Synthesis of the C21–C34 Segment of Aplyronine A toward Its Scalable Preparation

Abstract

The development of scalable synthesis of the C21–C34 segment, a key intermediate for aplyronine A and its analogs, is reported. Marshall propargylation and Noyori asymmetric hydrogen transfer served as key reactions for the stereoselective construction of the desired C21–C34 segment on a gram scale. Further transformation of the segment successfully afforded the side chain analog that exhibited actin depolymerization activity similar to that previously reported.

Introduction

Aplyronine A (ApA) (1) is a 24-membered macrolide that was isolated from Aplysia kurodai in 1993 by Yamada et al. It exhibits extremely strong cytotoxicity against HeLa S₃ cells (IC₅₀ = 0.039 ng/mL)^1–12) and actin depolymerizing activity (K_D = 100 nM)¹³⁾ (Chart 1). Several analogs of 1 have also been isolated, and these analogs, except for ApB, ApC, and ApH, show potent cytotoxicity comparable to that of ApA (1).^1,6–8,10) The key structural difference between ApA (1) with the most potent cytotoxicity and its less analogs lie in the presence of a trimethylserine (TMSer) moiety at the C-7 position. Thus, the TMSer moiety at the specific position is considered crucial for the strong cytotoxicity. Because of its unique structure and potent biological activity, our group has extensively investigated the clarification of the mechanism of action of 1. It has been revealed that ApA initially forms a 1 : 1 complex with the globular actin (G-actin) and promotes the depolymerization of fibrous actin (F-actin).^13–18) In addition, we previously reported that ApA forms a 1 : 1 : 1 heterotrimeric complex with actin and tubulin, resulting in strong cytotoxicity against tumor cells through the inhibition of tubulin polymerization.¹⁹⁾ However, the detailed mechanism of action remains unclear due to the limited availability of ApA from the natural sources. Meanwhile, many research groups including us have challenged the total synthesis of ApA (1) toward a scalable preparation^20–25) and clarification of the structure–activity relationship.^22,26–28) Although most syntheses have been accomplished in a stereoselective manner and applied to the efficient synthesis of analogs, synthetic protocols for the efficient supply of ApA (1) are still in high demand. To facilitate further clarification of the mechanism of action of ApA, we planned to develop a synthetic method based on the total synthesis we previously reported. Here, we focused on a side-chain segment of ApA and its derivative, as these compounds can serve as useful biological tools to regulate actin depolymerization and assist in the clarification of the mechanism of action of ApA.^15,29,30) In this paper, we report the gram-scale synthesis of the C21–C34 segment of ApA (1) as part of our efforts toward its scalable preparation.

Chart 1. Structure of Aplyronine A (1)

Results and Discussion

For the scalable synthesis of the side-chain analog 2 of ApA, we designed the C21–C34 segment 3, whose retrosynthetic analysis is shown in Chart 2. In our previous synthesis, we employed the Julia–Kocienski olefination or the asymmetric Ni/Cr coupling to construct the structure of the side chain segment.^22,24,30) However, these coupling reactions presented several issues, including epimerization at the C29 position and the requirement for stoichiometric amounts of highly toxic Cr reagent. Therefore, we planned to apply a low-toxic transition-metal-catalyzed asymmetric reduction of an ynone moiety, considering ynone 4 as a precursor of the intermediate 3. The desired 3 could be obtained from 4 by Noyori asymmetric hydrogen transfer^31,32) followed by hydrogenation of the unsaturated bond. We envisioned that ynone 4 could be synthesized by anion coupling using two segments, alkyne 5 and Weinreb amide 6. The synthesis of these two segments was planned as follows: alkyne 5 would be prepared by Marshall propargylation^33,34) of aldehyde 7, whose stereocenters would be introduced through a syn-selective Evans asymmetric aldol reaction³⁵⁾ using 8. Weinreb amide 6 could be obtained through acetal formation after hydroboration of the alkyne 9 and subsequent conversion of the primary alcohol. The anti-configuration of homopropargyl alcohol 9 could be stereoselectively prepared by Marshall propargylation of aldehyde 10.

Chart 2. Retrosynthesis of C21–C34 Segment 2

We initially synthesized alkyne 5 from the known acylated oxazolidinone 8³⁵⁾ (Chart 3). The Evans aldol reaction of aldehyde 11 with 8 was performed at −78 to 0 °C to afford the desired alcohol 12 in 86% yield as a single diastereomer. After removal of the tert-butyldimethylsilyl (TBS) group, the resulting diol 13 was converted to the di-tert-butyl silylene acetal 14. Reductive cleavage of the chiral auxiliary in 14 was performed using LiBH₄ in the presence of MeOH³⁶⁾ to provide alcohol 15, which was subsequently oxidized by Swern oxidation to afford aldehyde 7. Marshall propargylation of 7 with (R)-mesylate 16 proceeded in the presence of Pd(OAc)₂/PPh₃ at −20 °C to furnish the propargylic alcohols with high stereoselectivity (dr = 12 : 1). The diastereomers were readily separated to afford the desired alcohol 17³⁷⁾ (83%). Finally, protection of the secondary hydroxyl group with a TBS group furnished the desired alkyne 5.

