Total Synthesis and Anti-inflammatory Activity of Stemoamide-Type Alkaloids Including Totally Substituted Butenolides and Pyrroles

Abstract

Totally substituted butenolide including two tetrasubstituted olefins is a distinct structural motif seen in Stemona alkaloids, but efficient methods for its synthesis are not well developed. As an ongoing program aimed at the collective total synthesis of the stemoamide group, we report a stereodivergent method to give either (E)- or (Z)-totally substituted butenolide from the same intermediate. While AgOTf­-mediated elimination via an E1-type mechanism results in the formation of the kinetic (Z)-tetrasubstituted olefin, subsequent TfOH-mediated isomerization gives the thermodynamic (E)-tetrasubstituted olefin. The pyrrole ring is another important structure found in Stemona alkaloids. The direct oxidation of pyrrolidine rings with MnO₂ and careful purification gives the pyrrole groups without isomerization of the stereocenter in the lactone group. These two methods enabled us to synthesize a series of stemoamide-type alkaloids including tricyclic, tetracyclic, and pentacyclic frameworks. The anti-inflammatory activities by inhibition of iNOS expression in macrophage cell line RAW264.7 indicate that the most potent anti-inflammatory compounds without cytotoxicity are protostemonines, which consist of pentacyclic frameworks including the totally substituted butenolide.

Key words

amides - anti-inflammatory agents - butenolides - DFT calculations - iNOS - pyrroles - Stemona alkaloids - total synthesis

Stemonaceae plants are a rich source of unique alkaloids, and their extracts have been utilized as traditional folk medicines in China and Japan.[2] Although the biological activities of each constituent have not been investigated for a long time, recent studies revealed that they show a variety of biological activities such as insecticidal and antitussive activities. In particular, the anti-inflammatory activity of protostemonine (1) is one of the hottest topics regarding these natural products (Figure [1]).[3] [4] The Shu group reported protective effects of protostemonine (1) on lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute liver failure in mice.[4b] The Qian group documented that protostemonine (1) effectively attenuates LPS-induced acute lung injury in mice. Administration of 1 significantly reduced LPS-induced­ inflammatory responses in vitro and in vivo such as production of pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production.[4d] These reports suggested protostemonine (1) could become a potential anti-inflammatory agent without detectable toxic effects on vital organs. In spite of the high potential utility, the systematic structure–activity relationship of Stemona alkaloids has not been demonstrated yet due to the scarcity of these natural products.

Figure 1 Protostemonine (1) as an anti-inflammatory agent

More than 200 Stemona alkaloids have been isolated from Stemonaceae plants to date.[2] Pilli proposed the classification of this large family into eight groups based on their structural features (Scheme [1]).[2a] [d] [5] In general, Stemona alkaloids share a 1-azabicyclo[5.3.0]decane core 2, and are comprised of various types of polycyclic skeletons. Among them, two unique structural motifs have attracted attention with respect to their biological roles. The first one is the totally substituted butenolide 3,[6] which is widely seen in Stemona alkaloids such as the stemofoline[7] [8] and stemocurtisine[9] groups (Scheme [1a] and 1b). This motif is sterically hindered and highly oxygenated, and includes two tetrasubstituted olefins. The second important motif is the pyrrole group 4,[10] which is produced by oxidation of the core structure 2 as seen in the stenine[11] [12] [13] and tuberostemospironine groups[14] [15] (Scheme [1c] and 1d). To reveal the biological roles of these two structural motifs, the stemoamide group[16] [17] is the ideal research target because both distinct structures are embedded in this single group (Scheme [1e]).[2] Unfortunately, no comprehensive biological study has been performed due to the lack of pure samples. Previously, our group documented a chemoselective assembly of five-membered building blocks for the collective synthesis of tricyclic, tetracyclic, and pentacyclic stemoamide groups.[18] In this article, we report the full details of our approach to polycyclic stemoamide-type alkaloids including the totally substituted butenolide and pyrrole groups.[19] The method gives either (E)- or (Z)-totally substituted butenolide from the same intermediate by simply changing the reaction conditions. In addition, we developed the direct oxidation of pyrrolidine-type natural products, leading to the formation of the corresponding pyrrole-type natural products. The comprehensive anti-inflammatory tests of synthetic samples using iNOS expression revealed the roles of structures including the number of rings, the totally substituted butenolide, and the pyrrole ring.

Scheme 1 Five representative groups in the Stemona alkaloid family including totally substituted butenolides and pyrroles

The first challenge toward the collective total synthesis of stemoamide-type alkaloids is the construction of the totally substituted butenolides from tricyclic stemoamide (12)[20] (Scheme [1e]).[6] Tetracyclic protostemonamide (14)[21] and isoprotostemonamide (15, unnatural compound) could be synthesized by a lactone-selective coupling reaction between 12 and butenolide anion equivalent 13, associated with the stereoselective construction of the tetrasubstituted olefins. The second challenge is the direct oxidation of the pyrrolidine groups to the pyrroles.[10] After lactam-selective nucleophilic addition using butenolide anion equivalent 16,[22] [23] the resulting pyrrolidine groups of protostemonine (1)[3] and isoprotostemonine (17) could be converted into the corresponding pyrroles [bisdehydroprotostemonine (18)[24] and isobisdehydroprotostemonine (19, unnatural compound)]. The key to success is the development of mild conditions to suppress undesired oxidations in the presence of the electron-rich tetrasubstituted olefins and the generated pyrroles. Thus, the development of two key methods would enable the collective total synthesis of stemoamide-type alkaloids.[25]

Several groups have reported their original approaches to totally substituted butenolides seen in Stemona alkaloids.[6] The Kende group accomplished the first total synthesis of isostemofoline (6), in which construction of the totally substituted butenolide was achieved by addition of a lithiated butenolide to an aldehyde, and subsequent dehydration.[6a] Unfortunately, the sequence gave the (E)-butenolide in low yield (12%) due to retro-aldol scission. Overman and co-workers solved the issue associated with the retro-aldol reaction by taking advantage of the Corey–Winter reaction.[6c] They accomplished the total synthesis of didehydrostemofoline and isodidehydrostemofoline by nucleophilic addition to an aldehyde and an olefin synthesis via cyclic thionocarbamates. Some research groups have developed more direct methods through the addition of butenolides to lactone equivalents. The Olivo group converted lactone 20 into orthoethyllactone 21 with the Meerwein­ reagent (Scheme [2a]). The resulting orthoethyllactone 21 underwent nucleophilic addition of lithiated butenolide derivative 22 in a one-pot process. Subsequent elimination of 23 with TiCl₄ and Hünig’s base provided the totally substituted butenolides 24 with Z/E = 2.0:1 selectivity.[6b] The Huang group documented a direct method through nucleophilic addition to thionolactone 26, and subsequent methylation and (Z)-selective elimination in a one-pot process (Scheme [2b]).[6f] Although these two approaches are highly efficient, pre-activation of the lactones is essential, which is not applicable to stemoamide (12) due to the presence of a more Lewis basic lactam than a lactone. Considering the collective total synthesis of the stemoamide group, the method to pre-activate the lactone carbonyl groups such as with the Meerwein reagent is not an option. To overcome this chemoselectivity issue, we envisioned the lactone-selective reductive nucleophilic addition of 13 to stemoamide (12) (Scheme [2c]). Subsequent installation of a leaving group to the resulting tetracyclic intermediate 29 would provide 30, which could undergo an elimination reaction to construct the tetrasubstituted olefin.

Scheme 2 Selected approaches to totally substituted butenolides using nucleophilic addition to lactones and their derivatives

Scheme 3 Development of the approach to the totally substituted butenolides

Our approach to the totally substituted butenolide was first evaluated using model lactone 31 [26] (Scheme [3]). DIBAL-H reduction of lactone 31 provided lactol 32, which was converted into acetate 33. 2-Siloxyfuran 34 [17s] as a nucleophilic form of the butenolide was added to 33 via the oxocarbenium ion in the presence of BF₃·OEt₂. Although a four diastereomer mixture of 35 was produced, all diastereomers could potentially be converted into the tetrasubstituted olefins­. Deprotonation of butenolide 35 with NaHMDS, followed­ by regioselective bromination,[27] gave a four diastereomer­ mixture of 36 in 63% yield (2 steps, dr = 4.0:3.2:1.4:1).

Table 1 Optimization of Silver-Mediated Elimination^a

Entry	AgX	Temp	Combined yield (%)b	Z/E c
1	AgOAc	rt	0	–
2	AgNO3	rt	0	–
3	AgPF6	rt	36	5.3:1
4	AgClO4	rt	45	5.8:1
5	AgOTf	rt	64	5.0:1
6d	AgOTf	40 °C	94 (82)	5.0:1 (5.0:1)

^a Reaction conditions: 36 (1.0 equiv), AgX (5.0 equiv), 3 Å MS (1000 wt%), CH₂Cl₂ (4.4 mM), 2 h.

^b Yields were determined by ¹H NMR using mesitylene as an internal standard.

^c The ratio was determined by ¹H NMR.

^d Yield and diastereoselectivity of isolated products after purification by column chromatography are given in parentheses.

With bromide 36 in hand, we then investigated the elimination reaction. First, attempted elimination of bromide 36 with bases did not give totally substituted butenolides 37. For example, while attempted elimination of 36 with DBU in CH₂Cl₂ at room temperature resulted in no reaction, the elimination with NaHMDS in THF at –78 to –20 °C led to decomposition. We then investigated silver-mediated elimination (Table [1]). A silver salt was added to a solution of bromide 36 in CH₂Cl₂ in the presence of 3 Å molecular sieves at room temperature. The nature of the silver salt proved to be crucial in this elimination. Addition of AgOAc and AgNO₃ resulted in no reaction (entries 1 and 2). More Lewis acidic silver salts such as AgPF₆ and AgClO₄ promoted the elimination to give a mixture of totally substituted butenolides 37, favoring the (Z)-stereoisomer (entries 3 and 4). AgOTf was evaluated as the best silver salt, giving (Z)- and (E)-37 in 64% combined yield with Z/E = 5.0:1 selectivity (entry 5).[28] Elimination at 40 °C gave a better yield while maintaining the stereoselectivity (entry 6). Thus, we developed a practical approach to the totally substituted butenolides including a coupling reaction with siloxyfuran 34 via the oxocarbenium ion, and subsequent silver-mediated elimination. It is noteworthy that the method begins with the DIBAL-H reduction of the lactone, which would be applicable to stemoamide (12) bearing a less reactive lactam moiety.

The ideal reaction conditions should be stereodivergent to give either the (Z)- or the (E)-isomer of the totally substituted butenolide. Therefore, we surveyed the isomerization of the enediol structure under acidic conditions (Table [2]). Addition of CSA to a solution of (E)-37 in CH₂Cl₂ at 40 °C promoted the isomerization, giving a 1.3:1 mixture of (Z)-37 and (E)-37 in 94% yield after 20 hours (entry 1). Use of more acidic CF₃CO₂H increased the Z/E ratio of the product (entry 2, Z/E = 2.3:1). When using either MsOH or TfOH, the reaction reached equilibrium, providing a 4.9:1 mixture of (Z)-37 and (E)-37 (entries 3 and 4). In contrast, when (Z)-37 was subjected to the same conditions with TfOH, a mixture of two diastereomers, with essentially the same diastereoselectivity as that derived from (E)-37, was produced (entry 5). These results indicated that (Z)-37 is the thermodynamic product. Conditions to give the (E)-isomer as the major product were not found when using model compound 36. However, we successfully developed a AgOTf-mediated elimination and acid-mediated isomerization of the totally substituted butenolides, which would be applicable to the natural product synthesis.

Table 2 Acid-Mediated Isomerization of the Totally Substituted Butenolide^a

Entry	Starting material	Acid	Combined yield (%)b	Z/E c
1	(E)-37	CSA	94	1.3:1
2	(E)-37	CF3CO2H	96	2.3:1
3	(E)-37	MsOH	95	4.9:1
4	(E)-37	TfOH	90	4.9:1
5	(Z)-37	TfOH	96	5.0:1

^a Reaction conditions: (E)-37 or (Z)-37 (1.0 equiv), acid (20 equiv), 3 Å MS (1000 wt%), CH₂Cl₂ (2.7 mM), 40 °C, 20 h.

