Oxidation of the Meroterpenoid (−)-Terreumol C from the Mushroom

Article Cite This: J. Nat. Prod. 2017, 80, 2652-2658

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Oxidation of the Meroterpenoid (−)-Terreumol C from the Mushroom Tricholoma terreum: Discovery of Cytotoxic Analogues Alex Frichert,† Peter G. Jones,‡ Mark Brönstrup,§ and Thomas Lindel*,† †

Institute of Organic Chemistry, Technical University Braunschweig, Hagenring 30, 38106 Braunschweig, Germany Institute of Inorganic and Analytical Chemistry, Technical University Braunschweig, Hagenring 30, 38106 Braunschweig, Germany § Helmholtz Centre for Infection Research and German Center for Infection Research (DZIF), Inhoffenstraße 7, 38124 Braunschweig, Germany ‡

S Supporting Information *

ABSTRACT: Aiming at the discovery of new cytotoxic meroterpenoids, the chemical reactivity of the natural product (−)-terreumol C from the edible mushroom Tricholoma terreum was investigated. A series of high-yielding oxygenations and brominations at the 10-membered ring were discovered. The regioselectivity of hydroxylation could be guided by installing protecting groups at the hydroquinone section. Dihydroxylation led to the stereoselective formation of a novel tricycle containing an 11-oxabicyclo[5.3.1]undecane system. Several of the compounds proved to be as cytotoxic against cancer cell lines as the natural products terreumols A and C in the single-digit micromolar range. Interestingly, functionalization of the southern rim formed by carbons C5−C6−C7−C8 is tolerated without much loss of cytotoxicity.

T

he terreumols A−D (1−4) are meroterpenoids that were isolated from the fruiting bodies of the mushroom Tricholoma terreum by Ji Kai Liu and co-workers.1 Compounds 1, 3, and 4 exhibited cytotoxic activity against five human cancer cell lines in the single- to double-digit micromolar range (IC50), comparable to cisplatin.1 T. terreum is considered edible, although there has been some debate on whether this is still appropriate.2,3 In addition to the terreumols (1−4), T. terreum has provided more than 20 triterpenoids of the saponaceolide and terreolide series,4 among them the cytotoxic saponaceolide B, which occurred in a concentration about 100 times higher than the terreumols. Recently, we reported the total synthesis of terreumols A (1) and C (3), which provided terreumol C (3) in quantities above 100 mg, sufficient for further chemical studies.5 Previously, only minor amounts were available (5.9 mg of 1 and 1.7 mg of 3 from 1 kg of dried fruiting body).1 The key step of our synthesis was a ring-closing metathesis, which led to the formation of the 10membered ring with full control of the (Z)-configuration of the trisubstituted double bond between C6 and C7.

chemical reactivity of a given scaffold. C−H functionalization of natural products has been investigated only rarely. Regarding terpenoids, examples include the allylic oxidation of the cembranolide sarcophine7 and of the triterpenoids sipholenol A and sipholenone A8 with selenium dioxide. In this paper, we report on the behavior of (−)-terreumol C (3) under oxidative conditions.5 We included the bis-(tert-butyldimethylsilyl) (TBS)-protected analogue 5, because the bulkiness of the TBS groups could alter the regioselectivity of certain reactions. Our goals were to gain insight into the reactivity of the positions of the southern rim and to assess the in vitro biological activities of new derivatives. The southern rim of (−)-terreumol C (3) exhibits seven allylic hydrogen atoms, with the diastereotopic H-5α and H-5β also being benzylic. Given the conformational restriction of the 10membered ring, it was not clear at which position an oxygenation could be achieved. Moreover, multiple oxidations could take place. Hydroxylated derivatives of (−)-terreumol C (3) might also undergo intramolecular cyclization. Encouragingly, epoxidation of (−)-terreumol C (3) to A (1) had proceeded with perfect diastereoselectivity.5 Regarding the oxidative conditions to be investigated, we focused on the reactions of 3 and 5 with SeO2, Pd(II)/p-benzoquinone or tert-butyl hydroperoxide (TBHP), PhSeBr/LiOAc, N-bromosuccinimide (NBS) under ionic and radical conditions, dihydroxylation, Mn(III)/O2/ TBHP, and ceric ammonium nitrate (CAN).

