Introduction

Various antibiotics and other antimicrobial agents have been developed and they have saved uncountable numbers of lives. However, the threat of antimicrobial resistance (AMR) has recently arisen and must be addressed urgently1,2. Even though the problem of morbidity and mortality associated with AMR is not new, AMR has recently been increasing at a significant rate due to bacteria that have acquired resistance to multiple groups of antimicrobial agents2. Moreover, the decline in the development and marketing of new antimicrobial agents worsens the situation. O'Neill’s report predicts that with the current increasing trend of AMR, an estimated 10 million lives per year may be lost to AMR-related disease by 20503. To avoid this worst-case scenario, it is necessary to develop new kinds of antimicrobial agents with chemical skeletons and/or antimicrobial mechanisms different from those of conventional drugs.

Molecules with a cyclopentenone framework are Michael acceptors for various cellular nucleophiles due to their highly reactive α,β-unsaturated carbonyl moiety4,5. Thus, the five-membered carbon ring framework of cyclopentenone is often used as the core of building blocks to synthesize natural products and related compounds6,7. Additionally, highly oxygenated cyclopentenoids are known to be effective as promising antimicrobials5. Recently, we have reported the synthesis of pentenomycin I (1; Fig. 1), which was isolated from a cultured strain of Streptomyces eurythermus8, and its analogues9. In addition, we tested their antimicrobial activity and determined some of the structural factors that are important for antimicrobial activity. According to the results of this evaluation, their antimicrobial activity was moderate and unsuitable for pharmaceutical lead compounds. Therefore, advancing investigations into highly effective antimicrobial cyclopentenoids would undoubtedly be valuable for pharmaceutical development. Between 2004 and 2017, the group of Arnold has reported the isolation and structural elucidation of new cyclopentenoids, which they named hygrophorones, derived from various Hygrophorus species10,11,12,13,14. Isolated natural products are highly substituted 2-cyclopentenones with two hydroxy groups at asymmetric centers C-4 and C-5 and a hydrocarbon chain that contains an additional hydroxylated asymmetric center bonded at C-5. The structures of representative hygrophorones are shown in Fig. 1. Hygrophorones A–D (2–8) consist of a 2-cyclopenten-1-one skeleton substituted with hydroxy or acetoxy groups at the C-4 and C-5 positions and an oxidized long hydrocarbon chain attached to C-5. The structures and the stereochemistry of hygrophorones have been determined via extensive spectroscopic studies, including 2D NMR spectroscopy experiments. Furthermore, 4,6-diacetylhygrophorone A12 (3) showed potent antimicrobial activity against several bacterial species in the sub- to low-micromolar range (MIC = 0.25–8 µg/mL)15. Interestingly, 3, which contains a long hydrocarbon chain, showed significantly higher antimicrobial activity than 18,9, which possesses an enone structure without an alkyl chain. Therefore, we hypothesize that other hygrophorones such as B type show high antibacterial activity. According to our survey of the literature, antimicrobial susceptibility tests of other hygrophorones toward bacterial-pathogen-caused human infectious diseases have not yet been reported, albeit in 2021 Westermann’s group disclosed the fungicidal activity of hygrophorone B12 (4) and its 6-deoxy analogue against plant pathogens16. Consequently, the development of efficient and flexible methods for the synthesis of the hygrophorone family, and the elucidation of their structure–activity relationship are desirable and useful from the viewpoint of medicinal/pharmaceutical chemistry.

Figure 1
figure 1

Chemical structures of pentenomycin I and representative hygrophorones.

Full size image

Due to their unique structural features, hygrophorones have been drawing increasing attention and become a target for total synthesis. Numerous efforts have been devoted to the total synthesis of this class of natural products, and so far, three methods have been reported for the total synthesis of hygrophorone-B-type compounds and hygrophorone A12 (2)17,18,19. Westermann et al. reported the asymmetric total synthesis of hygrophorone A12 (2) and B12 (4) using a biomimetic strategy, in which the stereo-controlled dihydroxylation of fatty acid ester 9 is the key step (9 → 10). (+)-2 and (−)-4 were obtained using AD-mix-alpha, and the enantiomers (−)-2 and (+)-4 using AD-mix-beta (Scheme 1a)17. Rao et al. achieved the stereoselective synthesis of (−)-6-epi-hygrophorone B14 (6) from D-mannose (11) using a stereoselective Grignard reaction for the insertion of the hydrocarbon chain, the Bernet–Vasella protocol (11 → 12), and a ring-closing metathesis (Scheme 1b)18. More recently, the enantioselective syntheses of (−)-Hygrophorone A12 (2) and (+)-Hygrophorone B12 (4) from 13 has been achieved by Gholap et al. (Scheme 1c)19. The common intermediate 15 was prepared starting from 13 by a Grignard reaction for the insertion of the carbon chain, followed by a Barton–McCombie reaction (13 → 14), and finally a diastereoselective intramolecular aldol reaction (14 → 15) . The deprotection of acetonide in 15 produced (–)-Hygrophorone A12 (2). In addition, Hygrophorone B12 (4) was obtained by epimerization at C-4 via the Mitsunobu inversion. All strategies are excellent methods for the stereoselective synthesis of the targeted hygrophorones. However, in order to synthesize hygrophorone analogues, an arbitrary carbon chain needs to be present from the beginning, or needs to be inserted at the earliest stage of the synthetic plan. In addition, there are so far no reports on the evaluation of the antimicrobial activity of such B-type hygrophorones with side chains of different length. Therefore, a new modular synthetic strategy for the preparation of a variety of hygrophorone analogues starting from a common intermediate would be useful to investigate the structure–activity relationship for hygrophorones. In this study, we focus on hygrophorone B12 (4) and we describe the enantioselective total synthesis of 4 and its analogues from cyclopentenone, as well as the evaluation of their antimicrobial activity.

