1,4-Benzodioxane Lignans: An Efficient, Asymmetric Synthesis of

Article Cite This: J. Nat. Prod. 2018, 81, 2630−2637

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1,4-Benzodioxane Lignans: An Efficient, Asymmetric Synthesis of Flavonolignans and Study of Neolignan Cytotoxicity and Antiviral Profiles Lisa I. Pilkington,† Jessica Wagoner,‡ Toni Kline,§ Stephen J. Polyak,*,‡,§,⊥ and David Barker*,†,∥ †

School of Chemical Sciences, University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand Department of Laboratory Medicine, §Department of Microbiology, and ⊥Department of Global Health, University of Washington, Seattle, Washington 98195, United States ∥ The MacDiarmid Institute of Advanced Materials and Nanotechnology, New Zealand

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ABSTRACT: 1,4-Benzodioxane lignans are a class of bioactive compounds that have received much attention through the years. Herein research pertaining to both 1,4-benzodioxane flavonolignans and 1,4-benzodioxane neolignans is presented. A novel synthesis of both traditional 1,4-benzodioxane flavonolignans and 3-deoxyflavonolignans is described. The antiviral and cytotoxic activities of 1,4-benzodioxane neolignans were then investigated; eusiderins A, B, G, and M, deallyl eusiderin A, and nitidanin, which contain the 1,4-benzodioxane motif but lack the chromanone motif found in the known antiviral flavonolignans, were tested. Notably, it was found that all eusiderin 1,4-benzodioxane neolignans exhibited greater antiviral activity than the potent and well-known silybin flavonolignans. While most modifications of the C-1′ side chain did not significantly alter the cytotoxicity or antiviral activity, eusiderin M and nitidanin, which contain an allylic alcohol side chain, had lower cytotoxicity. All the eusiderins had similar antiviral activities, with eusiderin B having the best selectivity index. These results show that the chromanone moiety of the flavonolignans is not essential for bioactivity. ilymarin is a complex mixture of flavonolignans, a notable class of bioactive compounds, isolated from the seeds of Silybum marianum. This extract is a popular liver protectant that has been used in traditional medicine for centuries and has demonstrated antiviral effects (specifically anti-HCV) as well as in vitro inhibition of T-cell proliferation.1−3 There are eight main components of silymarin, one of which is taxifolin (1), while four are 1,4-benzodioxane flavonolignans and the remaining three compounds are related, non-1,4benzodioxane flavonolignans, with a dihydroflavonol nucleus.4 The most abundant compound in silymarin is the 1,4benzodioxane flavonolignans silybin 2 (frequently referred to as silibinin in many sources), which is an approximately equimolar diastereomeric mixture of (+)-silybin A (3) and (−)-silybin B (4).4−8 Silybin 2 is the major component of silmarin, accounting for approximately 45% of this extract,9 while their regioisomers, isosilybin A and isosilybin B, are also

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© 2018 American Chemical Society and American Society of Pharmacognosy

prevalent in this mixture, constituting ca. 15% of this complex extract.6,9,10 Related to these flavonolignans are isosilandrins A (5) and B (6), which are the flavanolignan analogues of 3 and 4. Interestingly, 5 and 6 are found in the white-flowering variety of Silybum marianum, while silymarin is traditionally sourced from the purple-flowering variety.11−13 When the hepatoprotective properties of the individual components of silymarin were analyzed, it was found that the most active compounds were those that contain the 1,4benzodioxane moiety (i.e., 1,4-benzodioxane flavonolignans) (Figure 1, red).3 While the antiviral activity of these flavonolignans in silymarin is well documented,2 until recently there had been no studies of 1,4-benzodioxane neolignans, Received: May 23, 2018 Published: November 28, 2018 2630

DOI: 10.1021/acs.jnatprod.8b00416 J. Nat. Prod. 2018, 81, 2630−2637

Journal of Natural Products

Article

Figure 1. Structures of taxifolin 1, silybin 2, silybins A (3) and B (4), isosilandrin A (5), isosilandrin B (6), eusiderin A (7), and trans-rodgersinine A (8). The 1,4-benzodioxane core is highlighted red in trans-rodgersinine A, eusiderin A, and silybin A, with the dihydroflavonol nucleus in the blue rectangle and the chromanone in the green rectangle. Silybin/sibinin 2 is a diastereomeric mixture of silybins A (3) and B (4).

antiviral activity, suggesting that the chromanone group may not be required for activity in these classes of compounds.16 The rodgersinines are a small subset of this class of natural products, with the eusiderins [e.g., eusiderin A (7)] being the major family of 1,4-benzodioxane neolignans.17 Contradictory claims exist as to the hepatoprotective properties of 1,4benzodioxane neolignans; natural 1,4-benzodioxane neolignans have been suggested to demonstrate hepatoprotective activities,18,19 due to their similarity in structure to silybin A (3), while there have also been claims that the antihepatitis C virus (anti-HCV) properties of silybin 1 and other flavonolignans are a result of the dihydroflavonol moiety and not due to the 1,4-benzodioxane scaffold; that is, 1,4benzodioxane neolignans would not have these biological activities.20 Consequently, we wished to further assess the antiviral properties of 1,4-benzodioxane neolignans by testing members and analogues of the eusiderin family. It was envisaged that these results, along with those previously obtained from the rodgersinines, could verify the requirement for the chromanone functionality for antiviral activity in 1,4-benzodioxanecontaining natural products. Furthermore, we also wished to establish a new method to access the 1,4-benzodioxane flavonolignan scaffold in an effort to improve existing routes to synthesize these notable compounds. Silybin 2 and other 1,4-benzodioxane flavonolignans were originally synthesized through nonspecific oxidative dimerization methods.21,22 Subsequent total syn-

which are non-chromanone-containing-1,4-benzodioxane natural products. These 1,4-benzodioxane neolignans, such as eusiderin A (7) (Figure 2) and trans-rodgersinine A (8), are a significant group of natural products that have been shown to exhibit other interesting pharmacological properties.14,15 Recently we reported that four members of the rodgersinine family of 1,4-benzodioxane neolignans, including 5, exhibit

