Completion of the Total Synthesis of Several Bioactive Sarpagine/Macroline Alkaloids including the Important NF-κB Inhibitor N4-Methyltalpinine

The unification of the general synthetic strategy regarding the important and emerging group of C-19 methyl-substituted sarpagine/macroline alkaloids has culminated in the completion of the total synthesis of several bioactive alkaloids. Key transformations include an ACE-Cl mediated late-stage N(4)-demethylation and an anhydrous acid-mediated intramolecular quaternary hemiaminal formation between a tertiary amine and an aldehyde function to allow efficient access to several biologically important alkaloids from this group. Herein, the enantiospecific total synthesis of the first known sarpagine/macroline alkaloid with NF-κB inhibitory activity, N(4)-methyltalpinine (as a chloride salt), as well as the anticancer alkaloids talpinine, O-acetyltalpinine, and macrocarpines F–G, are described.


Introduction
The sarpagine/macroline/ajmaline family represents an important class of biosynthetically related monoterpene indole alkaloids [1][2][3]. Sarpagine, ajmaline, and macroline individually are eponymous with respect to their corresponding subclass of alkaloids. To date, this family comprises more than 300 monomeric and about 100 bisindole alkaloids [2,[4][5][6]. A number of alkaloids from this family have been reported to possess important biological properties, such as anticancer, antileishmanial, antiarrhythmic, and antimalarial activities, which is not surprising since these alkaloids occur primarily in various plant species of the Apocynaceae family that have been used in traditional or folk medicines for centuries [4,[7][8][9][10][11].
The C-19 methyl-substituted sarpagine/ajmaline sub-family of alkaloids is a growing ensemble of alkaloids with a common structural feature, i.e., alkaloids having a stereogenic methyl group at the C-19 position of their biogenetic architecture. To date, this group has been populated with more than seventy alkaloids [8,12]. Despite this common structural feature, notable diversity in substitution patterns, and oxidation states, very few syntheses have been reported for this sub-group [13][14][15]. In addition, as usual, the paucity of isolated material has impeded the study of medicinal properties.
A general and unified synthetic approach regarding this emerging group has been of interest due to the important biological activities as well as the interesting structures of a number of alkaloids from the Apocynaceae [8]. One of the important outcomes of the general strategy developed in Milwaukee is the improved and readier access to the core sarpagine/ajmaline architecture via an ambidextrous Pictet-Spengler/Dieckmann protocol,

Results and Discussion
Since the Na-CH3, Nb-CH3 alkaloids, and macrocarpines A-B (1-2) were in hand from previous work, [15] it was felt the Nb-H substitution in macrocaprines F (6) and G (7) would be accessible via an Nb-demethylation process from 1 [21] and 2 [21], respectively (Scheme 1). Talpinine (10) [22] is a macroline-derived sarpagine-type alkaloid containing a hemiaminal (carbinolamine) function in which the N(4) atom of the sarpagine architecture is directly connected to the C(21) atom of the macroline system. This provides an additional ring, as well as increasing the rigidity of the system due to the quinuclidine moiety. Retrosynthetically, the hemiaminal function at C-21 would originate from an intramolecular cyclization between the secondary Nb-nitrogen atom (as in 15) with the C-20α formyl function (Scheme 1). The secondary amine 15 would be available from the tertiary amine 9 via dealkylation. The same Nb-demethylation reaction employed for the synthesis of 6 and 7 should also be useful in this case. On the other hand, for the important quaternary ammonium alkaloid 12 containing a methyl function at the Nb-nitrogen atom of talpinine (10), the most obvious precursor would be 10 itself, which would be accessible by means of an Nb-methylation process with a methyl halide (Scheme 1). Although the counter anion for the quaternary ammonium ion in the natural sample was not known, [23] the synthetic N4-methyltalpinine would have the counter anion corresponding to the methyl halide used. Another possible way of accessing N4-methyltalpinine would be the intramolecular reaction between the tertiary Nb-amine nitrogen atom with the C-21 carbonyl function of N4-methyl-N4,21-secotalpinine 9. However, potential steric hindrance and conformational restriction of the tertiary Nb-nitrogen atom could deter it from reacting with the aldehyde function to furnish the hemiaminal (carbinolamine) function present in the desired N4-methyltalpinine. The carbonyl function in 9 could be further activated towards nucleophilic addition by means of a Lewis or Brønsted acid catalyst.

