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Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product
Accepted Manuscript Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product Ken Ishigami, Munetaka Kobayashi, Motoki Takagi, Kazuo Shin-ya, Hidenori Watanabe PII:
S0040-4020(15)30042-9
DOI:
10.1016/j.tet.2015.09.013
Reference:
TET 27110
To appear in:
Tetrahedron
Received Date: 21 August 2015 Revised Date:
3 September 2015
Accepted Date: 3 September 2015
Please cite this article as: Ishigami K, Kobayashi M, Takagi M, Shin-ya K, Watanabe H, Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product, Tetrahedron (2015), doi: 10.1016/j.tet.2015.09.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract (K. Ishigami et al.)
Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product *
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Ken Ishigami , Munetaka Kobayashi, Motoki Takagi, Kazuo Shin-ya, Hidenori Watanabe
1
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Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product
*
Ken Ishigamia , Munetaka Kobayashia, Motoki Takagib, Kazuo Shin-yac, Hidenori
a
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Watanabea
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
b
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Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Medical-industrial Translational Research Center, Fukushima Medical University, 11-
c
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23 Sakaemachi, Fukushima, Fukushima 960-8031, Japan
National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi,
Koto-ku, Tokyo 135-0064, Japan
Abstract
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Four possible stereoisomers of topsentolide A1, a cytotoxic oxylipin against human solid tumor cell lines, were efficiently synthesized in a stereoselective manner in order to determine the stereochemistry of natural product. The absolute configuration of
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topsentolide A1 was determined to be 8R,11R,12S by comparing NMR spectra and specific rotations of the synthetic isomers and the natural product. Cytotoxicity of the
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synthetic isomers was also examined.
Key Words
topsentolide A1; 9-membered lactone; determination of the absolute configuration;
marine oxylipin
1. Introduction *
Corresponding author. Fax: +81-3-5841-8019; e-mail: [email protected]
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A number of oxylipins have been isolated as secondary metabolites of marine organisms, such as cyanobacteria, alga, hydrozoa and sponges. These oxylipins have interesting activities and synthetic studies on them are meaningful, as well as biological studies, because their bioactivities are frequently concerned with structure. Halicholactone
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(2, isolated from Halichondoria okadai)1 is an inhibitor of lipoxygenase, mueggelone (3, isolated from Aphanizomenon flos-aquae)2 is an inhibitor of fish development, and
solandelactone D (4, isolated from Solanderia secunda)3 is an inhibitor of farnesyl protein
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transferase. Previously in our group, the syntheses of these marine oxylipins had been
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Figure 1. Bioactive marine oxylipins.
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achieved in stereoselective manner and the results had been already reported.4-8
In 2006, Jung’s group isolated topsentolide A1 (1)9 from the extract of a marine
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sponge Topsentia sp. as a cytotoxic oxylipin against human solid tumor cell lines. Topsentolide A1 has a nine-membered lactone with an epoxide side chain. Stereochemistry
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of the epoxide was proposed to be cis by spectroscopic analysis, however, the relative as well as the absolute configuration of the three stereocenters remained unknown. Therefore, we undertook the synthesis of possible stereoisomers of topsentolide A1 in order to determine the absolute configuration of the natural product. We have already published a rapid communication10, in which we succeeded in the enantioselective synthesis of the enantiomer of topsentolide A1, (8S,11S,12R)-isomer, and determined the stereochemistry of natural product to be 8R,11R,12S as shown in Figure 1. Now we achieved the synthesis of natural form of topsentolide A1 and here, we wish to report a full account of our work. 3
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2. Results and discussion Our synthetic strategy of all the four possible stereoisomers of topsentolide A1 (1, 5,
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6, 7) is illustrated in Scheme 1. We decided to utilize a key intermediate (8R)- and (8S)-A, which could be transformed into both of (11R,12S)-isomers (1 and 5) and (11S,12R)-
isomers (6 and 7) stereoselectively via epoxides formation, where the stereochemistry of the epoxide could be controlled by changing elimination direction.4,5 Compound A would
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be constructed by Horner-Wadsworth-Emmons (HWE) reaction of a side chain moiety B with a lactone moiety C. Both of the lactones (S)- and (R)-C would be prepared from
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optically active hydroxycarboxylic acid D via Yamaguchi lactonization or Mitsunobu lactonization. The phosphonate B and the hydroxycarboxylic acid D would be derived from
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the known aldehyde 8,11,12 which could be easily prepared from L-malic acid.
Scheme 1. Retrosynthetic analysis of four possible stereoisomers of topsentolide A1.
Synthesis of the phosphonate 17 (= B) is shown in Scheme 2. The known aldehyde
811,12 was subjected to Wittig reaction with the known phosphonium salt 913 to afford Zolefin 10 selectively. Z-Selectivity was confirmed to be >98% by GC analysis. The Z-olefin 10 was simply transformed into the corresponding primary alcohol 14 in four steps, which 4
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was then oxidized to give aldehyde 15. Oxidation of the aldehyde 15 with NaClO2 and subsequent esterification using CH2N2 afforded the desired methyl ester 16 only in poor yield (~20%), and undesired migration of TBS group was observed. On the other hand,
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oxidation of 15 with NIS14 in methanol proceeded smoothly to give the ester 16 in good yield without any side reactions. Finally, the ester 16 was reacted with anion of dimethyl methylphosphonate to afford the desired β-ketophosphonate 17, one of the substrates for
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HWE reaction.
Scheme 2. Synthesis of the phosphonate 17. (a) KHMDS, THF, –78 °C to rt, 84%; (b) 1N
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HCl, THF, rt, 90%; (c) PivCl, Pyr., 0 °C to rt, 89%; (d) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C to rt, 96%; (e) DIBAL, CH2Cl2, –50 °C, 97%; (f) (COCl)2, DMSO, Et3N, CH2Cl2, –78 °C to rt, 96%; (g) NIS, K2CO3, MeOH, rt, 78%; (h) (MeO)2P(O)Me, n-BuLi, THF, –78 °C,
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52%.
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Synthesis of the lactone 25 (= C), another substrate for HWE reaction, is shown in Scheme 3. Wittig reaction of the known aldehyde 811,12 and a commercially available phosphonium salt and subsequent treatment with CH2N2 afforded Z-olefin 19 selectively (E-isomer was not observed in 1H and 13C NMR spectra), which was transformed into the corresponding hydroxycarboxylic acid 22 in four steps. As described in synthetic strategy, we supposed both of the lactone (S)- and (R)-25 would be prepared from hydroxycarboxylic acid 22 via Yamaguchi lactonization and Mitsunobu lactonization, respectively. Yamaguchi lactonization15 of 22 successfully provide nine-membered lactone 5
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23 in satisfactory yield, together with a small amount of a dimeric lactone (3%). After the removal of PMB group, 24 was oxidized into the desired aldehyde (S)-25. On the other hand, Mitsunobu lactonization of 22 afforded the desired nine-membered lactone only in
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poor yield (~20%) even under high dilution conditions. So enantiomeric (R)-25 was prepared from ent-8 in the same manner as the synthesis of (S)-25 via Yamaguchi
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lactonization.
Scheme 3. Synthesis of the lactone 25. (a) (4-carboxybutyl)triphenylphosphonium bromide, NaHMDS, THF, –78 °C to rt; (b) CH2N2, Et2O, MeOH, 0 °C, 69% in 2 steps; (c)
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1N HCl, THF, rt, 97%; (d) PMBOC(NH)CCl3, TsOH·H2O, CH2Cl2, rt, 63%; (e) LiOH·H2O, H2O, MeOH, rt, 98%; (f) 2,4,6-trichlorobenzoyl chloride, Et3N, THF, rt, then DMAP, toluene, reflux, 81%; (g) DDQ, H2O, CH2Cl2, rt, 87%; (h) (COCl)2, DMSO, Et3N,
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CH2Cl2, –78 °C to rt, 69%.
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Now that both of the side chain moiety 17 and the lactone moieties (R)- and (S)-25 were obtained enantioselectively, HWE reaction was examined. At first, syntheses of (8R)isomers 1 and 6 are shown in Scheme 4. HWE reaction of the β-ketophosphonate 17 and the aldehyde (R)-25 under Masamune conditions employing DBU-LiCl16 proceeded smoothly to give unsaturated ketone (8R)-26, with excellent E selectivity without any epimerization, and other isomers were not observed in 1H NMR spectrum. Reduction of the ketone (8R)-26 using Luche conditions17 successfully proceeded under Felkin-Anh control, and stereoselectively afforded the key intermediate (8R)-27, a precursor for epoxide 6
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the epoxide was controlled as described below by changing elimination direction.4,5 At first, alcohol (8R)-27 was converted to the corresponding mesylate (8R)-28, which was
treated with TBAF to provide β-epoxide, whereby the synthesis of natural topsentolide A1 [(8R,11R,12S)-isomer (1)] was completed. On the other hand, alcohol (8R)-27 was
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converted to another mesylate (8R)-31 by changing the position of the leaving group. After removing its EE group, mesylate was treated with base to afford α-epoxide, (8R,11S,12R)-
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isomer (6), successfully. Next, (8S)-isomers 5 and 7 were synthesized in the same manner as for (8R)-isomers. HWE reaction of the β-ketophosphonate 17 and the aldehyde (S)-25 was followed by stereoselective reduction to give alcohol (8S)-27. Stereochemistry at C-11 was confirmed to be S by modified Mosher’s method18 after conversion to the corresponding MTPA esters (Table 1). The alcohol (8S)-27 was transformed into both of β-
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epoxide 5 and α-epoxide 7 in two and five steps, respectively.
Scheme 4. Synthesis of possible stereoisomers of topsentolide A1. (a) DBU, LiCl, MeCN, rt, 75%; (b) NaBH4, CeCl3·7H2O, MeOH, rt, 91%; (c) MsCl, Et3N, DMAP, CH2Cl2, rt; (d) TBAF, THF, rt, 98% in 2 steps; (e) EVE, PPTS, CH2Cl2, rt, 98%; (f) TBAF, THF, rt, 92%; (g) 0.5N HCl, THF, rt; (h) TBAF, THF, rt, 51% in 3 steps. 7
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Table 1. 1H NMR (300 MHz, CDCl3) data for (S)- and (R)-MTPA esters of (8R)- and
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(8S)-27.
With the four possible stereoisomers of topsentolide A1 in hand, we analyzed their specific rotations and NMR spectra carefully in order to confirm the absolute configuration of the natural product. About the specific rotations of the synthetic stereoisomers, [α]D24 –
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98 (c 0.10, MeOH) for 5 and [α]D24 –88 (c 0.11, MeOH) for 7, showed opposite sign to [α]D24 +59.4 (c 0.11, MeOH)9 for the natural material. On the other hand, stereoisomers 1 and 6 showed identical sign of the specific rotation to the natural product [1: [α]D24 +88 (c
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0.22, MeOH), 6: [α]D24 +96 (c 0.18, MeOH)], and it was thought that either (8R)-isomers 1 or 6 should have the same stereochemistry as natural topsentolide A1. As for the 13C NMR,
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there was no difference between the diastereomers (1 and 6) to distinguish them. In the 1H NMR, slight differences of the chemical shifts and the shapes of peaks were observed between the diastereomers. 1H NMR spectra of natural topsentolide A1 and synthesized (8R)-isomers 1 and 6 were partially shown in Figure 2. 1H NMR spectrum of (8R,11R,12S)isomer (1) was completely identical with that of natural topsentolide A1, while some signals of (8R,11S,12R)-isomer (6) were not identical with those of natural product [especially at 2.74-2.86 ppm (16-H), 2.32-2.42 ppm (2-H and 13-Hb), 2.23-2.30 (13-Ha) and 2.03-2.12 ppm (3-H, 4-H and 19-H)]. On the basis of these results, the (8R,11R,12S)-stereochemistry 8
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for natural topsentolide A1 (1) was unambiguously determined as shown in Figure 3. Actually, Jung’s group also isolated topsentolide C1 (33) and C2 (34) together with 1.9 They suspected these methyl ether analogues to be artifacts formed during the process of
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extraction with MeOH, and determined the absolute configuration at C-12 as S by Mosher’s method. Their determination of stereochemistry at C-12 supports our results. In addition, Kuwahara’s group recently reported the synthesis and the stereochemical assignment of
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topsentolide C219, which also fully supports our results.
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Figure 2. Partial 1H NMR spectra of natural topsentolide A1 and synthesized (8R)-isomers
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(500 MHz, in CD3OD).
Figure 3. Absolute configuration of topsentolide A1.
Having succeeded in the total syntheses of four stereoisomers and in the determination of the absolute configuration of natural topsentolide A1, our next interest 9
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came to structure–activity relationship of topsentolide A1. Jung’s group reported that topsentolide A1 showed moderate and broad cytotoxicity against human solid tumor cell lines. We examined the cytotoxic activity of topsentolide A1 and its stereoisomers against
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HeLa cells and human lymphoblastoid namalva cells. Unfortunately, these compounds exhibited no activity against HeLa cells at less than 1 mM. Against human lymphoblastoid namalva cells, topsentolide A1 and its stereoisomers showed weak cytotoxicity, but
cytotoxicity of four stereoisomers were much the same (IC50 1: 137 µM, 5: 198 µM, 6: 148
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µM, 7: 182 µM).
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In summary, we succeeded in the synthesis of topsentolide A1 (1) and its stereoisomers (5-7) efficiently, and determined the stereochemistry of natural product to be 8R,11R,12S. Synthetic topsentolide A1 showed week cytotoxicity as well as other stereoisomers. We wish our study on these oxylipins, which have various activities, would
3. Experimental 3.1. General
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offer any useful information for further investigation on the related fields.
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Optical rotations were recorded with a JASCO DIP-1000 polarimeter. IR spectra were measured with a JASCO FT/IR-230 spectrophotometer. 1H and 13C NMR were recorded on JEOL JNM AL300 or JEOL JNM GSX500. Chemical shifts (δ) were
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referenced to the residual solvent peaks as the internal standard (CDCl3: δH = 7.26, δC = 77.23, CD3OD: δH = 3.30, δC = 49.00). Refractive indexes were measured with an Atago 1T refractometer. Mass spectra were recorded on JEOL JMS SX102. Column chromatography was performed using Kanto silica gel 60N (0.060-0.200 mm). TLC was carried out on Merck glass plates precoated with silica gel 60 F254 (0.25 mm).
3.2. Synthetic studies 10
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3.2.1 (S)-2,2-Dimethyl-4-[(2Z,5Z)-octa-2,5-dienyl]-1,3-dioxolane (10) To a cooled (–78 °C) suspension of phosphonium salt 913 (11.8 g, 25.1 mmol) in dry THF (100 ml) under argon atmosphere was added KHMDS (50 ml, 0.5 M in toluene, 25
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mmol) and the mixture was stirred for 10 min. To the resulting solution was added a solution of aldehyde 8 (3.60 g, 25.0 mmol) in THF (10 ml) via cannula at –78 °C. The
mixture was stirred for 30 min at –78 °C, then gradually warmed to room temperature, and stirred for 2 h at ambient temperature. The reaction mixture was quenched with water and
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extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1
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hexanes/EtOAc) gave olefin 10 as a colorless oil (4.41 g, 21.0 mmol, 84%). nD18 = 1.4591. [α]D20 = +25.4 (c 1.1, CHCl3). IR (film): ν = 2965, 2934, 2874, 1369, 1216, 1065 cm-1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.2 Hz), 1.36 (3H, s), 1.43 (3H, s), 2.07 (2H, m), 2.33 (1H, m), 2.43 (1H, m), 2.80 (2H, br t, J = 6.9 Hz), 3.56 (1H, dd, J = 7.8, 7.2 Hz), 4.03 (1H, dd, J = 7.8, 6.0 Hz), 4.13 (1H, m), 5.26-5.53 (4H, m). 13C NMR (125
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MHz, CDCl3): δ (ppm) = 14.4, 20.8, 25.8, 25.9, 27.1, 31.7, 69.2, 75.8, 109.1, 124.3, 126.9, 131.3, 132.4. ESI-HRMS m/z calcd. for C13H22NaO2 [M+Na]+ 233.1512, found 233.1517.