^b Yields were determined by ¹H NMR using mesitylene as an internal standard.

^c The ratio was determined by ¹H NMR.

With the method to construct the totally substituted butenolides in hand, we turned our attention to the application to stemoamide (12) (Scheme [4]). In contrast to the model compound, we developed a one-pot process for the reductive vinylogous aldol-type reaction to give tetracyclic compound 29 (Table [3]). Addition of DIBAL-H to stemoamide (12) at –78 °C initiated lactone-selective reduction to generate aluminum alkoxide 39. Unfortunately, addition of 2-siloxyfuran­ 34 and BF₃·OEt₂ in a one-pot process did not provide tetracyclic compound after the subsequent aldol-type reaction (entry 1). Methanol was added to consume excess DIBAL-H prior to the vinylogous aldol-type reaction (entry 2). However, the reaction resulted in a complex mixture, although the products were included. Finally, we found that use of benzaldehyde to consume the remaining DIBAL-H enabled the following coupling reaction with 2-siloxyfuran 34 in the presence of BF₃·OEt₂ via the oxocarbenium ion (entry 3). The resulting tetracyclic compounds 29 were obtained in 70% combined yield as a mixture of four diastereomers (dr = 2.2:2.1:1.8:1), which were used without further separation.

Scheme 4 Total syntheses of protostemonamide (14) and isoprotostemonamide (15) through silver-mediated elimination under kinetic conditions

Table 3 Reductive Vinylogous Aldol-Type Reaction of Stemoamide (12)^a

Entry	Additive	Resultsb
1	none	29: 0
2	MeOH	complex mixture
3	PhCHO	29: 70% (dr = 2.2:2.1:1.8:1)c

^a Reaction conditions: 12 (1.0 equiv), DIBAL-H (2.5 to 3.0 equiv), CH₂Cl₂ (0.04 M), –78 °C, 1 h; additive (2 equiv), –78 °C to rt, 40 min; 2-siloxyfuran 34 (6 equiv), BF₃·OEt₂ (4 equiv), rt, 24 h.

^b Yields of isolated products after purification by column chromatography.

^c The ratio was determined by ¹H NMR.

The next challenge was the regioselective installation of bromide as a leaving group to tetracyclic intermediate 29 (Scheme [4]). Regioselective deprotonation of the butenolide was possible with NaHMDS in the presence of the lactam. Subsequent addition of bromine provided bromides 38 as a mixture of four diastereomers in 96% combined yield (38a/38b/38c/38d = 1:2.8:1.1:3.1). The optimized AgOTf-mediated elimination of the diastereomeric mixture of bromides 38 in the presence of 3 Å molecular sieves[29] at 40 °C successfully constructed the totally substituted butenolide, affording protostemonamide (14) and isoprotostemonamide (15) in 85% combined yield with 3.0:1 diastereoselectivity. It is noteworthy that both products are unstable under aerobic atmosphere.[30] For example, a solution of protostemonamide (14) in EtOAc was maintained under an air atmosphere at room temperature for 10 hours, providing a 6.5:1 mixture of 14 and stemoamide (12). Although the mechanism has yet to be clarified, the enediol structure in the totally substituted butenolide could be oxidatively cleaved with molecular oxygen.

The stereochemistries of the totally substituted butenolides were confirmed as follows (Figure [2]). While NOESY experiments (400 MHz, CDCl₃) with synthetic protostemonamide (14) showed correlations of the C18 methoxy group with both the C16 and C17 methyl groups, isoprotostemonamide (15) exhibited correlation of the corresponding C18 methoxy group only with the C16 methyl group. These experiments strongly suggested that our synthetic protostemonamide (14) and isoprotostemonamide (15) have (Z)- and (E)-configuration, respectively. The Huang group reported an empirical rule by comparison of ¹³C NMR spectra to determine the configuration of the totally substituted butenolides.[6f] They found that the resonances for C11 and C12 of (E)-isomers tend to be located downfield to those of (Z)-isomers. That tendency is applicable in our case. In ¹³C NMR spectra (100 MHz, CDCl₃), both the C11 and C12 resonances of isoprotostemonamide (15) were observed downfield to those of protostemonamide (14).

Figure 2 Stereochemical confirmation of the totally substituted butenolides

We found that the stereoselectivity of the AgOTf-mediated elimination depended on the stereochemistry at the C11 stereocenter (Scheme [5]). After separation of the four diastereomers 38a–d by HPLC, each diastereomer was individually subjected to the AgOTf-mediated elimination. Although the stereochemistry at C12 was not determined, the reactions of both 38a and 38b with α-H at C11 provided protostemonamide (14) and isoprotostemonamide (15) with 5.8:1 diastereoselectivity. In contrast, 38c and 38d with β-H at C11 showed the same diastereoselectivity, giving a 2.0:1 mixture of 14 and 15. Although the factors to exhibit the (Z)-selectivity have not been elucidated, these experimental results indicated that the AgOTf-mediated elimination proceeds through E1-type elimination via cationic intermediates 40a and 40b.

Scheme 5 Mechanistic study to support E1-type elimination

Having protostemonamide (14) with (Z)-configuration as the kinetically favored product, we turned our attention to acid-mediated isomerization of the tetrasubstituted olefin (Scheme [6a]). Protostemonamide (14) was exposed to TfOH at 40 °C, providing a 1:3.0 mixture of 14 and isoprotostemonamide (15) in 88% yield. Isomerization of isoprotostemonamide (15) under the identical conditions led to the same results, giving 14 and 15 in 90% yield with 1:3.3 diastereoselectivity. In contrast to model compound 37, acid-mediated isomerization revealed that isoprotostemonamide (15) with the (E)-tetrasubstituted olefin is the thermodynamic product.

The unexpected results inconsistent with the model studies of 37 prompted us to explore the origin of the thermodynamic preference of protostemonamide (14) and isoprotostemonamide (15). We optimized the structures of 14 and 15 at the B3LYP-D3BJ/6-311+G(d,p)-IEFPCM//B3LYP-D3BJ/ 6-31G(d)-IEFPCM level of theory (Scheme [6b]). The Gibbs free energy of 14 is higher than that of 15 by 1.1 kcal/mol, which is consistent with the experimental results that isoprotostemonamide (15) was the main product in the isomerization (Scheme [6a]).

The energy gap between 14 and 15 is attributable to the (Z)-stereochemistry of the C11–C12 olefin. Specifically, there would be a repulsive steric interaction of the methyl group at C10 with the methoxy substituent at C13 on the butenolide in 14, whereas no such interaction is included in (E)-15. The superimposition of the optimized structures 14 and 15 with hypothetical molecules deMe-14 and deMe-15, which have no substituents at C10, clearly demonstrates the steric impact of the methyl group (Scheme [6c]). The superimposed structure of 14 and deMe-14 shows a significant distortion of the hydrofuran ring of 14 as highlighted in blue [root mean square distance (RMSD): 1.04 Å]. In contrast, the geometries of 15 and deMe-15 are comparable (RMSD: 0.26 Å). These RMSD values support the methyl group at C10 as being the cause of the repulsive interaction with the substituent at C13 in 14, resulting in the higher energy of 14 than 15. Accordingly, we conclude that the methyl group at C10 plays a crucial role in determining the thermodynamic stability of 14 and 15. Since the model substrate 37 did not incorporate the methyl group at C10, the stereoselectivity of the reaction using 37 was not reflected in the reactions in Scheme [4].[31]

Scheme 6 (a) Isomerization of protostemonamide (14) and isoprotostemonamide (15). (b) The global minimum structures of 14 and 15 and their relative Gibbs free energy. (c) The superimposed picture of deMe-14 and deMe-15 with 14 and 15, respectively, where the structures of 14 and 15 are colored blue. RMSD means the root mean square distance of heavy atoms except for the Me at C10. (d) Total synthesis of isoprotostemonamide (15) through silver-mediated elimination and isomerization under thermodynamic conditions.

Synthesis of isoprotostemonamide (15) as the major product was investigated from bromides 38 (Scheme [6d]). After AgOTf-mediated elimination of bromides 38, addition of TfOH in a one-pot process initiated the isomerization of protostemonamide (14), giving isoprotostemonamide (15) including the (E)-totally substituted butenolide as the thermodynamically more stable product (14/15 = 1:4.0). Thus, we have developed a stereodivergent method to construct the totally substituted butenolides in three steps from lactones. The reductive aldol-type reaction enabled the direct coupling of the butenolide without pre-activation of the lactone carbonyl group of stemoamide (12). In addition, the stereodivergent conditions gave either protostemonamide (14) or isoprotostemonamide (15) as the major product. While the AgOTf-mediated elimination afforded 14 possessing the (Z)-totally substituted butenolide as the kinetic product, elimination and subsequent TfOH-mediated isomerization gave 15 having the (E)-totally substituted butenolide as the thermodynamic product. Thus, the total syntheses of protostemonamide (14) and isoprotostemonamide (15) were accomplished in 10 steps from commercially available ethyl 4-bromobutanoate.

The next challenge in the unified total synthesis of stemoamide-type alkaloids was the direct oxidation of the pyrrolidine ring to the pyrrole ring[10] often found in stemoamide-type alkaloids (Table [4]). First, we evaluated the direct oxidation of the pyrrolidine ring using tetracyclic stemonine (41).[32] [33] In this transformation, the instability of the pyrrole-type natural products is known to be problematic.[34] For example, when the reaction conditions were not mild enough, didehydrostemonine (42)[35] underwent quick epimerization at the C13 carbon center, probably through elimination of the carboxylate and recyclization, to give 13-epi-didehydrostemonine (43). In addition, silica gel column chromatography caused the epimerization, which prevented the isolation of 42 in pure form. Thus, the development of mild reaction conditions and the appropriate procedure for the purification were essential for this direct oxidation.

Table 4 Optimization of Direct Oxidation of Stemonine (41)

Entry	Conditions	Yield (%)a
		42	43	41
1	Ag2O (12 equiv), acetone, 40 °C	0	0	54
2	DDQ (5 equiv), THF, 70 °C	0	0	0
3	IBX (5 equiv), CH2Cl2, 40 °C	9	9	33
4	MnO2 (10 equiv), THF, 40 °C	13	2	57
5	MnO2 (200 equiv), THF, 40 °C	28	10	3
6	MnO2 (200 equiv), THF, –20 °C	46	0	30
7b	MnO2 (648 equiv), THF, –20 °C	63	0	0

^a Yields were determined by ¹H NMR using mesitylene as an internal standard.

^b Yields of isolated products after purification by gel permeation chromatography.

Oxidation of stemonine (41) with Ag₂O[3d] or DDQ did not provide didehydrostemonine (42) (Table [4], entries 1 and 2). The reaction with IBX provided a 1:1 diastereomeric mixture of 42 and 13-epi-didehydrostemonine (43) in 18% combined yield (entry 3). Use of MnO₂ [3a] [10d] reduced the epimerization during the reaction, although stemonine (41) was recovered in 57% yield (entry 4). Addition of a large amount of MnO₂ enabled consumption of 41 (entry 5). Epimerization was completely suppressed when the reaction temperature was lowered to –20 °C (entry 6). Finally, pure didehydrostemonine (42) was isolated in 63% yield after filtration through a pad of basified silica gel, and subsequent purification by gel permeation chromatography (entry 7). Thus, we achieved the first total synthesis of didehydrostemonine (42) by direct oxidation with MnO₂.

With optimized conditions in hand, we turned our attention to direct oxidation of pyrrolidines in the presence of totally substituted butenolide, which is highly oxygenated and possesses the electron-rich tetrasubstituted olefin, rendering the direct oxidation more challenging (Scheme [7]). First, pentacyclic protostemonine (1) was synthesized through lactam-selective nucleophilic addition (Scheme [7a]).[22] [23] Thus, treatment of a solution of protostemonamide (14) in toluene with the Vaska complex [IrCl(CO)(PPh₃)₂] (1 mol%) and (Me₂HSi)₂O[36] initiated hydrosilylation of the lactam carbonyl group. Subsequent addition of 2-siloxyfuran 44 [37] in the presence of 2-nitrobenzoic acid and MeCN afforded pentacyclic compounds 45 and 46 in 56% combined yield. It is noteworthy that the lactam-selective reaction took place in the presence of the totally substituted butenolide. While the stereochemistry at the C3 carbon center was completely controlled, no stereoselectivity at the C18 carbon center was observed. However, the isomerization of unfavorable diastereomer 45 was possible with DBU. The total synthesis of protostemonine (1) was achieved by both site-selective and stereoselective hydrogenation of 46 with Rh/Al₂O₃ (20 wt%) in EtOH in 93% yield. The stage was set for the crucial oxidation of the pyrrolidine. Gratifyingly, the direct oxidation of protostemonine (1) using excess MnO₂ proceeded without affecting the totally substituted butenolide, affording bisdehydroprotostemonine (18) in 53% yield. The same reaction sequence from isoprotostemonamide (15) including the (E)-totally substituted butenolide resulted in the total syntheses of isoprotostemonine (17) and isobisdehydroprotostemonine (19) (Scheme [7b]). Thus, the total syntheses of bisdehydroprotostemonine (18) and isobisdehydroprotostemonine (19) were achieved in 13 steps from commercially available ethyl 4-bromobutanoate.

