Bioactive Sesterterpenoids from a Korean Sponge Monanchora sp

Jan 29, 2013 - Research Institute of Oceanography, Seoul National University, Korea. §. Queensland Museum, Queensland, Australia. ⊥. College of ...
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Bioactive Sesterterpenoids from a Korean Sponge Monanchora sp. Weihong Wang,† Bora Mun,† Yehee Lee,† Mallepally Venkat Reddy,† Youngmin Park,† Jihye Lee,† Hiyoung Kim,† Dongyup Hahn,† Jungwook Chin,†,‡ Merrick Ekins,§ Sang-Jip Nam,*,⊥ and Heonjoong Kang*,†,‡ †

Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul, 151-747, Korea ‡ Research Institute of Oceanography, Seoul National University, Korea § Queensland Museum, Queensland, Australia ⊥ College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, Suncheon, 540-950, Korea S Supporting Information *

ABSTRACT: Chemical investigation of a Korean marine sponge, Monanchora sp., yielded nine new sesterterpenoids (1−9) along with phorbaketals A−C (10−12). The planar structures were established on the basis of NMR and MS analysis, and the absolute configurations of 1−9 were defined using the modified Mosher’s method and CD spectroscopic data analysis. Compounds 1−8, designated as phorbaketals D−K, possess a spiroketal-modified benzopyran moiety such as phorbaketal A, and their structural variations are due to oxidation and/or reduction of the tricyclic core or the side chain. Compound 9, designated as phorbin A, has a monocyclic structure and is proposed to be a possible biogenetic precursor of the phorbaketals. Compounds 1−9 were evaluated for cytotoxicity against four human cancer cell lines (A498, ACHN, MIA-paca, and PANC-1), and a few of them were found to exhibit cytotoxic activity.

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arine sponges of the genus Monanchora have been proven to be rich sources of novel secondary metabolites, exhibiting diverse biological activities. The major group of these metabolites is guanidine alkaloids, which were isolated from different Monanchora species including M. arbuscula,1−6 M. unguiculata,3 M. sp.,7−9 M. dianchora,10,11 M. unguifera,12−14 M. calla,5 and M. pulchra.15−17 These metabolites showed a broad range of biological activities including cytotoxic,5,10,13,15,17,18 apoptosis-inducing,15 antifungal,8 antibacterial,8,14 antiviral,7,14 antiprotozoal,5,8,14 antimalarial,5,8 and antitumor properties.6,16 Marine sponges of the genus Monanchora have also yielded a small number of non-nitrogenous metabolites, such as sterols.18 During the course of our search for new secondary metabolites from Korean marine organisms, we encountered one Monanchora species (class Demospongiae, order Poecilosclerida, family Crambeidae). Chemical investigation of this sponge led to the discovery of nine new sesterterpenoids (1−9) along with phorbaketals A−C (10−12),19 which represent the first examples of nonsterol terpenoids isolated from marine sponges of the genus Monanchora. Herein, we report the isolation, structure elucidation, and biological properties of nine sesterterpenoids with a phorbaketal carbon skeleton.

+ Na]+ in the high-resolution FABMS spectrum. Absorption bands at 3394 and 1678 cm−1 in the IR spectrum of 1 indicated the presence of hydroxy and α,β-unsaturated ketone carbonyl

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RESULTS AND DISCUSSION Phorbaketal D (1) has the molecular formula C25H34O6, as determined by a pseudomolecular ion peak at m/z 453.2250 [M © 2013 American Chemical Society and American Society of Pharmacognosy

Received: August 21, 2012 Published: January 29, 2013 170

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Figure 1. Circular dichroism (CD) spectra of phorbaketals A−C, compounds 1−9, and the synthetic standard compound of 6 (c = 4.0 × 10−4 M, MeOH, 23 °C).

groups, respectively. The NMR data of 1 were comparable to those of phorbaketal A,19 suggesting that the natural product likewise possesses a modified benzopyran-containing tricyclic ring system. However, differences were observed in the unsaturated side chain of the molecule. The 1H NMR spectrum of 1 revealed the presence of two coupled olefinic signals (J = 15.9 Hz) centered at δ 5.61 and 5.66 due to a 1,2-disubstituted double bond (Δ21E) instead of a triplet assigned to the trisubstituted double bond (Δ22E) in phorbaketal A. Accordingly, a doubly allylic proton signal was observed at δ 2.77, at higher field than the two allylic proton signals in phorbaketal A. Compared with phorbaketal A, an additional oxygenated quaternary signal (δ 82.3) was observed in the 13C NMR spectrum. The HMBC correlations from the two olefinic protons (H-21 and H-22) and a six-proton aliphatic methyl signal (δ 1.29) to this quaternary carbon placed it at C-23. Analysis of the NMR data of the side chain and comparison with those of quadrangularic acid F20 suggested the presence of a hydroperoxy group at C-23, which was further confirmed by hydroperoxide reduction of 1 to 1a with triphenylphosphine. The 1H and 13C NMR spectra of 1a were almost identical to those of 1 except for the upfield-shifted carbon chemical shift of C-23 (δ 71.0). The relative configuration of 1 was assigned by the analysis of ROESY cross-peaks (Figure 2) and coupling constants. The cis A/B ring junction was derived from the small coupling constant (J = 3.4 Hz)21 between H-1 and H-7 and the strong NOE correlation of H-1 with H-7. A clear ROESY correlation between olefinic proton H-12 in ring C and proton H-6b in ring A indicated the relative configuration of the spiro junction of rings B and C, while the ROESY correlations of Me-19/H-1, Me-19/ H-2, and Me-19/H-16 showed the orientation of the side chain at C-16 as shown. High-resolution FABMS of phorbaketal E (2) provided the molecular formula C25H34O5. The 1H NMR spectrum revealed

Figure 2. Key NOE correlations observed for phorbaketal D (1).

that it is closely related to compound 1. The obvious difference is the presence of a carbonyl group as revealed by 13C NMR data (δ 216.6) and the absence of a pair of olefinic proton signals compared with compound 1. The location of the carbonyl group at C-22 was determined by the three-bond HMBC correlations of H3-24/H3-25 (δ 1.08) and H-20 (δ 2.28) and by the two-bond HMBC correlations from H-23 (δ 2.68) and H-21 (δ 2.66) to C22 (δ 216.6). The absolute configurations of compounds 1 and 2 could be established by comparison of the CD data with that of phorbaketal A (10), as they share the same tricyclic ring moiety. Both compounds 1 and 2 showed a positive Cotton effect at 212 nm (π → π*) and a negative Cotton effect at 234 nm (π → π*), having the same sign as that of phorbaketal A (10), which 171

