Synthesis, Structure, and Cytotoxicity Studies of Some Fungal

Sep 13, 2013 - Cooperative Research Center of Life Sciences, Kobe Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan. ⊥ Department of Applied ...
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Synthesis, Structure, and Cytotoxicity Studies of Some Fungal Isochromanes Kouji Kuramochi,*,† Kazunori Tsubaki,† Isoko Kuriyama,‡ Yoshiyuki Mizushina,‡,§ Hiromi Yoshida,‡ Toshifumi Takeuchi,⊥ Shinji Kamisuki,⊥ Fumio Sugawara,⊥ and Susumu Kobayashi∥ †

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan Laboratory of Food & Nutritional Sciences, Faculty of Nutrition, Kobe Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan § Cooperative Research Center of Life Sciences, Kobe Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan ⊥ Department of Applied Biological Science, University Tokyo of Sciences, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ∥ Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ‡

S Supporting Information *

ABSTRACT: Ustusorane D and penicisochromans B−D are natural isochromans isolated from Aspergillus ustus 094102 and Penicillium sp. PSU-F40, respectively. Herein, we report the syntheses of (−)-ustusorane D and (+)-penicisochroman B and the structures of penicisochromans C and D. The relative configuration of natural ustusorane D and the absolute configuration of natural penicisochroman B were determined. Two plausible structures for penicisochroman C were evaluated through synthesis, but their 1H and 13C NMR data were not in agreement with those of the natural product. The structural revision and the determination of the absolute configuration of natural penicisochroman D were achieved. Structure−activity relationship studies of the synthetic compounds as well as a series of related isochromans indicated that the enone of the furanone moiety was essential for the cytotoxicity of these compounds toward HCT116 human colon cancer cells. Pseudodeflectusin, the related natural isochroman, suppressed cell growth and induced apoptosis in HCT116 cells.

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isolated from Aspergillus ustus 094102 by Lu et al.,2 whereas penicisochromans B (4), C (5), and D (6) were isolated from the sea fan-derived fungus Penicillium sp. PSU-F40 by Trisuwan et al.3 The 1H and 13C NMR spectra of 3 are different from those of 4, suggesting that these compounds could be a pair of diastereomers. The relative and absolute configurations of compounds 3−6 have not yet been determined. The total syntheses of pseudodeflectusin and ustusorane C were accomplished by our group4,5 and successfully revealed the absolute configurations of these natural products. In the current paper, we report the syntheses of (−)-ustusorane D and (+)-penicisochroman B, as well as the proposed structures for penicisochromans C and D. The relative configuration of ustusorane D and absolute configuration of penicisochroman B have been also reported. The NMR data for natural penicisochroman C were similar to those of the two plausible structures of penicisochroman C synthesized in the current study. The proposed structure for natural penicisochroman D has been revised, and its absolute configuration was determined. Furthermore, the cytotoxicities of these synthetic

seudodeflectusin (1) is an isochroman derivative that was first isolated from the culture broth of Aspergillus pseudodef lectus by our group in 2004 (Figure 1).1 This compound exhibits cytotoxicity toward several human cancer cell lines, including those derived from the stomach (NUGC3), cervix (HeLa-S3), and peripheral blood (HL-60). The related natural products, ustusoranes C (2) and D (3), were

Figure 1. Chemical structures of pseudodeflectusin (1), ustusoranes C (2) and D (3), and penicisochromans B (4), C (5), and D (6). © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 10, 2013

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treated with p-TsOH·H2O in MeOH to afford the corresponding methyl acetal with concomitant deprotection of the TMS group to give 13 as a sole product in 68% yield over the two steps. Unfortunately, NMR analysis of this material did not reveal any NOESY correlations, and it was therefore not possible to confirm the relative configuration at the C-9 position in 13. It was possible, however, to confirm the absolute configuration at the C-9 position by comparing the energies of 13 and 9-epi-13. Given that the acetal formation is a reversible process, the thermodynamically more stable product should be obtained. The lowest conformations and the sum of electronic and zeropoint energies of 13 and 9-epi-13 were determined by density functional theory (DFT) calculations at the B3LYP/6-31G(d,p) level. These calculations indicated that the lowest energy conformation of 13 was predicted to be more stable than that of 9-epi-13 by 2.88 kcal/mol (Figure 3A).8 The methoxy group at the C-9 position should adopt a pseudoaxial orientation because of the anomeric effect, as well as repulsion (1,3-allylic strain) from the adjacent aromatic ring. The oxidation of 13 with MnO2 gave ketone 14 in 51% yield.8 The 1H and 13C NMR spectra of 14 were identical to those of ustusorane D (3), indicating that the relative configuration of 14 was identical to that of 3. The specific rotation of 14 was determined to be [α]17D −67.9 (c 0.61, MeOH) and was therefore much lower than that of 3, which has been reported to be [α]20D −1 (c 0.1, MeOH).2 These results suggested that the sample of natural 3 isolated by Lu et al.2 could contain impurities that could lead to a reduction in the observed optical rotation or that the material may be nearly racemic. The acidic hydration of 14 with p-TsOH·H2O in THF−H2O (1:1, v/v) afforded 15 in 92% yield. The absolute configuration of the C-9 position in 15 was determined to be S by DFT calculations at the B3LYP/6-31G(d,p) level. The calculations showed that the lowest energy conformation of 15 was more stable than that of 9-epi-15 by 2.51 kcal/mol (Figure 3B). The 1 H NMR data for 15 in CDCl3 were similar to those reported for natural penicisochroman C (5) (Table 1). All of the 1H NMR signals of 15 were shifted downfield by a difference in chemical shift values (ΔδH) of 0.06−0.08 ppm in comparison with those reported for 5. In contrast, all of the 13C NMR signals of 15 were shifted upfield by a difference in chemical shift values (ΔδC) of 0.3−1.2 ppm in comparison with those reported for 5. The specific rotation of 15 was determined to be [α]18D −167.2 (c 0.25, CHCl3), whereas the specific rotation of 5 was reported to be [α]25D −55 (c 0.08, CHCl3).3 These results suggested that natural penicisochroman C could not be identical to our synthetic 15. Isomerization of the C-2 position in 14 with NaOMe in MeOH gave 16 in 46% yield, together with recovered 14 in 46% yield (Scheme 3). All of the 1H NMR signals of 16 were shifted downfield with differences in chemical shift values (ΔδH) of 0.06−0.07 ppm in comparison with those reported for natural penicisochroman B (4) except for the signal derived from the methoxy group (ΔδH = 0.16 ppm) (Table 2). The 13C NMR spectrum of 16 was identical with that of 4 (ΔδC = 0−0.2 ppm) except for the signal derived from the C-3 position (ΔδC = 1.0 ppm). The absolute configuration of the C-9 position in 16 was confirmed to be S on the basis of a NOESY correlation between the H-7 proton and the methoxy group. The specific rotation of 16 was determined to be [α]22D +54.9 (c 0.21, CHCl3) and was the opposite of that reported for natural 4,

compounds toward HCT116 human colon cancer cells have been evaluated.