Results and Discussion
Since the N a -CH 3 , N b -CH 3 alkaloids, and macrocarpines A-B (1-2) were in hand from previous work, [15] it was felt the N b -H substitution in macrocaprines F (6) and G (7) would be accessible via an N b -demethylation process from 1 [21] and 2 [21], respectively (Scheme 1). Talpinine (10) [22] is a macroline-derived sarpagine-type alkaloid containing a hemiaminal (carbinolamine) function in which the N(4) atom of the sarpagine architecture is directly connected to the C(21) atom of the macroline system. This provides an additional ring, as well as increasing the rigidity of the system due to the quinuclidine moiety. Retrosynthetically, the hemiaminal function at C-21 would originate from an intramolecular cyclization between the secondary N b -nitrogen atom (as in 15) with the C-20α formyl function (Scheme 1). The secondary amine 15 would be available from the tertiary amine 9 via dealkylation. The same N b -demethylation reaction employed for the synthesis of 6 and 7 should also be useful in this case. On the other hand, for the important quaternary ammonium alkaloid 12 containing a methyl function at the N b -nitrogen atom of talpinine (10), the most obvious precursor would be 10 itself, which would be accessible by means of an N b -methylation process with a methyl halide (Scheme 1). Although the counter anion for the quaternary ammonium ion in the natural sample was not known, [23] the synthetic N 4 -methyltalpinine would have the counter anion corresponding to the methyl halide used. Another possible way of accessing N 4 -methyltalpinine would be the intramolecular reaction between the tertiary N b -amine nitrogen atom with the C-21 carbonyl function of N 4 -methyl-N 4 ,21-secotalpinine 9. However, potential steric hindrance and conformational restriction of the tertiary N b -nitrogen atom could deter it from reacting with the aldehyde function to furnish the hemiaminal (carbinolamine) function present in the desired N 4methyltalpinine. The carbonyl function in 9 could be further activated towards nucleophilic addition by means of a Lewis or Brønsted acid catalyst. As planned, the focus was initially on the search for and optimization of a facile and robust method for the Nb-dealkylation process. Late-stage transformation towards important alkaloids sometimes occurs with limited amounts of the required precursors in hand (which albeit are alkaloids themselves), therefore, as mentioned, the Nb-demethylation process must be robust. The Nb-methyl function is inert to many chemical transformations and aggressive reagents. Consequently, it is considered to be a persistent protecting group for amines [27,28]. In addition, it is an additional challenge to remove a methyl group on amines in functionally rich and sterically hindered systems, such as 1 and 2. In order to remove the Nb-methyl group regioselectively, it was decided to attempt a number of available methods present in the literature. The use of cyanogen bromide (Von Braun reaction) [29] and carbonochloridates [30] (chloroformates) are well-known for regioselective dealkylation of alkylamines. It was also reported that the chloroformates are one of the best methods for this purpose due to better selectivity, as well as cleaner and milder reaction conditions. In addition, one looked for inspiration from similar transformations in related systems. Inspired by the methods in the total synthesis of the oxindole alkaloid isoalstonisine by Fonseca [31], fortunately, the chloroformate was successfully used to Ndealkylate the Nb-methyl group in a congested system at the final stage of the synthesis. It was then decided to employ the well-known N-dealkylation process developed by Olofson et al. [32]. This process employs an excess of ACE-Cl in 1,2-DCE at reflux. The so formed quaternary ammonium carbamate, upon refluxing in methanol, followed by a basic work-up, would provide the desired Nb-demethylated products, i.e., 6 and 7.
As planned, macrocarpine A (1) and B (2) (individually) were reacted with 10 equivalents of ACE-Cl in refluxing 1,2-dichloroethane. After that, the reaction mixtures were dissolved in dry methanol and heated at reflux, and this was followed by a basic work-up with cold aq 1 N NaOH (Scheme 2). The C-20β hydroxymethyl compound, macrocarpine A (1), furnished the Nb-demethylated secondary amine, macrocarpine F (6, LRMS: [M + H] + = 327), along with unreacted starting material 1, which resulted in a 90% yield (based on recovered starting material). An LC-MS analysis of the reaction mixture indicated that the desired product 6 and the starting material 1 were present in a ratio of 88:12. On the other hand, it was observed that the C-20α hydroxymethyl compound, macrocarpine B (2), remained unchanged (LRMS: [M + H] + = 341) even after prolonged heating at reflux (up to 72 h), i.e., the Nb-methyl base 2 did not form the quaternary ammonium carbamate salt, hence the subsequent decarboxylation did not proceed. Consequently, the O-acetyl variant of 2, macrocarpine C (3, MW = 382.5), was subjected to the same conditions used Scheme 1. Retrosynthetic strategy for the synthesis of C-19 methyl-substituted alkaloids.
As planned, the focus was initially on the search for and optimization of a facile and robust method for the N b -dealkylation process. Late-stage transformation towards important alkaloids sometimes occurs with limited amounts of the required precursors in hand (which albeit are alkaloids themselves), therefore, as mentioned, the N b -demethylation process must be robust. The N b -methyl function is inert to many chemical transformations and aggressive reagents. Consequently, it is considered to be a persistent protecting group for amines [27,28]. In addition, it is an additional challenge to remove a methyl group on amines in functionally rich and sterically hindered systems, such as 1 and 2. In order to remove the N b -methyl group regioselectively, it was decided to attempt a number of available methods present in the literature. The use of cyanogen bromide (Von Braun reaction) [29] and carbonochloridates [30] (chloroformates) are well-known for regioselective dealkylation of alkylamines. It was also reported that the chloroformates are one of the best methods for this purpose due to better selectivity, as well as cleaner and milder reaction conditions. In addition, one looked for inspiration from similar transformations in related systems. Inspired by the methods in the total synthesis of the oxindole alkaloid isoalstonisine by Fonseca [31], fortunately, the chloroformate was successfully used to N-dealkylate the N b -methyl group in a congested system at the final stage of the synthesis. It was then decided to employ the well-known N-dealkylation process developed by Olofson et al. [32]. This process employs an excess of ACE-Cl in 1,2-DCE at reflux. The so formed quaternary ammonium carbamate, upon refluxing in methanol, followed by a basic work-up, would provide the desired N b -demethylated products, i.e., 6 and 7.
