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Synthesis of the 2-methylene analogue of the HRV 3C protease inhibitor thysanone (2-carbathysanone)† Katrin Schünemann,a,b Daniel P. Furkert,a Eun Cho Choi,a Stephen Connelly,c John D. Fraser,b Jonathan Sperrya and Margaret A. Brimble*a The Human Rhinovirus (HRV) is the major aetiological agent for the common cold, for which only symptomatic treatment is available. HRV maturation and replication is entirely dependent on the activity of a virally encoded 3C protease that represents an attractive target for the development of therapeutics to treat the common cold. Herein we report the synthesis and biological evaluation of the 2-methylene ana-
Received 26th September 2013, Accepted 9th December 2013 DOI: 10.1039/c3ob41951g www.rsc.org/obc
logue of the HRV 3C protease inhibitor (−)-thysanone (1) namely 2-carbathysanone (2), in an attempt to decipher the structural features in the natural product that are responsible for the 3C protease activity. 2-Carbathysanone (2) (and related analogues (±)-cis-23, (±)-cis-30, (±)-31) did not inhibit HRV 3C protease, indicating that the lactol functionality present in (−)-thysanone (1) is a critical structural feature required for inhibition.
Introduction The Human Rhinovirus (HRV) is the major cause of the common cold, which typically infects nasal epithelia cells in the upper respiratory tract.1 The virus contains a positive strand RNA genome, which is directly translated into a viral polyprotein precursor of 200 kDa. In order to produce active viral proteins and enzymes the polyprotein undergoes a series of proteolytic cleavages. These cleavages are governed by two virally encoded proteases designated as 2A and 3C. These proteases are therefore interesting targets for drug development against the common cold. (−)-Thysanone (1) was isolated from the fungus Thysanophora penicilloides by the process of screening microbial extracts for bioactive lead structures.2 (−)-Thysanone (1) was found to be an effective inhibitor of HRV 3C protease with an IC50 of 13 µg mL−1 (47 µM).2 To date, two total syntheses and one formal synthesis of the natural product (−)-thysanone (1) have been published as well as syntheses of several different analogues 3–8.3–17 Given our continued interest in synthesizing (−)-thysanone (1) and its
a School of Chemical Sciences, The University of Auckland, 23 Symonds St., Auckland, New Zealand. E-mail: [email protected] b Department of Molecular Medicine and Pathology, The University of Auckland, 85 Park Road, Auckland, New Zealand c Department of Molecular Biology, The Scripps Research Institute, BCC 265, 10550 Nr. Torrey Pines Rd., La Jolla, CA 92037, USA † Electronic supplementary information (ESI) available: 1H and 13C NMR data for all new compounds and experimental data for compounds 10, 13, 14, 17–21, 23 and 24. See DOI: 10.1039/c3ob41951g
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Fig. 1 Structure of (−)-thysanone (1), (±)-2-carbathysanone (2) and analogues 3–8 previously synthesised by our group.6–12,15
analogues (Fig. 1),7–15 we herein report the synthesis and biological evaluation of 2-carbathysanone (2) prepared from substituted naphthalene (±)-9 that is assembled via Hauser–Kraus annulation of cyanophthalide 10 with cyclohexenone (±)-11 (Scheme 1).
Results and discussion Despite the fact that our earlier work suggested that Hauser–Kraus annulation using a 4,6-dimethoxy substituted
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Scheme 3 Reagents and conditions: (i) KOtBu, tBuOH, 0 °C → 90 °C, 20 h, 27%.20
Scheme 1 Retrosynthesis of 2-carbathysanone (2) via Hauser–Kraus annulation.
Table 1 Investigation of the Hauser–Kraus annulation. Reagents and conditions: (i) (a) base, solvent, r.t., 1 h; (b) Me2SO4, KOH, 60 °C, 16 h
Entry 1 2 3 4 Scheme 2 Reagents and conditions: (i) H2SO4, MeOH, reflux, 5 d, quant.; (ii) Cs2CO3, nBu4NI (TBAI), 2-bromopropane, acetone–DMF (10 : 1), reflux, 17 h, quant.; (iii) AlMe3, Et2NH, PhMe, reflux, quant.; (iv) t BuLi, tetramethylethylenediamine (TMEDA), DMF, −78 °C → r.t., 17 h, quant.; (v) (a) TMSCN, KCN, 18-crown-6, CH2Cl2, 3 h, 0 °C → r.t.; (b) AcOH, 17 h, r.t., 53% over two steps.
cyanophthalide did not result in successful annulation with a variety of electrophiles,15 we nevertheless decided to start our synthesis of 2-carbathysanone (2) by reinvestigation of this approach. We therefore synthesized cyanophthalide 10 from 2,4-dihydroxybenzoic acid (12) (Scheme 2). 2,4-Dihydroxybenzoic acid (12) was readily converted to the diisopropylprotected methyl benzoate 13 in two steps using sulfuric acid in methanol to effect the methyl esterification followed by protection of both hydroxyl groups as isopropyl ethers.18 Methyl benzoate 13 was then converted to benzaldehyde 14 by treatment with trimethylaluminium and diethylamine followed by formylation using tBuLi, TMEDA and DMF. Cyanophthalide 10 was synthesized following a general literature procedure by reaction of benzaldehyde 14 with trimethylsilyl cyanide (TMSCN) in presence of catalytic amounts of KCN and 18-crown-6, followed by treatment of the α-trimethylsilyloxybenzyl cyanide intermediate with glacial acetic acid.19 Cyanophthalide 10 was obtained in a moderate 53% yield over two steps. Hauser–Kraus annulation partner cyclohexenone (±)-11 was synthesized following a one-pot procedure described by Yudin and co-workers (Scheme 3).20 Hauser–Kraus annulation between cyanophthalide 10 and enone (±)-11 requires investigation of a variety of different bases and solvents. Due to the instability of the initial naphthalenediol annulation product it was decided to perform
906 | Org. Biomol. Chem., 2014, 12, 905–912
Base t
KO Bu KOtBu KHMDS DBU
Solvent
Yield
THF DMSO THF THF
42% 19% 35% Decomposition
a one-pot Hauser–Kraus annulation, methylation strategy. Selected examples of this reaction are shown in Table 1. The optimum conditions involving reaction of cyanophthalide 10 with potassium tert-butoxide in THF at 0 °C followed by the addition of enone (±)-11 (entry 1) afforded naphthol (±)-17 in a moderate 42% yield after methylation using dimethyl sulfate and potassium hydroxide. The nature of the leaving group on the phthalide can substantially influence the yield obtained in Hauser–Kraus annulations.21–26 We therefore decided to prepare phosphonylphthalide 18, phenylthiophthalide 19 and phenylsulfonylphthalide 20 from the common intermediate benzaldehyde 14 (Scheme 3). Phosphonylphthalide 18 was obtained following a procedure published by Van der Veken and co-workers.27 Treatment of benzaldehyde 14 with trimethylphosphite in the presence of oxalic acid gave phosphonylphthalide 18 in 81% yield. Phenylthiophthalide 19 was prepared in a two-step, onepot procedure.28 Potassium hydroxide was used first to generate an unstable hydroxyphthalide from benzaldehyde 14, which was then treated in situ with thiophenol to form thiophenylphthalide 19 in moderate 36% yield over two steps. The phenylthiophthalide 19 was then oxidized with m-CPBA to afford phenylsulphonylphthalide 20 in 97% yield (Scheme 4). Disappointingly, use of all three phthalides 18–20 in the key-Hauser–Kraus annulation with cyclohexenone (±)-11 using potassium tert-butoxide in different solvents (e.g. DMSO and THF) led to decomposition of both starting materials (Scheme 5). It was therefore decided to continue the synthesis with cyanophthalide 10 using the previously established Hauser– Kraus annulation conditions. Reduction of the ketone in the
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Fig. 2
Observed nOe signals and proposed configuration.
Scheme 4 Reagents and conditions: (i) oxalic acid, P(OEt)3, reflux, 16 h, 81%; (ii) (a) KOH, H2O, THF, r.t., 18 h; (b) PhSH, PhMe, p-TsOH, 18 h, 36% over two steps; (iii) m-CPBA, CH2Cl2, 0 °C → r.t., 97%.
Scheme 6
Scheme 5 Unsuccessful Hauser–Kraus annulation using phthalides 18, 19 and 20.
annulation product (±)-17 using borane-dimethyl sulfide complex gave an inseparable mixture of (±)-cis-alcohol 21 and (±)-trans-alcohol 21 in excellent yield (cis–trans, 5 : 1, Table 2), with some of the over-reduced product (±)-22 also isolated. In an attempt to improve the diastereoselectivity of the reduction some sterically hindered boranes possessing two hydrides were investigated. Pleasingly, use of both cyclohexylborane and isopropoxyborane did result in the diastereoselective formation of the (±)-cis-alcohol 21, however the amount of over-reduced product 22 also increased. The observed nOe between H-1 and H-3 established the cis-stereochemistry in (±)-cis-alcohol 21, consistent with the C-1 hydroxyl and the C-3 methyl groups occupying pseudoequatorial positions (Fig. 2). Despite not being able to isolate a diastereomerically pure sample of the trans-product 21 possessing the cyclohexyl substituents in the same relationship as observed in thysanone, we aimed to complete the synthesis using the cis-product 21 and the next step required its oxidation to the corresponding
Table 2
Reagents and conditions: (i) CAN, MeCN–H2O, 74%.
