Synthesis, Biological Investigation, and Structural Revision of

Mar 19, 2018 - Department of Chemistry, P.O. Box 1033 Blindern, University of Oslo , N-0315 Oslo , Norway. J. Nat. ... *E-mail: [email protected]...
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Synthesis, Biological Investigation, and Structural Revision of Sielboldianin A Renate Kristianslund,† Marius Aursnes,† Jørn E. Tungen,† Carl H. Görbitz,‡ and Trond V. Hansen*,† †

School of Pharmacy, Department of Pharmaceutical Chemistry, P.O. Box 1068 Blindern, University of Oslo, N-0316 Oslo, Norway Department of Chemistry, P.O. Box 1033 Blindern, University of Oslo, N-0315 Oslo, Norway

‡

S Supporting Information *

ABSTRACT: The two ar-bisabol sesquiterpenoids (+)-sielboldianin A (1) and (+)-sielboldianin B (2) were isolated from the stem bark of the plant Fraxinus sielboldiana and belong to a medicinally interesting class of natural products used in traditional Chinese medicine. Herein the total synthesis of the proposed structure of (+)-sielboldianin A (1) is reported using an organocatalyzed enantioselective bromolactonization protocol. X-ray analysis of a key intermediate together with specific rotation values and NOESY data of the synthesized product enabled the revision of the absolute configuration of the natural product (+)-sielboldianin A to (7R,10R). Studies on the antioxidant effects using two cell-based assays were conducted. These studies revealed that the enantiomer of 1 exhibited antioxidant effects with IC50 values of 18 ± 3 μM in a cellular lipid peroxidation antioxidant activity assay. Moreover, (−)-1 showed strong protective effects against reactive oxygen species in a cell-based antioxidant activity assay (IC50 = 31 ± 5 μM). In addition, the two ar-sesquiterpenoids (−)-boivinianin B and (−)-gossoronol showed no effect in either assay. No cytotoxic activity in the K562 cancer cell line was observed for the three sesquiterpenoids tested (IC50 > 50 μM). atural products continue to be an inspiring field of research and an important source of biologically active compounds toward drug development.1 Total synthesis is still important for the exact configurational and structural assignment2 of naturally occurring compounds and for providing sufficient material for extensive biological studies.3 Moreover, these endeavors continue to inspire the development of new synthetic reactions.4 In 2011 Shi and co-workers reported the structural elucidation of the two ar-bisabol sesquiterpenoids (+)-sielboldianin A (1) and (+)-sielboldianin B (2).5,6 These two novel natural products were isolated from the stem bark of the plant Fraxinus sielboldiana and belong, together with (−)-boivinianin A (3) and (−)-α-bisabol oxide (4), to a small, but medicinally interesting class of natural products.7

N

Extracts from F. sielboldiana were reported to display diuretic, antifebrile, analgesic, antirheumatic, and anti-inflammatory activities.5,7,8 Owing to our interest in naturally occurring compounds with anti-inflammatory properties,9 the sesquiterpenoid 1 attracted our attention. In order to perform further biological studies, access to sufficient material was needed. No synthesis of (+)-sielboldianin A (1) has been reported. Moreover, Shi and co-workers assigned the structure of 1 and its relative configuration by applying several spectroscopic techniques (MS, IR, and various NMR experiments), while the absolute configuration was tentatively assigned based on biosynthetic considerations and in analogy with the literature5,8 with the risk of misassignment.2 Hence, access to enantiomerically enriched synthesized material would allow the configurational assignment of 1 and aid further biological investigations. We have recently reported enantioselective protocols for iodo- and bromolactonizations of aryl-substituted δ-unsaturated carboxylic acids.10 These protocols use chiral squaramide organocatalysts with hydrogen-bonding properties resulting in moderate to high enantioinduction.11 Owing to the seminal work of Rawal and co-workers,12 chiral squaramide organocatalysts have recently been employed in a number of synthetic transformations,13 but have witnessed only a few applications in total synthesis of natural products.10b Chiral squaramides are Received: January 5, 2018

© XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.8b00020 J. Nat. Prod. XXXX, XXX, XXX−XXX

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easy to prepare, store, and use in various enantioselective applications.13 Hence, we became interested in using the recently disclosed protocol in the total synthesis of the arbisabol sesquiterpenoid 1. Our retrosynthetic analysis is depicted in Scheme 1.

Scheme 2. Toward the Synthesis of 1

Scheme 1. Retrosynthetic Analysis of (+)-Sielboldianin A (1)