Figure 2. Eusiderins A (7), B (17), G (18), and M (19), deallyl eusiderin A (20), and nitidanin (21). 2631



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Scheme 1. Synthesis of Flavonolignans

catalyzed deprotection and concomitant cyclization to give an inseparable mixture of trans- and cis-1,4-benzodioxanes 12a and 12b in a 5:1 ratio. The mixture of 1,4-benzodioxanes 12 was then diprotected with methoxymethyl chloride (MOMCl), to give pure trans-1,4-benzodioxane 13 with no cis isomer obtained, indicating epimerization of C-3 during this basepromoted protection procedure. The next step required for the synthesis of the targeted flavonolignans was the addition of a formyl moiety to bromide 13 through formation of the corresponding lithiate, followed by the addition of dimethylformamide (DMF), giving 14 in a near-quantitative yield. Aldehyde 14 then underwent a condensation reaction with aryl ketone 1527 using NaOH in EtOH, providing chalcone 16. Using methods reported by Zhao et al.28 and Tanaka23 chalcone 16 was converted, via epoxidation and acid-catalyzed deprotection/cyclization, to give an equimolar mixture of entsilybin A, ent-(3), and silybin B (4). Chalcone 16 was separately converted, using the methods reported by Samu et al.,11 via acid-catalyzed deprotection followed by base-induced cyclization, to ent-isosilandrin A, ent-(5), and isosilandrin B (6). These two transformations gave mixtures of two diastereoisomers, which are the same at C-2′ and C-3′ but are different on the dihydroflavonol fragment. This is due to the nonselective nature of the dihydroflavonol/flavanone ring formation, which is not effected by the fixed stereochemistry at the distant C-2′ and C-3′. These diastereoisomers 3/4 and 5/6 are inseparable using standard chromatography but have previously been separated using reversed-phase HPLC.12,23,29

theses of these compounds include the synthesis of silybin 2 through the condensation of a racemic α-bromophenone and a phenol23 and the formal stereoselective synthesis of 3 and 4 through the condensation of an enantiopure epoxide precursor.24 These previous synthetic approaches have inherent drawbacks, particularly associated with lack of flexibility as well as difficulties concerning the synthesis of analogous compounds. Additionally, the commonly used oxidative dimerization methods often give mixtures of isomers, thereby providing only an unselective method toward these compounds. Herein, we demonstrate that the previously described25,26 route to synthesize a number of naturally occurring 1,4-benzodioxane neolignans could also be used in the asymmetric synthesis of these flavonolignans, providing a novel, flexible, and stereoselective route to access these highly sought after, potent, natural products.

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RESULTS AND DISCUSSION Synthesis of Flavonolignans. To extend the reported methods on 1,4-benzodioxane neolignans, the stereoselective synthesis of 3-aryl-1,4-benzodioxanes silybin B (4) and isosilandrin B (6) was targeted. We aimed to demonstrate the utility of our previous methods toward the stereoselective synthesis of flavonolignans, both typical flavonolignans and 3flavanolignans. To aldehyde 926 was added the lithiate of aromatic bromide 10, giving a 1.2:1 diastereomeric mixture of anti 11a and syn 11b (Scheme 1). The resulting alcohols 11 underwent an acid2632



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This method allows for the synthesis of flavonolignans where control of the benzodioxane stereochemistry is required and complements the work of McDonald et al.,30 who prepared the silybin regioisomer isosilybin A using a biomimetic approach. This method contrasts with previous synthesis of these compounds, which, while shorter and potentially more scalable, have either been completely racemic11,23,28 or begin with the configuration of the dihydroflavonol fixed (e.g., using taxifolin 1 as the starting material),28,31,32 resulting in flavonolignans with various regio- and stereoisomers in the added benzodioxane moiety. Antiviral Testing of 1,4-Benozdioxane Neolignans. Testing of the antiviral properties of 1,4-benzodioxane flavonoligans was extended to a larger set of 1,4-benzodioxane neolignans than previously examined. The four eusiderins, A (7), B (17), G (18), and M (19) (Figure 2), along with the deallyl eusiderin A (20) and nitidanin33 (21) (a related neolignan originally isolated from Xanthoxylum nitidum), were tested for their ability to protect liver cells from HCV. Eusiderins 7 and 17−20 were obtained using our previously reported procedures.25 It was envisaged that eusiderin A (7) and deallyl eusiderins A (20), G (18), and M (19) would permit the establishment of the importance of the C-1′ side chain (C-7′ to C-9′) on activity. Eusiderin B (17) would indicate if the presence of a methylenedioxy substitution on the non-benzodioxane moiety alters activity compared to trimethoxy substitution. Nitidanin (21) shares many structural similarities with the eusiderin compounds, particularly with eusiderin M (19), but also contains the hydroxymethyl group present in the analogous position on silybin A (3). In contrast, the methylenedioxy moiety of eusiderin B (17) is absent in the known flavonolignans. We initially tested the cytotoxicity profile of the compounds 7 and 17−21 and determined their cytotoxic concentration (CC50, Table 1). The CC50 values of the tested compounds are