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3.2.2 (2S,4Z,7Z)-Deca-4,7-diene-1,2-diol (11) To a stirred solution of compound 10 (10.5 g, 50.0 mmol) in THF (150 ml) was added
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1 N HCl (50 ml) and the mixture was stirred at room temperature for 15 h. The reaction mixture was quenched with saturated NaHCO3 solution and extracted with EtOAc. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (2 : 1 hexanes/EtOAc) gave diol 11 as a colorless oil (7.64 g, 44.9 mmol, 90%). nD19 = 1.4868. [α]D22 = +13.9 (c 0.5, CHCl3). IR (film): ν = 3333, 2964, 1068 cm-1; 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.8 Hz,), 1.88 (2H, br), 2.02-2.11 (2H, m), 2.20-2.37 (2H, m), 2.81 (2H, br t, J = 7.2 Hz), 3.49 (1H, dd, J = 11.1, 6.9 Hz), 3.69 (1H, dd, J = 11.1, 3.3 Hz), 3.76 (1H, m), 5.27-5.61 (4H, m). 13C 11
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3.2.3 (2S,4Z,7Z)-2-Hydroxydeca-4,7-dienyl pivalate (12) To a cooled (0 °C) solution of diol 11 (2.46 g, 14. 5 mmol) in pyridine (60 ml) was added PivCl (2.1 ml, 18 mmol) under argon atmosphere, and the mixture was stirred at
room temperature for 1.5 h. The reaction mixture was quenched with water and extracted
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with Et2O. The combined organic layer was washed with saturated NaHCO3 solution, water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel
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(25 : 1 hexanes/EtOAc) gave ester 12 as a colorless oil (3.27 g, 12.9 mmol, 89%). nD18 = 1.4657. [α]D24 = +8.3 (c 0.61, CHCl3). IR (film): ν = 3445, 2964, 1732, 1160 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.5 Hz), 1.23 (9H, s), 2.02-2.12 (3H, m), 2.32 (2H, br t, J = 6.9 Hz), 2.81 (2H, br t, J = 7.2 Hz), 3.88 (1H, m), 4.02 (1H, dd, J = 11.1, 6.6 Hz), 4.16 (1H, dd, J = 11.1, 2.1 Hz), 5.27-5.58 (4H, m). 13C NMR (125 MHz, CDCl3): δ
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(ppm) = 14.4, 20.8, 25.8, 27.4, 31.7, 39.1, 68.0, 70.0, 124.3, 126.7, 132.1, 132.5, 178.9. ESI-HRMS m/z calcd. for C15H26NaO3 [M+Na]+ 277.1774, found 277.1777.
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3.2.4 (2S,4Z,7Z)-2-(t-Butyldimethylsilyloxy)deca-4,7-dienyl pivalate (13) To a cooled (0 °C) solution of alcohol 12 (2.71 g, 10.7 mmol) in dry CH2Cl2 (100
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ml) was added 2,6-lutidine (3.3 g, 31 mmol) and TBSOTf (4.01 g, 15.2 mmol) at room temperature under argon atmosphere, and the mixture was stirred for 10 min. The reaction mixture was quenched with water and extracted with CH2Cl2. The combined organic layer was dried with MgSO4, and then evaporated. Purification of the residue on silica gel (50 : 1 hexanes/EtOAc) gave silyl ether 13 as a colorless oil (3.78 g, 10.3 mmol, 96%). nD18 = 1.4553. [α]D23 = +7.9 (c 0.45, CHCl3). IR(film): ν = 3011, 2959, 1731, 1156 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.07 (6H, s), 0.88 (9H, s), 0.97 (3H, t, J = 7.5 Hz), 1.23 (9H, s), 2.01-2.09 (2H, m), 2.24-2.38 (2H, m), 2.78 (2H, br t, J = 6.6 Hz), 3.86-4.03 (3H, m), 12
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calcd. for C21H40NaO3Si [M+Na]+ 391.2639, found 391.2628.
3.2.5 (2S,4Z,7Z)-2-(t-Butyldimethylsilyloxy)deca-4,7-dien-1-ol (14)
To a cooled (–50 °C) solution of ester 13 (3.78 g, 10.3 mmol) in dry CH2Cl2 (100 ml) was added DIBAL-H (1.03 M in hexane, 22 ml, 23 mmol) under argon atmosphere, and
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the mixture was stirred for 20 min. To the resulting solution was added saturated potassium sodium tartrate solution, and the mixture was stirred 3 h at room temperature. The resulting
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biphasic mixture was extracted with Et2O and the combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave alcohol 14 as a colorless oil (2.82 g, 9.92 mmol, 97%). nD17 = 1.4666. [α]D25 = +20.5 (c 0.48, CHCl3). IR (film): ν = 2957, 1255, 1105 cm–1. 1
H NMR (300 MHz, CDCl3): δ (ppm) = 0.10 (6H, s), 0.91 (9H, s), 0.97 (3H, t, J = 7.2 Hz),
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1.65 (1H, br), 2.07 (2H, qui, J = 7.2 Hz), 2.24-2.38 (2H, m), 2.79 (2H, br t, J = 6.9 Hz), 3.44 (1H, dd, J = 11.1, 5.4 Hz), 3.56 (1H, dd, J = 11.1, 3.9 Hz), 3.77 (1H, m), 5.27-5.47 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.4, -4.2, 14.5, 18.3, 20.8, 26.0, 26.1,
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31.2, 66.2, 72.9, 125.1, 127.0, 130.8, 132.3. ESI-HRMS m/z calcd. for C16H32NaO2Si
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[M+Na]+ 307.2064, found 307.2078.
3.2.6 (2S,4Z,7Z)-2-(t-Butyldimethylsilyloxy)deca-4,7-dienal (15) To a cooled (–78 °C) solution of oxalyl chloride (1.5 ml, 18 mmol) in CH2Cl2 (10
ml) was added dimethyl sulfoxide (2.49 ml, 35.1 mmol) under argon atmosphere, and the mixture was stirred for 20 min. To the resulting solution was added a solution of alcohol 14 (2.49 g, 8.76 mmol) in dry CH2Cl2 (1 ml), and the mixture was stirred 20 min at –78 °C. After addition of Et3N (7.34 ml, 52.6 mmol), the reaction mixture was stirred for 50 min while gradually warming to room temperature. The reaction mixture was quenched with 13
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saturated NaHCO3 solution and extracted with Et2O. The combined organic layer was washed with saturated NaHCO3 solution, water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (20 : 1 hexanes/EtOAc) gave aldehyde 15 as a colorless oil (2.38 g, 8.42 mmol, 96%). nD19 = 1.4646. [α]D22 = –18.5 (c 1.0, CHCl3).
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IR (film): ν = 3011, 2957, 2931, 1738, 1254, 1112 cm–1. 1H NMR (300 MHz, CDCl3): δ
(ppm) = 0.08 (3H, s), 0.10 (3H, s), 0.91 (9H, s), 0.97 (3H, t, J = 7.8 Hz), 2.00-2.09 (2H, m), 2.43 (2H, m), 2.78 (2H, br), 4.01 (1H, dt, J = 1.5, 6.3 Hz), 5.26-5.53 (4H, m), 9.61 (1H, d, J
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= 1.5 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.6, –4.5, 14.5, 18.4, 20.8, 25.9, 26.0, 31.1, 77.7, 123.7, 126.8, 131.7, 132.5, 204.1. ESI-HRMS m/z calcd. for C16H30NaO2Si
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[M+Na]+ 305.1907, found 305.1889.
3.2.7 Methyl (2S,4Z,7Z)-2-(t-butyldimethylsilyloxy)deca-4,7-dienoate (16) To a solution of NIS (3.90 g, 17.3 mmol) in methanol (90 ml) was added potassium carbonate (2.67 g, 19.3 mmol) and the mixture was stirred for 20 min at room temperature.
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To the resulting suspension was added a solution of aldehyde 15 (2.44 g, 8.64 mmol) in methanol (20 ml), and the mixture was stirred 2 h at room temperature. The resulting mixture was quenched with aqueous sodium thiosulfate solution and extracted with Et2O.
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The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave ester 16 as
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a colorless oil (2.11 g, 6.76 mmol, 78%). nD17 = 1.4618. [α]D24 = –1.5 (c 0.82, CHCl3). IR (film): ν = 3011, 2956, 2858, 1759, 1136 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.06 (3H, s), 0.08 (3H, s), 0.90 (9H, s), 0.97 (3H, t, J = 7.8 Hz), 2.01-2.10 (2H, m), 2.50 (2H, m), 2.78 (2H, br), 3.72 (3H, s,), 4.24 (1H, t, J = 6.3 Hz), 5.26-5.53 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –5.0, –4.8, 14.5, 18.5, 20.8, 25.8, 25.9, 33.5, 52.0, 72.4, 124.4, 127.1, 131.4, 132.3, 174.0. ESI-HRMS m/z calcd. for C17H32NaO3Si [M+Na]+ 335.2013, found 335.1994.
14
ACCEPTED MANUSCRIPT
3.2.8 Dimethyl (3S,5Z,8Z)-3-(t-butyldimethylsilyloxy)-2-oxoundeca-5,8-dienylphosphonate (17) To a cooled (–78 °C) solution of dimethyl methylphosphonate (360 mg, 2.90 mmol)
RI PT
in dry THF (30 ml) was added n-BuLi (2.64 M in hexane, 1.1 ml, 2.9 mmol) under argon atmosphere and the mixture was stirred for 15 min. To the resulting mixture was added a solution of ester 16 (880 mg, 2.82 mmol) in dry THF (3 ml), and the mixture was stirred for 40 min at –78 °C. The resulting mixture was quenched with saturated ammonium chloride
SC
solution and extracted with EtOAc. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (1 : 1
M AN U
hexanes/EtOAc) gave phosphonate 17 as a colorless oil (0.62 g, 1.48 mmol, 52%). nD19 = 1.4728. [α]D24 = +12.9 (c 1.0, CHCl3). IR (film): ν = 3445, 3274, 3010, 2957, 2857, 1724, 1257 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.08 (6H, s), 0.92 (9H, s), 0.97 (3H, t, J = 7.5 Hz), 2.01-2.08 (2H, m), 2.39 (1H, m), 2.49 (1H, m), 2.76 (2H, br), 3.23 (1H, dd, J = 19.8, 15.3 Hz) , 3.29 (1H, dd, J = 19.8, 15.3 Hz), 3.79 (6H, d, J = 11.1 Hz), 4.17 (1H, t, J =
TE D
6.0 Hz), 5.23-5.53 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.7, –4.6, 14.5, 18.3, 20.8, 25.8, 26.0, 32.5, 35.5 (d, J = 135 Hz), 53.2 (d, J = 6 Hz), 78.7, 123.7, 126.8, 131.7, 132.5, 204.7. ESI-HRMS m/z calcd. for C19H37NaO5PSi [M+Na]+ 427.2040, found
EP
427.2060.
AC C
3.2.9 Methyl (Z)-7-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]hept-5-enoate (19) (a) (S)-19: To a cooled (0 °C) suspension of (4-carboxybutyl)triphenylphosphonium
bromide (22.3 g, 50.3 mmol) in dry THF (200 ml) under argon atmosphere was added NaHMDS (1.0 M in THF, 100 ml, 100 mmol) over 20 min, and the mixture was stirred for 20 min at 0 °C. To the resulting solution was added a solution of aldehyde 8 (7.20 g, 50.0 mmol) in dry THF (20 ml) at –78 °C. The mixture was stirred for 1.5 h, gradually warmed to room temperature, and stirred for 2 h. Water and ether were added to the reaction mixture, and it was extracted with water. The combined aqueous layer was saturated with 15
ACCEPTED MANUSCRIPT
ammonium sulfate and extracted with EtOAc. The combined organic layer was dried with MgSO4, and then evaporated to give crude carboxylic acid 18. Crude 18 was dissolved in methanol and treated with a solution of CH2N2 in ether (prepared from N-nitroso-N-
RI PT
methylurea). The reaction mixture was quenched by acetic acid, neutralized with saturated NaHCO3 solution, and extracted with ether. The combined organic layer was washed with saturated NaHCO3 solution and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (4 : 1 hexanes/EtOAc) gave ester (S)-19 as a colorless oil (8.30
SC
g, 34.3 mmol, 69%). nD20 = 1.4558. [α]D24 = +23 (c 0.10, CHCl3). IR (film): ν = 2958, 2950, 2873, 1739, 1218, 1063 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.35 (3H, s), 1.42
M AN U
(3H, s,), 1.70 (2H, qui, J = 6.9 Hz), 2.09 (2H, q, J = 6.9 Hz), 2.21-2.42 (4H, m), 3.55 (1H, dd, J = 7.8, 6.9 Hz), 3.67 (3H, s), 4.03 (1H, dd, J = 7.8, 6.0 Hz), 4.11 (1H, m), 5.43 (1H, dt, J = 10.5, 6.9 Hz) 5.47 (1H, dt, J = 10.5, 6.9 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 24.9, 25.8, 26.9, 27.1, 31.7, 33.6, 51.7, 69.2, 75.8, 109.1, 125.3, 131.7, 174.2. ESI-HRMS m/z calcd. for C13H22NaO4 [M+Na]+ 265.1410, found 265.1366.
TE D
(b) (R)-19: In the same manner as the synthesis of (S)-19 described above, ent-8 (10.8 g, 75.0 mmol) was converted into (R)-19 (12.2 g, 50.6 mmol, 67%, colorless oil). nD20 = 1.4557. [α]D21 = –24 (c 0.06, CHCl3). Its IR and NMR spectra were identical with those of
EP
(S)-19. ESI-HRMS m/z calcd. for C13H22NaO4 [M+Na]+ 265.1410, found 265.1439.
AC C
3.2.10 Methyl (5Z,8S)-8,9-dihydroxynon-5-enoate (20) (a) (S)-20: To a stirred solution of compound (S)-19 (2.63 g, 10.9 mmol) in dry THF
(20 ml) was added 1 N HCl (5 ml) and the mixture was stirred at room temperature for 18 h. The resulting mixture was neutralized with saturated NaHCO3 solution and extracted with EtOAc. The combined organic layer was dried with MgSO4 and then evaporated. Purification of the residue on silica gel (1 : 1 hexanes/EtOAc) gave diol (S)-20 as a colorless oil (2.14 g, 10.6 mmol, 97%). nD20 = 1.4770. [α]D23 = +5.6 (c 0.02, CHCl3). IR (film). ν = 3401, 2950, 1738, 1220, 1034 cm-1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 16
ACCEPTED MANUSCRIPT
1.71 (2H, qui, J = 7.2 Hz), 1.79 (2H, br), 2.11 (2H, q, J = 7.2 Hz), 2.17-2.31 (2H, m), 2.33 (2H, t, J = 7.2 Hz), 3.49 (1H, dd, J = 11.1, 6.6 Hz), 3.67 (3H, s), 3.68 (1H, dd, J = 11.1, 2.7 Hz), 3.75 (1H, m), 5.41-5.57 (2H, m). 13C NMR (125 MHz, CDCl3) δ (ppm) = 24.9, 26.8, 31.5, 33.6, 51.8, 66.4, 72.0, 125.9, 132.2, 174.5. ESI-HRMS m/z calcd for C10H18NaO4
RI PT
[M+Na]+ 225.1097, found 225.1104.
(b) (R)-20: In the same manner as the synthesis of (S)-20 described above, (R)-19 (1.95 g, 8.06 mmol) was converted into (R)-20 (1.21 g, 5.97 mmol, 74%, colorless oil). nD20
SC
= 1.4767. [α]D21 = –6.1 (c 0.2, CHCl3). Its IR and NMR spectra were identical with those of
M AN U
(S)-20. ESI-HRMS m/z calcd for C10H18NaO4 [M+Na]+ 225.1097, found 225.1068.