Scheme 7 Total syntheses of bisdehydroprotostemonines by direct oxidation of pyrrolidines

iNOS expression is one of the LPS-induced inflammatory responses, resulting in production of NO, which is essential for host innate immune responses with cytotoxic activity against pathogens.[38] However, excessive expression of iNOS causes inflammatory diseases such as septic shock, and it is vital to find anti-inflammatory agents without toxicity. Using a series of synthetic samples of stemoamide-type alkaloids, we investigated the structure–activity relationship on their anti-inflammatory effects through inhibitory activities on LPS-induced iNOS expression in macrophage cell line RAW264.7[4d] [39] (Figure [3]). The anti-inflammatory activities of tricyclic and tetracyclic stemoamide-type alkaloids indicated that protostemonamide (14) and isoprotostemonamide (15) having the totally substituted butenolides were more potent than other tetracyclic compounds (Figure [3a] and 3b). In general, the number of rings was important, and pentacyclic stemoamide-type alkaloids exhibited better biological profiles than tetracyclic derivatives (Figure [3c] and 3d). The pyrrole derivatives including bisdehydroprotostemonine (18) and isobisdehydroprotostemonine (19) exhibited almost complete inhibition of iNOS expression.[40] However, the results were derived from the cytotoxicity against macrophage cell line RAW264.7 because these two compounds showed cytotoxicity against human colon adenocarcinoma HCT-116 (Table [5]). The most potent anti-inflammatory compounds without cytotoxicity were protostemonine (1) and isoprotostemonine (17), which consist of pentacyclic frameworks including a totally substituted butenolide.

Figure 3 Inhibitory effects of stemoamide-type alkaloids on LPS-induced iNOS expression. (a) and (c) RAW264.7 cells were treated with stemoamide-type alkaloids or indomethacin (IMC) at 100 μM in DMF for 1 hour before stimulation with LPS (1 mg/mL). After a 24-hour incubation, cells were collected and western blotting was performed using anti-iNOS and anti-α-tubulin antibodies. (b) and (d) Signal intensities of western blotting were quantified by Image J software.

Table 5 Cytotoxicity of Synthetic Stemoamide-Type Alkaloids Against Human Colon Adenocarcinoma HCT-116^a

Inhibition activity (%)
12	29a	29b	49	50	41	51	52	14	15	12	29a	46	48	1	17	18 b	19 b	53	Nonec	CPTd
NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	82.0	67.6	NA	NA	17.9	32.3	93.0	NA	8.7

^a Antiproliferative effects of tested compounds (100 μM in DMF) against HCT-116 cells in a growth inhibitory assay (MTT assay). Inhibition activity (%) is defined as ((compound-treated cell number)/(DMF-treated cell number)) × 100. NA = The compound did not show inhibition activity (more than 95%).

^b Bisdehydroprotostemonine (18) and isobisdehydroprotostemonine (19) were examined as an epimeric mixture at C18 (β-H/α-H = 2.5:1).

^c DMF without containing any compound was tested as a control experiment.

^d Camptothecin (CPT) in DMF (1 μM) was tested as a control experiment.

In conclusion, we have developed two useful methods to gain access to the important structural motifs widely observed in the Stemona alkaloids. First, a stereodivergent method to provide either (E)- or (Z)-totally substituted butenolide was investigated. While the kinetic conditions in AgOTf-mediated elimination resulted in the formation of the (Z)-totally substituted butenolide, subsequent isomerization of the tetrasubstituted olefin using TfOH produced the (E)-totally substituted butenolide as the thermodynamic major product. Direct oxidation of the pyrrolidines using excess MnO₂ was highly general to afford the pyrrole-type alkaloids even in the presence of the electron-rich totally substituted butenolides. Combination of the two developed methods with our chemoselective assembly of five-membered rings enabled the supply of a series of stemoamide-type alkaloids. A comprehensive survey of the anti-inflammatory activities revealed an important role of the pentacyclic framework including the totally substituted butenolide.

Reactions were performed in oven-dried glassware fitted with rubber septa under an argon atmosphere. Toluene was distilled from CaH₂. All distilled solvents, EtOH, MeCN, and CH₂Cl₂ were dried over activated 3 Å molecular sieves. THF (dehydrated, stabilizer free) was purchased from KANTO CHEMICAL CO., INC. Manganese(IV) oxide (MnO₂, 99.5%) was purchased from Wako Pure Chemical Industries (133-09681). Other commercial reagents were used without further purification. TLC was performed on Merck TLC silica gel 60 F₂₅₄; plates were visualized by exposure to UV light (254 nm) or stained by submersion in ethanolic ninhydrin or ethanolic phosphomolybdic acid solution followed by heating on a hot plate. Flash column chromatography was performed on silica gel (Silica Gel 60 N, 63–210 or 40–50 mesh, KANTO­ CHEMICAL CO., INC.). For basic products, basified silica gel was used. [Preparation: A mixture of silica gel (500 cc), Et₃N (300 mL), and hexane (500 mL) was vigorously stirred for 24 h at rt, and concentrated.] Preparative layer chromatography was performed on Merck PLC silica gel 60 F₂₅₄. ¹H NMR spectra were recorded at 500 MHz with a JEOL ECA-500 spectrometer, 400 MHz with a JEOL ECS-400 spectrometer, or 400 MHz with a JEOL ECZ-400 spectrometer, and ¹³C NMR spectra at 125 MHz with a JEOL ECA-500 spectrometer, 100 MHz with a JEOL ECS-400 spectrometer, or 100 MHz with a JEOL ECZ-400 spectrometer. Chemical shifts are reported in ppm with reference to solvent signals [¹H NMR: CDCl₃ (7.26), ¹³C NMR: CDCl₃ (77.16)]. Signal patterns are indicated as brs, broad peak; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. MPLC was performed on a Yamazen, YFLC AI-580 system. HPLC and GPC were performed on an SSC-3462, 5410 system (Senshu Scientific Co., Ltd.) with a recycle unit (SSC-1322). IR spectra were recorded using a BRUKER ALPHA FT-IR spectrometer. Mass spectra (ESI-TOF) were measured with a Waters, LCT Premier XE spectrometer. Melting points were measured with a Yanaco model MP-S3 apparatus.

(5S)-5-(Methoxymethyl)tetrahydrofuran-2-ol (32)

DIBAL-H (1.0 M in hexane, 2.5 mL, 2.5 mmol) was added to a solution of lactone 31 [26] (256 mg, 1.97 mmol, 94% ee) and CH₂Cl₂ (39 mL) at –78 °C. The resulting solution was maintained for 1 h at –78 °C, and quenched with aqueous saturated (+)-potassium sodium tartrate (40 mL). The mixture was vigorously stirred for 1 h, and extracted with CHCl₃ (3 × 40 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (EtOAc) to give 215 mg of lactols 32 (colorless oil, mixture of two diastereomers, 83%, dr = 1.4:1).

IR (film): 3413, 2930, 1460, 1200, 1125, 1068, 1007 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ (1.4:1 mixture of two diastereomers) = 5.58 (m, 5/12 H), 5.42 (br, 7/12 H), 4.42–4.34 (m, 5/12 H), 4.30–4.22 (m, 7/12 H), 3.80–3.72 (m, 7/12 H), 3.52 (ddd, J = 10.1, 3.2, 1.4 Hz, 7/12 H), 3.45–3.32 (m, 17/12 H), 3.41 (d, J = 1.4 Hz, 21/12 H), 3.37 (d, J = 1.4 Hz, 15/12 H), 3.28–3.14 (br, 5/12 H), 2.10 (dddd, J = 12.4, 7.8, 7.8, 7.8 Hz, 5/12 H), 2.04–1.81 (m, 38/12 H), 1.67–1.56 (m, 5/12 H).

¹³C NMR (100 MHz, CDCl₃): δ (1.4:1 mixture of two diastereomers) = 99.0 (CH), 98.8 (CH), 78.7 (CH), 77.0 (CH), 75.5 (CH₂), 75.1 (CH₂), 59.4 (CH₃), 59.3 (CH₃), 34.6 (CH₂), 32.8 (CH₂), 25.8 (CH₂), 24.6 (CH₂).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₆H₁₂O₃Na⁺: 155.0684; found: 155.0683.

(5S)-5-(Methoxymethyl)tetrahydrofuran-2-yl Acetate (33)

Ac₂O (530 μL, 5.6 mmol, 4 equiv) was added to a solution of lactols 32 (184 mg, 1.39 mmol), Et₃N (2.3 mL, 17 mmol, 12 equiv), and CH₂Cl₂ (20 mL) at room temperature. The solution was warmed to 40 °C, stirred for 17 h at 40 °C, quenched with aqueous saturated NaHCO₃ (20 mL), and extracted with CH₂Cl₂ (3 × 20 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/hexane, 1:5 to 1:4) to give 215 mg of acetals 33 (colorless oil, mixture of two diastereomers, 89%, 33a/33b = 1.4:1). For analytical samples, the two diastereomers were separated by HPLC with a recycle unit (PEGASIL Silica 120-5, 250 × 20 mm, iPrOH/hexane 1:9, 10 mL/min, 3 cycles; 33a: t _R = 35 min, 33b: t _R = 42 min).

Acetal 33a (Less Polar)

[α]_D ²² +81.8 (c 1.0, CHCl₃); 94% ee.

IR (film): 2985, 2929, 1743, 1456, 1376, 1239, 1112, 1004, 953, 861 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 6.33 (dd, J = 5.2, 1.2 Hz, 1 H), 4.38 (dddd, J = 8.1, 8.1, 5.2, 5.2 Hz, 1 H), 3.42 (dd, J = 10.3, 4.3 Hz, 1 H), 3.39 (dd, J = 10.3, 5.2 Hz, 1 H), 3.37 (s, 3 H), 2.19–2.06 (m, 2 H), 2.02 (s, 3 H), 2.00–1.93 (m, 1 H), 1.79–1.73 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.6 (C), 99.4 (CH), 79.0 (CH), 74.6 (CH₂), 59.5 (CH₃), 31.7 (CH₂), 25.2 (CH₂), 21.5 (CH₃).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₈H₁₄O₄Na⁺: 197.0790; found: 197.0789.

Acetal 33b (Polar)

[α]_D ²⁴ –51.6 (c 0.68, CHCl₃); 94% ee.

IR (film): 2984, 2928, 1741, 1459, 1376, 1239, 1099, 1000, 958, 868, 849 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 6.32–6.27 (m, 1 H), 4.34–4.25 (m, 1 H), 3.48 (dd, J = 10.0, 6.6 Hz, 1 H), 3.44 (dd, J = 10.0, 4.6 Hz, 1 H), 3.39 (s, 3 H), 2.09–2.01 (m, 3 H), 2.02 (s, 3 H), 1.83–1.72 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.5 (C), 99.2 (CH), 80.5 (CH), 76.2 (CH₂), 59.4 (CH₃), 32.7 (CH₂), 25.7 (CH₂), 21.6 (CH₃).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₈H₁₄O₄Na⁺: 197.0790; found: 197.0796.

