NAD(P)H Quinone Oxidoreductase 1 (NQO1)-Bioactivated

Feb 20, 2013 - Christopher E. Blunt , Canan Torcuk , Yang Liu , William Lewis , David Siegel , David Ross , Christopher J. Moody. Angewandte Chemie 20...
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NAD(P)H Quinone Oxidoreductase 1 (NQO1)-Bioactivated Pronqodine A Regulates Prostaglandin Release from Human Synovial Sarcoma Cells Koichi Nakae,* Hayamitsu Adachi, Ryuichi Sawa, Nobuo Hosokawa, Masaki Hatano, Masayuki Igarashi, Yoshio Nishimura, Yuzuru Akamatsu, and Akio Nomoto Institute of Microbial Chemistry (BIKAKEN), 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan S Supporting Information *

ABSTRACT: Natural products have contributed to the elucidation of biological mechanisms as well as drug discovery research. Even now, the expectation for natural products is undiminished. We screened prostaglandin release inhibitors that had no effect on in vitro cyclooxygenase activity derived from natural product sources and discovered pronqodine A. Using spectral analysis and total synthesis, the structure of pronqodine A was shown to be a benzo[d]isothiazole-4,7-dione analogue. Evaluation of the biological activity of pronqodine A revealed that the NAD(P)H dehydrogenase quinone 1 (NQO1) converted pronqodine A into a twoelectron reductive form. The reductive form underwent autoxidation and reversed to its native form immediately with the generation of reactive oxygen species. Further investigations proved that pronqodine A inhibited cyclooxygenase enzyme activity only in the presence of NQO1. Pronqodine A acts as a potential bioreductive compound, inhibiting prostaglandin release in selectively activated NQO1-expressing cells.

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ioactive natural products have greatly enhanced the understanding of biological mechanisms. Although processes such as isolation, structure elucidation, total synthesis, and biological exploration, which are used to investigate new natural products, remain challenging, natural products are still a potential source of biological probes.1−3 To discover potential bioactive compounds from natural resources, we conducted a screening program for inhibitors of prostaglandin release. Prostaglandins are bioactive lipid mediators composed of 20-carbon unsaturated fatty acids. Arachidonic acid is enzymatically converted to various kinds of prostaglandins via cyclooxygenase (COX) and prostaglandin synthase. Each prostaglandin binds to its specific G proteincoupled receptor and activates second messengers.4 A number of research findings have already demonstrated that prostaglandins play an important role in several diseases such as inflammatory disorders, sensory neuron related disorders, and cancers, as well as in maintaining physiological homeostasis.5−7 Although these results led to the development of many prostaglandin-release inhibitors such as nonsteroidal antiinflammatory drugs (NSAIDs), for example, indomethacin, that inhibit COX activity, the development of new compounds with unique mechanisms of action are still needed.8,9 In this study, we report the screening of natural products and the isolation, physicochemical properties, structural determination, and biological activities of pronqodine A. © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION During primary screening, we examined the inhibitory activity of samples on prostaglandin E2 (PGE2) release induced by bradykinin in human synovial sarcoma SW982 cells, which possess the ability to release prostaglandins by induction of bradykinin. In order to characterize positive samples, we investigated the inhibitory activity on 6-keto-prostaglandin F1α (stable prostacyclin (PGI2) metabolite) production, interleukin6 (IL-6) release, and in vitro COX-2 enzyme activity. After screening 25 400 samples derived from microorganisms and plants, we discovered a positive sample from the culture medium of Streptomyces sp. MK832-95F2. It possessed potent inhibitory activity on PGE2 release and 6-keto-prostaglandin F1α production but had no inhibitory effects against IL-6 release and in vitro COX-2 activity. Fermentation and Isolation of Pronqodine A. Streptomyces sp. MK832-95F2 was isolated from a soil sample Received: September 19, 2012 Published: February 20, 2013 510