Scheme 1
scheme 1

a: Key step in the synthesis of ( +)-hygrophorone B12 (4) by Westermann et al. b: Key step in the synthesis of (−)-6-epi-hygrophorone B14 (6) by Rao et al. c: Key step in the synthesis of (−)-hygrophorone A12 (2) and ( +)-4 by Gholap et al.

Full size image

Results and discussion

The outline of our synthetic plan is shown in Scheme 2. The most important component of our plan is the use of a key intermediate, i.e., formyl enone 18, which is suitably functionalized and oxidized. The production of a wide variety of hygrophorone analogues from 18 could theoretically be achieved via a convenient and general method. It is assumed that a simple sequence could be proposed to insert any hydrocarbon chains and aromatic groups using organometallic reagents (18 → 17). After the insertion of the hydrocarbon chain, conversion into the hygrophorone skeleton would be accomplished by a stereoselective dihydroxylation followed by the formation of an enone moiety (17 → 16 → 4). The key intermediate 18 would be obtained from optically active cyclopentenone 20 via 19.

Scheme 2
scheme 2

Retrosynthesis of ( +)-hygrophorone B12 (4). TBS = tert-butyldimethylsilyl; TIPS = triisopropylsilyl.

Full size image

The synthesis of key intermediate 18 is shown in Scheme 3. The starting material, cyclopentenone 20, was obtained from D-glucose (21) according to published procedures20,21. First, 22 was synthesized from 20 by the selective protection of hydroxyl group by catalyst-free method using tert-butyldimethylsilylchloride (TBSCl) and triethylamine9, and then 19 was obtained by treatment with triisopropylsilyl trifluoromethanesulfonate (TIPSOTf) and 2,6-lutidine, followed by the regioselective desilylation of primary TBS under mild conditions using scandium (III) triflate as a catalyst (19 → 23). We next investigated the oxidation to obtain the desired formyl enone 18 (see the Table in Scheme 3). We examined oxidation methods to transform the primary hydroxyl group into a formyl group; however, 18 was not obtained under any of the attempted conditions. When the direct oxidation of the C2-hydroxymethyl group of 23 into a formyl group was attempted, the substrate decomposed, maybe due to the instability of the resulting enone with a formyl group at the C-2 position, which is highly electron-deficient. Therefore, it was crucial to control the electron density of the enone moiety by conversion of the ketone into an oxime. The electron-donating features of the oxime are able to reduce the electron deficiency of the enone moiety. O-(tert-butyldimethylsilyl)hydroxylamine (TBSONH2), which enabled easy access to oxime,“from the resulting O-silyl oxime” was used. The cleavage of oxime has been reported under a wide variety of conditions, and we expected that it would enable the conversion of the relatively hard enone–oxime into a ketone22,23,24,25,26,27. So, 23 was converted to 24 using TBSONH2, which was prepared from TBSCl and hydroxylamine28, with an acidic catalyst in the presence of anhydrous magnesium sulfate as water scavenger29. The resulting compound 24 was selectively obtained as E steroisomer avoiding the steric hindrance between the TBSO group and the hydroxymethyl group at the C-2 position of the enone. In the subsequent step, the Dess–Martin oxidation of the crude product 24 gave aldehyde 25 in 79% yield over two steps (Scheme 4). With key intermediate 25 in hand, we proceeded with the synthesis of (+)-4 through the insertion of the hydrocarbon chain followed by the suitable ring modifications to obtain hygrophorone skeleton (Scheme 5). The coupling reaction of 25 with the hydrocarbon chain to construct the desired carbon skeleton of (+)-4 was investigated intensively. Initial attempts with commercially available dodecylmagnesium bromide (C12H25MgBr) in tetrahydrofuran (THF) at − 78 to − 20 °C30 resulted in the formation of 24 as the major product, while compound 26 was barely produced. We assumed that this result is due to the reduction of the β-hydride via a six-membered-ring transition state between the formyl group of 25 and C12H25MgBr as a hydride source, similar to the Meerwein–Ponndorf–Verley (MPV) protocol (for details, see the Supporting Information, Scheme S1)31,32,33,34,35. To test this hypothesis, we used an unsaturated hydrocarbon chain, 1-dodecyne, without a hydride in the beta position. The organolithium obtained in situ from the treatment of 1-dodecyne with n-BuLi was allowed to react with intermediate 25 at − 40 °C to give 27 in moderate yield (57%) without forming 24. Moreover, 27 was obtained in satisfactory yield (92%) as an inseparable diastereomeric mixture (α/β = 1:1) with regard to the C-6 position by using 2-methyl-THF, a less hygroscopic solvent than THF (for details, see the Supporting Information, Table S1). The scandium-catalyzed treatment of 27 under mild reaction conditions removed the TBS group to produce the oxime 28, and a subsequent reductive deoximation by titanium (III) chloride in the presence of ammonium acetate36 gave 29 and epi-29 as a mixture separable via column chromatography on silica gel. Compound 30 was prepared as a single stereoisomer from 29 by treatment with an osmium catalyst and N-methylmorpholine oxide at 70 °C; the formation of the osmate ester intermediate was stereoselective due to the steric hindrance of the TIPS group at the C-4 position. The catalytic hydrogenation of 30 over 10% Pd/C was performed in methanol at room temperature. Finally, the desilylation of the resulting intermediate and the subsequent elimination of the hydroxyl group to construct the enone moiety were efficiently achieved through treatment with 1 M aqueous HCl in ethanol at 90 °C37, affording the targeted (+)-hygrophorone B12 (4). The experimental evidences (1H and 13C NMR spectra and high-resolution mass spectrum) of synthetic (+)-4 were identical to those of natural hygrophorone B12 (4). The optical rotation of synthetic (+)-4 ([α]D25 =  + 23.0; c = 0.10 in MeOH) matched that reported for natural 4 ([α]D27 =  + 20.7; c = 0.135, MeOH)17. Furthermore, we prepared two analogues of 4 starting from key intermediate 30. Alkyne 31 was synthesized through direct treatment of 30 with 1 M aqueous HCl in ethanol at 90 °C, while the reduced analogue 32 was obtained by the Pd catalyzed hydrogenation of the endocyclic double bond of (+)-4.