As planned, macrocarpine A (1) and B (2) (individually) were reacted with 10 equivalents of ACE-Cl in refluxing 1,2-dichloroethane. After that, the reaction mixtures were dissolved in dry methanol and heated at reflux, and this was followed by a basic work-up with cold aq 1 N NaOH (Scheme 2). The C-20β hydroxymethyl compound, macrocarpine A (1), furnished the N b -demethylated secondary amine, macrocarpine F (6, LRMS: [M + H] + =327), along with unreacted starting material 1, which resulted in a 90% yield (based on recovered starting material). An LC-MS analysis of the reaction mixture indicated that the desired product 6 and the starting material 1 were present in a ratio of 88:12. On the other hand, it was observed that the C-20α hydroxymethyl compound, macrocarpine B (2), remained unchanged (LRMS: [M + H] + = 341) even after prolonged heating at reflux (up to 72 h), i.e., the N b -methyl base 2 did not form the quaternary ammonium carbamate salt, hence the subsequent decarboxylation did not proceed. Consequently, the O-acetyl variant of 2, From these observations, it was concluded that the C-20α hydroxymethyl group in tertiary amine 2 was too close to the amine function, which created some steric congestion and probably hindered the amine function from reacting with the chloroformate. More importantly, from molecular modeling (see Figure 2), it was clear that a hydrogen bond between the tertiary amine function in 2 with the primary alcohol would also retard the amine function from reacting with the chloroformate. This was evident from the fact that both macrocarpine A 1 and the O-acetyl version of macrocarpine B (macrocarpine C, 3) did react to form the desired Nb-demethylated products 6 and 7, respectively. The spectroscopic properties of 6 and 7 were in excellent agreement with the natural alkaloids [20] (see Section 3). From these observations, it was concluded that the C-20α hydroxymethyl group in tertiary amine 2 was too close to the amine function, which created some steric congestion and probably hindered the amine function from reacting with the chloroformate. More importantly, from molecular modeling (see Figure 2), it was clear that a hydrogen bond between the tertiary amine function in 2 with the primary alcohol would also retard the amine function from reacting with the chloroformate. This was evident from the fact that both macrocarpine A 1 and the O-acetyl version of macrocarpine B (macrocarpine C, 3) did react to form the desired N b -demethylated products 6 and 7, respectively. The spectroscopic properties of 6 and 7 were in excellent agreement with the natural alkaloids [20] (see Section 3).
Since the desired demethylation was executed effectively, the C-20α aldehyde 9 was also subjected to the Olofson [32] N b -demethylation conditions, which furnished the demethylated secondary amine 15 in situ, and it underwent cyclization, subsequently, in an intramolecular fashion, to form the desired hemiaminal present in talpinine 10. During the initial trials it was observed that the starting tertiary amine 9 was somewhat unreactive with the chloroformate (ACE-Cl) and the conversion was very slow. It was felt that using a bulky and non-nucleophilic base, such as pempidine (1,2,2,6,6-pentamethylpiperidine) [33], would facilitate the carbamate formation at the initial stage of the demethylation process by scavenging any residual protons present in the reaction solution. Using a stoichiometric amount of pempidine and an excess of ACE-Cl in DCE at reflux (for 18 h), it was observed that the corresponding carbamates ( tertiary amine nitrogen atom with the ACE-Cl carbonyl function, was present as the major product in the reaction mixture (after 18 h), while the N b -demethylated carbamate 17 was present as the minor product. Gratifyingly, this observation indicated that the reaction was progressing, albeit slowly. Accordingly, the reaction mixture was subjected to prolonged heat (up to 42 h), which completed the conversion, as indicated by the absence of the starting material 9 upon analysis by LC-MS. After the subsequent decarboxylation reaction in refluxing methanol was followed by an alkaline work-up, this process furnished the secondary amine (see 15 in Scheme 1). The amine N b -nitrogen atom reacted with the C-21 formyl function, as above, ultimately to form the hemiaminal present in talpinine 10 (Scheme 4) in 75% yield. Examination of the 1 H NMR spectrum confirmed the absence of the formyl function (at δ 9.44 ppm) and the presence of the H-21 proton (at carbinolamine carbon, C-21) at δ 4.71 ppm.