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5 with R-(−)- and S-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride yielded (S)- and (R)-MTPA esters, respectively. The C-5 configuration was assigned as R on the basis of the Δδ values of the (S)- and (R)-MTPA esters, which showed negative values for H-2 and H3-4 and positive values for H-6, H-7, H-9, and H-10 (Figure 3).

suggested that they had the same absolute configuration as phorbaketal A (Figure 1). A molecular formula of C 27 H 38 O 7 was deduced for phorbaketal F (3) from the interpretation of its HRFABMS data. The NMR features of 3 closely resemble those of compound 1. The difference is the presence of an acetoxy group instead of the carbonyl group at C-5. The presence of characteristic NMR signals (δH 2.08, δC 21.1; δC 172.6) and the three-bond HMBC correlations from a methyl singlet H-4 and an olefinic proton H-2 to the oxygenated carbon C-5 permitted the placement of an acetoxy group at C-5 (δC 73.3). The large coupling constants of H-6b [δ 1.56 (ddd, 12.1, 11.6, 11.6 Hz)] with its two adjacent protons revealed the axial position of both H-5 and H-7, which was also supported by the strong NOE correlation between H-5 and H-7. Further analysis of the ROESY data allowed the assignment of the same relative configurations as that of phorbaketal B (11). Phorbaketal G (4) has the same molecular formula, C27H38O7, as 3 on the basis of HRFABMS data and was deduced to be a C-5 epimer of the latter. Accordingly, H-5 was coupled to H-6a and H-6b by 2.4 and 4.0 Hz, respectively, which was typical for an equatorial proton coupled with two other equatorial or axial protons. Due to a steric-compression effect, the 13C NMR signals of C-5 and C-7 were upfield shifted by 2.1 and 3.7 ppm, respectively, compared with those of compound 3. The absolute configurations of C-1, C-7, C-11, and C-16 for compounds 3 and 4 were determined as 1R, 7R, 11R, and 16S by comparing the shape of the CD curves with those of phorbaketals B (11) and C (12), respectively (Figure 1). High-resolution FABMS of phorbaketal H (5) established its molecular formula as C25H34O4, which is identical to that of phorbaketal A. However, differences were observed in the 1H and 13 C NMR data of the tricyclic ring moiety. Further investigation of the 2D NMR data revealed that the primary hydroxy group at C-9 in ring B was oxidized into an aldehyde group and the ketone group in ring A was replaced by a hydroxy group. The downfieldshifted resonance at δ 9.49, a typical aldehyde proton signal, showed HMBC correlations with three neighboring carbons, C-7 (δ 31.6), C-8 (δ 144.6), and C-10 (δ 148.7). The oxymethine proton (δ 4.11) attached to C-5 (δ 70.3) via an HSQC correlation showed HMBC correlations with five neighboring carbons, C-2 (δ 122.7), C-3 (δ 146.0), C-4 (δ 19.3), C-6 (δ 33.0), and C-7 (δ 31.6). The analysis of coupling constants and NOESY correlations suggested that compound 5 had the same relative configurations for C-1, C-7, C-11, and C-16 as those of phorbaketal A. Strong NOE correlations between H-1/H-7 indicated the cis configuration of the A/B ring junction. The same configuration of the spiro junction of rings B and C with phorbaketal B (11) for 5 was also determined by NOE correlations from the olefinic proton H10 and the methylene proton H-6b to the olefinic proton H-12. Furthermore, the NOE correlation between H-17 and H-15a (δ 2.06) indicated that H-15a had an anti relationship with H-16. Lastly, the large coupling constants for H-6b [δH 1.45 (ddd, 12.3, 11.6, 11.6 Hz)] and the NOE correlation between H-7 and H-5 suggested that H-6b should be positioned trans to H-5 and H-7. In order to determine the absolute configuration of 5, we first applied CD spectroscopic data analysis. But it is difficult to determine the configuration of 5 because 5 had a different CD Cotton effect compared to that of phorbaketal B (11) possibly due to the influence of the α,β-unsaturated aldehyde group. Therefore, the absolute configuration of 5 was elucidated by application of the modified Mosher’s method.22 Esterification of

Figure 3. Delta values (ΔδS−R) in ppm for (S)- and (R)-MTPA esters of compound 5 in CDCl3.

The molecular formula of phorbaketal I (6) was deduced as C25H32O4 on the basis of the HRFABMS data. The examination of the 1H NMR spectrum revealed that it is comparable to that of phorbaketal A. The difference is the presence of an aldehyde group, which was indicated by the 13C NMR resonance at δ 194.3 and 1H NMR resonance at δ 9.51. The placement of the aldehyde group at C-9 was corroborated by the HMBC correlations from the aldehyde proton to carbons C-7 (δ 31.9), C-8 (δ 143.4), and C-10 (δ 148.6). The downfield shift of H-7 to δ 2.96 (δ 2.59 in phorbaketal A19) can be explained by the anisotropic influence of the C-9 aldehyde group. Determination of the absolute configuration of compound 6 was secured by comparison of the CD spectrum with that of the synthetic standard compound prepared by oxidation of phorbaketal A using Dess-Martin periodinane. The molecular formula of phorbaketal J (7) was established as C27H36O5 on the basis of the HRFABMS data and assigned as the C-5 acetate ester of compound 5, which was supported by a series of NMR experiments. High-resolution FABMS of phorbaketal K (8) provided the molecular formula C27H36O5. Compound 8 was shown to be the C-5 epimer of 7 by the 1H and 13C NMR data. The upfield shifts of C-5 and C-7 relative to those of compound 7 were observed for compound 8, which is similar to the case of compound 4 relative to compound 3. The absolute configurations of compounds 7 and 8 were established by comparison of the CD data with that of compound 5 (Figure 1). The HRFABMS data for phorbin A (9) gave a pseudomolecular ion peak at m/z 409.2709 [M + Na]+ corresponding to C25H38O3Na. All 25 carbons and 36 protons attached to carbons were observed in the 13C and 1H NMR spectra. The presence of a ketone group was indicated by the 13C NMR resonance at δ 202.5 and the IR absorption at 1677 cm−1. The IR spectrum also revealed a broad band attributable to hydroxy groups at 3414 cm−1. The 1H NMR spectrum revealed the presence of four trisubstituted olefinic signals at δ 6.85, 5.17, 5.12, and 5.10 and two terminal methylene signals at δ 5.06 and 4.88. One ketone group and five double bonds accounted for six of the seven sites of unsaturation required by the molecular formula and indicated that compound 9 contained one ring. Compound 9 had the same basic carbon skeleton of phorbaketal, and the major difference was that it did not further cyclize to form the tricyclic ring moiety of phorbaketal after the initial cyclization of the isoprenoid precursor. Further analysis of the 2D NMR data established the planar structure as shown. The small coupling constant (J = 1.8 Hz) between the protons H-1 (δ 4.43) and H-7 (δ 2.81) indicated the cis configuration of 172

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compound in searching for new drug leads to meet the urgent need of developing new drugs against pancreatic cancer.