Scheme 3
scheme 3

Toward the synthesis of aldehyde 18. THF = tetrahydrofuran; TBS = tert-butyldimethylsilyl; TIPS = triisopropylsilyl; DMSO = dimethyl sulfoxide; rt: room temperature: temp. = temperature; for details, see the Supporting Information.

Full size image
Scheme 4
scheme 4

Synthesis of oxime 25. TBS = tert-butyldimethylsilyl; TIPS = triisopropylsilyl; PPTS = pyridinium p-toluenesulfonate; rt: room temperature; for details, see the Supporting Information.

Full size image
Scheme 5.
scheme 5

Synthesis of ( +)-hygrophorone B12 (4) and analogues 31/32. THF = tetrahydrofuran; TBS = tert-butyldimethylsilyl; TIPS = triisopropylsilyl; NMO = N-methylmorpholine oxide; rt: room temperature; for details, see the Supporting Information.

Full size image

The antimicrobial activity of the synthesized (+)-hygrophorone B12 (4) and its analogues 31 and 32 were evaluated to gauge their potency. Antimicrobial susceptibility testing was performed with the broth dilution method according to the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines9,38. Initially, the compounds were tested with six bacterial species (Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii) that represent the most common pathogens in clinical settings. Gentamicin was used as a positive control in this assay, and the minimum inhibitory concentration (MIC) values are summarized in Table 1. (+)-4 and 31 showed antimicrobial activity toward five of the bacterial species, excluding P. aeruginosa. In particular, they were significantly more effective against E. faecium than gentamicin, while the antimicrobial activity of 32 was remarkably lower against many species. These results indicate that the expression of antimicrobial activity depends on the α,β-unsaturated carbonyl framework in the hygrophorone B class of compounds. Moreover, the antimicrobial activities of (+)-4 and 31 were investigated against six antimicrobial-resistant (AMR) bacteria isolates (methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, carbapenem-resistant Escherichia coli, carbapenem-resistant Enterobacterales, multidrug-resistant Pseudomonas aeruginosa, and multidrug- resistant Acinetobacter baumannii) (Table 2). Intriguingly, (+)-4 and 31 were notably effective in suppressing the growth of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecium (VRE). Moreover, 31 with a carbon–carbon triple bond in its hydrocarbon chain suppressed the growth of multidrug-resistant A. baumannii (MDRA) compared to (+)-4. However, these compounds were not effective against carbapenem-resistant Enterobacterales (E. coli or K. pneumoniae) (CRE) or multidrug-resistant P. aeruginosa (MDRP). The aforementioned results show that hygrophorone-B-type compounds including 4 and 31 can be expected to exhibit higher antimicrobial activity against specific bacterial species, especially gram-positive bacteria. Detailed antimicrobial activity data, including MIC values for hygrophorone B12 (4) and its analogues 31/32 against bacterial pathogens causing human infectious diseases, have not been reported previously in the literature, while the MIC value of hygrophorone-A-type compound (3) has been reported elsewhere15. The results of our evaluation suggest that their chemical structure (e.g., the presence of triple bonds or enone moiety) have a substantial influence on the microbial activity of hygrophorones. Therefore, our strategy for a new synthesis of hygrophorone-B-type compounds from key intermediate 25 with a suitable side chain is useful. These findings demonstrated that hygrophorone B is an accessible lead compound for designing novel antimicrobial drugs.