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RESULTS AND DISCUSSION The optically active (R)-lactone 74 was hydrogenated for 2 h in the presence of a catalytic amount of Pd(OH)2 to give 8 and 9. Compound 8 was formed in a 50% yield, whereas 9 was formed in 41% yield as an inseparable 2:1 mixture of diastereomers derived from the chiral center at the C-2 position (Scheme 1). Scheme 1. Reduction of Compound 7

An increase in the reaction time of this transformation led to the deoxygenation of the hydroxyl group in 8. The relative configuration of 8 was determined from its NMR data. The occurrence of a NOESY correlation between the H-2 and H-3 protons indicated that these two protons existed in a synrelationship (Figure 2A). The relative configuration of 10,

Figure 2. Determination of the relative and absolute configurations of 8. (A) Determination of relative configuration of 8 by NOSEY correlations between H-2 and H-3 and comparison of the coupling constants of H-2/H-3 and H-2/H-11 in 8 with those in 10 reported by Kohno et al.6 (B) Determination of the absolute configuration of 8 using a modified Mosher’s method. Preparation of (S)- and (R)MTPA esters (11a and 11b). The chemical shift differences (ΔδH = δS − δR in ppm) between (S)- and (R)-MTPA esters have been indicated.

which has a similar skeleton to 8, was determined by Kohno et al.6 using NOE experiments. The syn-relationship between the H-2 and H-3 protons in 8 was also confirmed through a comparison of the coupling constants between H-2 and H-3, and H-2 and H-11, in 8 with those in 10. The absolute configuration of the secondary alcohol in 8 was determined to be S using a modified version of Mosher’s method7 involving the (S)- and (R)-MTPA esters (11a and 11b) (Figure 2B). The hydroxyl group in 8 was protected with TMSCl to give the corresponding TMS ether 12 (Scheme 2). Reduction of the lactone in 12 with DIBAL-H gave a hemiacetal, which was B

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Scheme 2. Synthesis of Compounds 14 and 15

3). In particular, the signal for the H-2 proton (δH 4.46) overlapped with that of the H-7 proton (δH 4.46) in 17, whereas the corresponding peaks in compound 5 did not overlap {H-2 (δH 4.46) and H-7 (δH 4.40)}. With the exception of the signal for the H-2 proton, all of the 1H NMR signals of 17 were shifted downfield (Δδ = 0.06−0.09 ppm) relative to those reported for 5. All of the 13C NMR signals of 17 were shifted upfield relative to those for 5 (Δδ = 0.4−1.7 ppm). The specific rotation of 17 was found to be [α]25D +93.4 (c 0.23, CHCl3), whereas the reported specific rotation of 5 was [α]25D −55 (c 0.08, CHCl3).3 These results suggested that natural penicisochroman C was different from our synthetic 17. According to the 1H NMR data reported by Trisuwan et al.,3 the signal reported for the hydroxyl group at the C-9 position of natural penicisochroman C was not observed. The NMR spectra of 15 and 17 were therefore measured in CDCl3 containing different amounts of D2O or CD3OD. Although the spectra were not in agreement with the reported data, the NMR data for both 15 and 17 in a 1:1 (v/v) mixture of CD3OD and CDCl3 were similar to the data from the literature (Table 4). On the basis of these results, it remains difficult to determine whether our synthetic 15 or 17 is the same material as natural penicisochroman C. The structure and the absolute configuration of natural penicisochroman C therefore remain unknown. The reduction of (R)-(−)-mellein (18) with BH3 gave isochroman 19 and alcohol 20 in yields of 14% and 80%, respectively (Scheme 4). The molecular formula of compound 19 was determined to be C10H12O2 by high-resolution electrospray ionization mass spectrometry (HRESIMS). HMBC correlations from H-6 to C-8 and from H-8 to C-6 in 19 indicated the presence of a six-membered cyclic ether ring. The 1H and 13C NMR spectra of 19 were different from those reported for natural penicisochroman D (6) (Table 5).3 The 1H and 13C NMR spectra of 20, however, were in agreement with those reported for penicisochroman D. The molecular formula of compound 20 was determined to be C10H14O3 by HRESIMS. Acetylation of 20 with acetic anhydride in pyridine gave a triacetate, which confirmed the presence of three hydroxyl groups in 20 (Supporting Information). The specific rotation of 20 was determined to be [α]24D −26.7 (c 0.30, CHCl3) and was almost identical to the value of [α]25D −26 (c 0.21, CHCl3) reported for penicisochroman D. On the basis of these results, the absolute configuration of natural penicisochroman D was determined to be R, and natural penicisochroman D was therefore determined to be (R)-2-(hydroxymethyl)-(2′-hydroxypropyl)phenol.

Figure 3. Lowest energy conformations of 13, 9-epi-13, 15, 9-epi-15, 17, and 9-epi-17 as determined by DFT calculation at the B3LYP/631G(d,p) level. The differences in the sum of electronic and zero-point energies (ΔE) between 13 and 9-epi-13, 14 and 9-epi-14, and 17 and 9-epi-17 have been indicated in kcal/mol.

which was [α]25D −57 (c 0.08, CHCl3).3 These results indicated that natural penicisochroman B (4) is the enantiomer of our synthetic 16. Thus, the absolute configuration of natural penicisochroman B (4) was determined to be 2S,7S,9R. The acidic hydration of 16 with p-TsOH·H2O in THF−H2O (1:1, v/v) afforded 17 in 92% yield. The absolute configuration of the C-9 position in 17 was deduced to be S using DFT calculations at the B3LYP/6-31G(d,p) level. Compound 17 was predicted to be more stable than 9-epi-17 by 2.95 kcal/mol (Figure 3C). The 1H and 13C NMR data for 17 in CDCl3 were not in agreement with those for penicisochroman C (5) (Table C

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Table 1. NMR Data for Natural Penicisochroman C (5)3 and Synthetic 15 5 position

δC, type

15 δH (J in Hz)

2 3 3a 4 5 5a 6

91.0, 202.0, 121.5, 124.5, 122.5, 146.0, 36.9,

CH C C CH CH C CH2

7 9 9a 9b 10 11 12 13 OH

63.5, 88.8, 121.0, 172.5, 21.9, 32.0, 19.5, 16.7,

CH CH C C CH3 CH CH3 CH3

4.46 d (3.5)

7.46 d (8.5) 6.76 d (8.5) 2.72 2.62 4.40 6.18

dd (16.5, 2.5) dd (16.5, 10.0) m s

1.33 2.29 1.08 0.81

d (6.0) m d (6.0) d (6.5)

δC, type

δH (J in Hz)

90.3, 201.0, 120.3, 123.5, 122.2, 145.5, 35.9,

CH C C CH CH C CH2

62.8, 87.7, 120.1, 170.7, 21.1, 31.1, 18.8, 15.7,

CH CH C C CH3 CH CH3 CH3

4.54 d (3.6)

7.53 d (8.0) 6.83 d (8.0) 2.79 2.70 4.47 6.26

dd (17.2, 3.2) dd (17.2, 10.8) m d (4.0)

1.40 2.37 1.15 0.87 3.01

d (6.8) dh (3.6, 6.8) d (6.8) d (6.8) brs

ΔδC (ppm)

ΔδH (ppm)

