Naphthablins B and C, Meroterpenoids Identified ... - ACS Publications

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Naphthablins B and C, Meroterpenoids Identified from the Marine Sediment-Derived Streptomyces sp. CP26-58 Using HeLa Cell-Based Cytological Profiling Hana Martucci,† Scott E. Campit,† Stephanie R. Gee,† Walter M. Bray,‡ Trevor Gokey,† A. King Cada,† Ten-Yang Yen,† Katsuhiko Minoura,§ Anton B. Guliaev,† R. Scott Lokey,‡ and Taro Amagata*,† †

Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132, United States ‡ Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High Street, Santa Cruz, California, 95064, United States § Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan S Supporting Information *

ABSTRACT: HeLa cell-based cytological profiling (CP) was applied to an extract library of marine sediment-derived actinomycetes to discover new cytotoxic secondary metabolites. Among the hit strains, Streptomyces sp. CP26-58 was selected for further investigation to identify its cytotoxic metabolites. CP revealed that the known ionophore tetronasin (1) was responsible for the cytotoxic effect found in the extract. Furthermore, three naphthoquinone meroterpenoids, naphthablin A (2) and two new derivatives designated as naphthablins B (3) and C (4), were isolated from other cytotoxic fractions. The structures of the new compounds were elucidated based on analysis of their HRESIMS and comprehensive NMR data. The absolute configurations of the new compounds were deduced by simulating ECD spectra and calculating potential energies for the model compounds using density function theory (DFT) calculations. Compound 1 showed a significant cytotoxic effect against HeLa cells with an IC50 value of 0.23 μM, and CP successfully clustered 1 with calcium ionophores.

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As part of our program to identify new anticancer lead compounds from marine sediment-derived actinomycetes, we have recently screened an organic extract library of marine sediment-derived actinomycetes against the HeLa cell-based CP. Among the hit strains, the strain Streptomyces sp. CP26-58 was selected for further investigation to elucidate cytotoxic substances. The application of CP identified tetronasin (1)8 as a compound responsible for the cytotoxic effect against HeLa cells. We have also identified three naphthoquinone meroterpenoids, naphthablin A (2, originally reported as naphthablin)9 and two new compounds designated as naphthablins B (3) and C (4), from weaker cytotoxic fractions. Described below is the structure elucidation for 3 and 4 as well as the biological evaluation, including CP-based target identification for 1.

igh-content screening (HCS) is an unbiased image-based whole cell screening method recognized as a promising screening tool for drug discovery.1,2 This screening technique has been applied to a wide array of therapeutic areas, including oncology, infectious diseases, cardiovascular diseases, and neurodegenerative diseases.3 High-content analysis (HCA) of HeLa cells treated by a number of cytotoxic compounds revealed that HeLa cells showed 12 unique cell morphologies based on inhibition of molecular targets and cell cycle arrest.4 Using the morphological characteristic of HeLa cells, cytological profiling (CP) has been recently introduced as a “Function First” approach.5 In this screening methodology, the multiparametric phenotypic responses based on the treatment of a small molecule drug are converted as an inherent fingerprint.6 This HeLa cell-based CP has successfully identified molecular targets of bioactive secondary metabolites with a small quantity of extract,5 which is an advantage of CP, eliminating the time-consuming target identification required for classical drug discovery. More recently, CP has been integrated into untargeted metabolomics as a next generation screening approach designated as “Compound Activity Mapping”.7 © 2017 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Phil Crews Received: October 29, 2016 Published: January 27, 2017 684

DOI: 10.1021/acs.jnatprod.6b00996 J. Nat. Prod. 2017, 80, 684−691

Journal of Natural Products

Article

The most potent cytotoxic fraction (27.0−28.0 min, 89% cytotoxicity, Figure 2) contained a compound with the

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RESULTS AND DISCUSSION We have screened a chemical library containing 474 organic extracts of marine sediment-derived actinomycetes against HeLa cells. The screening results selected 40 strains (8.4%) that exhibited over 95% cytotoxicity at 60 μg/mL. The strain Streptomyces sp. CP26-58 (95% cytotoxicity) was selected for further investigation due to a unique chemical profile observed in the HRLCMS analysis. The peak library (PL) generated from 20 mg of the extract was applied to CP (Figure 1A). The nonpolar fractions were clustered with the known ionophores, ionomycin10 at 0.295 μM and A2318711 at 0.382 μM, whereas the medium polar fractions were clustered with the adenylyl cyclase inhibitor SQ2253612 at 0.195 μM and the acetylcholinesterase inhibitor huperzine A13 at 0.165 μM (Figure 1B).