3.2.11 Methyl (5Z,8S)-8-hydroxy-9-(p-methoxybenzyloxy)non-5-enoate (21) (a) (S)-21: To a stirred solution of diol (S)-20 (841 mg, 4.16 mmol) in CH2Cl2 (40 ml) was added PMBOC(NH)CCl3 (1.23 g, 4.36 mmol) and p-TsOH (40.0 mg, 0.21 mmol ). After stirring at room temperature for 16 h, the resulting mixture was neutralized with
TE D
saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was dried with MgSO4, and then evaporated. Purification of the residue on silica gel (2 : 1 hexanes/EtOAc) gave ether (S)-21 as a yellow oil (790 mg, 2.62 mmol, 63%). nD20 = 1.5162. [α]D23 = +2.0 (c 0.2, CHCl3). IR (film): ν = 3460, 2950, 1732, 1248, 1035 cm–1. 1H
EP
NMR (300 MHz, CDCl3): δ (ppm) = 1.57 (1H, br), 1.69 (2H, qui, J = 7.2 Hz), 2.08 (2H, q,
AC C
J = 7.2 Hz), 2.20-2.25 (2H, m), 2.31 (2H, t, J = 7.2 Hz), 3.33 (1H, dd, J = 9.3, 7.2 Hz), 3.48 (1H, dd, J = 9.3, 3.3 Hz), 3.66 (3H, s), 3.81 (3H, s), 3.85 (1H, m), 4.48 (2H, s), 5.42-5.53 (2H, m), 6.89 (2H, d, J = 8.7 Hz), 7.25 (2H, d, J = 8.7 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 24.9, 26.8, 31.5, 33.6, 51.7, 55.4, 70.4, 73.2, 73.8, 114.0, 126.0, 129.6, 130.2, 131.5, 159.5, 174.2. ESI-HRMS m/z calcd for C18H26NaO5 [M+Na]+ 345.1672, found 345.1700. (b) (R)-21: In the same manner as the synthesis of (S)-21 described above, (R)-20 (1.21 g, 5.97 mmol) was converted into (R)-21 (986 mg, 3.06 mmol, 51%, yellow oil). nD20 17
ACCEPTED MANUSCRIPT = 1.5162. [α]D21 = –2.7 (c 0.32, CHCl3). Its IR and NMR spectra were identical with those of (S)-21. ESI-HRMS m/z calcd for C18H26NaO5 [M+Na]+ 345.1672, found 345.1709.
RI PT
3.2.12 (5Z,8S)-8-Hydroxy-9-(p-methoxybenzyloxy)non-5-enoic acid (22) (a) (S)-22: To a stirred solution of ester (S)-21 (841 mg, 4.16 mmol) in methanol (10 ml) was added water (2 ml) and LiOH (332 mg, 7.92 mmol). After stirring for 14 h at room temperature, the resulting mixture was neutralized with saturated ammonium chloride
SC
solution and extracted with EtOAc three times. The aqueous layer was acidified by addition of acetic acid and extracted again with EtOAc. The combined organic layer was dried with
M AN U
MgSO4, and then evaporated. Purification of the residue on silica gel (1 : 1 hexanes/EtOAc) gave carboxylic acid (S)-22 as a yellow oil (742 mg, 2.56 mmol, 98%). nD20 = 1.5250. [α]D24 = +0.97 (c 0.4, CHCl3). IR (film) : ν = 3420, 2936, 1713, 1248, 1035 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.25 (1H, br), 1.71 (2H, qui, J = 7.5 Hz), 2.07-2.14 (2H, m), 2.20-2.27 (2H, m), 2.35 (2H, t, J = 7.5 Hz), 3.34 (1H, dd, J = 9.3, 7.5 Hz), 3.48 (1H, dd, J =
TE D
9.3, 3.3 Hz), 3.81 (3H, s), 3.84 (1H, m), 4.48 (2H, s), 5.45-5.51 (2H, m), 6.89 (2H, d, J = 8.7 Hz), 7.26 (2H, d, J = 8.7 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 24.6, 26.7, 31.4, 33.5, 55.5, 70.5, 73.2, 73.8, 114.0, 126.2, 129.6, 130.2, 131.4, 159.5, 179.2. ESI-HRMS m/z
EP
calcd for C17H24NaO5 [M+Na]+ 331.1516, found 331.1541. (b) (R)-22: In the same manner as the synthesis of (S)-22 described above, (R)-21
AC C
(986 mg, 3.06 mmol) was converted into (R)-22 (682.3 mg, 2.215 mmol, 72%, yellow oil). nD20 = 1.5246. [α]D21 = –0.26 (c 0.1, CHCl3). Its IR and NMR spectra were identical with those of (S)-22. ESI-HRMS m/z calcd for C17H24NaO5 [M+Na]+ 331.1516, found 331.1507.
3.2.13 (5Z,8S)-9-(p-Methoxybenzyloxy)non-5-en-8-olide (23) (a) (S)-23: To a cooled (0 °C) solution of carboxylic acid (S)-22 (2.01 g, 6.91 mmol) in dry THF (10 ml) was added Et3N (1.1 ml, 7.9 mmol) and 2,4,6-trichlorobenzoyl chloride (1.08 ml, 6.91 mmol). After stirring for 2 h at room temperature, the resulting mixture was 18
ACCEPTED MANUSCRIPT
filtered through Celite and the filtrate was diluted with toluene (50 ml). The resulting solution was slowly added via syringe pump (during 8 h) to a refluxed solution of DMAP (16.4 g, 134 mmol) in toluene (2.0 l). After stirring for additional 1 h, the resulting mixture
RI PT
was evaporated, mixed with water and extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (9 : 1, then 4 : 1 hexanes/EtOAc) gave lactone (S)-23 as a yellow oil (1.53 g, 5.61 mmol, 81%). nD24 = 1.5344. [α]D25 = –57 (c 0.08, CHCl3). IR (film): ν =
SC
2948, 1740, 1249, 1034 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.78 (1H, m), 2.022.09 (3H, m), 2.22-2.55 (4H, m), 3.52 (1H, dd, J = 10.5, 4.8 Hz), 3.60 (1H, dd, J =10.5, 6.0
M AN U
Hz), 3.81 (3H, s), 4.48 (1H, d, J = 11.7 Hz), 4.54 (1H, d, J = 11.7 Hz), 4.97 (1H, m), 5.455.53 (2H, m), 6.88 (2H, d, J = 8.7 Hz), 7.27 (2H, d, J = 8.7 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 25.5, 26.7, 30.8, 33.7, 55.4, 71.2, 71.9, 73.0, 114.0, 124.6, 129.5, 130.2, 135.0, 159.4, 174.4. ESI-HRMS m/z calcd for C17H22NaO4 [M+Na]+ 313.1410, found 313.1441.
TE D
(b) (R)-23: In the same manner as the synthesis of (S)-23 described above, (R)-22 (9.0 mg, 0.031 mmol) was converted into (R)-23 (6.4 mg, 0.024 mmol, 76%, yellow oil). nD24 = 1.5344. [α]D21 = +63 (c 0.15, CHCl3). Its IR and NMR spectra were identical with
EP
those of (S)-23. ESI-HRMS m/z calcd for C17H22NaO4 [M+Na]+ 313.1410, found 313.1407.
AC C
3.2.14 (5Z,8S)-9-Hydroxynon-5-en-8-olide (24) (a) (S)-24: To a stirred solution of compound (S)-23 (246 mg, 0.903 mmol) in
CH2Cl2 (3 ml) was added water (0.3 ml) and DDQ (0.29 g, 1.28 mmol) and the mixture was stirred vigorously for 1 h at room temperature. The resulting mixture was poured into saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (1 : 1 hexanes/EtOAc) gave alcohol (S)-24 as a colorless oil (134 mg, 0.787 mmol, 87%). nD24 = 1.4985. [α]D25 = – 86 (c 0.18, CHCl3). IR (film): ν = 3444, 2948, 1744, 19
ACCEPTED MANUSCRIPT 1715 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.61 (1H, br), 1.81 (1H, m), 1.99-2.13 (3H, m), 2.25-2.55 (4H, m), 3.76-3.82 (2H, m), 4.88 (1H, m), 5.45-5.56 (2H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) 25.4, 26.7, 30.2, 33.7, 64.8, 74.1, 124.4, 134.9, 174.6. ESI-
RI PT
HRMS m/z calcd for C9H14NaO3 [M+Na]+ 193.0835, found 193.0831. (b) (R)-24: In the same manner as the synthesis of (S)-24 described above, (R)-23 (2.53 g, 9.32 mmol) was converted into (R)-24 (1.22 g, 7.19mmol, 77%, colorless oil). nD24 = 1.4985. [α]D21 = +84 (c 0.14, CHCl3). Its IR and NMR spectra were identical with those
M AN U
3.2.15 (5Z,8S)-8-Formyloct-5-en-8-olide (25)
SC
of (S)-24. ESI-HRMS m/z calcd for C9H14NaO3 [M+Na]+ 193.0835, found 193.0793.
(a) (S)-25: To a cooled (–78 °C) solution of oxalyl chloride (2.16 g, 17.0 mmol) in dry CH2Cl2 (20 ml) was added dimethyl sulfoxide (2.4 ml, 34 mmol) under argon atmosphere and the mixture was stirred for 15 min. To the resulting solution was added a solution of alcohol (S)-24 (1.45 g, 8.53 mmol) in CH2Cl2 (5 ml) and the mixture was stirred
TE D
for 20 min at –78 °C. After addition of Et3N (7.0 ml, 50.2 mmol), the resulting mixture was stirred for 30 min while gradually warming to room temperature, then poured into saturated NaHCO3 solution, and extracted with EtOAc. The combined organic layer was
EP
dried with MgSO4, and then evaporated. Purification of the residue on silica gel (3 : 1 hexanes/EtOAc) gave aldehyde (S)-25 as a colorless oil (983 mg, 5.85 mmol, 69%). nD24 =
AC C
1.5056. [α]D24 = –163 (c 0.05, CHCl3). IR (film): ν = 2946, 1746 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.82 (1H, m), 2.08-2.52 (7H, m), 5.17 (1H, ddd, J = 9.3, 4.2, 1.2 Hz), 5.46-5.61 (2H, m), 9.74 (1H, d, J = 1.2 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 25.4, 26.5, 28.7, 33.3, 76.9, 123.2, 136.1, 173.8, 198.8. ESI-HRMS m/z calcd for C9H12NaO3 [M+Na]+ 191.0679, found 191.0667. (b) (R)-25: In the same manner as the synthesis of (S)-25 described above, (R)-24 (975 mg, 5.88 mmol) was converted into (R)-25 (889 mg, 5.29mmol, 90%, colorless oil).
20
ACCEPTED MANUSCRIPT nD20 = 1.5041. [α]D21 = +167 (c 0.05, CHCl3). Its IR and NMR spectra were identical with those of (S)-25. ESI-HRMS m/z calcd for C9H12NaO3 [M+Na]+ 191.0679, found 191.0683.
RI PT
3.2.16 (5Z,8R,9E,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-oxoicosa-5,9,14,17-tetraen8-olide [(8R)-26]
To a stirred solution of phosphonate 17 (422 mg, 1.00 mmol) in dry acetonitrile (1.5 ml) was added DBU (0.15 ml, 1.0 mmol), lithium chloride (80.4 mg, 1.89 mmol) and
SC
aldehyde (R)-25 (169 mg, 1.00 mmol) successively under argon atmosphere and the
mixture was stirred for 1.5 h at room temperature. The reaction mixture was poured into
M AN U
water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave compound (8R)-26 as a colorless oil (336 mg, 0.751 mmol, 75%). nD18 = 1.4979. [α]D22 = + 118.4 (c 0.03, CHCl3). IR (film): ν = 2954, 2929, 1747, 1698, 1632 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.02 (3H, s), 0.06 (3H, s), 0.90 (9H, s),
TE D
0.97 (3H, t, J = 7.5 Hz), 1.82 (1H, m), 2.01-2.53 (11H, m), 2.77 (2H, br t, J = 6.3 Hz, 16H), 4.14 (1H, dd, J = 6.9, 5.4 Hz), 5.21-5.57 (7H, m), 6.84 (1H, dd, J = 15.6, 1.5 Hz), 6.94 (1H, dd, J = 15.6, 3.9 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.7, –4.6, 14.5, 18.4,
EP
20.8, 25.6, 25.9, 26.6, 29.9, 33.2, 33.4, 33.9, 71.8, 78.5, 123.8, 124.1, 124.2, 127.0, 131.4, 132.4, 135.8, 144.1, 173.6, 201.4. ESI-HRMS m/z calcd for C26H43O4Si [M+H]+ 447.2925,
AC C
found 447.2905.
3.2.17 (5Z,8S,9E,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-oxoicosa-5,9,14,17-tetraen8-olide [(8S)-26]
In the same manner as the synthesis of (8R)-26 described above, phosphonate 17
(1.41 g, 3.34 mmol) and (S)-25 (509 mg, 3.0 mmol) were converted into (8S)-26 (900 mg, 2.02 mmol, 67%, colorless oil). nD19 = 1.4980. [α]D22 = –92 (c 0.16, CHCl3). IR (film): ν = 2954, 2929, 1748, 1698, 1632 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.04 (3H, s), 21
ACCEPTED MANUSCRIPT
0.07 (3H, s), 0.94 (9H, s), 0.96 (3H, t, J = 7.2 H), 1.82 (1H, m), 2.01-2.49 (11H, m,), 2.76 (2H, br t, J = 6.9 Hz), 4.14 (1H, m), 5.22-5.61 (7H, m), 6.86 (1H, dd, J = 15.9, 0.9 Hz), 6.94 (1H, dd, J = 15.9, 3.3 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.7, –4.6, 14.4, 18.4, 22.9, 25.5, 26.0, 26.5, 31.8, 33.2, 33.6, 33.9, 71.7, 78.5, 123.8, 124.1, 124.2, 127.0,
RI PT
131.4, 132.4, 135.8, 144.0, 173.7, 201.5. ESI-HRMS m/z calcd for C26H43O4Si [M+H]+ 447.2925, found 447.2994.
SC
3.2.18 (5Z,8R,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-hydroxyicos-5,9,14,17tetraen-8-olide [(8R)-27]
M AN U
To a stirred solution of ketone (8R)-26 (336 mg, 0.751 mmol) in methanol (6 ml) was added cerium chloride heptahydrate (417 mg, 1.12 mmol) and sodium borohydride (42.2 mg, 1.12 mmol) and the mixture was stirred for 5 min at room temperature. The reaction mixture was quenched with water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the
TE D
residue on silica gel (25 : 1 hexanes/EtOAc) gave compound (8R)-27 as a colorless oil (306 mg, 0.683 mmol, 91%). nD18 = 1.4974. [α]D18 = + 53.7 (c 0.09, CHCl3). IR (film): ν = 3480, 2929, 1743 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.06 (3H, s), 0.09 (3H, s), 0.90
EP
(9H, s), 0.97 (3H, t, J = 7.2 Hz), 1.80 (1H, m), 2.02-2.51 (11H, m), 2.80 (2H, br t, J = 6.9 Hz), 3.61 (1H, m), 4.00 (1H, br t, J = 3.6 Hz), 5.25-5.52 (7H, m), 5.73-5.87 (2H, m, 9-H). C NMR (125 MHz, CDCl3): δ (ppm) = –4.4, –4.0, 14.5, 18.3, 20.8, 25.6, 25.9, 26.1, 26.6,
AC C
13
32.1, 33.7, 34.6, 72.5, 72.7, 75.1, 124.7, 124.9, 127.0, 129.5, 131.2, 132.5, 133.0, 135.2, 173.9. ESI-HRMS m/z calcd for C26H45O4Si [M+H]+ 449.3082, found 449.3035.
3.2.19 (5Z,8S,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-hydroxyicos-5,9,14,17tetraen-8-olide [(8S)-27] In the same manner as the synthesis of (8R)-27 described above, (8S)-26 (91.3 mg, 0.205 mmol) was converted into (8S)-27 (88.1 mg, 0.197 mmol, 96%, colorless oil). nD18 = 22
ACCEPTED MANUSCRIPT 1.4982. [α]D16 = –72 (c 0.11, CHCl3). IR (film): ν = 3469, 2930, 1743 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.07 (3H, s), 0.09 (3H, s), 0.90 (9H, s), 0.97 (3H, t, J = 7.5 Hz), 1.80 (1H, m), 2.02-2.51 (11H, m), 2.80 (2H, br t, J = 6.9 Hz), 3.62 (1H, m), 4.02 (1H, br t,
RI PT
J = 3.6 Hz), 5.26-5.52 (7H, m), 5.74-5.87 (2H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.4, –4.0, 14.5, 18.3, 20.8, 25.5, 25.9, 26.1, 26.6, 32.0, 33.7, 34.5, 72.5, 72.8, 75.2, 124.7, 124.9, 127.0, 129.6, 131.2, 132.4, 133.0, 135.2, 173.8. ESI-HRMS m/z calcd for
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C26H44NaO4Si [M+Na]+ 471.2901, found 471.2904.