+0.7 +1.0 +1.2 +1.0 +0.3 +0.5 +1.0

−0.08

+0.7 +1.1 +0.9 +1.8 +0.8 +0.8 +0.7 +1.0

−0.07 −0.07 −0.07 −0.08 −0.07 −0.08

−0.07 −0.08 −0.07 −0.06

on the growth of the cells at concentrations of 100 μM or less. These results clearly indicated that the enone moiety on the furanone ring is important for the observed cytotoxicity. To examine whether pseudodeflectusin {(+)-1}, which has the strongest cytotoxic effect among the compounds tested, induced apoptosis, we analyzed the extent of DNA fragmentation in the cells before and after treatment using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method (Figure 4). Significant levels of DNA fragmentation were detected when the HCT116 cells were treated with (+)-1 at 5.0 μM for 72 h. The percentage of apoptotic cells produced by the treatment with (+)-1 was 2-fold higher than the value achieved after an equivalent treatment with etoposide, which is a known apoptosis inducer and anticancer agent. In conclusion, we have successfully synthesized ustusorane D and penicisochroman B and proposed structures for

Scheme 3. Synthesis of Compounds 16 and 17

The cytotoxicities of synthetic (+)-1,4a (+)-2,4b 7, 14, 15, 16, 17, and 20 were tested using HCT116 human colon cancer cells (Table 6). The WST-8 assay was used to measure the cell viability of the HCT116 cells following 72 h of treatment with each compound.9 Compounds (+)-1, (+)-2, and 7 displayed cytotoxicity activity toward HCT116 cells, with IC50 values of 5.0 ± 0.1, 5.9 ± 0.1, and 9.8 ± 0.1 μM, respectively. In contrast, compounds 14, 15, 16, 17, and 20 had no discernible impact

Table 2. NMR Data for Natural Penicisochroman B (4)3 and Synthetic 16 4 position

a

δC, type

16 δH (J in Hz)

2 3 3a 4 5 5a 6

90.2, 200.2, 120.1, 123.4, 122.0, 145.4, 35.9,

CH C C CH CH C CH2

7a 9 9a 9b 10 11 12 13 OCH3a

62.6, 94.7, 120.0, 171.0, 21.1, 31.1, 19.0, 15.8, 55.8,

CH CH C C CH3 CH CH3 CH3 C

4.35 d (4.0)

7.43 d (8.0) 6.72 d (8.0) 2.68 2.62 4.25 5.61

dd (17.0, 3.5) dd (17.0, 11.0) m s

1.32 2.28 1.11 0.80 3.45

d (6.5) m d (7.0) d (7.0) s

δC, type

δH (J in Hz)

90.2, 201.2, 120.1, 123.3, 121.9, 145.2, 35.8,

CH C C CH CH C CH2

62.6, 94.7, 119.9, 171.0, 21.1, 31.1, 19.0, 15.8, 55.8,

CH CH C C CH3 CH CH3 CH3 C

4.42 d (4.0)

7.50 d (8.0) 6.79 d (8.0) 2.75 2.68 4.32 5.68

dd (17.2, 3.6) dd (17.2, 10.4) m s

1.39 2.35 1.18 0.87 3.61

d (6.0) dh (4.0, 6.8) d (6.8) d (6.8) s

ΔδC (ppm)

ΔδH (ppm)

0.0 +1.0 0.0 +0.1 +0.1 +0.2 +0.1

−0.07

0.0 0.0 +0.1 0.0 0.0 0.0 0.0 0.0 0.0

−0.07 −0.07 −0.07 −0.06 −0.07 −0.07

−0.07 −0.07 −0.07 −0.07 −0.16

NOESY correlations between H-7 and OCH3 in 4 were reported previously. The same NOESY correlations were observed for 16. D

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Table 3. NMR Data for Natural Penicisochroman C (5)3 and Synthetic 17 5 position

17

δC, type

δH (J in Hz)

2 3 3a 4 5 5a 6

91.0, 202.0, 121.5, 124.5, 122.5, 146.0, 36.9,

CH C C CH CH C CH2

7 9 9a 9b 10 11 12 13 OH

63.5, 88.8, 121.0, 172.5, 21.9, 32.0, 19.5, 16.7,

CH CH C C CH3 CH CH3 CH3

4.46 d (3.5)

7.46 d (8.5) 6.76 d (8.5) 2.72 2.62 4.40 6.18

dd (16.5, 2.5) dd (16.5, 10.0) m s

1.33 2.29 1.08 0.81

d (6.0) m d (6.5) d (6.0)

δC, type

δH (J in Hz)

90.3, 201.0, 120.4, 123.4, 122.1, 145.4, 36.0,

CH C C CH CH C CH2

62.7, 87.8, 120.2, 170.8, 21.2, 31.1, 18.9, 15.7,

CH CH C C CH3 CH CH3 CH3

ΔδC (ppm)

4.46 d (4.0)

7.53 d (8.0) 6.82 d (8.0) 2.78 2.69 4.46 6.24

dd (17.6, 3.6) dd (17.6, 10.8) m d (3.2)

1.40 2.37 1.17 0.90 3.05

d (6.5) dh (3.6, 6.8) d (6.8) d (6.8) brs

ΔδH (ppm)

+0.7 +1.0 +1.1 +1.1 +0.4 +0.6 +0.9

0.00

−0.07 −0.06 −0.06 −0.07 −0.06 −0.06

+0.8 +1.0 +0.8 +1.7 +0.7 +0.9 +0.6 +1.0

−0.07 −0.08 −0.09 −0.09

Table 4. NMR Data Reported for Natural Penicisochroman C (5) in CDCl3 and NMR Data for the Two Plausible Structures (15 and 17) in a 1:1 (v/v) Mixture of CD3ODa and CDCl3 5

a

position

δC

2 3 3a 4 5 5a 6

91.0 202.0 121.5 124.5 122.5 146.0 36.9

7 9 9a 9b 10 11 12 13

63.5 88.8 121.0 172.5 21.9 32.0 19.5 16.7

15 δH (J in Hz)

4.46 d (3.5)

7.46 d (8.5) 6.76 d (8.5) 2.72 2.62 4.40 6.18

dd (16.5, 2.5) dd (16.5, 10.0) m s

1.33 2.29 1.08 0.81

d (6.0) m d (6.0) d (6.5)

δC

17 δH (J in Hz)

90.8 202.8 121.6 123.5 123.0 146.7 36.5

δC

4.51 d (4.0)

7.46 d (8.0) 6.83 d (8.0)

62.9 87.6 120.4 171.7 21.3 31.6 19.0 15.8

2.78 2.64 4.41 6.11

dd (17.6, 2.8) dd (17.6, 11.2) m s

1.34 2.30 1.12 0.82

d (6.0) dh (3.2, 7.2) d (7.2) d (7.2)

91.0 203.0 121.9 123.5 122.9 146.6 36.5 62.9 87.7 120.5 171.9 21.4 31.7 19.0 15.8

δH (J in Hz) 4.46 d (4.0)

7.46 d (8.0) 6.82 d (8.0) 2.77 2.63 4.40 6.09

dd (17.6, 2.8) dd (17.6, 11.2) m s

1.34 2.31 1.14 0.85

d (6.0) dh (4.0, 6.8) d (6.8) d (6.8)