(S)-4-Methoxy-5-(5-(methoxymethyl)dihydrofuran-2(3H)-ylidene)-3-methylfuran-2(5H)-one (37)

BF₃·OEt₂ (130 μL, 1.0 mmol, 1.5 equiv) was added to a solution of acetals 33 (116 mg, 666 μmol, 1.0 equiv), triisopropyl((4-methoxy-3-methylfuran-2-yl)oxy)silane (34)[17s] (568 mg, 2.00 mmol, 3.0 equiv), and CH₂Cl₂ (11 mL) at room temperature. After stirring for 30 min at room temperature, the resulting solution was quenched with aqueous saturated NaHCO₃ (11 mL), and extracted with CH₂Cl₂ (5 × 11 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was filtered through a pad of silica gel (EtOAc/hexane, 1:3.5 to 1:1) to give a mixture of bicyclic compounds 35, which was used in the next reaction without further purification.

NaHMDS (1.0 M in THF, 1.2 mL, 1.2 mmol, 1.2 equiv) was added to a solution of bicyclic compounds 35 (247 mg, 1.02 mmol, 1.0 equiv) and THF (15 mL) at –78 °C. The orange suspension was stirred for 30 min at –78 °C. Bromine (150 μL, 2.9 mmol, 2.8 equiv) was added dropwise to the suspension at –78 °C. After stirring for 15 min at –78 °C, the resulting orange solution was allowed to warm to room temperature. After stirring for 1 h at room temperature, the solution was quenched with aqueous saturated Na₂S₂O₃ (15 mL) and aqueous saturated NH₄Cl (15 mL), and extracted with CH₂Cl₂ (3 × 15 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/hexane, 1:6 to 1:3) to give an inseparable mixture of bromides 36 [135 mg, 63% combined yield over 2 steps, dr = 4.0:3.2:1.4:1.0 (determined by ¹H NMR)].

In a glovebox, 3 Å molecular sieves (204 mg, 1000 wt%) were added to a solution of bromides 36 (20.4 mg, dr = 4.0:3.2:1.4:1.0, 63.5 μmol, 1.0 equiv) and CH₂Cl₂ (3.2 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature. Then, AgOTf (81.6 mg, 318 μmol, 5.0 equiv) was added to the mixture at room temperature. The flask was removed from the glovebox. The mixture was warmed to 40 °C, stirred for 2 h at 40 °C, quenched with aqueous saturated NaHCO₃ (3 mL), and extracted with CHCl₃ (4 × 5 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/hexane, 1:1 to 3:1) to give a mixture of totally substituted butenolides (Z)-37 and (E)-37 [colorless oil, 12.5 mg, 82% combined yield, (Z)-37/(E)-37 = 5.0:1 (determined by ¹H NMR)]. For analytical samples, (Z)-37 and (E)-37 were separated by HPLC with a recycle unit [PEGASIL Silica 120-5, 250 × 10 mm, EtOAc/hexane 1.5:1, 10 mL/min, 6 cycles; (E)-37: t _R = 90.1 min, (Z)-37: t _R = 93.1 min].

Totally Substituted Butenolide (Z)-37

[α]_D ²⁴ –47.0 (c 1.0, CHCl₃); 94% ee.

IR (film): 2931, 2881, 1734, 1695, 1614, 1461, 1400, 1298, 1156, 1120, 1052, 1015 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 4.64–4.56 (m, 1 H), 4.11 (s, 3 H), 3.59 (dd, J = 10.5, 4.1 Hz, 1 H), 3.53 (dd, J = 10.5, 4.6 Hz, 1 H), 3.38 (s, 3 H), 3.01 (ddd, J = 17.4, 9.2, 5.5 Hz, 1 H), 2.84 (ddd, J = 17.4, 8.7, 8.7 Hz, 1 H), 2.23–2.13 (m, 1 H), 2.05 (s, 3 H), 2.08–1.96 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃): δ = 170.5 (C), 164.1 (C), 144.4 (C), 122.7 (C), 95.9 (C), 83.3 (CH), 73.7 (CH₂), 59.7 (CH₃), 59.0 (CH₃), 28.0 (CH₂), 26.4 (CH₂), 9.2 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₂H₁₇O₅ ⁺: 241.1076; found: 241.1078.

Totally Substituted Butenolide (E)-37

[α]_D ²⁵ +15.5 (c 0.68, CHCl₃); 94% ee.

IR (film): 2922, 2851, 1733, 1694, 1612, 1459, 1401, 1196, 1160, 1024, 1008 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 4.73–4.66 (m, 1 H), 4.12 (s, 3 H), 3.60 (dd, J = 10.5, 4.1 Hz, 1 H), 3.53 (dd, J = 10.5, 4.6 Hz, 1 H), 3.40 (s, 3 H), 3.04 (ddd, J = 17.4, 9.2, 5.5 Hz, 1 H), 2.91 (ddd, J = 17.4, 9.2, 8.2 Hz, 1 H), 2.14 (dddd, J = 12.4, 9.2, 7.3, 5.5 Hz, 1 H), 2.02 (s, 3 H), 1.95 (dddd, J = 12.4, 9.2, 8.2, 6.9 Hz, 1 H).

¹³C NMR (100 MHz, CDCl₃): δ = 171.1 (C), 163.3 (C), 148.4 (C), 124.8 (C), 98.2 (C), 85.0 (CH), 73.9 (CH₂), 59.7 (CH₃), 59.6 (CH₃), 29.0 (CH₂), 25.4 (CH₂), 8.9 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₂H₁₇O₅ ⁺: 241.1076; found: 241.1073.

Isomerization of Totally Substituted Butenolide (E)-37

In a glovebox, 3 Å molecular sieves (16 mg, 1000 wt%) were added to a solution of butenolide (E)-37 (1.3 mg, 5.4 μmol, 1.0 equiv) and CH₂Cl₂ (2.0 mL) at room temperature. The mixture was stirred at room temperature for 20 min. The flask was removed from the glovebox. Triflic acid (12 μL, 130 μmol) was added to the mixture at room temperature. The resulting mixture was heated to 40 °C, stirred for 20 h at 40 °C, quenched with aqueous saturated NaHCO₃ (2 mL), and extracted with CHCl₃ (3 × 2 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The yield and ratio were determined by ¹H NMR using mesitylene as an internal standard, to give (Z)-37 and (E)-37 [90% combined yield, (Z)-37/(E)-37 = 4.9:1].

Isomerization of Totally Substituted Butenolide (Z)-37

In a glovebox, 3 Å molecular sieves (16 mg, 1000 wt%) were added to a solution of butenolide (Z)-37 (1.8 mg, 7.5 μmol, 1.0 equiv) and CH₂Cl₂ (2.0 mL) at room temperature. The mixture was stirred at room temperature for 20 min. The flask was removed from the glovebox. Triflic acid (12 μL, 130 μmol) was added to the mixture at room temperature. The resulting mixture was heated to 40 °C, stirred for 20 h at 40 °C, quenched with aqueous saturated NaHCO₃ (2 mL), and extracted with CHCl₃ (3 × 2 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The yield and ratio were determined by ¹H NMR using mesitylene as an internal standard, to give (Z)-37 and (E)-37 [96% combined yield, (Z)-37/(E)-37 = 5.0:1].

11,12-Dihydroprotostemonamides 29a–d

DIBAL-H (1.0 M in hexane, 1.1 mL, 1.1 mmol, 3.0 equiv) was added to a solution of stemoamide[17p] [s] [v] (12) (81.3 mg, 360 μmol, 1.0 equiv) and CH₂Cl₂ (9.1 mL) at –78 °C. The solution was maintained for 1 h at –78 °C. Benzaldehyde (74 μL, 730 μmol, 2.0 equiv) was added to the solution at –78 °C. The resulting solution was allowed to warm to –50 °C, maintained for 40 min at –50 °C, then allowed to warm to room temperature. A solution of triisopropyl((4-methoxy-3-methylfuran-2-yl)oxy)silane (34) (621 mg, 2.3 mmol, 6.4 equiv) and CH₂Cl₂ (4.6 mL) was added to the solution of 39 via cannula at room temperature. Then, BF₃·OEt₂ (190 μL, 1.5 mmol, 4.2 equiv) was added to the solution at room temperature. After stirring for 24 h at room temperature, the resulting mixture was quenched with aqueous saturated NaHCO₃ (15 mL), and extracted with CHCl₃ (5 × 15 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (MeOH/EtOAc, 1:49 to 1:19) to give a mixture of 11,12-dihydroprotostemonamides (29a–d) [85.5 mg, 70% combined yield, 29a/29b/29c/29d = 1.8:2.1:1.0:2.2 (determined by ¹H NMR)]. For analytical samples, the four diastereomers of 29 were separated by HPLC with a recycle unit (PEGASIL Silica 120-5, 250 × 10 mm, MeOH/EtOAc 1:12, 10 mL/min; 29a: 3 cycles, t _R = 44.0 min; 29b: 3 cycles, t _R = 45.5 min; 29c: 3 cycles, t _R = 48.7 min; 29d: 2 cycles, t _R = 37.9 min).

Tetracyclic Compound 29a

White amorphous solid.

[α]_D ²³ –113.6 (c 0.5, CHCl₃).

IR (film): 2933, 2872, 1751, 1667, 1323, 1071, 1043, 756 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.83 (dq, J = 2.9, 1.4 Hz, 1 H), 4.11–4.06 (m, 1 H), 4.10 (s, 3 H), 4.00 (dd, J = 7.7, 2.9 Hz, 1 H), 3.96–3.85 (m, 2 H), 2.60 (dd, J = 13.0, 11.4 Hz, 1 H), 2.41–2.27 (m, 2 H), 2.20–2.14 (m, 1 H), 2.12–2.02 (m, 2 H), 2.01 (d, J = 1.4 Hz, 3 H), 2.00–1.93 (m, 1 H), 1.79–1.68 (m, 2 H), 1.50–1.34 (m, 2 H), 1.02 (d, J = 6.0 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.8 (C), 174.3 (C), 171.0 (C), 99.1 (C), 84.4 (CH), 79.9 (CH), 78.7 (CH), 59.0 (CH₃), 56.2 (CH), 55.7 (CH), 40.6 (CH₂), 36.0 (CH₂), 35.1 (CH), 30.9 (CH₂), 26.0 (CH₂), 22.6 (CH₂), 17.4 (CH₃), 8.7 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₆NO₅ ⁺: 336.1811; found: 336.1805.

Tetracyclic Compound 29b

White solid; mp 240–241 °C.

[α]_D ²³ –11.9 (c 0.5, CHCl₃).

IR (film): 2934, 2872, 1750, 1672, 1342, 1308, 1083, 1005, 756 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.52 (dq, J = 1.2, 1.2 Hz, 1 H), 4.12 (s, 3 H), 4.09–4.04 (m, 1 H), 3.91 (ddd, J = 10.6, 6.3, 6.3 Hz, 1 H), 3.89 (dd, J = 8.6, 1.2 Hz, 1 H), 3.79 (ddd, J = 10.3, 10.3, 2.9 Hz, 1 H), 2.59 (ddd, J = 13.3, 11.9, 1.4 Hz, 1 H), 2.45 (ddq, J = 10.3, 8.6, 6.6 Hz, 1 H), 2.39–2.31 (m, 2 H), 2.16–2.10 (m 1 H), 2.07 (ddd, J = 10.3, 10.3, 6.3 Hz, 1 H), 2.01 (d, J = 1.2 Hz, 3 H), 2.02–1.96 (m, 1 H), 1.87–1.76 (m, 1 H), 1.74–1.68 (m, 1 H), 1.46–1.28 (m, 2 H), 1.13 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 175.2 (C), 174.3 (C), 171.1 (C), 98.4 (C), 82.3 (CH), 80.5 (CH), 77.0 (CH), 59.1 (CH₃), 56.3 (CH), 55.4 (CH), 40.6 (CH₂), 37.6 (CH), 36.2 (CH₂), 30.9 (CH₂), 26.0 (CH₂), 22.7 (CH₂), 16.1 (CH₃), 8.8 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₆NO₅ ⁺: 336.1811; found: 336.1808.

Tetracyclic Compound 29c

White solid; mp 198–199 °C.

[α]_D ²³ –158.7 (c 0.5, CHCl₃).