3.2.20 (5Z,8R,9E,11R,12S,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (1)
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To a stirred solution of alcohol (8R)-27 (10 mg, 0.022 mmol) in CH2Cl2 (4 ml) was added Et3N (29 mg, 0.29 mmol), DMAP (8.6 mg, 0.070 mmol) and MsCl (13.4 mg, 0.12 mmol) successively at room temperature under argon atmosphere, and the mixture was stirred for 10 min at room temperature. The reaction mixture was quenched with water and extracted with ether. The combined organic layer was washed with water, saturated
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ammonium chloride solution, saturated NaHCO3 solution and brine. After drying with MgSO4, evaporation gave crude mesylate (8R)-28. Crude (8R)-28 was dissolved with dry THF (5 ml). After addition of hydrated TBAF (30 mg), the reaction mixture was stirred for
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2 h at room temperature. The reaction mixture was poured into water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then
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evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave an compound 1 as a colorless oil (7.4 mg, 0.022 mmol, 98%). nD16 = 1.5144. [α]D24 = +88 (c 0.22, MeOH). IR (film): ν = 2961, 1743, 1218, 1137, 969 cm–1. 1H NMR (500 MHz, CD3OD): δ (ppm) = 0.97 (3H, t, J = 7.5 Hz, 20-H), 1.76 (1H, m, 3-Ha), 2.03-2.30 (7H, m, 2-Ha, 3-Hb, 4-Ha, 7-Ha, 13-Ha, 19-H), 2.32-2.55 (4H, m, 2-Hb, 4-Hb, 7-Hb, 13-Hb), 2.74-2.86 (2H, m, 16-H), 3.12 (1H, dt, J = 4.5, 6.5 Hz, 12-H), 3.48 (1H, br dd, J = 7.0, 4.5 Hz, 11-H), 5.25-5.32 (2H, m, 8-H, 17-H), 5.35-5.52 (5H, m, 5-H, 6-H, 14-H, 15-H, 18-H), 5.76 (1H, ddd, J = 15.5, 7.0, 1.5 Hz, 10-H), 6.04 (1H, ddd, J = 15.5, 5.5, 1.0 Hz, 9-H). 13C NMR (125 23
ACCEPTED MANUSCRIPT MHz, CD3OD): δ (ppm) = 14.7, 21.5, 26.3, 26.7, 27.1, 27.5, 34.4, 35.3, 57.3, 59.4, 73.8, 124.9, 125.5, 127.3, 127.9, 132.0, 133.0, 135.3, 136.3, 175.6. ESI-HRMS m/z calcd for
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C20H28NaO3 [M+Na]+ 339.1931, found 339.1910.
3.2.21 (5Z,8S,9E,11R,12S,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (5)
In the same manner as the synthesis of 1 described above, (8S)-27 (195 mg, 0.434 mmol) was converted into 5 (125 mg, 0.368 mmol, 85%, colorless oil). nD16 = 1.5156.
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[α]D20 = –98 (c 0.10, MeOH). IR (film): ν = 2962, 1742, 1218, 1136, 969 cm–1. 1H NMR
(500 MHz, CD3OD): δ (ppm) = 0.96 (3H, t, J = 7.5 Hz, 20-H), 1.76 (1H, m, 3-Ha), 2.03-
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2.29 (7H, m, 2-Ha, 3-Hb, 4-Ha, 7-Ha, 13-Ha, 19-H), 2.32-2.55 (4H, m, 2-Hb, 4-Hb, 7-Hb, 13Hb), 2.73-2.85 (2H, m, 16-H), 3.13 (1H, dt, J = 4.5, 6.5 Hz, 12-H), 3.47 (1H, br dd, J = 6.5, 4.5 Hz, 11-H), 5.24-5.31 (2H, m, 8-H, 17-H), 5.35-5.52 (5H, m, 5-H, 6-H, 14-H, 15-H, 18H), 5.76 (1H, ddd, J = 15.5, 7.0, 1.5 Hz, 10-H), 6.03 (1H, ddd, J = 15.5, 5.5, 1.0 Hz, 9-H). C NMR (125 MHz, CD3OD): δ (ppm) = 14.7, 21.5, 26.3, 26.6, 27.1, 27.5, 34.4, 35.4,
13
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57.2, 59.4, 73.9, 124.9, 125.5, 127.2, 127.9, 132.0, 133.0, 135.2, 136.3, 175.6. ESI-HRMS m/z calcd for C20H28NaO3 [M+Na]+ 339.1931, found 339.1942.
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3.2.22 (5Z,8R,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-(1-ethoxyethoxy)icosa5,9,14,17-tetraen-8-olide [(8R)-29]
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To a stirred solution of alcohol (8R)-27 (7.5 mg, 0.017 mmol) in CH2Cl2 (4 ml) was added ethyl vinyl ether (1.0 ml, 10 mmol) and PPTS (1.4 mg, 0.0056 mmol), and the mixture was stirred for 50 min at room temperature. The reaction mixture was poured into saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was washed with water, saturated ammonium chloride solution, saturated NaHCO3 solution and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (20 : 1 hexanes/EtOAc) gave an compound (8R)-29 as a colorless oil (8.5 mg, 0.016 mmol, 98%). nD16 = 1.4848. [α]D18 = + 64.0 (c 0.17, CHCl3). IR (film): ν = 2956, 2929, 1746 cm–1. 24
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H NMR (300 MHz, CDCl3): δ (ppm) = 0.05-0.09 (6H, m), 0.89 (9H, br), 0.96 (3H, t, J =
7.8 Hz), 1.17, 1.19 (3H, two t, J = 6.9 Hz), 1.30, 1.33 (3H, two d, J = 5.4 Hz), 1.71-1.87 (1H, m), 2.01-2.52 (11H, m), 2.73-2.82 (2H, m), 3.41-3.80 (3H, m), 3.91-4.08 (1H, m) 4.69-4.76 (1H, m), 5.25-5.51 (7H, m), 5.72-5.84 (2H, m). ESI-HRMS m/z calcd for
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C30H52NaO5Si [M+Na]+ 543.3476, found 543.3452
3.2.23 (5Z,8S,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-(1-ethoxyethoxy)icosa-
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5,9,14,17-tetraen-8-olide [(8S)-29]
In the same manner as the synthesis of (8R)-29 described above, (8S)-27 (389 mg,
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0.867 mmol) was converted into (8S)-29 (374 mg, 0.718 mmol, 87%, colorless oil). nD18 = 1.4838. [α]D18 = –51.0 (c 0.24, CHCl3). IR (film): ν = 2956, 2930, 1746 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) 0.05 (3H, s), 0.08 (3H, s), 0.887, 0.891 (9H, two s), 0.96 (3H, t, J = 7.8 Hz), 1.17, 1.19 (3H, two t, J = 6.9 Hz), 1.29, 1.31 (3H, two d, J = 5.4 Hz), 1.731.87 (1H, m), 2.01-2.53 (11H, m), 2.73-2.79 (2H, m), 3.40-3.78 (3H, m), 4.00-4.10 (1H, m)
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4.69, 4.76 (1H, two q, J = 5.4 Hz), 5.25-5.54 (7H, m), 5.73-5.84 (2H, m). ESI-HRMS m/z calcd for C30H52NaO5Si [M+Na]+ 543.3476, found 543.3452.
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3.2.24 (5Z,8R,9E,11S,12S,14Z,17Z)-11-(1-Ethoxyethoxy)-12-hydroxyicosa-5,9,14,17tetraen-8-olide [(8R)-30]
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To a stirred solution of (8R)-29 (8.5 mg, 0.016 mmol) in THF (4 ml) was added hydrated TBAF (30 mg) and the mixture was stirred for 30 min at room temperature. The reaction mixture was poured into water and extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (2 : 1 hexanes/EtOAc) gave alcohol (8R)-30 as a colorless oil (6.1 mg, 0.015 mmol, 92%). nD16 = 1.4989. [α]D18 = +99 (c = 0.03, CHCl3). IR (film): ν = 3482, 2973, 1743 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.5 Hz), 1.18, 1.21 (3H, two t, J = 6.9 Hz), 1.32-1.34 (3H, m), 1.72-1.87 (1H, m), 2.01-2.54 25
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(11H, m), 2.78 (2H, br t, J = 5.7 Hz), 3.37-3.71 (3H, m), 3.78 (0.5H, t, J = 6.3 Hz), 3.96 (0.5H, t, J = 7.5 Hz), 4.69-4.74 (1H, m), 5.27-5.53 (7H, m), 5.62-5.87 (2H, m). ESI-HRMS
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m/z calcd for C24H38NaO5+ [M+Na] 429.2611, found 429.2655.
3.2.25 (5Z,8S,9E,11S,12S,14Z,17Z)-11-(1-Ethoxyethoxy)-12-hydroxyicosa-5,9,14,17tetraen-8-olide [(8S)-30]
In the same manner as the synthesis of (8R)-30 described above, (8S)-29 (364 mg,
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0.699 mmol) was converted into (8S)-30 (260 mg, 0.64 mmol, 91%, colorless oil). nD16 = 1.4989. [α]D18 = –90 (c 0.05, CHCl3). IR (film): ν = 3481, 2971, 2932, 1743 cm–1. 1H NMR
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(300 MHz, CDCl3): δ (ppm) = 0.96 (3H, t, J = 7.8 Hz), 1.17, 1.21 (3H, two t, J = 6.9 Hz), 1.31-1.34 (3H, m), 1.73-1.88 (1H, m), 2.03-2.54 (11H, m), 2.78 (2H, br), 3.37-3.70 (3H, m), 3.78 (0.5H, t, J = 6.6 Hz), 3.96 (0.5H, t, J = 7.2 Hz), 4.67-4.73 (1H, m), 5.28-5.53 (7H, m), 5.62-5.86 (2H, m). ESI-HRMS m/z calcd for C24H38NaO5 [M+Na]+ 429.2611, found
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429.2591.
3.2.26 (5Z,8R,9E,11S,12R,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (6) To a stirred solution of alcohol (8R)-30 (6.1 mg, 0.015 mmol) in CH2Cl2 (3 ml) was
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added Et3N (23.4 mg, 0.231 mmol), DMAP (9.4 mg, 0.77 mmol) and MsCl (0.008 ml, 0.103 mmol) successively at room temperature under argon atmosphere, and the mixture
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was stirred for 30 min at room temperature. The reaction mixture was quenched with water and extracted with ether. The combined organic layer was washed with water, saturated ammonium chloride solution, saturated NaHCO3 solution and brine, dried with MgSO4, and then evaporated to afford crude mesylate (8R)-31. Crude (8R)-31 was dissolved in THF (5 ml), and treated with 0.5 N HCl (5 ml). After stirring for 3 h at room temperature, the reaction mixture was neutralized with saturated NaHCO3 solution and extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated to give crude alcohol (8R)-32. Crude (8R)-32 was dissolved in THF (2 ml) and 26
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treated with hydrated TBAF (19 mg). After stirring for10 min at room temperature, the reaction mixture was poured into water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave epoxide 6 as a colorless oil (2.6 mg,
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0.0077 mmol, 51%). nD16 = 1.5153. [α]D24 = +96 (c 0.18, MeOH). Its IR and NMR spectra were identical with those of 5. ESI-HRMS m/z calcd for C20H28NaO3 [M+Na]+ 339.1931,
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found 339.1925.
3.2.27 (5Z,8S,9E,11S,12R,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (7)
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In the same manner as the synthesis of 6 described above, (8S)-30 (256 mg, 0.63 mmol) was converted into 7 (140 mg, 0.182 mmol, 29%, colorless oil). nD16 = 1.5151. [α]D20 = –88 (c 0.11, MeOH). Its IR and NMR spectra were identical with those of 1. ESIHRMS m/z calcd for C20H28NaO3 [M+Na]+ 339.1931, found 339.1933.
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3.3. Biological studies
Cytotoxicity against human cervical carcinoma HeLa cells and human lymphoblastoid namalva cells were determined by a colorimetric assay using WST-8 [2-(2-
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methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt]. Cells were cultured in DMEM medium (Wako Pure Chemical
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Industries, Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (GIBCO, Carlsbad, CA), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 °C in a humidified incubator under a 5% CO2 atmosphere. The 384-well plates were seeded with aliquots of a 20-µL medium containing 1.0 × 103 cells per well and were incubated overnight before being treated with compounds at various concentrations for 48 h. Plates were incubated for 1 h at 37 °C after the addition of 2-µL WST-8 reagent solution (Cell Counting Kit; Dojindo, Kumamoto, Japan) per well. The absorption of formazan dye was measured at 450 nm. The vehicle solvent (DMSO) was used as a negative control. 27
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Acknowledgement The authors sincerely thank Professor J. H. Jung, Pusan National University, for a
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kind gift of the spectral charts of natural topsentolide A1. We sincerely thank Mr. Y. Osano and Mr. T. Imaoka, University of Tokyo, for mass spectroscopy. This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of
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Science to K.I (Grant Number 22580115).
References
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1. Niwa, H.; Wakamatsu, K.; Yamada, K. Tetrahedron Lett. 1989, 30, 4543-4546. 2. Papendorf, O.; König, G. M.; Wright, A. D.; Chorus, I.; Oberemm, A. J. Nat. Prod. 1997, 60, 1298-1300.
3. Seo, Y.; Cho, K. W.; Rho, J.-R.; Shin, J.; Kwon, B.-M.; Bok, S.-H.; Song, J.-I. Tetrahedron 1996, 52, 10583-10596.
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4. Ishigami, K.; Motoyoshi, H.; Kitahara, T. Tetrahedron Lett. 2000, 41, 8897-8901. 5. Motoyoshi, H.; Ishigami, K.; Kitahara, T. Tetrahedron 2001, 57, 3899-3908. 6. Takahashi, T.; Watanabe, H.; Kitahara, T. Heterocycles 2002, 58, 99-104.
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7. Takahashi, T.; Takahashi, T.; Watanabe, H.; Kitahara, T. Abstracts of Papers, 44th Symposium on the Chemistry of Natural Products, Tokyo, Japan, 2002, 1-6.
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8. Ishigami, K. Biosci. Biotechnol. Biochem. 2009, 73, 971-979. 9. Luo, X.; Li, F.; Hong, J.; Lee, C.-O.; Sim, C. J.; Im, K. S.; Jung, J. H. J. Nat. Prod. 2006, 69, 567-571.
10. Kobayashi, M.; Ishigami, K.; Watanabe, H. Tetrahedron Lett. 2010, 51, 2762-2764. 11. Mori, K.; Takigawa, T.; Matsuo, T. Tetrahedron 1979, 35, 933-940. 12. Saito, S.; Hasegawa, T.; Inaba, M.; Nishida, R.; Fujii, T.; Nomizu, S.; Moriwake, T. Chem. Lett. 1984, 13, 1389-1392. 13. Kojima, K.; Koyama, K.; Amemiya, S. Tetrahedron 1985, 41, 4449-4462. 28
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14. McDonald, C.; Holcomb, H.; Kennedy, K.; Kirkpatrick, E.; Leathers, T.; Vanemon, P. J. Org. Chem. 1989, 54, 1213-1215. 15. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn.
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1979, 52, 1989-1993. 16. Blanchette, M. A.; Choy, W.; Davis, J. T.; Essenfeld, A. P.; Masamune, S.; Roush, W. R.; Sakai, T. Tetrahedron Lett. 1984, 25, 2183-2186. 17. Luche, J.-L. J. Am. Chem. Soc. 1978, 100, 2226-2227.
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18. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 40924096.
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19. Towada, R.; Kurashina, Y.; Kuwahara, S. Tetrahedron Lett. 2013, 54, 6878-6881.
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S0040-4020(15)30042-9
DOI:
10.1016/j.tet.2015.09.013
Reference:
TET 27110
To appear in:
Tetrahedron
Received Date: 21 August 2015 Revised Date:
3 September 2015
Accepted Date: 3 September 2015
Please cite this article as: Ishigami K, Kobayashi M, Takagi M, Shin-ya K, Watanabe H, Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product, Tetrahedron (2015), doi: 10.1016/j.tet.2015.09.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract (K. Ishigami et al.)
Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product *
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Ken Ishigami , Munetaka Kobayashi, Motoki Takagi, Kazuo Shin-ya, Hidenori Watanabe
1
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Synthesis of marine oxylipin topsentolide A1 and its stereoisomers, and determination of the absolute configuration of the natural product
*
Ken Ishigamia , Munetaka Kobayashia, Motoki Takagib, Kazuo Shin-yac, Hidenori
a
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Watanabea
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
b
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Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Medical-industrial Translational Research Center, Fukushima Medical University, 11-
c
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23 Sakaemachi, Fukushima, Fukushima 960-8031, Japan
National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi,
Koto-ku, Tokyo 135-0064, Japan
Abstract
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Four possible stereoisomers of topsentolide A1, a cytotoxic oxylipin against human solid tumor cell lines, were efficiently synthesized in a stereoselective manner in order to determine the stereochemistry of natural product. The absolute configuration of
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topsentolide A1 was determined to be 8R,11R,12S by comparing NMR spectra and specific rotations of the synthetic isomers and the natural product. Cytotoxicity of the
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synthetic isomers was also examined.
Key Words
topsentolide A1; 9-membered lactone; determination of the absolute configuration;
marine oxylipin
1. Introduction *
Corresponding author. Fax: +81-3-5841-8019; e-mail: [email protected]
2
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A number of oxylipins have been isolated as secondary metabolites of marine organisms, such as cyanobacteria, alga, hydrozoa and sponges. These oxylipins have interesting activities and synthetic studies on them are meaningful, as well as biological studies, because their bioactivities are frequently concerned with structure. Halicholactone
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(2, isolated from Halichondoria okadai)1 is an inhibitor of lipoxygenase, mueggelone (3, isolated from Aphanizomenon flos-aquae)2 is an inhibitor of fish development, and
solandelactone D (4, isolated from Solanderia secunda)3 is an inhibitor of farnesyl protein
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transferase. Previously in our group, the syntheses of these marine oxylipins had been
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Figure 1. Bioactive marine oxylipins.
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achieved in stereoselective manner and the results had been already reported.4-8
In 2006, Jung’s group isolated topsentolide A1 (1)9 from the extract of a marine
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sponge Topsentia sp. as a cytotoxic oxylipin against human solid tumor cell lines. Topsentolide A1 has a nine-membered lactone with an epoxide side chain. Stereochemistry
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of the epoxide was proposed to be cis by spectroscopic analysis, however, the relative as well as the absolute configuration of the three stereocenters remained unknown. Therefore, we undertook the synthesis of possible stereoisomers of topsentolide A1 in order to determine the absolute configuration of the natural product. We have already published a rapid communication10, in which we succeeded in the enantioselective synthesis of the enantiomer of topsentolide A1, (8S,11S,12R)-isomer, and determined the stereochemistry of natural product to be 8R,11R,12S as shown in Figure 1. Now we achieved the synthesis of natural form of topsentolide A1 and here, we wish to report a full account of our work. 3
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2. Results and discussion Our synthetic strategy of all the four possible stereoisomers of topsentolide A1 (1, 5,
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6, 7) is illustrated in Scheme 1. We decided to utilize a key intermediate (8R)- and (8S)-A, which could be transformed into both of (11R,12S)-isomers (1 and 5) and (11S,12R)-
isomers (6 and 7) stereoselectively via epoxides formation, where the stereochemistry of the epoxide could be controlled by changing elimination direction.4,5 Compound A would
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be constructed by Horner-Wadsworth-Emmons (HWE) reaction of a side chain moiety B with a lactone moiety C. Both of the lactones (S)- and (R)-C would be prepared from
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optically active hydroxycarboxylic acid D via Yamaguchi lactonization or Mitsunobu lactonization. The phosphonate B and the hydroxycarboxylic acid D would be derived from
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the known aldehyde 8,11,12 which could be easily prepared from L-malic acid.
Scheme 1. Retrosynthetic analysis of four possible stereoisomers of topsentolide A1.
Synthesis of the phosphonate 17 (= B) is shown in Scheme 2. The known aldehyde
811,12 was subjected to Wittig reaction with the known phosphonium salt 913 to afford Zolefin 10 selectively. Z-Selectivity was confirmed to be >98% by GC analysis. The Z-olefin 10 was simply transformed into the corresponding primary alcohol 14 in four steps, which 4
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was then oxidized to give aldehyde 15. Oxidation of the aldehyde 15 with NaClO2 and subsequent esterification using CH2N2 afforded the desired methyl ester 16 only in poor yield (~20%), and undesired migration of TBS group was observed. On the other hand,
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oxidation of 15 with NIS14 in methanol proceeded smoothly to give the ester 16 in good yield without any side reactions. Finally, the ester 16 was reacted with anion of dimethyl methylphosphonate to afford the desired β-ketophosphonate 17, one of the substrates for
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HWE reaction.
Scheme 2. Synthesis of the phosphonate 17. (a) KHMDS, THF, –78 °C to rt, 84%; (b) 1N
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HCl, THF, rt, 90%; (c) PivCl, Pyr., 0 °C to rt, 89%; (d) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C to rt, 96%; (e) DIBAL, CH2Cl2, –50 °C, 97%; (f) (COCl)2, DMSO, Et3N, CH2Cl2, –78 °C to rt, 96%; (g) NIS, K2CO3, MeOH, rt, 78%; (h) (MeO)2P(O)Me, n-BuLi, THF, –78 °C,
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52%.
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Synthesis of the lactone 25 (= C), another substrate for HWE reaction, is shown in Scheme 3. Wittig reaction of the known aldehyde 811,12 and a commercially available phosphonium salt and subsequent treatment with CH2N2 afforded Z-olefin 19 selectively (E-isomer was not observed in 1H and 13C NMR spectra), which was transformed into the corresponding hydroxycarboxylic acid 22 in four steps. As described in synthetic strategy, we supposed both of the lactone (S)- and (R)-25 would be prepared from hydroxycarboxylic acid 22 via Yamaguchi lactonization and Mitsunobu lactonization, respectively. Yamaguchi lactonization15 of 22 successfully provide nine-membered lactone 5
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23 in satisfactory yield, together with a small amount of a dimeric lactone (3%). After the removal of PMB group, 24 was oxidized into the desired aldehyde (S)-25. On the other hand, Mitsunobu lactonization of 22 afforded the desired nine-membered lactone only in
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poor yield (~20%) even under high dilution conditions. So enantiomeric (R)-25 was prepared from ent-8 in the same manner as the synthesis of (S)-25 via Yamaguchi
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lactonization.
Scheme 3. Synthesis of the lactone 25. (a) (4-carboxybutyl)triphenylphosphonium bromide, NaHMDS, THF, –78 °C to rt; (b) CH2N2, Et2O, MeOH, 0 °C, 69% in 2 steps; (c)
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1N HCl, THF, rt, 97%; (d) PMBOC(NH)CCl3, TsOH·H2O, CH2Cl2, rt, 63%; (e) LiOH·H2O, H2O, MeOH, rt, 98%; (f) 2,4,6-trichlorobenzoyl chloride, Et3N, THF, rt, then DMAP, toluene, reflux, 81%; (g) DDQ, H2O, CH2Cl2, rt, 87%; (h) (COCl)2, DMSO, Et3N,
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CH2Cl2, –78 °C to rt, 69%.
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Now that both of the side chain moiety 17 and the lactone moieties (R)- and (S)-25 were obtained enantioselectively, HWE reaction was examined. At first, syntheses of (8R)isomers 1 and 6 are shown in Scheme 4. HWE reaction of the β-ketophosphonate 17 and the aldehyde (R)-25 under Masamune conditions employing DBU-LiCl16 proceeded smoothly to give unsaturated ketone (8R)-26, with excellent E selectivity without any epimerization, and other isomers were not observed in 1H NMR spectrum. Reduction of the ketone (8R)-26 using Luche conditions17 successfully proceeded under Felkin-Anh control, and stereoselectively afforded the key intermediate (8R)-27, a precursor for epoxide 6
ACCEPTED MANUSCRIPT formation. Stereochemistry at C-11 was confirmed to be S by modified Mosher’s method18 after conversion to the corresponding MTPA esters (Table 1). In order to construct both of α- and β-epoxide from (8R)-27, the stereochemistry of
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the epoxide was controlled as described below by changing elimination direction.4,5 At first, alcohol (8R)-27 was converted to the corresponding mesylate (8R)-28, which was
treated with TBAF to provide β-epoxide, whereby the synthesis of natural topsentolide A1 [(8R,11R,12S)-isomer (1)] was completed. On the other hand, alcohol (8R)-27 was
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converted to another mesylate (8R)-31 by changing the position of the leaving group. After removing its EE group, mesylate was treated with base to afford α-epoxide, (8R,11S,12R)-
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isomer (6), successfully. Next, (8S)-isomers 5 and 7 were synthesized in the same manner as for (8R)-isomers. HWE reaction of the β-ketophosphonate 17 and the aldehyde (S)-25 was followed by stereoselective reduction to give alcohol (8S)-27. Stereochemistry at C-11 was confirmed to be S by modified Mosher’s method18 after conversion to the corresponding MTPA esters (Table 1). The alcohol (8S)-27 was transformed into both of β-
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epoxide 5 and α-epoxide 7 in two and five steps, respectively.
Scheme 4. Synthesis of possible stereoisomers of topsentolide A1. (a) DBU, LiCl, MeCN, rt, 75%; (b) NaBH4, CeCl3·7H2O, MeOH, rt, 91%; (c) MsCl, Et3N, DMAP, CH2Cl2, rt; (d) TBAF, THF, rt, 98% in 2 steps; (e) EVE, PPTS, CH2Cl2, rt, 98%; (f) TBAF, THF, rt, 92%; (g) 0.5N HCl, THF, rt; (h) TBAF, THF, rt, 51% in 3 steps. 7
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Table 1. 1H NMR (300 MHz, CDCl3) data for (S)- and (R)-MTPA esters of (8R)- and
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(8S)-27.
With the four possible stereoisomers of topsentolide A1 in hand, we analyzed their specific rotations and NMR spectra carefully in order to confirm the absolute configuration of the natural product. About the specific rotations of the synthetic stereoisomers, [α]D24 –
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98 (c 0.10, MeOH) for 5 and [α]D24 –88 (c 0.11, MeOH) for 7, showed opposite sign to [α]D24 +59.4 (c 0.11, MeOH)9 for the natural material. On the other hand, stereoisomers 1 and 6 showed identical sign of the specific rotation to the natural product [1: [α]D24 +88 (c
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0.22, MeOH), 6: [α]D24 +96 (c 0.18, MeOH)], and it was thought that either (8R)-isomers 1 or 6 should have the same stereochemistry as natural topsentolide A1. As for the 13C NMR,
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there was no difference between the diastereomers (1 and 6) to distinguish them. In the 1H NMR, slight differences of the chemical shifts and the shapes of peaks were observed between the diastereomers. 1H NMR spectra of natural topsentolide A1 and synthesized (8R)-isomers 1 and 6 were partially shown in Figure 2. 1H NMR spectrum of (8R,11R,12S)isomer (1) was completely identical with that of natural topsentolide A1, while some signals of (8R,11S,12R)-isomer (6) were not identical with those of natural product [especially at 2.74-2.86 ppm (16-H), 2.32-2.42 ppm (2-H and 13-Hb), 2.23-2.30 (13-Ha) and 2.03-2.12 ppm (3-H, 4-H and 19-H)]. On the basis of these results, the (8R,11R,12S)-stereochemistry 8
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for natural topsentolide A1 (1) was unambiguously determined as shown in Figure 3. Actually, Jung’s group also isolated topsentolide C1 (33) and C2 (34) together with 1.9 They suspected these methyl ether analogues to be artifacts formed during the process of
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extraction with MeOH, and determined the absolute configuration at C-12 as S by Mosher’s method. Their determination of stereochemistry at C-12 supports our results. In addition, Kuwahara’s group recently reported the synthesis and the stereochemical assignment of
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topsentolide C219, which also fully supports our results.
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Figure 2. Partial 1H NMR spectra of natural topsentolide A1 and synthesized (8R)-isomers
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(500 MHz, in CD3OD).
Figure 3. Absolute configuration of topsentolide A1.
Having succeeded in the total syntheses of four stereoisomers and in the determination of the absolute configuration of natural topsentolide A1, our next interest 9
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came to structure–activity relationship of topsentolide A1. Jung’s group reported that topsentolide A1 showed moderate and broad cytotoxicity against human solid tumor cell lines. We examined the cytotoxic activity of topsentolide A1 and its stereoisomers against
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HeLa cells and human lymphoblastoid namalva cells. Unfortunately, these compounds exhibited no activity against HeLa cells at less than 1 mM. Against human lymphoblastoid namalva cells, topsentolide A1 and its stereoisomers showed weak cytotoxicity, but
cytotoxicity of four stereoisomers were much the same (IC50 1: 137 µM, 5: 198 µM, 6: 148
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µM, 7: 182 µM).
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In summary, we succeeded in the synthesis of topsentolide A1 (1) and its stereoisomers (5-7) efficiently, and determined the stereochemistry of natural product to be 8R,11R,12S. Synthetic topsentolide A1 showed week cytotoxicity as well as other stereoisomers. We wish our study on these oxylipins, which have various activities, would
3. Experimental 3.1. General
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offer any useful information for further investigation on the related fields.
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Optical rotations were recorded with a JASCO DIP-1000 polarimeter. IR spectra were measured with a JASCO FT/IR-230 spectrophotometer. 1H and 13C NMR were recorded on JEOL JNM AL300 or JEOL JNM GSX500. Chemical shifts (δ) were
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referenced to the residual solvent peaks as the internal standard (CDCl3: δH = 7.26, δC = 77.23, CD3OD: δH = 3.30, δC = 49.00). Refractive indexes were measured with an Atago 1T refractometer. Mass spectra were recorded on JEOL JMS SX102. Column chromatography was performed using Kanto silica gel 60N (0.060-0.200 mm). TLC was carried out on Merck glass plates precoated with silica gel 60 F254 (0.25 mm).
3.2. Synthetic studies 10
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3.2.1 (S)-2,2-Dimethyl-4-[(2Z,5Z)-octa-2,5-dienyl]-1,3-dioxolane (10) To a cooled (–78 °C) suspension of phosphonium salt 913 (11.8 g, 25.1 mmol) in dry THF (100 ml) under argon atmosphere was added KHMDS (50 ml, 0.5 M in toluene, 25
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mmol) and the mixture was stirred for 10 min. To the resulting solution was added a solution of aldehyde 8 (3.60 g, 25.0 mmol) in THF (10 ml) via cannula at –78 °C. The
mixture was stirred for 30 min at –78 °C, then gradually warmed to room temperature, and stirred for 2 h at ambient temperature. The reaction mixture was quenched with water and
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extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1
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hexanes/EtOAc) gave olefin 10 as a colorless oil (4.41 g, 21.0 mmol, 84%). nD18 = 1.4591. [α]D20 = +25.4 (c 1.1, CHCl3). IR (film): ν = 2965, 2934, 2874, 1369, 1216, 1065 cm-1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.2 Hz), 1.36 (3H, s), 1.43 (3H, s), 2.07 (2H, m), 2.33 (1H, m), 2.43 (1H, m), 2.80 (2H, br t, J = 6.9 Hz), 3.56 (1H, dd, J = 7.8, 7.2 Hz), 4.03 (1H, dd, J = 7.8, 6.0 Hz), 4.13 (1H, m), 5.26-5.53 (4H, m). 13C NMR (125
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MHz, CDCl3): δ (ppm) = 14.4, 20.8, 25.8, 25.9, 27.1, 31.7, 69.2, 75.8, 109.1, 124.3, 126.9, 131.3, 132.4. ESI-HRMS m/z calcd. for C13H22NaO2 [M+Na]+ 233.1512, found 233.1517.