The residual solvent peak of CD3OD was used as an internal standard (3.30 ppm for 1H NMR and 49.0 ppm for 13C NMR).

penicisochroman B was determined to be 2R,7R,9S. The structure and the absolute configuration of natural penicisochroman C remain unclear because the 1H and 13C NMR data for the two plausible structures of penicisochroman C were not identical to those of the natural product. The structural revision and determination of the absolute configuration of natural penicisochroman D has been achieved. Evaluation of the cytotoxicities of the synthetic benzofuranoids indicated that the presence of the enone moiety on the furanone ring is critical to the cytotoxic activity of these compounds. Apoptosis is the process of programmed cell death that occurs in multicellular organisms.10 Biochemical events can trigger characteristic morphological changes and cell death. One characteristic feature of apoptotic cells is a marked elevation in the levels of DNA fragmentation. Pseudodeflectusin induced apoptosis as detected by the TUNEL assay. Given that apoptosis inducers often display anticancer activity, this compound and related

Scheme 4. Reduction of 18 with BH3

penicisochromans C and D. The relative configuration of natural ustusorane D was determined through a comparison of the NMR data of our synthetic ustusorane D with those of the natural product. Although both natural and synthetic ustusorane D have negative optical rotations, the specific rotation value of natural ustusorane D is almost zero {[α]20D −1 (c 0.1, MeOH)}. Thus, it remains unclear whether natural ustusorane is optically active or racemic. Given that the specific rotation of natural penicisochroman B was the opposite of that of our synthetic product, the absolute configuration of natural E

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Table 5. NMR Spectroscopic Data for Natural Penicisochroman D (6) and Synthetic 19 and 20 6 position

δH (J in Hz)

1 2 3 4 4a 5

156.2, 115.0, 129.0, 122.5, 137.7, 41.8,

C CH CH CH C CH2

6 8

69.1, 58.5,

CH CH2

124.9, 23.4,

C CH3

8a 9 1-OH

19

δC, type

6.74 d (7.8) 7.11 t (7.8) 6.71 d (7.8) 2.75 2.74 3.92 4.85 4.79

dd (12.5, 2.4) dd (12.5, 10.0) d br q (10.0, 6.0) d (13.2) d (13.2)

1.24 d (6.0)

δC, type

20 δH (J in Hz)

151.3, 112.1, 126.9, 120.9, 135.5, 35.6,

C CH CH CH C CH2

70.5, 64.3,

CH CH

121.7, 21.5,

C CH3

6.56 d (8.0) 7.03 t (8.0) 6.69 d (8.0) 2.69 d (6.4) 3.80 h (6.4) 4.81 d (12.4) 4.75 d (12.4) 1.36 d (6.4) 5.12 brs

δC, type

δH (J in Hz)

156.0, 114.8, 129.0, 122.4, 138.0, 41.7,

C CH CH CH C CH2

69.1, 57.9,

CH CH2

125.0, 23.3,

C CH3

6.70 d (7.6) 7.03 t (7.6) 6.69 d (7.6) 2.72−2.74 m 3.88 h (6.4) 4.80 d (12.8) 4.73 d (12.8) 1.24 d (6.4)

Table 6. IC50 Values of Synthetic Derivatives against the Viability of HCT116 Cellsa compound (+)-1 (+)-2 7 14 15 16 17 20 camptothecinb

IC50 (μM) 5.0 5.9 9.8 >100 >100 >100 >100 >100 0.016

± 0.1 ± 0.1 ± 0.1

± 0.004

These data represent mean values ± SD (n = 3). Cytotoxicity was measured by cell death assay (WST-8). Briefly, cells were seeded at 5 × 103/well in a 96-well plate in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ mL), and streptomycin (100 μg/mL). The compound was added to the medium, and the cells were incubated at 37 °C for 72 h. Cell viability was then measured using WST-8. Each assay was performed in triplicate. IC50 is the concentration of compound that reduced cell viability by 50% compared with untreated cells. bCamptothecin was used as a positive control. a

synthetic isochroman derivatives represent potential anticancer chemotherapy agents.11

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting point data were determined with a Yanaco MP-3S instrument (Kyoto, Japan) and were uncorrected. Optical rotations were recorded on a JASCO P-1010 digital polarimeter (Tokyo, Japan). UV spectra were measured on a JASCO UV V-650 spectrophotometer (Tokyo, Japan). IR spectra were recorded on a Horiba FT210 spectrometer (Kyoto, Japan), using NaCl (neat) or KBr pellets (solid). 1H and 13C NMR spectra were recorded on a Bruker Biospin Avance 400 (400 and 100 MHz, respectively) spectrometer (Rheinstetten, Germany) using CDCl3 as the solvent. Unless otherwise noted, the chemical shift values have been expressed in δ (ppm) with TMS and CDCl3 used as the internal references for the 1H and 13C NMR, respectively. The NMR data have been reported as follows: chemical shifts, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet); coupling constants (J in Hz); and integration. Signals marked with an asterisk are from the minor diastereomer. Electrospray-ionization mass spectra (ESIMS) were obtained on an Applied Biosystems mass spectrometer (APIQSTAR Pulsar i, Foster, CT, USA) under conditions of high resolution, using poly(ethylene glycol) as the internal standard. Analytical TLC was performed on silica gel 60 F254 plates (0.5 mm,

Figure 4. Apoptotic effect of pseudodeflectusin {(+)-1} on HCT116 cells. (A) Apoptotic cell number. Apoptotic cells treated with (+)-1 (5.0 μM, based on the IC50 value) or 5.0 μM etoposide, which is a positive control, were individually counted from a total of at least 200 cells (for the different conditions). Values are shown as the mean ± SD from three independent experiments. (B) Photographs of apoptotic cells stained and detected by the TUNEL assay using an ApopTag Red in Situ apoptosis detection kit. The HCT116 cells were incubated for 72 h in the absence (control) or presence of 5.0 μM (+)-1. The white bar is 10 μm.

Merck). Flash column chromatography was performed on a SilicaFlash F60 column (230−400 mesh). Hydrogenation of Compound 7. A solution of 74 (557 mg, 2.16 mmol) and 20% Pd(OH)2/C (61 mg) in THF (55 mL) was stirred at rt under H2 atmosphere for 2 h. The reaction mixture was then filtered through Celite and washed with EtOAc. The filtrate was concentrated, and the resulting residue was purified by flash column chromatography F