H-1 and H-7, which was also supported by the strong ROESY correlation between H-1 and H-7. The absolute configurations for C-1 and C-11 in compound 9 were determined by modified Mosher’s method. Treatment of 9 with an excess of MTPA-Cl in pyridine resulted in bis-MTPA esters; however, inconsistencies were observed for ΔδS−R values (data not shown). Furthermore, we observed that the bis-MTPA esters slowly decomposed at room temperature. One of the products was identified as the 1-OMTPA ester of 9. The calculation of the ΔδS−R values of 1-OMTPA esters of 9 permitted assignment of the R configuration for C-1 (Figure 4). The mono-Mosher’s esters, 11-O-MTPA

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured in MeOH using a 1.0 cm cell on a Rudolph Research Autopol III. UV spectra were obtained with a Hitachi JP/U-3010 UV spectrophotometer. CD spectra were taken in MeOH using a Chirascan plus circular dichroism detector. IR spectra were acquired on a JASCO FT/IR 4200 spectrophotometer. All NMR spectra were recorded on Bruker Avance DPX-500 or DPX-600 or Ascend 700 spectrometers using methanol-d4 or CDCl3 as the solvent. Chemical shifts were reported with reference to the respective solvent peaks [δH 3.31 and δC 49.0 for CD3OD; δH 7.26 and δC 77.2 for CDCl3]. Electrospray ionization source (ESI) low-resolution mass spectra were recorded on an Agilent Technologies 6120 Quadrupole mass spectrometer coupled with an Agilent Technologies 1260 series HPLC. High-resolution mass data were collected on a Jeol JMS-700 double-focusing (B/E configuration) instrument. Animal Material. Specimens of the sponge Monanchora sp. (QM G331990) were collected by scuba diving at 15−20 m depth off the shore of Gageo Island, southwestern Korea, in July 2009. The specimens were massive (120 mm ×100 mm × 80 mm) with an undulating surface covered in small papillae, with numerous scattered oscula (0.5−4 mm in diameter). The sponge was bright orange externally and pale orange internally and turned dark brown in alcohol. The texture of the sponge was soft and compressible and could easily be torn or cut. The ectosomal skeleton was composed of a thin spongy layer encrusted with anchorate isochelae (30−40 μm). The choanosomal skeleton was composed of sponging containing spicules of tylostyles to subtylostyles (300−400 μm) as well as anchorate and unguiferous isochelae (30−40 μm), interspersed with aquiferous channels lined with unidirectional styles (300−400 μm). The specimen was taxonomically identified as Monanchora sp. 4831 (class Demospongiae, order Poecilosclerida, family Crambeidae) by Dr. Merrick Ekins from the Queensland Museum, Australia. A voucher specimen (QM G331990) is deposited at the Queensland Museum, Australia, and at the Center for Marine Natural Products and Drug Discovery, Seoul National University, Korea (registered as CMDD09A0401). Extraction and Isolation. The lyophilized specimens (dry wt 400 g) were cut into small pieces and extracted three times with 50% MeOH in CH2Cl2 at room temperature (rt). These extracts were combined and partitioned three times between H2O and CH2Cl2. The CH2Cl2 layer was further partitioned between aqueous MeOH (90%) and n-hexane to afford an aqueous MeOH-soluble fraction (17 g) and an n-hexanesoluble fraction (15.8 g). The aqueous MeOH fraction was subjected to reversed-phase silica gel flash column chromatography (YMC Gel ODSA, 120 Å, 40−60 μm), eluting with a step gradient solvent system of 50% to 0% H2O/MeOH and washing with acetone, to afford 14 fractions (1− 14). Compounds 1 (6.5 mg) and 2 (3.5 mg) were separated on a reversed-phase preparative HPLC column (Phenomenex Luna 10 μ C18 (2) 100 Å AXIA I 250 × 21.20 mm, 8.0 mL/min, 210 nm) eluting with 50% CH3CN, followed by purification using reversed-phase HPLC (Shiseido MGII C18 5 μm, 250 × 10 mm, 2.0 mL/min, UV detection at 210 nm) eluting with 48% CH3CN (retention time for 1: 50 min; retention time for 2: 57 min). Compound 10 (2.5 g) was purified on the above-mentioned reversed-phase preparative HPLC column (9.0 mL/ min, 210 nm) eluting with 65% CH3CN with a retention time of 43 min. Compounds 3 (2.0 mg), 4 (1.6 mg), and 9 (2.1 mg) were separated on the above-mentioned reversed-phase preparative HPLC column (8.0 mL/min, 210 nm) eluting with 50% CH3CN with retention times of 59, 48, and 75 min, respectively. Compound 5 (1.5 mg) was obtained by separation on the above-mentioned reversed-phase preparative HPLC column (8.0 mL/min, 210 nm) eluting with 60% CH3CN, followed by purification using reversed-phase HPLC (Phenomenex Polar-RP, 250 × 10 mm, 4 μm, 80 Å, 2.0 mL/min, 210 nm) eluting with 60% CH3CN (retention time: 20 min). Compounds 6 (1.6 mg), 7 (1.4 mg), and 8 (1.4 mg) were separated on the above-mentioned reversed-phase preparative HPLC column (8.0 mL/min, 210 nm) eluting with 65%

Figure 4. Delta values (ΔδS−R) in ppm for 1-O-(S)- and 1-O-(R)-MTPA esters of compound 9 in CDCl3.

esters of compound 9, were prepared by treatment of 9 with two equivalents of MTPA-Cl at 4 °C for 24 h and purified by RPHPLC. An analysis of the ΔδS−R values of 1H NMR data allowed the S configuration for C-11 to be assigned (Figure 5). Compound 9 ([α]20D −61) also shared the same 1R,7S configuration with that of revised phorbasin C ([α]22D −131), which was established by synthetic methods.23

Figure 5. Delta values (ΔδS−R) in ppm for 11-O-(S)- and 11-O-(R)MTPA esters of compound 9 in CDCl3.