Table 1 Antimicrobial evaluation of the synthesized hygrophorones (+)-4, 31, and 32 using clinically relevant bacterial pathogens.
Full size table
Table 2 Antimicrobial evaluation of synthesized (+)-4 and 31 against antimicrobial-resistant (AMR) bacterial isolates.
Full size table

Conclusion

In conclusion, we have achieved the enantioselective total synthesis of (+)-hygrophorone B12 (4) and its analogues 31 and 32 in 4.5–4.8% overall yield over a linear sequence of 10–12 steps starting from cyclopentenone 20, which can be obtained from d-glucose (21). The main advantage of our synthetic method is expected to contribute the provision of a wide variety of hygrophorone B analogues starting from the common key intermediate 25 via the insertion of various hydrocarbon chains or aryl groups. As ongoing research, we are investigating other side chains including aryl groups that can be introduced, and studying toward the synthesis of other analogues. The antimicrobial evaluation of (+)-4 and its analogues revealed their potency, and the structure—activity relationship of hygrophorone-B-type compounds was disclosed. Moreover, the synthesized hygrophorones are highly effective in preventing the proliferation of chemical-sensitive bacteria and AMR bacteria (MRSA, VRE, and MDRA), especially gram-positive bacteria. These results can be expected to be useful for the design and development of new antimicrobial agents. Further studies concerning the synthesis of other hygrophorone B analogues and the evaluation of their antimicrobial activity, as well as an investigation of their side effects on human normal cells, are currently in progress in our group. In the near future, the bacterial intracellular target(s) that interact with hygrophorones will be revealed in detail, and we will provide accurate information on the mechanism of the expression of their activity.

Methods

Instruments

Optical rotations were recorded on an Anton Paar MCP-100. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE–400 III and AVANCE-600 III spectrometer and calibrated using residual undeuterated solvent as an internal reference (CDCl3 at δ 7.26 ppm for 1H, δ 77.16 for 13C NMR). High–resolution mass spectrometry (HR–MS) was performed using a Bruker MicrOTOF–Q II–S1 using electrospray ionization (ESI) technique.

General organic synthetic methods

Reactions were monitored by analytical thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60 F254). Visualization of the developed chromatogram was performed by UV absorbance and aqueous cerium ammonium molybdate. Flash chromatography was performed on Kanto Chemical silica gel 60 N (spherical, neutral, 40–50 µm) with the indicated solvent systems.

Materials

Compound 20 (> 99% ee) and pyridinium p-toluenesulfonate (PPTS) stored in the freezer were used. trimethylamine (Et3N), 2,6-lutidine, anhydrous magnesium sulfate (MgSO4), ethylenediamine, ammonium acetate (NH4OAc), titanium(III) chloride (20% aqueous solution), potassium osmate(VI) dihydrate (K2OsO2(OH)2), palladium on carbon (10%, Pd/C), ethanol (EtOH), methanol (MeOH) and anhydrous solvents for organic synthesis, including CH2Cl2, tetrahydrofuran (THF) and acetonitrile (MeCN) were purchased from FUJIFILM Wako Pure Chemical Co. Triisopropylsilyl trifluoromethanesulfonate (TIPSOTf), scandium(III) trifluoromethanesulfonate (Sc(OTf)3) and 1-dodecyne were purchased from Tokyo Chemical Industry Co. n-butyllithium solution and methyllithium solution were purchased Kanto Chemical Co. tert-Butyldimethylsilyl chloride (TBSCl), dodecylmagnesium bromide solution (C12H25MgBr), N-methylmorpholine oxide (NMO), Gentamicin sulfate, anhydrous 2-methyl-THF for organic synthesis and phenylmagnesium bromide solution (PhMgBr) were purchased from Sigma-Aldrich Co. Gram–positive and Gram–negative bacterial reference strains, including Stapylococcus aureus ATCC 29,213, Enterococcus faecium ATCC 35,667, Escherichia coli DH5 α, Klebsiella pneumoniae ATCC 10,031, Pseudomonas aeruginosa PAO1, and Acinetobacter baumannii ATCC 17,978, S. aureus MRY04-1385, E. faecium MRY05-0006, E. coli MRY13-0331, K. pneumoniae MRY12-0017, P. aeruginosa MRY09-1249, A. baumannii MRY12-0277 were purchased from the American Type Culture Collection. Silica gel plates (60F–254) for thin layer chromatography were purchased from Merck. Silica gel 60 N (230–400 mesh) for flash chromatography was purchased from Kanto Chemical. All reagents were used without further purification. All reactions were carried out in flame–dried glassware under a nitrogen atmosphere with dry solvents. Unless stated otherwise, commercial grade reagents were used without further purification.