IR (film): 2934, 2864, 1752, 1668, 1454, 1422, 1341, 1322, 1083, 1040, 756 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.71 (d, J = 5.4 Hz, 1 H), 4.12 (s, 3 H), 4.07 (ddd, J = 14.0, 2.6, 2.6 Hz, 1 H), 3.98 (dd, J = 7.2, 5.4 Hz, 1 H), 3.92 (ddd, J = 10.6, 6.6, 6.6 Hz, 1 H), 3.72 (ddd, J = 10.0, 9.7, 2.3 Hz, 1 H), 2.67 (dd, J = 14.0, 12.9 Hz, 1 H), 2.46 (ddq, J = 9.5, 7.2, 7.2 Hz, 1 H), 2.41–2.32 (m, 2 H), 2.24 (ddd, J = 9.7, 9.5, 6.6 Hz, 1 H), 2.21–2.15 (m, 1 H), 2.03–1.94 (m, 1 H), 1.98 (s, 3 H), 1.80–1.72 (m, 1 H), 1.71–1.40 (m, 3 H), 1.09 (d, J = 7.2 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.7 (C), 174.3 (C), 172.9 (C), 99.0 (C), 81.4 (CH), 80.0 (CH), 78.4 (CH), 59.3 (CH₃), 56.5 (CH), 53.7 (CH), 40.4 (CH₂), 38.8 (CH), 35.3 (CH₂), 31.0 (CH₂), 26.0 (CH₂), 22.2 (CH₂), 14.1 (CH₃), 8.6 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₆NO₅ ⁺: 336.1811; found: 336.1808.

Tetracyclic Compound 29d

White solid; mp 185–186 °C.

[α]_D ²³ –104.0 (c 0.5, CHCl₃).

IR (film): 2935, 2866, 1753, 1671, 1454, 1344, 1324, 1083, 755 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.49 (brs, 1 H), 4.30 (d, J = 7.7 Hz, 1 H), 4.13 (s, 3 H), 4.05–3.99 (m, 1 H), 3.91 (ddd, J = 10.6, 6.0, 6.0 Hz, 1 H), 3.74 (ddd, J = 9.7, 9.7, 2.6 Hz, 1 H), 2.68 (dd, J = 13.8, 10.9 Hz, 1 H), 2.58–2.43 (m, 2 H), 2.40–2.33 (m, 2 H), 2.02 (d, J = 0.9 Hz, 3 H), 1.99–1.91 (m, 2 H), 1.76–1.59 (m, 2 H), 1.45–1.34 (m, 2 H), 1.18 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 175.4 (C), 174.1 (C), 171.4 (C), 98.5 (C), 80.4 (CH), 78.9 (CH), 77.5 (CH), 59.2 (CH₃), 56.3 (CH), 51.4 (CH), 40.5 (CH₂), 38.1 (CH), 35.1 (CH₂), 31.1 (CH₂), 25.9 (CH₂), 22.1 (CH₂), 13.0 (CH₃), 8.7 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₆NO₅ ⁺: 336.1811; found: 336.1812.

Bromides 38

NaHMDS (1.0 M in THF, 480 μL, 480 μmol, 1.2 equiv) was added to a solution of tetracyclic compounds 29 (29a/29b/29c/29d = 1.8:2.1:1.0:2.2, 131 mg, 390 μmol, 1.0 equiv) and THF (5.8 mL) at –78 °C. The orange suspension was stirred for 30 min at –78 °C. Bromine (36 μL, 700 μmol, 1.8 equiv) was added dropwise to the suspension at –78 °C. After stirring for 15 min at –78 °C, the resulting orange solution was allowed to warm to room temperature. After stirring for 1 h at room temperature, the solution was quenched with aqueous saturated Na₂S₂O₃ (5 mL) and aqueous saturated NH₄Cl (5 mL), and extracted with CHCl₃ (5 × 15 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography (MeOH/EtOAc, 1:49 to 1:12) to give a mixture of bromides 38 [155 mg, 96% combined yield, 38a/38b/38c/38d = 1:2.8:1.1:3.1 (determined by ¹H NMR)]. For analytical samples, the four diastereomers of 38 were separated by HPLC with a recycle unit (PEGASIL Silica 120-5, 250 × 10 mm, MeOH/EtOAc 1:19, 10 mL/min, 2 cycles; 38d: t _R = 50.7 min, 38b: t _R = 52.9 min, 38c: t _R = 54.9 min, 38a: t _R = 58.6 min).

Bromide 38a

White solid; mp 191–192 °C.

[α]_D ²⁶ –5.7 (c 0.5, CHCl₃).

IR (film): 2938, 2870, 1776, 1667, 1456, 1331, 1031, 1012, 733 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.36 (d, J = 7.2 Hz, 1 H), 4.23 (s, 3 H), 4.06 (ddd, J = 14.0, 3.4, 3.4 Hz, 1 H), 3.90–3.79 (m, 2 H), 2.69 (ddd, J = 14.0, 12.5, 1.4 Hz, 1 H), 2.53–2.43 (m, 2 H), 2.39–2.33 (m, 2 H), 2.30–2.23 (m, 1 H), 2.04 (s, 3 H), 1.92–1.74 (m, 3 H), 1.68–1.58 (m, 1 H), 1.52–1.41 (m, 1 H), 0.97 (d, J = 6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.1 (C), 173.3 (C), 170.6 (C), 98.1 (C), 94.1 (C), 83.2 (CH), 80.0 (CH), 59.9 (CH₃), 56.2 (CH), 51.9 (CH), 40.5 (CH₂), 39.8 (CH), 35.1 (CH₂), 30.9 (CH₂), 26.0 (CH₂), 21.6 (CH₂), 12.3 (CH₃), 8.6 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₅NO₅Br⁺: 414.0916; found: 414.0909.

Bromide 38b

White solid; mp 181–182 °C.

[α]_D ²⁷ –98.4 (c 0.5, CHCl₃).

IR (film): 2937, 2866, 1777, 1671, 1456, 1327, 1044, 1021, 970, 822, 751 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.47 (d, J = 7.7 Hz, 1 H), 4.20 (s, 3 H), 4.02 (ddd, J = 12.9, 3.2, 3.2 Hz, 1 H), 3.92 (ddd, J = 10.6, 6.3, 6.3 Hz, 1 H), 3.66 (ddd, J = 10.3, 9.9, 2.9 Hz, 1 H), 2.64 (dd, J = 12.9, 12.9 Hz, 1 H), 2.61 (dqd, J = 12.0, 7.7, 7.2 Hz, 1 H), 2.48 (ddd, J = 12.0, 9.9, 6.3 Hz, 1 H), 2.40–2.32 (m, 2 H), 2.03 (s, 3 H), 2.00–1.92 (m, 2 H), 1.72–1.66 (m, 2 H), 1.47 (d, J = 7.2 Hz, 3 H), 1.43–1.31 (m, 1 H), 1.12 (dddd, J = 12.9, 12.9, 10.3, 4.9 Hz, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.1 (C), 173.8 (C), 171.9 (C), 97.5 (C), 90.5 (C), 82.9 (CH), 80.0 (CH), 59.9 (CH₃), 56.2 (CH), 51.9 (CH), 40.4 (CH₂), 39.0 (CH), 35.4 (CH₂), 31.0 (CH₂), 25.8 (CH₂), 21.8 (CH₂), 14.4 (CH₃), 8.4 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₅NO₅Br⁺: 414.0916; found: 414.0910.

Bromide 38c

White solid; mp 171–172 °C.

[α]_D ²⁷ –69.6 (c 0.5, CHCl₃).

IR (film): 2937, 2872, 1774, 1667, 1453, 1422, 1333, 1030, 1015, 750 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.22 (s, 3 H), 4.12–4.07 (m, 1 H), 4.04 (ddd, J = 10.3, 10.0, 2.9 Hz, 1 H), 3.91 (d, J = 7.5 Hz, 1 H), 3.90 (ddd, J = 11.2, 6.6, 6.0 Hz, 1 H), 2.60 (dd, J = 13.5, 12.0 Hz, 1 H), 2.42–2.32 (m, 2 H), 2.31–2.22 (m, 2 H), 2.13 (ddd, J = 10.3, 10.3, 6.6 Hz, 1 H), 2.06 (s, 3 H), 2.04–1.94 (m, 1 H), 1.82–1.72 (m, 2 H), 1.50–1.35 (m, 2 H), 1.15 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.3 (C), 172.4 (C), 170.4 (C), 98.6 (C), 93.0 (C), 88.3 (CH), 80.7 (CH), 59.9 (CH₃), 56.2 (CH), 55.9 (CH), 40.6 (CH₂), 38.5 (CH), 35.9 (CH₂), 30.9 (CH₂), 26.0 (CH₂), 22.8 (CH₂), 18.3 (CH₃), 8.8 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₅NO₅Br⁺: 414.0916; found: 414.0920.

Bromide 38d

White solid; mp 168–169 °C.

[α]_D ²⁵ +49.1 (c 0.5, CHCl₃).

IR (film): 2939, 2872, 1783, 1671, 1453, 1422, 1333, 1027, 966, 910, 733 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.20 (s, 3 H), 4.12 (d, J = 6.3 Hz, 1 H), 4.07 (ddd, J = 13.2, 3.2, 3.2 Hz, 1 H), 3.92 (ddd, J = 10.6, 6.3, 6.3 Hz, 1 H), 3.80 (ddd, J = 10.3, 10.3, 2.9 Hz, 1 H), 2.76 (dqd, J = 10.6, 6.6, 6.3 Hz, 1 H), 2.58 (dd, J = 13.2, 13.2 Hz, 1 H), 2.44–2.29 (m, 2 H), 2.16–2.05 (m, 3 H), 2.04 (s, 3 H), 1.99–1.88 (m, 1 H), 1.71–1.56 (m, 1 H), 1.47–1.33 (m, 1 H), 1.36 (d, J = 6.6 Hz, 3 H), 1.32–1.21 (m, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.5 (C), 171.9 (C), 171.0 (C), 98.3 (C), 91.8 (C), 86.0 (CH), 81.7 (CH), 59.8 (CH₃), 56.7 (CH), 56.4 (CH), 40.5 (CH₂), 38.5 (CH), 35.8 (CH₂), 30.9 (CH₂), 25.8 (CH₂), 23.0 (CH₂), 21.1 (CH₃), 8.7 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₅NO₅Br⁺: 414.0916; found: 414.0915.

Protostemonamide (14) and Isoprotostemonamide (15)[4a]

In a glovebox, 3 Å molecular sieves (320 mg, 1000 wt%) were added to a solution of tetracyclic compounds 38 (31.6 mg, 38a/38b/38c/38d = 1:2.8:1.1:3.1, 76.3 μmol, 1.0 equiv) and CH₂Cl₂ (3.8 mL) at room temperature. The resulting mixture was stirred for 17 h at room temperature. Then, AgOTf (98.0 mg, 381 μmol, 5.0 equiv) was added to the mixture at room temperature. The flask was removed from the glovebox. The mixture was warmed to 40 °C, stirred for 1 h at 40 °C, quenched with aqueous saturated NaHCO₃ (5 mL), and extracted with CHCl₃ (5 × 6 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was filtered through a pad of silica gel (MeOH/EtOAc, 1:4), and purified by preparative layer chromatography (MeOH/EtOAc, 1:9) to give 16.2 mg of pure protostemonamide (14, 64%), and impure isoprotostemonamide (15) which was purified by HPLC with a recycle unit (PEGASIL Silica 120-5, 250 × 10 mm, MeOH/EtOAc 1:9, 10 mL/min, 10 cycles; 15: t _R = 117.5 min) to afford 5.4 mg of pure 15 (21%).

Protostemonamide (14)

White solid; mp 138–139 °C (Lit.[4a] 140–142 °C).

[α]_D ²⁷ +111.3 (c 0.6, CHCl₃) [Lit.[4a] [α]_D ²⁵ +240 (c 0.6, CHCl₃)]; 98% ee.

IR (film): 2924, 2852, 1736, 1677, 1599, 1459, 1417, 1218, 1066, 1014, 822, 732 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 4.15 (s, 3 H), 4.15–4.00 (m, 2 H), 4.03 (ddd, J = 10.5, 6.4, 6.4 Hz, 1 H), 3.11 (dq, J = 8.5, 6.6 Hz, 1 H), 2.67 (dd, J = 14.0, 11.5 Hz, 1 H), 2.46 (m, 1 H), 2.42–2.36 (m, 2 H), 2.34 (ddd, J = 9.9, 8.5, 6.4 Hz, 1 H), 2.13 (m, 1 H), 2.08 (s, 3 H), 1.83 (m, 1 H), 1.74–1.46 (m, 3 H), 1.40 (d, J = 6.6 Hz, 3 H).