Figure 2. HRLCMS data of the CP26-58 extract [MeCN/H2O linear gradient (1:9 to 1:0) over 20 min] and the cytotoxic effects for the selected PL fractions.

molecular formula C35H54O8, which was dereplicated as the known ionophore tetronasin (1).8,14 The molecular formula of the main compound in the mildly cytotoxic fraction (22.0−23.0 min, 73% cytotoxicity) was suggested to be C29H36O9 based on the HRLCMS data (Figure 2). This compound showed a characteristic UV pattern (λmax 210, 270, 315, 420 nm, Figure S6), indicating the presence of a 5-hydroxynaphthoquinone

Figure 1. (A) CP results for the peak library (PL) fractions of the CP26-58 extract with 480 compounds at four dilutions. (B) Selected cluster data for the PL fractions. The CP fingerprint is generated by 12 scans for nuclear stain images and 16 scans for cytoskeleton stain images. The cluster data were obtained using Cluster 3.0, which was visualized by Java TreeView.5 685



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Table 1. NMR Data for 3 and 4 in CD3OD (600 MHz for 1H and 150 MHz for 13C) 3

a

no.

δC, type

δH, mult. (J in Hz)

1ax 1eq 2 3 4ax 4eq 4a 5 6a 7 7a 8 9 10 11 11a 12 12a 12b 13 14 15 1′ 2′ 3′a 3′b 4′ 5′ 1″ 2″ 3″ 4″α 4″β 5″

30.0, CH2

1.73, 2.80, 1.45, 4.95, 1.32, 2.00, 1.92,

31.5, CH 73.7, CH 29.0, CH2 37.7, 81.6, 157.1, 178.2, 108.4, 163.1, 125.4, 161.3, 110.0, 137.1, 185.7, 120.9, 31.5, 18.3, 25.9, 25.4, 178.0, 43.1, 28.0,

CH C C C C C C C CH C C C CH CH3 CH3 CH3 C CH CH2

12.1, 17.4, 52.9, 101.3, 103.4, 76.9,

CH3 CH3 C CH CH CH2

20.5, CH3

td (13.5, 4.7) brd (14.1) m br s td (14.7, 3.0) dt (14.7, 3.3) dt (12.8, 4.4)

4

gCOSY 1eq, 2, 12b 1ax, 3, 12b 1ax, 13 1eq, 4ax, 4eq 3, 4eq, 4a 3, 4ax 4ax, 12b

7.06, s

NOESY

δC

1eq, 4a, 12b, 13 1ax, 2, 12b, 13 1eq, 3, 4ax, 13 2, 4ax, 4eq, 13 2, 3, 4eq, 14 3, 4ax, 4a, 14 1ax, 4eq, 12b, 14, 15

29.9

gHMBC 2, 3, 12a, 12b

4a 4, 12a, 12b

7a, 9, 10, 11a, 12, 1″

3.20, 0.80, 1.41, 1.29,

br s d (6.7) s s

1eq, 1ax, 4a 2

1, 2, 12a 1, 2, 3 4a, 5, 6a, 15 4a, 5, 14

1ax, 1eq, 4a, 15 1eq, 1ax, 2, 3 4ax, 4eq, 4a 4a, 12

2.42, 1.53, 1.69, 0.93, 1.17,

sextet (6.9) m m t (7.5) d (7.1)

5′ 3′b, 4′ 3′a, 4′ 3′a, 3′b 2′

1, 3′, 4′, 5′ 1′, 2′, 4′, 5′ 1′, 2′, 4′, 5′ 2′, 3′ 1′, 2′, 3′

3a′, 4′, 5′ 2′, 3′b, 4′, 5′ 3′a, 4′, 5′ 2′, 3′a, 3′b 3′a, 3′b

4.84, 5.47, 4.21, 4.04, 1.63.

