Scheme 2. Introduction of substituent at C11 by Stille coupling, leading to total synthesis of (+)-SEA (9) by Du Bois' group.

Carbon-Carbon Bond Formation at C11 by C-Alkylation, As Applied for The Synthesis of (+)-SEA by Looper's Group
In 2019, Looper and co-workers successfully constructed a carbon-carbon bond at C11 in STX, and reported a total synthesis of SEA (9) [69]. They initially examined C-alkylation with ketone 28 and electrophiles in the presence of variety of bases, such as lithium bis(trimethylsilyi)amide (LHMDS), lithium diisopropyl amide (LDA), potassium bis(trimethylsilyi)amide (KHMDS) and sodium bis(trimethylsilyi)amide (NaHMDS). In addition, they examined various electrophiles (haloacetates, allylic halides, and propargylic halides), but no reaction took place, as Nagasawa and co-workers had found (Scheme 3) [67]. On the other hand, they found that C-alkylation took place upon reaction of ketone 28 and tertbutyl bromoacetate via the generation of zinc enolate by reaction with LiHMDS in the presence of Et2Zn, affording a mixture of 29a and its Boc-deprotected derivative 29b with 60% yield (based on the starting material) (Scheme 4). By means of this alkylation strategy, they succeeded in synthesizing Scheme 2. Introduction of substituent at C11 by Stille coupling, leading to total synthesis of (+)-SEA (9) by Du Bois' group. Another approach for the construction of the carbon-carbon bond at C11 in STX was explored by Du Bois and co-workers, who employed Stille coupling reaction conditions [68]. They firstly examined the coupling reaction of zinc enolate of ethyl acetate or the stannane enolate of ethyl acetate-type agents with vinyl halide 17, which was prepared from 20, developed by their group (Scheme 2), in the presence of palladium catalyst ( Table 3, entries 1 and 2) [78][79][80][81][82][83]. Under the conditions examined, decomposition of the starting substrate was observed in the case of zinc agent, and no reaction occurred with the stannane agent. Then they examined the Stille coupling reaction, using vinyl stannane for the construction of the carbon-carbon bond at C11 [84]. A Stille coupling reaction of vinyl iodide 17 with tributyl(vinyl)tin was examined in the presence of a catalytic amount of Pd(PPh3)4, with CuI as an additive (a standard condition). Unfortunately, only a trace amount of the corresponding coupling product of 18c was obtained (entry 3). Then, they changed vinyl stannane to cis-tributyl (2-ethoxyvinyl) tin, and included LiCl as an additional additive. Under these conditions, the corresponding coupling product 18d was obtained with 67% yield (entry 4) [85,86]. Interestingly, poor reproducibility or low yield of the coupling reaction was observed when they used a highly oxidized vinyl stannane agent, tributyl(2,2-diethoxyvinyl)stannane (entry 5). This issue was successfully overcome by switching from CuI to copper(I) thiophene-2-carboxylate (CuTC), and 19 was obtained with 60% yield and with good reproducibility (entry 6) [87]. Based upon the Stille coupling strategy, Du Bois and co-workers achieved a total synthesis of SEA (9), as shown in Scheme 2, including the synthesis of vinyl halide 17 as a substrate for the Stille coupling reaction. Firstly, vinyl halide 17 was synthesized from 20 via Mislow-Evans [2,3] rearrangement: bisguanidine 20 was converted to N,S-acetal 21 by reaction with benzenethiol in the presence of BF3·Et2O with 84% yield. Upon treatment of 21 with urea-hydrogen peroxide (UHP), the Mislow-Evans [2,3] rearrangement reaction [88,89] took place under heating in the presence of sodium benzenthiolate, and allylic alcohol 23 was obtained with 81% yield in two steps. After oxidation of the alcohol with Dess-Martin periodinate, the resulting enone 24 was reacted with iodine in the presence of pyridine to give vinyl iodide 17 [