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was then quenched by the addition of H2O, and the mixture was diluted with EtOAc to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by preparative TLC (hexane−EtOAc, 2:1, v/v) to give 11b (19.1 mg, 73%) as an amorphous solid: [α]25D +45.6 (c 0.71, CHCl3); UV (MeOH) λmax (log ε) 209 (4.49), 314 (3.77) nm; IR (film) νmax 3019, 2976, 1734, 1724, 1610, 1448, 1385, 1348, 1271, 1241, 1174, 1120, 1057 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.67 (1H, d, J = 7.6 Hz, H-4), 7.40−7.28 (5H, m, Ph MTPA), 6.77 (1H, d, J = 7.6 Hz, H-5), 6.30 (1H, d, J = 5.6 Hz, H-3), 4.59 (1H, m, H-7), 4.21 (1H, dd, J = 10.4, 5.6 Hz, H-2), 3.38 (3H, brq, JH−F = 1.2 Hz, OCH3), 2.95 (1H, dd, J = 16.4, 10.8 Hz, H-6a), 2.88 (1H, dd, J = 16.4, 3.6 Hz, H-6b), 2.20 (1H, m, H-11), 1.51 (3H, d, J = 6.4 Hz, H-10), 1.20 (3H, d, J = 6.8 Hz, H-12), 0.77 (3H, d, J = 6.4 Hz, H-13); 13C NMR (CDCl3, 100 MHz) δ 166.4 (C, CO2 MTPA), 162.3 (C, C-9), 162.2 (C, C-9b), 143.1 (C, C-5a), 132.2 (CH, C-4), 131.9 (C, Ph MTPA), 129.6 (CH, Ph MTPA), 128.3 (2C, CH, Ph MTPA), 126.9 (2C, CH, Ph MTPA), 126.7 (C, C-3a), 123.2 (q, JC−F = 288 Hz, C, CF3), 119.5 (CH, C-5), 109.5 (C, C-9a), 92.6 (CH, C-2), 84.0 (q, JC−F = 28 Hz, C, CF3C), 74.7 (CH, C-7), 74.1 (CH, C-3), 55.4 (CH3, OCH3), 35.5 (CH2, C-6), 27.0 (CH, C-11), 20.8 (CH3, C-10), 20.0 (CH3, C-12), 18.6 (CH3, C-13); HRESIMS m/z 501.1486 (calcd for C25H25O6F3Na, 501.1495). (2S,3S,7R)-6,9-Dihydro-7-methyl-2-(1-methylethyl)-3-trimethylsilyloxy-7H-furo[3,2-h][2]benzopyran-9(2H)-one (12). Trimethylsilyl chloride (110 μL, 0.87 mmol) was added to a stirred solution of 8 (174 mg, 0.66 mmol) and imidazole (104 mg, 1.53 mmol) in CH2Cl2 (5 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 30 min. The reaction was then quenched by the addition of H2O, and the mixture was diluted with CHCl3 to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by flash column chromatography (hexane−EtOAc, 4:1, v/v) to give 12 (197 mg, 89%) as white solids: mp 80−82 °C; [α]17D −25.6 (c 1.0, CHCl3); UV (MeOH) λmax (log ε) 211 (4.41), 316 (3.83) nm; IR (KBr) νmax 2958, 2906, 2875, 1724, 1612, 1473, 1446, 1389, 1346, 1279, 1250, 1173, 1126, 1086, 1061, 1023, 1022 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.40 (1H, d, J = 7.6 Hz, H-4), 6.71 (1H, d, J = 7.6 Hz, H-5), 5.12 (1H, d, J = 5.2 Hz, H-3), 4.57 (1H, m, H-7), 4.03 (1H, dd, J = 9.2, 5.2 Hz, H-2), 2.91 (1H, dd, J = 16.0, 9.6 Hz, H-6a), 2.86 (1H, dd, J = 16.0, 4.4 Hz, H-6b), 2.39 (1H, m, H-11), 1.49 (3H, d, J = 6.4 Hz, H-10), 1.25 (3H, d, J = 6.4 Hz, H-12), 1.07 (3H, d, J = 6.8 Hz, H-13), 0.11 (9H, s, TMS); 13C NMR (CDCl3, 100 MHz) δ 162.6 (C, C-9), 161.7 (C, C-4b), 141.3 (C, C-5a), 131.3 (C, C-3a), 130.3 (CH, C-4), 118.6 (CH, C-5), 109.1 (C. C-9a), 94.7 (CH, C-2), 74.6 (CH, C-7), 70.9 (CH, C-3), 35.5 (CH2, C-6), 26.9 (CH, C-11), 20.8 (CH3, C-10), 19.6 (CH3, C-12), 19.2 (CH3, C-13), 0.6 (3C, CH3, TMS); HRESIMS m/z 357.1489 (calcd for C18H26O4SiNa, 357.1492). (2S,3S,7R,9S)-6,9-Dihydro-3-hydroxy-9-methoxy-7-methyl-2-(1methylethyl)-2H,7H-furo[3,2-h][2]benzopyrane (13). DIBAL-H (600 μL of 1.02 M solution in toluene, 0.60 mmol) was added to a stirred solution of 12 (169 mg, 0.50 mmol) in CH2Cl2 (5 mL) at −78 °C, and the resulting mixture was stirred at −78 °C for 30 min. The reaction was then quenched by the addition of a saturated aqueous solution of Na2SO4. The mixture was then filtered through Celite and washed with CHCl3. The filtrate was concentrated. p-TsOH·H2O (10 mg, 53 μmol) was added to a stirred solution of the residue (155 mg) in MeOH (5 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 30 min. The reaction was then quenched by the addition of H2O, and the mixture was diluted with EtOAc to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by flash column chromatography (hexane−EtOAc, 5:1, v/v) to give 13 (96 mg, 68%) as white solids: mp 157 °C; [α]18D +18.4 (c 0.50, CHCl3); UV (MeOH) λmax (log ε) 287 (3.72) nm; IR (KBr) νmax 3437, 2956, 2924, 2827, 1626, 1599, 1450, 1413, 1389, 1363, 1313, 1294, 1274, 1194, 1155, 1122, 1097, 1051, 1026 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.25 (1H, d, J = 7.6 Hz, H-4), 6.57 (1H, d, J = 7.6 Hz, H-5), 5.41 (1H, s, H-9), 4.98 (1H, dd, J = 9.2, 5.2 Hz, H-3), 4.11 (1H, brm, H-7), 4.00 (1H, dd, J = 10.4, 5.6 Hz, H-2), 3.52 (3H, s,