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skeleton. The bathochromic effect observed in the UV−vis spectrum (480 nm) showed that a nitrogen atom should be adjacent to a quinone skeleton. Connectivity of the methyl amino group to the quinone ring in the active compound was anticipated on the basis of the structure of caulibugulone A.11 Caulibugulone A has a methyl amine substituted quinone skeleton in which the 13C value of the connecting carbon between the methyl amino group and the quinone was δC 150.7. This was almost identical to the 13C value (δC 149.8) and four quinonoid carbons (δC 149.8, 98.8, 133.4, and 167.9) of the active compound. Therefore, a methyl amino group of the active compound should be attached to the quinone skeleton. The remaining unit, which consisted of one carbon, one nitrogen, and one sulfur, was predicted to be a five-membered thiazole ring connected to the quinonoid carbons (δC 133.4 and 167.9) as shown in Figure S1a. Six possible structures, 1 and 4− 8, of the active compound were speculated (Figure S1b). The 1,3-thiazole ring proton as shown in compounds 5 and 8 appears generally at δC 9.5−9.6,12 whereas the active compound showed a proton signal at δC 8.87. These results indicated that compounds 5 and 8 would be excluded as structural candidates. Aulosirazole, with the quinone skeleton conjugated 1,2-thiazole ring, shows the 13C signal adjacent to sulfur and the 13C signal vicinal to a thiazole ring carbon at δC 166.4 and at δC 122.1, respectively.13 The 13C(sp2) signal adjacent to nitrogen of the N-methyl group of the active compound appeared at δC 149.8, shifted higher field than the 13C signal (δC 166.4) vicinal to sulfur as shown in aulosirazole. These observations suggested that nitrogen would not be connected to the quinone ring, ruling out compounds 4 and 6 from the candidates. A comparison of 13C chemical shift values for quinone sp2 carbons of the active compound with those of aulosirazole indicated that the sequence of atoms of the thiazole ring would fit compound 1 rather than that of compound 7. On the basis of these findings, we assumed compound 1 to be the most likely structure of the active compound, and its total synthesis was designed as outlined in Scheme 1. Synthesis of compound

collected at Saru-gun, Hokkaido, Japan. MK832-95F2 formed well-branched substrate mycelia. This strain formed straight to flexuous aerial mycelia without spirals. Mature spore chains were moderately short but usually contained 10 to 20 or more spores per chain. The spores were oval with a smooth surface and 0.5 × 0.6−0.4 × 0.8 μm in size. The substrate mycelia were pale yellowish-brown to pale brown, and the aerial mycelia were grayish-white to light olive-gray. The partial 16S rRNA gene sequence (1420 bp) of MK832-95F2 showed high similarity with 16S rRNA sequences in the genus Streptomyces, such as Streptomyces sampsonii (ATCC 25495T, 1419/1420 bp, T: Type strain, 99.9%), S. felleus (NBRC 12766T, 1415/1415 bp, 100%), and S. limosus (NBRC 12790T, 1414/1414 bp, 100%). These phenotypic and genotypic data suggested that strain MK83295F2 belongs to the genus Streptomyces; hence the strain was tentatively designated as Streptomyces sp. MK832-95F2. Streptomyces sp. MK832-95F2 was cultured on barley medium (1.44 kg) by solid-state fermentation for 14 days at 30 °C. The barley medium was then extracted with EtOH (6.7 L). The extract was concentrated, and the resulting aqueous solution was extracted with EtOAc (6 L). The extract was dried over anhydrous Na2SO4 and evaporated to give a red oil (2.0 g). The oil was chromatographed on a column of silica gel eluted with CHCl3. The resulting active residue (0.4 g) was further chromatographed on a Sephadex LH-20 column. The active fraction (4.0 mg) in a small volume of MeOH was subjected to HPLC using 87% aqueous acetonitrile containing 0.01% trifluoroacetic acid (TFA) as the mobile phase. The active fractions were then combined and concentrated in vacuo to give 0.3 mg of pronqodine A (compound 1) as a red powder. Table 1. NMR Data for Pronqodine A (CDCl3)a

a

position

δC

3 3a 4 5 5-NH 5-NHCH3 6 7 7a

155.4 133.4 176.0 149.8

CH C CO C

δH (multiplicity, J, Hz)

29.8 98.8 176.6 167.9

CH3 CH CO C

8.87 (s)

Scheme 1 6.10 (br) 2.97 (d, 5.4) 5.58 (s)

Chemical shifts in ppm from TMS as an internal standard.