Carbon-Carbon Bond Formation at C11 by C-Alkylation, As Applied for The Synthesis of (+)-SEA by Looper's Group
In 2019, Looper and co-workers successfully constructed a carbon-carbon bond at C11 in STX, and reported a total synthesis of SEA (9) [69]. They initially examined C-alkylation with ketone 28 and electrophiles in the presence of variety of bases, such as lithium bis(trimethylsilyi)amide (LHMDS), lithium diisopropyl amide (LDA), potassium bis(trimethylsilyi)amide (KHMDS) and sodium bis(trimethylsilyi)amide (NaHMDS). In addition, they examined various electrophiles (haloacetates, allylic halides, and propargylic halides), but no reaction took place, as Nagasawa and co-workers had found (Scheme 3) [67]. and reported a total synthesis of SEA (9) [69]. They initially examined C-alkylation with ketone 28 and electrophiles in the presence of variety of bases, such as lithium bis(trimethylsilyi)amide (LHMDS), lithium diisopropyl amide (LDA), potassium bis(trimethylsilyi)amide (KHMDS) and sodium bis(trimethylsilyi)amide (NaHMDS). In addition, they examined various electrophiles (haloacetates, allylic halides, and propargylic halides), but no reaction took place, as Nagasawa and co-workers had found (Scheme 3) [67]. On the other hand, they found that C-alkylation took place upon reaction of ketone 28 and tertbutyl bromoacetate via the generation of zinc enolate by reaction with LiHMDS in the presence of Et2Zn, affording a mixture of 29a and its Boc-deprotected derivative 29b with 60% yield (based on the starting material) (Scheme 4). By means of this alkylation strategy, they succeeded in synthesizing On the other hand, they found that C-alkylation took place upon reaction of ketone 28 and tert-butyl bromoacetate via the generation of zinc enolate by reaction with LiHMDS in the presence of Et 2 Zn, affording a mixture of 29a and its Boc-deprotected derivative 29b with 60% yield (based on the starting material) (Scheme 4). By means of this alkylation strategy, they succeeded in synthesizing ZTX (9) as follows. Deprotection of TBDPS ether in 29 with TBAF followed by carbamoylation of the resulting alcohol resulted in 30. Finally, total synthesis of SEA (9) was achieved by reaction with TFA to hydrolyze the ester and deprotect the Boc and DPM groups.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 9 of 19 ZTX (9) as follows. Deprotection of TBDPS ether in 29 with TBAF followed by carbamoylation of the resulting alcohol resulted in 30. Finally, total synthesis of SEA (9) was achieved by reaction with TFA to hydrolyze the ester and deprotect the Boc and DPM groups.

NaV-Inhibitory Activity of Synthesized, C11-Substituted Saxitoxin Analogs
Based on the method described above for constructing a carbon-carbon bond at C11 in STX, Nagasawa and co-workers synthesized a series of STX analogs bearing substituents at C11, and evaluated the NaV-inhibitory activity of these analogs at the cellular level [67].

Na V -Inhibitory Activity of Synthesized, C11-Substituted Saxitoxin Analogs
Based on the method described above for constructing a carbon-carbon bond at C11 in STX, Nagasawa and co-workers synthesized a series of STX analogs bearing substituents at C11, and evaluated the Na V -inhibitory activity of these analogs at the cellular level [67].

Stereoselective Synthesis of The Isoxazolidine Moiety of ZTX (8), And Its Introduction at C13 in A Model Compound
As described in the introduction, ZTX (8) has a characteristic macrolactam structure from C6 to C11, involving an isoxazolidine ring system. Thus, stereoselective synthesis of the di-substituted isoxazolidine unit in ZTX (8) has been examined. In the paper reporting the isolation of 8 in 2004, the amide carbonyl group in ZTX (8) at C13 appeared at 156.5 ppm in the 13 C nuclear magnetic resonance (NMR) spectrum, which is a higher chemical shift compared to other amide carbonyls [49]. This interesting observation might be attributed to the unusual macrolactam structure in ZTX (8), and synthetic studies of model compounds have been carried out to understand the origin of this unusual chemical shift. In the following section, we discuss the stereoselective isoxazolidine syntheses reported by Nishikawa's [93] and Lopper's groups [94].