(hexane−EtOAc, 2:1, v/v) to give 8 (281 mg, 50%) and 9 (229.6 mg, 41%), which were isolated as a 2:1 diastereomeric mixture. (2S,3S,7R)-6,9-Dihydro-3-hydroxy-7-methyl-2-(1-methylethyl)7H-furo[3,2-h][2]benzopyran-9(2H)-one (8): white solids; mp 125− 126 °C; [α]25D −102.2 (c 0.57, CHCl3); UV (MeOH) λmax (log ε) 211 (4.39), 320 (3.81) nm; IR (KBr) νmax 3383, 3016, 2979, 2873, 1710, 1612, 1473, 1446, 1387, 1348, 1279, 1217, 1174, 1122, 1061, 1022 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.55 (1H, d, J = 7.6 Hz, H-4), 6.77 (1H, d, J = 7.6 Hz, H-5), 5.04 (1H, dd, J = 8.8, 5.2 Hz, H-3), 4.58 (1H, m, H-7), 4.05 (1H, dd, J = 10.4, 5.2 Hz, H-2), 2.94 (1H, dd, J = 16.4, 9.6 Hz, H-6a), 2.86 (1H, dd, J = 16.4, 4.0 Hz, H-6b), 2.37 (1H, m, H-11), 1.60 (1H, brs, OH), 1.50 (3H, d, J = 6.4 Hz, H-10), 1.30 (3H, d, J = 6.8 Hz, H-12), 1.14 (3H, d, J = 6.8 Hz, H-13); 13C NMR (CDCl3, 100 MHz) δ 162.8 (C, C-9), 161.3 (C, C-9b), 141.7 (C, C5a), 131.1 (C, C-3a), 130.9 (CH, C-4), 119.1 (CH, C-5), 108.8 (C, C9a), 94.8 (CH, C-2), 74.7 (CH, C-7), 70.4 (CH, C-3), 35.4 (CH2, C6), 27.1 (CH, C-11), 20.7 (CH3, C-10), 20.0 (CH3, C-12), 19.0 (CH3, C-13); HRESIMS m/z 285.1089 (calcd for C15H18O4Na, 285.1097). 6,9-Dihydro-7-methyl-2-(1-methylethyl)-7H-furo[3,2-h][2]benzopyran-3,9(2H)-dione (9) (2:1 diastereomeric mixture): IR (KBr) νmax 3076, 2970, 2933, 2875, 1716, 1604, 1466, 1434, 1383, 1328, 1280, 1224, 1203, 1171, 1128, 1058, 1022 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.80 (1H, d, J = 7.6 Hz, H-4)*, 7.79 (1H, d, J = 7.6 Hz, H-4), 6.97 (1H, d, J = 7.6 Hz, H-5)*, 6.97 (1H, d, J = 7.6 Hz, H-5), 4.76−4.72 (1H, m, H-7)*, 4.73−4.68 (1H, m, H-7), 4.67 (1H, d, J = 4.0 Hz, H-2)*, 4.60 (1H, d, J = 4.0 Hz, H-2), 3.04 (2H, m, H6)*, 3.03 (2H, m, H-6), 2.43 (1H, m, H-11), 2.43 (1H, m, H-11)*, 1.56 (3H, d, J = 6.4 Hz, H-10)*, 1.55 (3H, d, J = 6.0 Hz, H-10), 1.18 (3H, d, J = 7.2 Hz, H-12), 1.16 (3H, d, J = 8.0 Hz, H-12)*, 0.98 (3H, d, J = 7.2 Hz, H-13), 0.94 (3H, d, J = 6.8 Hz, H-13)*; 13C NMR (CDCl3, 100 MHz) δ 200.2 (C, C-3), 200.1 (C, C-3)*, 173.0 (C, C9), 173.0 (C-9)*, 161.3 (C, C-9b)*, 161.1 (C, C-9b), 150.1 (C-5a)*, 150.0 (C-5a), 129.1 (CH, C-4)*, 129.0 (CH, C-4), 122.8 (C, C-3a), 122.8 (C, C-3a)*, 120.8 (CH, C-5), 120.8 (CH, C-5)*, 111.2 (C, C9a), 111.1 (C, C-9a)*, 90.8 (CH, C-2), 90.8 (CH, C-2)*, 74.3 (CH, C-7)*, 74.2 (CH, C-7), 35.9 (CH2, C-6)*, 35.8 (CH2, C-6), 31.1 (CH, C-11)*, 31.1 (CH, C-11), 20.6 (CH3, C-10), 20.6 (CH3, C-10)*, 18.2 (CH3, C-12), 18.2 (CH3, C-12)*, 15.9 (CH3, C-13), 15.9 (CH3, C13)*; HRESIMS m/z 283.0940 (calcd for C15H16O4Na, 283.0940). (S)-MTPA Ester 11a. Et3N (12 μL, 86.1 μmol), (R)-MTPA chloride (12 μL, 64.1 μmol), and DMAP (0.8 mg, 6.5 μmol) were added sequentially to a stirred solution of 8 (11.7 mg, 44.6 μmol) in CH2Cl2 (2 mL) at rt, and the resulting mixture was stirred for 1 h. The reaction was then quenched by the addition of H2O, and the mixture was diluted with EtOAc to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by preparative TLC (hexane−EtOAc, 2:1, v/v) to give 11a (14.8 mg, 69%) as an amorphous solid: [α]25D +37.2 (c 0.56, CHCl3); UV (MeOH) λmax (log ε) 210 (4.50), 315 (3.82) nm; IR (film) νmax 3016, 2975, 2938, 2877, 1745, 1728, 1614, 1446, 1389, 1350, 1275, 1169, 1122, 1061 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.64 (1H, d, J = 7.6 Hz, H-4), 7.40−7.30 (5H, m, Ph MTPA), 6.74 (1H, d, J = 7.6 Hz, H-5), 6.32 (1H, d, J = 5.6 Hz, H-3), 4.57 (1H, m, H-7), 4.23 (1H, dd, J = 10.4, 5.6 Hz, H-2), 3.39 (3H, brq, JH−F = 1.2 Hz, OCH3), 2.94 (1H, dd, J = 16.0, 9.6 Hz, H-6a), 2.87 (1H, dd, J = 16.0, 4.0 Hz, H-6b), 2.33 (1H, m, H-11), 1.49 (3H, d, J = 6.4 Hz, H-10), 1.26 (3H, d, J = 6.4 Hz, H12), 0.95 (3H, d, J = 6.4 Hz, H-13); 13C NMR (CDCl3, 100 MHz) δ 166.6 (C, CO2 MTPA), 162.2 (C, C-9), 162.1 (C, C-9b), 143.1 (C, C5a), 132.1 (CH, C-4), 131.2 (C, Ph MTPA), 129.7 (CH, Ph MTPA), 128.4 (2C, CH, Ph MTPA), 127.5 (2C, CH, Ph MTPA), 126.1 (C, C3a), 123.1 (q, JC−F = 288 Hz, C, CF3), 119.5 (CH, C-5), 109.4 (C, C9a), 92.4 (CH, C-2), 84.6 (q, JC−F = 28 Hz, C, CF3C), 74.7 (CH, C-7), 74.4 (CH, C-3), 55.3 (CH3, OCH3), 35.5 (CH2, C-6), 27.2 (CH, C11), 20.8 (CH3, C-10), 20.0 (CH3, C-12), 18.9 (CH3, C-13); HRESIMS m/z 501.1502 (calcd for C25H25O6F3Na, 501.1495). (R)-MTPA Ester 11b. Et3N (12 μL, 86.1 μmol), (S)-MTPA chloride (12 μL, 64.1 μmol), and DMAP (0.6 mg, 4.9 μmol) were added sequentially to a stirred solution of 8 (14.4 mg, 54.9 μmol) in CH2Cl2 (2 mL) at rt, and the resulting mixture was stirred for 2 h. The reaction G