Additionally, in analyzing the cytotoxicity data for the compounds, it is apparent that the nature, or presence, of a side chain per se for the 3,4,5-trisubstituted phenyl compounds 7 and 18−20 does not significantly affect the cytotoxicity (cf. 7, 18, and 20). The exception to this is the hydroxypropenyl side chain, present in 19 and 21, which seems to be associated with greatly decreased cytotoxicity. Nitidanin (21) was the least cytotoxic of the compounds tested, with the main difference between 21 and the other compounds being the presence of a hydroxymethyl group in 21 as opposed to a methyl group in all other compounds. Comparing the activity of eusiderins A (7) and B (17), which are different only in the substitution on the non-benzodioxane ring, it is clear that methylenedioxy substitution causes a much lower degree of cytotoxicity than trimethoxy substitution. These distinctions cannot be simply attributed to changes in properties such as lipophilicity and suggest instead the influence of these structural features on specific interactions at a molecular target. Compounds 7 and 17−21 were also tested for their antiHCV activity (Figure 3) by analyzing the reduction in expression of HCV nonstructural proteins NS3 and NS5A. Specifically, the eusiderins were tested for inhibition of fully infectious virus, using the JFH-1 isolate34 to infect human hepatoma Huh7.5.1 liver cells, which are highly permissive for JFH-1 infection (i.e., the cells permit high-level infection).35 As human liver cells (i.e., hepatocytes) are the primary cell type that is infected by HCV in vivo (i.e., in humans), JFH-1 infection of Huh7.5.1 cells is an accepted model for in vitro cell culture infectivity studies. The principal reason for using an infectious virus in the antiviral assay is that silymarin and purified flavonolignans do not inhibit HCV replication in noninfectious HCV replicon cell lines.36,37 For each compound, three concentrations were tested up to concentrations that approached the IC50, and the antiviral IC50 value was estimated by examining the pixel intensity of viral protein expression (determined by Western blot analysis). From these antiviral results, it is immediately apparent that all of the eusiderins, naturally occurring and analogues, boast a greater or equal antiviral potency compared with the mixtures of silymarin or silibinin 2 (IC50 ≤ 50 μM) and have comparable activity to the single components of silymarin which have been individually analyzed.3 Such a result is significant; not only does it show that the dihydroflavonol moiety is nonessential for anti-HCV activity, but it shows that these 1,4-benzodioxane neolignans in general have greater activity than the commonly used silymarin mixture and exhibit similar, or better, antiviral activity to pure samples of 1,4benzodioxane flavonolignans. Most of the 1,4-benzodioxane neolignans also exhibit better selectivity than the flavonolignans, silybin A (3) and silybin B (4). Only nitidanin (21) displayed poorer antiviral properties than 3 and 4 (IC50 200 μM vs 50 and 50 μM, respectively). Eusiderin B (17) appeared to have the most potent anti-HCV activity, with an estimated antiviral IC50 of approximately 20 μM and a selectivity index (SI) of >6 (Table 1). The beneficial effects of the methylenedioxy ring in eusiderin B (17) is interesting from a structural perspective since, unlike the hydroxy/methoxy analogues, the ring cannot rotate out of the plane of the aromatic ring, bestowing unique electronic and steric properties on it. In summary, this paper shows that our previously developed method for the synthesis of 1,4-benzodioxane neolignans is applicable to the synthesis of both typical 1,4-benzodioxane

Table 1. Cytotoxicity CC50 and Antiviral IC50 Values for Compounds 7 and 17−21 compound silymarin38 silibinin 236 silybin A (3)39 silybin B (4)39 7 17 18 19 20 21

cytotoxicity CC50 (μM)

antiviral IC50 (μM)

selectivity index (SI)a

100 80 80

80 60 50

1.25 1.33 1.60

68.7

50

1.37

36.2 >125 47.6 128.7 50.2 464.4

30 20 25 50 40 200

1.21 >6.25 1.91 2.57 1.25 2.32

a

Selectivity index is CC50/IC50.

variable, ranging from 36 to 464 μM. Compared to the reported values for silymarin, silybin 2, silybin A (3), and silybin B (4), eusiderins A (8) and G (18) and deallyl eusiderin A (20) exhibit lower CC50 values, thus greater cytotoxicity. From this, it is clear that the presence of a dihydroflavonol moiety is not necessary for a 1,4-benzodioxane to exhibit cytotoxic properties. In contrast, eusiderins B (17) and M (19) and nitidanin (21) have lower cytotoxicities. 2633



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Figure 3. Anti-HCV profile of compounds 7 and 17−21. Huh7.5.1 cells were infected with HCV at a multiplicity of infection of 0.05 for 5 h. Virus inoculum was removed and replaced with fresh media containing the indicated concentrations (in μM) of each compound.

flavonolignans and a common structural variant, the flavanolignans. These findings highlight the versatility of this method and provide an attractive alternative to less-selective traditional methods used to synthesize 1,4-benzodioxane flavonolignans. While previously it has been proposed that the anti-HCV properties of silybin A (3) and other similar flavonolignans are linked to the presence of the dihydroflavonol nucleus,20 these data indicate that compounds containing the 1,4-benzodioxane scaffold alone can affect cellular viability and can also inhibit HCV infection. Furthermore, it was shown that all of the eusiderins exhibited greater or equal antiviral potency compared with silymarin and silibinin (IC50 ≤ 50 μM). Flavones, and in particular the chromanone functionality, are known to have promiscuous cellular interactions40 and are well-known to have antioxidant41 and metal-binding properties.42 Further investigation and use of anti-HCV 1,4-benzodioxane-containing compounds which lack a chromanone component may therefore reveal novel cellular and anti-infective functions of this scaffold and provide a template for comparison of the biological activities of other polyphenolic natural products. Thus, while flavonoid compounds in general are often regarded as too promiscuous to be viable therapeutic scaffolds, trimming away the chromanone unit may provide lead compounds for optimization as anti-infectives.