s s d (8.8) d (8.8) s

8, 9, 1″, 4″, 5″ 1″, 4″ 9, 1″, 2″, 3″ 9, 5″ 9, 1″, 2″, 4″

5″

4″β 4″α

4″β, 5″ 4″α, 5″ 2″, 4″α, 4″β

δH, mult. (J in Hz)

31.5 73.7 29.0 37.7 81.6 157.2 178.5 107.8 163.2 125.3 160.8a 110.9 137.0 186.0 120.8 31.6 18.2 25.9 25.4 178.0 35.7 19.5 19.6 52.9 101.2 103.4 77.0 20.6

1.74, 2.79, 1.46, 4.95, 1.32, 1.99, 1.93,

td br m br td dt dt

(13.5, 4.7) d (14.0) s (14.5, 2.7) (14.5, 3.6) (12.8, 5.0)

7.03, s

3.20, 0.80, 1.41, 1.29,

br s d (6.9) s s

2.61, hept (7.1) 1.186, d (7.0) 1.194, d (7.0)

4.82, 5.47, 4.22, 4.04, 1.62,

s s d (8.8) d (8.8) s

Detected at 125 MHz.

Figure 3. Selected 2D NMR correlations and the planar structure of 3.

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structure has been firmly determined by X-ray crystal analysis.18 Interestingly, compound 3 was obtained as a single epimer though it had a hemiacetal group in ring F. This phenomenon was also observed for a series of the synthetic lyxofuranoses possessing a fused 2,3-γ-butyrolactone.19 The synthesis of the lyxofuranoses with a fused γ-butyrolactone only furnished a single anomer on the hemiacetal carbon for each synthesized compound, and the stereostructure of (1S,5S,6S,8S)-5-methyl6-pivaloyloxymethyl-2,7-dioxycyclo[3.3.0]octane-2-one (5) was firmly confirmed by X-ray crystal analysis.20 This synthetic study and our findings indicate that an equilibrium on the hemiacetal group is not observed for hexahydrofuro[3,4b]furan-6-ol (E/F rings in 3). Potential energy simulated using density functional theory (DFT) calculations for the epimers on C-3″ discussed below also supported the assignment of C-3″. The relative configurations of the stereogenic centers and conformations for rings A and B were deduced based on the coupling constants and NOESY correlations (Figure 4). Ring A