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3.2.2 (2S,4Z,7Z)-Deca-4,7-diene-1,2-diol (11) To a stirred solution of compound 10 (10.5 g, 50.0 mmol) in THF (150 ml) was added
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1 N HCl (50 ml) and the mixture was stirred at room temperature for 15 h. The reaction mixture was quenched with saturated NaHCO3 solution and extracted with EtOAc. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (2 : 1 hexanes/EtOAc) gave diol 11 as a colorless oil (7.64 g, 44.9 mmol, 90%). nD19 = 1.4868. [α]D22 = +13.9 (c 0.5, CHCl3). IR (film): ν = 3333, 2964, 1068 cm-1; 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.8 Hz,), 1.88 (2H, br), 2.02-2.11 (2H, m), 2.20-2.37 (2H, m), 2.81 (2H, br t, J = 7.2 Hz), 3.49 (1H, dd, J = 11.1, 6.9 Hz), 3.69 (1H, dd, J = 11.1, 3.3 Hz), 3.76 (1H, m), 5.27-5.61 (4H, m). 13C 11
ACCEPTED MANUSCRIPT NMR (125 MHz, CDCl3): δ (ppm) = 14.4, 20.8, 25.8, 31.5, 66.5, 72.0, 124.7, 126.8, 132.0, 132.5. ESI-HRMS m/z calcd. for C10H18NaO2 [M+Na]+ 193.1199, found 193.1192.
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3.2.3 (2S,4Z,7Z)-2-Hydroxydeca-4,7-dienyl pivalate (12) To a cooled (0 °C) solution of diol 11 (2.46 g, 14. 5 mmol) in pyridine (60 ml) was added PivCl (2.1 ml, 18 mmol) under argon atmosphere, and the mixture was stirred at
room temperature for 1.5 h. The reaction mixture was quenched with water and extracted
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with Et2O. The combined organic layer was washed with saturated NaHCO3 solution, water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel
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(25 : 1 hexanes/EtOAc) gave ester 12 as a colorless oil (3.27 g, 12.9 mmol, 89%). nD18 = 1.4657. [α]D24 = +8.3 (c 0.61, CHCl3). IR (film): ν = 3445, 2964, 1732, 1160 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.5 Hz), 1.23 (9H, s), 2.02-2.12 (3H, m), 2.32 (2H, br t, J = 6.9 Hz), 2.81 (2H, br t, J = 7.2 Hz), 3.88 (1H, m), 4.02 (1H, dd, J = 11.1, 6.6 Hz), 4.16 (1H, dd, J = 11.1, 2.1 Hz), 5.27-5.58 (4H, m). 13C NMR (125 MHz, CDCl3): δ
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(ppm) = 14.4, 20.8, 25.8, 27.4, 31.7, 39.1, 68.0, 70.0, 124.3, 126.7, 132.1, 132.5, 178.9. ESI-HRMS m/z calcd. for C15H26NaO3 [M+Na]+ 277.1774, found 277.1777.
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3.2.4 (2S,4Z,7Z)-2-(t-Butyldimethylsilyloxy)deca-4,7-dienyl pivalate (13) To a cooled (0 °C) solution of alcohol 12 (2.71 g, 10.7 mmol) in dry CH2Cl2 (100
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ml) was added 2,6-lutidine (3.3 g, 31 mmol) and TBSOTf (4.01 g, 15.2 mmol) at room temperature under argon atmosphere, and the mixture was stirred for 10 min. The reaction mixture was quenched with water and extracted with CH2Cl2. The combined organic layer was dried with MgSO4, and then evaporated. Purification of the residue on silica gel (50 : 1 hexanes/EtOAc) gave silyl ether 13 as a colorless oil (3.78 g, 10.3 mmol, 96%). nD18 = 1.4553. [α]D23 = +7.9 (c 0.45, CHCl3). IR(film): ν = 3011, 2959, 1731, 1156 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.07 (6H, s), 0.88 (9H, s), 0.97 (3H, t, J = 7.5 Hz), 1.23 (9H, s), 2.01-2.09 (2H, m), 2.24-2.38 (2H, m), 2.78 (2H, br t, J = 6.6 Hz), 3.86-4.03 (3H, m), 12
ACCEPTED MANUSCRIPT 5.27-5.47 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.45, –4.4, 14.5, 18.2, 20.8, 25.9, 26.0, 27.4, 32.8, 39.0, 67.8, 70.3, 125.1, 127.1, 130.8, 132.3, 178.7. ESI-HRMS m/z
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calcd. for C21H40NaO3Si [M+Na]+ 391.2639, found 391.2628.
3.2.5 (2S,4Z,7Z)-2-(t-Butyldimethylsilyloxy)deca-4,7-dien-1-ol (14)
To a cooled (–50 °C) solution of ester 13 (3.78 g, 10.3 mmol) in dry CH2Cl2 (100 ml) was added DIBAL-H (1.03 M in hexane, 22 ml, 23 mmol) under argon atmosphere, and
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the mixture was stirred for 20 min. To the resulting solution was added saturated potassium sodium tartrate solution, and the mixture was stirred 3 h at room temperature. The resulting
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biphasic mixture was extracted with Et2O and the combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave alcohol 14 as a colorless oil (2.82 g, 9.92 mmol, 97%). nD17 = 1.4666. [α]D25 = +20.5 (c 0.48, CHCl3). IR (film): ν = 2957, 1255, 1105 cm–1. 1
H NMR (300 MHz, CDCl3): δ (ppm) = 0.10 (6H, s), 0.91 (9H, s), 0.97 (3H, t, J = 7.2 Hz),
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1.65 (1H, br), 2.07 (2H, qui, J = 7.2 Hz), 2.24-2.38 (2H, m), 2.79 (2H, br t, J = 6.9 Hz), 3.44 (1H, dd, J = 11.1, 5.4 Hz), 3.56 (1H, dd, J = 11.1, 3.9 Hz), 3.77 (1H, m), 5.27-5.47 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.4, -4.2, 14.5, 18.3, 20.8, 26.0, 26.1,
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31.2, 66.2, 72.9, 125.1, 127.0, 130.8, 132.3. ESI-HRMS m/z calcd. for C16H32NaO2Si
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[M+Na]+ 307.2064, found 307.2078.
3.2.6 (2S,4Z,7Z)-2-(t-Butyldimethylsilyloxy)deca-4,7-dienal (15) To a cooled (–78 °C) solution of oxalyl chloride (1.5 ml, 18 mmol) in CH2Cl2 (10
ml) was added dimethyl sulfoxide (2.49 ml, 35.1 mmol) under argon atmosphere, and the mixture was stirred for 20 min. To the resulting solution was added a solution of alcohol 14 (2.49 g, 8.76 mmol) in dry CH2Cl2 (1 ml), and the mixture was stirred 20 min at –78 °C. After addition of Et3N (7.34 ml, 52.6 mmol), the reaction mixture was stirred for 50 min while gradually warming to room temperature. The reaction mixture was quenched with 13
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saturated NaHCO3 solution and extracted with Et2O. The combined organic layer was washed with saturated NaHCO3 solution, water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (20 : 1 hexanes/EtOAc) gave aldehyde 15 as a colorless oil (2.38 g, 8.42 mmol, 96%). nD19 = 1.4646. [α]D22 = –18.5 (c 1.0, CHCl3).
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IR (film): ν = 3011, 2957, 2931, 1738, 1254, 1112 cm–1. 1H NMR (300 MHz, CDCl3): δ
(ppm) = 0.08 (3H, s), 0.10 (3H, s), 0.91 (9H, s), 0.97 (3H, t, J = 7.8 Hz), 2.00-2.09 (2H, m), 2.43 (2H, m), 2.78 (2H, br), 4.01 (1H, dt, J = 1.5, 6.3 Hz), 5.26-5.53 (4H, m), 9.61 (1H, d, J
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= 1.5 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.6, –4.5, 14.5, 18.4, 20.8, 25.9, 26.0, 31.1, 77.7, 123.7, 126.8, 131.7, 132.5, 204.1. ESI-HRMS m/z calcd. for C16H30NaO2Si
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[M+Na]+ 305.1907, found 305.1889.
3.2.7 Methyl (2S,4Z,7Z)-2-(t-butyldimethylsilyloxy)deca-4,7-dienoate (16) To a solution of NIS (3.90 g, 17.3 mmol) in methanol (90 ml) was added potassium carbonate (2.67 g, 19.3 mmol) and the mixture was stirred for 20 min at room temperature.
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To the resulting suspension was added a solution of aldehyde 15 (2.44 g, 8.64 mmol) in methanol (20 ml), and the mixture was stirred 2 h at room temperature. The resulting mixture was quenched with aqueous sodium thiosulfate solution and extracted with Et2O.
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The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave ester 16 as
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a colorless oil (2.11 g, 6.76 mmol, 78%). nD17 = 1.4618. [α]D24 = –1.5 (c 0.82, CHCl3). IR (film): ν = 3011, 2956, 2858, 1759, 1136 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.06 (3H, s), 0.08 (3H, s), 0.90 (9H, s), 0.97 (3H, t, J = 7.8 Hz), 2.01-2.10 (2H, m), 2.50 (2H, m), 2.78 (2H, br), 3.72 (3H, s,), 4.24 (1H, t, J = 6.3 Hz), 5.26-5.53 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –5.0, –4.8, 14.5, 18.5, 20.8, 25.8, 25.9, 33.5, 52.0, 72.4, 124.4, 127.1, 131.4, 132.3, 174.0. ESI-HRMS m/z calcd. for C17H32NaO3Si [M+Na]+ 335.2013, found 335.1994.
14
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3.2.8 Dimethyl (3S,5Z,8Z)-3-(t-butyldimethylsilyloxy)-2-oxoundeca-5,8-dienylphosphonate (17) To a cooled (–78 °C) solution of dimethyl methylphosphonate (360 mg, 2.90 mmol)
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in dry THF (30 ml) was added n-BuLi (2.64 M in hexane, 1.1 ml, 2.9 mmol) under argon atmosphere and the mixture was stirred for 15 min. To the resulting mixture was added a solution of ester 16 (880 mg, 2.82 mmol) in dry THF (3 ml), and the mixture was stirred for 40 min at –78 °C. The resulting mixture was quenched with saturated ammonium chloride
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solution and extracted with EtOAc. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (1 : 1
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hexanes/EtOAc) gave phosphonate 17 as a colorless oil (0.62 g, 1.48 mmol, 52%). nD19 = 1.4728. [α]D24 = +12.9 (c 1.0, CHCl3). IR (film): ν = 3445, 3274, 3010, 2957, 2857, 1724, 1257 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.08 (6H, s), 0.92 (9H, s), 0.97 (3H, t, J = 7.5 Hz), 2.01-2.08 (2H, m), 2.39 (1H, m), 2.49 (1H, m), 2.76 (2H, br), 3.23 (1H, dd, J = 19.8, 15.3 Hz) , 3.29 (1H, dd, J = 19.8, 15.3 Hz), 3.79 (6H, d, J = 11.1 Hz), 4.17 (1H, t, J =
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6.0 Hz), 5.23-5.53 (4H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.7, –4.6, 14.5, 18.3, 20.8, 25.8, 26.0, 32.5, 35.5 (d, J = 135 Hz), 53.2 (d, J = 6 Hz), 78.7, 123.7, 126.8, 131.7, 132.5, 204.7. ESI-HRMS m/z calcd. for C19H37NaO5PSi [M+Na]+ 427.2040, found
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427.2060.
AC C
3.2.9 Methyl (Z)-7-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]hept-5-enoate (19) (a) (S)-19: To a cooled (0 °C) suspension of (4-carboxybutyl)triphenylphosphonium
bromide (22.3 g, 50.3 mmol) in dry THF (200 ml) under argon atmosphere was added NaHMDS (1.0 M in THF, 100 ml, 100 mmol) over 20 min, and the mixture was stirred for 20 min at 0 °C. To the resulting solution was added a solution of aldehyde 8 (7.20 g, 50.0 mmol) in dry THF (20 ml) at –78 °C. The mixture was stirred for 1.5 h, gradually warmed to room temperature, and stirred for 2 h. Water and ether were added to the reaction mixture, and it was extracted with water. The combined aqueous layer was saturated with 15
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ammonium sulfate and extracted with EtOAc. The combined organic layer was dried with MgSO4, and then evaporated to give crude carboxylic acid 18. Crude 18 was dissolved in methanol and treated with a solution of CH2N2 in ether (prepared from N-nitroso-N-
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methylurea). The reaction mixture was quenched by acetic acid, neutralized with saturated NaHCO3 solution, and extracted with ether. The combined organic layer was washed with saturated NaHCO3 solution and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (4 : 1 hexanes/EtOAc) gave ester (S)-19 as a colorless oil (8.30
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g, 34.3 mmol, 69%). nD20 = 1.4558. [α]D24 = +23 (c 0.10, CHCl3). IR (film): ν = 2958, 2950, 2873, 1739, 1218, 1063 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.35 (3H, s), 1.42
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(3H, s,), 1.70 (2H, qui, J = 6.9 Hz), 2.09 (2H, q, J = 6.9 Hz), 2.21-2.42 (4H, m), 3.55 (1H, dd, J = 7.8, 6.9 Hz), 3.67 (3H, s), 4.03 (1H, dd, J = 7.8, 6.0 Hz), 4.11 (1H, m), 5.43 (1H, dt, J = 10.5, 6.9 Hz) 5.47 (1H, dt, J = 10.5, 6.9 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 24.9, 25.8, 26.9, 27.1, 31.7, 33.6, 51.7, 69.2, 75.8, 109.1, 125.3, 131.7, 174.2. ESI-HRMS m/z calcd. for C13H22NaO4 [M+Na]+ 265.1410, found 265.1366.
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(b) (R)-19: In the same manner as the synthesis of (S)-19 described above, ent-8 (10.8 g, 75.0 mmol) was converted into (R)-19 (12.2 g, 50.6 mmol, 67%, colorless oil). nD20 = 1.4557. [α]D21 = –24 (c 0.06, CHCl3). Its IR and NMR spectra were identical with those of
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(S)-19. ESI-HRMS m/z calcd. for C13H22NaO4 [M+Na]+ 265.1410, found 265.1439.
AC C
3.2.10 Methyl (5Z,8S)-8,9-dihydroxynon-5-enoate (20) (a) (S)-20: To a stirred solution of compound (S)-19 (2.63 g, 10.9 mmol) in dry THF
(20 ml) was added 1 N HCl (5 ml) and the mixture was stirred at room temperature for 18 h. The resulting mixture was neutralized with saturated NaHCO3 solution and extracted with EtOAc. The combined organic layer was dried with MgSO4 and then evaporated. Purification of the residue on silica gel (1 : 1 hexanes/EtOAc) gave diol (S)-20 as a colorless oil (2.14 g, 10.6 mmol, 97%). nD20 = 1.4770. [α]D23 = +5.6 (c 0.02, CHCl3). IR (film). ν = 3401, 2950, 1738, 1220, 1034 cm-1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 16
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1.71 (2H, qui, J = 7.2 Hz), 1.79 (2H, br), 2.11 (2H, q, J = 7.2 Hz), 2.17-2.31 (2H, m), 2.33 (2H, t, J = 7.2 Hz), 3.49 (1H, dd, J = 11.1, 6.6 Hz), 3.67 (3H, s), 3.68 (1H, dd, J = 11.1, 2.7 Hz), 3.75 (1H, m), 5.41-5.57 (2H, m). 13C NMR (125 MHz, CDCl3) δ (ppm) = 24.9, 26.8, 31.5, 33.6, 51.8, 66.4, 72.0, 125.9, 132.2, 174.5. ESI-HRMS m/z calcd for C10H18NaO4
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[M+Na]+ 225.1097, found 225.1104.
(b) (R)-20: In the same manner as the synthesis of (S)-20 described above, (R)-19 (1.95 g, 8.06 mmol) was converted into (R)-20 (1.21 g, 5.97 mmol, 74%, colorless oil). nD20
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= 1.4767. [α]D21 = –6.1 (c 0.2, CHCl3). Its IR and NMR spectra were identical with those of
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(S)-20. ESI-HRMS m/z calcd for C10H18NaO4 [M+Na]+ 225.1097, found 225.1068.