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RESULTS AND DISCUSSION Based on the retrosynthetic analysis, the synthesis of 1 began with known 8, which was converted into the aryl-disubstituted δ-unsaturated acid 7 in 87% yield (Scheme 2).10a,b The intermediate 7 was subjected to the recently disclosed enantioselective bromolactonization protocol, which afforded 6 in 91% isolated yield as a solid material. The enantiomeric excess was determined to be 90% using chiral-phase HPLC analysis (Supporting Information). An analytical sample was recrystallized for X-ray structural analysis14 (see Figure 1), with ee = 98%. This established the S-configuration in 6. The conversion of 6 into the natural product 1 involved several challenges. Of note, compound 1 contains a tertiary alcohol moiety prone to water elimination and a benzylic tetrahydrofuran function that may open or decompose under harsh acidic conditions. Also, the electron-rich phenolic moiety in 1 may undergo radical phenolic oxidative couplings or aromatic substitutions. There are several well-established methods available for reducing the nitro group in 6 to get access to 11.15 However, all efforts using some of the classical methods failed.16 A mild and metal-free HSiCl3 method has recently been reported to reduce the nitro functionality in the presence of various functional groups.17 The aniline derivative 11 was obtained in 68% yield with ee = 90% using this method. Attempts to reduce the bromomethyl group in compounds 6 and 11 using different conditions also failed. However, Bocprotection of 11 to afford 12, followed by reduction of the bromide, gave 13 (90% ee) in 91% yield over two steps. Addition of excess MeMgBr to 13 gave diol 14 (82% yield, ee = 90%), which was reacted with POCl3 in pyridine to afford a 3:2 mixture of regioisomers 15a and 15b. In the presence of Rh(III)Cl3·H2O and K2CO3 in EtOH, the ratio of 15a:15b was increased to 7:1. Shi-epoxidation18 followed by acid-catalyzed opening of the resulting epoxide afforded a 4:1 ratio (72% yield) of diasteromers of tetrahydrofuran 16 with the major isomer depicted (Scheme 3). The remaining steps toward 1 involved a Boc-deprotection followed by diazotization and hydrolysis. In spite of the presence of the tertiary alcohol moiety and the benzylic tetrahydrofuran unit, the deprotection of 16 went smoothly using tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF). However, the conversion of the aniline functionality in this intermediate proved

Figure 1. Single-crystal X-ray structure obtained from 6 at 298 K (50% probability displacement ellipsoids).

problematic. The regular diazotization protocol using 20% aqueous H2SO4 and NaNO2 did not afford product 1. When a milder hydrolysis of the diazonium salt was attempted using Cu(NO3)2 and Cu2O,19 compound 17 was obtained in 42% isolated yield. A successful transformation of the aniline to the phenol had occurred albeit accompanied by nitration of the B

DOI: 10.1021/acs.jnatprod.8b00020 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 3. Final Steps in the Synthesis of 1

Figure 2. Structural revision of (+)-sielboldianin A. Nuclear Overhauser effect (NOE) observed for the tetrahydrofuran moiety in synthesized (−)-sielboldianin A with the (S,S)-configuration. The revised structure of naturally occurring (+)-sielboldianin A, the specific rotation, and the NOE effects observed by Shi and co-workers are depicted to the right.5

was more active than quercetin, IC50 = 31 ± 5 μM for (−)-1 and 65 ± 10 μM for quercetin, respectively. Furthermore, the three sesquiterpenoids were subjected to the K562 leukemia cancer cell line assay for evaluation of cytotoxic effects. No significant cytotoxicity was observed for any of the three compounds (IC50 > 50 μM), in contrast to quercetin (IC50 = 2.5 ± 0.5 μM). The antifebrile and analgesic activities reported for extracts from F. sielboldiana may arise from dampening multiple inflammatory processes governed by enzymatically driven mechanisms.22,23 The cyclooxygenase enzymes participate in the biosynthesis of pro-inflammatory mediators.24,25 It has been reported that enzymes of this oxygenase class are inhibited by phenolic natural produts.24 The only difference between (−)-(1) and (−)-boivinanin B is that the former contains a phenolic functional group, which proved essential for antioxidant effects.

activated para-position of the phenol. One obvious nitration source is Cu(NO3)2. Substituting Cu(NO3)2 with CuSO4 yielded target 1 in 45% yield over the two steps and chromatographic separation of diastereomers (Scheme 3).20 The UV, NMR, and MS data of synthesized material of 1 matched the literature data.5,20 Comparison of the specific rotation data reported by Shi and co-workers {[α]20 D +13.2 (c 0.36, MeOH)} with the data for synthesized 1 {[α]20 D −12.5 (c 0.4, MeOH)} showed a good numerical match, but with an opposite sign. The X-ray structure of the intermediate 6 provided direct evidence for assigning an S absolute configuration. The synthetic sequence does not involve any configurational changes at this carbon atom. Moreover, no racemization processes were detected by HPLC analyses of compounds 11, 12, and 14 using chiral-phase HPLC columns (Supporting Information). These observations combined with the data from the NOESY experiment of 1 permitted definition of the absolute configuration of the synthesized 1 as (7S,10S). Hence, the absolute configuration of naturally occurring (+)-sielboldianin A should be revised as (7R,10R) (Figure 2). With multi-milligram amounts of synthesized material available, the antioxidant and cytotoxic effects of (−)-1 were evaluated by using the cellular lipid peroxidation antioxidant activity assay (CLPAA).21 Compound (−)-1 was active, with an IC50 value of 18 ± 3 μM. A similar IC50 value (12 ± 3 μM) was observed for the potent and established antioxidant quercetin.22 In the same assay, significantly weaker inhibitory activity (IC50 > 100 μM) was observed for (−)-boivinianin B and (−)-gossoronol.10b The presence of a phenolic moiety seems to be important for inhibition of the free radicals formed in the CLPAA assay, in accord with the literature.21 Significant antioxidant effects were also observed in the cellular antioxidant activity (CAA) assay. In this assay, the more lipophilic (−)-1

Table 1. Evaluation of Antioxidant and Cytotoxic Effects

a

The IC50 value was determined based on three experiments in the CLPAA assay. bThe IC50 value was determined based on three experiments in the CAA assay. cThe IC50 value was determined based on three experiments in the cell proliferation assay.