moiety.15 Dereplication suggested that this compound was the known naphthoquinone meroterpenoid naphthablin A (2).9 In the other two mildly cytotoxic fractions (16.0−18.0 min, Figure 2), there were two additional meroterpenoinds 3 and 4 with a similar UV profile to that of 2. Though the HRLCMS data suggested the molecular formulas of 3 and 4 to be C30H36O9 and C29H34O9, respectively, they were not dereplicated. Thus, we conducted a scale-up culture to elucidate the structures of compounds 3 and 4 and their cytotoxic effects. An organic extract obtained from the culture was subjected to HP20 column chromatography to fractionate the secondary metabolites produced by this actinomycete strain. Compounds 1−4 accumulated in the MeOH/H2O (4:1) fraction, each of which was purified by reversed-phase HPLC. As suggested by dereplication, compounds 1 and 2 were confirmed as tetronasin and naphthablin A, respectively, based on their spectroscopic data. The molecular formula of naphthablin B (3) was elucidated as C30H36O9 based on the HRESIMS data. The 13C NMR spectra of 3 showed 29 carbon signals, one of which was two overlapping signals at δC 31.5 to yield a total of 30 carbons (Table 1). These carbon signals were classified into the following groups based on HSQC and DEPT experiments: (1) three carbonyl groups (C-7, C-12, C-1′), (2) four pairs of olefinic carbons (C-6a, C-7a, C-8, C-9, C-10, C-11, C-11a, C12a), one of which has a hydrogen (C-11), (3) seven sp3 methines (C-2, O-C-3, C-4a, C-12b, C-2′, O-C-2″, O-C-3″), including three carbons bonded to oxygens and one α carbon to an ester (O-C-3), (4) four sp3 methylenes (C-1, C-4, C-3′, O-C-4″), including one oxygen bearing methylene, (5) six methyl groups (C-13, C-14, C-15, C-4′, C-5′, C-5″) and (6) two remaining sp3 quaternary carbons (C-5, C-1″). Analysis of the COSY and HMBC data provided three substructures, including 2-methylbutanoic acid, p-menthane-2,8-diol, and 4methyl-tetrahydrofuran-2,3-diol. The 2-methylbutanoic acid moiety was connected as an ester on the C-3 position of the p-menthanediol moiety due to its proton and carbon chemical shifts (δH 4.95, δC 73.7) (Figure 3). The naphthoquinone core (rings C and D) had a low H/C ratio (0.1), indicating that it would be difficult to assemble this moiety with HMBC correlations based on the Crews rule.16,17 HMBC correlations from H-11 (δH 7.06) to C-7a (δC 108.4), C-9 (δC 125.4), C-10 (δC 161.3), C-11a (δC 137.1), and C-12 (δC 185.7) partially established the naphthoquinone core. The remaining four carbons in the ring system, C-6a (δC 157.1), C-7 (δC 178.2), C8 (δC 163.1), and C-12a (δC 120.9), were assigned based on the comparison of the carbons signals of C-6a (δC 157.3), C-7 (δC 183.4), C-8 (δC 165.8), and C-12a (δC 121.8) in 2, which was coisolated from the same culture (Table S2). The menthanediol moiety was connected to the naphthoquinone core through HMBC correlations from H-1ax (δH 1.73), H-4a (δH 1.92), and H-12b (δH 3.20) to C-12a (δC 120.9) as well as a relatively strong long-range HMBC correlation from CH3-14 (δH 1.41) to C-6a (δC 157.1). The tetrahydrofuran moiety was attached on the naphthoquinone benzene based on HMBC correlation from H-2″ (δH 4.84), CH2-4″ (δHα 4.21, δHβ 4.04), and H-5″ (δH 1.63) to C-9 (δC 125.4) and from H-2″ to C-8. Thus, the planar structure of 3 was determined to be a fused hexacyclic naphthoquinone. The possibility of the structural isomer obtained by flipping the C-6a (δ 157.1)/C-12a (δ 120.9) double bond was eliminated based on the similar carbon chemical shifts (δ 153.5 and 123.3 in CDCl3) of the biosynthetically related meroterpenoid naphtherpin, whose

Figure 4. Key NOE correlations for 3.

was deduced to exist in a chair conformation due to the following two observations: (1) NOEs between H-1ax and H-4a as well as H-2 and H-4ax and (2) large coupling between H-1ax and H-2 (3J = 13.5 Hz) as well as H-4a and H-4ax (3J = 12.8 Hz). These observations suggested CH3-13 on C-2 was in an equatorial arrangement. The small coupling constant of H-3 (br s) implied this proton was in an equatorial arrangement and oriented cis to H-2, and H-4ax, which indicated that the ester functional group on C-3 was in an axial arrangement and oriented cis to H-1ax, H-4a, and CH3-13. The small coupling constant of H-12b (br s) implied that this proton was also in an equatorial arrangement and oriented cis to H-1ax and H-4a, which indicated a cis-fused A/B ring system. Ring B was deduced to exist in a twist-chair conformation based on observation of NOE between H-12b and CH3-15. This NOE suggested CH3-15 was cis relative to H-4a and H-12b, which was further supported by NOEs between H-4ax and CH3-14 as well as H-4eq and CH3-14. NOEs between H-2″ and CH3-5″ indicated that ring E existed in a planar conformation and was cis-fused with ring F. Neither spin−spin coupling nor NOE correlation between H-2″ and H-3″ suggested 3″-OH was oriented cis to H-2″ and CH3-5″. This configurational assignment for C-3″ was supported by the synthetic γbutyrolactone fused lyxofuranose 5, which has an identical stereochemical relationship to that of 3, in which no spin−spin coupling was observed between the hemiacetal methine and the oxymethine.15 Thus, the relative configurations except C-2′ were deduced as either 2R*,3S*,4aS*,12bR*,1″S*,2″S*,3″S* or 2R*,3S*,4aS*,12bR*,1″R*,2″R*,3″R* because there are no 687



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Figure 5. (A) Simulated ECD spectra for 4a−4d in the gas phase. (B) Experimental ECD spectrum of 4. (C) Simulated ECD spectra for 4a and 4b in MeOH and experimental ECD spectrum 4.

Figure 6. CP results for 1 and the clustered compounds.