3.2.11 Methyl (5Z,8S)-8-hydroxy-9-(p-methoxybenzyloxy)non-5-enoate (21) (a) (S)-21: To a stirred solution of diol (S)-20 (841 mg, 4.16 mmol) in CH2Cl2 (40 ml) was added PMBOC(NH)CCl3 (1.23 g, 4.36 mmol) and p-TsOH (40.0 mg, 0.21 mmol ). After stirring at room temperature for 16 h, the resulting mixture was neutralized with
TE D
saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was dried with MgSO4, and then evaporated. Purification of the residue on silica gel (2 : 1 hexanes/EtOAc) gave ether (S)-21 as a yellow oil (790 mg, 2.62 mmol, 63%). nD20 = 1.5162. [α]D23 = +2.0 (c 0.2, CHCl3). IR (film): ν = 3460, 2950, 1732, 1248, 1035 cm–1. 1H
EP
NMR (300 MHz, CDCl3): δ (ppm) = 1.57 (1H, br), 1.69 (2H, qui, J = 7.2 Hz), 2.08 (2H, q,
AC C
J = 7.2 Hz), 2.20-2.25 (2H, m), 2.31 (2H, t, J = 7.2 Hz), 3.33 (1H, dd, J = 9.3, 7.2 Hz), 3.48 (1H, dd, J = 9.3, 3.3 Hz), 3.66 (3H, s), 3.81 (3H, s), 3.85 (1H, m), 4.48 (2H, s), 5.42-5.53 (2H, m), 6.89 (2H, d, J = 8.7 Hz), 7.25 (2H, d, J = 8.7 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 24.9, 26.8, 31.5, 33.6, 51.7, 55.4, 70.4, 73.2, 73.8, 114.0, 126.0, 129.6, 130.2, 131.5, 159.5, 174.2. ESI-HRMS m/z calcd for C18H26NaO5 [M+Na]+ 345.1672, found 345.1700. (b) (R)-21: In the same manner as the synthesis of (S)-21 described above, (R)-20 (1.21 g, 5.97 mmol) was converted into (R)-21 (986 mg, 3.06 mmol, 51%, yellow oil). nD20 17
ACCEPTED MANUSCRIPT = 1.5162. [α]D21 = –2.7 (c 0.32, CHCl3). Its IR and NMR spectra were identical with those of (S)-21. ESI-HRMS m/z calcd for C18H26NaO5 [M+Na]+ 345.1672, found 345.1709.
RI PT
3.2.12 (5Z,8S)-8-Hydroxy-9-(p-methoxybenzyloxy)non-5-enoic acid (22) (a) (S)-22: To a stirred solution of ester (S)-21 (841 mg, 4.16 mmol) in methanol (10 ml) was added water (2 ml) and LiOH (332 mg, 7.92 mmol). After stirring for 14 h at room temperature, the resulting mixture was neutralized with saturated ammonium chloride
SC
solution and extracted with EtOAc three times. The aqueous layer was acidified by addition of acetic acid and extracted again with EtOAc. The combined organic layer was dried with
M AN U
MgSO4, and then evaporated. Purification of the residue on silica gel (1 : 1 hexanes/EtOAc) gave carboxylic acid (S)-22 as a yellow oil (742 mg, 2.56 mmol, 98%). nD20 = 1.5250. [α]D24 = +0.97 (c 0.4, CHCl3). IR (film) : ν = 3420, 2936, 1713, 1248, 1035 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.25 (1H, br), 1.71 (2H, qui, J = 7.5 Hz), 2.07-2.14 (2H, m), 2.20-2.27 (2H, m), 2.35 (2H, t, J = 7.5 Hz), 3.34 (1H, dd, J = 9.3, 7.5 Hz), 3.48 (1H, dd, J =
TE D
9.3, 3.3 Hz), 3.81 (3H, s), 3.84 (1H, m), 4.48 (2H, s), 5.45-5.51 (2H, m), 6.89 (2H, d, J = 8.7 Hz), 7.26 (2H, d, J = 8.7 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 24.6, 26.7, 31.4, 33.5, 55.5, 70.5, 73.2, 73.8, 114.0, 126.2, 129.6, 130.2, 131.4, 159.5, 179.2. ESI-HRMS m/z
EP
calcd for C17H24NaO5 [M+Na]+ 331.1516, found 331.1541. (b) (R)-22: In the same manner as the synthesis of (S)-22 described above, (R)-21
AC C
(986 mg, 3.06 mmol) was converted into (R)-22 (682.3 mg, 2.215 mmol, 72%, yellow oil). nD20 = 1.5246. [α]D21 = –0.26 (c 0.1, CHCl3). Its IR and NMR spectra were identical with those of (S)-22. ESI-HRMS m/z calcd for C17H24NaO5 [M+Na]+ 331.1516, found 331.1507.
3.2.13 (5Z,8S)-9-(p-Methoxybenzyloxy)non-5-en-8-olide (23) (a) (S)-23: To a cooled (0 °C) solution of carboxylic acid (S)-22 (2.01 g, 6.91 mmol) in dry THF (10 ml) was added Et3N (1.1 ml, 7.9 mmol) and 2,4,6-trichlorobenzoyl chloride (1.08 ml, 6.91 mmol). After stirring for 2 h at room temperature, the resulting mixture was 18
ACCEPTED MANUSCRIPT
filtered through Celite and the filtrate was diluted with toluene (50 ml). The resulting solution was slowly added via syringe pump (during 8 h) to a refluxed solution of DMAP (16.4 g, 134 mmol) in toluene (2.0 l). After stirring for additional 1 h, the resulting mixture
RI PT
was evaporated, mixed with water and extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (9 : 1, then 4 : 1 hexanes/EtOAc) gave lactone (S)-23 as a yellow oil (1.53 g, 5.61 mmol, 81%). nD24 = 1.5344. [α]D25 = –57 (c 0.08, CHCl3). IR (film): ν =
SC
2948, 1740, 1249, 1034 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.78 (1H, m), 2.022.09 (3H, m), 2.22-2.55 (4H, m), 3.52 (1H, dd, J = 10.5, 4.8 Hz), 3.60 (1H, dd, J =10.5, 6.0
M AN U
Hz), 3.81 (3H, s), 4.48 (1H, d, J = 11.7 Hz), 4.54 (1H, d, J = 11.7 Hz), 4.97 (1H, m), 5.455.53 (2H, m), 6.88 (2H, d, J = 8.7 Hz), 7.27 (2H, d, J = 8.7 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 25.5, 26.7, 30.8, 33.7, 55.4, 71.2, 71.9, 73.0, 114.0, 124.6, 129.5, 130.2, 135.0, 159.4, 174.4. ESI-HRMS m/z calcd for C17H22NaO4 [M+Na]+ 313.1410, found 313.1441.
TE D
(b) (R)-23: In the same manner as the synthesis of (S)-23 described above, (R)-22 (9.0 mg, 0.031 mmol) was converted into (R)-23 (6.4 mg, 0.024 mmol, 76%, yellow oil). nD24 = 1.5344. [α]D21 = +63 (c 0.15, CHCl3). Its IR and NMR spectra were identical with
EP
those of (S)-23. ESI-HRMS m/z calcd for C17H22NaO4 [M+Na]+ 313.1410, found 313.1407.
AC C
3.2.14 (5Z,8S)-9-Hydroxynon-5-en-8-olide (24) (a) (S)-24: To a stirred solution of compound (S)-23 (246 mg, 0.903 mmol) in
CH2Cl2 (3 ml) was added water (0.3 ml) and DDQ (0.29 g, 1.28 mmol) and the mixture was stirred vigorously for 1 h at room temperature. The resulting mixture was poured into saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (1 : 1 hexanes/EtOAc) gave alcohol (S)-24 as a colorless oil (134 mg, 0.787 mmol, 87%). nD24 = 1.4985. [α]D25 = – 86 (c 0.18, CHCl3). IR (film): ν = 3444, 2948, 1744, 19
ACCEPTED MANUSCRIPT 1715 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.61 (1H, br), 1.81 (1H, m), 1.99-2.13 (3H, m), 2.25-2.55 (4H, m), 3.76-3.82 (2H, m), 4.88 (1H, m), 5.45-5.56 (2H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) 25.4, 26.7, 30.2, 33.7, 64.8, 74.1, 124.4, 134.9, 174.6. ESI-
RI PT
HRMS m/z calcd for C9H14NaO3 [M+Na]+ 193.0835, found 193.0831. (b) (R)-24: In the same manner as the synthesis of (S)-24 described above, (R)-23 (2.53 g, 9.32 mmol) was converted into (R)-24 (1.22 g, 7.19mmol, 77%, colorless oil). nD24 = 1.4985. [α]D21 = +84 (c 0.14, CHCl3). Its IR and NMR spectra were identical with those
M AN U
3.2.15 (5Z,8S)-8-Formyloct-5-en-8-olide (25)
SC
of (S)-24. ESI-HRMS m/z calcd for C9H14NaO3 [M+Na]+ 193.0835, found 193.0793.
(a) (S)-25: To a cooled (–78 °C) solution of oxalyl chloride (2.16 g, 17.0 mmol) in dry CH2Cl2 (20 ml) was added dimethyl sulfoxide (2.4 ml, 34 mmol) under argon atmosphere and the mixture was stirred for 15 min. To the resulting solution was added a solution of alcohol (S)-24 (1.45 g, 8.53 mmol) in CH2Cl2 (5 ml) and the mixture was stirred
TE D
for 20 min at –78 °C. After addition of Et3N (7.0 ml, 50.2 mmol), the resulting mixture was stirred for 30 min while gradually warming to room temperature, then poured into saturated NaHCO3 solution, and extracted with EtOAc. The combined organic layer was
EP
dried with MgSO4, and then evaporated. Purification of the residue on silica gel (3 : 1 hexanes/EtOAc) gave aldehyde (S)-25 as a colorless oil (983 mg, 5.85 mmol, 69%). nD24 =
AC C
1.5056. [α]D24 = –163 (c 0.05, CHCl3). IR (film): ν = 2946, 1746 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 1.82 (1H, m), 2.08-2.52 (7H, m), 5.17 (1H, ddd, J = 9.3, 4.2, 1.2 Hz), 5.46-5.61 (2H, m), 9.74 (1H, d, J = 1.2 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = 25.4, 26.5, 28.7, 33.3, 76.9, 123.2, 136.1, 173.8, 198.8. ESI-HRMS m/z calcd for C9H12NaO3 [M+Na]+ 191.0679, found 191.0667. (b) (R)-25: In the same manner as the synthesis of (S)-25 described above, (R)-24 (975 mg, 5.88 mmol) was converted into (R)-25 (889 mg, 5.29mmol, 90%, colorless oil).
20
ACCEPTED MANUSCRIPT nD20 = 1.5041. [α]D21 = +167 (c 0.05, CHCl3). Its IR and NMR spectra were identical with those of (S)-25. ESI-HRMS m/z calcd for C9H12NaO3 [M+Na]+ 191.0679, found 191.0683.
RI PT
3.2.16 (5Z,8R,9E,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-oxoicosa-5,9,14,17-tetraen8-olide [(8R)-26]
To a stirred solution of phosphonate 17 (422 mg, 1.00 mmol) in dry acetonitrile (1.5 ml) was added DBU (0.15 ml, 1.0 mmol), lithium chloride (80.4 mg, 1.89 mmol) and
SC
aldehyde (R)-25 (169 mg, 1.00 mmol) successively under argon atmosphere and the
mixture was stirred for 1.5 h at room temperature. The reaction mixture was poured into
M AN U
water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave compound (8R)-26 as a colorless oil (336 mg, 0.751 mmol, 75%). nD18 = 1.4979. [α]D22 = + 118.4 (c 0.03, CHCl3). IR (film): ν = 2954, 2929, 1747, 1698, 1632 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.02 (3H, s), 0.06 (3H, s), 0.90 (9H, s),
TE D
0.97 (3H, t, J = 7.5 Hz), 1.82 (1H, m), 2.01-2.53 (11H, m), 2.77 (2H, br t, J = 6.3 Hz, 16H), 4.14 (1H, dd, J = 6.9, 5.4 Hz), 5.21-5.57 (7H, m), 6.84 (1H, dd, J = 15.6, 1.5 Hz), 6.94 (1H, dd, J = 15.6, 3.9 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.7, –4.6, 14.5, 18.4,
EP
20.8, 25.6, 25.9, 26.6, 29.9, 33.2, 33.4, 33.9, 71.8, 78.5, 123.8, 124.1, 124.2, 127.0, 131.4, 132.4, 135.8, 144.1, 173.6, 201.4. ESI-HRMS m/z calcd for C26H43O4Si [M+H]+ 447.2925,
AC C
found 447.2905.
3.2.17 (5Z,8S,9E,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-oxoicosa-5,9,14,17-tetraen8-olide [(8S)-26]
In the same manner as the synthesis of (8R)-26 described above, phosphonate 17
(1.41 g, 3.34 mmol) and (S)-25 (509 mg, 3.0 mmol) were converted into (8S)-26 (900 mg, 2.02 mmol, 67%, colorless oil). nD19 = 1.4980. [α]D22 = –92 (c 0.16, CHCl3). IR (film): ν = 2954, 2929, 1748, 1698, 1632 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.04 (3H, s), 21
ACCEPTED MANUSCRIPT
0.07 (3H, s), 0.94 (9H, s), 0.96 (3H, t, J = 7.2 H), 1.82 (1H, m), 2.01-2.49 (11H, m,), 2.76 (2H, br t, J = 6.9 Hz), 4.14 (1H, m), 5.22-5.61 (7H, m), 6.86 (1H, dd, J = 15.9, 0.9 Hz), 6.94 (1H, dd, J = 15.9, 3.3 Hz). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.7, –4.6, 14.4, 18.4, 22.9, 25.5, 26.0, 26.5, 31.8, 33.2, 33.6, 33.9, 71.7, 78.5, 123.8, 124.1, 124.2, 127.0,
RI PT
131.4, 132.4, 135.8, 144.0, 173.7, 201.5. ESI-HRMS m/z calcd for C26H43O4Si [M+H]+ 447.2925, found 447.2994.
SC
3.2.18 (5Z,8R,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-hydroxyicos-5,9,14,17tetraen-8-olide [(8R)-27]
M AN U
To a stirred solution of ketone (8R)-26 (336 mg, 0.751 mmol) in methanol (6 ml) was added cerium chloride heptahydrate (417 mg, 1.12 mmol) and sodium borohydride (42.2 mg, 1.12 mmol) and the mixture was stirred for 5 min at room temperature. The reaction mixture was quenched with water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the
TE D
residue on silica gel (25 : 1 hexanes/EtOAc) gave compound (8R)-27 as a colorless oil (306 mg, 0.683 mmol, 91%). nD18 = 1.4974. [α]D18 = + 53.7 (c 0.09, CHCl3). IR (film): ν = 3480, 2929, 1743 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.06 (3H, s), 0.09 (3H, s), 0.90
EP
(9H, s), 0.97 (3H, t, J = 7.2 Hz), 1.80 (1H, m), 2.02-2.51 (11H, m), 2.80 (2H, br t, J = 6.9 Hz), 3.61 (1H, m), 4.00 (1H, br t, J = 3.6 Hz), 5.25-5.52 (7H, m), 5.73-5.87 (2H, m, 9-H). C NMR (125 MHz, CDCl3): δ (ppm) = –4.4, –4.0, 14.5, 18.3, 20.8, 25.6, 25.9, 26.1, 26.6,
AC C
13
32.1, 33.7, 34.6, 72.5, 72.7, 75.1, 124.7, 124.9, 127.0, 129.5, 131.2, 132.5, 133.0, 135.2, 173.9. ESI-HRMS m/z calcd for C26H45O4Si [M+H]+ 449.3082, found 449.3035.