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4-Bromo-2-methoxy-1-(methoxymethyloxy)benzene, 10. To 4-bromo-2-methoxyphenol43 (2.88 g, 0.014 mol) in CH2Cl2 (50 mL) at 0 °C under an atmosphere of nitrogen was added N,Ndiisopropylethylamine (DIPEA, 10.1 mL, 0.057 mol) slowly. After a few minutes, methoxymethyl chloride (3.8 mL, 0.035 mol) was added dropwise, and the solution allowed to warm to room temperature. After 3 days of stirring, saturated aqueous NH4Cl (25 mL) was added, the layers were separated, and the aqueous layer was extracted with CH2Cl2 (3 × 30 mL). The combined organic layers were dried (MgSO4) and the solvent was removed in vacuo. The crude product was purified by flash chromatography (4:1 n-hexane/EtOAc) to yield the title product 10 (3.20 g, 91%) as a colorless oil: Rf (3:1 n-hexane/ EtOAc) 0.81; IR (film) νmax 2990, 2970, 2902, 1591, 1498, 1464, 1444, 1395, 1252, 1224, 1200, 1156, 1130, 1078, 996, 923, 854, 808 cm−1; 1H NMR (400 MHz; CDCl3; Me4Si) δ 3.50 (3H, s, −OCH2OCH3), 3.86 (3H, s, 2-OMe), 5.19 (2H, s, −OCH2OCH3) and 7.00−7.02 (3H, m, H-3, H-5, and H-6); 13C NMR (100 MHz; CDCl3) δ 56.1 (OMe), 56.2 (OMe), 95.6 (OCH2OCH3), 114.6 (C4), 115.3 (C-3), 117.8 (C-6), 123.6 (C-5), 145.7 (C-1), and 150.6 (C-2); EIMS m/z 271 [81BrMNa]+ (100), 269 [79BrMNa]+ (100), 191 (45); HREIMS m/z 270.9775 (calcd for C9H1181BrNaO3, 270.9764), 268.9793 (calcd for C9H1179BrNaO3, 268.9784). (1R,2S)-2-[4′-Bromo-2′-(methoxymethoxy)phenoxy]-1-[3″methoxy-4′′-(methoxymethoxy)phenyl]-3-[(triisopropylsilyl)oxy]propan-1-ol, 11a, and (1S,2S)-2-[4′-Bromo-2′(methoxymethoxy)phenoxy]-1-[3″-methoxy-4′′(methoxymethoxy)phenyl]-3-[(triisopropylsilyl)oxy]propan-1ol, 11b. To a stirred solution of bromide 10 (0.360 g, 1.30 mmol) in dry tetrahydrofuran (THF; 25 mL), under an atmosphere of nitrogen at −78 °C, was added tBuLi (1.4 M in THF, 1.93 mL, 2.71 mmol). After 2 min, a solution of aldehyde 926 (0.500 g, 1.08 mmol) in THF (15 mL) was added slowly, and the mixture stirred at −78 °C for 1 h and then allowed to warm to room temperature and left for a further 23 h. Saturated aqueous NH4Cl (20 mL) was added, and the aqueous mixture extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried (MgSO4) and the solvent was removed in vacuo. The crude product was purified by flash chromatography (4:1 nhexane/EtOAc) to yield the title products 11a and 11b (0.253 g, 37%) in a 1.2:1 anti 11a to syn 11b inseparable mixture of diastereomers as a yellow oil: Rf (4:1 n-hexane/EtOAc) 0.17; IR (film) νmax 3483, 2942, 2866, 1591, 1491, 1253, 1190, 1154, 1133, 1078, 983, 922, 822, 683 cm−1; 11a: 1H NMR (400 MHz; CDCl3; Me4Si) δ 0.99−1.08 (21H, m, Si(CH(CH3)2)3 and Si(CH(CH3)2)3), 3.47 (3H, s, −OCH2OCH3), 3.50 (3H, s, −OCH2OCH3), 3.81 (1H, dd, J = 5.6 and 10.8 Hz, H-3a), 3.85 (3H, s, OMe), 3.89 (1H, br s, OH), 3.96 (1H, dd, J = 5.6 and 10.8 Hz, H-3b), 4.29 (1H, q, J = 4.8 Hz, H-2), 4.95 (1H, d, J = 4.4 Hz, H-1), 5.13 (2H, s, OCH2OCH3), 5.20 (2H, s, OCH2OCH3), 6.88 (1H, dd, J = 1.6 and 8.4 Hz, H-6″), 6.90 (1H, d, J = 8.4 Hz, H-6′), 7.03 (1H, d, J = 1.6 Hz, H-2″), 7.03 (1H, dd, J = 1.6 and 8.4 Hz, H-5′), 7.06 (1H, d, J = 8.4 Hz, H-5″), and 7.25 (1H, d, J = 1.6 Hz, H-3′); 13C NMR (100 MHz; CDCl3) δ