The construction of libraries based on natural products needs to be intensified6 and relies on the detailed understanding of the

Received: March 17, 2017 Published: September 28, 2017

© 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Reaction with Selenium-Based Electrophiles. To obtain an impression of the overall reactivity of (−)-terreumol C (3), we treated 3 with SeO2 (0.4 equiv)/TBHP (3 equiv) in dichloromethane (DCM) between 0 and 23 °C. We isolated (5S)hydroxyterreumol C (6), which was formed in perfect diastereoselectivity (Scheme 1), in a good yield of 53%. All

of the methyl group attached to C6, obtaining the primary allylic alcohol 7 in a good yield of 64%. Thus, it was possible to alter the regiochemical outcome of the SeO2-mediated oxidation by protecting the hydroquinone moiety. We desilylated 7 (Et3N· 3HF) to obtain 15-hydroxyterreumol C (8). When the Riley oxidation was performed a second time using allylic alcohol 7 as starting material, we obtained the corresponding α,β-unsaturated aldehyde (see the Supporting Information). Mechanistically, the regioselectivity of the Riley oxidation showed the expected pattern in the absence of the TBS groups. In the first step, SeO2 must have undergone an ene reaction on the accessible face of the trisubstituted (Z)-double bond of 5. In the presence of the bulky TBS groups, the initial ene reaction must have taken place at the (15-H)−C15−C6−C7 system, followed by [2,3]-sigmatropic rearrangement and elimination of Se(OH)2. It is known that SeO2-mediated oxidation occurs at the higher substituted side in trisubstituted olefins, with a preference for CH2 rather than CH3 groups and for endo- rather than exocyclic positions.9 The oxygenation pattern of terreumol D (4), carrying a hydroxy group at C8, was not observed, although there are a few known cases where SeO2-mediated oxidation occurs at the less substituted side of a trisubstituted double bond, albeit only as a side reaction.10 Treatment of 5 with catalytic amounts of Pd(OAc)2 (0.1 equiv) in the presence of p-benzoquinone (2 equiv) in acetic acid11 afforded the TBS-protected bis(acetoxy) compound, which was desilylated to afford the masked aldehyde 9 (51% over two steps). Geminal bisacetoxylation in the allylic position is unprecedented. Probably, the π-allyl-Pd(II) complex was formed diastereoselectively on the accessible side with C15−C6−C7 of the trisubstituted double bond, followed by nucleophilic attack by acetate from the back side, which was only possible at C15, not at C7, followed by repetition of the same reaction. Pd-catalyzed acetoxylation has not always preferred the methyl side chain. In the case of limonene derivatives, allylic oxidation at the ring carbons was observed.11 Other methods, which had allowed the conversion of alkenes to α,β-unsaturated ketones, were less effective. Treatment of 5 with TBHP (5.0 equiv)/Pd(OH)2/C (0.1 equiv) in the presence of Cs2CO312 led to epoxidation of the C6C7 double bond, affording TBS-protected terreumol A (14%, 73% brsm). The same epoxide was obtained in 9% yield (69% brsm) when using O2, TBHP (10 equiv), and Mn(OAc)313 (0.5 equiv, see the Supporting Information). We also tried to react TBS-protected terreumol C (5) with PhSeBr (Scheme 3). In the presence of AgOAc, allylselenium compound 10 was formed, with the phenylselenyl group placed at the exocyclic methylene group C15. Silver bromide had precipitated. Oxidation of 10 with H2 O 2 /pyridine and desilylation afforded allylic alcohol 11 as a 1:1 mixture of diastereomers. A similar observation was made by Kobayashi et al. in the PhSeCl-mediated allylic oxidation of prenyl partial structures.14 When using LiOAc instead of AgOAc, a bromo substituent was introduced at the former methyl carbon C15. Subsequent desilylation provided the allyl bromide 12. In the absence of added acetate, treatment of 5 with PhSeBr in acetic acid led to decomposition. A scheme of the possible mechanisms leading to 10, 11, and 12 is given in the Supporting Information. In the presence of silver acetate, PhSeOAc is formed in situ,15 which is probably attacked by the trisubstituted C6C7 double bond of 5, forming a seleniranium ion or the corresponding tertiary carbenium ion. Deprotonation by acetate would afford the secondary

Scheme 1. Riley Oxidation of (−)-Terreumol C (3) Leads to Hydroxylation at C5a

a

Key NOESY correlations and distances of selected hydrogen pairs (Spartan software, MMFF94 force field) are given.