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31.5 μmol) was added to a stirred solution of 16 (6.7 mg, 24.3 μmol) in THF and H2O (1:1, v/v, 2 mL), and the resulting mixture was stirred for 19 h. The reaction was then quenched by the addition of H2O, and the mixture was diluted with EtOAc to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by preparative TLC (hexane−EtOAc, 3:1, v/v) to give 17 (5.6 mg, 88%) as white solids: mp 132−135 °C; [α]25D +93.4 (c 0.23, CHCl3); UV (MeOH) λmax (log ε) 215 (4.38), 259 (4.03), 328 (3.70) nm; IR (KBr) νmax 3406, 3018, 2969, 2930, 1703, 1615, 1497, 1438, 1367, 1338, 1216, 1114, 1087, 1022 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Table 3; 1H NMR (CDCl3−CD3OD, 1:1, 400 MHz) and 13C NMR (CDCl3−CD3OD, 1:1, 100 MHz) see Table 4; HRESIMS m/z 285.1092 (calcd for C15H18O4Na, 285.1097). Reduction of (R)-Mellein. BH3 (0.7 mL, 1.1 M solution in THF, 0.77 mmol) was added to a stirred solution of (R)-mellein (18)4b (51.1 mg, 0.29 mmol) in THF (3.0 mL) at 0 °C, and the resulting mixture was stirred at rt for 15 h. The reaction was then quenched by the addition of H2O, and the mixture was diluted with EtOAc and a 1 M aqueous HCl solution to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by flash column chromatography (hexane−EtOAc, 3:1, v/v, then CHCl3− MeOH, 5:1, v/v) to give 19 (7.8 mg, 14%) and 20 (51 mg, 80%). (R)-3,4-Dihydro-3-methyl-1H-2-benzopyran-8-ol (19): white solids; mp 148 °C; [α]24D −96.8 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 216 (3.99), 274 (3.30) nm; IR (KBr) νmax 3238, 2976, 2947, 2919, 2892, 2850, 1591, 1469, 1429, 1390, 1346, 1305, 1252, 1219, 1196, 1141, 1117, 1072, 1049, 1016, 989 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Table 5; HRESIMS m/z 163.0768 (calcd for C10H11O2, 163.0764). (R)-2-(Hydroxymethyl)(2′-hydroxypropyl)phenol (20): colorless oil; [α]24D −26.7 (c 0.30, CHCl3); UV (MeOH) λmax (log ε) 218 (1.18), 281 (3.42) nm; IR (neat) νmax 3305, 2970, 2927, 1608, 1587, 1464, 1373, 1352, 1288, 1269, 1188, 1120, 1061, 993 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Table 5; HRESIMS m/z 205.0830 (calcd for C10H14O3Na, 205.0835). Computational Details. Conformational analyses of 13, 9-epi-13, 15, 9-epi-15, 17, and 9-epi-17 were performed using the conformational search algorithm implemented in version 1.4.2 of the BARISTA software (Conflex Corp., Tokyo, Japan).12 The lower energy conformers of each compound, which differed from the most stable conformer by less than 10 kcal/mol, were optimized using DFT calculations at the B3LYP/6-31G(d,p) level, that were implemented in the Gaussian 09 program package.13 The lowest energy conformations of 13, 9-epi-13, 15, 9-epi-15, 17, and 9-epi-17 were determined by comparing the sum of the electronic and zero-point energies of each conformer. Cell Viability. HCT 116 cells were purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK) and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL). The growth of the HCT116 cells was evaluated using the cell counting kit according to the manufacturer’s instructions based on the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay.9 For the assay, the cells were cultured in a 96-well plate, with each well containing 5000 cells in a total volume of 100 μL. The concentration of DMSO in the cell cultures was 0.1% (v/v). The plates also included blank wells (0 cells/100 μL) and control wells (5000 cells/100 μL). The plates were incubated with each compound for 72 h. Ten microliters of the solution provided with the kit was then added, and the resulting mixture incubated for 2 h at 37 °C. The absorbance values were then measured at 450 nm with a 96-well plate reader (Amersham Pharmacia Biotech or Perkin-Elmer). Camptothecin (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control and exhibited an IC50 value of 0.016 ± 0.004 μM. Apoptosis Assay Using Immunofluorescence Microscopy. Aliquots of 2.5 × 104 cells were plated in each well of an eight-well

OCH3), 2.79 (1H, brs, OH), 2.49 (1H, brd, J = 16.4 Hz, H-6a), 2.31 (1H, m, H-11), 2.22 (1H, brm, H-6b), 1.25 (3H, d, J = 6.0 Hz, H-10), 1.24 (3H, d, J = 6.0 Hz, H-12), 1.12 (3H, d, J = 6.4 Hz, H-13); 13C NMR (CDCl3, 100 MHz) δ 157.2 (C, C-9b), 136.7 (C, C-5a), 127.9 (C, C-3a), 125.3 (CH, C-4), 120.2 (CH, C-5), 116.8 (C, C-9a), 95.3 (CH, C-9), 93.6 (CH, C-2), 71.5 (CH, C-3), 63.2 (CH, C-7), 55.6 (CH3, OCH3), 35.1 (CH2, C-6), 27.5 (CH, C-11), 20.9 (CH3, C-10), 19.6 (CH3, C-12), 19.3 (CH3, C-13); HRESIMS m/z 301.1403 (calcd for C16H22O4Na, 301.1410). (2S,7R,9S)-6,9-Dihydro-9-methoxy-7-methyl-2-(1-methylethyl)7H-furo[3,2-h][2]benzopyran-3(2H)-one (14). MnO2 (646 mg, 85%, 646 mmol) was added to a solution of 13 (87.9 mg, 0.32 mmol) in CH2Cl2 (5 mL), and the resulting mixture was stirred for 10 h. MnO2 (655 mg, 85%, 6.40 mmol) was then added to the mixture, and the resulting mixture was stirred for 5 h. The reaction mixture was then filtered through Celite and washed with CHCl3. The filtrate was concentrated, and the resulting residue was purified by flash column chromatography (hexane−EtOAc, 5:1, v/v) to give 14 (44.3 mg, 51%) as a colorless oil: [α]17D −67.9 (c 0.61, MeOH); UV (MeOH) λmax (log ε) 214 (4.41), 258 (4.09), 326 (3.87) nm; IR (neat) νmax 2968, 2931, 2900, 2827, 1714, 1618, 1603, 1495, 1464, 1439, 1389, 1369, 1336, 1290, 1220, 1188, 1155, 1099, 1051, 1028 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.51 (1H, d, J = 7.6 Hz, H-4), 6.81 (1H, d, J = 7.6 Hz, H-5), 5.68 (1H, s, H-9), 4.54 (1H, d, J = 3.6 Hz, H-2), 4.33 (1H, m, H-7), 3.61 (3H, s, OCH3), 2.78 (1H, dd, J = 17.2, 3.2 Hz, H6a), 2.69 (1H, dd, J = 17.2, 10.8 Hz, H-6b), 2.36 (1H, dh, J = 4.0, 6.8 Hz, H-11), 1.40 (3H, d, J = 6.0 Hz, H-10), 1.15 (3H, d, J = 6.8 Hz, H13), 0.86 (3H, d, J = 6.8 Hz, H-12); 13C NMR (CDCl3, 100 MHz) δ 201.1 (C, C-2), 170.9 (C, C-9b), 145.5 (C, C-5a), 123.5 (C, C-4), 122.1 (CH, C-5), 120.2 (CH, C-3a), 119.6 (C, C-9a), 94.3 (CH, C-9), 90.1 (CH, C-2), 62.5 (CH, C-7), 55.5 (CH3, OCH3), 35.9 (CH2, C6), 31.1 (CH, C-11), 21.1 (CH3, C-10), 18.7 (CH3, C-13), 15.7 (CH3, C-12); HRESIMS m/z 299.1267 (calcd for C16H20O4Na, 299.1253). (2S,7R,9S)-6,9-Dihydro-9-hydroxy-7-methyl-2-(1-methylethyl)7H-furo[3,2-h][2]benzopyran-3(2H)-one (15). p-TsOH·H2O (7.6 mg, 40.0 μmol) was added to a stirred solution of 14 (9.8 mg, 35.5 μmol) in THF and H2O (1:1, v/v, 2 mL), and the resulting mixture was stirred for 12 h. The reaction was then quenched by the addition of H2O, and the mixture was diluted with EtOAc to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by preparative TLC (hexane−EtOAc, 4:1, v/v) to give 15 (8.6 mg, 92%) as white solids: mp 125−127 °C; [α]17D −117.2 (c 0.25, MeOH); [α]18D −167.2 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 215 (4.36), 259 (4.03), 327 (3.80) nm; IR (KBr) νmax 3350, 2970, 2929, 2881, 1680, 1614, 1498, 1437, 1367, 1336, 1311, 1284, 1215, 1173, 1145, 1117, 1088, 1065, 1024 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Table 1; 1H NMR (CDCl3−CD3OD, 1:1, 400 MHz) and 13C NMR (CDCl3−CD3OD, 1:1, 100 MHz) see Table 4; HRESIMS m/z 285.1096 (calcd for C15H18O4Na, 285.1097). (2R,7R,9S)-6,9-Dihydro-9-methoxy-7-methyl-2-(1-methylethyl)7H-furo[3,2-h][2]benzopyran-3(2H)-one (16). NaOMe (42 μL of a 0.44 M solution in MeOH, 0.18 mmol) was added to a stirred solution of 14 (5.0 mg, 0.18 mmol) in MeOH (1 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 20 min. The reaction was then quenched by the addition of 1 M aqueous HCl solution, and the mixture was diluted with EtOAc to give a biphasic solution. The organic layer was then collected and washed with brine before being dried over Na2SO4 and concentrated to a residue, which was purified by preparative TLC (hexane−EtOAc, 5:1, v/v) to give 16 (2.3 mg, 46%) and recovered 14 (2.3 mg, 46%). 16: white solids; mp 70−72 °C; [α]22D +54.9 (c 0.21, CHCl3); UV (MeOH) λmax (log ε) 215 (4.31), 258 (3.96), 327 (3.74) nm; IR (KBr) νmax 2966, 2927, 2875, 1716, 1618, 1599, 1495, 1462, 1441, 1371, 1331, 1290, 1245, 1217, 1192, 1163, 1146, 1117, 1095, 1051, 1028 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Table 2; HRESIMS m/z 299.1249 (calcd for C16H20O4Na, 299.1253). (2R,7R,9S)-6,9-Dihydro-9-hydroxy-7-methyl-2-(1-methylethyl)7H-furo[3,2-h][2]benzopyran-3(2H)-one (17). p-TsOH·H2O (6.0 mg, H