In conclusion, the total synthesis of the enantiomer of naturally occurring (+)-sielboldianin A (1) is reported. The overall yield of (−)-1 was 6% over 13 steps. The enantiomeric excess was determined to be >90% based on specific rotation values. The synthesis features a modified and mild protocol for converting anilines into phenols as well as an extension of an organocatalyzed enantioselective bromolactonization protocol. C

DOI: 10.1021/acs.jnatprod.8b00020 J. Nat. Prod. XXXX, XXX, XXX−XXX

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(Chiralcel OD-H, hexanes/iPrOH, 90:10, 1 mL/min, 254 nm): tR(major) = 22.44 min and tR(minor) = 25.18. (S)-6-(3-Amino-4-methylphenyl)-6-(bromomethyl)tetrahydro-2H-pyran-2-one (11). The bromolactone 6 (3.37 g, 10.3 mmol, 1.00 equiv) and diisopropylethylamine (6.63 g, 51.3 mmol, 5.00 equiv) were dissolved in CH2Cl2 (80 mL), and the reaction flask was evacuated and filled with argon. The solution was cooled to 0 °C before a solution of HSiCl3 (4.96 g, 36.2 mmol, 3.57 equiv) in CH2Cl2 (20 mL) was added dropwise over a period of 15 min. The reaction was stirred overnight from 0 °C to room temperature. Saturated aqueous NaHCO3 (50 mL) was added, and the biphasic mixture was stirred for 30 min and extracted with EtOAc (3 × 50 mL). The combined organic layer was dried (Na2SO4), filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica (50% EtOAc in heptane) to afford aniline 11: light brown oil 1 (1.60 g, 52%); [α]20 D +28.0 (c 2.0, CHCl3); H NMR (400 MHz, CDCl3) δ 7.06 (d, J = 7.9 Hz, 1H), 6.74 (d, J = 2.0 Hz, 1H), 6.64 (dd, J = 7.8, 2.0 Hz, 1H), 3.62 (dd, J = 11.3 Hz, 2H), 2.42−2.17 (m, 4H), 2.10 (s, 3H), 1.79−1.68 (m, 1H), 1.63−1.48 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 170.8, 144.6, 139.2, 131.0, 122.8, 115.4, 112.1, 85.1, 41.7, 29.9, 29.1, 17.0, 16.2; HRESIMS m/z 320.0257 [M + Na]+ (calcd for C13H16BrNNaO2 320.0257); TLC (50% EtOAc in heptane) Rf 0.20, visualized with KMnO4; the enantiomeric excess was determined by chiral-phase HPLC analysis (Chiralcel OD-H, hexanes/iPrOH, 90:10, 1 mL/min, 254 nm): tR(major) = 25.77 min and tR(minor) = 45.16, ee: 90%. tert-Butyl (S)-(5-(2-(Bromomethyl)-6-oxotetrahydro-2Hpyran-2-yl)-2-methylphenyl)carbamate (12). The aniline 11 (1.55 g, 5.18 mmol, 1.00 equiv) was dissolved in THF (52 mL) before addition of Boc2O (1.70 g, 7.77 mmol, 1.50 equiv). The resulting solution was stirred at reflux until completion as monitored by TLC. The solvent was removed in vacuo, and the residue was purified by column chromatography on silica (30% EtOAc in heptane) to afford Boc-protected aniline 12: white solid (2.00 g, 97%); mp 1 172−174 °C; [α]20 D −6.9 (c 1.2, CHCl3); H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 7.08 (dd, J = 7.9, 2.1 Hz, 1H), 6.32 (s, 1H), 3.67 (s, 2H), 2.49−2.43 (m, 2H), 2.43−2.32 (m, 2H), 2.24 (s, 3H), 1.87−1.77 (m, 1H), 1.69−1.55 (m, 1H), 1.52 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 170.6, 152.7, 139.0, 137.0, 130.9, 127.1, 120.7, 116.8, 85.1, 80.8, 41.7, 30.1, 29.1, 28.3, 17.4, 16.3; HRESIMS m/z 420.0781 [M + Na]+ (calcd for C18H24BrNNaO4 420.0781); TLC (30% EtOAc in heptane) Rf 0.21, visualized with KMnO4; the enantiomeric excess was determined by chiral-phase HPLC analysis (Chiralcel OD-H, hexanes/iPrOH, 93:7, 1 mL/min, 254 nm): tR(major) = 13.75 min and tR(minor) = 12.62, ee: 90%. tert-Butyl (S)-(2-Methyl-5-(2-methyl-6-oxotetrahydro-2Hpyran-2-yl)phenyl)carbamate (13). The Boc-protected aniline 12 (2.00 g, 5.02 mmol, 1.00 equiv) was dissolved in toluene (110 mL) before tris(trimethylsilyl)silane (3.37 g, 13.6 mmol, 2.70 equiv) and 2,2′-azobisisobutyronitrile (0.27 g, 1.66 mmol, 0.33 equiv) were added, and the reaction mixture was heated to 80 °C overnight. The reaction mixture was cooled and concentrated in vacuo, and the residue was purified by column chromatography on silica (30% EtOAc in heptane) to give 13: white solid (1.50 g, 94%); mp 175−179 °C; [α]25 D −24.0 (c 5.9, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H), 7.13 (d, J = 7.9 Hz, 1H), 7.03 (dd, J = 7.9, 2.0 Hz, 1H), 6.28 (s, 1H), 2.53−2.39 (m, 2H), 2.38−2.29 (m, 1H), 2.23 (s, 3H), 2.06−1.92 (m, 1H), 1.85− 1.73 (m, 1H), 1.67 (s, 3H), 1.65−1.58 (m, 1H), 1.52 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 171.6, 152.9, 143.3, 136.6, 130.5, 126.2, 119.8, 116.6, 85.3, 80.6, 34.2, 31.2, 29.0, 28.3, 17.3, 16.6; HRESIMS m/z 342.1675 [M + Na]+ (calcd for C18H25NNaO4 342.1676) TLC (30% EtOAc in heptane) Rf 0.20, visualized with KMnO4. tert-Butyl (S)-(5-(2,6-Dihydroxy-6-methylheptan-2-yl)-2methylphenyl)carbamate (14). The lactone 13 (0.65 g, 2.04 mmol, 1.00 equiv) was suspended in Et2O (82 mL) and cooled to −78 °C before a solution of MeMgBr (1.0 M in dibutyl ether, 41 mL, 20.0 equiv) was added dropwise. The reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was added slowly to saturated aqueous NH4Cl (30 mL) at 0 °C, extracted with EtOAc (4 × 25 mL), dried (MgSO4), filtered, and concentrated in