Structure Determination of Pronqodine A. 1H and 13C NMR data of pronqodine A are summarized in Table 1. The 13 C and DEPT135 spectra revealed that pronqodine A has eight carbons including two carbonyls, three quaternary sp2 and two sp2 methines, and one methyl carbon. The signal at δH 6.10 was observed from an exchangeable proton without correlation in the HMQC spectrum. In the 1H−1H COSY spectrum, only one correlation was observed between the methyl proton (δH 2.97, d J = 5.4 Hz) and the exchangeable proton, which should be the NH at δH 6.10 (br s). The HMBC spectrum of pronqodine A revealed the long-range couplings from a methyl proton to a quaternary sp2 carbon (δC 149.8), from the sp2 methine proton (δH 5.58, s) to two quaternary sp2 carbons (δC 167.9 and 176.0), and from another sp2 methine at d δH 8.87 to δC 133.4 and 167.9. Two carbonyl signals (δC 176.0 and 176.6) and the 1684 cm−1 signal in the IR spectrum suggested a quinone

1 began with the known compound 3.14 Treatment of compound 3 with phenyliodine bis(trifluoroacetate) (PIFA) gave a quinone intermediate, which was subjected to the next reaction without isolation. Subsequent methylamination of the resulting quinone intermediate produced compound 1. 1H and 13 C NMR of compound 1 were consistent with those of the active compound (Figures S2 and S3). Thus, the structure of the active compound was confirmed to be compound 1. Biological Property of Pronqodine A. The biological activity of pronqodine A was investigated as follows. Pronqodine A inhibited bradykinin-induced prostaglandin 511

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examined the mechanism of action of pronqodine A. The clue for this analysis was that the red-purple color of pronqodine A in buffer solution disappeared in a few minutes after addition of the SW982 cell lysate and NADH. This phenomenon together with the quinone nature of pronqodine A suggested that pronqodine A was converted to a reduced form, probably the semiquinone or hydroquinone form of pronqodine A. Therefore we analyzed the mass spectra of pronqodine A in cell lysate by LC-HRESIMS, focusing on one-electron reduction, which would form the semiquinone (positive mode; C8H8O2N2S calculated, m/z 196.030 (M + H)+), and two-electron reduction, which would form the hydroquinone (positive mode; C8H9O2N2S calculated, m/z 197.038 (M + H)+). As a result, the existence of pronqodine A itself and the twoelectron-reduced form of pronqodine A was detected at 30 min (Figure 1b). It is known that NAD(P)H dehydrogenase quinone 1 (NQO1) catalyzes the quinone structure into its two-electronreduced form.15 We measured the expression of NQO1, and its expression was not affected by pronqodine A in SW982 cells (Figure 2a). Next, we examined the effect of pronqodine A on NQO1 enzyme activity. Figure 2b shows the change in NADH absorbance at 340 nm in the NQO1 activity assay with pronqodine A or menadione. Menadione is known to be converted to the two-electron reductive form menadiol (2methyl-1,4-naphthalenediol) by NQO1.16 NADH functions as an electron donor. Pronqodine A and menadione decreased the absorbance at 340 nm in a concentration-dependent manner, clarifying that both compounds act as electron acceptors. Furthermore, we observed a change in the absorbance at 492 nm of pronqodine A by NQO1, as the red-purple color of a pronqodine A solution with cell lysate became transiently colorless and then returned to the original color in the upper layer after 10 min (Figure 2c). As shown in Figure 2d, when NQO1 was added in the reaction mixture, the absorbance was transiently decreased and then recovered after 8.5 min. To identify whether the recovery of absorbance was from pronqodine A itself or another metabolite, an LC-HRESIMS investigation was conducted. Figure 2e showed the existence of the two-electron reductive form of pronqodine A converted by NQO1 observed at 2 min. This signal was diminished at 30 min, while the signal of pronqodine A itself returned, suggesting that the hydroquinone type of pronqodine A was oxidized back to the quinone type. It is reported that menadiol formed from menadione by an NQO1-mediated reduction undergoes re-formation to menadione with concomitant formation of reactive oxygen species (ROS) in human embryonic kidney HEK293 cells expressing NQO1, because of the instability of the two-electron reductive form.16 The two-electron reductive form of pronqodine A was also identified to be unstable and immediately autoxidized to the native form. Pronqodine A caused ROS production intracellularly in SW982 cells (Figure 3a), and pronqodine A induced ROS production at a lower concentration than that of menadione (Figure 3b). Effect of Pronqodine A on the Arachidonic Acid Cascade. It is well documented that COX and prostaglandin synthases convert arachidonic acid into prostaglandins such as PGE2, PGI2, and PGD2. Arachidonic acid induced the release of PGE2 from SW982 cells, which was inhibited by pronqodine A in a concentration-dependent manner (Figure 4a and b). Hence, we examined whether pronqodine A with NQO1 directly affected arachidonic acid. The arachidonic acid

release in a concentration-dependent manner, but did not affect the release of IL-6 from SW982 cells (Figure 1a). The IC50 values of pronqodine A against PGE2 production, 6-ketoprostaglandin F1α production, and prostaglandin D2 (PGD2) production were 9, 19, and 7 nM, respectively. Next, we