The C-20β aldehyde function containing indole base 8 was also subjected to the same conditions to check whether the stereochemistry at the C-20 function played a significant role in the rate of initial carbamate formation. It was found that talcarpine 8 also underwent demethylation and furnished the same product, talpinine 10 in 75% yield. This indicated that the C-20 aldehyde undergoes epimerization under these conditions to the α-stereochemistry and then cyclizes (Scheme 4). Simple acetylation of talpinine 10 using standard methods furnished O-acetyltalpinine 11 in 85% yield. The spectral and optical properties of synthetic 10 and 11 were in excellent agreement with the values reported in the literature [22,34] for the corresponding natural products.

Scheme 2.
Completion of the total synthesis of 6 and 7 via the late-stage demethylation process.
From these observations, it was concluded that the C-20α hydroxymethyl group in tertiary amine 2 was too close to the amine function, which created some steric congestion and probably hindered the amine function from reacting with the chloroformate. More importantly, from molecular modeling (see Figure 2), it was clear that a hydrogen bond between the tertiary amine function in 2 with the primary alcohol would also retard the amine function from reacting with the chloroformate. This was evident from the fact that both macrocarpine A 1 and the O-acetyl version of macrocarpine B (macrocarpine C, 3) did react to form the desired Nb-demethylated products 6 and 7, respectively. The spectroscopic properties of 6 and 7 were in excellent agreement with the natural alkaloids [20] (see Section 3).   (2). The C-21 atom in macrocarpine B is generating greater steric congestion to the oncoming ACE-Cl electrophile toward the N(4) nitrogen atom. In addition, the C-21 in 2 is closer than the C-21 in 1 to the N(4) nitrogen atom, which suggests stronger intra-molecular H-bonding between the C-21 (OH) with the N(4) nitrogen atom in 2.
Molecules 2022, 27, x FOR PEER REVIEW 6 of 18 the oncoming ACE-Cl electrophile toward the N(4) nitrogen atom. In addition, the C-21 in 2 is closer than the C-21 in 1 to the N(4) nitrogen atom, which suggests stronger intra-molecular H-bonding between the C-21 (OH) with the N(4) nitrogen atom in 2.
Since the desired demethylation was executed effectively, the C-20α aldehyde 9 was also subjected to the Olofson [32] Nb-demethylation conditions, which furnished the demethylated secondary amine 15 in situ, and it underwent cyclization, subsequently, in an intramolecular fashion, to form the desired hemiaminal present in talpinine 10. During the initial trials it was observed that the starting tertiary amine 9 was somewhat unreactive with the chloroformate (ACE-Cl) and the conversion was very slow. It was felt that using a bulky and non-nucleophilic base, such as pempidine (1,2,2,6,6-pentamethylpiperidine) [33], would facilitate the carbamate formation at the initial stage of the demethylation process by scavenging any residual protons present in the reaction solution. Using a stoichiometric amount of pempidine and an excess of ACE-Cl in DCE at reflux (for 18 h), it was observed that the corresponding carbamates ( . This indicated that the first intermediate, 16, which was formed by the reaction between the tertiary amine nitrogen atom with the ACE-Cl carbonyl function, was present as the major product in the reaction mixture (after 18 h), while the Nb-demethylated carbamate 17 was present as the minor product. Gratifyingly, this observation indicated that the reaction was progressing, albeit slowly. Accordingly, the reaction mixture was subjected to prolonged heat (up to 42 h), which completed the conversion, as indicated by the absence of the starting material 9 upon analysis by LC-MS. After the subsequent decarboxylation reaction in refluxing methanol was followed by an alkaline workup, this process furnished the secondary amine (see 15 in Scheme 1). The amine Nb-nitrogen atom reacted with the C-21 formyl function, as above, ultimately to form the hemiaminal present in talpinine 10 (Scheme 4) in 75% yield. Examination of the 1 H NMR spectrum confirmed the absence of the formyl function (at δ 9.44 ppm) and the presence of the H-21 proton (at carbinolamine carbon, C-21) at δ 4.71 ppm.   At this point, as planned, talpinine 10 was treated with iodomethane in methanol at room temperature in the dark for 16 h (Scheme 5). A 1 H NMR spectrum of the reaction mixture in deuterated methanol indicated the disappearance of the aldehydic proton (at δ 9.44 ppm), whereas a broad multiplet at δ 5.0-4.9 ppm appeared, which was expected for the hemiaminal proton (H-21 on the carbinolamine carbon, C-21). This result was encouraging; however, the NMR spectrum of the crude reaction mixture was not clean enough for full characterization and for comparison with the natural alkaloid [23]. Consequently, chromatography (silica gel, CH 2 Cl 2 /MeOH/28%NH 4 OH (aq.); 94:5:1) was attempted in order to obtain a pure sample of synthetic N 4 -methyltalpinine (12). Unfortunately, the compound that was isolated by chromatography lacked the hemiaminal proton peak at δ 5 ppm as well as the aldehydic proton peak at δ 9.44 ppm. Intrigued by this result, attempts were made to identify this product. The product was found to be identical to N 4 -methyl-N 4 ,21-secotalpinine 9 in deuterated methanol. One important observation was made. In deuterated methanol the aldehyde peak of 9 (Figure 3c The C-20β aldehyde function containing indole base 8 was also subjected to the sa conditions to check whether the stereochemistry at the C-20 function played a signific role in the rate of initial carbamate formation. It was found that talcarpine 8 also und went demethylation and furnished the same product, talpinine 10 in 75% yield. This in cated that the C-20 aldehyde undergoes epimerization under these conditions to the stereochemistry and then cyclizes (Scheme 4). Simple acetylation of talpinine 10 us standard methods furnished O-acetyltalpinine 11 in 85% yield. The spectral and opti properties of synthetic 10 and 11 were in excellent agreement with the values reported the literature [22,34] for the corresponding natural products.