¹³C NMR (100 MHz, CDCl₃): δ = 174.3 (C), 170.1 (C), 163.0 (C), 148.2 (C), 124.9 (C), 97.5 (C), 83.6 (CH), 59.0 (CH₃), 55.8 (CH), 55.6 (CH), 40.4 (CH₂), 37.5 (CH), 34.6 (CH₂), 30.8 (CH₂), 25.6 (CH₂), 23.1 (CH₂), 21.1 (CH₃), 9.4 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₄NO₅ ⁺: 334.1654; found: 334.1647.

Isoprotostemonamide (15)

White solid; mp 129–130 °C.

[α]_D ²³ –66.3 (c 0.6, CHCl₃).

IR (film): 2938, 2878, 1733, 1677, 1610, 1197, 1165, 1145, 996, 754 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 4.23–4.07 (m, 2 H), 4.12 (s, 3 H), 4.02 (ddd, J = 10.5, 6.6, 6.6 Hz, 1 H), 3.22 (dq, J = 10.3, 6.6 Hz, 1 H), 2.67 (dd, J = 14.2, 11.9 Hz, 1 H), 2.46–2.36 (m, 3 H), 2.27 (ddd, J = 10.3, 10.3, 6.6 Hz, 1 H), 2.09 (m, 1 H), 2.04 (s, 3 H), 1.84 (m, 1 H), 1.74–1.52 (m, 3 H), 1.48 (d, J = 6.6 Hz, 3 H).

¹³C NMR (100 MHz, CDCl₃): δ = 174.3 (C), 170.7 (C), 163.7 (C), 150.2 (C), 126.1 (C), 98.1 (C), 84.3 (CH), 59.6 (CH₃), 55.6 (CH), 54.2 (CH), 40.4 (CH₂), 39.8 (CH), 35.0 (CH₂), 30.8 (CH₂), 25.6 (CH₂), 23.0 (CH₂), 17.9 (CH₃), 8.9 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₄NO₅ ⁺: 334.1654; found: 334.1653.

Protostemonamide (14) and Isoprotostemonamide (15)[4a]

In a glovebox, 3 Å molecular sieves (390 mg) were added to a solution of tetracyclic compounds 38 (39.7 mg, 38a/38b/38c/38d = 1:2.8:1.1:3.1, 95.8 μmol, 1.0 equiv) and CH₂Cl₂ (4.8 mL) at room temperature. The resulting mixture was stirred for 20 h at room temperature. Then, AgOTf (123 mg, 479 μmol, 5.0 equiv) was added to the mixture at room temperature. The flask was removed from the glovebox. The mixture was warmed to 40 °C, and stirred for 2.5 h at 40 °C. Triflic acid (130 μL, 1.4 mmol, 15 equiv) was added dropwise to the mixture at 40 °C, which was stirred for 20 h at 40 °C, quenched with aqueous saturated NaHCO₃ (7 mL), and extracted with CHCl₃ (5 × 7 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was filtered through a pad of silica gel (MeOH/EtOAc, 1:4), and purified by preparative layer chromatography (MeOH/EtOAc, 1:9) to give 20.4 mg of isoprotostemonamide (15, 64%) and 5.2 mg of protostemonamide (14, 16%).

Oxidative Cleavage of the Enediol Structure under Aerobic Conditions

A solution of protostemonamide (14) (5.3 mg, 15.9 μmol) in EtOAc (1.2 mL) was stirred for 10 h at room temperature without a septum. The solution was concentrated to give a mixture of 14 and stemoamide (12) (14/12 = 6.5:1), which was confirmed by ¹H NMR.[17p] [s] [v]

A solution of protostemonamide (14) (5.0 mg, 15.0 μmol) in CHCl₃ (2.5 mL) was stirred for 12 h at room temperature without a septum. The solution was concentrated to give a mixture of 14 and stemoamide (12) (14/12 = 2.0:1), which was confirmed by ¹H NMR.[17p] [s] [v]

Didehydrostemonine (42)[35]

MnO₂ (1.10 g, 12.7 mmol, 648 equiv) was added to a solution of stemonine (41) (6.0 mg, 19.5 μmol) and THF (5.0 mL) at –20 °C. After stirring for 2 h at –20 °C, the resulting mixture was filtered through a pad of basified silica gel (10 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was purified by gel permeation chromatography with a recycle unit (Shodex GPC H-2002, 500 × 20 mm, 254 nm, CHCl₃, 3.6 mL/min, 8 cycles; 42: t _R = 185.4 min) to give 3.7 mg of 42 (63%).

White amorphous solid.

[α]_D ²⁶ –9.2 (c 0.4, CHCl₃); 98% ee.

IR (film): 2936, 1767, 1200, 1165, 991, 755 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 6.15 (d, J = 3.7 Hz, 1 H), 5.94 (d, J = 3.7 Hz, 1 H), 5.38 (dd, J = 11.0, 5.0 Hz, 1 H), 4.36 (dd, J = 14.6, 6.0 Hz, 1 H), 3.89 (ddd, J = 10.1, 9.6, 3.7 Hz, 1 H), 3.73 (dd, J = 14.6, 10.5 Hz, 1 H), 3.06 (dd, J = 12.4, 9.6 Hz, 1 H), 2.97 (dq, J = 12.4, 6.9 Hz, 1 H), 2.81 (m, 1 H), 2.72 (ddd, J = 11.9, 8.2, 5.0 Hz, 1 H), 2.55 (m, 1 H), 2.21 (ddd, J = 11.9, 11.4, 11.0 Hz, 1 H), 2.16 (m, 1 H), 1.85–1.66 (m, 2 H), 1.42 (d, J = 6.9 Hz, 3 H), 1.36 (d, J = 6.9 Hz, 3 H).

¹³C NMR (100 MHz, CDCl₃): δ = 179.0 (C), 178.1 (C), 132.3 (C), 129.5 (C), 107.3 (CH), 103.9 (CH), 81.3 (CH), 71.5 (CH), 49.3 (CH), 45.5 (CH₂), 39.5 (CH), 36.1 (CH), 34.9 (CH₂), 34.5 (CH₂), 25.9 (CH₂), 15.2 (CH₃), 13.9 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₇H₂₂NO₄ ⁺: 304.1549; found: 304.1553.

19,20-Dehydroprotostemonine (46) and 18-epi-19,20-Dehydroprotostemonine (45)

Preparation of a stock solution of IrCl(CO)(PPh₃)₂ in toluene (0.27 mM): In a glovebox, toluene (8.0 mL) was added to IrCl(CO)(PPh₃)₂ (1.6 mg, 2.1 μmol) at room temperature.

In a glovebox, 1,1,3,3-tetramethyldisiloxane (21 μL, 120 μmol, 1.4 equiv) was added to a mixture of protostemonamide (14) (29.0 mg, 87.0 μmol, 1.0 equiv), IrCl(CO)(PPh₃)₂ (0.27 mM in toluene, 3.4 mL, 0.87 μmol, 1.0 mol%), and toluene (7.5 mL) at room temperature. After the solution was maintained for 1 h at room temperature, MeCN (29 mL) was added. The flask was removed from the glovebox. Triisopropyl((3-methylfuran-2-yl)oxy)silane (44)[37] (78 μL, 260 μmol, 3.0 equiv) and 2-nitrobenzoic acid (80.8 mg, 435 μmol, 5.0 equiv) were added to the solution at room temperature. After being maintained for 40 h at room temperature, the solution was filtered through a pad of activated alumina (10 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was filtered through a pad of basified silica gel (EtOAc/Et₃N, 1:0.01), and concentrated. The mixture of pentacyclic products 46 and 45 was purified by preparative layer chromatography (EtOAc/Et₃N, 1:0.01) to give 13.5 mg of 46 (37%) and 6.7 mg of 45 (19%).

Pentacyclic Compound 46

White solid; mp 171–172 °C.

[α]_D ²³ +70.7 (c 0.8, MeOH).

IR (film): 3011, 2935, 2871, 1741, 1677, 1617, 1459, 1215, 1092, 1062, 1015, 754 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 6.98 (dq, J = 1.7, 1.7 Hz, 1 H), 4.80 (ddq, J = 5.7, 1.7, 1.7 Hz, 1 H), 4.13 (s, 3 H), 4.05 (ddd, J = 10.3, 10.3, 3.4 Hz, 1 H), 3.71–3.64 (m, 1 H), 3.38–3.27 (m, 2 H), 3.00–2.86 (m, 2 H), 2.40–2.34 (m, 1 H), 2.19 (ddd, J = 10.3, 8.9, 5.2 Hz, 1 H), 2.07 (s, 3 H), 1.98–1.84 (m, 2 H), 1.93 (dd, J = 1.7, 1.7 Hz, 3 H), 1.61–1.45 (m, 5 H), 1.34 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.2 (C), 170.2 (C), 163.3 (C), 149.2 (C), 146.6 (CH), 131.3 (C), 124.7 (C), 97.1 (C), 85.2 (CH), 84.4 (CH), 63.2 (CH), 59.0 (CH₃), 58.3 (CH), 56.0 (CH), 46.6 (CH₂), 39.7 (CH), 34.0 (CH₂), 27.8 (CH₂), 26.9 (CH₂), 21.0 (CH₃), 20.9 (CH₂), 10.9 (CH₃), 9.3 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₀NO₆ ⁺: 416.2073; found: 416.2078.

Pentacyclic Compound 45

White solid; mp 177–178 °C.

[α]_D ²³ +239.2 (c 0.54, MeOH).

IR (film): 3014, 2933, 2871, 1739, 1677, 1617, 1459, 1215, 1064, 1016, 754 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 7.01 (dq, J = 1.7, 1.7 Hz, 1 H), 4.93 (ddq, J = 4.0, 1.7, 1.7 Hz, 1 H), 4.13 (s, 3 H), 4.15–4.06 (m, 1 H), 3.64 (ddd, J = 8.6, 5.7, 5.7 Hz, 1 H), 3.42 (ddd, J = 6.9, 6.9, 4.0 Hz, 1 H), 3.09 (dd, J = 15.8, 3.2 Hz, 1 H), 2.99–2.88 (m, 2 H), 2.44–2.31 (m, 1 H), 2.16 (ddd, J = 10.0, 9.2, 5.7 Hz, 1 H), 2.06 (s, 3 H), 1.93 (dd, J = 1.7, 1.7 Hz, 3 H), 1.87–1.83 (m, 1 H), 1.77–1.70 (m, 1 H), 1.56–1.40 (m, 5 H), 1.33 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.5 (C), 170.2 (C), 163.3 (C), 149.3 (C) 147.0 (CH), 131.0 (C), 124.6 (C), 97.0 (C), 84.5 (CH), 82.0 (CH), 63.0 (CH), 59.0 (CH₃), 57.7 (CH), 55.8 (CH), 46.0 (CH₂), 39.9 (CH), 34.1 (CH₂), 27.4 (CH₂), 24.8 (CH₂), 22.4 (CH₂), 20.9 (CH₃), 11.0 (CH₃), 9.3 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₀NO₆ ⁺: 416.2073; found: 416.2071.

Isomerization of 18-epi-19,20-Dehydroprotostemonine (45)

1,8-Diazabicyclo[5.4.0]undec-7-ene (50 μL, 360 μmol, 14 equiv) was added to a solution of pentacyclic compound 45 (9.2 mg, 22.1 μmol, 1.0 equiv) and CH₂Cl₂ (3.0 mL) at room temperature. After being maintained for 6 h at room temperature, the solution was filtered through a pad of basified silica gel (EtOAc/Et₃N, 1:0.01), and concentrated. The residue was purified by preparative layer chromatography (EtOAc/Et₃N, 1:0.01) to give 4.2 mg of 46 (46%) and 3.5 mg of 45 (38%).

19,20-Isodehydroprotostemonine (48) and 18-epi-19,20-Isodehydroprotostemonine (47)

Preparation of a stock solution of IrCl(CO)(PPh₃)₂ in toluene (0.27 mM): In a glovebox, toluene (8.0 mL) was added to IrCl(CO)(PPh₃)₂ (1.6 mg, 2.1 μmol) at room temperature.