These efforts also allowed minor correction of spectroscopic data of (+)-sielboldianin A (1) and resulted in the revision of the absolute configuration to (7R,10R). In addition, potent antioxidant effects of (−)-1 are reported.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a 0.7 mL cell with a 1.0 dm path length on an Anton Paar MCP 100 polarimeter. The UV/vis spectra from 190 to 900 nm were recorded using an Agilent Technologies Cary 8485 UV/ vis spectrophotometer using quartz cuvettes. NMR spectra were recorded on a Bruker AVII400 or a Bruker AVIII HD 400 spectrometer at 400 MHz or a Bruker AVII600 spectrometer at 600 MHz for 1H NMR and at 100 or 150 MHz for 13C NMR. Spectra are referenced relative to the central residual protium solvent resonance in 1 H NMR (CDCl3 δH = 7.26, acetone-d6 δH = 2.05, and methanol-d4 δH = 3.31) and the central carbon solvent resonance in 13C NMR (CDCl3 δC = 77.00, acetone-d6 δC = 29.84, and methanol-d4 δC = 49.00). Mass spectra were recorded at 70 eV on a Waters Prospec Q or Micromass QTOF 2W spectrometer using ESI as the method of ionization. Highresolution mass spectra were recorded at 70 eV on a Waters Prospec Q or Micromass QTOF 2W spectrometer using ESI as the method of ionization. Thin-layer chromatography was performed on silica gel 60 F254 aluminum-backed plates fabricated by Merck (Darmstadt, Germany). Flash column chromatography was performed on silica gel 60 (40−63 μm) produced by Merck (Darmstadt, Germany). Determination of enantiomeric excess was performed by HPLC on an Agilent Technologies 1200 Series instrument with a diode array detector set at the wavelength stated and equipped with a chiral stationary phase (Chiralpak AD-H, 4.6 × 250 mm, particle size 5 μm or Chiralcel OD-H, 4.6 × 250 mm, particle size 5 μm, both from Daicel Chemical Ind., Ltd), applying the conditions stated. Achiral HPLC analyses were performed using a C18 stationary phase (Eclipse XDB-C18, 4.6 × 250 mm, particle size 5 μm, from Agilent Technologies), applying the conditions stated. Unless stated otherwise, all commercially available reagents and solvents were used in the form they were supplied without any further purification. All reactions were performed under an argon atmosphere, unless otherwise stated. The stated yields are based on isolated material. Liquid chromatography-grade solvents were purchased from Fisher Scientific (Oslo, Norway). The squaramide catalyst was prepared as reported.10 Diastereomeric ratios reported have not been validated by calibration; see Wernerova and Hudlicky for discussions and guidelines.26 (S)-6-(Bromomethyl)-6-(4-methyl-3-nitrophenyl)tetrahydro2H-pyran-2-one (6). A solution of 5-(4-methyl-3-nitrophenyl)hex-5enoic acid (7) (3.00 g, 12.0 mmol, 1.00 equiv) and the squaramide catalyst 1010 (1.86 g, 3.61 mmol, 0.30 equiv) was dissolved in acetone (60 mL) and cooled to −78 °C. Subsequently, a solution of Nbromophthalimide (4.08 g, 18.1 mmol, 1.50 equiv) dissolved in acetone (60 mL) was added, and the mixture was stirred at −78 °C for 10.5 h. The reaction mixture was treated with saturated aqueous Na2S2O3 (50 mL) while still in the cooling bath and allowed to equilibrate to ambient temperature. EtOAc (50 mL) was added, the phases were separated, and the organic phase was washed with aqueous NaOH (2 × 50 mL, 1.00 M) and brine (50 mL). The organic phase was dried (MgSO4), filtered, and evaporated in vacuo. The residue was purified by column chromatography on silica (40% EtOAc in heptane) to afford the bromolactone 6: white solid (3.60 g, 91%, 1 90% ee); mp 147−153 °C; [α]20 D +20 (c 1.4, CHCl3); H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 2.1 Hz, 1H), 7.61 (dd, J = 8.1, 2.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 3.63 (dd, J = 11.2 Hz, 2H), 2.61 (s, 3H), 2.56−2.45 (m, 2H), 2.44−2.38 (m, 2H), 1.98−1.85 (m, 1H), 1.67− 1.53 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 169.3, 149.4, 140.0, 134.0, 133.5, 130.1, 121.6, 84.3, 40.7, 30.1, 29.2, 20.1, 16.3; HRESIMS m/z 349.9998 [M + Na]+ (calcd for C13H14BrNNaO4 349.9998); TLC (40% EtOAc in heptane) Rf 0.31, visualized with KMnO4; the enantiomeric excess was determined by chiral-phase HPLC analysis D