Figure 1. (a) Effect of pronqodine A on 1 nM bradykinin-induced PGE2, 6-keto-prostaglandin F1α and PGD2, and IL-6 release from SW982 cells. (b) Positive LC-HRESIMS spectra in the mixture of pronqodine A and cell lysate of SW982 at m/z 195.0202−195.0242 (native form of pronqodine A), 197.0359−197.0399 (two-electron reductive form of pronqodine A), and 196.0280−196.0320 (oneelectron reductive form of pronqodine A) at 0 and 30 min. For the preparation of cell lysate, SW982 cells were harvested in 25 mM TrisHCl, then sonicated on ice and centrifuged (1700g, 10 min). Supernatant was used as the reaction source. Reaction mixtures contained 25 mM Tris-HCl (pH 7.6), 1.6 mM NADH, and 5 μM FAD, and SW982 cell lysate with 180 μM pronqodine A at 37 °C was analyzed by positive LC-HRESIMS at 0 and 30 min. 512

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Figure 2. (a) Effect of pronqodine A on the expression of NQO1 in SW982 cells over 30 min (Western blotting). (b) Effect of pronqodine A and menadione as an electron acceptor in the NQO1 activity assay. (c) Mixture of pronqodine A, SW982 cell lysate, and NADH in a glass vial. At 0 min, both solutions (140 μM pronqodine A with or without SW982 cell lysate) in a glass vial were red-purple (i). A few minutes later, the color of the solution with cell lysate became transparent, and 10 min later, the color of the upper layer with cell lysate became red-purple again (ii, left). The solution without cell lysate did not exhibit any change in color (ii, right). (d) Monitoring absorbance at 492 nm of pronqodine A with or without NQO1. Reaction mixtures contained 25 mM Tris-HCl (pH 7.6), 1 mM NADH, 3 μM FAD, and 140 μg/mL NQO1 with or without 150 μM pronqodine A at room temperature. (e) Positive LC-HRESIMS spectra for pronqodine A and NQO1 reaction mixtures at m/z 195.0202−195.0242 (native form of pronqodine A) and 197.0359−097.0399 (two-electron reductive form of pronqodine A) at 0 min (upper), 2 min (middle), and 30 min (lower). Reaction mixtures contained 25 mM Tris-HCl (pH 7.6), 1 mM NADH, 3 μM FAD, and 140 μg/mL NQO1 with 180 μM pronqodine A at 37 °C.

derivative 7-amino-4-methylcoumarin-arachidonamide (AMCAA) was used for quantitative analysis by HPLC. Pronqodine A with NQO1 had no effect on AMC-AA (Figure 4c). Subsequently the effect of pronqodine A on the expression of COX-1 and COX-2 was investigated. In SW982 cells, the expression of COX-1 was not detectable, and pronqodine A did not affect the expression of COX-2 (Figure 4d). We then examined the effect of pronqodine A on COX-2 activity. Treatment with pronqodine A alone did not affect COX-2 activity (Figure 4e), but in the presence of NQO1, pronqodine A inhibited COX-2 enzyme activity (Figure 4f).

NQO1, widely expressed in most tissues, is a two-electron reductase that catalyzes the conversion of the quinone into hydroquinone. This conversion is reported to act in detoxication against quinolone compounds. NQO1 knockout mice show no detectable phenotype and are indistinguishable from wild-type mice, except for their sensitivity against menadione.17 However, some quinones are bioactivated and cause redox cycling with concomitant production of ROS that depends on the stability of the resulting hydroquinone produced by NQO1. We clarified that pronqodine A is converted into a two-electron reduction form, the hydroquinone structure of pronqodine A, by NQO1. This structure is 513