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at 20 °C on the sodium D line (λ = 589 nm, 0.1 dm cell). NMR spectra were recorded on a 400 MHz spectrometer, with chemical shifts reported relative to the solvent peak of CHCl3 (δ 7.26 for 1H and δ 77.0 for 13C). 1H NMR data are reported as shift (δ), relative integral, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad peak; qd, quartet of doublets), coupling constant (J, Hz), and the assignment of the atom. 13C NMR data are reported as shift (δ) and assignment of the atom. NMR assignments were performed using HSQC and HMBC experiments. High-resolution mass spectroscopy (HRMS) was carried out by either chemical ionization (CI) or electrospray ionization (ESI) on a MicroTOF-Q mass spectrometer. All reactions were carried out under a nitrogen atmosphere in dry, freshly distilled solvents unless otherwise noted. All chemicals were obtained from Sigma-Aldrich and used without additional purification. Eusiderins A (8), B (17), G (18), and M (19) and deallyl eusiderin A (20) were prepared as reported.25 Nitidanin (21), isolated from Croton laevigatus, was purchased from Quality Phytochemicals, NJ, USA. 2634



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was purified by flash chromatography (3:1 n-hexane/EtOAc) to yield the title product 13 (67.0 mg, 99%) as a colorless oil: Rf (4:1 nhexane/EtOAc) 0.24; [α]D −6 (c 0.3, CHCl3); IR (film) νmax 2924, 2852, 1515, 1486, 1464, 1263, 1153, 1077, 995, 919, 885, 806 cm−1; 1 H NMR (400 MHz; CDCl 3 ; Me 4 Si) δ 3.35 (3H, s, −CH 2 OCH 2 OCH 3 ), 3.48 (1H, dd, J = 4.4 and 11.2 Hz, CHaOMOM), 3.53 (3H, s, 4′-OCH2OCH3), 3.70 (1H, dd, J = 4.0 and 12.0 Hz, CHbOMOM), 3.91 (3H, s, 3′-OMe), 4.13−4.17 (1H, m, H-2), 4.60 (2H, dd, J = 8.0 and 20 Hz, −CH2OCH2OCH3), 4.97 (1H, d, J = 8.0 Hz, H-3), 5.26 (2H, s, 4′-OCH2OCH3), 6.86 (1H, d, J = 8.0 Hz, H-8), 6.94 (1H, br s, H-2′), 6.96 (1H, dd, J = 2.4 and 8.0 Hz, H-6′), 6.98 (1H, dd, J = 2.4 and 8.0 Hz, H-7), 7.11 (1H, d, J = 2.4 Hz, H-5), and 7.18 (1H, d, J = 8.0 Hz, H-5′); 13C NMR (100 MHz; CDCl3) δ 55.4 (−CH2OCH2OCH3), 56.0 (3′-OMe), 56.2 (4′OCH2OCH3), 66.3 (CH2OMOM), 76.3 (C-3), 77.1 (C-2), 95.4 (4′OCH2OCH3), 96.9 (−CH2OCH2OCH3), 110.7 (C-2′), 112.9 (C-6), 116.2 (C-5′), 118.4 (C-8), 120.1 (C-5), 120.3 (C-6′), 124.4 (C-7), 130.0 (C-1′), 142.7 (C-8a), 144.5 (C-4a), 147.2 (C-4′), and 150.0 (C-3′); EIMS m/z 479 [81BrMNa]+ (80), 477 [79BrMNa]+ (81), 338 (100); HREIMS m/z 479.05010 (calcd for C20H2381BrNaO7, 479.0500), 477.0515 (calcd for C20H2379BrNaO3, 477.0519). (2S,3S)-2-(Methoxymethoxy)methyl-3-(4′-methoxymethoxy-3′-methoxyphenyl)-6-formyl-1,4-benzodioxane, 14. To a solution of bromide 13 (16.0 mg, 0.035 mmol) in THF (2.5 mL) under an atmosphere of nitrogen at −78 °C was added tBuLi (1.4 M in THF, 0.050 