NOESY correlations of 6 (600 MHz, CDCl3) agree with the calculated conformation (Spartan software, MMFF94 force field) given in Scheme 1. In particular, we observed an intense NOE between H-5α and the epoxide methine 11-H. Thus, the hydroxy group had been introduced in the 5β-position. The conformation of 6 remained unchanged when compared to (−)-terreumol C (3). The regioselectivity changed when bis-TBS-protected terreumol C (5) was reacted (Scheme 2). Now we found hydroxylation Scheme 2. Oxidation of Bis-TBS-Protected (−)-Terreumol C (5) by SeO2 and by Catalytic Pd(II)/Benzoquinone

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Scheme 3. Effect of Lithium and Silver Acetate on the Reaction of TBS-Protected (−)-Terreumol C (5) with Phenylselenyl Bromide

Scheme 4. Bromination of TBS-Protected (−)-Terreumol C under Ionic and Radical Conditions

allylphenylselane, which could undergo [1,3]-sigmatropic rearrangement to allylphenylselane 10. Diastereomeric allylic alcohols 11 would be formed after oxidation of 10 to the allylphenylselenoxide, which could undergo rotation around the C6−C15 bond prior to [2,3]-sigmatropic rearrangement, as has been observed starting from alkylidene cyclohexane derivatives.16 In this way the selenoxide moiety would be placed on either side of the allyl system, explaining the formation of both diastereomers of 11. Formation of the primary allyl bromide 12 when using LiOAc instead of AgOAc may take place by reaction of the allylphenylselane 10 or of its 1,3-rearranged precursor with then-present PhSeBr. Tiecco et al. have proposed that selenylation of allylphenylselane occurs via diselanium bromides.17 Reaction with another equivalent of PhSeBr would lead to dibromoselanes being in equilibrium with phenylselenonium bromides. The concluding step would be an allylic nucleophilic attack of bromide at a primary or secondary phenylselenonium bromide.18 Bromination. Scheme 4 outlines the bromination of TBSprotected (−)-terreumol C under ionic and radical conditions. Treatment of 5 with NBS in acetone/water afforded a mixture of allylic bromide 13 and the brominated quinone 14. Desilylation of 13 and oxidation to the quinone occurred slowly, and it was possible to obtain the allylic bromide 13 almost quantitatively when quenching the reaction mixture with Na2S2O3 after 25 min. After desilylation, the allyl bromide 15 was obtained, of which we were able to obtain crystals suitable for an X-ray analysis that confirmed the absolute configuration.19 The compound crystallized as a dichloromethane solvate. The bromo substituent and the exocyclic methylidene group of allyl bromide 15 subtend a dihedral angle of −112° (C15−C6−C7−Br, Figure 1). The molecule exhibits an intramolecular hydrogen bond O1−H···O5. We did not observe the bromohydrin, which is expected to be formed in reactions of alkenes with aqueous NBS, but which may nevertheless be an intermediate undergoing dehydration to the exocyclic methylidene group of 15. The reaction of 5 with NBS under radical conditions (AIBN, CCl4, 40 °C to reflux) was less clean and afforded three main products, accounting for about half of the theoretical yield (Scheme 4). The methyl side chain and the position C7 were always brominated (products 16, 17, and 18). The product 16 (17%) contains three bromine substituents, two of them in a

Figure 1. Thermal ellipsoid plot (50% probability level) of compound 15 in the crystal.

geminal arrangement at the former methyl carbon C15. The endocyclic double bond has moved into conjugation with the arene ring (C5C6). The configuration of the C5C6 double bond was established on the basis of NOE data for both 16 and 17. Compound 16 was inseparable from the dibrominated analogue 17 (12%). The third product 18 was isolated as a pure substance (17% yield) and contained an exocyclic bromomethylidene group, to which we assigned the (Z)-configuration, because the transannular NOE correlation between 11-H and 15H is consistent with a conformation corresponding to that of 15 (Figure 1). Dihydroxylation. We also investigated the possibility of accessing a 6,7-dihydroxyterreumol C, which was found recently in the plant Salvia chinensis.20 The alkene moiety of 5 underwent a clean electrophilic addition under conditions of cis-dihydroxylation (cat. K2OsO2(OH)4, K3Fe(CN)6). Here we could isolate the tricyclic product 20 in a good yield of 74%. Compound 21 was obtained after double desilylation of 20 (80%, NEt3·3HF). We observed strong NOESY correlations between H-5β, H-8β, and H-11α, which are in full agreement with the low-energy (Spartan, MMFF94) conformation shown in Scheme 5. The axial position of 7-OH can be derived from NOESY correlations of H2654

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5α with H-7α. The tricycle 21 appears to be favored compared to 6,7-dihydroxyterreumol C. Scheme 5. Cis-Dihydroxylation of TBS-Protected (−)-Terreumol C (5) Afforded the Novel Tricycle 21 after Desilylation, the Structure of Which Was Established by NOESY Correlations

Figure 2. p-Benzoquinone Analogues 22 and 23 of Terreumol C (3) and Terreumol A (1).

