Asymmetric Total Syntheses, Stereostructures, and Cytotoxicities of Marine Bromotriterpenoids Aplysiol B (Laurenmariannol) and Saiyacenol A.

There are marine cytotoxic bromotriterpenoids, named the thyrsiferol family that are structurally characterized by some tetrahydropyran (THP) and tetrahydrofuran (THF) rings. The thyrsiferol family belongs to natural products that are often difficult to determine their stereostructures even by the current, highly advanced spectroscopic methods, especially in acyclic systems including stereogenic tetrasubstituted carbon centers. In such cases, it is effective to predict and synthesize the possible stereostructures. Herein, to elucidate ambiguous stereostructures and unassigned absolute configurations of aplysiol B, laurenmariannol, and saiyacenol A, members of the thyrsiferol family, we carried out their asymmetric chemical syntheses featuring 6-exo and 5-exo oxacyclizations of epoxy alcohol precursors and 6-endo bromoetherification of a bishomoallylic alcohol. In this paper, we report total assignments of their stereostructures through their asymmetric chemical syntheses and also their preliminary cytotoxic activities against some tumor cells. These results could not have been achieved without depending on asymmetric total synthesis.

Aplysiol B with feeding‐deterrence and ichthyotoxicity properties, a marine bromotriterpenoid structurally related to the cytotoxic thyrsiferol family that possess as a partial structure a common dioxabicyclo[4.4.0]decane ring system with a bromine‐containing tetrahydropyranyl ring at C7 (ABC ring system), was isolated from the mantle of the sea hare Aplysia dactylomela by Manzo and co‐workers in 2007. The original structure 1 bearing threo configurations at C14−C15 and C18−C19 was determined based on NMR analysis and an integrated NMR‐QM (Quantum Mechanical) approach (Scheme 1). Afterwards, the original structure 1 was revised to a structure 2 bearing erythro configurations by Bowden and co‐workers in 2010 based on the biogenetic considerations from a squalene polyepoxide precursor. However, taking account of the biogenetic epoxide‐opening cascade triggered by a bromo cation as shown in 4, we think the stereostructure at C18−C19 should be further revised to 3 because squalene tetraepoxide 5 has been proposed as a plausible biogenetic precursor for many triterpenoids.[ , ] The ambiguity of the stereostructure would be due to technical limitations of current spectroscopic methods for acyclic systems that include stereogenic tetrasubstituted carbon centers.

Scheme 1

Proposed stereostructures and biosynthetic pathway of aplysiol B.

On the other hand, cytotoxic laurenmariannol (IC50=0.99 μM against P388 tumor cells) has also been isolated from the marine red alga Laurencia mariannensis by Wang and co‐workers in 2008 as a member of the thyrsiferol family. The original structure 6 with unknown stereochemistries at C19 and C22 was suggested by means of spectroscopic analyses (Figure 1). Saiyacenol A (7) exhibiting moderate cytotoxicities against some human tumor cells was isolated from the red alga Laurencia viridis by Fernández and co‐workers in 2012. Since the ABC ring system (C1−C14) in these compounds is common to the thyrsiferol family, it has been thought that the absolute configuration of the system is the same as that of the first member thyrsiferol (8). However, we recently discovered that isodehydrothyrsiferol (9), a member of the thyrsiferol family, possesses the enantiomeric ABC ring system. The phenomenon of enantiodivergence in the ABC ring system common to the thyrsiferol family would be of great interest in the fields of natural product chemistry and biosynthesis. Thus, to elucidate ambiguous stereostructures and unknown absolute configurations of aplysiol B, laurenmariannol, and saiyacenol A and whether or not other members showing the opposite chirality for the ABC ring system than 9 exist, we performed asymmetric chemical syntheses of these molecules. In this contribution, we report that the relative and absolute configurations of aplysiol B and saiyacenol A are shown in 3 and 7, respectively, and spectroscopic data of aplysiol B (3) are identical to those of laurenmariannol through their asymmetric chemical syntheses. Further, preliminary cytotoxicities of synthetic compounds were also evaluated against some tumor cells.