At this point, as planned, talpinine 10 was treated with iodomethane in methano room temperature in the dark for 16 h (Scheme 5). A 1 H NMR spectrum of the react mixture in deuterated methanol indicated the disappearance of the aldehydic proton δ 9.44 ppm), whereas a broad multiplet at δ 5.0-4.9 ppm appeared, which was expec for the hemiaminal proton (H-21 on the carbinolamine carbon, C-21). This result was couraging; however, the NMR spectrum of the crude reaction mixture was not cle enough for full characterization and for comparison with the natural alkaloid [23]. Co sequently, chromatography (silica gel, CH2Cl2/MeOH/28%NH4OH (aq.); 94:5:1) was tempted in order to obtain a pure sample of synthetic N4-methyltalpinine (12). Unfor nately, the compound that was isolated by chromatography lacked the hemiaminal p ton peak at ~δ 5 ppm as well as the aldehydic proton peak at δ 9.44 ppm. Intrigued by t result, attempts were made to identify this product. The product was found to be identi to N4-methyl-N4,21-secotalpinine 9 in deuterated methanol. One important observat was made. In deuterated methanol the aldehyde peak of 9 (Figure 3c) was not well defin in comparison to the 1 H NMR spectrum of 8 in CD3OD (Figure 2a). On the other hand CDCl3 the aldehyde peak of both 8 ( Figure 3b) and 9 (Figure 3d) was well-defined.  This result indicated that while the C-20β aldehyde in talcarpine 8 remained as aldehyde moiety in methanol, there was an equilibrium mixture in the case of N4-methy N4,21-secotalpinine 9 ( Figure 3). Furthermore, it was observed that the aldehyde pe broadened or sharpened in a temperature-dependent manner. At higher temperature the aldehyde peak was found to be broader than the corresponding aldehyde peak lower temperatures (not shown here).
Furthermore, it was determined that the aldehyde peak broadened after the epime ization of talcarpine 8 into 9. Talcarpine 8 was treated with triethylamine in methanol room temperature. It was observed that the aldehyde peak of 8 (at δ 9.9 ppm) gradual diminished and a broad peak corresponding to the C-20α aldehyde proton appeared a ~9.3 ppm. This experiment indicated that the β-aldehyde function in talcarpine 8 epime ized in the presence of a base and gradually formed the corresponding α-aldehyde which is the thermodynamically more stable epimer. As soon as the α-aldehyde 9 w formed, it interacted with the tertiary amine, which was in the vicinity and formed equilibrium favoring the cyclized form. As a result, this altered the sharp peak for t aldehyde to a broader peak (Scheme 6). This result indicated that while the C-20β aldehyde in talcarpine 8 remained as an aldehyde moiety in methanol, there was an equilibrium mixture in the case of N 4 -methyl-N 4 ,21-secotalpinine 9 ( Figure 3). Furthermore, it was observed that the aldehyde peak broadened or sharpened in a temperature-dependent manner. At higher temperatures, the aldehyde peak was found to be broader than the corresponding aldehyde peak at lower temperatures (not shown here).
Furthermore, it was determined that the aldehyde peak broadened after the epimerization of talcarpine 8 into 9. Talcarpine 8 was treated with triethylamine in methanol at room temperature. It was observed that the aldehyde peak of 8 (at δ 9.9 ppm) gradually diminished and a broad peak corresponding to the C-20α aldehyde proton appeared at δ~9.3 ppm. This experiment indicated that the β-aldehyde function in talcarpine 8 epimerized in the presence of a base and gradually formed the corresponding α-aldehyde 9, which is the thermodynamically more stable epimer. As soon as the α-aldehyde 9 was formed, it interacted with the tertiary amine, which was in the vicinity and formed an equilibrium favoring the cyclized form. As a result, this altered the sharp peak for the aldehyde to a broader peak (Scheme 6).
diminished and a broad peak corresponding to the C-20α aldehyde proton appeared at δ ~9.3 ppm. This experiment indicated that the β-aldehyde function in talcarpine 8 epimerized in the presence of a base and gradually formed the corresponding α-aldehyde 9, which is the thermodynamically more stable epimer. As soon as the α-aldehyde 9 was formed, it interacted with the tertiary amine, which was in the vicinity and formed an equilibrium favoring the cyclized form. As a result, this altered the sharp peak for the aldehyde to a broader peak (Scheme 6). Scheme 6. Epimerization of talcarpine 8 into N4-methyl-N4,21-secotalpinine 9 under basic conditions. Scheme 6. Epimerization of talcarpine 8 into N 4 -methyl-N 4 ,21-secotalpinine 9 under basic conditions.