MIC value of compound 12 against MRSA RKI was 13 μg/mL. For terreumols A (1) and C (3) we even found a slight growth promotion (above 25% at 50 μM) toward MRSA. More promising results were obtained when testing for antiproliferative activity. The natural products terreumols A (1) and C (3) had already been tested by Liu and co-workers following isolation of the substances, who found single- or double-digit micromolar activities. We tested our synthetic samples of 1 and 3 against the mouse fibroblast cell line L929, the human cervix carcinoma cell line KB-3-1, the human breast cancer cell line MCF-7, and the conditional immortalized human fibroblast cell line FS4-LTM using a WST-1 assay that quantifies the metabolic activity of the cell population. Although this parameter reflects various cellular processes, we use the term cytotoxicity throughout the article for this bioactivity. We found that 1 and 3 were equipotent and can generally confirm the findings by Liu et al.1 (see Table 1). For example, we found IC50 values of 17 and 15 μM against MCF-7, respectively, compared to 16.1 and 24.6 μM reported by Liu.

The tetrahydropyran ring of the 11-oxabicyclo[5.3.1]undecane moiety of 20 displays a chair conformation. The longest bridge of the bicycle is attached in the 2,6-diaxial positions of the tetrahydropyran ring. It can be assumed that the (Z)-double bond of TBS-protected (−)-terreumol C (5) had been attacked from the same side as for the epoxidation of (−)-terreumol C (3) to terreumol A (1). Thus, the (6S,7R,10R,11S) diol 19 becomes an intermediate. From 19, the relative configuration of the product 21 can only be reached by attack of the tertiary alcohol at the tertiary epoxide carbon with inversion at C10, which means that the product has (6S,7R,10S,11S) configuration. Apart from compound 21, only a few compounds contain an oxabicyclo[5.3.1]undecane-2,8-diol partial structure. The parent compound has been accessed by double epoxidation of (E,Z)-1,5-cyclodecadiene and hydrolysis.21 Among natural products, the corresponding partial structure has been found in a sesquiterpene from Cremanthodium ellisii (Asteraceae), lacking the anellated benzene ring but including the two methyl substituents.22 Furthermore, an oxabicyclo[5.3.1]undecane-2,8-diol partial structure occurs in the diterpenoids australins A and D from the soft coral Cladiella australis.23 Biological Activity. Having synthesized an interesting series of oxidized derivatives directly from the natural product (−)-terreumol C (3), we were able to assess antimicrobial effects and in vitro cytotoxicity. We tested terreumols A (1) and C (3) and all newly oxidized (−)-terreumol C derivatives with unprotected hydroquinone moieties. We also included the quinone analogue 22 of (−)-terreumol C (3), which had been obtained quantitatively by oxidation of (−)-terreumol C (3) with CAN in MeCN/H2O (see the Supporting Information), and the quinone analogue 23 of terreumol A (1, Figure 2).5 Antimicrobial activities were investigated against the Grampositive bacteria MRSA (methicillin-resistant Staphylococcus aureus, DCM 11822, ICB25701, RKI) and Enterococcus faecium, against the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, and against the yeast Candida albicans. All compounds proved to be inactive, with the exception of 12, 8, 9, and 22, which showed EC50 values against the MRSA RKI in the double-digit micromolar range (22, 50, 54, and 64 μM, respectively). The

Table 1. Cytotoxicity of Terreumols A (1) and C (3) and of Our Postsynthesis Oxidation Products against Four Cancer Cell Linesa

a

compound

L929

KB-3-1

MCF-7

FS4-LTM

1 3 6 8 9 12 15 21 22 23 auranofin staurosporine

9.9 9.7 4.0 40 2.7 10 2.5 >100 13 18 1.2 100 14 20 1.2 100 27 24 2.8 1.8

29 28 29 42 2.5 29 14 >100 28 31 3.1 2.8

IC50 values are given in μM.