Phorbaketals D−K (1−8) possess a spiroketal-modified benzopyran moiety as seen for phorbaketal A, and the structural variations arise from the oxidation and/or reduction of the tricyclic core or the side chain. Interestingly, phorbin A (9) has a monocyclic substructure and a linear side chain in the molecule. We propose that compound 9 is a possible biogenetic precursor of phorbaketals based on structural analogy. Compounds 1−9 were evaluated for cytotoxic activity against four human cancer cell lines (Table 3; renal cancer A498 and ACHN; pancreatic cancer MIA-paca and PANC-1). Compounds 5 and 6 showed weak cytotoxicity against the A498 human renal cancer cell line. Compound 9 showed moderate activity against all four human cancer cell lines, while compounds 1−4, 7, and 8 were inactive. Considering structure−activity relationships, a ketone group at C-5 of ring A is much more favorable than a hydroxy group for activity, as exemplified by compound 6, which was more active than compounds 5, 7, and 8. Meanwhile, the hydroperoxy group in the side chain is harmful to the cytotoxicity, as compounds 1, 3, and 4 did not display any activity against the four human cancer cell lines. Among the compounds tested, compound 9 showed the most potent activity, which suggested that the spiroketal-modified benzopyran moiety was not the compulsory substructure for cytotoxicity. Compound 9 showed potent cytotoxicity against MIA-paca and PANC-1 human pancreatic cancer cell lines, similar to or better than the positive control, which suggested the significance of this 173

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Table 1. 1H and 13C NMR Data for Compounds 1−4 in MeOH-d4 1a position

δC

1 2 3 4 5 6a 6b 7 8 9a 9b 10 11 12 13 14 15a 15b 16 17 18 19 20 21 22 23 24 25 COCH3 COCH3

64.6 141.6 139.4 15.9 200.5 38.8 34.6 143.6 63.7 124.6 96.0 123.0 138.5 22.9 36.3 66.8 126.4 140.8 17.0 43.3 128.4 137.8 82.3 25.0 25.0

δH (J in Hz) 4.49, dd (5.6, 3.4) 6.70, dq (5.6, 0.8) 1.81, s 2.57, m 2.43, dd (16.4, 13.9) 2.59, m 4.07, d (15.4) 4.04, d (15.4) 5.54, s 5.29, s 1.75, s 2.04, dd (17.3, 10.8) 1.87, dd (17.3, 3.2) 4.75, ddd (10.8, 8.3, 3.7) 5.27, d (8.3) 1.76, s 2.77, d (5.8) 5.61, dd (15.8, 5.9) 5.66, d (15.8) 1.29, s 1.29, s

2a δC 64.8 141.6 139.6 15.8 200.7 38.8 34.7 143.7 63.8 124.7 96.1 123.0 138.7 22.8 36.3 66.8 126.1 141.1 17.0 34.2 39.6 216.6 41.9 18.6 18.6

3b

δH (J in Hz)

δC

4.49, dd (5.3, 3.3) 6.70, dq (5.3, 1.3) 1.81, s 2.57, m 2.43, dd (16.6, 13.9) 2.59, m 4.07, d (15.1) 4.04, d (15.1) 5.53, s 5.29, s 1.75, s 2.02, dd (17.3, 10.8) 1.84, dd (17.3, 3.0) 4.73, ddd (10.8, 8.3, 3.7) 5.23, d (8.3) 1.78, s 2.28, t (7.4) 2.66, t (7.4) 2.68, septet (6.9) 1.08, d (6.9) 1.08, d (6.9)

65.2 125.6 141.6 19.9 73.3 29.5 33.8 144.1 63.9 124.9 95.8 123.7 138.5 23.0 36.5 66.8 126.6 141.2 17.1 43.5 128.7 138.0 82.5 25.0 25.0 172.6 21.1

4a

δH (J in Hz) 4.27, br s 5.66, br d (6.3) 1.73, s 5.38, dd (11.5, 5.3) 2.13, ddd (11.5, 5.3, 2.4) 1.56, ddd (12.1, 11.6, 11.6) 2.18, ddd (12.1, 2.4, 2.4) 4.08, d (14.2) 4.05, d (14.2) 5.52, s 5.24, s 1.76, s 2.03, dd (17.7, 11.2) 1.86, dd (17.7, 3.1) 4.74, ddd (11.2, 8.3, 3.1) 5.26, d (8.3) 1.75, s 2.77, d (5.8) 5.61, dd (15.5, 5.9) 5.65, d (15.5) 1.30, s 1.30, s 2.08, s

δC 65.1 127.3 138.4 21.1 71.2 29.5 30.1 144.0 64.0 124.8 95.6 123.6 138.3 22.8 36.4 66.7 126.5 141.0 16.9 43.4 128.6 137.8 82.4 24.9 24.9 172.7 21.0

δH (J in Hz) 4.32, br s 5.75, dq (5.3, 1.2) 1.75, s 5.22, br s 1.95, ddd(14.2, 2.4, 2.4) 1.73, ddd (14.2, 12.1, 4.0) 2.29, ddd (12.1, 2.4, 2.4) 4.06, d (15.3) 4.02, d (15.3) 5.50, s 5.23, s 1.74, s 2.02, dd (17.3, 10.1) 1.84, dd (17.3, 3.1) 4.73, ddd (10.1, 8.2, 3.1) 5.26, d (8.2) 1.75, s 2.76, d (5.2) 5.61, dd (15.7, 5.2) 5.66, d (15.7) 1.29, s 1.29, s 2.06, s