3.2.19 (5Z,8S,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-hydroxyicos-5,9,14,17tetraen-8-olide [(8S)-27] In the same manner as the synthesis of (8R)-27 described above, (8S)-26 (91.3 mg, 0.205 mmol) was converted into (8S)-27 (88.1 mg, 0.197 mmol, 96%, colorless oil). nD18 = 22
ACCEPTED MANUSCRIPT 1.4982. [α]D16 = –72 (c 0.11, CHCl3). IR (film): ν = 3469, 2930, 1743 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.07 (3H, s), 0.09 (3H, s), 0.90 (9H, s), 0.97 (3H, t, J = 7.5 Hz), 1.80 (1H, m), 2.02-2.51 (11H, m), 2.80 (2H, br t, J = 6.9 Hz), 3.62 (1H, m), 4.02 (1H, br t,
RI PT
J = 3.6 Hz), 5.26-5.52 (7H, m), 5.74-5.87 (2H, m). 13C NMR (125 MHz, CDCl3): δ (ppm) = –4.4, –4.0, 14.5, 18.3, 20.8, 25.5, 25.9, 26.1, 26.6, 32.0, 33.7, 34.5, 72.5, 72.8, 75.2, 124.7, 124.9, 127.0, 129.6, 131.2, 132.4, 133.0, 135.2, 173.8. ESI-HRMS m/z calcd for
SC
C26H44NaO4Si [M+Na]+ 471.2901, found 471.2904.
3.2.20 (5Z,8R,9E,11R,12S,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (1)
M AN U
To a stirred solution of alcohol (8R)-27 (10 mg, 0.022 mmol) in CH2Cl2 (4 ml) was added Et3N (29 mg, 0.29 mmol), DMAP (8.6 mg, 0.070 mmol) and MsCl (13.4 mg, 0.12 mmol) successively at room temperature under argon atmosphere, and the mixture was stirred for 10 min at room temperature. The reaction mixture was quenched with water and extracted with ether. The combined organic layer was washed with water, saturated
TE D
ammonium chloride solution, saturated NaHCO3 solution and brine. After drying with MgSO4, evaporation gave crude mesylate (8R)-28. Crude (8R)-28 was dissolved with dry THF (5 ml). After addition of hydrated TBAF (30 mg), the reaction mixture was stirred for
EP
2 h at room temperature. The reaction mixture was poured into water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then
AC C
evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave an compound 1 as a colorless oil (7.4 mg, 0.022 mmol, 98%). nD16 = 1.5144. [α]D24 = +88 (c 0.22, MeOH). IR (film): ν = 2961, 1743, 1218, 1137, 969 cm–1. 1H NMR (500 MHz, CD3OD): δ (ppm) = 0.97 (3H, t, J = 7.5 Hz, 20-H), 1.76 (1H, m, 3-Ha), 2.03-2.30 (7H, m, 2-Ha, 3-Hb, 4-Ha, 7-Ha, 13-Ha, 19-H), 2.32-2.55 (4H, m, 2-Hb, 4-Hb, 7-Hb, 13-Hb), 2.74-2.86 (2H, m, 16-H), 3.12 (1H, dt, J = 4.5, 6.5 Hz, 12-H), 3.48 (1H, br dd, J = 7.0, 4.5 Hz, 11-H), 5.25-5.32 (2H, m, 8-H, 17-H), 5.35-5.52 (5H, m, 5-H, 6-H, 14-H, 15-H, 18-H), 5.76 (1H, ddd, J = 15.5, 7.0, 1.5 Hz, 10-H), 6.04 (1H, ddd, J = 15.5, 5.5, 1.0 Hz, 9-H). 13C NMR (125 23
ACCEPTED MANUSCRIPT MHz, CD3OD): δ (ppm) = 14.7, 21.5, 26.3, 26.7, 27.1, 27.5, 34.4, 35.3, 57.3, 59.4, 73.8, 124.9, 125.5, 127.3, 127.9, 132.0, 133.0, 135.3, 136.3, 175.6. ESI-HRMS m/z calcd for
RI PT
C20H28NaO3 [M+Na]+ 339.1931, found 339.1910.
3.2.21 (5Z,8S,9E,11R,12S,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (5)
In the same manner as the synthesis of 1 described above, (8S)-27 (195 mg, 0.434 mmol) was converted into 5 (125 mg, 0.368 mmol, 85%, colorless oil). nD16 = 1.5156.
SC
[α]D20 = –98 (c 0.10, MeOH). IR (film): ν = 2962, 1742, 1218, 1136, 969 cm–1. 1H NMR
(500 MHz, CD3OD): δ (ppm) = 0.96 (3H, t, J = 7.5 Hz, 20-H), 1.76 (1H, m, 3-Ha), 2.03-
M AN U
2.29 (7H, m, 2-Ha, 3-Hb, 4-Ha, 7-Ha, 13-Ha, 19-H), 2.32-2.55 (4H, m, 2-Hb, 4-Hb, 7-Hb, 13Hb), 2.73-2.85 (2H, m, 16-H), 3.13 (1H, dt, J = 4.5, 6.5 Hz, 12-H), 3.47 (1H, br dd, J = 6.5, 4.5 Hz, 11-H), 5.24-5.31 (2H, m, 8-H, 17-H), 5.35-5.52 (5H, m, 5-H, 6-H, 14-H, 15-H, 18H), 5.76 (1H, ddd, J = 15.5, 7.0, 1.5 Hz, 10-H), 6.03 (1H, ddd, J = 15.5, 5.5, 1.0 Hz, 9-H). C NMR (125 MHz, CD3OD): δ (ppm) = 14.7, 21.5, 26.3, 26.6, 27.1, 27.5, 34.4, 35.4,
13
TE D
57.2, 59.4, 73.9, 124.9, 125.5, 127.2, 127.9, 132.0, 133.0, 135.2, 136.3, 175.6. ESI-HRMS m/z calcd for C20H28NaO3 [M+Na]+ 339.1931, found 339.1942.
EP
3.2.22 (5Z,8R,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-(1-ethoxyethoxy)icosa5,9,14,17-tetraen-8-olide [(8R)-29]
AC C
To a stirred solution of alcohol (8R)-27 (7.5 mg, 0.017 mmol) in CH2Cl2 (4 ml) was added ethyl vinyl ether (1.0 ml, 10 mmol) and PPTS (1.4 mg, 0.0056 mmol), and the mixture was stirred for 50 min at room temperature. The reaction mixture was poured into saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was washed with water, saturated ammonium chloride solution, saturated NaHCO3 solution and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (20 : 1 hexanes/EtOAc) gave an compound (8R)-29 as a colorless oil (8.5 mg, 0.016 mmol, 98%). nD16 = 1.4848. [α]D18 = + 64.0 (c 0.17, CHCl3). IR (film): ν = 2956, 2929, 1746 cm–1. 24
ACCEPTED MANUSCRIPT 1
H NMR (300 MHz, CDCl3): δ (ppm) = 0.05-0.09 (6H, m), 0.89 (9H, br), 0.96 (3H, t, J =
7.8 Hz), 1.17, 1.19 (3H, two t, J = 6.9 Hz), 1.30, 1.33 (3H, two d, J = 5.4 Hz), 1.71-1.87 (1H, m), 2.01-2.52 (11H, m), 2.73-2.82 (2H, m), 3.41-3.80 (3H, m), 3.91-4.08 (1H, m) 4.69-4.76 (1H, m), 5.25-5.51 (7H, m), 5.72-5.84 (2H, m). ESI-HRMS m/z calcd for
RI PT
C30H52NaO5Si [M+Na]+ 543.3476, found 543.3452
3.2.23 (5Z,8S,9E,11S,12S,14Z,17Z)-12-(t-Butyldimethylsiloxy)-11-(1-ethoxyethoxy)icosa-
SC
5,9,14,17-tetraen-8-olide [(8S)-29]
In the same manner as the synthesis of (8R)-29 described above, (8S)-27 (389 mg,
M AN U
0.867 mmol) was converted into (8S)-29 (374 mg, 0.718 mmol, 87%, colorless oil). nD18 = 1.4838. [α]D18 = –51.0 (c 0.24, CHCl3). IR (film): ν = 2956, 2930, 1746 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) 0.05 (3H, s), 0.08 (3H, s), 0.887, 0.891 (9H, two s), 0.96 (3H, t, J = 7.8 Hz), 1.17, 1.19 (3H, two t, J = 6.9 Hz), 1.29, 1.31 (3H, two d, J = 5.4 Hz), 1.731.87 (1H, m), 2.01-2.53 (11H, m), 2.73-2.79 (2H, m), 3.40-3.78 (3H, m), 4.00-4.10 (1H, m)
TE D
4.69, 4.76 (1H, two q, J = 5.4 Hz), 5.25-5.54 (7H, m), 5.73-5.84 (2H, m). ESI-HRMS m/z calcd for C30H52NaO5Si [M+Na]+ 543.3476, found 543.3452.
EP
3.2.24 (5Z,8R,9E,11S,12S,14Z,17Z)-11-(1-Ethoxyethoxy)-12-hydroxyicosa-5,9,14,17tetraen-8-olide [(8R)-30]
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To a stirred solution of (8R)-29 (8.5 mg, 0.016 mmol) in THF (4 ml) was added hydrated TBAF (30 mg) and the mixture was stirred for 30 min at room temperature. The reaction mixture was poured into water and extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (2 : 1 hexanes/EtOAc) gave alcohol (8R)-30 as a colorless oil (6.1 mg, 0.015 mmol, 92%). nD16 = 1.4989. [α]D18 = +99 (c = 0.03, CHCl3). IR (film): ν = 3482, 2973, 1743 cm–1. 1H NMR (300 MHz, CDCl3): δ (ppm) = 0.97 (3H, t, J = 7.5 Hz), 1.18, 1.21 (3H, two t, J = 6.9 Hz), 1.32-1.34 (3H, m), 1.72-1.87 (1H, m), 2.01-2.54 25
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(11H, m), 2.78 (2H, br t, J = 5.7 Hz), 3.37-3.71 (3H, m), 3.78 (0.5H, t, J = 6.3 Hz), 3.96 (0.5H, t, J = 7.5 Hz), 4.69-4.74 (1H, m), 5.27-5.53 (7H, m), 5.62-5.87 (2H, m). ESI-HRMS
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m/z calcd for C24H38NaO5+ [M+Na] 429.2611, found 429.2655.
3.2.25 (5Z,8S,9E,11S,12S,14Z,17Z)-11-(1-Ethoxyethoxy)-12-hydroxyicosa-5,9,14,17tetraen-8-olide [(8S)-30]
In the same manner as the synthesis of (8R)-30 described above, (8S)-29 (364 mg,
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0.699 mmol) was converted into (8S)-30 (260 mg, 0.64 mmol, 91%, colorless oil). nD16 = 1.4989. [α]D18 = –90 (c 0.05, CHCl3). IR (film): ν = 3481, 2971, 2932, 1743 cm–1. 1H NMR
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(300 MHz, CDCl3): δ (ppm) = 0.96 (3H, t, J = 7.8 Hz), 1.17, 1.21 (3H, two t, J = 6.9 Hz), 1.31-1.34 (3H, m), 1.73-1.88 (1H, m), 2.03-2.54 (11H, m), 2.78 (2H, br), 3.37-3.70 (3H, m), 3.78 (0.5H, t, J = 6.6 Hz), 3.96 (0.5H, t, J = 7.2 Hz), 4.67-4.73 (1H, m), 5.28-5.53 (7H, m), 5.62-5.86 (2H, m). ESI-HRMS m/z calcd for C24H38NaO5 [M+Na]+ 429.2611, found
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429.2591.
3.2.26 (5Z,8R,9E,11S,12R,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (6) To a stirred solution of alcohol (8R)-30 (6.1 mg, 0.015 mmol) in CH2Cl2 (3 ml) was
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added Et3N (23.4 mg, 0.231 mmol), DMAP (9.4 mg, 0.77 mmol) and MsCl (0.008 ml, 0.103 mmol) successively at room temperature under argon atmosphere, and the mixture
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was stirred for 30 min at room temperature. The reaction mixture was quenched with water and extracted with ether. The combined organic layer was washed with water, saturated ammonium chloride solution, saturated NaHCO3 solution and brine, dried with MgSO4, and then evaporated to afford crude mesylate (8R)-31. Crude (8R)-31 was dissolved in THF (5 ml), and treated with 0.5 N HCl (5 ml). After stirring for 3 h at room temperature, the reaction mixture was neutralized with saturated NaHCO3 solution and extracted with ether. The combined organic layer was washed with water and brine, dried with MgSO4, and then evaporated to give crude alcohol (8R)-32. Crude (8R)-32 was dissolved in THF (2 ml) and 26
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treated with hydrated TBAF (19 mg). After stirring for10 min at room temperature, the reaction mixture was poured into water and extracted with ether. The combined organic layer was washed with brine, dried with MgSO4, and then evaporated. Purification of the residue on silica gel (25 : 1 hexanes/EtOAc) gave epoxide 6 as a colorless oil (2.6 mg,
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0.0077 mmol, 51%). nD16 = 1.5153. [α]D24 = +96 (c 0.18, MeOH). Its IR and NMR spectra were identical with those of 5. ESI-HRMS m/z calcd for C20H28NaO3 [M+Na]+ 339.1931,
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found 339.1925.
3.2.27 (5Z,8S,9E,11S,12R,14Z,17Z)-11,12-Epoxyicosa-5,9,14,17-tetraen-8-olide (7)
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In the same manner as the synthesis of 6 described above, (8S)-30 (256 mg, 0.63 mmol) was converted into 7 (140 mg, 0.182 mmol, 29%, colorless oil). nD16 = 1.5151. [α]D20 = –88 (c 0.11, MeOH). Its IR and NMR spectra were identical with those of 1. ESIHRMS m/z calcd for C20H28NaO3 [M+Na]+ 339.1931, found 339.1933.
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3.3. Biological studies
Cytotoxicity against human cervical carcinoma HeLa cells and human lymphoblastoid namalva cells were determined by a colorimetric assay using WST-8 [2-(2-
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methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt]. Cells were cultured in DMEM medium (Wako Pure Chemical
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Industries, Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (GIBCO, Carlsbad, CA), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 °C in a humidified incubator under a 5% CO2 atmosphere. The 384-well plates were seeded with aliquots of a 20-µL medium containing 1.0 × 103 cells per well and were incubated overnight before being treated with compounds at various concentrations for 48 h. Plates were incubated for 1 h at 37 °C after the addition of 2-µL WST-8 reagent solution (Cell Counting Kit; Dojindo, Kumamoto, Japan) per well. The absorption of formazan dye was measured at 450 nm. The vehicle solvent (DMSO) was used as a negative control. 27
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Acknowledgement The authors sincerely thank Professor J. H. Jung, Pusan National University, for a
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kind gift of the spectral charts of natural topsentolide A1. We sincerely thank Mr. Y. Osano and Mr. T. Imaoka, University of Tokyo, for mass spectroscopy. This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of
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Science to K.I (Grant Number 22580115).
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1. Niwa, H.; Wakamatsu, K.; Yamada, K. Tetrahedron Lett. 1989, 30, 4543-4546. 2. Papendorf, O.; König, G. M.; Wright, A. D.; Chorus, I.; Oberemm, A. J. Nat. Prod. 1997, 60, 1298-1300.
3. Seo, Y.; Cho, K. W.; Rho, J.-R.; Shin, J.; Kwon, B.-M.; Bok, S.-H.; Song, J.-I. Tetrahedron 1996, 52, 10583-10596.
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4. Ishigami, K.; Motoyoshi, H.; Kitahara, T. Tetrahedron Lett. 2000, 41, 8897-8901. 5. Motoyoshi, H.; Ishigami, K.; Kitahara, T. Tetrahedron 2001, 57, 3899-3908. 6. Takahashi, T.; Watanabe, H.; Kitahara, T. Heterocycles 2002, 58, 99-104.
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7. Takahashi, T.; Takahashi, T.; Watanabe, H.; Kitahara, T. Abstracts of Papers, 44th Symposium on the Chemistry of Natural Products, Tokyo, Japan, 2002, 1-6.
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14. McDonald, C.; Holcomb, H.; Kennedy, K.; Kirkpatrick, E.; Leathers, T.; Vanemon, P. J. Org. Chem. 1989, 54, 1213-1215. 15. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn.
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1979, 52, 1989-1993. 16. Blanchette, M. A.; Choy, W.; Davis, J. T.; Essenfeld, A. P.; Masamune, S.; Roush, W. R.; Sakai, T. Tetrahedron Lett. 1984, 25, 2183-2186. 17. Luche, J.-L. J. Am. Chem. Soc. 1978, 100, 2226-2227.
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18. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 40924096.
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19. Towada, R.; Kurashina, Y.; Kuwahara, S. Tetrahedron Lett. 2013, 54, 6878-6881.
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