dx.doi.org/10.1021/np400460m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

chamber slide (Nunc, Rochester, NY, USA). HCT116 cells were incubated with synthetic pseudodeflectusin {(+)-1} (5.0 μM, based on their IC50 value) or 5.0 μM etoposide (Sigma-Aldrich, St. Louis, MO, USA), as a positive control, for 72 h at 37 °C. TUNEL is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. The percentage of apoptotic cells was determined using the ApopTag Red in Situ apoptosis detection kit (CHEMICON, Temecula, CA, USA). The culture dishes were stained, and the percentage of apoptotic cells was examined under an Olympus IX70 fluorescence microscope (Tokyo, Japan).

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(11) For a recent review on isolations, biological activities, and syntheses of tricyclic benzofurans and related natural products, see: Simonetti, S. O.; Larghi, E. L.; Bracca, A. B.; Kaufman, T. S. Nat. Prod. Rep. 2013, DOI: 10.1039/C3NP70014C. (12) (a) Goto, H.; Osawa, E. J. Am. Chem. Soc. 1989, 111, 8950− 8951. (b) Goto, H.; Osawa, E. Tetrahedron Lett. 1992, 33, 1343−1346. (c) Goto, H.; Osawa, E. J. Chem. Soc., Perkin Trans. 2 1993, 187−198. (13) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision C.01; Gaussian, Inc.: Wallingford, CT, 2009.

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of all new compounds, selected NOESY correlations of compounds 8 and 16, selected HMBC correlations of compound 19, and procedure for acetylation of compound 20. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81-75-703-5603. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. V. Rukachaisirikul (Prince of Songkla University) for checking 1H and 13C NMR spectra of our synthetic compounds.

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DEDICATION This paper is dedicated to Professor Teruaki Mukaiyama in celebration of the 40th anniversary of the Mukaiyama aldol reaction.

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REFERENCES

(1) Ogawa, A.; Murakami, C.; Kamisuki, S.; Kuriyama, I.; Yoshida, H.; Sugawara, F.; Mizushina, Y. Bioorg. Med. Chem. Lett. 2004, 14, 3539−3543. (2) Lu, Z.; Wang, Y.; Miao, C.; Liu, P.; Hong, K.; Zhu, W. J. Nat. Prod. 2009, 72, 1761−1767. (3) Trisuwan, K.; Rukachaisirikul, V.; Sukpondma, Y.; Phongpaichit, S.; Preedanon, S.; Sakayaroj, J. Tetrahedron 2010, 66, 4484−4489. (4) (a) Saito, F.; Kuramochi, K.; Nakazaki, A.; Mizushina, Y.; Sugawara, F.; Kobayashi, S. Eur. J. Org. Chem. 2006, 4796−4799. (b) Kuramochi, K.; Saito, F.; Nakazaki, A.; Takeuchi, T.; Tsubaki, K.; Sugawara, F.; Kobayashi, S. Biosci. Biotechnol. Biochem. 2010, 74, 1635−1640. (c) Sato, Y.; Kuramochi, K.; Suzuki, T.; Nakazaki, A.; Kobayashi, S. Tetrahedron Lett. 2011, 52, 626−629. (5) Efficient syntheses of (±)-1 have been reported by other groups; see: (a) Tobe, M.; Tashiro, T.; Sasaki, M.; Takikawa, H. Tetrahedron 2007, 63, 9333−9337. (b) Maegawa, T.; Otake, K.; Hirosawa, K.; Goto, H.; Fujioka, H. Org. Lett. 2012, 14, 4798−4801. (6) Kohno, J.; Sakurai, M.; Kameda, N.; Nishio, M.; Kawano, K.; Kishi, N.; Okuda, T.; Komatsubara, S. J. Antibiot. 1999, 52, 913−916. (7) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (8) The absolute configuration at C-9 in 13 and 14 was also confirmed to be S by NOESY correlations between H-7 and the methoxy group at C-9 in 16. (9) Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. Anal. Commun. 1999, 36, 47−50. (10) Green, D. R. Means to an End: Apoptosis and Other Cell Death Mechanisms; Cold Spring Harbor Laboratory Press: New York, 2011. I

dx.doi.org/10.1021/np400460m | J. Nat. Prod. XXXX, XXX, XXX−XXX