mL, 0.071 mmol). After 2 min, dry DMF (0.043 mL, 0.56 mmol) was added and the mixture stirred at −78 °C for 30 min and then allowed to warm to room temperature and left for a further 15 min. Saturated aqueous NH4Cl (2 mL) was added and the aqueous mixture extracted with EtOAc (3 × 3 mL). The combined organic extracts were dried (MgSO4), and the solvent was removed in vacuo. The crude product was purified by flash chromatography (2:1 n-hexane/EtOAc) to yield the title product 14 (13.5 mg, 96%) as a colorless oil: Rf (2:1 n-hexane/EtOAc) 0.27; [α]D +4 (c 0.6, CHCl3); IR (film) νmax 2958, 2919, 2853, 1690, 1605, 1584, 1503, 1464, 1282, 1154, 1135, 1107, 1076, 1037, 821, 667 cm−1; 1H NMR (400 MHz; CDCl3; Me4Si) δ 3.35 (3H, s, −CH2OCH2OCH3), 3.51 (1H, dd, J = 4.4 and 11.6 Hz, CHaOMOM), 3.53 (3H, s, 4′-OCH2OCH3), 3.75 (1H, dd, J = 2.8 and 11.6 Hz, CHbOMOM), 3.91 (3H, s, 3′-OMe), 4.23−4.27 (1H, m, H-2), 4.61 (2H, dd, J = 6.4 and 18.0 Hz, −CH2OCH2OCH3), 5.00 (1H, d, J = 8.0 Hz, H-3), 5.26 (2H, s, 4′OCH2OCH3), 6.96 (1H, d, J = 1.6 Hz, H-2′), 6.96 (1H, dd, J = 1.6 and 8.0 Hz, H-6′), 7.10 (1H, J = 8.0 Hz, H-8), 7.19 (1H, d, J = 8.8 Hz, H-5′), 7.46 (1H, dd, 1.6 and 8.0 Hz, H-7), 7.49 (1H, d, J = 1.6 Hz, H-5), and 9.85 (1H, s, CHO); 13C NMR (100 MHz; CDCl3) δ 55.5 (−CH2OCH2OCH3), 56.0 (3′-OMe), 56.3 (4′-OCH2OCH3), 66.1 (CH2OMOM), 76.1 (C-3), 77.8 (C-2), 95.4 (4′-OCH2OCH3), 96.9 (−CH2OCH2OCH3), 110.7 (C-2′), 116.2 (C-5′), 117.6 (C-8), 118.4 (C-5), 120.3 (C-6′), 124.3 (C-7), 129.7 (C-1′), 130.7 (C-6), 144.1 (C-4a), 147.3 (C-4′), 149.0 (C-8a), 150.1 (C-3′), and 190.7 (CO); EIMS m/z 427 [MNa]+ (100), 393 (25), 337 (50); HREIMS 427.1379 (calcd for C21H24NaO8, 427.1363). (E)-3-{(2′S,3′S)-3′-[3″-Methoxy-4′′-(methoxymethoxy)phenyl]-2′-[(methoxymethoxy)methyl]-2′,3′-dihydrobenzo[b][1′,4′]dioxin-6′-yl}-1-[2‴,4′′′,6′′′-tris(methoxymethoxy)phenyl]prop-2-en-1-one, 16. A mixture of aldehyde 14 (9.00 mg, 0.022 mmol), ketone 1527 (7.00 mg, 0.022 mmol), and NaOH (10.0 mg, 0.22 mmol) was stirred in absolute EtOH (1.5 mL) under an atmosphere of nitrogen at room temperature for 22 h. Water (1 mL) was added, and the aqueous phase was extracted with CH2Cl2 (3 × 3 mL). The combined organic layers were washed with water (5 mL) and dried (MgSO4), and the solvent was removed in vacuo. The crude product was purified by flash chromatography (1:1 n-hexane/ EtOAc) to yield the title product 16 (8.00 mg, 52%) as a yellow oil: Rf (2:1 n-hexane/EtOAc) 0.17; [α]D +7 (c 1.2, CDCl3); IR (film) νmax 2921, 2853, 1648, 1605, 1506, 1451, 1265, 1152, 1111, 1083, 1048, 1016, 922, 822, 765 cm−1; 1H NMR (400 MHz; CDCl3; Me4Si) δ 3.34 (3H, s, −CH2OCH2OCH3), 3.39 (6H, s, 2‴- and 6″′OCH2OCH3), 3.50 (3H, s, 4‴-OCH2OCH3), 3.50−3.53 (1H, m, CHaOMOM), 3.53 (3H, s, 4″-OCH2OCH3), 3.71 (1H, dd, J = 2.4