(±)-cis-naphthoquinone 23. (±)-cis-Naphthol 21 was oxidized using ceric ammonium nitrate (CAN) in aqueous acetonitrile affording (±)-cis-naphthoquinone 23 in moderate 74% yield (Scheme 6). At this stage completion of the synthesis of 2-carbathysanone (2) only required one step, namely cleavage of the isopropyl protecting groups. Disappointingly, treatment of (±)-cis-23 with AlCl3, BCl3 or Bi(OTf )3 only resulted in decomposition of the starting material.29–31 Due to the inability to deprotect the isopropyl ethers in 23 we next decided to change the isopropyl protecting groups to methoxymethyl (MOM)-groups. The synthesis of MOMprotected cyanophthalide 27 is shown in Scheme 7. Methyl benzoate 24 was readily converted to TBS-protected benzaldehyde 25 in three steps using sodium hydride and tertbutyldimethylsilyl chloride (TBSCl) to afford TBS-protected methyl benzoate, which was then converted to the corresponding amide using trimethylaluminium and diethylamine.32 Formylation of the amide using tBuLi, TMEDA and DMF afforded the desired benzaldehyde 25 in 82% yield over three steps. The synthesis of MOM-protected phthalide 27 from benzaldehyde 25 was first attempted using TMSCN in the presence of a catalytic amount of KCN and 18-crown-6, followed by treatment of the α-trimethylsilyloxybenzyl cyanide
Reduction of ketone (±)-17 using different boranes. Reagents and conditions: (i) borane, THF, 0 °C → r.t., 1 h
Entry
Borane
(±)-cis-21 : (±)-trans-21
(±)-22
1 2 3
BH3·SMe2 (C6H11)BH2 i PrOBH2
93% (5 : 1) 72% (1 : 0) 56% (1 : 0)
7% 25% 32%
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Scheme 7 Reagents and conditions: (i) NaH, TBSCl, 0 °C → r.t., 2 h, quant.; (ii) AlMe3, Et2NH, PhMe, reflux, quant.; (iii) tBuLi, TMEDA, DMF, THF, −78 °C → r.t., 18 h, 82%; (iv) (a) TMSCN, KCN, 18-crown-6, CH2Cl2, 0 °C → r.t., 3 h; (b) AcOH, 18 h; (c) P4O10, CH2(OMe)2, CH2Cl2, 0 °C → r.t., 3 h, 37%.
intermediate with glacial acetic acid, which afforded cyanophthalide 26. Disappointingly, the isolation of 26 was difficult due its high solubility in water. Furthermore, the subsequent protection of 26 using MOMCl and Hünig’s base resulted in alkylation at C-1 of the resulting cyanophthalide. To circumvent these issues, we developed a one-pot procedure (Scheme 7). Benzaldehyde 25 was treated with TMSCN in the presence of a catalytic amount of KCN and 18-crown-6 followed by glacial acetic acid, delivering cyanophthalide 26 that had undergone concomitant desilylation under the reaction conditions. Subsequent treatment of 26 with phosphorus pentoxide and dimethoxymethane afforded cyanophthalide 27 in 37% yield over three steps. Disappointingly, the previously established conditions used to effect Hauser–Kraus annulation of diisopropyl-protected cyanophthalide 10 with enone (±)-11 using potassium tertbutoxide followed by methylation using dimethyl sulfate and potassium hydroxide, were unsuccessful when applied to MOM-protected cyanophthalide 27. Fortunately, changing the base to sodium hydride followed by methylation using dimethyl sulfate and sodium hydride afforded dimethoxynaphthalene (±)-28 in 79% yield over two steps (Scheme 8). In this case, methylation of the 10-naphthol group occurred as the sodium hydride was able to deprotonate the hydrogen bonded C-10-hydroxyl-group. Although attempted reduction of (±)-28 using cyclohexylborane only resulted in decomposition, employing iBu2AlH exclusively afforded the desired (±)-cis-
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alcohol 29 in excellent yield. Subsequent oxidative demethylation of (±)-cis-29 using CAN afforded (±)-cis-naphthoquinone 30 in 33% yield. Removal of the MOM protecting groups was first attempted using 1 M aq. hydrochloric acid; however, only decomposition was observed and milder conditions were therefore investigated. Bismuth triflate has been shown to cleave MOM-acetals in a THF–water mixture at room temperature.31 However, under these conditions, only starting material was observed after two hours. However, heating the reaction mixture to 80 °C did effect deprotection, affording the desired 2-carbathysanone (2) in 87% yield. The synthesis of 2-carbathysanone (2) described above also resulted in two analogues (±)-23 and (±)-cis-30, good candidates to be screened for HRV 3C protease activity. (±)-Naphthol 22 was also oxidised to (±)-naphthoquinone 31 using CAN in 81% yield in order to provide an additional analogue to evaluate for inhibition of HRV 3C protease (Scheme 9). The cytotoxicity of 2-carbathysanone (2) and the three analogues (±)-cis-23, (±)-cis-30 and (±)-31 was also investigated. The HRV 3C protease screening was carried out using our previously reported solid phase based fluorescent substrate assay.33 The cytotoxicity (LD50) of the compounds was evaluated using a cell-proliferation assay in which the human lung adenocarcinoma epithelia cell-line NCI-H441 was grown in the presence of the synthesized inhibitors 2-carbathysanone (2), (±)-cis-23, (±)-cis-30 and (±)-31 and treated with 3H-thymidine to detect cell-proliferation. The results are summarized in Table 3. All analogues did not inhibit HRV 3C protease, inferring the 2-oxa-lactol functionality to be a key structural feature in the mechanism of inhibition of HRV 3C protease by thysanone (1). All four analogues also exhibited high cytotoxicity against NCI-H441.
Scheme 9 81%.
Reagents and conditions: (i) CAN, MeCN–H2O, r.t., 10 min,
Scheme 8 Reagents and conditions: (i) (a) NaH, THF, 0 °C → r.t., 1 h; (b) Me2SO4, NaH, 0 °C → r.t., 3 h, 79%; (ii) iBu2AlH, CH2Cl2, 0 °C → r.t., 87%, (iii) CAN, MeCN–H2O, 10 min, 33%; (iv) Bi(OTf )3, THF–H2O, 80 °C, 87%.
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Table 3 study
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IC50 and LD50 values of thysanone analogues prepared in this
Compound
R1
R2
IC50 [µM]
LD50 [µM]
2-Carbathysanone (2) (±)-cis-23 (±)-cis-30 (±)-31
OH OiPr OMOM OiPr
OH OH OH H
n.i.d. n.i.d. n.i.d. n.i.d.
93.4 ± 36 52.0 ± 0 28.4 ± 11 37.4 ± 0
n.i.d. = no inhibition detected.
Conclusion In conclusion, we have developed a synthesis of 2-carbathysanone (2) and closely related analogues using a Hauser–Kraus annulation. 2-Carbathysanone (2), and analogues (±)-cis-23, (±)-cis-30, (±)-31 did not inhibit HRV 3C protease thus indicating that the lactol functionality present in thysanone (1) is a critical structural feature required for inhibition of HRV 3C protease.
Experimental General details Unless otherwise stated, all non-aqueous reactions and distillations were performed under a nitrogen or argon atmosphere in oven- or flame-dried glassware. Tetrahydrofuran was freshly distilled from sodium/benzophenone. Dichloromethane (CH2Cl2) and toluene were freshly distilled from calcium hydride. TMEDA was distilled from calcium hydride and stored under a nitrogen atmosphere over MgSO4. Reactions performed at low temperature were either cooled in an acetone-dry ice bath for temperatures below 0 °C or using a water-ice bath for 0 °C. Flash chromatography was carried out using 0.063–0.1 mm silica gel (Davisil R LC60A 40-63 Micron) with the indicated solvent. Preparatory TLC was carried out on 500 μm Uniplate™ (Analtech) silica gel (20 × 20 cm) thin layer chromatography plates. Infrared spectra were recorded using a Perkin Elmer R Spectrum 1000 Fourier Transform Infrared spectrometer. Values are expressed in wavenumbers (cm−1) and recorded in a range of 4000 to 450 cm−1. NMR spectra were recorded at 21 °C in CDCl3 or C6D6 on either a Bruker® Avance 300 spectrometer operating at 300 MHz for 1H nuclei and 75 MHz for 13C nuclei or on a Bruker® DRX400 or Bruker® 400 spectrometer operating at 400 MHz for 1H nuclei and 100 MHz for 13C nuclei. All chemical shifts are reported in parts per million ( ppm) from tetramethylsilane (δ = 0) and were measured relative to the solvent in which the sample was analysed (CDCl3: TMS δ = 0.00 for 1H NMR and CDCl3 δ = 77.16 for 13C NMR; acetone-d6: δ = 2.05 for 1H NMR and δ =