Figure 1

Proposed structures 6 and 7 for laurenmariannol and saiyacenol A, respectively, and enantiodivergency in the ABC ring system common to the thyrsiferol family.

The retrosynthetic analysis of our target structure 3 for aplysiol B as a representative of these compounds is indicated in Scheme 2. The A ring could be cyclized by bromoetherification from bishomoallylic alcohol 10. The D ring would be formed from diene 11 by regioselective Shi epoxidation of a trisubstituted alkene at C18 followed by 5‐exo oxacyclization. Disconnection at the C16−C17 bond in 11 generates the known C10 unit 13 and the common fused BC ring system 12, both tetrahydropyran (THP) rings of which could be constructed by 6‐exo oxacyclization from the corresponding epoxy alcohol.

Scheme 2

Retrosynthetic analysis of target structure 3.

We embarked on the asymmetric chemical synthesis of the target molecule 3 with known compounds chiral epoxide 14 and allylic sulfide 15 (Scheme 3). The lithiation of 15 and alkylation of the lithio derivative with epoxide 14 were carried out in situ in the presence of TMEDA, and subsequent desulfurization and selective TES protection of the secondary hydroxy group afforded alcohol 16. Diastereoselective epoxidation of the bishomoallylic alcohol 16 provided epoxy alcohol 17 in 79% yield. An epoxy alcohol obtained by manipulation of protecting groups was treated with KOt‐Bu in DMSO to give THP product 18 in a 6‐exo selective manner and quantitative yield. Demethoxymethylation of 18 yielded a triol, the stoichiometric Sharpless asymmetric epoxidation[ , ] of which was accompanied with stepwise elevation of the reaction temperature to successfully achieve 6‐exo oxacyclization, that led to the C ring adopting a twist‐boat conformation, in 79% yield. Selective mesylation of the primary hydroxy group in triol 19 and the basic treatment furnished terminal epoxide 12. Attachment of the C10 unit 13 to the terminal epoxide 12 provided diene 11, wherein Shi asymmetric epoxidation using D‐ketone 20 regioselectively proceeded for the trisubstituted double bond to afford epoxy alcohol 21 in high yield. Construction of the D ring was performed in 96% yield by 5‐exo oxacyclization of the epoxy alcohol 21 with CSA. Cross‐metathesis of the terminal olefin 22 with Grubbs second generation catalyst 23 generated a trisubstituted alkene, and bromoetherification of the resulting bishomoallylic alcohol 10 with bromodiethylsulfonium bromopentachloroantimonate (BDSB) in MeNO2 gave the desired 6‐endo product 24 along with 5‐exo byproduct 25 and recovered 10. Finally, removal of an acetonide protecting group in 24 yielded our target structure 3. The 1H‐ and 13C‐NMR spectra of synthetic 3, [α]28 D −7.8 (c 0.12, CHCl3), were identical to those of the natural product, [α]27 D −9.0 (c 0.7, CHCl3), kindly provided by Manzo. Surprisingly, it was also found that the spectral data of synthetic 3 are consistent with those reported for laurenmariannol, [α]18 D −15.7 (c 0.41, CHCl3).

Scheme 3

Asymmetric chemical syntheses of our target structure 3 and Bowden structure 2. Piv=pivaloyl, TBS=t‐butyldimethylsilyl, TMEDA=N,N,N',N'‐tetramethylethylenediamine, THF=tetrahydrofuran, TES=triethylsilyl, MS=molecular sieves, TBHP=t‐butyl hydroperoxide, rsm=recovered starting material, MOM=methoxymethyl, TBAF=tetrabutylammonium fluoride, DMSO=dimethyl sulfoxide, DET=diethyl tartrate, Ms=methanesulfonyl, CSA=(±)‐10‐camphorsulfonic acid, Mes=mesityl.