4′ and the HMBC correlation from CH3-3′ to C-1′ (Table S3). The relative stereostructure of 4 was deduced to be the same as that of 3 due to the closely identical NMR chemical shifts and splitting patterns of the stereogenic centers between 4 and 3. The absolute stereostructure of 4 was determined by ab initio calculations of electronic circular dichroism (ECD) spectra.21 Four possible diastereomers (4a−4d) were subjected to timedependent density functional theory (TD-DFT) calculations to obtain simulated ECD spectra in the gas phase (Figure 5A). The experimental ECD data of 4 in MeOH (Figure 5B) were similar to those of 4a and 4b. To distinguish between 4a and 4b, we refined the geometries in MeOH and recalculated their ECD spectra (Figure 5C). The simulated ECD spectrum of 4a

stereochemical interactions between A/B and E/F rings. Lastly, the absolute configurations were determined as 2R,3S,4aS,12bR,1″S,2″S, 3″S due to the identical ECD spectrum of 3 to that of 4 (Figure S30), where the absolute stereostructure is elucidated below. The molecular formula of naphthablin C (4) was established as C29H34O9 based on the HRESIMS data, which differed from that of 3 by CH2. The general features of the 1H and 13C NMR spectra of 4 closely resemble those of 3 except for the two terminal carbons (C-3′ and C-4′) in the 2-methylbutyrate moiety in 3, which were replaced by a doublet methyl group (C-3′) in 4 (Table 1). This change was supported by COSY correlations between CH3-3′ and H-2′ as well as H-2′ and CH3688



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were recorded on an Agilent NMR system 600 DD2 at 600 MHz (1H) and 150 MHz (13C) and a Bruker Avance spectrometer operated at 500 MHz (1H) and 125 MHz (13C), with TMS as an internal reference. High-resolution MS was obtained on a Marinar ESITOFMS and Thermo Q Exactive. High-resolution LCMS was obtained in a Thermo Q Exactive. HPLC was performed on an Agilent 1200 LC system equipped with a diode array detector. Collection and Identification of Actinomycete Strain. The actinomycete strain (CP26−58) was separated from a sediment sample collected at the San Francisco Bay tidal flat in August 2008, using a seawater-based ISP3 agar plate. The CP26-58 strain was then purified on a seawater-based ISP2 agar plate. The 16S rRNA sequence of this strain was close to that of Streptomyces sp. CNQ509 with 99.6% identity. The sequence data has been deposited to GenBank (accession no. KX986151). Isolation of Compounds 1 − 4. The strain CP26-58 was grown in a seed liquid medium (pH 7.4, 50 mL) containing glucose (2%), soluble starch (1%), yeast extract (0.5%), peptone (0.5%), meat extract (0.5%) and CaCO3 (0.3%) in artificial seawater with trace element mix (0.1% v/v) for 7 days at 30 °C at 240 rpm.17 This seed culture (10 mL) was inoculated in a production liquid (pH 7.4, 1 L x 32) containing soluble starch (1%), yeast extract (0.4%), peptone (0.2%), CaCO3 (0.1%), FeSO4·7H2O (0.004%) in artificial seawater with trace element mix (0.1% v/v). The production medium was incubated for 7 days at 30 °C at 240 rpm. The culture was centrifuged (6000 G, 30 min) to separate culture broth from the actinomycete pellet. To the separated culture broth, HP-20 resin (50 mL/L) was added and the suspension was stirred for 30 min to absorb organic substances. The resin was filtered out and rinsed thoroughly with deionized H2O, and was then eluted with MeOH. The pellet separated earlier through centrifugation was soaked in MeOH overnight three times. The combined MeOH solution from the HP-20 resin and pellet was dried under reduced pressure. The residue was partitioned between EtOAc (500 mL) and deionized H2O (500 mL) and the EtOAc layer was washed with deionized H2O three times. The EtOAc layer was dried under reduced pressure to give an extract (5.0 g). The EtOAc extract was partitioned through HP20 column chromatography with stepwise gradient (MeOH/H2O, 1:4, 2:3, 3:2, 4:1, 1:0). The MeOH/H2O (4:1) fraction (1.6 g) was subjected to reversed-phase HPLC using a Phenomenex Synergi Hydro-RP (80 Å, 4 μm, 250 mm × 10 mm i.d.) with MeCN/H2O linear gradient (1:1 to 1:0 over 20 min, then 10 min post gradient with 1:0 for 10 min, flow rate 3.0 mL/min) to afford three semipure fractions H1 (34.4 mg, tR 15.0−18.0 min), H2 (91.7 mg, tR 22.0−23.0 min) and H3 (225 mg, tR 26.5−28.0 min). H1 was further purified with revered-phase HPLC using a Phenomenex Synergi Hydro-RP (80 Å, 4 μm, 150 mm × 4.6 mm i.d.) with MeCN/ H2O liner gradient (3:7 to 4:1 over 20 min, flow rate 1.0 mL/mL) to afford 4 (1.5 mg, tR 13.4 min) and 3 (3.9 mg, tR 14.0 min). H2 was purified with reversed-phase HPLC using a Phenomenex Synergi Hydro-RP (80 Å, 4 μm, 150 mm × 4.6 mm i.d.) with MeCN/H2O liner gradient (1:1 to 1:0 over 20 min, flow rate 1.0 mL/min) to afford 2 (15.1 mg, tR 13.1 min). Similarly, H3 was purified using a Phenomenex Synergi Hydro-RP (80 Å, 4 μm, 150 mm × 4.6 mm i.d.) with MeCN/H2O liner gradient (4:1 to 1:0 over 20 min) to afford 1 (22.3 mg, tR 14.0 min). Tetronasin (1). Colorless amorphous solid; 1H and 13C NMR data, see Table S1; HRESI-TOFMS m/z 625.3688 [M + Na]+ (calcd for C35H54O8Na, 625.3711). Naphthablin A (2). Orange amorphous solid; [α]24D −262 (c 0.7 MeOH), [α]24D −166 (c 0.2 CHCl3);9 UV (MeOH) λmax (log ε) 210(4.49), 270 (4.18), 315 (4.02), 420 (3.61) nm; 1H and 13C NMR data, Table S2; HRESI-TOFMS m/z 513.2489 [M + H]+ (calcd for C29H37O8, 513.2483). Naphthablin B (3). Orange amorphous solid; [α]24D −107 (c 2.1 MeOH); UV (MeOH) λmax (log ε) 215 (4.69), 265 (4.43), 310 (4.29), 390 (3.84) nm; ECD (5.0 × 10−5 M, MeOH) λmax (Δε) 212 (+2.48), 228 (+1.08), 261 (−1.21), 280 (−0.27), 310 (−1.32), 360 (+0.24), 439 (−0.35), 546 (+0.29) nm; 1H and 13C NMR data, Table 1; HRESI-TOFMS m/z 563.2268 [M + Na]+ (calcd for C30H36O9Na, 563.2252).