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29.84 ± 0.01 for 13C NMR). Coupling constants ( J) are reported in Hertz (Hz). 1H-NMR data is reported as chemical shift in ppm, followed by the relative integral, the multiplicity (“s” singlet, “d” doublet, “dd” doublet of doublets, “ddd”, doublet of doublets of doublets, “dt” doublet of triplets, “t” triplet, “q” quartet, “quint.” quintuplet, “sept.” septet, “m” multiplet, “b” broad) and coupling constant where applicable and relative integral. 13C NMR spectra are reported as chemical shift in ppm. High resolution mass spectra were recorded using a Bruker microTOF-QII mass spectrometer. 2,4-Bis((tert-butyldimethylsilyl)oxy)-N,N-diethyl-6formylbenzamide 25 To a solution of methyl 2,4-dihydroxybenzoate 24 (1.5 g, 8.9 mmol) in THF (45 mL) was added sodium hydride (0.9 g, 22.3 mmol) at 0 °C. The reaction mixture was stirred for 30 min and TBSCl (2.8 g, 22.3 mmol) was added. The reaction mixture was stirred for 2 h then satd. aq. NH4Cl solution (20 mL) was added. The aq. layer was extracted with EtOAc (3 × 30 mL) and the combined organic layers were washed with brine (30 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (9 : 1) as eluent to afford methyl 2,4-bis((tert-butyldimethylsilyl)oxy)-benzoate (3.5 g, 8.8 mmol, quant.) as a yellow oil. Rf (hexanes–ethyl acetate = 9 : 1): 0.81. 1H NMR (400 MHz, CDCl3): δ = 7.72 (1H, d, J = 8.8 Hz), 6.40 (1H, dd, J = 8.8 Hz, 2.4 Hz), 4.46 (1H, d, J = 2.4 Hz, H-3), 3.83 (3H, s), 1.01 (9H, s), 0.97 (9H, s), 0.21 (6H, s), 0.21 (6H, s). 13C NMR (100 MHz, CDCl3): δ = 167.0, 160.3, 157.1, 133.4, 116.0, 113.6, 112.8, 51.7, 25.8, 25.8, 18.5, 18.4. The spectroscopic data were in agreement with that reported in the literature.34 To a solution of trimethylaluminum (2 M in toluene, 7 mL, 14 mmol) in toluene (10 mL) was added diethylamine (1.5 mL, 14 mmol) at −6 °C under an atmosphere of nitrogen. After 10 min the reaction mixture was warmed to r.t. and methyl 2,4-bis((tertbutyldimethylsilyl)oxy)-benzoate (1.4 g, 4.2 mmol) was added. The resulting mixture was heated at reflux overnight. The mixture was cooled to 0 °C then 10% aq. HCl (10 mL) was added. The layers were separated and the organic layer was washed with 10% aq. HCl (20 mL). The combined aq. layers were extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexane–EtOAc (4 : 1) as eluent to afford 2,4-Bis((tert-butyldimethylsilyl)oxy)-N,N-diethylbenzamide (1.54 g, 4.2 mmol, quant.) as a yellow oil. Rf (hexane– ethyl acetate = 4 : 1): 0.42. 1H NMR (400 MHz, CDCl3): δ = 7.04 (1H, d, J = 8.3 Hz), 6.47 (1H, dd, J = 8.3 Hz), 6.31 (1H, d, J = 2.2 Hz), 3.69–3.02 (4H, bm), 1.22 (3H, t, J = 7.1 Hz), 0.99 (3H, t, J = 7.1 Hz), 0.98 (9H, s), 0.98 (9H, s), 0.21 (6H, s), 0.19 (6H, s). 13C NMR (100 MHz, CDCl3): δ = 169.3, 156.9, 152.3, 128.6, 123.4, 113.6, 111.4, 43.0, 39.4, 25.8, 25.8, 18.4, 18.3, 14.2, 13.4. The spectroscopic data were in agreement with that reported in the literature.34 A solution of 2,4-bis((tert-butyldimethylsilyl)oxy)N,N-diethylbenzamide (2.5 g, 6.8 mmol) and TMEDA (1.1 mL, 7.5 mmol) in THF (10 mL) was cooled to −78 °C under an
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atmosphere of nitrogen and t-BuLi (6.4 mL, 10.2 mmol) was added slowly. The solution turned yellow and was stirred for a further 10 min. DMF (1.1 mL, 13.6 mmol) was added and the mixture warmed to r.t. overnight. Satd. aq. NH4Cl solution (20 mL) was added and the mixture stirred for 30 min. The aq. layer was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (4 : 1) as eluent to afford the title compound (2.2 g, 5.5 mmol, 82%) as a yellow oil. Rf (hexanes–EtOAc = 4 : 1): 0.42. νmax (film/cm−1): 2956, 2934, 2894, 2861, 1699, 1633, 1594, 1570, 1332, 1162, 783, 733. 1H NMR (400 MHz, CDCl3): δ = 9.86 (1H, s, H-CHO), 6.97 (1H, d, J = 2.3 Hz), 6.53 (1H, d, J = 2.3 Hz), 3.70 (1H, dq. J = 7.0 Hz, 14.2 Hz), 3.93 (1H, dq, J = 7.0 Hz, 14.2 Hz), 3.14 (1H, dq, J = 7.3 Hz, 14.4 Hz), 3.07 (1H, dq, J = 7.3 Hz, 14.4 Hz), 1.24 (3H, t, J = 7.3 Hz), 0.98 (3H, t, J = 7.0 Hz), 0.95 (9H, s), 0.94 (9H, s), 0.22–0.19 (12H, m). 13C NMR (100 MHz, CDCl3): δ = 190.4, 166.2, 156.7, 153.1, 134.8, 125.8, 116.9, 113.0, 43.2, 39.6, 25.7, 25.6, 18.3, 18.2, 14.0, 13.0. HRMS (ESI): Calcd for C24H44NO4Si2 [MH]+: 466.2803, found: 466.2817; calcd for C24H43NNaO4Si2 [MNa]+: 488.2623, found: 488.2625.
4,6-Bis(methoxymethoxy)-3-oxo-1,3-dihydroisobenzofuran1-carbonitrile 27 To a solution of 2,4-bis((tert-butyldimethylsilyl)oxy)-N,Ndiethyl-6-formylbenzamide 25 (4.0 g, 10 mmol) in CH2Cl2 (20 mL) was added KCN (13 mg, 0.2 mmol) and 18-crown-6 (53 mg, 0.2 mmol) under an atmosphere of nitrogen. The reaction mixture was cooled to 0 °C and TMSCN (1.9 mL, 15 mmol) was added dropwise. The solvent was removed under reduced pressure after 15 min and the residue taken up in acetic acid (20 mL) and the mixture stirred overnight at 80 °C. The solvent was removed under reduced pressure and the residue taken up in CH2Cl2–dimethoxymethane (1 : 1, 80 mL) and phosphorus pentoxide (10 g, 70 mmol) was added at 0 °C. The mixture was warmed to r.t., stirred for 3 h then poured into ice cold satd. aq. Na2CO3 solution (100 mL). The aq. layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (9 : 1) as eluent to afford the title compound (1.03 g, 3.7 mmol, 37%) as a colourless oil. Rf (hexanes–EtOAc = 1 : 1): 0.70. νmax (film/ cm−1): 2980, 2938, 1785, 1697, 1614, 1598, 1319, 1150, 972, 920, 686. 1H NMR (400 MHz, CDCl3): δ = 6.95 (1H, d, J = 1.6 Hz), 6.92 (1H, d, J = 1.6 Hz), 5.92 (1H, s), 5.36 (2H, s), 5.30 (1H, d, J = 7.2 Hz), 5.23 (1H, d, J = 7.2 Hz), 3.54 (3H, s), 3.51 (3H, s). 13C NMR (100 MHz, CDCl3): δ = 165.3, 157.9, 146.0, 114.2, 103.7, 105.8, 102.5, 95.1, 94.8, 64.8, 57.0, 56.8. HRMS (ESI): Calcd for C13H14NO6 [MH]+: 280.0816, found: 280.0823; calcd for C13H13NNaO6 [MNa]+: 302.0635, found: 302.0631.
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9,10-Dimethoxy-6,8-bis(methoxymethoxy)-3-methyl-3,4dihydroanthracen-1(2H)-one (±)-28 To a solution of 4,6-bis(methoxymethoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carbonitrile 27 (120 mg, 0.43 mmol) and cyclohexenone (±)-1119 (95 mg, 0.86 mmol) in THF (1 mL) was added NaH (19 mg, 60% in mineral oil, 0.47 mmol) in THF (0.5 mL) under an atmosphere of nitrogen. The reaction mixture was stirred for 1 h then dimethyl sulfate (0.41 mL, 4.3 mmol) and NaH (38 mg, 0.95 mmol) were added. The mixture was stirred at r.t. for 3 h then satd. aq. NH4Cl solution (5 mL) was added. The aq. layer was extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with brine (10 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (7 : 3) as eluent to afford the title compound (132 mg, 0.34 mmol, 79%) as a yellow oil. Rf (hexanes–EtOAc = 7 : 3): 0.54. νmax (film/cm−1): 3071, 2954, 2933, 2859, 1715, 1613, 1529, 1335, 1150, 930, 765. 1H NMR (400 MHz, CDCl3): δ = 7.27 (1H, d, J = 2.4 Hz), 6.87 (1H, d, J = 2.4 Hz), 5.31 (2H, d, J = 0.9 Hz), 5.30 (2H, s), 3.90 (3H, s), 3.84 (3H, s), 3.58 (3H, s), 3.52 (3H, s), 3.32 (1H, dq, J = 16.2 Hz, 2.0 Hz), 2.75 (1H, dq, J = 16.2 Hz, 2.0 Hz), 2.52 (1H, dd, J = 16.2 Hz, 10.6 Hz), 2.34 (1H, dd, J = 16.2 Hz, 11.5 Hz), 2.28–2.19 (1H, m), 1.16 (3H, d, J = 6.4 Hz). 13C NMR (100 MHz, CDCl3): δ = 197.8, 158.0, 147.7, 134.9, 131.4, 121.7, 117.1, 105.2, 98.3, 96.3, 94.5, 63.1, 60.8, 56.7, 56.5, 49.4, 32.4, 29.3, 21.5. HRMS (ESI): Calcd for C21H27O7 [MH]+: 391.1751, found: 391.1738; calcd for C21H26NaO7 [MNa]+: 413.1571, found: 413.1557; calcd for C21H26KO7 [MK]+: 429.1310, found: 429.1295.
cis-9,10-Dimethoxy-6,8-bis(methoxymethoxy)-3-methyl-1,2,3,4tetrahydroanthracen-1-ol (±)-cis-29 To a solution of 9,10-dimethoxy-6,8-bis(methoxymethoxy)3-methyl-3,4-dihydroanthracen-1(2H)-one (±)-28 (60 mg, 0.15 mmol) in CH2Cl2 (1 mL) was added at −78 °C DIBAL-H (170 µL, 1 M in cyclohexane, 0.17 mmol) under an atmosphere of nitrogen and the mixture stirred at −78 °C for 30 min then allowed to warm to r.t. Methanol (1 mL) and half-satd. Rochelle’s salt (3 mL) were added and the reaction was stirred for 20 min. The aq. layer was extracted with Et2O (3 × 5 mL) and the combined organic extracts were dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (7 : 3) as eluent to afford the title compound (40 mg, 0.10 mmol, 87%) as a yellow oil. Rf (hexane–EtOAc = 7 : 3): 0.39. νmax (film/cm−1): 3505, 2934, 2833, 1751, 1619, 1595, 1452, 1335, 1227, 1147, 1024, 928. 1H NMR (400 MHz, CDCl3): δ = 7.27 (1H, d, J = 2.3 Hz), 6.88 (1H, d, J = 2.3 Hz), 5.32 (2H, s), 5.29 (2H, s), 5.33–5.24 (1H, m), 4.68 (1H, bs), 3.90 (3H, s), 3.81 (3H, s), 3.59 (3H, s), 3.53 (3H, s), 3.13 (1H, dq, J = 16.6 Hz, 2.2 Hz), 2.36–2.29 (2H, m), 1.86–1.76 (1H, m), 1.52–1.44 (1H, m), 1.17 (3H, d, J = 6.5 Hz). 13C NMR (100 MHz, CDCl3): δ = 155.6, 154.5, 151.4, 148.5, 131.2, 129.2, 128.9, 115.9, 104.7, 98.6, 96.3, 94.7, 67.5, 61.9, 60.6, 56.7, 56.4, 40.0, 33.2, 27.7, 22.3. HRMS (ESI): Calcd
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for C21H28NaO7 [MNa]+: 415.1727, found: 415.1729; calcd for C21H28KO7 [MK]+: 431.1467, found: 431.1474.