DOI: 10.1021/acs.jnatprod.8b00020 J. Nat. Prod. XXXX, XXX, XXX−XXX

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vacuo. The crude product was purified by column chromatography on silica (50% EtOAc in heptane) to afford the diol 14: colorless oil (586 1 mg, 82%); [α]25 D −13.0 (c 2.2, CHCl3); H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 7.06 (dd, J = 7.9, 1.9 Hz, 1H), 6.25 (s, 1H), 2.22 (s, 3H), 1.85−1.76 (m, 2H), 1.55 (s, 3H), 1.47− 1.24 (m, 4H), 1.16 (d, J = 2.0 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ 153.0, 146.9, 136.0, 130.1, 125.5, 120.1, 117.7, 80.4, 74.7, 71.0, 44.3, 44.0, 30.3, 29.3, 29.2, 28.4, 18.8, 17.3; HRESIMS m/z 374.2303 [M + Na]+ (calcd for C20H33NNaO4 374.2302; TLC (50% EtOAc in heptane) Rf = 0.15, visualized with KMnO4; the enantiomeric excess was determined by chiral-phase HPLC analysis (Chiralpak AD-H, hexanes/iPrOH, 90:10, 1 mL/min, 254 nm): tR(major) = 15.76 min and tR(minor) = 12.68, ee: 90%. tert-Butyl (S)-(5-(2-Hydroxy-6-methylhept-5-en-2-yl)-2methylphenyl)carbamate (15a). The diol 14 (586 mg, 1.67 mmol, 1.00 equiv) was azeotroped with 2-methyltetrahydrofuran (3×), dissolved in dry pyridine (30 mL), and cooled to 0 °C. Phosphorus oxychloride (155 μL, 1.67 mmol, 1.00 equiv) was next added in a dropwise manner over a period of 10 min. The reaction mixture was allowed to slowly warm to room temperature over 6.5 h, then carefully added to a stirred solution of saturated aqueous NaHCO3 (30 mL). The resulting mixture was extracted with ether (3 × 25 mL), the combined organic extracts were dried (Na2SO4) and concentrated in vacuo, and the crude material thus obtained, ∼3:2 in favor of the targeted olefinic regioisomer, was used in the isomerization reaction without further purification. To the mixture of 15a and 15b (763 mg, 2.29 mmol, 1.00 equiv) dissolved in EtOH (24 mL) was added Rh(III)Cl3 hydrate (130 mg, 0.573 mmol, 0.250 equiv) and K2CO3 (142 mg, 1.03 mmol, 0.450 equiv), and the reaction mixture was heated at reflux for 4 h. The regioisomeric ratio was improved to ∼7:1 in favor of the targeted olefinic regioisomer as determined by 1H NMR. The solvent was removed in vacuo, and the residue was purified by column chromatography on silica (20% EtOAc in heptane) to give 15a: colorless oil (243 mg, 44%); [α]25 D −8.7 (c 8.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.11 (d, J = 7.9 Hz, 1H), 7.06 (dd, J = 7.9, 1.9 Hz, 1H), 6.24 (s, 1H), 5.14−5.05 (m, 1H), 2.23 (s, 3H), 2.02−1.79 (m, 4H), 1.65 (s, 3H), 1.56−1.48 (m, 15H); 13C NMR (101 MHz, CDCl3) δ 153.0, 146.9, 136.1, 132.0, 130.0, 125.4, 124.3, 120.1, 117.5, 80.3, 74.9, 43.6, 30.4, 28.4, 25.7, 22.9, 17.7, 17.3; HRESIMS m/z 356.2197 [M + Na]+ (calcd for C20H31NNaO3 356.2196); TLC (30% EtOAc in heptane) Rf 0.30, visualized with CAM stain. tert-Butyl (5-((2S,5S)-5-(2-Hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl)-2-methylphenyl)carbamate (16). Compound 15a (69 mg, 0.21 mmol, 1.0 equiv), D-epoxone (16 mg, 0.063 mmol, 0.30 equiv), tetrabutylammonium hydrogen sulfate (3.0 mg, 0.0084 mmol, 0.04 equiv), and Na2B4O7·10H2O (0.050 M) in aqueous Na2EDTA (4 × 10−4 M, 2.2 mL) were dissolved in a mixture of dimethoxymethane and MeCN (2:1, 3.2 mL). The reaction mixture was cooled to 0 °C. An aqueous solution of K2CO3 (0.17 g, 1.2 mmol, in 1.4 mL water) and Oxone (0.18 g, 1.2 mmol, dissolved in aqueous Na2EDTA, 4 × 10−4 M, 1.4 mL) were simultaneously added dropwise over a period of 2 h. When the addition was completed, the reaction mixture was stirred overnight at 0 °C. The reaction mixture was diluted with water and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with brine (10 mL), dried (Na2SO4), and filtered, and the solvent was evaporated in vacuo. The residue was dissolved in CHCl3, a catalytic amount of pTsOH·H2O was added, and the flask was swirled a couple of times. The solution was left alone for 30 min; then a few drops of saturated aqueous NaHCO3 were added, and the reaction mixture was concentrated in vacuo. The diastereomeric ratio (∼4:1) was determined by 1H NMR. The crude material was purified by column chromatography on silica (30% EtOAc in hexane) to yield 16 as a colorless oil comprising two diasteromers (52.0 mg, 72%, dr 4:1). The diastereomers could not be separated by column chromatography on silica at this stage, and the next step was performed using the isomeric mixture. However, the diastereomers could be separated after Boc-deprotection in the next 1 step. [α]20 D −11.0 (c 1.8, MeOH, dr 4:1); H NMR (400 MHz, methanol-d4) δ 7.39 (s, 1H), 7.16−7.07 (m, 2H), 3.82 (t, J = 7.2 Hz,