In a glovebox, 1,1,3,3-tetramethyldisiloxane (12.0 μL, 67 μmol, 1.3 equiv) was added to a mixture of isoprotostemonamide (15) (17.1 mg, 51 μmol, 1.0 equiv), IrCl(CO)(PPh₃)₂ (0.27 mM in toluene, 1.0 mL, 0.26 μmol, 0.5 mol%), and toluene (5.4 mL) at room temperature. After stirring for 1 h at room temperature, MeCN (17 mL) was added to the solution. The flask was removed from the glovebox. Triisopropyl((3-methylfuran-2-yl)oxy)silane (44)[37] (150 μL, 510 μmol, 10 equiv) and 2-nitrobenzoic acid (47.6 mg, 256 μmol, 5.0 equiv) were added to the solution at room temperature. After being maintained for 40 h at room temperature, the solution was filtered through a pad of activated alumina (10 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was filtered through a pad of basified silica gel (EtOAc­/Et₃N, 1:0.01), and concentrated. The mixture of pentacyclic products 48 and 47 was purified by preparative layer chromatography (EtOAc/Et₃N, 1:0.01) to give 7.0 mg of 48 (33%) and 6.1 mg of 47 (29%).

Pentacyclic Compound 48

White solid; mp 179–180 °C.

[α]_D ²² –67.7 (c 0.8, MeOH).

IR (film): 3002, 2935, 2875, 1737, 1680, 1611, 1459, 1400, 1161, 1065, 1000, 771, 754 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 6.98 (dq, J = 1.7, 1.7 Hz, 1 H), 4.80 (ddq, J = 6.0, 1.7, 1.7 Hz, 1 H), 4.18 (ddd, J = 10.6, 10.6, 3.4 Hz, 1 H), 4.11 (s, 3 H), 3.66 (ddd, J = 9.0, 6.0, 5.4 Hz, 1 H), 3.40 (dd, J = 15.8, 4.3 Hz, 1 H), 3.29 (ddd, J = 8.0, 7.2, 6.0 Hz, 1 H), 3.02 (dq, J = 10.6, 6.6 Hz, 1 H), 2.94 (dd, J = 15.8, 10.6 Hz, 1 H), 2.38–2.31 (m, 1 H), 2.13 (ddd, J = 10.6, 10.6, 5.4 Hz, 1 H), 2.03 (s, 3 H), 1.94 (dd, J = 1.7, 1.7 Hz, 3 H), 1.92–1.84 (m, 2 H), 1.56–1.46 (m, 5 H), 1.43 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.2 (C), 170.9 (C), 163.8 (C), 151.3 (C), 146.6 (CH), 131.4 (C), 125.7 (C), 97.8 (C), 85.2 (CH), 85.0 (CH), 63.2 (CH), 59.6 (CH₃), 57.9 (CH), 54.3 (CH), 46.6 (CH₂), 41.9 (CH), 34.4 (CH₂), 27.5 (CH₂), 26.8 (CH₂), 20.9 (CH₂), 17.7 (CH₃), 10.9 (CH₃), 8.9 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₀NO₆ ⁺: 416.2073; found: 416.2077.

Pentacyclic Compound 47

White solid; mp 185–186 °C.

[α]_D ²³ +70.2 (c 0.5, MeOH).

IR (film): 3008, 2934, 2871, 1737, 1680, 1611, 1459, 1400, 1194, 1161, 1069, 1001, 771, 754 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 7.02 (dq, J = 1.7, 1.7 Hz, 1 H), 4.92 (ddq, J = 4.0, 1.7, 1.7 Hz, 1 H), 4.21 (ddd, J = 10.3, 10.3, 4.0 Hz, 1 H), 4.11 (s, 3 H), 3.61 (ddd, J = 8.9, 5.4, 5.4 Hz, 1 H), 3.43 (ddd, J = 6.9, 6.9, 4.0 Hz, 1 H), 3.12–2.99 (m, 1 H), 3.04 (dq, J = 10.3, 6.6 Hz, 1 H), 2.97–2.88 (m, 1 H), 2.41–2.29 (m, 1 H), 2.08 (ddd, J = 10.3, 10.3, 5.4 Hz, 1 H), 2.03 (s, 3 H), 1.94 (dd, J = 1.7, 1.7 Hz, 3 H), 1.88–1.74 (m, 2 H), 1.57–1.45 (m, 5 H), 1.43 (d, J = 6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 174.4 (C), 170.9 (C), 163.7 (C), 151.4 (C), 146.9 (CH), 131.1 (C), 125.7 (C), 97.8 (C), 85.1 (CH), 82.5 (CH), 63.1 (CH), 59.7 (CH₃), 57.6 (CH), 54.1 (CH), 46.5 (CH₂), 42.1 (CH), 34.5 (CH₂), 27.3 (CH₂), 25.3 (CH₂), 22.5 (CH₂), 17.7 (CH₃), 11.0 (CH₃), 8.9 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₀NO₆ ⁺: 416.2073; found: 416.2062.

Isomerization of 18-epi-19,20-Isodehydroprotostemonine (47)

1,8-Diazabicyclo[5.4.0]undec-7-ene (42 μL, 280 μmol, 14 equiv) was added to a solution of pentacyclic compound 47 (8.4 mg, 20.2 μmol, 1.0 equiv) and CH₂Cl₂ (3.5 mL) at room temperature. After being maintained for 6 h at room temperature, the solution was filtered through a pad of basified silica gel (EtOAc/Et₃N, 1:0.01), and concentrated. The residue was purified by preparative layer chromatography (EtOAc/Et₃N, 1:0.01) to give 4.2 mg of 48 (50%) and 3.2 mg of 47 (38%).

Protostemonine (1)[3]

Rhodium on alumina (1.5 mg, 20 wt%) was added to a solution of pentacyclic compound 46 (7.0 mg, 17.0 μmol) and EtOH (1.0 mL). The flask was purged with hydrogen. The mixture was stirred under hydrogen atmosphere (1 atm) at room temperature for 1.5 h. The resulting mixture was filtered through a pad of Celite (0.2 cc) and basified silica gel (1 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was purified by preparative layer chromatography (EtOAc­/Et₃N, 1:0.01) to give 6.5 mg of 1 (93%).

White solid; mp 177–178 °C (Lit.[3b] 172–173 °C; Lit.[3d] 170–172 °C).

[α]_D ²² +103.3 (c 0.47, EtOH) (the optical rotation of the natural sample was not reported); 98% ee.

IR (film): 2932, 2871, 1770, 1737, 1667, 1618, 1459, 1155, 1062, 1015 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.15 (m, 1 H), 4.13 (s, 3 H), 4.08 (ddd, J = 10.3, 9.5, 3.4 Hz, 1 H), 3.74 (ddd, J = 9.5, 5.4, 5.4 Hz, 1 H), 3.49 (dd, J = 16.0, 4.3 Hz, 1 H), 3.28 (ddd, J = 8.6, 7.2, 6.0 Hz, 1 H), 2.93 (m, 1 H), 2.91 (m, 1 H), 2.60 (m, 1 H), 2.37 (m, 1 H), 2.35 (ddd, J = 12.6, 8.3, 5.5 Hz, 1 H), 2.21 (ddd, J = 9.5, 9.5, 5.4 Hz, 1 H), 2.07 (s, 3 H), 1.94 (m, 1 H), 1.85 (m, 1 H), 1.66 (m, 1 H), 1.59–1.37 (m, 5 H), 1.34 (d, J = 6.6 Hz, 3 H), 1.26 (d, J = 7.2 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 179.6 (C), 170.2 (C), 163.3 (C), 149.3 (C), 124.6 (C), 97.1 (C), 84.5 (CH), 83.6 (CH), 64.2 (CH), 59.0 (CH₃), 58.7 (CH), 56.1 (CH), 46.6 (CH₂), 39.6 (CH), 35.1 (CH), 34.4 (CH₂), 34.0 (CH₂), 27.7 (CH₂), 26.9 (CH₂), 21.0 (CH₂), 20.2 (CH₃), 15.1 (CH₃), 9.3 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₂NO₆ ⁺: 418.2230; found: 418.2231.

Isoprotostemonine (17)[3]

Rhodium on alumina (1.0 mg, 20 wt%) was added to a solution of pentacyclic compound 48 (5.1 mg, 12.0 μmol) and EtOH (2.0 mL). The flask was purged with hydrogen. The mixture was stirred under hydrogen atmosphere (1 atm) at room temperature for 1.5 h. The resulting mixture was filtered through a pad of Celite (0.2 cc) and basified silica gel (1 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was purified by preparative layer chromatography (EtOAc­/Et₃N, 1:0.01) to give 5.0 mg of 17 (98%).

White solid; mp 171–172 °C (Lit.[3c] [d] 165–167 °C).

[α]_D ²⁵ –12.5 (c 0.47, EtOH) [Lit.[3c] [d] [α]_D ²⁰ –23.6 (c 0.47, EtOH)]; 98% ee.

IR (film): 2935, 2874, 1770, 1736, 1678, 1612, 1459, 1400, 1193, 1159, 1075, 1002, 732 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 4.20 (ddd, J = 10.6, 10.6, 3.4 Hz, 1 H), 4.15 (m, 1 H), 4.11 (s, 3 H), 3.72 (ddd, J = 10.3, 5.4, 5.4 Hz, 1 H), 3.52 (dd, J = 15.8, 4.6 Hz, 1 H), 3.27 (ddd, J = 9.2, 7.7, 6.3 Hz, 1 H), 3.02 (dq, J = 10.6, 6.6 Hz, 1 H), 2.91 (dd, J = 15.8, 11.5 Hz, 1 H), 2.61 (ddq, J = 12.4, 8.3, 6.9 Hz, 1 H), 2.37 (ddd, J = 12.9, 8.3, 7.2 Hz, 1 H), 2.34 (m, 1 H), 2.14 (ddd, J = 10.6, 10.6, 5.4 Hz, 1 H), 2.03 (s, 3 H), 1.94–1.85 (m, 2 H), 1.64 (m, 1 H), 1.58–1.43 (m, 5 H), 1.42 (d, J = 6.6 Hz, 3 H), 1.26 (d, J = 6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 179.7 (C), 170.9 (C), 163.8 (C), 151.4 (C), 125.7 (C), 97.8 (C), 85.1 (CH), 83.7 (CH), 64.3 (CH), 59.7 (CH₃), 58.3 (CH), 54.4 (CH), 46.6 (CH₂), 41.9 (CH), 35.1 (CH), 34.5 (CH₂), 34.5 (CH₂), 27.5 (CH₂), 26.8 (CH₂), 20.3 (CH₂), 17.7 (CH₃), 15.0 (CH₃), 8.9 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₂NO₆ ⁺: 418.2230; found: 418.2219.

Bisdehydroprotostemonine (18)[3]

MnO₂ (528 mg, 6.07 mmol, 487 equiv) was added to a solution of protostemonine (1) (5.2 mg, 12.0 μmol) and THF (4.0 mL) at –20 °C. After stirring for 1 h at –20 °C, the resulting mixture was filtered through a pad of basified silica gel (10 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was purified by gel permeation chromatography with a recycle unit (Shodex GPC H-2002, 500 × 20 mm, 254 nm, CHCl₃, 3.6 mL/min, 15 cycles; 18: t _R = 352.7 min, 1: t _R = 356.2 min) to give 2.7 mg of 18 (53%) and 1.2 mg of 1 (23%).

White solid; mp 188–189 °C (Lit.[3c] [d] 192–194 °C).

[α]_D ²⁵ +166.1 (c 0.31, EtOH) [Lit.[3c] [α]_D ²⁰ +169.5 (c 0.81, EtOH); Lit.[3d] [α]_D ²⁰ +169 (c 0.81, EtOH)]; 98% ee.