cis-1-Hydroxy-6,8-bis(methoxymethoxy)-3-methyl-1,2,3,4tetrahydroanthracene-9,10-dione (±)-cis-30 To a solution of cis-9,10-dimethoxy-6,8-bis(methoxymethoxy)3-methyl-1,2,3,4-tetrahydro-anthracen-1-ol (±)-cis-29 (10 mg, 25 μmol) in acetonitrile (0.5 mL) was slowly added a solution of CAN (28 mg, 51 μmol) in water (0.5 mL) at 0 °C. The mixture was stirred at 0 °C for 5 min then warmed to r.t., stirred for 5 min then poured into ice cold water (10 mL). The aq. layer was extracted with EtOAc (3 × 10 mL) and the combined organic extracts were dried over MgSO4. The solvent was removed in vacuo and the residue purified by preparative TLC using hexanes–EtOAc (7 : 1) as eluent to afford the title compound (3 mg, 8 μmol, 33%) as a yellow oil. Rf (hexanes–EtOAc = 7 : 3): 0.27. νmax (film/cm−1): 3521, 2954, 2922, 2877, 2855, 1740, 1711, 1648, 1595, 1569, 1458, 1295, 1149, 1026, 927. 1 H NMR (300 MHz, acetone-d6): δ = 7.37 (1H, d, J = 2.4 Hz), 7.12 (1H, d, J = 2.4 Hz), 5.37 (2H, s), 5.34 (2H, d, J = 0.9 Hz), 4.97–4.88 (1H, m), 3.51 (3H, s), 3.48 (3H, s), 2.76–2.71 (1H, m), 2.18–2.08 (1H, m), 2.02–1.19 (1H, m), 1.85–1.71 (1H, m), 1.41–1.31 (1H, m), 1.10 (3H, d, J = 6.6 Hz). 13C NMR (75 MHz, acetone-d6): δ = 185.4, 183.1, 162.9, 160.1, 146.8, 143.1, 136.6, 110.3, 107.7, 96.2, 95.2, 66.9, 56.8, 56.7, 39.6, 32.2, 27.3, 21.9. HRMS (ESI): Calcd for C19H22NaO7 [MNa]+: 385.1258, found: 385.1253.
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5,7-Diisopropoxy-2-methyl-1,2,3,4-tetrahydroanthracene-9,10dione (±)-31 To a solution of 6,8-diisopropoxy-10-methoxy-3-methyl-1,2,3,4tetrahydroanthracen-9-ol (±)-22 (13 mg, 36 μmol) in acetonitrile (0.5 mL) was slowly added a solution of CAN (40 mg, 72 μmol) in water (0.5 mL) at 0 °C. The mixture was stirred at 0 °C for 5 min. then warmed to r.t., stirred for 5 min. then poured into ice cold water (10 mL). The aq. layer was extracted with EtOAc (3 × 10 mL) and the combined organic extracts were dried over MgSO4. The solvent was removed in vacuo and the residue purified by preparative TLC using hexanes–EtOAc (7 : 1) as eluent to afford title compound (10 mg, 29 μmol, 81%) as a yellow oil. Rf (hexanes–EtOAc = 7 : 3): 0.83. νmax (film/ cm−1): 2980, 2932, 2859, 1727, 1652, 1591, 1457, 1310, 1266, 1156, 1109. 1H-NMR (300 MHz, acetone-d6): δ = 7.15 (1H, d, J = 2.5 Hz), 6.83 (1H, d, J = 2.5 Hz), 4.85 (1H, sept., J = 6.0 Hz), 4.74 (1H, sept., J = 6.0 Hz), 2.73–2.67 (2H, m), 2.45–2.29 (1H, m), 2.01–1.91 (1H, m), 1.91–1.81 (1H, m), 1.78–1.64 (1H, m), 1.64–1.55 (1H, m), 1.37 (6H, d, J = 6.0 Hz), 1.36 (6H, d, J = 6.0 Hz), 1.07 (3H, d, J = 6.6 Hz). 13C-NMR (75 MHz, acetoned6): δ = 184.4, 181.7, 162.4, 160.3, 146.0, 140.6, 136.2, 107.5, 105.0, 71.6, 70.3, 30.7, 28.8, 27.4, 23.4, 21.4, 21.2, 20.8. HRMS (ESI): Calcd for C21H27O4 [MH]+: 343.1904, found: 343.1878; calcd for C21H26NaO4 [MNa]+: 365.1723, found: 365.1712; calcd for C21H26KO4 [MK]+: 381.1463, found: 381.1466.
Acknowledgements The authors are grateful to the Maurice Wilkins Centre for Molecular Biodiscovery for financial support.
cis-1,6,8-Trihydroxy-3-methyl-1,2,3,4-tetrahydroanthracene9,10-dione (2) (2-carbathysanone) To a solution of cis-1-hydroxy-6,8-bis(methoxymethoxy)3-methyl-1,2,3,4-tetrahydroanthracene-9,10-dione (±)-cis-30 (4.5 mg, 12.3 µmol) in THF–H2O (1 : 1, 1 mL) was added Bi(OTf)3 (1.6 mg, 2.5 µmol). The reaction mixture was heated at 80 °C for 3 h. Satd. aq. NH4Cl solution (5 mL) was added and the aq. layer was extracted with EtOAc (3 × 10 mL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification of the residue by preparative TLC using hexanes–EtOAc (19 : 1) as eluent then afforded the title compound (2.9 mg, 10.7 μmol, 87%) as an unstable yellow oil; Rf (hexanes–EtOAc = 1 : 1): 0.51. νmax (film/cm−1): 3352, 2976, 1924, 1854, 1650, 1623, 1596, 1579, 1447, 1393, 1359, 1311, 1276, 1231, 1208, 1179, 1167, 1142, 1081, 1071, 1051, 1021, 1008, 984, 875, 857, 788, 744. 1H NMR (400 MHz, CDCl3): δ = 12.08 (1H, s, H-OH), 7.09 (1H, d, J = 2.4 Hz), 6.60 (1H, d, J = 2.4 Hz), 6.43 (1H, bs), 5.03–4.96 (1H, m), 4.06 (1H, bd, J = 1.3 Hz), 2.85–2.76 (1H, m), 2.78–1.97 (2H, m), 1.81–1.70 (1H, m), 1.45–1.34 (1H, m), 1.13 (3H, d, J = 6.7 Hz). 13C NMR (75 MHz, CDCl3): δ = 190.1, 184.4, 164.7, 163.4, 146.3, 143.9, 134.1, 109.7, 108.9, 108.5, 66.7, 38.3, 32.2, 26.6, 21.7. HRMS (ESI): Calcd for C15H14NaO5 [MNa]+: 297.0733, found: 297.0743.
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Notes and references 1 P. L. Callahan, S. Mizutani and R. J. Colonno, Proc. Natl. Acad. Sci. U. S. A., 1985, 82, 732–736. 2 S. B. Singh, M. G. Cordingley, R. G. Ball, J. L. Smith, A. W. Dombrowski and M. A. Goetz, Tetrahedron Lett., 1991, 32, 5279–5282. 3 C. D. Donner and M. Gill, Tetrahedron Lett., 1999, 40, 3921–3924. 4 C. D. Donner and M. Gill, J. Chem. Soc., Perkin Trans. 1, 2002, 938–948. 5 R. T. Sawant and S. B. Waghmode, Tetrahedron, 2009, 65, 1599–1602. 6 R. T. Sawant, S. G. Jadhav and S. B. Waghmode, Eur. J. Org. Chem., 2010, 4442–4449. 7 J. Sperry, T. Y. Yuen and M. A. Brimble, Synthesis, 2009, 2561–2569. 8 J. Sperry and M. A. Brimble, Synlett, 2008, 1910–1912. 9 M. A. Brimble, R. J. Elliott and J. F. McEwan, Aust. J. Chem., 2000, 53, 571–576. 10 M. A. Brimble and R. J. R. Elliott, Tetrahedron, 2002, 58, 183–189.
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11 D. Barker, M. A. Brimble, P. Do and P. Turner, Tetrahedron, 2003, 59, 2441–2449. 12 A. D. Wadsworth, J. Sperry and M. A. Brimble, Synthesis, 2010, 2604–2608. 13 P. Bachu, J. Sperry and M. A. Brimble, Tetrahedron, 2008, 64, 4827–4834. 14 M. A. Brimble, P. Bachu and J. Sperry, Synthesis, 2007, 2887–2893. 15 M. A. Brimble, S. I. Houghton and P. D. Woodgate, Tetrahedron, 2007, 63, 880–887. 16 V. J. Bulbule, P. S. Koranne, V. H. Deshpande and H. B. Borate, Tetrahedron, 2007, 63, 166–177. 17 G. A. Kraus and H. Ogutu, Tetrahedron, 2002, 58, 7391– 7395. 18 K. Tangdenpaisal, S. Sualek, S. Ruchirawant and P. Ploypadith, Tetrahedron, 2009, 65, 4316–4325. 19 K. Okazaki, K. Nomura and E. Yoshii, Synth. Commun., 1987, 17, 1021–1027. 20 I. D. G. Watson and A. K. Yudin, J. Am. Chem. Soc., 2005, 127, 17516–17529. 21 G. Kraus and H. Sugimoto, Synth. Commun., 1977, 7, 505– 508. 22 F. M. Hauser, P. Hewawasam and V. M. Baghdanov, J. Org. Chem., 1988, 53, 223–224.