1H), 2.26−2.17 (m, 5H), 2.04−1.95 (m, 1H), 1.94−1.81 (m, 1H), 1.79−1.66 (m, 1H), 1.51 (s, 9H), 1.49 (s, 3H), 1.24 (s, 3H), 1.20 (s, 3H); 13C NMR (101 MHz, methanol-d4) δ 156.5, 147.9, 137.2, 131.3, 131.2, 122.7, 122.6, 86.7, 85.8, 80.8, 72.6, 40.4, 30.6, 28.7, 27.5, 25.8, 25.8, 17.6; HRESIMS m/z 372.2145 [M + Na]+ (calcd for C20H31NNaO4 372.2145); TLC (30% EtOAc in hexane) Rf = 0.25, visualized with CAM stain. 2-[(2S,5S)-5-(3-Amino-4-methylphenyl)-5-methyltetrahydrofuran-2-yl)]propan-2-ol (19). The Boc-protected aniline 16 together with its minor isomer (43 mg, 0.11 mmol, 1.0 equiv) was dissolved in THF (1 mL) before addition of TBAF (1.0 M in THF, 0.55 mL, 5.0 equiv). The reaction mixture was stirred at reflux overnight, then cooled and concentrated in vacuo. The residue was purified by column chromatography on silica (50% EtOAc in hexane) to give 19: dark yellow oil (24 mg, 88%); [α]20 D = −7.3 (c 1.5, MeOH); 1 H NMR (400 MHz, methanol-d4) δ 6.93 (d, J = 7.6 Hz, 1H), 6.81 (d, J = 1.8 Hz, 1H), 6.67 (dd, J = 7.8, 1.9 Hz, 1H), 3.81 (t, J = 7.5 Hz, 1H), 2.25−2.15 (m, 1H), 2.13 (s, 3H), 1.99−1.80 (m, 2H), 1.77−1.64 (m, 1H), 1.46 (s, 3H), 1.23 (s, 3H), 1.20 (s, 3H); 13C NMR (101 MHz, methanol-d4) δ 148.1, 146.0, 131.0, 122.1, 116.1, 113.2, 86.6, 86.1, 72.6, 40.4, 30.6, 27.5, 25.8, 25.8, 17.1; HRESIMS m/z 272.1621 [M + Na]+ (calcd for C15H23NNaO2 272.1621); TLC (50% EtOAc in hexane) Rf = 0.16, visualized with CAM stain. 5-[(2S,5S)-5-(2-Hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl]-2-methylphenol [(−)-(1), (−)-Sielboldianin A (1)]. A suspension of aniline 19 (8.0 mg, 0.036 mmol, 1.0 equiv) in H2O (0.20 mL) and HBF4 (∼48−50% aqueous solution, 0.20 mL) was evacuated, filled with argon (×3), and stirred at room temperature for 5 min. The suspension was cooled to 0 °C before dropwise addition of NaNO2 in H2O (0.058 M, 0.61 mL, 1.1 equiv). Stirring was continued at 0 °C for 30 min, followed by addition of a saturated aqueous solution of copper(II) sulfate (3.6 mL) and solid Cu2O (15 mg, 0.10 mmol, 3.2 equiv). The resulting reaction mixture was stirred at room temperature for 30 min, then extracted with CH2Cl2 (3 × 5 mL), dried (MgSO4), filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica (30% EtOAc in hexane) to give 4.0 5 mg (50%) of (−)-1: colorless oil; [α]20 D = −12.5 (c 0.40, MeOH) lit. [α]20 = +13.2 (c 0.36, MeOH); UV (MeOH) λ (log ε) 205 (4.1), D max 216 (sh), 276 (0.7) nm; 1H NMR (600 MHz, acetone-d6) δ 8.02 (s, 1H), 7.01 (d, J = 7.7 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 6.75 (dd, J = 7.7, 1.8 Hz, 1H), 3.76 (t, J = 7.1 Hz, 1H), 3.07 (s, 1H), 2.19−2.12 (m, 4H), 2.00−1.87 (m, 2H), 1.72−1.65 (m, 1H), 1.43 (s, 3H), 1.20 (s, 3H), 1.14 (s, 3H); 13C NMR (151 MHz, acetone-d6 δ 155.9, 148.6, 131.2, 122.6, 116.6, 112.0, 86.4, 85.1, 71.3, 40.2, 30.7, 27.0, 26.4, 26.3, 15.8; HRESIMS m/z 273.1462 [M + Na]+ (calcd for C15H22NaO3 273.1461); TLC (30% EtOAc in hexane) Rf = 0.19, visualized with CAM stain. The chemical purity (>97%) was determined by HPLC analysis (Eclipse XDB-C18, MeOH/H2O, 60:40, 1 mL/min, 216 nm): tR(major) = 15.43). All other spectroscopic and physical data were in agreement with those reported in the literature.5 Antioxidant (CLPAA and CAA) Assays Using HepG2 Cells. Human hepatocellular carcinoma (HepG2) cells were cultivated in minimum essential medium Earle’s (MEM Earle’s) supplemented with gentamycin, L-alanyl-L-glutamine, nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum (FBS) inside 175 cm3 flasks in a humidified 5% CO2 atmosphere at 37 °C. A total of 80 000 HepG2 cells were seeded per microplate well 24 h before experiments, giving ∼50% confluence at the time-point of the experiment. The evaluation of antioxidant effects on all compounds employing the CLPAA and CAA assays was performed as previously described.21 Quercetin was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), dissolved in 1 M NaOH, and diluted in medium without FBS. The sesquiterpenoids were dissolved in DMSO and diluted in medium (FBS) containing 5% DMSO, performing final HepG2 incubations (also controls) in 1% DMSO (obtained by adding 20 μL of the diluted compound into 80 μL of medium). From dose−response testing the half-maximal inhibitory concentrations (IC50) were determined by curve fitting using the Hill slope model with Prism 5.0 (GraphPad Software, San Diego, CA, USA). Values are expressed as means ± SD. E