To confirm that the structure 2 proposed by Bowden et al. is not that of aplysiol B, we intended to synthesize the structure 2 bearing another erythro configuration at C18−C19 different from that of 3 as well. The synthesis of 2 was carried out in the same way as that from the synthetic intermediate 11 to 3 except for Shi asymmetric epoxidation of 11 using L‐ketone ent‐20 instead of 20 (Scheme 3). Predictably, it was confirmed that the spectral data (1H‐ and 13C‐NMR) of synthetic 2 are inconsistent with those reported for the natural products aplysiol B and laurenmariannol. Thus, it has been revealed that aplysiol B and laurenmariannol are the same compound and the correct stereostructure is not 1, 2, and 6 but 3.

Next, we commenced the asymmetric chemical synthesis of the stereostructure 7 proposed for another target saiyacenol A. The synthetic intermediate 12 to aplysiol B (3) was extended to diene 26 by alkylating with the known C10 unit ent‐13, an enantiomer of allylic sulfide 13, followed by desulfurization (Scheme 4). Deprotection of an acetonide group in 26 and epoxidation of the resulting vicinal diol afforded a (22S)‐epoxide, which was subjected to regioselective Shi asymmetric epoxidation using D‐ketone 20 at C18 alkene to yield a double cyclization precursor diepoxide 27. The double oxacyclization of the diepoxy alcohol 27 smoothly proceeded in a 5‐exo selective manner to provide the desirable bisTHF product 28 in a quantitative yield. The construction of the A ring was performed through cross‐metathesis of the terminal olefin 28 and subsequent bromoetherification by BDSB to accomplish the chemical synthesis of the target structure 7. The spectral data (1H‐ and 13C‐NMR) of synthetic 7, [α]25 D +1.6 (c 0.12, CHCl3), were identical to those reported for saiyacenol A, [α]25 D +1.53 (c 0.37, CHCl3). Thus, it was confirmed that the absolute configuration of saiyacenol A is shown as 7.

Scheme 4

Asymmetric chemical synthesis of saiyacenol A (7).

With these synthetic aplysiol B (3), compound 2, and saiyacenol A (7) in hand, we evaluated their cell growth inhibitory activities against some tumor cell lines. The results were shown in terms of IC50 (50% inhibitory concentration) values against P388, HT‐29, and HeLa (Table 1). All three compounds specifically indicated stronger activity for P388 than that for HT‐29 and HeLa. The synthetic aplysiol B (3) which was a naturally occurring compound inhibited cell growth stronger than compound 2 on every cell line. These results suggested the importance of 18S and 19R stereochemistries on biological activities. The activity of synthetic saiyacenol A (7) was lower than that of synthetic aplysiol B (3) in all cell lines, indicating that the formation of THF ring (E ring) reduces the biological activity.

Table 1

In vitro growth inhibitory activities of synthetic compounds 3, 2, and 7 on tumor cell lines.

Compound	IC50 [μM]
	P388	HT‐29	HeLa
3	0.10 (0.99)[a]	31	51
2	2.0	81	>100
7	5.4	85	>100 (27.5)[a]

[a] Numerals in parentheses are IC50 (μM) values cited from ref. [6] for natural 3 and ref. [7] for natural 7.

In conclusion, we have accomplished the asymmetric total syntheses of marine cytotoxic bromotriterpenoids aplysiol B (3), laurenmariannol (3), and saiyacenol A (7) with ambiguous stereostructures and unknown absolute configurations and determined their correct relative and absolute configurations. In addition, we have also revealed that aplysiol B and laurenmariannol reported as different compounds possess the same stereostructure 3. These results could not have been achieved without depending on asymmetric total synthesis. Exploration of other members exhibiting the ABC ring system enantiomeric to that of thyrsiferol (8), the first member of the thyrsiferol family, and more detailed cytotoxicities of synthetic compounds are under investigation in our laboratory.

Experimental Section

Experimental procedures, spectroscopic data, and copies of 1H‐ and 13C‐NMR spectra are available in the Supporting Information (SI).

Conflict of interest

The authors declare no conflict of interest.

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Supporting Information

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