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Figure 3. Effect of pronqodine A with NQO1 on ROS production. (a) Confocal images of ROS production induced by pronqodine A in SW982 cells: (i) control, (ii) 300 nM pronqodine A at 30 min. (b) Quantitation of ROS production induced by pronqodine A and menadione in SW982 cells. (c) Effect of pronqodine A on cytotoxicity in HEK293T cells transiently expressing NQO1.

unstable and undergoes autoxidation to revert to the native form with concomitant production of ROS. In general, overproduction of ROS inflicts severe damage, causing cytotoxicity, but restricted ROS production would regulate cell function.18,19 The IC50 value of pronqodine A on cytotoxicity in SW982 cells is about 30-fold higher than the IC50 value on prostaglandin release (data not shown). In addition, menadione is known to be not only a substrate of NQO1 but also a phosphatase inhibitor.20 We confirmed that menadione, but not pronqodine A, inhibited EGFR dephosphorylate activity (data not shown). These results show that pronqodine A would selectively generate ROS and regulate prostaglandin release in NQO1-expressing cells. Our results suggested that pronqodine A inhibits prostaglandin release by interrupting the conversion of arachidonic acid to prostaglandins by acting as a potential bioreductive compound. NQO1-bioactivated compounds could regulate the release of prostaglandins in NQO1-expressing cells

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Figure 4. (a) Effect of arachidonic acid on PGE2 release from SW982 cells for 30 min. (b) Effect of pronqodine A on 10 μg/mL arachidonic acid-induced PGE2 release from SW982 cells. (c) Effect of pronqodine A with NQO1 on the consumption of AMC-AA for 10 min. (d) Effect of pronqodine A on COX-1 and COX-2 expression in SW982 cells. The concentration of bradykinin was 1 nM and pronqodine A was 100 nM for 30 min. (e) Effect of pronqodine A on COX-2 enzyme activity. (f) Effect of pronqodine A with NQO1 on COX-2 enzyme activity.

(Greiner), plasmid of human NQO1-pCMV6-XL5 (Origene), polyfectamine transfection reagent (Qiagen), and cell counting kit-8 (Dojindo) were purchased. Characterization of the Microorganism. Characterization of the strain MK832-95F2 was conducted by observing growth on yeast extract−malt extract agar (ISP medium No.2), oatmeal agar (ISP medium No.3), inorganic salts−starch agar (ISP medium No.4), and glycerol−asparagine agar (ISP medium No.5). The type of diaminopimelic acid isomers in whole-cell hydrolysates of the strain MK832-95F2 was determined to be the LL-form by the method of Staneck and Roberts.10 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of MK832-95F2 is AB693874. Physicochemical Properties of Pronqodine A. The physicochemical properties of pronqodine A are as follows: UV λmax (MeOH) 217, 281, 480 nm (ε 97 000, 40 800, 12 400); IR spectrum νmax (KBr) cm−1 3276, 3093, 3060, 3010, 1684, 1620, 1576, 1520, 1504, 1414, 1373, 1346, 1225, 1105, 1092. The molecular formula of pronqodine A was determined to be C8H7N2O2S by HRESIMS (positive ion mode; m/z 195.0223 (M + H)+ Δ 0.1 mDa).

EXPERIMENTAL SECTION

General Experimental Procedures. The HPLC column (Develosil RPAQEOUS 10 × 250 mm) for isolation of pronqodine A was obtained from Nomura Chemical. Bradykinin, purified human NQO1, and flavin adenine dinucleotide disodium salt hydrate (FAD) were obtained from Sigma, and NADH was purchased from Amresco. Prostaglandin E 2 HTRF kits were purchased from Cisbio. Prostaglandin D2, 6-keto-prostaglandin F1α, IL-6, COX-1 polyclonal antiserum, COX-2 polyclonal antiserum, and ovine COX-2 enzyme assay kit were purchased from Cayman Chemical. NQO1 monoclonal antibody (Abnova), CM-H2DCFDA (Invitrogen), glass bottom dishes 514

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and 13C NMR spectra of natural pronqodine A and synthetic pronqodine A (Figure S3) are available free of charge via the Internet at http://pubs.acs.org.