Synthesis of The Isoxazolidine Part of Zetekitoxin (8) from D-ribose by Nishikawa And Co-workers
In 2009, Nishikawa and co-workers reported the stereoselective synthesis of isoxazolidine 42 from D-ribose (34) (Scheme 5) [93]. They firstly synthesized nitroolefin 36 from aldehyde 35, which was derived from D-ribose (34) by means of a Henry reaction followed by dehydration with mesylation. After reduction of the double bond in 36 with NaBH 4 , the resulting nitroalkane 37 was treated with Boc 2 O to produce dihydrooxazole 39a and 39b with 86% yield, as a diastereomeric mixture at C16 in a ratio of 3:1. In this reaction, nitrile oxide 38 was generated first, and a 1,3-dipolar cyclization reaction occurred simultaneously. The major transition state model is shown in Scheme 5. The major diastereomer 39a was reduced stereoselectively with NaBH 3 CN to isoxazolidine 40. After acetylation of the amine in 40, isoxazolidine 42, which has the same stereochemistry as ZTX at C15 and C16, was obtained in three steps: (1) deprotection of acetonide with TFA, (2) oxidative cleavage of diol with NaIO 4 , and (3) reduction of the resulting aldehyde with NaBH 4 .

Stereoselective Synthesis of The Isoxazolidine Moiety of ZTX (8), And Its Introduction at C13 in A Model Compound
As described in the introduction, ZTX (8) has a characteristic macrolactam structure from C6 to C11, involving an isoxazolidine ring system. Thus, stereoselective synthesis of the di-substituted isoxazolidine unit in ZTX (8) has been examined. In the paper reporting the isolation of 8 in 2004, the amide carbonyl group in ZTX (8) at C13 appeared at 156.5 ppm in the 13 C nuclear magnetic resonance (NMR) spectrum, which is a higher chemical shift compared to other amide carbonyls [49]. This interesting observation might be attributed to the unusual macrolactam structure in ZTX (8), and synthetic studies of model compounds have been carried out to understand the origin of this unusual chemical shift. In the following section, we discuss the stereoselective isoxazolidine syntheses reported by Nishikawa's [93] and Lopper's groups [94].

Synthesis of The Isoxazolidine Part of Zetekitoxin (8) from D-ribose by Nishikawa And Co-workers
In 2009, Nishikawa and co-workers reported the stereoselective synthesis of isoxazolidine 42 from D-ribose (34) (Scheme 5) [93]. They firstly synthesized nitroolefin 36 from aldehyde 35, which was derived from D-ribose (34) by means of a Henry reaction followed by dehydration with mesylation. After reduction of the double bond in 36 with NaBH4, the resulting nitroalkane 37 was treated with Boc2O to produce dihydrooxazole 39a and 39b with 86% yield, as a diastereomeric mixture at C16 in a ratio of 3:1. In this reaction, nitrile oxide 38 was generated first, and a 1,3-dipolar cyclization reaction occurred simultaneously. The major transition state model is shown in Scheme 5. The major diastereomer 39a was reduced stereoselectively with NaBH3CN to isoxazolidine 40. After acetylation of the amine in 40, isoxazolidine 42, which has the same stereochemistry as ZTX at C15 and C16, was obtained in three steps: (1) deprotection of acetonide with TFA, (2) oxidative cleavage of diol with NaIO4, and (3) reduction of the resulting aldehyde with NaBH4.