in MeOH agreed well with the experimental ECD spectrum of 4 in terms of the general features and the relative intensities of the Cotton effects. Thus, the absolute configurations for the stereogenic centers of 4 were proposed as 2R,3S,4aS,12bR,1″S,2″S,3″S. The potential energies for 4 and its epimer at C-3″ were simulated using DFT calculations to clarify the stereochemical assignment for C-3″. The calculation results indicated that 4 was thermodynamically more stable than the C-3″ epimer by 1.51 kcal/mol, which supported the assignment of C-3″. Cytotoxic effects of the four compounds isolated in this study were evaluated against HeLa cells. The known ionophore tetronasin (1), which was the main cytotoxic compound produced by the strain CP26-58, showed significant cytotoxicity with an IC50 value of 0.23 μM. The meroterpenoids 2−4 exhibited weak HeLa cell growth inhibition with 19%, 25%, and 32% inhibition at 33 μM, respectively. These data implied that the cytotoxic effects observed in the peak library fractions containing these meroterpenoids likely came from other minor components. CP results clustered tetronasin (1) at four dilutions with the calcium ionophores ionomycin10 at 0.295 μM and A2381711 at 0.382 μM (Figure 6), suggesting CP functioned effectively. As depicted in Figure 6, these known inophores exhibited different CP profiles at higher concentrations, which indicates that cluster data imply relative inhibitory activities to the standard bioactive compounds used for CP. The cluster data of the PL fraction containing 1 described above were identical to that of 1 purified from the extract. These cluster results verified the most important feature of the HeLa cell-based CP “Function First” previously reported by Linington and co-workers.5