Total Synthesis of Pronqodine A. To a solution of compound 3 (50 mg, 0.33 mmol) in a mixture of ethyl alcohol (0.5 mL) and distilled water (0.5 mL) was added phenyliodine bis(trifluoroacetate) (284 mg, 0.66 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with ethyl acetate, washed with water, dried over MgSO4, and filtered. The filtrate was evaporated to give solids, which were used in the next reaction without purification. The solids were dissolved in ethanol (1 mL). Cerium chloride (306 mg, 1.65 mmol) and 40% methyl amine aqueous solution (0.275 mL, 3.3 mmol) were added to the solution, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, washed with water, dried over MgSO4, and filtered. The filtrate was evaporated to give solids, which were subjected to preparative TLC developed with a mixture of hexane and ethyl acetate (1:1) to give reddish-purple solids. The solids were crystallized from hot chloroform, and recrystallization was repeated four times to give compound 1 (15 mg, 23% yield in two steps). Measurement of Prostaglandins and IL-6 Release. Human synovial sarcoma cells, SW982, were incubated for 24 h in a 96-well plate (10 000 cells/well) and then washed with assay buffer (Hank’s balanced salt solution, 17 mM HEPES pH 7.4, 0.1% BSA). Cells were treated with 1 nM bradykinin at 37 °C. To detect prostaglandins and IL-6, cells were incubated for 30 min and 3 h, respectively. The release of prostaglandins and IL-6 into the assay buffer was quantified using an assay kit. Detection of Native, One-Electron Reductive, and TwoElectron Reductive Forms of Pronqodine A. Native and reductive forms of pronqodine A were analyzed by reversed-phase LCHRESIMS (Orbitrap ion trap; Thermo Fischer). The reaction mixture (10 μL) was eluted with a linear gradient between (A) CH3CN containing 0.01% TFA and (B) 0.01% aqueous TFA from 5% to 50% over 15 min at a flow rate of 0.28 mL/min using an ODS column (CAPCELL PAK C18 UG 5 μm, 2.0 × 150 mm; Shiseido). NQO1 Activity Assay. NQO1 activity was determined spectrophotometrically by monitoring the decrease of NADH absorbance at 340 nm using Envision (PerkinElmer).21 NADH was used as an electron donor, and pronqodine A and menadione were evaluated as electron acceptors. SW982 cells were harvested in 25 mM Tris-HCl (pH 7.6), 1 mM EDTA, and protease inhibitor cocktail (Roche Diagnostics K.K.) followed by sonication on ice. The centrifuged supernatant (1700g, 10 min) was used as the enzyme source. Reaction mixtures contained 25 mM Tris-HCl (pH 7.5), 200 μM NADH, 25 μM FAD, 0.7 mg/mL BSA, 0.01% Tween 20, SW982 cell lysate, and each concentration of pronqodine A with (condition A) or without (condition B) 30 μM dicoumarol at 37 °C. The specific activity of NQO1 was determined by the difference between conditions A and B. Visualization of Generating ROS in SW982 Cells by Confocal Microscopy and Quantification of ROS Production in 96-Well Plates. ROS loading buffer was prepared by mixing 10 μM CMH2DCFDA in the ROS assay buffer (17 mM HEPES pH 7.4, 0.1% BSA, Hank’s balanced salt solution). Cells were seeded in glass-bottom dishes and incubated in ROS loading buffer at 37 °C for 20 min, then washed with ROS assay buffer. Fluorescence images were obtained using laser-scanning confocal microscopy (LSM 5 Live; Zeiss); emission was collected through a 505−550 nm band-pass filter. Cells were imaged on the stage of an inverted microscope (Axiovert 200M) with a 63× Zeiss Plan-Neofluar objective. For the quantification of ROS production, SW982 cells were seeded in 96well plates and incubated for 16 h. ROS loading buffer was treated as described above, and fluorescence was measured using Envision (excitation at 485 nm, emission at 535 nm).

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-3-3441-4173. Fax: +81-3-3441-7589. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI, Grant Number 22710227. The authors would like to thank M. Kawada, H. Inoue, S Ohba, T. Masuda, S. Wada, M. Yamasaki, Y. Kubota, N. Kinoshita, and I. Kurata for providing technical assistance and helpful discussions.

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ASSOCIATED CONTENT

S Supporting Information *

The estimated partial structure of the active compound from IR, UV−vis HMBC, and MS spectra, six estimated possible structures of pronqodine A (Figure S1), 1H NMR spectra of natural pronqodine A and synthetic pronqodine A (Figure S2), 515

dx.doi.org/10.1021/np300643f | J. Nat. Prod. 2013, 76, 510−515