Given the experiments described above, it was felt that the indole base 9 would stay principally in a Zwitterionic form with 16 in methanol (Scheme 6), while in chloroform the cyclized form 16 would be present in a small amount while the equilibrium favored mostly the open form 9 (Scheme 7). In the presence of dry HCl in solution, the oxygen atom would be protonated and the chloride ion would act as the counter anion for the quaternary ammonium nitrogen atom, shifting the equilibrium towards the closed form 16 (i.e., 12). This would lead to an irreversible formation of the desired stable N 4 -methyltalpinine 12 as a chloride salt (Scheme 7) if silica gel chromatography was avoided. Consequently, the base 9 was stirred with anhydrous HCl (4.0 M solution in dioxane) at room temperature. Then deuterated chloroform was used as the solvent instead of CD 3 OD to avoid any peak overlap with the peak at δ 4.87 ppm (residual moisture) with the desired H-21 peaks at δ 5.00-4.95 ppm. After adding a catalytic amount of dry HCl, a small broad peak at δ 5.0 ppm appeared, which indicated that the conversion had begun, albeit in very small amounts (Figure 4b). After adding 2 equivalents of anhydrous HCl it was observed that the multiplet at δ 5 ppm increased in intensity, while the aldehydic peak at δ 9.44 ppm began to diminish in intensity (Figure 4c). After standing at room temperature for an additional 2 h, examination of the 1 H NMR spectrum indicated that the aldehydic peak was completely gone and the spectrum appeared much cleaner (Figure 4d). After that, the solvent was removed under reduced pressure and the alkaloid was dissolved in deuterated methanol for comparison with the literature values (see Section 3). A 1 H NMR spectrum of the compound in CD 3 OD was found to be identical to one found in the literature [23]. All other spectroscopic and optical rotation values were in excellent agreement with the literature values natural [23] for the desired alkaloid N 4 -methyltalpinine 12. (Caution: in Zwitterionic molecules, such as 12, it is best to avoid silica gel chromatography).
Molecules 2022, 27, x FOR PEER REVIEW 9 of 18 Given the experiments described above, it was felt that the indole base 9 would stay principally in a Zwitterionic form with 16 in methanol (Scheme 6), while in chloroform the cyclized form 16 would be present in a small amount while the equilibrium favored mostly the open form 9 (Scheme 7). In the presence of dry HCl in solution, the oxygen atom would be protonated and the chloride ion would act as the counter anion for the quaternary ammonium nitrogen atom, shifting the equilibrium towards the closed form 16 (i. e., 12). This would lead to an irreversible formation of the desired stable N4-methyltalpinine 12 as a chloride salt (Scheme 7) if silica gel chromatography was avoided. Consequently, the base 9 was stirred with anhydrous HCl (4.0 M solution in dioxane) at room temperature. Then deuterated chloroform was used as the solvent instead of CD3OD to avoid any peak overlap with the peak at δ 4.87 ppm (residual moisture) with the desired H-21 peaks at δ 5.00-4.95 ppm. After adding a catalytic amount of dry HCl, a small broad peak at δ 5.0 ppm appeared, which indicated that the conversion had begun, albeit in very small amounts (Figure 4b). After adding 2 equivalents of anhydrous HCl it was observed that the multiplet at δ 5 ppm increased in intensity, while the aldehydic peak at δ 9.44 ppm began to diminish in intensity (Figure 4c). After standing at room temperature for an additional 2 h, examination of the 1 H NMR spectrum indicated that the aldehydic peak was completely gone and the spectrum appeared much cleaner (Figure 4d). After that, the solvent was removed under reduced pressure and the alkaloid was dissolved in deuterated methanol for comparison with the literature values (see Section 3). A 1 H NMR spectrum of the compound in CD3OD was found to be identical to one found in the literature [23. All other spectroscopic and optical rotation values were in excellent agreement with the literature values natural [23] for the desired alkaloid N4-methyltalpinine 12. (Caution: in Zwitterionic molecules, such as 12, it is best to avoid silica gel chromatography). ( 1 H NMR, CDCl3, 300 MHz) to form N4-methyltalpinine (12). 1 H NMR of (a) 9, (b) 9 + HCl (cat.), (c) 9 + HCl (2 eq) immediately after addition, and (d) 9 + HCl (2 eq) after 2 h. The star symbols mark the positions of changes in important signals. ( 1 H NMR, CDCl 3 , 300 MHz) to form N 4 -methyltalpinine (12). 1 H NMR of (a) 9, (b) 9 + HCl (cat.), (c) 9 + HCl (2 eq) immediately after addition, and (d) 9 + HCl (2 eq) after 2 h. The star symbols mark the positions of changes in important signals.