Stereoselective Synthesis of The Isoxazolidine Part from Methyl α-d-glucopyranoside by Lopper and Co-Workers
In 2015, Lopper and co-workers reported a synthesis of isoxazolidine 59 (Scheme 6) [94]. Aldehyde 45 was synthesized from commercially available methyl α-d-glucopyranoside (43) by the iodination of 44 with iodine and PPh 3 , acetylation of the hydroxyl group, and reductive cleavage of the pyran ring with zinc in acetic acid [95]. Then, intramolecular 1,3-dipolar reaction of the terminal olefin with nitrone, which was generated from aldehyde 45 by reaction with hydroxylamine 46, took place stereoselectively to afford 48 via 47 with 52% yield. After deprotection of acetate in 48 with sodium methoxide [96,97], the resulting triol 49 was treated with NaOI 4 followed by LiAlH 4 to give diol 51 with 70% yield in two steps [98,99]. The isoxazolidine synthon 59 in ZTX (8) was synthesized from diol 51 in seven steps by selective functionalization of the two hydroxyl groups, followed by N-acylation.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 12 of 19 olefin with nitrone, which was generated from aldehyde 45 by reaction with hydroxylamine 46, took place stereoselectively to afford 48 via 47 with 52% yield. After deprotection of acetate in 48 with sodium methoxide [96,97], the resulting triol 49 was treated with NaOI4 followed by LiAlH4 to give diol 51 with 70% yield in two steps [98,99]. The isoxazolidine synthon 59 in ZTX (8) was synthesized from diol 51 in seven steps by selective functionalization of the two hydroxyl groups, followed by Nacylation.
Scheme 6. Synthesis of N-acyl isoxazolidine 59 by Looper's group. (8) with Those in Some Synthetic Models.

Comparison of the Chemical Shift at C13 in Zetekitoxin
As discussed above, the 13 C NMR chemical shift of the carbonyl group at C13 in ZTX (8) has been observed at 156.5 ppm [49], which is a higher value compared with usual amide carbonyl groups (170-175 ppm). To address the issue, Nishikawa's and Looper's groups independently examined the 13 C chemical shifts of the carbonyl group at C13 in some model compounds ( Figure 9) [93,94]. Simple N-acyl isoxazolidine models 60, 42, and 59 showed chemical shifts of 171.0, 172.7, 171.0 ppm, respectively, which are quite similar to those of regular cyclic N-acyl amides. However, model compounds 61-63 bearing alpha-guanidinoacetyl amide groups showed chemical shifts of 166.0, 168.3, and 167.0 ppm, respectively, being shifted ca. 5 ppm upfield compared to the other simple models.
Nagasawa and co-workers examined the chemical shift at C13 of 70, which has an STX skeleton; its synthesis is depicted in Scheme 7 [100]. They firstly aimed to obtain carboxylic acid 66 from alcohol 64 by oxidation. They examined various oxidants and conditions, but it appeared that the hydroxyl group in 64 was unreactive due to its axial orientation, and no reaction occurred, or unexpected side reactions proceeded. Finally, they found that 2-azaadamantane N-oxyl (AZADO)-NaClO and Scheme 6. Synthesis of N-acyl isoxazolidine 59 by Looper's group.

Comparison of the Chemical Shift at C13 in Zetekitoxin (8) with Those in Some Synthetic Models
As discussed above, the 13 C NMR chemical shift of the carbonyl group at C13 in ZTX (8) has been observed at 156.5 ppm [49], which is a higher value compared with usual amide carbonyl groups (170-175 ppm). To address the issue, Nishikawa's and Looper's groups independently examined the 13 C chemical shifts of the carbonyl group at C13 in some model compounds ( Figure 9) [93,94]. Simple N-acyl isoxazolidine models 60, 42, and 59 showed chemical shifts of 171.0, 172.7, 171.0 ppm, respectively, which are quite similar to those of regular cyclic N-acyl amides. However, model compounds 61-63 bearing alpha-guanidinoacetyl amide groups showed chemical shifts of 166.0, 168.3, and 167.0 ppm, respectively, being shifted ca. 5 ppm upfield compared to the other simple models.