11.7 (Si(CH(CH3)2)3), 17.8 (Si(CH(CH3)2)3), 55.7 (OMe), 56.0 (2′-OCH2OCH3), 56.2 (4″-OCH2OCH3), 62.9 (C-3), 73.8 (C-1), 85.1 (C-2), 95.4 (2′-OCH2OMe), 95.5 (4″-OCH2OMe), 110.2 (C2″), 114.6 (C-4′), 116.0 (C-5′′), 119.0 (C-6′′), 120.1 (C-3′), 120.3 (C-6′), 125.3 (C-5′), 134.5 (C-1′′), 145.8 (C-4′′), 147.6 (C-1′), 149.0 (C-2′), and 149.5 (C-3′′). 11b: 1H NMR (400 MHz; CDCl3; Me4Si) δ 0.99−1.08 (21H, m, Si(CH(CH3)2)3 and Si(CH(CH3)2)3), 3.49 (3H, s, −OCH2OCH3), 3.50 (3H, s, −OCH2OCH3), 3.81 (1H, dd, J = 5.6 and 10.8 Hz, H-3a), 3.85 (3H, s, OMe), 3.88 (1H, br s, OH), 3.96 (1H, dd, J = 5.6 and 10.8 Hz, H-3b), 4.16−4.22 (1H, m, H2), 4.86 (1H, d, J = 6.8 Hz, H-1), 5.16−5.20 (2H, m, OCH2OCH3), 5.20 (2H, s, OCH2OCH3), 6.87 (1H, dd, J = 1.6 and 8.4 Hz, H-6″), 6.90 (1H, d, J = 8.4 Hz, H-6′), 6.97 (1H, d, J = 1.6 Hz, H-2″), 7.04 (1H, dd, J = 1.6 and 8.4 Hz, H-5′), 7.08 (1H, d, J = 8.4 Hz, H-5″), and 7.27 (1H, d, J = 1.6 Hz, H-3′); 13C NMR (100 MHz; CDCl3) δ 11.7 (Si(CH(CH3)2)3), 17.8 (Si(CH(CH3)2)3), 55.7 (OMe), 56.0 (2′-OCH2OCH3), 56.4 (4″-OCH2OCH3), 62.9 (C-3), 73.4 (C-1), 87.8 (C-2), 95.4 (2′-OCH2OMe), 95.6 (4″-OCH2OMe), 110.3 (C2″), 114.5 (C-4′), 116.1 (C-5′′), 119.5 (C-6′′), 120.0 (C-3′), 120.2 (C-6′), 125.3 (C-5′), 134.4 (C-1′′), 146.1 (C-4′′), 148.4 (C-1′), 148.5 (C-2′), and 149.7 (C-3′′); EIMS m/z 653 [81BrMNa]+ (15), 651 [79MNa]+ (13), 573 (100), 557 (40), 555 (38), 477 (75); HREIMS m/z 653.1951 (calcd for C29H4581BrNaO8Si, 653.1941); 651.1971 (calcd for C29H4579BrNaO8Si, 651.1959). (2S,3S)-2-Hydroxymethyl-3-(3′-methoxy-4′-hydroxyphenyl)-6-bromo-1,4-benzodioxane, 12a, and (2S,3R)-2-Hydroxymethyl-3-(3′-methoxy-4′-hydroxyphenyl)-6-bromo-1,4-benzodioxane, 12b. A 1.2:1 mixture of alcohols 11a and 11b (0.330 g, 0.52 mmol) and Amberlyst 15 (0.220 g) in dry toluene (20 mL), under an atmosphere of nitrogen, was heated at 80 °C for 18 h. The solution was cooled and filtered, and the solvent removed in vacuo. The crude product was purified by flash chromatography (2:1 nhexane/EtOAc) to yield the title products 12a and 12b (0.144 g, 75%) in a 5:1 trans 12a to cis 12b inseparable mixture as an orange oil: Rf (1:1 n-hexane/EtOAc) 0.36; IR (film) νmax 3520, 2926, 2849, 1518, 1486, 1264, 1237, 1201, 1122, 1032, 907, 878, 728, 703 cm−1; 12a (trans): 1H NMR (400 MHz; CDCl3; Me4Si) δ 3.50 (1H, dd, J = 4.0 and 12.4 Hz, −OCH2OH), 3.75 (1H, dd, J = 2.4 and 12.4 Hz, −OCH2OH), 3.88 (3H, s, OMe), 3.97−4.00 (1H, m, H-2), 4.89 (1H, d, J = 8.0 Hz, H-3), 5.95 (1H, br s, 4′-OH), 6.80 (1H, d, J = 8.8 Hz, H-8), 6.89 (1H, br s, H-2′), 6.89 (1H, dd, J = 1.6 and 8.0 Hz, H-6′), 6.92 (1H, d, J = 8.0 Hz, H-5′), 6.96 (1H, dd, J = 2.4 and 8.8 Hz, H-7) and 7.10 (1H, d, J = 2.4 Hz, H-5); 13C NMR (100 MHz; CDCl3) δ 55.9 (3′-OMe), 61.5 (CH2OH), 76.3 (C-3), 78.1 (C-2), 109.5 (C-2′), 113.0 (C-6), 114.7 (C-5′), 118.2 (C-8), 120.2 (C-5), 120.7 (C-6′), 124.3 (C-7), 127.5 (C-1′), 142.5 (C-8a), 144.6 (C-4a), 146.4 (C-3′), and 146.9 (C-4′). 12b (cis): 1H NMR (400 MHz; CDCl3; Me4Si) δ 3.50 (1H, dd, J = 4.0 and 12.4 Hz, −OCH2OH), 3.75 (1H, dd, J = 2.4 and 12.4 Hz, −OCH2OH), 3.84 (3H, s, OMe), 4.47−4.51 (1H, m, H2), 5.18 (1H, d, J = 2.8 Hz, H-3), 5.91 (1H, br s, 4′-OH), 6.83 (1H, d, J = 8.8 Hz, H-8), 6.90 (1H, br s, H-2′), 6.90 (1H, dd, J = 1.6 and 8.0 Hz, H-6′), 6.92 (1H, d, J = 8.0 Hz, H-5′), 6.98 (1H, dd, J = 2.4 and 8.8 Hz, H-7), and 7.12 (1H, d, J = 2.4 Hz, H-5); 13C NMR (100 MHz; CDCl3) δ 59.1 (3′-OMe), 60.4 (CH2OH), 75.3 (C-3), 77.0 (C-2), 108.5 (C-2′), 113.2 (C-6), 114.7 (C-5′), 118.2 (C-8), 120.4 (C-5), 121.5 (C-6′), 124.8 (C-7), 127.2 (C-1′), 140.8 (C-8a), 143.9 (C-4a), 145.8 (C-3′), and 146.8 (C-4′); EIMS m/z 391 [81BrMNa]+ (75), 289 [79MNa]+ (65), 311 (100), 203 (75); HREIMS m/z 390.9984 (calcd for C16H1581BrNaO5, 390.9975), 388.9997 (calcd for C16H1579BrNaO5, 388.9995). (2S,3S)-2-(Methoxymethoxy)methyl-3-(4′-methoxymethoxy-3′-methoxyphenyl)-6-bromo-1,4-benzodioxane, 13. To a solution of a 5:1 mixture of diols 12a and 12b (55.0 mg, 0.15 mmol) in CH2Cl2 (2 mL) under an atmosphere of nitrogen at room temperature were added DIPEA (0.067 mL, 0.37 mmol) and MOMCl (0.067 mL, 0.60 mmol) dropwise, and the mixture was stirred at this temperature for 48 h. Saturated aqueous NH4Cl (3 mL) was added and the layers were separated. The aqueous layer was further extracted with CH2Cl2 (3 × 3 mL), the combined organic layers were dried (MgSO4), and the solvent was removed in vacuo. The crude product 2635