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23 F. M. Hauser and R. P. Rhee, J. Org. Chem., 1978, 43, 178–180. 24 D. Mal, S. Ray and I. Sharma, J. Org. Chem., 2007, 72, 4981– 4984. 25 M. Watanabe, H. Morimoto, K. Nogami, S. Ijichi and S. Furukawa, Chem. Pharm. Bull., 1993, 41, 968–970. 26 D. Mal and S. Ray, Eur. J. Org. Chem., 2008, 3014–3020. 27 P. Van der Veken, I. El Sayed, J. Joossens, C. V. Stevens, K. Augustyns and A. Haemers, Synthesis, 2005, 634–638. 28 M. A. Bates, P. G. Sammes and G. A. Thomson, J. Chem. Soc., Perkin Trans. 1, 1988, 3037–3045. 29 M. Gerecke, R. Borer and A. Brossi, Helv. Chim. Acta, 1976, 59, 2551–2557. 30 P. R. Brooks, M. C. Wirtz, M. G. Vetelino, D. M. Rescek, G. F. Woodworth, B. P. Morgan and J. W. Coe, J. Org. Chem., 1999, 64, 9719–9721. 31 S. V. Reddy, R. J. Rao, U. S. Kumar and J. M. Rao, Chem. Lett., 2003, 32, 1038–1039. 32 K. Tatsuta, Y. Tanaka, M. Kojima and H. Ikegami, Chem. Lett., 2002, 31, 14–15. 33 K. Schünemann, S. Connelly, R. Kowalczyk, J. Sperry, I. A. Wilson, J. D. Fraser and M. A. Brimble, Bioorg. Med. Chem. Lett., 2012, 22, 5018–5024. 34 A. Kalivretenos, J. K. Stille and L. S. Hegedus, J. Org. Chem., 1991, 50, 2883–2894.
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Synthesis of the 2-methylene analogue of the HRV 3C protease inhibitor thysanone (2-carbathysanone)† Katrin Schünemann,a,b Daniel P. Furkert,a Eun Cho Choi,a Stephen Connelly,c John D. Fraser,b Jonathan Sperrya and Margaret A. Brimble*a The Human Rhinovirus (HRV) is the major aetiological agent for the common cold, for which only symptomatic treatment is available. HRV maturation and replication is entirely dependent on the activity of a virally encoded 3C protease that represents an attractive target for the development of therapeutics to treat the common cold. Herein we report the synthesis and biological evaluation of the 2-methylene ana-
Received 26th September 2013, Accepted 9th December 2013 DOI: 10.1039/c3ob41951g www.rsc.org/obc
logue of the HRV 3C protease inhibitor (−)-thysanone (1) namely 2-carbathysanone (2), in an attempt to decipher the structural features in the natural product that are responsible for the 3C protease activity. 2-Carbathysanone (2) (and related analogues (±)-cis-23, (±)-cis-30, (±)-31) did not inhibit HRV 3C protease, indicating that the lactol functionality present in (−)-thysanone (1) is a critical structural feature required for inhibition.
Introduction The Human Rhinovirus (HRV) is the major cause of the common cold, which typically infects nasal epithelia cells in the upper respiratory tract.1 The virus contains a positive strand RNA genome, which is directly translated into a viral polyprotein precursor of 200 kDa. In order to produce active viral proteins and enzymes the polyprotein undergoes a series of proteolytic cleavages. These cleavages are governed by two virally encoded proteases designated as 2A and 3C. These proteases are therefore interesting targets for drug development against the common cold. (−)-Thysanone (1) was isolated from the fungus Thysanophora penicilloides by the process of screening microbial extracts for bioactive lead structures.2 (−)-Thysanone (1) was found to be an effective inhibitor of HRV 3C protease with an IC50 of 13 µg mL−1 (47 µM).2 To date, two total syntheses and one formal synthesis of the natural product (−)-thysanone (1) have been published as well as syntheses of several different analogues 3–8.3–17 Given our continued interest in synthesizing (−)-thysanone (1) and its
a School of Chemical Sciences, The University of Auckland, 23 Symonds St., Auckland, New Zealand. E-mail: [email protected] b Department of Molecular Medicine and Pathology, The University of Auckland, 85 Park Road, Auckland, New Zealand c Department of Molecular Biology, The Scripps Research Institute, BCC 265, 10550 Nr. Torrey Pines Rd., La Jolla, CA 92037, USA † Electronic supplementary information (ESI) available: 1H and 13C NMR data for all new compounds and experimental data for compounds 10, 13, 14, 17–21, 23 and 24. See DOI: 10.1039/c3ob41951g
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Fig. 1 Structure of (−)-thysanone (1), (±)-2-carbathysanone (2) and analogues 3–8 previously synthesised by our group.6–12,15
analogues (Fig. 1),7–15 we herein report the synthesis and biological evaluation of 2-carbathysanone (2) prepared from substituted naphthalene (±)-9 that is assembled via Hauser–Kraus annulation of cyanophthalide 10 with cyclohexenone (±)-11 (Scheme 1).
Results and discussion Despite the fact that our earlier work suggested that Hauser–Kraus annulation using a 4,6-dimethoxy substituted
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Scheme 3 Reagents and conditions: (i) KOtBu, tBuOH, 0 °C → 90 °C, 20 h, 27%.20
Scheme 1 Retrosynthesis of 2-carbathysanone (2) via Hauser–Kraus annulation.
Table 1 Investigation of the Hauser–Kraus annulation. Reagents and conditions: (i) (a) base, solvent, r.t., 1 h; (b) Me2SO4, KOH, 60 °C, 16 h
Entry 1 2 3 4 Scheme 2 Reagents and conditions: (i) H2SO4, MeOH, reflux, 5 d, quant.; (ii) Cs2CO3, nBu4NI (TBAI), 2-bromopropane, acetone–DMF (10 : 1), reflux, 17 h, quant.; (iii) AlMe3, Et2NH, PhMe, reflux, quant.; (iv) t BuLi, tetramethylethylenediamine (TMEDA), DMF, −78 °C → r.t., 17 h, quant.; (v) (a) TMSCN, KCN, 18-crown-6, CH2Cl2, 3 h, 0 °C → r.t.; (b) AcOH, 17 h, r.t., 53% over two steps.
cyanophthalide did not result in successful annulation with a variety of electrophiles,15 we nevertheless decided to start our synthesis of 2-carbathysanone (2) by reinvestigation of this approach. We therefore synthesized cyanophthalide 10 from 2,4-dihydroxybenzoic acid (12) (Scheme 2). 2,4-Dihydroxybenzoic acid (12) was readily converted to the diisopropylprotected methyl benzoate 13 in two steps using sulfuric acid in methanol to effect the methyl esterification followed by protection of both hydroxyl groups as isopropyl ethers.18 Methyl benzoate 13 was then converted to benzaldehyde 14 by treatment with trimethylaluminium and diethylamine followed by formylation using tBuLi, TMEDA and DMF. Cyanophthalide 10 was synthesized following a general literature procedure by reaction of benzaldehyde 14 with trimethylsilyl cyanide (TMSCN) in presence of catalytic amounts of KCN and 18-crown-6, followed by treatment of the α-trimethylsilyloxybenzyl cyanide intermediate with glacial acetic acid.19 Cyanophthalide 10 was obtained in a moderate 53% yield over two steps. Hauser–Kraus annulation partner cyclohexenone (±)-11 was synthesized following a one-pot procedure described by Yudin and co-workers (Scheme 3).20 Hauser–Kraus annulation between cyanophthalide 10 and enone (±)-11 requires investigation of a variety of different bases and solvents. Due to the instability of the initial naphthalenediol annulation product it was decided to perform
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Base t
KO Bu KOtBu KHMDS DBU
Solvent
Yield
THF DMSO THF THF
42% 19% 35% Decomposition
a one-pot Hauser–Kraus annulation, methylation strategy. Selected examples of this reaction are shown in Table 1. The optimum conditions involving reaction of cyanophthalide 10 with potassium tert-butoxide in THF at 0 °C followed by the addition of enone (±)-11 (entry 1) afforded naphthol (±)-17 in a moderate 42% yield after methylation using dimethyl sulfate and potassium hydroxide. The nature of the leaving group on the phthalide can substantially influence the yield obtained in Hauser–Kraus annulations.21–26 We therefore decided to prepare phosphonylphthalide 18, phenylthiophthalide 19 and phenylsulfonylphthalide 20 from the common intermediate benzaldehyde 14 (Scheme 3). Phosphonylphthalide 18 was obtained following a procedure published by Van der Veken and co-workers.27 Treatment of benzaldehyde 14 with trimethylphosphite in the presence of oxalic acid gave phosphonylphthalide 18 in 81% yield. Phenylthiophthalide 19 was prepared in a two-step, onepot procedure.28 Potassium hydroxide was used first to generate an unstable hydroxyphthalide from benzaldehyde 14, which was then treated in situ with thiophenol to form thiophenylphthalide 19 in moderate 36% yield over two steps. The phenylthiophthalide 19 was then oxidized with m-CPBA to afford phenylsulphonylphthalide 20 in 97% yield (Scheme 4). Disappointingly, use of all three phthalides 18–20 in the key-Hauser–Kraus annulation with cyclohexenone (±)-11 using potassium tert-butoxide in different solvents (e.g. DMSO and THF) led to decomposition of both starting materials (Scheme 5). It was therefore decided to continue the synthesis with cyanophthalide 10 using the previously established Hauser– Kraus annulation conditions. Reduction of the ketone in the
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Fig. 2
Observed nOe signals and proposed configuration.