The allyl bromide 15 and the diacetyl acetal 9 proved to be slightly more cytotoxic than the natural products 1 and 3. (5S)Hydroxyterreumol C (6) was of comparable cytotoxicity to 1. The quinones 22 and 23 were less active than their hydroquinone counterparts 3 and 1. The cytotoxicity of (−)-terreumol C was diminished when the methyl side chain C15 was hydroxylated (8) or brominated (12). The tricyclic compound 21 showed no cytotoxicity. Although it is difficult to derive firm structure−activity relationships (SARs) from the phenotypic biological data, it is striking that the derivatives with enhanced bioactivity (15, 9, and 6) are potential electrophiles, either through the formation of an aldehyde or through an activated allylic position (Figure 3). As the target and therefore the precise binding site of terreumols are unknown at this stage, we cannot conclude whether the increased 2655

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stirred at 0 °C for 30 min and then warmed to room temperature and stirred for a further 2 h. After filtration of the reaction mixture through a plug of Celite, the filtrate was concentrated under reduced pressure. Purification by column chromatography (petroleum ether/EtOAc, 5:1) afforded 7 (6.6 mg, 12.025 μmol, 64%) as a colorless oil. General Procedure for the Desilylation of the TBS-Protected Hydroquinones. To a solution of TBS-protected hydroquinone (1 equiv) in tetrahydrofuran (THF) (1 mL) was added Et3N·3HF (10 equiv). The mixture was stirred at room temperature for 16 h and concentrated under reduced pressure. (1aR,11aS,E)-7,10-Dihydroxy-5-(hydroxymethyl)-8-methoxy1a-methyl-1a,3,6,11a-tetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (8). The bicycle 7 (6.1 mg, 11.447 μmol) was treated according to the general procedure for the desilylation of the TBS-protected hydroquinones. Purification by column chromatography (petroleum ether/EtOAc, 1:2) afforded 8 (3.0 mg, 9.365 μmol, 82%) as a yellow oil. ((1aR,11aS,E)-7,10-Dihydroxy-8-methoxy-1a-methyl-11-oxo1a,2,3,6,11,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren5-yl)methylene diacetate (9). Pd(OAc)2 (0.2 mg, 0.938 μmol) and p-benzoquinone (2.0 mg, 18.766 μmol) were dissolved in AcOH (0.5 mL), and the mixture was stirred for 30 min at 80 °C. The mixture was cooled to room temperature, a solution of bicycle 5 (5.0 mg, 9.383 μmol) in AcOH (0.5 mL) was added dropwise, and the mixture was stirred at 80 °C for 5 h. Distilled water (5 mL) and tert.-butyl methyl ether (TBME) (5 mL) were added, and the layers were separated. The aqueous layer was extracted with TBME (3 × 5 mL), and the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was treated according to the general procedure for the desilylation of the TBS-protected hydroquinones. Purification by column chromatography (petroleum ether/EtOAc, 2:1) afforded 9 (2.4 mg, 4.756 μmol, 51%) as a yellow oil. (1aR,11aS,E)-7,10-Bis((tert-butyldimethylsilyl)oxy)-8-methoxy-1a-methyl-5-((phenylselanyl)methyl)-1a,3,6,11atetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (10). AgOAc (21.9 mg, 131.364 μmol) and PhSeBr (8.9 mg, 37.533 μmol) were added to a solution of bicycle 5 (10 mg, 18.766 μmol) in AcOH (1 mL). The resulting mixture was stirred at 60 °C for 15 h. The mixture was cooled to room temperature, and saturated aqueous NaHCO3 (10 mL) and DCM (10 mL) were added. Layers were separated and the aqueous layer was extracted with DCM (3 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/ EtOAc, 20:1 to 10:1) afforded 10 (8.3 mg, 12.065 μmol, 64%) as a colorless oil. (1aR,11aS)-7,10-Bis((tert-butyldimethylsilyl)oxy)-4-hydroxy8-methoxy-1a-methyl-5-methylene-1a,3,4,5,6,11ahexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one. Compound 10 (7.2 mg, 10.466 μmol) was dissolved in THF (0.5 mL), followed by addition of aqueous H2O2 (30%, 39 μL, 0.385 mmol) and pyridine (7.6 μL). The resulting mixture was stirred at room temperature for 2 h. Aqueous NaHCO3 (10 mL) and DCM (10 mL) were added. Layers were separated, and the aqueous layer was extracted with DCM (3 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/EtOAc, 10:1 to 3:1) afforded (1aR,11aS)-7,10-bis((tert-butyldimethylsilyl)oxy)-4-hydroxy8-methoxy-1a-methyl-5-ethylene-1a,3,4,5,6,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (4.6 mg, 8.381 μmol, 80%) as a 1:1 mixture of diastereomers as a colorless oil. (1aR,11aS)-4,7,10-Trihydroxy-8-methoxy-1a-methyl-5-methylene-1a,3,4,5,6,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (11). (1aR,11aS)-7,10-Bis((tertbutyldimethylsilyl)oxy)-4-hydroxy-8-methoxy-1a-methyl-5-ethylene1a,3,4,5,6,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)one (3.9 mg, 7.106 μmol) was treated according to the general procedure for the desilylation of the TBS-protected hydroquinones. Purification by column chromatography (petroleum ether/EtOAc, 1:2 to EtOAc) afforded 11 (1.9 mg, 5.931 μmol, 84%) as 1:1 mixture of diastereomers as a yellow solid.