Experimental procedures

Synthesis of 22. TBSCl (5.24 g, 23.2 mmol) and Et3N (6.5 mL, 46.2 mmol) were added to a solution of cyclopentenone 2020,21 (2.97 g, 23.2 mmol) in THF (77 mL). The reaction mixture was stirred at room temperature. After 24 h, the resulting mixture was quenched with saturated aqueous NH4Cl. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined extracts were washed with brine then dried with MgSO4. Concentration in vacuo afforded a residue, which was purified by column chromatography (hexane/EtOAc 3:1 → 1:1) to give 22 (4.86 g, 86%) as a colorless-pale yellow oil. TLC (Hexane:EtOAc, 1:3 v/v): Rf = 0.73; [α]D20 =  + 11.2 (c = 1.0 in CHCl3); 1H NMR (400 MHz, CDCl3): δ 0.080 (s, 3H), 0.085 (s, 3H), 0.92 (s, 9H), 1.91 (d, J = 5.2 Hz, 1H), 2.37 (dd, J = 2.0, 18.8 Hz, 1H), 2.86 (dd, J = 6.0, 18.8 Hz, 1H), 4.37–4.38 (m, 2H), 4.99 (brs, 1H), 7.37–7.38 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ − 5.35, − 5.32, 18.4, 26.0 (3C), 45.8, 58.0, 68.9, 148.4, 155.9, 204.8 ppm; HR–MS (ESI–TOF): m/z calcd. for C12H23O3Si ([M + H]+), 243.1411; found, 243.1415.

Synthesis of 19. TIPSOTf (1.80 mL, 6.72 mmol) was added to a stirred solution of 22 (1.36 g, 5.61 mmol) and 2,6-lutidine (0.97 mL, 8.42 mmol) in CH2Cl2 (19 mL) at room temperature under argon atmosphere. After 1.5 h, the resulting mixture was quenched with saturated aqueous NH4Cl. The resulting mixture was extracted with EtOAc (2 × 100 mL), and the extracts were washed with brine then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 25:1 → 15:1) to give 19 (2.23 g, 99%) as a colorless oil. TLC (Hexane:EtOAc, 2:1 v/v): Rf = 0.90; [α]D20 =  + 17.5 (c = 1.0 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.078 (s, 3H), 0.085 (s, 3H), 0.92 (s, 9H), 1.06–1.12 (m, 21H), 2.39 (dd, J = 2.0, 18.2 Hz, 1H), 2.81 (dd, J = 5.8, 18.2 Hz, 1H), 4.37 (t, J = 2.0 Hz, 2H), 4.99–5.03 (m, 2H), 7.32 (q, J = 2.2, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ − 5.31, − 5.30, 12.2 (3C), 18.04 (4C), 18.05 (3C), 18.4 (2C), 25.9, 46.8, 58.0, 69.2, 147.2, 157.2, 205.1 ppm; HR–MS (ESI–TOF) : m/z C21H43O3Si2 ([M + H]+) calcd. for 399.2745, found 399.2741.

Synthesis of 23. Sc(OTf)3 (173 mg, 0.352 mmol) and H2O (2.5 mL, 140 mmol) were added to a stirred solution of 19 (2.80 g, 7.04 mmol) in MeCN (70 mL) at room temperature, and the mixture was stirred at same temperature. After 5.5 h, the reaction mixture was quenched with saturated aqueous NaHCO3. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined extracts were washed with brine then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 4:1 → 2:1) to give 23 (1.53 g, 77%) as a colorless oil. TLC (Hexane:EtOAc, 4:1 v/v): Rf = 0.30; [α]D20 =  + 29.7 (c = 1.0 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.05–1.16 (m, 21H), 2.11–2.15 (m, 1H), 2.39 (dd, J = 2.1, 18.3 Hz, 1H), 2.83 (dd, J = 6.0, 18.6 Hz, 1H), 4.34–4.45 (m, 2H), 5.02–5.05 (m, 1H), 7.31–7.32 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 12.2 (3C), 18.03 (4C), 18.06 (2C), 46.5, 57.6, 69.3, 145.5, 157.7, 206.1 ppm; HR–MS (ESI–TOF) : m/z C15H28O3SiNa ([M + Na]+) calcd. for 307.1700, found 307.1697.

Synthesis of 25. TBSONH228 (775 mg, 5.26 mmol) and MgSO4 (169 mg, 1.40 mmol) were added to a stirred solution of 23 (1.0 g, 3.51 mmol) and PPTS (88 mg, 0.351 mmol) in toluene (3.5 mL) at room temperature, and the mixture was stirred at 100 °C. After 1.5 h, the reaction mixture was quenched with saturated aqueous NaHCO3. The resulting mixture was extracted with EtOAc (2 × 15 mL). The combined extracts were washed with brine then dried with MgSO4. Concentration under vacuum afforded a crude residue 24 (1.98 g), which was immediately used without any purification. crude residue 24: 1H NMR (400 MHz, CDCl3) δ 0.15 (d, J = 13.5 Hz, 3H), 0.18 (d, J = 12.9 Hz, 3H), 0.93 (s, 9H), 1.07–1.14 (m, 21H), 2.51 (dd, J = 2.2, 18.4 Hz, 1H), 2.73 (brs,1H), 3.15 (dd, J = 6.5, 18.4 Hz, 1H), 4.37 (dd, J = 6.5, 12.7 Hz, 1H), 4.45 (dd, J = 5.2, 14.2 Hz, 1H), 4.95–4.98 (1H, m), 6.30–6.31 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ − 5.1 (2C), 12.2 (3C), 18.1 (2C), 18.4 (4C), 26.2 (4C), 37.4, 59.2, 72.6, 141.8, 141.9, 167.3 ppm.