IR (film): 2934, 2873, 1765, 1739, 1670, 1619, 1157, 1016, 754 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 6.14 (d, J = 3.7 Hz, 1 H), 6.00 (d, J = 3.7 Hz, 1 H), 5.39 (dd, J = 10.9, 5.2 Hz, 1 H), 4.34 (dd, J = 14.6, 5.7 Hz, 1 H), 4.19 (s, 3 H), 3.77 (ddd, J = 10.3, 10.3, 3.7 Hz, 1 H), 3.72 (dd, J = 14.6, 11.2 Hz, 1 H), 3.52 (dq, J = 10.3, 6.6 Hz, 1 H), 2.98 (dd, J = 10.3, 10.3 Hz, 1 H), 2.81 (m, 1 H), 2.72 (ddd, J = 12.3, 8.3, 5.2 Hz, 1 H), 2.60 (m, 1 H), 2.21 (ddd, J = 12.3, 11.7, 10.9 Hz, 1 H), 2.13 (m, 1 H), 2.10 (s, 3 H), 1.83 (m, 1 H), 1.70 (m, 1 H), 1.51 (d, J = 6.6 Hz, 3 H), 1.35 (d, J = 6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 179.0 (C), 170.1 (C), 163.2 (C), 148.5 (C), 132.4 (C), 129.5 (C), 125.6 (C), 107.1 (CH), 103.7 (CH), 97.8 (C), 85.7 (CH), 71.6 (CH), 59.0 (CH₃), 52.0 (CH), 45.6 (CH₂), 39.6 (CH), 36.1 (CH), 34.9 (CH₂), 34.3 (CH₂), 25.8 (CH₂), 19.4 (CH₃), 15.2 (CH₃), 9.4 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈NO₆ ⁺: 414.1917; found: 414.1923.

Bisdehydroprotostemonine (18) was prone to undergo quick epimerization at C18 probably through elimination and recyclization, although the exact structure was not determined because the compound was inseparable from 18. Purification by silica gel chromatography causes the epimerization (18/epi-18 = 2.5:1), and use of gel permeation chromatography was essential to prevent the epimerization. CDCl₃ for NMR measurements was required to be purified through a pad of basic alumina before use.

Isobisdehydroprotostemonine (19)

MnO₂ (730 mg, 8.40 mmol, 687 equiv) was added to a solution of isoprotostemonine (17) (5.1 mg, 12.0 μmol) and THF (4.0 mL) at –20 °C. After stirring for 1 h at –20 °C, the resulting mixture was filtered through a pad of basified silica gel (10 cc), washed with EtOAc/Et₃N (1:0.01), and concentrated. The residue was purified by gel permeation chromatography with a recycle unit (Shodex GPC H-2002, 500 × 20 mm, 254 nm, CHCl₃, 3.6 mL/min; 19: 14 cycles, t _R = 308.5 min; 17: 11 cycles, t _R = 245.6 min) to give 2.6 mg of 19 (52%) and 0.5 mg of 17 (10%).

White solid; mp 179–180 °C.

[α]_D ²⁴ +16.1 (c 0.29, EtOH).

IR (film): 2935, 2877, 1763, 1735, 1684, 1615, 1160, 1004, 919, 730 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 6.15 (d, J = 3.7 Hz, 1 H), 5.98 (d, J = 3.7 Hz, 1 H), 5.39 (dd, J = 10.9, 5.2 Hz, 1 H), 4.34 (dd, J = 14.6, 5.7 Hz, 1 H), 4.14 (s, 3 H), 3.84 (ddd, J = 10.6, 10.6, 3.4 Hz, 1 H), 3.73 (dd, J = 14.6, 11.2 Hz, 1 H), 3.59 (dq, J = 10.6, 6.6 Hz, 1 H), 2.91 (dd, J = 10.6, 10.6 Hz, 1 H), 2.81 (m, 1 H), 2.72 (ddd, J = 12.3, 8.3, 5.2 Hz, 1 H), 2.57 (m, 1 H), 2.22 (ddd, J = 12.3, 12.3, 10.9 Hz, 1 H), 2.14 (m, 1 H), 2.06 (s, 3 H), 1.83 (m, 1 H), 1.71 (m, 1 H), 1.61 (d, J = 6.6 Hz, 3 H), 1.35 (d, J = 6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 179.1 (C), 170.8 (C), 163.7 (C), 150.3 (C), 132.1 (C), 129.3 (C), 126.5 (C), 107.2 (CH), 103.9 (CH), 98.0 (C), 86.5 (CH), 71.6 (CH), 59.6 (CH₃), 50.4 (CH), 45.6 (CH₂), 41.7 (CH), 36.1 (CH), 34.8 (CH₂), 34.6 (CH₂), 25.8 (CH₂), 17.0 (CH₃), 15.2 (CH₃), 9.0 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈NO₆ ⁺: 414.1917; found: 414.1920.

Isobisdehydroprotostemonine (19) was prone to undergo quick epimerization at C18 probably through elimination and recyclization, although the exact structure was not determined because the compound was inseparable from 19. Purification by silica gel chromatography causes the epimerization (19/epi-19 = 2.5:1), and use of gel permeation chromatography was essential to prevent the epimerization. CDCl₃ for NMR measurements was required to be purified through a pad of basic alumina before use.

Hemiacetal 51

Bromide 38d (3.0 mg, 7.2 μmol, 1.0 equiv) was dissolved in THF (1.0 mL) and H₂O (1.0 mL). The resulting solution was heated to 70 °C, maintained for 8 h at 70 °C, quenched with aqueous saturated NaHCO₃ (3 mL), and extracted with CHCl₃ (3 × 5 mL). The combined organic extracts were dried over Na₂SO₄, and concentrated. The residue was purified by preparative layer chromatography (MeOH/EtOAc, 1:9) to give 2.2 mg of 51 [white solid, 87% combined yield, inseparable mixture of two diastereomers, dr = 1.4:1 (determined by ¹H NMR)].

IR (film): 3211, 2936, 2871, 1754, 1673, 1461, 1453, 1335, 1273, 1114, 1025, 914, 735 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ (1.4:1 mixture of two diastereomers) = 4.58 (brs, OH, 7/12 H), 4.14 (s, 3 H), 4.09–3.97 (m, 2 H), 3.98 (d, J = 7.2 Hz, 7/12 H), 3.93 (d, J = 7.5 Hz, 5/12 H), 3.93–3.84 (m, 1 H), 3.81 (ddd, J = 10.3, 10.3, 3.2 Hz, 5/12 H), 2.68–2.54 (m, 10/12 H), 2.59 (dd, J = 13.2, 13.2 Hz, 7/12 H), 2.40–1.68 (m, 103/12 H), 2.02 (s, 21/12 H), 2.00 (s, 15/12 H), 1.48–1.20 (m, 2 H), 1.22 (d, J = 6.6 Hz, 15/12 H), 1.06 (d, J = 6.9 Hz, 21/12 H).

¹³C NMR (125 MHz, CDCl₃): δ (1.4:1 mixture of two diastereomers) = 174.53 (C), 174.50 (C), 172.3 (C), 172.0 (C), 169.5 (C), 168.0 (C), 102.6 (C), 101.3 (C), 100.5 (C), 98.9 (C), 86.1 (CH), 84.1 (CH), 81.0 (CH), 80.7 (CH), 59.3 (CH₃), 59.3 (CH₃), 56.5 (CH), 56.4 (CH), 56.2 (CH), 56.0 (CH), 40.6 (CH₂), 40.6 (CH₂), 37.2 (CH), 36.3 (CH), 36.0 (CH₂), 35.7 (CH₂), 31.0 (CH₂), 30.9 (CH₂), 26.0 (CH₂), 26.0 (CH₂), 22.9 (CH₂), 22.7 (CH₂), 19.1 (CH₃), 18.3 (CH₃), 8.44 (CH₃), 8.38 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₆NO₆ ⁺: 352.1760; found: 352.1757.

Computational Details

The computational study was performed using MacroModel implemented in the Maestro 12.8 software package[41] and the Gaussian 16, Revision C.01 program.[42] A part of these computations was conducted using the SuperComputer System, Institute for Chemical Research, Kyoto University. Molecular structures were visualized using the Maestro 12.8 software package.

Conformational sampling using 100000 steps of the Monte Carlo multiple minimum with the OPLS4 force field afforded five conformational isomers of 14. The geometries were then optimized at the B3LYP-D3BJ/6-31G(d)-IEFPCM(CH₂Cl₂) level of theory. Frequency calculations were carried out at the same level of theory to confirm the absence of imaginary frequencies and obtain thermal corrections to the Gibbs free energies at 313.15 K. After eliminating duplicated structures, the energy evaluation at the B3LYP-D3BJ/6-311+G(d,p)-IEFPCM(CH₂Cl₂) level of theory provided four low-lying conformers within 3.0 kcal/mol from the minimum Gibbs free energy. The most stable conformer was defined as the global minimum structure of 14 and analyzed in detail. In addition, we obtained 1, 18, and 40 low-lying conformers of 15, (Z)-37, and (E)-37 similarly from 20, 34, and 79 OPLS4-minimized structures, respectively.

The superimposed picture of 14 and deMe-14 was created as follows: First, the C10 methyl group in the above-obtained structure of 14 was replaced with a hydrogen atom. We then optimized the geometry at the B3LYP-D3BJ/6-31G(d)-IEFPCM(CH₂Cl₂) level of theory, affording the structure of deMe-14. The structure of deMe-14 and 14 was superposed based on the non-hydrogen atoms of the pyrroloazepine core. The RMSD value was calculated using all non-hydrogen atoms without the methyl group at C10. The superimposed picture of 15 and deMe-15 was created in the same way.

Biological Evaluation of Stemoamide-Type Alkaloids

Cell Culture

Human tumor cell line HCT-116 (colon adenocarcinoma) and murine cell line RAW264.7 (macrophage like) were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 5% (v/v) fetal bovine serum, 100 units/mL penicillin G, 100 mg/L kanamycin, 600 mg/L l-glutamine, and 2.5 g/L NaHCO₃, at 37 °C in 5% CO₂.[43]

Evaluation of iNOS Expression

To evaluate whether compounds inhibit iNOS expression, western blotting was performed. RAW264.7 cells were incubated with the compounds (100 μM in DMF) for 1 h, and stimulated with LPS (1 mg/mL) for 24 h. Then, the cells were lysed with RIPA buffer [25 mM HEPES–KOH, pH 7.8, 150 mM NaCl, 50 mM NaF, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride] at 4 °C with sonication, and the cell lysates were centrifuged at 13000 rpm for 10 min. The amount of protein in each lysate was measured with protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA). The sample buffer [350 mM Tris-HCl, pH 6.8, 30% (w/v) glycerol, 0.012% (w/v) bromophenol blue, 6% (w/v) SDS, and 30% (v/v) 2-mercaptoethanol] was added to each cell lysate, and the lysates were boiled at 98 °C for 3 min. The samples were electrophoresed on SDS–polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK) and immunoblotted with specific antibodies. Signals were detected with ECL using Western Lightning Plus-ECL (PerkinElmer, Inc., Waltham, MA) and Immobilon Western Chemiluminescent HRP substrates (Merck KGaA, Darmstadt, Germany). Protein bands were quantified by Image J software (National Institutes­ of Health, Bethesda, MD). The used primary antibodies were as follows: anti-iNOS (#2977S; Cell Signaling Technology, Inc., Danvers, MA) and anti-α-tubulin (#T5168, Merck KGaA) antibodies.[44]

MTT Assay of Stemoamide-Type Alkaloids

Cells were treated with 100 μM compound in DMF for 72 h. To carry out the MTT assay, cells were treated with 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT, Merck KGaA) and were incubated for 4 h at 37 °C. After incubation, the medium was removed, and the MTT formazan product was dissolved with DMSO (200 μL). The amount of the product was determined by measuring absorbance at 570 nm using a microplate reader (Multiskan FC).[45] [46]

Conflict of Interest

The authors declare no conflict of interest.

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For preparation of model lactone 31, see:
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• 29 The AgOTf-mediated elimination of bromides 38 in the absence of 3 Å molecular sieves indicated partial formation of hemiacetals such as 51 shown in Figure 3. Hemiacetal 51 was separately obtained by hydration of 38d in THF/H₂O (1:1) at 70 °C.
• 30 Although oxidative cleavage of model compounds 37, protostemonine (1), and isoprotostemonine (17) was not observed, all compounds including the totally substituted butenolides were stored in a glovebox.
• 31 The Gibbs free energy of (Z)-37 is 0.4 kcal/mol lower than that of (E)-37, which is consistent with the experimental trend for the isomerization; see the Supporting Information. The Huang group proposed the preferential formation of the (Z)-isomer without the methyl group might be understood due to repulsion by electron pair–electron pair interaction of the (E)-isomer; see ref. 6f.

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Publication History

Received: 22 August 2022

Accepted after revision: 13 September 2022

Accepted Manuscript online:
13 September 2022

Article published online:
17 October 2022