Figure 3. SAR regarding the cytotoxicity of terreumol C derivatives.

activity of 15, 9, and 6 stems from a covalent locking of the molecules in their primary binding site or from additional offtargets that are preferentially bound by the derivatives. Hydroxylation of C8, as prevalent in terreumol D (4), does not diminish the cytotoxicity, as observed by Liu.1 Functionalization of the methyl group C15 appears to be more delicate, as shown by the drop in activity of 8 and 12. The absence of cytotoxicity of tricycle 21 is probably a consequence of the different conformation of the 10-membered ring enforced by the oxygen bridge. In summary, we have discovered several selective oxygenations and brominations of the fungal natural product terreumol C (3), which led us to novel, natural-product-like analogues. A directed synthesis of each of the new derivatives would probably have been less efficient than by choosing terreumol C itself as starting material. The terreumols A (1) and C (3) did not exhibit convincing antimicrobial activity. However, the natural products themselves and several of the derivatives reached cytotoxicities in the single-digit micromolar range. As a guideline for further SAR studies, the hydroquinone moiety should not be oxidized to the quinone, and the conformation of the 10-membered ring should be retained.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded with a Bruker AV-II 600 (600 MHz for 1H, 150 MHz for 13C) spectrometer at 299 K. Chemical shifts are given in ppm (δ scale) and referenced to tetramethylsilane or the residual solvent peak. Mass spectra were obtained with a ThermoFisher Scientific (LTQ-Orbitrap Velos) spectrometer. IR spectra were recorded with a Bruker Tensor 27 spectrometer. UV/vis spectra were measured with a Varian Cary 100 Bio UV/vis spectrometer. Melting points were measured with a Büchi 530 melting point apparatus. Optical rotations were measured on a Dr. Kernchen Propol automatic polarimeter. Chemicals were purchased from commercial suppliers and used without further purification. Solvents were dried prior to use by using standard methods. Flash column chromatography was performed on Merck silica gel 60 (40−63 μm) and Merck RP-18 silica gel (40−63 μm). TLC was done on Merck silica gel 60 F254 and Merck silica gel 60 RP-18 F254S aluminum sheets. (1aR,6R,11aS,Z)-6,7,10-Trihydroxy-8-methoxy-1a,5-dimethyl-1a,3,6,11a-tetrahydro-benzo[4,5]cyclodeca[1,2-b]oxiren11(2H)-one (6). To a suspension of SeO2 (0.7 mg, 6.572 μmol) in DCM (0.5 mL) was added a solution of TBHP (5.5 M in decane, 9.0 μL, 49.287 μmol). After stirring for 5 min, a solution of terreumol C (3, 5 mg, 16.429 μmol) in DCM (1 mL) was added dropwise, and the resulting mixture was stirred at 0 °C for 30 min and then warmed to room temperature and stirred for a further 2 h. After filtration of the reaction mixture through a plug of Celite, the filtrate was concentrated under reduced pressure. Purification of the residue by column chromatography (petroleum ether/EtOAc, 1:1) afforded 6 (2.8 mg, 8.741 μmol, 53%) as a yellow oil. (1aR,11aS,E)-7,10-Bis((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-8-methoxy-1a-methyl-1a,3,6,11atetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (7). To a suspension of SeO2 (0.8 mg, 7.506 μmol) in DCM (0.5 mL) was added a solution of TBHP (5.5 M in decane, 10.2 μL, 56.298 μmol). After stirring for 5 min, a solution of the bicycle 5 (10 mg, 18.766 μmol) in DCM (1 mL) was added dropwise, and the resulting mixture was 2656