Dess-Martin periodinane (DMP) (1.78 g, 4.21 mmol) was added to a solution of a crude residue 24 in CH2Cl2 (18 mL) at room temperature, and the mixture was stirred at same temperature. After 0.5 h, the reaction mixture was quenched with aqueous NaHCO3 and Na2S2O3. The resulting mixture was extracted with CHCl3 (2 × 30 mL). The combined extracts were washed with brine then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 30:1) to give 25 (1.14 g, 79%, 2 steps) as a pale-yellow oil. TLC (Hexane:EtOAc, 6:1 v/v): Rf = 0.65; [α]D20 =  + 113.1 (c = 0.51 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.190 (s, 3H), 0.195 (s, 3H), 0.95 (s, 9H), 1.05–1.17 (m, 21H), 2.64 (dd, J = 2.9, 18.3 Hz, 1H), 3.26 (dd, J = 6.9, 18.5 Hz, 1H), 5.04–5.07 (m, 1H), 7.12–7.13 (d, J = 2.9 Hz 1H), 10.0 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ − 5.1 (2C), 12.2 (3C), 18.1 (4C), 18.4 (2C), 26.2 (4C), 37.6, 72.3, 138.9, 152.1, 163.8, 188.0 ppm; HR–MS (ESI–TOF) : m/z C21H42O3NSi2 ([M + H]+) calcd. for 412.2698, found 412.2703.

Synthesis of 27. n-BuLi (1.59 M in n-hexane, 1.14 mL, 1.82 mmol) was dropwised to a solution of 1-dodecyne (0.39 mL, 1.82 mmol) in 2-methyl-THF (6 mL) at − 20 °C under argon atmosphere, and the mixture was stirred at same temperature. After 0.5 h, a solution of aldehyde 25 (500 mg, 1.21 mmol) in 2-methyl-THF (4 mL) was added to the resulting solution, and the mixture was stirred further an hour at same temperature. the reaction mixture was quenched with saturated aqueous NH4Cl. The resulting mixture was extracted with EtOAc (2 × 20 mL). The combined extracts were washed with brine then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 40:1) to give 27 (644 mg, 92%) as a pale-yellow syrup. TLC (Hexane:EtOAc, 6:1 v/v): Rf = 0.65; 1H NMR (400 MHz, CDCl3) δ 0.168–0.17 (6H), 0.195 (s, 3H), 0.87 (t, J = 6.9 Hz, 3H), 0.93–0.94 (9H), 1.05–1.14 (m, 21H), 1.26–1.43 (m, 14H), 1.48–1.51 (m, 2H), 2.21–2.28 (m, 2H), 2.54 (dd, J = 2.2, 18.4 Hz, 1H), 3.15–3.22 (m, 1H), 3.63 (d, J = 5.3 Hz, 0.5H), 3.82 (d, J = 5.3 Hz, 0.5H), 4.94–4.98 (m, 1H), 5.19–5.20 (m, 0.5H), 5.28–5.29 (m, 0.5H), 6.45–6.46 (m, 0.5H), 6.49–6.50 (m, 0.5H) ppm; 13C NMR (100 MHz, CDCl3) δ − 5.2, 12.1, 12.2, 14.3, 18.1, 18.4, 18.89, 18.9, 22.8, 26.18, 22.19, 28.7, 29.1, 29.3, 29.5, 29.7, 29.8, 32.0, 37.7, 37.8, 59.5, 59.6, 72.21, 72.24, 77.7, 77.9, 86.9, 87.0, 141.7, 141.8, 143.2, 143.6, 166.2, 166.5 ppm; HR–MS (ESI–TOF) : m/z C33H64O3NSi2 ([M + H]+) calcd. for 578.4419, found 578.4416.

Synthesis of 28. Sc(OTf)3 (12.8 mg, 0.0259 mmol) and H2O (0.19 mL, 10.4 mmol) were added to a stirred solution of 27 (300 mg, 0.519 mmol) in MeCN (5.2 mL) at room temperature, and the mixture was stirred at same temperature. After an hour, the reaction mixture was quenched with saturated aqueous NaHCO3. The resulting mixture was extracted with CHCl3 (2 × 20 mL). The combined extracts were washed with brine then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 6:1 → 4:1) to give 28 (217 mg, 91%) as a pale-yellow syrup. TLC (Hexane:EtOAc, 4:1 v/v): Rf = 0.45; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.05–1.17 (m, 21H), 1.22–1.39 (m, 14H), 1.49–1.54 (m, 2H), 2.23–2.28 (m, 2H), 2.59 (dd, J = 2.3, 18.3 Hz, 1H), 3.15 (brs, 0.5H), 3.18–3.25 (m, 1H), 3.36 (brs, 0.5H), 4.34–4.45 (m, 2H), 4.97–5.02 (m, 1H), 5.20 (s, 0.5H), 5.27 (s, 0.5H), 6.53–6.54 (m, 0.5H), 6.55–6.56 (m 0.5H), 7.18 (brs, 0.5H), 7.22 (brs, 0.5H) ppm; 13C NMR (100 Mz, CDCl3) δ 12.2, 14.3, 18.1, 18.9, 19.0, 22.8, 28.7, 29.1, 29.3, 29.5, 29.7, 29.8, 32.0, 37.3, 58.7, 58.9, 72.2, 72.3, 77.7, 77.9, 87.3, 141.5, 141.6, 162.6, 162.9 ppm; HR–MS (ESI–TOF) : m/z C27H50O3NSi ([M + H]+) calcd. for 464.3554, found 464.3547.