a1

H NMR spectra were recorded at 500 MHz, while 13C NMR spectra were recorded at 125 MHz . b1H NMR spectra were recorded at 600 MHz, while 13C NMR spectra were recorded at 150 MHz . data, see Table 1; LRFABMS m/z 415, 457 [M − OH]+, 497 [M + Na]+; HRFABMS m/z 497.2490 (calcd for C27H38O7Na, 497.2515). Phorbaketal G (4): colorless oil; [α]20D −130 (c 0.3 MeOH); UV (MeOH) λmax (log ε) 197 (5.79) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 212 (13.82) nm; IR (film) νmax 3382, 2973, 2917, 1735, 1677, 1437, 1376, 1239, 1191, 1165, 1124, 1030, 1009, 975 cm−1; 1H and 13C NMR data, see Table 1; LRFABMS m/z 415, 457 [M − OH]+, 497 [M + Na]+; HRFABMS m/z 497.2491 (calcd for C27H38O7Na, 497.2515). Phorbaketal H (5): light yellowish oil; [α]20D −90 (c 0.2 MeOH); UV (MeOH) λmax (log ε) 204 (5.68) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 244 (5.63) nm; IR (film) νmax 3427, 2953, 2926, 1689, 1640, 1570, 1447, 1376, 1008, 984 cm−1; 1H and 13C NMR data, see Table 2; LRFABMS m/z 381, 399 [M + H]+, 421 [M + Na]+; HRFABMS m/z 399.2501 (calcd for C25H35O4, 399.2535). Phorbaketal I (6): light yellowish oil; [α]20D −27(c 0.3 MeOH); UV (MeOH) λmax (log ε) 199 (5.90) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 219 (−14.89), 243 (10.35) nm; IR (film) νmax 2962, 2923, 2855, 1735, 1688, 1563, 1447, 1377, 1247, 1197, 1171, 1115, 1007, 989 cm−1; 1 H and 13C NMR data, see Table 2; LRFABMS m/z 397 [M + H]+, 419 [M + Na]+; HRFABMS m/z 397.2377 (calcd for C25H33O4, 397.2379). Phorbaketal J (7): light yellowish oil; [α]20D −95 (c 0.2 MeOH); UV (MeOH) λmax (log ε) 198 (5.63), 230 (5.02) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 244 (10.63) nm; IR (film) νmax 2965, 2919, 2852, 1738, 1691, 1561, 1440, 1375, 1237, 1189, 1170, 1115, 1010, 987 cm−1; 1 H and 13C NMR data, see Table 2; LRFABMS m/z 381, 441 [M + H]+, 463 [M + Na]+; HRFABMS m/z 463.2458 (calcd for C27H36O5Na, 463.2460). Phorbaketal K (8): light yellowish oil; [α]20D −150 (c 0.2 MeOH); UV (MeOH) λmax (log ε) 198 (5.69), 230 (4.99) nm; CD (c 4.0 × 10−4

CH3CN, followed by purification using reversed-phase HPLC (Phenomenex Luna, 250 × 10 mm, 5 μm, 2.0 mL/min, 210 nm) eluting with 80% CH3CN (retention time for 6: 28 min; retention time for 7: 56 min; retention time for 8: 49 min). Compounds 11 (32.0 mg) and 12 (25.6 mg) were separated on the above-mentioned reversedphase preparative HPLC column (8.0 mL/min, 210 nm) eluting with 45% CH3CN for 42 min and then with 55% CH3CN for 60 min (retention time for 11: 80 min; retention time for 12: 95 min). Phorbaketal D (1): light yellowish oil; [α]20D −103 (c 1.0 MeOH); UV (MeOH) λmax (log ε) 204 (5.50), 229 (5.07) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 212 (9.46), 234 (−14.03) nm; IR (film) νmax 3394, 2975, 2916, 1678, 1445, 1420, 1378, 1360, 1170, 1123, 1045, 1004, 981 cm−1; 1H and 13C NMR data, see Table 1; LRFABMS m/z 379, 397, 413 [M − OH]+, 453 [M + Na]+; HRFABMS m/z 453.2250 (calcd for C25H34O6Na, 453.2253). Compound 1a: light yellowish oil; UV (MeOH) λmax (log ε) 203 (5.78), 228 (5.31) nm; LRFABMS m/z 397, 415 [M + H]+, 437 [M + Na]+; HRFABMS m/z 437.2302 (calcd for C25H34O5Na, 437.2304). Phorbaketal E (2): light yellowish oil; [α]20D −57 (c 0.2 MeOH); UV (MeOH) λmax (log ε) 203 (5.75), 228 (5.37) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 212 (9.43), 234 (−14.69) nm; IR (film) νmax 3450, 2967, 2923, 1709, 1680, 1447, 1417, 1379, 1170, 1121, 1068, 1043, 1004, 988 cm−1; 1H and 13C NMR data, see Table 1; LRFABMS m/z 379, 397, 415 [M + H]+, 437 [M + Na]+; HRFABMS m/z 437.2302 (calcd for C25H34O5Na, 437.2304). Phorbaketal F (3): colorless oil; [α]20D −110 (c 0.4 MeOH); UV (MeOH) λmax (log ε) 197 (5.80) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 211 (13.67) nm; IR (film) νmax 3402, 2971, 2930, 1734, 1678, 1446, 1376, 1239, 1182, 1125, 1008, 978, 937 cm−1; 1H and 13C NMR 174

dx.doi.org/10.1021/np300573m | J. Nat. Prod. 2013, 76, 170−177

Journal of Natural Products

Article

Table 2. 1H and 13C NMR Data for Compounds 5−8 in MeOH-d4 5a position

δC

1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14 15a 15b 16 17 18 19 20 21 22 23 24 25 COCH3 COCH3

65.3 122.7 146.0 19.3 70.3 33.0 31.6 144.6 194.8 148.7 95.4 121.6 140.0 22.9 36.4 66.8 125.6 142.4 16.8 40.5 27.5 125.0 132.6 25.8 17.7

6b

δH (J in Hz) 4.22, br s 5.53, br d (5.1) 1.83, s 4.11, dd (11.7, 5.6) 2.03, m 1.45, ddd (12.3, 11.6, 11.6) 2.44, ddd(12.3, 2.7, 2.7) 9.49, s 6.53, s 5.31, s 1.79, s 2.06, m 1.89, dd (17.2, 3.1) 4.73, ddd (11.1, 8.2, 3.1) 5.21, d (8.2) 1.75, s 2.05, m 2.12, m 5.10, br t (6.9) 1.66, s 1.60, s

δC 64.6 141.1 139.9 16.1 200.2 38.6 31.9 143.4 194.3 148.6 96.1 121.0 140.7 23.1 36.5 67.2 125.6 142.5 17.1 40.7 27.6 125.1 132.8 26.0 17.9

7b

δH (J in Hz)

δC

4.50, dd (5.3, 3.6) 6.73, dq (5.3, 1.3) 1.83, s 2.56, dd (14.9, 3.6) 2.38, dd (14.9, 13.2) 2.96, ddd (13.2, 3.6, 3.6) 9.51, s 6.60, s 5.40, s 1.82, s 2.10, m 1.94, dd (17.5, 3.1) 4.77, ddd (11.2, 8.1, 3.1) 5.24, dq (8.1, 1.0) 1.78, s 2.06, m 2.13, m 5.12, br t (6.9) 1.68, s 1.62, s

64.8 125.1 141.9 19.2 72.8 29.4 31.0 144.2 194.7 149.0 95.6 121.5 140.4 23.1 36.5 67.0 125.6 142.5 17.0 40.7 27.6 125.1 132.8 26.0 17.9 172.4 21.0