Synthesis of The Characteristic Macrocyclic Structure of ZTX (8) by Looper's Group
Looper and co-workers have reported macrocyclic compound 72 as a model for ZTX (8) (Scheme 8) [71]. After deprotection of the TBDPS ether group at C13 with TBAF, the resulting alcohol was reacted with iodoacetic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N,N-dimethyl-4-aminopyridine (DMAP) to give iodoester 71 with 58% yield. When iodoester 71 was treated with a strong base, tert-butylimino-tri(pyrrolidino)phosphorane (BTPP), intramolecular alkylation proceeded at C11, and the corresponding macrolactone 72 was obtained in 48% yield. It should be possible to construct the macrolactam structure of 8 via a similar strategy, and this should also resolve the chemical shift issue in ZTX (8). Nagasawa and co-workers examined the chemical shift at C13 of 70, which has an STX skeleton; its synthesis is depicted in Scheme 7 [100]. They firstly aimed to obtain carboxylic acid 66 from alcohol 64 by oxidation. They examined various oxidants and conditions, but it appeared that the hydroxyl group in 64 was unreactive due to its axial orientation, and no reaction occurred, or unexpected side reactions proceeded. Finally, they found that 2-azaadamantane N-oxyl (AZADO)-NaClO and NaClO 2 [101,102] were effective, resulting in carboxylic acid 66, which was obtained with 79% yield after TMSCHN 2 treatment of the crude carboxylic acid 66 to hydrolyze the methyl ester 67. Condensation of carboxylic acid 66 with isoxazolidine 40 [93] in the presence of 4-(4, 6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM,68) [103], followed by deprotection of the Boc group and acetal with TFA, gave amide 70 in 98% yield. Unfortunately, the chemical shift of the carbonyl group in 70 was observed at 166.1 ppm, slightly higher than that of 62 or 63, but still lower than that of ZTX (8). The chemical shift in ZTX (8) may reflect the characteristic spatial structure associated with the presence of the macrolactam moiety.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 13 of 19 NaClO2 [101,102] were effective, resulting in carboxylic acid 66, which was obtained with 79% yield after TMSCHN2 treatment of the crude carboxylic acid 66 to hydrolyze the methyl ester 67.

Synthesis of The Characteristic Macrocyclic Structure of ZTX (8) by Looper's Group
Looper and co-workers have reported macrocyclic compound 72 as a model for ZTX (8) (Scheme 8) [71]. After deprotection of the TBDPS ether group at C13 with TBAF, the resulting alcohol was reacted with iodoacetic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N,N-dimethyl-4-aminopyridine (DMAP) to give iodoester 71 with 58% yield. When iodoester 71 was treated with a strong base, tert-butylimino-tri(pyrrolidino)phosphorane (BTPP), intramolecular alkylation proceeded at C11, and the corresponding macrolactone 72 was obtained in 48% yield. It should be possible to construct the macrolactam structure of 8 via a similar strategy, and this should also resolve the chemical shift issue in ZTX (8).

Synthesis of The Characteristic Macrocyclic Structure of ZTX (8) by Looper's Group
Looper and co-workers have reported macrocyclic compound 72 as a model for ZTX (8) (Scheme 8) [71]. After deprotection of the TBDPS ether group at C13 with TBAF, the resulting alcohol was reacted with iodoacetic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N,N-dimethyl-4-aminopyridine (DMAP) to give iodoester 71 with 58% yield. When iodoester 71 was treated with a strong base, tert-butylimino-tri(pyrrolidino)phosphorane (BTPP), intramolecular alkylation proceeded at C11, and the corresponding macrolactone 72 was obtained in 48% yield. It should be possible to construct the macrolactam structure of 8 via a similar strategy, and this should also resolve the chemical shift issue in ZTX (8).

Conclusions
Here, we have reviewed recent progress towards the total synthesis of zetekitoxin AB (8, ZTX). Although this goal still remains elusive, there have been some significant synthetic advances in the construction of characteristic structures of ZTX, such as (i) the carbon-carbon bond at C11 in the STX structure, (ii) stereoselective construction of the substituted isoxazolidine moiety at C15 and C16, and (iii) the macrocyclic structure from C6 to C11. Since ZTX has potent inhibitory activity, even towards tetrodotoxin-resistant (TTX-r) NaVs, a total synthesis of ZTX and its analogs is expected to provide useful tools for chemical biological studies of NaVs, overcoming the severely restricted availability of natural ZTX.