Synthesis of 29/epi-29. A solution of NH4OAc (830 mg, 10.8 mmol) in H2O (1.5 mL) and TiCl3 (20% aq., 2.66 mL, 3.45 mmol) were added to a stirred solution of 28 (200 mg, 0.431 mmol) in THF (5.1 mL) at room temperature, and the mixture was stirred at 60 °C. After 30 min, the reaction mixture was quenched with H2O. The resulting mixture was extracted with EtOAc (2 × 20 mL). The combined extracts were washed with saturated aqueous NaHCO3 and brine then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 13:1 → 10:1) to give 29 (39.9 mg, 41%) and epi-29 (32.8 mg, 34%) as a pale-yellow viscous oil.

29; TLC (Hexane:EtOAc, 3:1 v/v): Rf = 0.55; [α]D20 =  + 12.0 (c = 1.0 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 6.8 Hz, 3H), 1.05–1.19 (m, 21H), 1.27–1.40 (m, 14H), 1.50–1.57 (m, 2H), 2.26 (td, J = 2.0, 7.1 Hz, 2H), 2.46 (dd, J = 2.1, 18.3, 1H), 2.85–2.91 (m, 2H), 3.36 (brs, 0.5H), 5.03–5.06 (m, 1H), 5.21–5.22 (m, 1H), 7.43 (q, J = 3.4 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 12.2, 14.3, 18.02, 18.05, 18.9, 22.8, 28.6, 29.0, 29.3, 29.4, 29.6, 29.7, 32.0, 46.8, 57.8, 68.8, 87.7, 145.4, 168.6, 204.9 ppm; HR–MS (ESI–TOF) : m/z C27H49O3Si ([M + H]+) calcd. for 449.3445, found 449.3441.

epi-29; TLC (Hexane:EtOAc, 3:1 v/v): Rf = 0.45; [α]D20 =  + 25.6 (c = 1.0 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.05–1.18 (m, 21H), 1.26–1.39 (m, 14H), 1.48–1.54 (m, 2H), 2.25 (dt, J = 2.0, 7.1 Hz, 2H), 2.43 (dd, J = 2.1, 18.3 Hz, 1H), 2.80 (dd, J = 5.8, 18.3 Hz, 1H), 3.05 (d, J = 4.3 Hz, 1H), 5.02–5.05 (m, 1H), 5.26 (brs, 1H), 7.43 (q, J = 3.5 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 12.1, 14.3, 18.02, 18.04, 18.9, 22.8, 28.6, 29.0, 29.3, 29.5, 29.7, 29.8, 32.0, 46.8, 58.1, 68.9, 87.7, 145.1, 168.9, 205.4 ppm; HR–MS (ESI–TOF) : m/z C27H48O3SiNa ([M + Na]+) calcd. for 471.3265, found 471.3251.

Synthesis of 30. K2OsO2(OH)4 (2.3 mg, 6.13 µmol) and NMO (42.8 mg, 0.306 mmol) were added to a stirred solution of 29 (55 mg, 0.122 mmol) in THF–H2O (2.5 mL, 10:1) at room temperature, and the mixture was stirred at 40 °C. After 3.5 h, the reaction mixture was quenched with aqueous Na2S2O3 (10%). The resulting mixture was extracted with EtOAc (2 × 15 mL). The combined extracts were washed with brine (20 mL) then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 4:1) to give 30 (24.3 mg, 41%) as a pale-yellow oil. TLC (Hexane:EtOAc, 2:1 v/v): Rf = 0.40; [α]D20 = − 39.0 (c = 0.10 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.1 Hz, 3H), 1.04–1.20 (m, 21H), 1.22–1.37 (m, 14H), 1.46–1.52 (m, 2H), 2.21 (dt, J = 2.0, 7.1 Hz, 2H), 2.44 (ddd, J = 1.0, 3.7, 20.6 Hz, 1H), 2.58 (brs, 1H), 2.85 (dd, J = 7.4, 19.6 Hz, 1H), 3.34 (brs, 1H), 3.72 (d, J = 10.6 Hz, 1H), 4.35 (d, J = 1.9 Hz), 1H), 4.45–4.48 (m, 1H), 4.55 (d, J = 9.8 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 12.0, 14.3, 179, 18.9, 22.8, 28.6, 29.0, 29.3, 29.5, 29.6, 29.7, 32.0, 44.5, 65.0, 71.5, 81.5, 89.3, 214.2 ppm; HR–MS (ESI–TOF) : m/z C27H49O5Si− ([M-H]−) calcd. for 481.3355, found 481.3346.