8b

δH (J in Hz) 4.27, dd (5.2, 3.6) 5.68, br (d5.2) 1.74, s 5.43, dd (11.5, 5.9) 2.10, m 1.49, ddd(12.4, 11.5, 11.5) 2.53, ddd (11.5, 3.6, 3.6) 9.49, s 6.56, s 5.33, s 1.81, s 2.08, m 1.91, dd (17.4, 3.1) 4.74, ddd (11.2, 8.2, 3.2) 5.22, d (8.2) 1.76, br d (0.8) 2.05, m 2.12, m 5.11, t (6.3) 1.67, s 1.61, s 2.07, s

δC 64.9 126.9 138.9 21.3 71.0 29.5 27.5 144.3 194.8 148.9 95.5 121.5 140.3 23.1 36.5 67.0 125.7 142.5 17.0 40.7 27.6 125.1 132.8 26.0 17.9 172.8 21.2

δH (J in Hz) 4.32, dd (5.3, 3.0) 5.78, dq (5.3, 1.2) 1.78, s 5.19, dd (3.3, 2.2) 1.94, ddd (13.9, 2.2, 2.2) 1.64, ddd (13.9, 13.9, 3.3) 2.65, ddd (13.9, 3.0, 3.0) 9.49, s 6.55, s 5.33, s 1.80, s 2.08, m 1.91, dd (17.6, 3.3) 4.74, ddd (11.2, 8.2, 3.3) 5.23, dq (8.2, 1.1) 1.77, br d (0.8) 2.05, m 2.10, m 5.12, t (6.9) 1.67, s 1.61, s 2.10, s

a1

H NMR spectra were recorded at 500 MHz, while 13C NMR spectra were recorded at 125 MHz . b1H NMR spectra were recorded at 600 MHz, while 13C NMR spectra were recorded at 150 MHz . (CD3OD, 600 MHz) δ 6.85 (1H, dq, J = 5.6, 1.3 Hz, H-2), 5.17 (1H, dq, J = 8.7, 1.0 Hz, H-12), 5.12 (1H, tq, J = 6.9, 1.0 Hz, H-17), 5.10 (1H, tsep, J = 7.0, 1.3 Hz, H-22), 5.06 (1H, s, H-9a), 4.88 (1H, s, H-9b), 4.51 (1H, dt, J = 8.5, 6.7 Hz, H-11), 4.43 (1H, dd, J = 5.6, 1.8 Hz, H-1), 2.82 (1H, m, H-6a), 2.81 (1H, m, H-7), 2.44 (1H, dd, J = 14.0, 6.8 Hz, H10a), 2.30 (1H, m, H-6b), 2.23 (1H, dd, J = 14.0, 6.6 Hz, H-10b), 2.11 (2H, m, H-16), 2.07 (2H, m, H-21), 2.02 (2H, t, J = 7.5 Hz, H-15), 1.98 (2H, t, J = 7.6 Hz, H-20), 1.78 (3H, s, H-4), 1.67 (3H, s, H-24), 1.66 (3H, d, J = 1.0 Hz, H-14), 1.61 (3H, s, H-19), 1.60 (3H, s, H-25); 13C NMR (CD3OD, 150 MHz) δ 202.5 (C, C-5), 146.5 (C, C-8), 145.5 (CH, C-2), 138.8 (C, C-13), 137.3 (C, C-3), 136.3 (C, C-18), 132.3 (C, C-23), 129.3 (CH, C-12), 125.6 (CH, C-22), 125.3 (CH, C-17), 115.0 (CH2, C-9), 68.8 (CH, C-11), 65.1(CH, C-1), 46.0 (CH, C-7), 44.5 (CH2, C-10), 41.0 (CH2, C-20), 40.9 (CH2, C-15), 38.6 (CH2, C-6), 28.0 (CH2, C-21), 27.6 (CH2, C-16), 26.1 (CH3, C-24), 17.9 (CH3, C25), 16.9 (CH3, C-14), 16.2 (CH3, C-19), 15.8 (CH3, C-4); LRESIMS m/z 351, 369 [M − OH]+, 409 [M + Na]+; HRFABMS m/z 409.2709 (calcd for C25H38O3Na, 409.2719). Phorbaketal A (10): [α]20D −99 (c 2.8 MeOH) [reported value19 [α]25D −118.1 (c 0.15 MeOH)]; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 214 (5.24), 234 (−17.49) nm. Phorbaketal B (11): [α]20D −100 (c 1.0 MeOH) [reported value19 [α]25D −115.1 (c 0.10 MeOH)]; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 212 (11.90) nm. Phorbaketal C (12): [α]20D −104 (c 0.7 MeOH) [reported value19 [α]25D −122.3 (c 0.10 MeOH)]; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 212 (13.97) nm. Hydroperoxide Reduction of Phorbaketal D (1) to 1a. To a solution of compound 1 (2 mg, 4.7 μmol) in CH2Cl2 (2 mL) was added triphenylphosphine (1.6 mg, 9.3 μmol) at ambient temperature, and the

Table 3. Potency of Isolated Compounds 1−9a cancer cell lines compound

A498

ACHN

MIA-paca

PANC-1

2 5 6 9 temb SUNb gemb 5-FUb

12.5 9.8 9.0 4.9 0.50

18.0 >50 22.3 10.0

16.7 >50 22.0 5.2

25.3 >50 12.8 7.8

2.5 3.0 50

a

Data are expressed in IC50 values (μM), and compounds 1, 3, 4, 7, and 8 are inactive, with IC50 values greater than 50 μM. A498: human renal cancer cell line; ACHN: human renal cancer cell line; MIA-paca: human pancreatic cancer cell line; PANC-1: human pancreatic cancer cell line. bTem (temsirolimus), SUN (sunitinib), gem (gemcitabine), and 5-FU (5-fluorouracil) were used as positive controls. M, MeOH) λmax (Δε) 242 (10.09) nm; IR (film) νmax 2965, 2921, 2853, 1736, 1691, 1563, 1438, 1371, 1237, 1191, 1169, 1115, 1013, 993, 977 cm−1; 1H and 13C NMR data, see Table 2; LRFABMS m/z 381, 441 [M + H]+, 463 [M + Na]+; HRFABMS m/z 463.2459 (calcd for C27H36O5Na, 463.2460). Phorbin A (9): light yellowish oil; [α]20D −61(c 0.4 MeOH); UV (MeOH) λmax (log ε) 206 (5.70), 230 (5.06) nm; CD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 234 (−6.98) nm; IR (film) νmax 3414, 2964, 2922, 2856, 1677, 1447, 1379, 1267, 1106, 1044, 986, 937 cm−1; 1H NMR 175