General Experimental Considerations
All reactions were carried out under an argon atmosphere with dry solvents using anhydrous conditions unless it is stated otherwise. The solvents (THF, DMF, toluene, DCM, MeCN, and MeOH) were dried using an Innovative Technology solvent purification system, Pure Solv TM . Occasionally, tetrahydrofuran was freshly distilled from Na/benzophenone ketyl prior to use. Dichloromethane was distilled from calcium hydride prior to use. Methanol was distilled over magnesium sulfate. Benzene was distilled over CaH 2 . Reagents were purchased of the highest commercial quality and used without further purification unless otherwise stated. Thin layer chromatography (TLC) was performed on UV active silica gel plates, 200 µm, aluminum-backed and UV active alumina N plates, 200 µm, F-254 aluminum-backed plates. Flash and gravity chromatography were performed using silica gel P60A, 40-63 µm, basic alumina (Act I, 50-200 µm), and neutral alumina (Brockman I,~150 mesh). TLC plates were visualized by exposure to short wavelength UV light (254 nm). Indoles were visualized with a saturated solution of ceric ammonium nitrate (CAN) in 50% phosphoric acid. The 1 H NMR data are reported as follows: chemical shift, multiplicity (br s = broad singlet, s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd = doublet of doublets, dt = doublet of triplets, ddd = doublet of doublet of doublets, td = triplet of doublets, qd = quartet of doublets, m = multiplet), integration, and coupling constants (Hz). The 13 C NMR data are reported in parts per million (ppm) on the δ scale. The low-resolution mass spectra (LRMS) were obtained as electron impact (EI, 70eV) and as chemical ionization (CI) using a magnetic sector (EBE) analyzer. HRMS was performed with electrospray ionization (ESI) using a TOF analyzer, electron impact (EI) using a trisector analyzer and atmospheric pressure chemical ionization (APCI) using a TOF analyzer. Optical rotations were measured on a JASCO Model DIP-370 polarimeter.

Macrocarpine F (6)
The indole 1 (3 mg, 0.009 mmol) was dissolved in dry 1,2-dichloroethane (2 mL) in a thick-walled vessel that could be sealed with a screw cap. The ACE-Cl (1-chloroethyl chloroformate, 12.6 mg, 0.09 mmol) was added to the above solution at 0 • C under argon. The reaction vessel was sealed and heated at 90 • C (oil bath) for 72 h. The reaction was then cooled to room temperature and the solvent was removed under reduced pressure. Then, distilled methanol (5 mL) was added to the residue and the solution that resulted was heated at reflux under argon for 6 h with stirring. After that, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc (5 mL) and brought to pH 8 with cold aq 1 N NaOH. The organic layer was separated and the aq layer was extracted with additional EtOAc (2 × 5 mL). The combined organic layers were washed with brine and dried (K 2 CO 3 ). The solvent was removed under reduced pressure to give a brown residue. The residue was purified by column chromatography (silica gel, CH 2 Cl 2 /MeOH; 20:1) to yield macrocarpine F 6 as a colorless residue (2.3 mg, 80%). The spectroscopic data for the synthetic alkaloid were in excellent agreement with those for the natural product [20]. For 1 H NMR results, see Table 1; for 13 C NMR results: see Table 1. (Note: complete structural assignment was carried out based on 1 H, 13 [20] macrocarpine F at H-12. # Merged with the chloroform peak; confirmed by COSY and HSQC NMRs. $ Overlapped peaks. § The quaternary carbon (C-13) atom was not visible in the 13 C NMR spectrum at this concentration.

Macrocarpine G (7)
The indole 3 (4 mg, 0.01 mmol) was dissolved in dry 1,2-dichloroethane (3 mL) in a thick-walled vessel that could be sealed with a screw cap. The ACE-Cl (1-chloroethyl chloroformate, 14.9 mg, 0.10 mmol) was added to the above solution at 0 • C under argon. The reaction vessel was sealed and heated at 90 • C (oil bath) for 72 h. The reaction mixture was then cooled to room temperature and the solvent was removed under reduced pressure. Then, distilled methanol (5 mL) was added to the residue and the solution that resulted was heated at reflux under argon for 6 h with stirring. After that, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc (5 mL) and brought to pH 8 with cold aq 1 N NaOH. The organic layer was separated and the aq layer was extracted with additional EtOAc (2 × 5 mL). The combined organic layers were washed with brine and dried (K 2 CO 3 ). The solvent was removed under reduced pressure to give a brown residue. The residue was purified by column chromatography (silica gel, CH 2 Cl 2 /MeOH; 20:1) to yield macrocarpine G (7) as a colorless residue (1.9 mg, 55%) accompanied by 2 (0.9 mg, 25%). The optical rotation and spectroscopic data were in agreement with those for the natural product [20]. For 1 H NMR results: see Table 2 (Note: A 13 C NMR measurement was attempted but due to the very small amount of sample available, it was not successful, even after a longer experiment time. The full structural assignment was carried out based on 1 H, COSY, and NOESY NMR spectroscopy (see Figure 5 for important NOE confirmation) and comparison of the spectra with those of the natural alkaloid 1 .) natural alkaloid 1 .)