Scheme 4 Reagents and conditions: (i) oxalic acid, P(OEt)3, reflux, 16 h, 81%; (ii) (a) KOH, H2O, THF, r.t., 18 h; (b) PhSH, PhMe, p-TsOH, 18 h, 36% over two steps; (iii) m-CPBA, CH2Cl2, 0 °C → r.t., 97%.
Scheme 6
Scheme 5 Unsuccessful Hauser–Kraus annulation using phthalides 18, 19 and 20.
annulation product (±)-17 using borane-dimethyl sulfide complex gave an inseparable mixture of (±)-cis-alcohol 21 and (±)-trans-alcohol 21 in excellent yield (cis–trans, 5 : 1, Table 2), with some of the over-reduced product (±)-22 also isolated. In an attempt to improve the diastereoselectivity of the reduction some sterically hindered boranes possessing two hydrides were investigated. Pleasingly, use of both cyclohexylborane and isopropoxyborane did result in the diastereoselective formation of the (±)-cis-alcohol 21, however the amount of over-reduced product 22 also increased. The observed nOe between H-1 and H-3 established the cis-stereochemistry in (±)-cis-alcohol 21, consistent with the C-1 hydroxyl and the C-3 methyl groups occupying pseudoequatorial positions (Fig. 2). Despite not being able to isolate a diastereomerically pure sample of the trans-product 21 possessing the cyclohexyl substituents in the same relationship as observed in thysanone, we aimed to complete the synthesis using the cis-product 21 and the next step required its oxidation to the corresponding
Table 2
Reagents and conditions: (i) CAN, MeCN–H2O, 74%.
(±)-cis-naphthoquinone 23. (±)-cis-Naphthol 21 was oxidized using ceric ammonium nitrate (CAN) in aqueous acetonitrile affording (±)-cis-naphthoquinone 23 in moderate 74% yield (Scheme 6). At this stage completion of the synthesis of 2-carbathysanone (2) only required one step, namely cleavage of the isopropyl protecting groups. Disappointingly, treatment of (±)-cis-23 with AlCl3, BCl3 or Bi(OTf )3 only resulted in decomposition of the starting material.29–31 Due to the inability to deprotect the isopropyl ethers in 23 we next decided to change the isopropyl protecting groups to methoxymethyl (MOM)-groups. The synthesis of MOMprotected cyanophthalide 27 is shown in Scheme 7. Methyl benzoate 24 was readily converted to TBS-protected benzaldehyde 25 in three steps using sodium hydride and tertbutyldimethylsilyl chloride (TBSCl) to afford TBS-protected methyl benzoate, which was then converted to the corresponding amide using trimethylaluminium and diethylamine.32 Formylation of the amide using tBuLi, TMEDA and DMF afforded the desired benzaldehyde 25 in 82% yield over three steps. The synthesis of MOM-protected phthalide 27 from benzaldehyde 25 was first attempted using TMSCN in the presence of a catalytic amount of KCN and 18-crown-6, followed by treatment of the α-trimethylsilyloxybenzyl cyanide
Reduction of ketone (±)-17 using different boranes. Reagents and conditions: (i) borane, THF, 0 °C → r.t., 1 h
Entry
Borane
(±)-cis-21 : (±)-trans-21
(±)-22
1 2 3
BH3·SMe2 (C6H11)BH2 i PrOBH2
93% (5 : 1) 72% (1 : 0) 56% (1 : 0)
7% 25% 32%
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Scheme 7 Reagents and conditions: (i) NaH, TBSCl, 0 °C → r.t., 2 h, quant.; (ii) AlMe3, Et2NH, PhMe, reflux, quant.; (iii) tBuLi, TMEDA, DMF, THF, −78 °C → r.t., 18 h, 82%; (iv) (a) TMSCN, KCN, 18-crown-6, CH2Cl2, 0 °C → r.t., 3 h; (b) AcOH, 18 h; (c) P4O10, CH2(OMe)2, CH2Cl2, 0 °C → r.t., 3 h, 37%.
intermediate with glacial acetic acid, which afforded cyanophthalide 26. Disappointingly, the isolation of 26 was difficult due its high solubility in water. Furthermore, the subsequent protection of 26 using MOMCl and Hünig’s base resulted in alkylation at C-1 of the resulting cyanophthalide. To circumvent these issues, we developed a one-pot procedure (Scheme 7). Benzaldehyde 25 was treated with TMSCN in the presence of a catalytic amount of KCN and 18-crown-6 followed by glacial acetic acid, delivering cyanophthalide 26 that had undergone concomitant desilylation under the reaction conditions. Subsequent treatment of 26 with phosphorus pentoxide and dimethoxymethane afforded cyanophthalide 27 in 37% yield over three steps. Disappointingly, the previously established conditions used to effect Hauser–Kraus annulation of diisopropyl-protected cyanophthalide 10 with enone (±)-11 using potassium tertbutoxide followed by methylation using dimethyl sulfate and potassium hydroxide, were unsuccessful when applied to MOM-protected cyanophthalide 27. Fortunately, changing the base to sodium hydride followed by methylation using dimethyl sulfate and sodium hydride afforded dimethoxynaphthalene (±)-28 in 79% yield over two steps (Scheme 8). In this case, methylation of the 10-naphthol group occurred as the sodium hydride was able to deprotonate the hydrogen bonded C-10-hydroxyl-group. Although attempted reduction of (±)-28 using cyclohexylborane only resulted in decomposition, employing iBu2AlH exclusively afforded the desired (±)-cis-
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alcohol 29 in excellent yield. Subsequent oxidative demethylation of (±)-cis-29 using CAN afforded (±)-cis-naphthoquinone 30 in 33% yield. Removal of the MOM protecting groups was first attempted using 1 M aq. hydrochloric acid; however, only decomposition was observed and milder conditions were therefore investigated. Bismuth triflate has been shown to cleave MOM-acetals in a THF–water mixture at room temperature.31 However, under these conditions, only starting material was observed after two hours. However, heating the reaction mixture to 80 °C did effect deprotection, affording the desired 2-carbathysanone (2) in 87% yield. The synthesis of 2-carbathysanone (2) described above also resulted in two analogues (±)-23 and (±)-cis-30, good candidates to be screened for HRV 3C protease activity. (±)-Naphthol 22 was also oxidised to (±)-naphthoquinone 31 using CAN in 81% yield in order to provide an additional analogue to evaluate for inhibition of HRV 3C protease (Scheme 9). The cytotoxicity of 2-carbathysanone (2) and the three analogues (±)-cis-23, (±)-cis-30 and (±)-31 was also investigated. The HRV 3C protease screening was carried out using our previously reported solid phase based fluorescent substrate assay.33 The cytotoxicity (LD50) of the compounds was evaluated using a cell-proliferation assay in which the human lung adenocarcinoma epithelia cell-line NCI-H441 was grown in the presence of the synthesized inhibitors 2-carbathysanone (2), (±)-cis-23, (±)-cis-30 and (±)-31 and treated with 3H-thymidine to detect cell-proliferation. The results are summarized in Table 3. All analogues did not inhibit HRV 3C protease, inferring the 2-oxa-lactol functionality to be a key structural feature in the mechanism of inhibition of HRV 3C protease by thysanone (1). All four analogues also exhibited high cytotoxicity against NCI-H441.
Scheme 9 81%.
Reagents and conditions: (i) CAN, MeCN–H2O, r.t., 10 min,
Scheme 8 Reagents and conditions: (i) (a) NaH, THF, 0 °C → r.t., 1 h; (b) Me2SO4, NaH, 0 °C → r.t., 3 h, 79%; (ii) iBu2AlH, CH2Cl2, 0 °C → r.t., 87%, (iii) CAN, MeCN–H2O, 10 min, 33%; (iv) Bi(OTf )3, THF–H2O, 80 °C, 87%.
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Table 3 study
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IC50 and LD50 values of thysanone analogues prepared in this
Compound
R1
R2
IC50 [µM]
LD50 [µM]
2-Carbathysanone (2) (±)-cis-23 (±)-cis-30 (±)-31
OH OiPr OMOM OiPr
OH OH OH H
n.i.d. n.i.d. n.i.d. n.i.d.
93.4 ± 36 52.0 ± 0 28.4 ± 11 37.4 ± 0
n.i.d. = no inhibition detected.
Conclusion In conclusion, we have developed a synthesis of 2-carbathysanone (2) and closely related analogues using a Hauser–Kraus annulation. 2-Carbathysanone (2), and analogues (±)-cis-23, (±)-cis-30, (±)-31 did not inhibit HRV 3C protease thus indicating that the lactol functionality present in thysanone (1) is a critical structural feature required for inhibition of HRV 3C protease.