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Journal of Natural Products

Article

(1aR,11aS,E)-5-(Bromomethyl)-7,10-bis((tertbutyldimethylsilyl)oxy)-8-methoxy-1a-methyl-1a,3,6,11atetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one. LiOAc (4.4 mg, 66.996 μmol) and PhSeBr (6.8 mg, 28.713 μmol) were added to a solution of bicycle 5 (5.1 mg, 9.571 μmol) in AcOH (1 mL). The resulting mixture was stirred at 60 °C for 4 h. The mixture was cooled to room temperature, and saturated aqueous NaHCO3 (10 mL) and DCM (10 mL) were added. Layers were separated, and the aqueous layer was extracted with DCM (3 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/ EtOAc, 10:1) afforded (1aR,11aS,E)-5-(bromomethyl)-7,10-bis((tertbuty ldimethylsilyl)oxy)-8-methoxy-1a-methyl-1a,3,6,11atetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (5.1 mg, 8.337 μmol, 87%) as a colorless oil. (1aR,11aS,E)-5-(Bromomethyl)-7,10-dihydroxy-8-methoxy1a-methyl-1a,3,6,11a-tetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (12). (1aR,11aS,E)-5-(Bromomethyl)-7,10-bis((tert-butyldimethylsilyl)oxy)-8-methoxy-1a-methyl-1a,3,6,11atetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (4.4 mg, 7.192 μmol) was treated according to the general procedure for the desilylation of the TBS-protected hydroquinones. Purification by column chromatography (petroleum ether/EtOAc, 2:1) afforded 12 (2.4 mg, 6.262 μmol, 87%) as a yellow solid. (1aR,4R,11aS)-4-Bromo-7,10-bis((tert-butyldimethylsilyl)oxy)-8-methoxy-1a-methyl-5-methylene-1a,3,4,5,6,11ahexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (13) and (1aR,4R,11aS)-4-bromo-8-methoxy-1a-methyl-5-methylene-1a,3,4,5,6,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxirene-7,10,11(2H)-trione (14). To a solution of compound 5 (5.4 mg, 10.134 μmol) in a mixture of acetone (1 mL) and water (0.25 mL) was added NBS (2.2 mg, 12.161 μmol) at room temperature, and the mixture was stirred for 3 h. Saturated aqueous Na2S2O3 (5 mL) and DCM (5 mL) were added, and the layers were separated. The aqueous layer was extracted with DCM (3 × 5 mL), and the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/ EtOAc, 5:1 to 1:1) afforded 13 (2.7 mg, 4.413 μmol, 44%) as a colorless oil and 14 (1.2 mg, 3.148 μmol, 31%) as a yellow solid. (1aR,4R,11aS)-4-Bromo-7,10-bis((tert-butyldimethylsilyl)oxy)-8-methoxy-1a-methyl-5-methylene-1a,3,4,5,6,11ahexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (13). Procedure A. To a solution of compound 5 (12.5 mg, 23.458 μmol) in a mixture of acetone (1 mL) and water (0.25 mL) was added NBS (5.0 mg, 28.150 μmol) at room temperature, and the mixture was stirred for 25 min. Saturated aqueous Na2S2O3 (5 mL) and DCM (5 mL) were added, and the layers were separated. The aqueous layer was extracted with DCM (3 × 5 mL), and the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/EtOAc, 5:1) afforded 13 (14.0 mg, 22.885 μmol, 98%) as a colorless oil. Procedure B. A solution of NaIO4 (5.5 mg, 25.522 μmol), LiBr (1.7 mg, 19.142 μmol), and compound 5 (6.8 mg, 12.761 μmol) in AcOH (1 mL) was stirred at 95 °C for 2 h. The mixture was cooled to room temperature, and distilled H2O (10 mL) and EtOAc (10 mL) were added. The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with saturated aqueous Na2S2O3 (1 × 10 mL), saturated aqueous NaHCO3 (1 × 10 mL), and brine (1 × 10 mL), dried over Na2SO4, and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/EtOAc, 5:1) afforded 13 (6.4 mg, 10.462 μmol, 82%) as a colorless oil. (1aR,4R,11aS)-4-Bromo-7,10-dihydroxy-8-methoxy-1amethyl-5-methylene-1a,3,4,5,6,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (15). The bicycle 13 (13.1 mg, 21.413 μmol) was treated according to the general procedure for the desilylation of the TBS-protected hydroquinones. Purification by column chromatography (petroleum ether/EtOAc, 3:1) afforded 15 (7.7 mg, 20.092 μmol, 94%) as a yellow solid. (1aR,4R,11aS,E)-4-Bromo-7,10-bis((tert-butyldimethylsilyl)oxy)-5-(dibromomethyl)-8-methoxy-1a-methyl-1a,3,4,11a-

tetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (16), (1aR,4R,11aS,Z)-4-bromo-5-(bromomethyl)-7,10-bis((tertbutyldimethylsilyl)oxy)-8-methoxy-1a-methyl-1a,3,4,11atetrahydrobenzo[4,5]cyclodeca[1,2-b]oxiren-11(2H)-one (17), and (1aR,4R,11aS)-4-bromo-5-(bromomethylene)-7,10-bis((tert-butyldimethylsilyl)oxy)-8-methoxy-1a-methyl1a,3,4,5,6,11a-hexahydrobenzo[4,5]cyclodeca[1,2-b]oxiren11(2H)-one (18). To a stirred solution of 5 (6.7 mg, 12.573 μmol) in CCl4 (0.7 mL) was added azobisisobutyronitrile (AIBN) (0.2 mg, 1.257 μm), and the mixture was heated to 40 °C. NBS (2.2 mg, 12.573 μm) was added in small portions within 30 min, and the mixture was stirred for 30 min at the same temperature. NBS (3.3 mg, 18.860 μmol) was additionally added, and the mixture was refluxed for a further 2 h, cooled to room temperature, and concentrated under reduced pressure. Purification of the residue by column chromatography (petroleum ether/EtOAc, 15:1) afforded 16 and 17 (2.6 mg) as an 1.4:1 mixture and 18 (1.5 mg, 2.172 μmol, 17%), both as colorless oils. (6S,7R,10S,11S)-1,4-Bis((tert-butyldimethylsilyl)oxy)-7,11-dihydroxy-3-methoxy-6,10-dimethyl-6,7,8,9,10,11-hexahydro6,10-epoxybenzo[10]annulen-12(5H)-one (20). To a solution of bicycle 5 (8.3 mg, 15.576 μmol) in a mixture of tBuOH (0.4 mL) and water (0.4 mL) were added sequentially K2CO3 (6.5 mg, 46.728 μmol), K2OsO2(OH)4 (0.1 mg, 0.271 μmol), MeSO2NH2 (1.5 mg, 15.768 μmol), 1,4-diazabicyclo[2.2.2]octane (DABCO) (0.1 mg, 0.892 μmol), and K3Fe(CN)6 (15.4 mg, 46.772 μmol). The reaction mixture was stirred at room temperature for 15 h. Saturated aqueous Na2S2O3 (5 mL) and EtOAc (5 mL) were added, and the layers were separated. The aqueous layer was extracted with EtOAc (3 × 5 mL), and the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (petroleum ether/ EtOAc, 5:1) afforded 20 (6.5 mg, 11.466 μmol, 74%) as a colorless oil. (6S,7R,10S,11S)-1,4,7,11-Tetrahydroxy-3-methoxy-6,10-dimethyl-6,7,8,9,10,11-hexahydro-6,10-epoxybenzo[10]annulen-12(5H)-one (21). The tricycle 20 (2.3 mg, 4.057 μmol) was treated according to the general procedure for the desilylation of the TBS-protected hydroquinones. Purification by column chromatography (petroleum ether/EtOAc, 1:1 to 1:2) afforded 21 (1.1 mg, 3.251 μmol, 80%) as a yellow solid.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00236. Physical data, experimental procedures, graphical NMR spectra, details regarding the X-ray analysis, biological data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +49 (0)531/3917300. Fax: +49 (0)531/391-7744. ORCID

Thomas Lindel: 0000-0002-7551-5266 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Merck KGaA (Darmstadt, Germany) for the generous gift of chromatography materials. BASF Group (Ludwigshafen, Germany) and Honeywell Specialty Chemicals Seelze GmbH (Seelze, Germany) are thanked for the donation of solvents. We thank B. Karge and B. Hinkelmann for conducting the assays on antimicrobial and antiproliferative activity. 2657

DOI: 10.1021/acs.jnatprod.7b00236 J. Nat. Prod. 2017, 80, 2652−2658

Journal of Natural Products

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00236 J. Nat. Prod. 2017, 80, 2652−2658