Talpinine 10
The indole 8 or 9 (6 mg, 0.018 mmol) was dissolved in dry 1,2-dichloroethane (4 mL) in a thick-walled vessel that could be sealed with a screw cap. The ACE-Cl (1-chloroethyl chloroformate, 25.3 mg, 0.18 mmol) and pempidine (2.7 mg, 0.018 mmol) were added to the above solution at 0 °C under argon. The reaction vessel was sealed and heated at 90 °C (oil bath) for 42 h. The reaction was then cooled to room temperature and the solvent was removed under reduced pressure. Then, distilled methanol (5 mL) was added to the residue and the solution that resulted was heated at reflux under argon for 6 h with stirring. After that, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc (5 mL) and brought to pH 8 with cold aq 1 N NaOH. The organic layer

Talpinine 10
The indole 8 or 9 (6 mg, 0.018 mmol) was dissolved in dry 1,2-dichloroethane (4 mL) in a thick-walled vessel that could be sealed with a screw cap. The ACE-Cl (1-chloroethyl chloroformate, 25.3 mg, 0.18 mmol) and pempidine (2.7 mg, 0.018 mmol) were added to the above solution at 0 • C under argon. The reaction vessel was sealed and heated at 90 • C (oil bath) for 42 h. The reaction was then cooled to room temperature and the solvent was removed under reduced pressure. Then, distilled methanol (5 mL) was added to the residue and the solution that resulted was heated at reflux under argon for 6 h with stirring. After that, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc (5 mL) and brought to pH 8 with cold aq 1 N NaOH. The organic layer was separated and the aq layer was extracted with additional EtOAc (2 × 5 mL). The combined organic layers were washed with brine and dried (K 2 CO 3 ). The solvent was removed under reduced pressure to give a brown residue. The residue was purified by chromatography (silica gel, CH 2 Cl 2 /MeOH; 20:1) to yield talpinine 10 as a colorless oil (4.3 mg, 75%). The spectral data were in excellent agreement with those of the natural product [22,34] Table 3; for 13 C NMR: see Table 3

O-Acetyltalpinine (11)
To a mixture of Ac 2 O and pyridine (1:1, 0.5 mL), talpinine 10 (1 mg, 0.003 mmol) was added at room temperature under argon. The solution that resulted was stirred at room temperature for 2 h. After that, a cold solution of saturated aq Na 2 CO 3 (2 mL) was added to the above reaction. The solution was extracted with CH 2 Cl 2 (3 × 3 mL). The combined organic layers were washed with brine. The solvent was removed under reduced pressure and the residue was purified by column chromatography (silica gel) in a Pasteur pipette with 0-3% MeOH in CH 2 Cl 2 to afford O-acetyltalpinine 11 (0.96 mg, 85%) as a colorless waxy solid. The spectral data for 11 were identical to those of the natural product [20]. For 1 H NMR results: see Table 4; for 13 C NMR: see Table 4;    (Note: There was an unidentified minor impurity in the synthetic 11, as indicated by examination of the 1 H NMR spectrum; the compound appeared as a single spot on TLC (silica gel). In addition, only the desired compound's (11) mass (LRMS [M + H] + = 367) was observed in the LC-MS spectrum. The minor impurity could not be removed after several chromatographic purifications. Further attempts for the synthesis or purification of this impurity could not be undertaken due to the lack of material. In spite of the presence of the impurity, the synthetic O-acetyltalpinine 11 was fully characterized and the structural assignments could be carried out to confirm the synthesis unambiguously by high resolution NMR spectroscopy.) 3.6. N 4 -Methyltalpinine (12) as the Chloride Salt (Preparation of the HCl solution for NMR titration: anhydrous HCl (0.3 mL, 4.0 M solution in dioxane) was dissolved in 5.0 mL of dry CDCl 3 . The solution, which resulted, was gradually added via a micropipette into the reaction vessel.) The indole 9 (1.0 mg, 0.003 mmol) was dissolved in dry CDCl 3 (1.0 mL) in an ovendried NMR tube (5 mm OD). The above HCl solution (25 µL in total) was gradually added to the NMR tube via a micropipette. The reaction that resulted was kept at room temperature for 2 h. After that, examination of the 1 H NMR spectrum indicated complete conversion of the aldehyde into the desired product. The solvent was removed under reduced pressure to afford N 4 -methyltalpinine 12 as a chloride salt (1.1 mg, 99%), a colorless solid. This residue was used for characterization without any purification. The optical rotation and spectroscopic data for the synthetic N 4 -methyltalpinine 12 were in excellent agreement with the values reported in the literature for the natural product [23] by Kinghorn et al. For 1 H NMR results: see Table 5; for 13 C NMR: see Table 5

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.