Experimental General details Unless otherwise stated, all non-aqueous reactions and distillations were performed under a nitrogen or argon atmosphere in oven- or flame-dried glassware. Tetrahydrofuran was freshly distilled from sodium/benzophenone. Dichloromethane (CH2Cl2) and toluene were freshly distilled from calcium hydride. TMEDA was distilled from calcium hydride and stored under a nitrogen atmosphere over MgSO4. Reactions performed at low temperature were either cooled in an acetone-dry ice bath for temperatures below 0 °C or using a water-ice bath for 0 °C. Flash chromatography was carried out using 0.063–0.1 mm silica gel (Davisil R LC60A 40-63 Micron) with the indicated solvent. Preparatory TLC was carried out on 500 μm Uniplate™ (Analtech) silica gel (20 × 20 cm) thin layer chromatography plates. Infrared spectra were recorded using a Perkin Elmer R Spectrum 1000 Fourier Transform Infrared spectrometer. Values are expressed in wavenumbers (cm−1) and recorded in a range of 4000 to 450 cm−1. NMR spectra were recorded at 21 °C in CDCl3 or C6D6 on either a Bruker® Avance 300 spectrometer operating at 300 MHz for 1H nuclei and 75 MHz for 13C nuclei or on a Bruker® DRX400 or Bruker® 400 spectrometer operating at 400 MHz for 1H nuclei and 100 MHz for 13C nuclei. All chemical shifts are reported in parts per million ( ppm) from tetramethylsilane (δ = 0) and were measured relative to the solvent in which the sample was analysed (CDCl3: TMS δ = 0.00 for 1H NMR and CDCl3 δ = 77.16 for 13C NMR; acetone-d6: δ = 2.05 for 1H NMR and δ =
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29.84 ± 0.01 for 13C NMR). Coupling constants ( J) are reported in Hertz (Hz). 1H-NMR data is reported as chemical shift in ppm, followed by the relative integral, the multiplicity (“s” singlet, “d” doublet, “dd” doublet of doublets, “ddd”, doublet of doublets of doublets, “dt” doublet of triplets, “t” triplet, “q” quartet, “quint.” quintuplet, “sept.” septet, “m” multiplet, “b” broad) and coupling constant where applicable and relative integral. 13C NMR spectra are reported as chemical shift in ppm. High resolution mass spectra were recorded using a Bruker microTOF-QII mass spectrometer. 2,4-Bis((tert-butyldimethylsilyl)oxy)-N,N-diethyl-6formylbenzamide 25 To a solution of methyl 2,4-dihydroxybenzoate 24 (1.5 g, 8.9 mmol) in THF (45 mL) was added sodium hydride (0.9 g, 22.3 mmol) at 0 °C. The reaction mixture was stirred for 30 min and TBSCl (2.8 g, 22.3 mmol) was added. The reaction mixture was stirred for 2 h then satd. aq. NH4Cl solution (20 mL) was added. The aq. layer was extracted with EtOAc (3 × 30 mL) and the combined organic layers were washed with brine (30 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (9 : 1) as eluent to afford methyl 2,4-bis((tert-butyldimethylsilyl)oxy)-benzoate (3.5 g, 8.8 mmol, quant.) as a yellow oil. Rf (hexanes–ethyl acetate = 9 : 1): 0.81. 1H NMR (400 MHz, CDCl3): δ = 7.72 (1H, d, J = 8.8 Hz), 6.40 (1H, dd, J = 8.8 Hz, 2.4 Hz), 4.46 (1H, d, J = 2.4 Hz, H-3), 3.83 (3H, s), 1.01 (9H, s), 0.97 (9H, s), 0.21 (6H, s), 0.21 (6H, s). 13C NMR (100 MHz, CDCl3): δ = 167.0, 160.3, 157.1, 133.4, 116.0, 113.6, 112.8, 51.7, 25.8, 25.8, 18.5, 18.4. The spectroscopic data were in agreement with that reported in the literature.34 To a solution of trimethylaluminum (2 M in toluene, 7 mL, 14 mmol) in toluene (10 mL) was added diethylamine (1.5 mL, 14 mmol) at −6 °C under an atmosphere of nitrogen. After 10 min the reaction mixture was warmed to r.t. and methyl 2,4-bis((tertbutyldimethylsilyl)oxy)-benzoate (1.4 g, 4.2 mmol) was added. The resulting mixture was heated at reflux overnight. The mixture was cooled to 0 °C then 10% aq. HCl (10 mL) was added. The layers were separated and the organic layer was washed with 10% aq. HCl (20 mL). The combined aq. layers were extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexane–EtOAc (4 : 1) as eluent to afford 2,4-Bis((tert-butyldimethylsilyl)oxy)-N,N-diethylbenzamide (1.54 g, 4.2 mmol, quant.) as a yellow oil. Rf (hexane– ethyl acetate = 4 : 1): 0.42. 1H NMR (400 MHz, CDCl3): δ = 7.04 (1H, d, J = 8.3 Hz), 6.47 (1H, dd, J = 8.3 Hz), 6.31 (1H, d, J = 2.2 Hz), 3.69–3.02 (4H, bm), 1.22 (3H, t, J = 7.1 Hz), 0.99 (3H, t, J = 7.1 Hz), 0.98 (9H, s), 0.98 (9H, s), 0.21 (6H, s), 0.19 (6H, s). 13C NMR (100 MHz, CDCl3): δ = 169.3, 156.9, 152.3, 128.6, 123.4, 113.6, 111.4, 43.0, 39.4, 25.8, 25.8, 18.4, 18.3, 14.2, 13.4. The spectroscopic data were in agreement with that reported in the literature.34 A solution of 2,4-bis((tert-butyldimethylsilyl)oxy)N,N-diethylbenzamide (2.5 g, 6.8 mmol) and TMEDA (1.1 mL, 7.5 mmol) in THF (10 mL) was cooled to −78 °C under an
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atmosphere of nitrogen and t-BuLi (6.4 mL, 10.2 mmol) was added slowly. The solution turned yellow and was stirred for a further 10 min. DMF (1.1 mL, 13.6 mmol) was added and the mixture warmed to r.t. overnight. Satd. aq. NH4Cl solution (20 mL) was added and the mixture stirred for 30 min. The aq. layer was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (4 : 1) as eluent to afford the title compound (2.2 g, 5.5 mmol, 82%) as a yellow oil. Rf (hexanes–EtOAc = 4 : 1): 0.42. νmax (film/cm−1): 2956, 2934, 2894, 2861, 1699, 1633, 1594, 1570, 1332, 1162, 783, 733. 1H NMR (400 MHz, CDCl3): δ = 9.86 (1H, s, H-CHO), 6.97 (1H, d, J = 2.3 Hz), 6.53 (1H, d, J = 2.3 Hz), 3.70 (1H, dq. J = 7.0 Hz, 14.2 Hz), 3.93 (1H, dq, J = 7.0 Hz, 14.2 Hz), 3.14 (1H, dq, J = 7.3 Hz, 14.4 Hz), 3.07 (1H, dq, J = 7.3 Hz, 14.4 Hz), 1.24 (3H, t, J = 7.3 Hz), 0.98 (3H, t, J = 7.0 Hz), 0.95 (9H, s), 0.94 (9H, s), 0.22–0.19 (12H, m). 13C NMR (100 MHz, CDCl3): δ = 190.4, 166.2, 156.7, 153.1, 134.8, 125.8, 116.9, 113.0, 43.2, 39.6, 25.7, 25.6, 18.3, 18.2, 14.0, 13.0. HRMS (ESI): Calcd for C24H44NO4Si2 [MH]+: 466.2803, found: 466.2817; calcd for C24H43NNaO4Si2 [MNa]+: 488.2623, found: 488.2625.
4,6-Bis(methoxymethoxy)-3-oxo-1,3-dihydroisobenzofuran1-carbonitrile 27 To a solution of 2,4-bis((tert-butyldimethylsilyl)oxy)-N,Ndiethyl-6-formylbenzamide 25 (4.0 g, 10 mmol) in CH2Cl2 (20 mL) was added KCN (13 mg, 0.2 mmol) and 18-crown-6 (53 mg, 0.2 mmol) under an atmosphere of nitrogen. The reaction mixture was cooled to 0 °C and TMSCN (1.9 mL, 15 mmol) was added dropwise. The solvent was removed under reduced pressure after 15 min and the residue taken up in acetic acid (20 mL) and the mixture stirred overnight at 80 °C. The solvent was removed under reduced pressure and the residue taken up in CH2Cl2–dimethoxymethane (1 : 1, 80 mL) and phosphorus pentoxide (10 g, 70 mmol) was added at 0 °C. The mixture was warmed to r.t., stirred for 3 h then poured into ice cold satd. aq. Na2CO3 solution (100 mL). The aq. layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (9 : 1) as eluent to afford the title compound (1.03 g, 3.7 mmol, 37%) as a colourless oil. Rf (hexanes–EtOAc = 1 : 1): 0.70. νmax (film/ cm−1): 2980, 2938, 1785, 1697, 1614, 1598, 1319, 1150, 972, 920, 686. 1H NMR (400 MHz, CDCl3): δ = 6.95 (1H, d, J = 1.6 Hz), 6.92 (1H, d, J = 1.6 Hz), 5.92 (1H, s), 5.36 (2H, s), 5.30 (1H, d, J = 7.2 Hz), 5.23 (1H, d, J = 7.2 Hz), 3.54 (3H, s), 3.51 (3H, s). 13C NMR (100 MHz, CDCl3): δ = 165.3, 157.9, 146.0, 114.2, 103.7, 105.8, 102.5, 95.1, 94.8, 64.8, 57.0, 56.8. HRMS (ESI): Calcd for C13H14NO6 [MH]+: 280.0816, found: 280.0823; calcd for C13H13NNaO6 [MNa]+: 302.0635, found: 302.0631.