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Cytotoxicity Assays Using K562 Cells. The evaluation of the cytotoxic effects assays was performed as previously described.27 K562 human chronic myelogeneous leukemia cells were cultivated in RMPI medium, free of antibiotics and containing 2-mercaptoethanol (2 μM) and L-glutamine (2 mM), supplemented with fetal calf serum (FCS) (10% v/v). The cells were adjusted to a concentration depending on their observed doubling time (ca. 40 000 cells/mL) in RPMI medium supplemented with FCS (10% v/v). The natural product was dissolved in DMSO. A solution of the natural product (100 μL) in medium was added to 100 μL of cell solution (40 000 cells/mL) in a 96-well microtiter test plate (4 μL of the mentioned solution diluted in medium in order to reach decreasing concentrations). This series of dilutions was continued to afford samples at different concentrations, leaving one cell solution free of drug acting as a control. The plates were incubated at 37 °C (5% CO2 in air) for 5 days. The plate was then removed from the incubator, and 50 μL of a solution of MTT (3 mg/mL in PBS) was added to each well. After incubation (37 °C, 5% CO2 in air, 3 h) the medium was carefully removed from each well by suction and the resulting formazan precipitate redissolved in 200 μL of DMSO. The optical density of each well was read at two wavelengths (λ 540 and 690 nm) using a Titertek Multiscan MCC/340 plate reader. After processing and analysis through the application of an inhouse software package, the results enabled the calculation of the drug dose required to inhibit cell growth by 50% (IC50 value), determined by graphical means as percentage of the control growth.