dx.doi.org/10.1021/np300573m | J. Nat. Prod. 2013, 76, 170−177

Journal of Natural Products

Article

1-O-(R)-MTPA ester of 9: 1H NMR (500 MHz, CDCl3) δ 7.419 (5H, m, Ar-H), 6.911 (1H, br d, J = 5.70 Hz, H-2), 5.773 (1H, dd, J = 5.7, 1.8 Hz, H-1), 5.112 (1H, t, J = 6.8 Hz, H-17), 5.093 (1H, t, J = 6.7 Hz, H-22), 5.082 (1H, d, J = 8.5 Hz, H-12), 4.612 (1H, m, H-11), 3.065 (1H, m, H7), 2.670 (1H, dd, J = 16.4, 12.7 Hz, H-6a), 2.443 (1H, dd, J = 13.8, 6.5 Hz, H-10a), 2.439 (1H, dd, J = 16.4, 5.0 Hz, H-6b), 2.153−1.966 (8H, m, H-15, 16, 20, 21), 2.142 (1H, dd, J = 13.8, 6.6 Hz, H-10b), 1.775 (3H, s, H-4), 1.654 (3H, s, H-14), 1.653 (3H, s, H-24), 1.585 (3H, s, H-19), 1.567 (3H, s, H-25); LRFABMS m/z 603 [M + H]+, 625 [M + Na]+. 11-O-(S)-MTPA ester of 9: 1H NMR (700 MHz, CDCl3) δ 7.480 (5H, m, Ar-H), 6.792 (1H, dd, J = 5.7, 1.4 Hz, H-2), 5.866 (1H, td, J = 8.5, 6.5 Hz, H-11), 5.214 (1H, s, H-9a), 5.123 (1H, t, J = 6.8 Hz, H-17), 5.113 (1H, t, J = 6.5 Hz, H-22), 5.043 (1H, s, H-9b), 5.090 (1H, d, J = 7.5 Hz, H-12), 4.393 (1H, m, H-1), 2.958 (1H, dd, J = 16.0, 13.5 Hz, H-6a), 2.791 (1H, m, H-7), 2.627 (1H, dd, J = 13.8, 6.4 Hz, H-10a), 2.405 (1H, dd, J = 13.8, 6.6 Hz, H-10b), 2.296 (1H, dd, J = 16.0, 5.0 Hz, H-6b), 2.069 (1H, m, H-16), 2.061 (1H, m, H-15), 2.210−1.974 (4H, m, H-20, 21), 1.851 (3H, s, H-4), 1.783 (3H, s, H-14), 1.705 (3H, s, H-24), 1.627 (3H, s, H-19), 1.624 (3H, s, H-25); LRFABMS m/z 603 [M + H]+, 625 [M + Na]+. 11-O-(R)-MTPA ester of 9: 1H NMR (700 MHz, CDCl3) δ 7.480 (5H, m, Ar-H), 6.754 (1H, dd, J = 5.7, 1.6 Hz, H-2), 5.899 (1H, td, J = 8.0, 6.5 Hz, H-11), 5.121 (1H, s, H-9a), 5.128 (1H, t, J = 6.5 Hz, H-17), 5.114 (1H, t, J = 6.7 Hz, H-22), 4.956 (1H, s, H-9b), 5.216 (1H, d, J = 7.3 Hz, H-12), 4.261 (1H, m, H-1), 2.783 (1H, dd, J = 15.5, 13.5 Hz, H-6a), 2.679 (1H, m, H-7), 2.573 (1H, dd, J = 13.1, 6.1 Hz, H-10a), 2.323 (1H, dd, J = 13.1, 6.0 Hz, H-10b), 2.258 (1H, dd, J = 15.5, 5.0 Hz, H-6b), 2.079 (1H, m, H-16), 2.074 (1H, m, H-15), 2.210−1.974 (4H, m, H-20, 21), 1.846 (3H, s, H-4), 1.808 (3H, s, H-14), 1.709 (3H, s, H-24), 1.628 (3H, s, H-19), 1.623 (3H, s, H-25); LRFABMS m/z 603 [M + H]+, 625 [M + Na]+. Preparation of Phorbaketal I (6) from Phorbaketal A (10). Phorbaketal A (10.0 mg, 0.025 mmol) in CH2Cl2 (2.0 mL) was treated with Dess-Martin periodinane (12.78 mg, 0.030 mmol) and stirred for 2.5 h at 0 °C. The reaction mixture was quenched with an aqueous Na2S2O3 solution and extracted with CH2Cl2 (5 mL). The organic layer was washed with a saturated aqueous NaHCO3 solution and brine. After removal of solvent, the crude residue was chromatographed over silica gel (hexane/EtOAc, 9:1) to give the aldehyde derivative 6 (9.4 mg, 94%) as a light yellowish oil. Cytotoxicity Assays. Cytotoxic activity was evaluated by the MTT method as described previously.24 Four human cancer cell lines, human renal cancer cell lines A498 and ACHN and human pancreatic cancer cell lines MIA-paca and PANC-1, were used. Temsirolimus, sunitinib, gemcitabine, and 5-fluorouracil were used as positive controls.