Synthesis of (+)-Hygrophorone B12 (4). 10% Pd/C (1.7 mg, 10 w/w%) were added to a solution of 30 (16.8 mg, 0.0348 mmol) in MeOH (1.7 mL), and the reaction mixture was stirred under hydrogen atmosphere (balloon). After 5.5 h, the reaction mixture was filtered through a pad of celite and concentrated under reduced pressure. An obtained crude product (16.7 mg) which was used without any purification was dissolved in EtOH (1.2 mL). 1 M HCl (0.6 mL) was added to the stirred solution at room temperature, and the mixture was stirred at 90 °C. After 30 min, the reaction mixture was quenched with saturated aqueous NaHCO3. The resulting mixture was extracted with EtOAc (2 × 15 mL). The combined extracts were washed with brine (20 mL) then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 1:1) to give (+)-hygrophorone B12 (4) (6.7 mg, 63%, 2 steps) as a white solid. TLC (Hexane:EtOAc, 1:1 v/v): Rf = 0.30; [α]D25 =  + 23.0 (c = 0.10 in MeOH), {ref.17 [α]D27 =  + 20.7 (c = 0.135, MeOH)}, 1H and 13C NMR, and MS spectra were identical to those of natural ( +)-hygrophorone B12; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.9 Hz, 3H), 1.25–1.36 (m, 20H), 1.52–1.62 (m, 2H), 2.07 (d, J = 8.3, 1H), 2.94 (d, J = 7.3, 1H), 3.58 (s, 1H), 3.77 (t, J = 9.6 Hz, 1H), 4.72 (ddd, J = 1.4, 2.2, 7.3 Hz, 1H), 6.30 (dd, J = 1.3, 6.1 Hz, 1H), 7.64 (dd, J = 2.3, 6.1 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 14.3, 22.8, 26.3, 29.5, 29.6, 29.67, 29.7, 29.77, 29.8, 31.4, 32.1, 71.6, 73.5, 76.0, 133.7, 163.7, 207.5 ppm; HR–MS (ESI–TOF) : m/z C18H31O4− ([M-H]−) calcd. for 311.2228, found 311.2219.

Synthesis of 31. 1 M aqueous HCl (0.75 mL) was added to a stirred solution of 30 (11.4 mg, 0.0236 mmol) in ethanol (1.5 mL) at room temperature, and the mixture was stirred at 90 °C. After 30 min, the reaction mixture was quenched with saturated aqueous NaHCO3. The resulting mixture was extracted with EtOAc (2 × 15 mL). The combined extracts were washed with brine (20 mL) then dried with MgSO4. Concentration under vacuum afforded a residue, which was purified by column chromatography (hexane/EtOAc 4:3) to give 31 (6.1 mg, 84%) as a colorless amorphous. TLC (Hexane:EtOAc, 1:1 v/v): Rf = 0.2; [α]D25 = − 13.0 (c = 0.10 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H), 1.25–1.31 (m, 15H), 1.41–1.46 (m, 2H), 2.16 (dt, J = 2.0, 7.1 Hz, 1H), 2.88 (d, J = 7.9, 1H), 2.91 (d, J = 8.8, 1H), 3.83 (s, 1H), 4.55 (dt, J = 1.9, 8.8 Hz, 1H), 4.85 (dt, J = 1.7, 7.6 Hz, 1H), 6.31 (dd, J = 1.5, 6.1 Hz, 1H), 7.64 (dd, J = 2.2, 6.1 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 14.3, 18.7, 22.8, 28.5, 28.9, 29.2, 29.5, 29.6, 29.7, 32.0, 64.9, 71.9, 75.0, 76.0, 89.5, 133.4, 163.9, 206.1 ppm; HR–MS (ESI–TOF) : m/z C18H27O4− ([M-H]−) calcd. for 307.1914, found 307.1915.

Synthesis of 32. 10% Pd/C (0.27 mg, 10 w/w%) were added to a solution of (+)-4 (2.7 mg, 8.64 µmol) in MeOH (1.0 mL), and the reaction mixture was stirred under hydrogen atmosphere (balloon). After an hour, the reaction mixture was filtered through a pad of celite and concentrated under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc 1:1) to give to 32 (2.7 mg, 99%) as a white solid. TLC (Hexane:EtOAc, 1:3 v/v): Rf. = 0.6; [α]D25 = − 26.0 (c = 0.10 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.8 Hz, 3H), 1.25–1.42 (m, 21H), 1.51–1.53 (m, 1H), 1.87 (d, J = 9.7 Hz, 1H), 2.04–2.12 (m, 1H), 2.23–2.54 (m, 4H), 3.13 (s, 1H), 3.60–3.65 (m, 1H), 4.40–4.41 (m, 1H) ppm; 13C NMR (150 MHz, CDCl3) δ 14.3, 22.8, 25.4, 26.0, 29.5, 29.56, 29.66, 29.71, 29.77 (2C), 29.8, 31.8, 32.1, 32.7, 71.3, 72.0, 83.4, 217.3 ppm; HR–MS (ESI–TOF) : m/z C18H33O4− ([M-H]−) calcd. for 313.2384, found 313.2354.