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(4) (a) Mohr, J. T.; Krout, M. R.; Stoltz, B. M. Nature 2008, 455, 323−332. (b) Nicolaou, K. C. Proc. R. Soc. A 470: 20130690. http:// dx.doi.org/10.1098/rspa.2013.0690. (5) Lin, S.; Zhang, Y.-L.; Liu, M.-T.; Zi, J.-C.; Gan, M.-L.; Song, W.X.; Fan, X.-N.; Wang, S.-J.; Yang, Y.-C.; Shi, J.-G. Acta Pharm. Sin. B 2011, 1, 89−92. (6) After fruitful discussions with Prof. Jian-Gong Shi we jointly propose the names (+)-sielboldianin A and (+)-sielboldianin B for these natural products. (7) Dewick, P. M. Medicinal Natural Products, 3rd ed.; Wiley: Sussex, 2009; pp 187−223. (8) Sun, J.; Shi, D.; Ma, M.; Li, S.; Wang, S.; Han, L.; Yang, Y.; Fan, X.; Shi, J.; He, L. J. Nat. Prod. 2005, 68, 915−919. (9) (a) Aursnes, M.; Tungen, J. E.; Vik, A.; Colas, R.; Cheng, C.-Y.; Dalli, J.; Serhan, C. N.; Hansen, T. V. J. Nat. Prod. 2014, 77, 910−916. (b) Tungen, J. E.; Aursnes, M.; Dalli, J.; Arnardottir, H.; Serhan, C. N.; Hansen, T. V. Chem. - Eur. J. 2014, 20, 14575−14578. (c) Tungen, J. E.; Aursnes, M.; Vik, A.; Ramon, S.; Colas, R.; Dalli, J.; Serhan, C. N.; Hansen, T. V. J. Nat. Prod. 2014, 77, 2241−2247. (d) Aursnes, M.; Tungen, J. T.; Vik, A.; Dalli, J.; Hansen, T. V. Org. Biomol. Chem. 2014, 12, 432−437. (e) Aursnes, M.; Tungen, J. E.; Colas, R. A.; Vlasakov, I.; Dalli, J.; Serhan, C. N.; Hansen, T. V. J. Nat. Prod. 2015, 78, 2924− 2931. (f) Primdahl, K. G.; Aursnes, M.; Walker, M. E.; Colas, R. A.; Serhan, C. N.; Dalli, J.; Hansen, T. V.; Vik, A. J. Nat. Prod. 2016, 79, 2693−2702. (g) Anwar, H. F.; Hansen, T. V. Org. Lett. 2009, 11, 587− 588. (h) Mohamed, Y. M. A.; Vik, A.; Hofer, T.; Andersen, J. H.; Hansen, T. V. Chem. Phys. Lipids 2013, 170−171, 41−45. (10) (a) Tungen, J. E.; Nolsøe, J. M. J.; Hansen, T. V. Org. Lett. 2012, 14, 5884−5887. (b) Aursnes, M.; Tungen, J. E.; Hansen, T. V. J. Org. Chem. 2016, 81, 8287−8295. (c) Kristianslund, R.; Aursnes, M.; Tungen, J. E.; Hansen, T. V. Tetrahedron Lett. 2016, 57, 5232−5236. (11) Han, X.; Zhou, H.-B.; Dong, C. Chem. Rec. 2016, 16, 897−906. (12) Malerich, J. P.; Hagihara, K.; Rawal, V. H. J. Am. Chem. Soc. 2008, 130, 14416−14417. (13) (a) Storer, R. I.; Aciro, C.; Jones, L. H. Chem. Soc. Rev. 2011, 40, 2330−2346. (b) Nolsøe, J. M.; Hansen, T. V. Eur. J. Org. Chem. 2014, 2014, 3051−3065. (14) Bruker D8 Venture diffractometer with Photon100 CMOS detector, Mo Kα radiation, T = 298 K, space group P212121, a = 7.5781(8) Å, b = 13.2292(14) Å, c = 13.2765(13) Å, Z = 4, Rint = 0.0512, Nrefl = 2681, Npar = 174, R = 0.0341, wR(F2) = 0.0876, H-atom parameters constrained, refined as a racemic twin with minor fraction = 0.012(14). Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre, CCDC 1555740 (https://www.ccdc.cam.ac.uk/). (15) Tafesh, A. M.; Weiguny, J. Chem. Rev. 1996, 96, 2035−2052. (16) Hydrogenation with either hydrogen or formic acid in the presence of Pd/C, as well as reductions with either Zn/HCl or Mg/ MeOH, failed. (17) Orlandi, M.; Tosi, F.; Bonsignore, M.; Benaglia, M. Org. Lett. 2015, 17, 3941−3943. (18) Tu, Y.; Wang, Z.-X.; Shi, Y. J. Am. Chem. Soc. 1996, 118, 9806− 9807. (b) Frohn, M.; Shi, Y. Synthesis 2000, 2000, 1979−2000. (19) Cohen, T.; Dietz, A. G., Jr.; Miser, J. R. J. Org. Chem. 1977, 42, 2053−2058. (20) We are grateful to Prof. Jian-Gong Shi for copies of NMR spectra of isolated (+)-1. In the original report cited in ref 5, the signal for carbon atom numbered 2 in the 13C NMR spectra reads 122.0 ppm. After inspection of spectra and personal communication with Prof. Jian-Gong Shi this chemical shift should read 112.0 ppm. (21) Hofer, T.; Eriksen, T. E.; Hansen, E.; Varmedal, I.; Jensen, I.-J.; Hammer-Andersen, J.; Olsen, R. L. Cellular and Chemical Assays for Discovery of Novel Antioxidants in Marine Organisms. In Studies on Experimental Models Springer Science series Oxidative Stress in Applied Basic Research and Clinical Practice; Basu, S.; Wiklund, L., Eds.; 2011; pp 637−658. (22) Dueñas, M.; González-Manzano, S.; González-Paramás, A.; Santos-Buelga, C. J. Pharm. Biomed. Anal. 2010, 51, 443−449.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00020. 1 H and 13C NMR spectra data, HRMS and UV/vis spectra, as well as chromatograms from HPLC analyses of synthetic intermediates and (−)-1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Trond V. Hansen: 0000-0001-5239-9920 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Helpful discussions with Dr. A. Vik are gratefully acknowledged. The Research Council of Norway is gratefully acknowledged for funding of a postdoctoral and a doctoral fellowship to M.A. and R.K., respectively (FRIPRO-FRINATEK 230470). We are thankful to the School of Pharmacy, University of Oslo, for a scholarship to J.E.T. as well as for the generous support from the Norwegian Ph.D. School in Pharmacy (Nasjonal forskerskole i farmasi, NFIF) for a travel grant to R.K.

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DEDICATION Dedicated to Professor Lars Skattebøl on the occasion of his 90th birthday. REFERENCES

(1) Koehn, F. E.; Carter, G. T. Nat. Rev. Drug Discovery 2005, 4, 206−220. (2) Nicolaou, K. C.; Snyder, S. A. Angew. Chem., Int. Ed. 2005, 44, 1012−1044. (3) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661. F

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(23) Williams, C. S.; Mann, M.; DuBois, R. N. Oncogene 1999, 18, 7908−7916. (24) Kelm, M. A.; Nair, M. G.; Strasburg, G. M.; DeWitt, D. L. Phytomedicine 2001, 7, 7−13. (25) Haeggström, J. Z.; Funk, C. D. Chem. Rev. 2011, 111, 5866− 5898. (26) Wernerova, M.; Hudlicky, T. Synlett 2010, 18, 2701−2707. (27) Odlo, K.; Hentzen, J.; Chabert, J. F.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.; Skrede, M.; Flørenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16, 4829−4838.

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DOI: 10.1021/acs.jnatprod.8b00020 J. Nat. Prod. XXXX, XXX, XXX−XXX