Chart 3. Synthesis of Alkyne 5

We next synthesized Weinreb amide 6 from known aldehyde 10³⁸⁾ (Chart 4). Marshall propargylation of 10 with (S)-mesylate 16 furnished the desired propargylic alcohol 9³⁹⁾ in 88% yield, along with a separable diastereomer in 8% yield. Direct conversion of the alkyne moiety in 9 into hemiacetal 18 was achieved via hydroboration using BH₃•SMe₂/2-methyl-2-butene, and the resulting 18 was treated with CSA/MeOH to afford a 3 : 2 diastereomeric mixture of acetal 19. Both diastereomers of 19 were separated by column chromatography, and the minor diastereomer was isomerized under acidic conditions to give the major diastereomer of 19 in 62% combined yield. Removal of the tert-butyldiphenylsilyl (TBDPS) group using ⁿBu₄NF afforded 20, and oxidation of the resulting alcohol in 20 was performed using AZADOL/NaClO₂/NaOCl⁴⁰⁾ to provide acid 21. Finally, the desired Weinreb amide 6 was successfully obtained by coupling with MeONHMe·HCl using a mixed anhydride method (PivCl/Et₃N) in 71% yield.

Chart 4. Synthesis of Weinreb Amide 6

With both segments 5 and 6 in hand, we investigated the synthesis of C21–C34 segment 3 (Chart 5). The ynone 4 was successfully obtained by anion coupling of 5 and 6 in 94% yield. Stereoselective reduction of the ynone moiety in 4 was performed by Noyori asymmetric hydrogen transfer using (S,S)-Ru catalyst 24, affording the desired propargylic alcohol 22 as a single diastereomer.⁴¹⁾ Hydrogenation of the alkyne in 22 was next investigated; however, the reaction unfortunately provided a 3 : 2 mixture of the desired product 23 and an undesired deoxygenated product 23′. This side reaction was presumed to occur through the acid-catalyzed hydrogenolysis of allyl and/or propargylic alcohols, followed by hydrogenation. Therefore, we next investigated the reaction in the presence of NaHCO₃ to suppress the above side reaction. Gratifyingly, the addition of NaHCO₃ effectively promoted the desired hydrogenation and improved the yield of the desired 23 up to a quantitative yield. Finally, protection of the secondary hydroxyl group with a [(3,4-dimethoxybenzyl)oxy]methyl group²²⁾ furnished the C21–C34 segment 3 on a gram scale.

Chart 5. Synthesis of C21–C34 Segment 3

Since a sufficient amount of the C21–C34 segment 3 was successfully prepared, we attempted the synthesis of the side chain analog of ApA 2 we previously reported²⁷⁾ (Chart 6). Acidic hydrolysis of the methyl acetal in 3 produced hemiacetal 26 without loss of the silyl groups, and subsequent reduction of 26 provided diol 27 in quantitative yield. Selective protection of the primary hydroxyl group with a Trt group, followed by acetylation of the secondary hydroxyl group in one-pot, afforded acetate 28, whose spectral data, including specific rotation, were identical to those of our intermediate in the second-generation total synthesis of ApA.²⁴⁾ With the intermediate 28 in hand, we next investigated its conversion to the side chain analog 2. After removal of the Trt group using HCOOH, the resulting alcohol 29 was oxidized with Dess–Martin periodinane to afford aldehyde 30. Construction of the enamide moiety was accomplished by condensation of N-methylformamide under acidic conditions,^22,42) providing the desired compound 31 in 65% yield. Subsequent removal of the [(3,4-dimethoxybenzyl)oxy]methyl group using 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and esterification of the resulting hydroxy group in 32 with N,N-dimethyl-l-alanine provided ester 33 (S/R = 7 : 1). Finally, removal of the silylene acetal smoothly proceeded using ⁿBu₄NF/AcOH at −5 °C leading to 34, which was treated with HF·Py at room temperature to remove the TBS group, furnishing the desired side chain analog 2. The spectral data of the synthetic 2 were in good agreement with those previously reported.²⁷⁾ In addition, the actin-depolymerizing activity of side chain analog 2 was evaluated in vitro by the ultracentrifugation method. Compound 2 exhibited actin-depolymerizing activity with EC₅₀ = 15.7 µM, whose activity was comparable to the EC₅₀ value we previously reported.^43,44)

Chart 6. Synthesis of Side-Chain Segment 2

In summary, we have demonstrated a scalable synthesis of key intermediate C21–C34 segment 3 toward the synthesis of aplyronine A and its analogs. A convergent strategy based on anion coupling of 5 and 6 was employed to prepare the ynone intermediate 4, and both segments were prepared utilizing Marshall propargylation in a stereoselective manner. The Noyori asymmetric hydrogen transfer was found to be effective in controlling the absolute configuration at the C29 position on a gram scale, enabling the successful preparation of the desired C21–C34 segment 3. Further transformation of 3 led to the side chain analog 2, whose spectra, including the specific rotation, were identical to those previously reported. The total number of steps from a commercially available substrate to compounds 28 and 2 was reduced compared with the previous synthesis (28: 16 steps → 11 steps, 2: 35 steps → 18 steps), and their overall yields were also improved (28: 16.7% → 17.3%, 2: 1.84% → 3.35%). Therefore, the synthetic method described herein provides stereoselective access to the key intermediate on a gram scale, thereby facilitating the chemical biology studies on aplyronine A.

Acknowledgments

This work was supported by the JSPS KAKENHI Grants 23K23474 (M.Y.), and 23K26783 (H.K.), as well as the Naito Foundation. This study was also partially supported by Terumo Life Science Foundation and the Novartis Foundation.

Declarations

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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