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and 11.6 Hz, CHbOMOM), 3.90 (3H, s, 3″-OMe), 4.18−4.22 (1H, m, H-2′), 4.60 (2H, dd, J = 6.4 and 18.4 Hz, −CH2OCH2OCH3), 4.97 (1H, d, J = 8.0 Hz, H-3′), 5.10 (4H, s, 2‴- and 6″′OCH2OCH3), 5.17 (2H, s, 4‴-OCH2OCH3), 5.25 (2H, s, 4″OCH2OCH3), 6.55 (2H, s, H-3‴ and H-5″′), 6.81 (1H, d, J = 16.0 Hz, H-2), 6.94 (1H, br s, H-2″), 6.95 (1H, dd, J = 2.0 and 8.0 Hz, H6″), 6.96 (1H, d, J = 8.4 Hz, H-8′), 7.06 (1H, dd, J = 2.0 and 8.4 Hz, H-7′), 7.16 (1H, d, J = 2.0 Hz, H-5′), 7.18 (1H, d, J = 8.0 Hz, H-5″), and 7.25 (1H, d, J = 16.0 Hz, H-3); 13C NMR (100 MHz; CDCl3) δ 55.5 (−CH2OCH2OCH3), 56.0 (3″-OMe), 56.3 (4′-, 2‴-, 4′′′-, and 6′′′-OCH2OCH3), 66.2 (CH2OMOM), 76.2 (C-3′), 77.5 (C-2′), 94.6 (2‴-, 4″′-, and 6′′′-OCH2OCH3), 95.4 (4″-OCH2OCH3), 96.9 (−CH2OCH2OCH3), 97.2 (C-3‴ and C-5″′), 110.7 (C-2′′), 115.0 (C-1′′′), 116.2 (C-6′′), 116.7 (C-5′), 117.5 (C-8′), 120.3 (C-5′′), 122.7 (C-7′), 127.5 (C-2), 128.5 (C-6′), 130.1 (C-1′′), 143.9 (C4a′), 144.9 (C-3), 145.6 (C-8a′), 147.3 (C-4′′), 150.0 (C-3′′), 155.8 (C-2′′′ and C-6′′′), 159.5 (C-4′′′), and 194.2 (C-1); EIMS m/z 725 [MK]+ (40), 709 [MNa]+ (100); HREIMS 709.2477 (calcd for C35H42NaO14, 709.2467). ent-Silybin A (ent-(3)) and Silybin B (4). Using the method of Tanaka et al.23 and Zhao et al,28 a solution of 30% H2O2 (0.14 mL) and 5% NaOH (0.14 mL) was added to a solution of chalcone 16 (13.0 mg, 0.019 mmol) in MeOH (0.7 mL), and the mixture was stirred at room temperature for 4 h. Water (1.0 mL) was added and the aqueous mixture was extracted with EtOAc (3 × 2 mL). The combined organic extracts were washed with water (2 mL) and dried (MgSO4), and the solvent was removed in vacuo, to give the crude epoxide, which was immediately suspended in MeOH (0.5 mL), to which a mixture of MeOH (0.15 mL) and concentrated HCl (0.04 mL) was added. The reaction mixture was heated at 70 °C for 15 min and cooled to room temperature. Ice−water (1 mL) was added, and the mixture was extracted with EtOAc (3 × 2 mL). The combined organic extracts were washed with water (3 mL) and dried (MgSO4), and the solvent was removed in vacuo. The crude product was purified by flash chromatography (19:1 CH2Cl2/MeOH) to yield the title products as an inseparable mixture (2.6 mg, 28%) as an off-white semisolid. The NMR spectroscopic data matched the reported data.29 ent-Isosilandrin A (ent-(5)) and Isosilandrin B (6). Using a method reported by Samu et al.11 and Zhao et al,28 to a solution of chalcone 16 (5.0 mg, 7.28 μmol) in MeOH (1 mL) was added 10% HCl (0.1 mL), and the mixture was heated at reflux for 1.5 h. NaOAc (60 mg, 3.64 mmol) was added, and the mixture heated at reflux for a further 5 h. The mixture was cooled to room temperature before water (10 mL) was added and acidified to pH 5. The mixture was extracted with EtOAc (3 × 10 mL), the organic extracts were washed with water until a neutral pH and dried (MgSO4), and the solvent was removed in vacuo. The crude product was purified by flash chromatography (19:1 CH2Cl2/MeOH) to yield the title products as an inseparable mixture (1.4 mg, 43%) as an off-white semisolid. The 1H NMR spectroscopic data matched the reported data.11 Cytotoxicity Testing. Cytotoxicity measurements were performed in human hepatoma Huh7.5.1 cells. Compounds were solubilized in DMSO added to cells at the indicated concentrations, and 72 h later, ATP levels were measured using the ATPlite assay (PerkinElmer). Control cell cultures were treated with DMSO, which was kept constant at a final concentration of 0.5% in all wells. All samples were measured in triplicate for each concentration. Doses were log transformed and fitted to a variable slope four-parameter regression model in Prism 6. Antiviral Testing. Antiviral measurements were performed in Huh7.5.1 cells that were infected with HCV at a multiplicity of infection of 0.05 for 5 h. Huh7.5.1 cells are a human hepatoma cell line derived from the parental line Huh7, and the cells are highly permissive for HCV infection.35 Virus inoculum was removed and replaced with fresh media containing the indicated concentrations (in μM) of each compound. DMSO was the solvent control and was added at no more than 0.5% final concentration. Cellular protein lysates were analyzed by SDS-PAGE and Western blotting to detect HCV nonstructural proteins NS3 and NS5A. Blots were stripped and

reprobed for actin expression as a control for equal loading of cellular proteins. Western blots were performed as described.38

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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.8b00416. 1 H and 13C NMR spectra of synthesized compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D. Barker). *E-mail: [email protected] (S. Polyak). ORCID

Lisa I. Pilkington: 0000-0002-9292-3261 David Barker: 0000-0002-3425-6552 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Royal Society of New Zealand, Marsden Fund, for funding this research and the University of Auckland for additional financial assistance and a doctoral scholarship (L.I.P.). This research is also partially supported by grant R01AT006842 from the National Institutes of Health (USA) to S.J.P.

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REFERENCES

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1,4-Benzodioxane Lignans: An Efficient, Asymmetric Synthesis of

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