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9,10-Dimethoxy-6,8-bis(methoxymethoxy)-3-methyl-3,4dihydroanthracen-1(2H)-one (±)-28 To a solution of 4,6-bis(methoxymethoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carbonitrile 27 (120 mg, 0.43 mmol) and cyclohexenone (±)-1119 (95 mg, 0.86 mmol) in THF (1 mL) was added NaH (19 mg, 60% in mineral oil, 0.47 mmol) in THF (0.5 mL) under an atmosphere of nitrogen. The reaction mixture was stirred for 1 h then dimethyl sulfate (0.41 mL, 4.3 mmol) and NaH (38 mg, 0.95 mmol) were added. The mixture was stirred at r.t. for 3 h then satd. aq. NH4Cl solution (5 mL) was added. The aq. layer was extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with brine (10 mL) and dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (7 : 3) as eluent to afford the title compound (132 mg, 0.34 mmol, 79%) as a yellow oil. Rf (hexanes–EtOAc = 7 : 3): 0.54. νmax (film/cm−1): 3071, 2954, 2933, 2859, 1715, 1613, 1529, 1335, 1150, 930, 765. 1H NMR (400 MHz, CDCl3): δ = 7.27 (1H, d, J = 2.4 Hz), 6.87 (1H, d, J = 2.4 Hz), 5.31 (2H, d, J = 0.9 Hz), 5.30 (2H, s), 3.90 (3H, s), 3.84 (3H, s), 3.58 (3H, s), 3.52 (3H, s), 3.32 (1H, dq, J = 16.2 Hz, 2.0 Hz), 2.75 (1H, dq, J = 16.2 Hz, 2.0 Hz), 2.52 (1H, dd, J = 16.2 Hz, 10.6 Hz), 2.34 (1H, dd, J = 16.2 Hz, 11.5 Hz), 2.28–2.19 (1H, m), 1.16 (3H, d, J = 6.4 Hz). 13C NMR (100 MHz, CDCl3): δ = 197.8, 158.0, 147.7, 134.9, 131.4, 121.7, 117.1, 105.2, 98.3, 96.3, 94.5, 63.1, 60.8, 56.7, 56.5, 49.4, 32.4, 29.3, 21.5. HRMS (ESI): Calcd for C21H27O7 [MH]+: 391.1751, found: 391.1738; calcd for C21H26NaO7 [MNa]+: 413.1571, found: 413.1557; calcd for C21H26KO7 [MK]+: 429.1310, found: 429.1295.
cis-9,10-Dimethoxy-6,8-bis(methoxymethoxy)-3-methyl-1,2,3,4tetrahydroanthracen-1-ol (±)-cis-29 To a solution of 9,10-dimethoxy-6,8-bis(methoxymethoxy)3-methyl-3,4-dihydroanthracen-1(2H)-one (±)-28 (60 mg, 0.15 mmol) in CH2Cl2 (1 mL) was added at −78 °C DIBAL-H (170 µL, 1 M in cyclohexane, 0.17 mmol) under an atmosphere of nitrogen and the mixture stirred at −78 °C for 30 min then allowed to warm to r.t. Methanol (1 mL) and half-satd. Rochelle’s salt (3 mL) were added and the reaction was stirred for 20 min. The aq. layer was extracted with Et2O (3 × 5 mL) and the combined organic extracts were dried over MgSO4. The solvent was removed in vacuo and the residue purified by flash chromatography using hexanes–EtOAc (7 : 3) as eluent to afford the title compound (40 mg, 0.10 mmol, 87%) as a yellow oil. Rf (hexane–EtOAc = 7 : 3): 0.39. νmax (film/cm−1): 3505, 2934, 2833, 1751, 1619, 1595, 1452, 1335, 1227, 1147, 1024, 928. 1H NMR (400 MHz, CDCl3): δ = 7.27 (1H, d, J = 2.3 Hz), 6.88 (1H, d, J = 2.3 Hz), 5.32 (2H, s), 5.29 (2H, s), 5.33–5.24 (1H, m), 4.68 (1H, bs), 3.90 (3H, s), 3.81 (3H, s), 3.59 (3H, s), 3.53 (3H, s), 3.13 (1H, dq, J = 16.6 Hz, 2.2 Hz), 2.36–2.29 (2H, m), 1.86–1.76 (1H, m), 1.52–1.44 (1H, m), 1.17 (3H, d, J = 6.5 Hz). 13C NMR (100 MHz, CDCl3): δ = 155.6, 154.5, 151.4, 148.5, 131.2, 129.2, 128.9, 115.9, 104.7, 98.6, 96.3, 94.7, 67.5, 61.9, 60.6, 56.7, 56.4, 40.0, 33.2, 27.7, 22.3. HRMS (ESI): Calcd
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for C21H28NaO7 [MNa]+: 415.1727, found: 415.1729; calcd for C21H28KO7 [MK]+: 431.1467, found: 431.1474.
cis-1-Hydroxy-6,8-bis(methoxymethoxy)-3-methyl-1,2,3,4tetrahydroanthracene-9,10-dione (±)-cis-30 To a solution of cis-9,10-dimethoxy-6,8-bis(methoxymethoxy)3-methyl-1,2,3,4-tetrahydro-anthracen-1-ol (±)-cis-29 (10 mg, 25 μmol) in acetonitrile (0.5 mL) was slowly added a solution of CAN (28 mg, 51 μmol) in water (0.5 mL) at 0 °C. The mixture was stirred at 0 °C for 5 min then warmed to r.t., stirred for 5 min then poured into ice cold water (10 mL). The aq. layer was extracted with EtOAc (3 × 10 mL) and the combined organic extracts were dried over MgSO4. The solvent was removed in vacuo and the residue purified by preparative TLC using hexanes–EtOAc (7 : 1) as eluent to afford the title compound (3 mg, 8 μmol, 33%) as a yellow oil. Rf (hexanes–EtOAc = 7 : 3): 0.27. νmax (film/cm−1): 3521, 2954, 2922, 2877, 2855, 1740, 1711, 1648, 1595, 1569, 1458, 1295, 1149, 1026, 927. 1 H NMR (300 MHz, acetone-d6): δ = 7.37 (1H, d, J = 2.4 Hz), 7.12 (1H, d, J = 2.4 Hz), 5.37 (2H, s), 5.34 (2H, d, J = 0.9 Hz), 4.97–4.88 (1H, m), 3.51 (3H, s), 3.48 (3H, s), 2.76–2.71 (1H, m), 2.18–2.08 (1H, m), 2.02–1.19 (1H, m), 1.85–1.71 (1H, m), 1.41–1.31 (1H, m), 1.10 (3H, d, J = 6.6 Hz). 13C NMR (75 MHz, acetone-d6): δ = 185.4, 183.1, 162.9, 160.1, 146.8, 143.1, 136.6, 110.3, 107.7, 96.2, 95.2, 66.9, 56.8, 56.7, 39.6, 32.2, 27.3, 21.9. HRMS (ESI): Calcd for C19H22NaO7 [MNa]+: 385.1258, found: 385.1253.
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5,7-Diisopropoxy-2-methyl-1,2,3,4-tetrahydroanthracene-9,10dione (±)-31 To a solution of 6,8-diisopropoxy-10-methoxy-3-methyl-1,2,3,4tetrahydroanthracen-9-ol (±)-22 (13 mg, 36 μmol) in acetonitrile (0.5 mL) was slowly added a solution of CAN (40 mg, 72 μmol) in water (0.5 mL) at 0 °C. The mixture was stirred at 0 °C for 5 min. then warmed to r.t., stirred for 5 min. then poured into ice cold water (10 mL). The aq. layer was extracted with EtOAc (3 × 10 mL) and the combined organic extracts were dried over MgSO4. The solvent was removed in vacuo and the residue purified by preparative TLC using hexanes–EtOAc (7 : 1) as eluent to afford title compound (10 mg, 29 μmol, 81%) as a yellow oil. Rf (hexanes–EtOAc = 7 : 3): 0.83. νmax (film/ cm−1): 2980, 2932, 2859, 1727, 1652, 1591, 1457, 1310, 1266, 1156, 1109. 1H-NMR (300 MHz, acetone-d6): δ = 7.15 (1H, d, J = 2.5 Hz), 6.83 (1H, d, J = 2.5 Hz), 4.85 (1H, sept., J = 6.0 Hz), 4.74 (1H, sept., J = 6.0 Hz), 2.73–2.67 (2H, m), 2.45–2.29 (1H, m), 2.01–1.91 (1H, m), 1.91–1.81 (1H, m), 1.78–1.64 (1H, m), 1.64–1.55 (1H, m), 1.37 (6H, d, J = 6.0 Hz), 1.36 (6H, d, J = 6.0 Hz), 1.07 (3H, d, J = 6.6 Hz). 13C-NMR (75 MHz, acetoned6): δ = 184.4, 181.7, 162.4, 160.3, 146.0, 140.6, 136.2, 107.5, 105.0, 71.6, 70.3, 30.7, 28.8, 27.4, 23.4, 21.4, 21.2, 20.8. HRMS (ESI): Calcd for C21H27O4 [MH]+: 343.1904, found: 343.1878; calcd for C21H26NaO4 [MNa]+: 365.1723, found: 365.1712; calcd for C21H26KO4 [MK]+: 381.1463, found: 381.1466.
Acknowledgements The authors are grateful to the Maurice Wilkins Centre for Molecular Biodiscovery for financial support.
cis-1,6,8-Trihydroxy-3-methyl-1,2,3,4-tetrahydroanthracene9,10-dione (2) (2-carbathysanone) To a solution of cis-1-hydroxy-6,8-bis(methoxymethoxy)3-methyl-1,2,3,4-tetrahydroanthracene-9,10-dione (±)-cis-30 (4.5 mg, 12.3 µmol) in THF–H2O (1 : 1, 1 mL) was added Bi(OTf)3 (1.6 mg, 2.5 µmol). The reaction mixture was heated at 80 °C for 3 h. Satd. aq. NH4Cl solution (5 mL) was added and the aq. layer was extracted with EtOAc (3 × 10 mL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification of the residue by preparative TLC using hexanes–EtOAc (19 : 1) as eluent then afforded the title compound (2.9 mg, 10.7 μmol, 87%) as an unstable yellow oil; Rf (hexanes–EtOAc = 1 : 1): 0.51. νmax (film/cm−1): 3352, 2976, 1924, 1854, 1650, 1623, 1596, 1579, 1447, 1393, 1359, 1311, 1276, 1231, 1208, 1179, 1167, 1142, 1081, 1071, 1051, 1021, 1008, 984, 875, 857, 788, 744. 1H NMR (400 MHz, CDCl3): δ = 12.08 (1H, s, H-OH), 7.09 (1H, d, J = 2.4 Hz), 6.60 (1H, d, J = 2.4 Hz), 6.43 (1H, bs), 5.03–4.96 (1H, m), 4.06 (1H, bd, J = 1.3 Hz), 2.85–2.76 (1H, m), 2.78–1.97 (2H, m), 1.81–1.70 (1H, m), 1.45–1.34 (1H, m), 1.13 (3H, d, J = 6.7 Hz). 13C NMR (75 MHz, CDCl3): δ = 190.1, 184.4, 164.7, 163.4, 146.3, 143.9, 134.1, 109.7, 108.9, 108.5, 66.7, 38.3, 32.2, 26.6, 21.7. HRMS (ESI): Calcd for C15H14NaO5 [MNa]+: 297.0733, found: 297.0743.
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