mixture was stirred for 10 h. Solvent was removed under reduced pressure, and the crude was purified using reversed-phase HPLC (Shiseido MGII C18 5 μm, 250 × 10 mm) eluting with 43% CH3CN to obtain the desired product 1a (1.50 mg, 75%) as a light yellowish oil: 1H NMR (CD3OD, 700 MHz) δ 6.69 (1H, dq, J = 5.5, 0.7 Hz, H-2), 5.66 (1H, d, J = 15.6 Hz, H-22), 5.60 (1H, dt, J = 15.6, 6.3 Hz, H-21), 5.53 (1H, s, H-10), 5.29 (1H, s, H-12), 5.27 (1H, d, J = 8.3 Hz, H-17), 4.75 (1H, ddd, J = 11.1, 8.3, 3.0 Hz, H-16), 4.49 (1H, dd, J = 4.5, 3.0 Hz, H-1), 4.07 (1H, d, J = 15.4 Hz, H-9a), 4.04 (1H, d, J = 15.4 Hz, H-9b), 2.75 (2H, d, J = 6.3 Hz, H-20), 2.58 (1H, m, H-7), 2.57 (1H, m, H-6a), 2.43 (1H, dd, J = 16.4, 13.9 Hz, H-6b), 2.04 (1H, dd, J = 17.2, 11.1 Hz, H15a), 1.86 (1H, dd, J = 17.2, 3.0 Hz, H-15b), 1.81 (3H, s, H-4), 1.76 (3H, s, H-19), 1.75 (3H, s, H-14), 1.27 (6H, s, H-24/25); 13C NMR (CD3OD, 175 MHz) 200.5 (C, C-5), 143.6 (C, C-8), 141.6 (CH, C-2), 141.4 (CH, C-22), 141.0 (C, C-18), 139.4 (C, C-3), 138.5 (C, C-13), 126.3 (CH, C-17), 125.0 (CH, C-21), 124.6 (CH, C-10), 123.0 (CH, C12), 96.0 (C, C-11), 71.0 (C, C-23), 66.8 (CH, C-16), 64.6 (CH, C-1), 63.7 (CH2, C-9), 43.1 (CH2, C-20), 38.8 (CH2, C-6), 36.3 (CH2, C-15), 34.6 (CH, C-7), 30.0 (CH3, C-24), 30.0 (CH3, C-25), 22.9 (CH3, C-14), 17.0 (CH3, C-19), 15.9 (CH3, C-4); LRESIMS m/z 379, 397 [M − OH]+, 437 [M + Na]+. Preparation of MTPA Esters. Compound 5 (0.4 mg, 1.0 μmol) was treated with (R)-(−)-and (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (4 μL, 21 μmol) in dry pyridine (25 μL) for 24 h at rt to afford (S)-(−)-MTPA ester and (R)-(+)-MTPA ester, respectively. Each mixture was allowed to stand at rt for 24 h. The reaction was monitored by TLC (ODS, MeOH). After removal of solvent, the product was purified by reversed-phase HPLC on an Xterra column (250 × 4.6 mm, 5 μm, 100 Å) and analyzed by 1H NMR. The procedure for preparation of 1,11-di-O-MTPA esters of compound 9 was the same as that for compound 5. Partial deacetylation occurred when the 1,11-di-O-MTPA esters of compound 9 were left standing at room temperature for three weeks. The 1-O-MTPA esters of 9 were obtained by purification of the deacetylation mixtures using the same HPLC conditions. The 11-O-MTPA esters of 9 were prepared by treating compound 9 (0.5 mg, 1.2 μmol) with 2 equiv of MTPA-Cl (0.46 μL, 2.4 μmol) at 4 °C for 24 h. The resulting products were purified by reversed-phase HPLC on an Xterra column (250 × 4.6 mm, 5 μm, 100 Å). (S)-MTPA ester of 5: 1H NMR (500 MHz, CDCl3) δ 9.501 (1H, s, H9), 7.523 (2H, m, Ar-H), 7.425 (3H, m, Ar-H), 6.574 (1H, s, H-10), 5.668 (1H, br d, J = 5.3 Hz, H-2), 5.646 (1H, m, H-5), 5.330 (1H, s, H12), 5.202 (1H, d, J = 9.0 Hz, H-17), 5.096 (1H, t, J = 6.7 Hz, H-22), 4.710 (1H, ddd, J = 11.1, 9.0, 3.0 Hz, H-16), 4.276 (1H, br s, H-1), 3.543 (3H, s, OMe), 2.589 (1H, dt, J = 12.3, 3.2 Hz, H-7), 2.266 (1H, dt, J = 13.1, 3.2 Hz, H-6a), 2.121 (2H, m, H-21), 2.074 (1H, m, H-15a), 2.035 (2H, m, H-20), 1.883 (1H, m, H-15b), 1.578 (1H, dt, J = 13.1, 11.7 Hz, H-6b), 1.794 (3H, s, H-14), 1.733 (3H, s, H-21), 1.659 (3H, s, H-25), 1.592 (3H, s, H-24), 1.509 (3H, s, H-4); LRFABMS m/z 615 [M + H]+, 637 [M + Na]+. (R)-MTPA ester of 5: 1H NMR (500 MHz, CDCl3) δ 9.492 (1H, s, H9), 7.499 (2H, m, Ar-H), 7.426 (3H, m, Ar-H), 6.555 (1H, s, H-10), 5.712 (1H, br d, J = 4.9 Hz, H-2), 5.624 (1H, m, H-5), 5.272 (1H, s, H12), 5.198 (1H, d, J = 8.2 Hz, H-17), 5.096 (1H, t, J = 6.2 Hz, H-22), 4.704 (1H, ddd, J = 11.1, 8.2, 3.0 Hz, H-16), 4.267 (1H, br s, H-1), 3.512 (3H, s, OMe), 2.576 (1H, dt, J = 12.9, 2.8 Hz, H-7), 2.226 (1H, dt, J = 13. 0, 2.8 Hz, H-6a), 2.111 (2H, m, H-21), 2.058 (1H, m, H-15a), 2.040 (2H, m, H-20), 1.887 (1H, m, H-15b), 1.777 (3H, s, H-14), 1.754 (3H, s, H-4), 1.737 (3H, s, H-21), 1.658 (3H, s, H-25), 1.594 (3H, s, H-24), 1.408 (1H, dt, J = 13.0, 11.1 Hz, H-6b); LRFABMS m/z 615 [M + H]+, 637 [M + Na]+. 1-O-(S)-MTPA ester of 9: 1H NMR (500 MHz, CDCl3) δ 7.419 (5H, m, Ar-H), 6.947 (1H, dd, J = 5.7, 1.3 Hz, H-2), 5.802 (1H, m, H-1), 5.110 (1H, t, J = 6.8 Hz, H-17), 5.090 (1H, t, J = 6.7 Hz, H-22), 5.086 (1H, d, J = 8.0 Hz, H-12), 4.613 (1H, m, H-11), 3.051 (1H, m, H-7), 2.663 (1H, dd, J = 15.8, 13.5 Hz, H-6a), 2.381 (1H, dd, J = 14.2, 6.5 Hz, H-10a), 2.379 (1H, dd, J = 15.8, 4.9 Hz, H-6b), 2.151−1.965 (8H, m, H15, 16, 20, 21), 2.137 (1H, dd, J = 14.2, 6.6 Hz, H-10b), 1.831 (3H, s, H4), 1.676 (3H, s, H-24), 1.662 (3H, s, H-14), 1.603 (3H, s, H-19), 1.587 (3H, s, H-25); LRFABMS m/z 603 [M + H]+, 625 [M + Na]+.

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra for phorbaketals A−C (10−12), compounds 1a and 1−9, and the synthetic standard compound of 6. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 82-2-880-5730. Fax: 82-2-883-9289. E-mail: sjnam@ sunchon.ac.kr; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime Affairs of Korea. B.M., Y.L., J.L., H.K., and D.H. were in part supported by the 2012 BK21 Program, Ministry of Education, Science and Technology, Korea. 176

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Journal of Natural Products

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REFERENCES

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