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CONCLUSIONS This study verified that the HeLa cell-based CP was an excellent screening tool to measure cytotoxic effects of small organic molecules as well as identifying their potential molecular targets. Ionophore antibiotics such as tetronasin (1) and monensin have been widely used as antibiotic growth promoters for livestock,22 but they have also recently received increased attention as promising anticancer drug candidates. In fact, anticancer properties for the known ionophores, including salinomycin,23 monensin,24 and alborixin,25 have been reported. Contrary to the extensive study for these ionophores, this is the first report for 1 in regard to its cytotoxic effects against cancer cells. Compounds 2−4 isolated in this study are categorized as naphthoquinone meroterpenoids, which are relatively common secondary metabolites produced by actinomycetes. The structural variation of this class of compounds depends on the cyclization pattern of the isoprene units. The biosynthetic gene clusters for this class of compounds, including napyradiomycins26,27 and merochlorins,28 have been elucidated. The key biosynthetic enzymes for these naphthoquinone meroterpenoids are vanadium-dependent chloroperoxidases that mediate both halogenation and cyclization of the terpene unit. We predict that these enzymes are also involved in the biosynthesis of 2−4.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a JASCO P-2000 polarimeter. UV spectra were measured in a Varian 50 Scan Cary 1 UV−visible spectrometer with a path length of 1 cm. ECD spectra were collected in a JASCO J-820 CD spectrometer with a path length of 0.3 cm. 1D and 2D NMR spectra 689



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Naphthablin C (4). Orange amorphous solid; [α]24D −140 (c 0.3 MeOH); UV (MeOH) λmax (log ε) 215 (4.43), 265 (4.05), 310 (3.88), 385 (3.43) nm; ECD (2.3 × 10−4 M (MeOH) λmax (Δε) 213 (+15.0), 228 (+4.05), 260 (−9.32), 280 (−3.10), 310 (−9.52), 369 (−0.75), 430 (−2.41), 547 (+1.47); 1H and 13C NMR data, Table 1; HRESIMS m/z 525.2130 [M-H]− (calcd for C29H33O9, 525.2119). DFT Calculations for 4. All DFT calculations were performed using Gaussian0929 based on the procedure that we previously reported.21 Briefly, gas phase geometry optimizations and ECD spectra for 4a - 4d were calculated using the B3LYP functional with 6-311+G(d,p) basis set. Frequency analysis was performed to calculate the zero-point corrected energies for 4 and its epimer on C-3″. To distinguish 4a and 4b, geometries and ECD spectra were further refined in MeOH implicit solvent using SCRF-B3LYP/aug-cc-pVDZ. The energies for 4 and its C-3″ epimer were calculated as −1802.509784 H and −1802.507378 H, respectively. Cytological Profiling. Cytological profiling was performed at the chemical screening center, University of California Santa Cruz. The detailed methods for culturing and staining HeLa cells and evaluation of assay results have been reported previously.5−7

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(4) Futamura, Y.; Kawatani, M.; Kazami, S.; Tanaka, K.; Muroi, M.; Shimizu, T.; Tomita, K.; Watanabe, N.; Osada, H. Chem. Biol. 2012, 19, 1620−1630. (5) Schulze, C. J.; Bray, W. M.; Woerhmann, M. H.; Stuart, J.; Lokey, R. S.; Linington, R. G. Chem. Biol. 2013, 20, 285−295. (6) Woehrmann, M. H.; Bray, W. M.; Durbin, J. K.; Nisam, S. C.; Michael, A. K.; Glassey, E.; Stuart, J. M.; Lokey, R. S. Mol. BioSyst. 2013, 9, 2604−2617. (7) Kurita, K. L.; Glassey, E.; Linington, R. G. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 11999−12004. (8) Kellerjuslen, C.; King, H. D.; Kuhn, M.; Loosli, H. R.; Pache, W.; Petcher, T. J.; Weber, H. P.; Vonwartburg, A. J. Antibiot. 1982, 35, 142−150. (9) Umezawa, K.; Masuoka, S.; Ohse, T.; Naganawa, H.; Kondo, S.; Ikeda, Y.; Kinoshita, N.; Hamada, M.; Sawa, T.; Takeuchi, T. J. Antibiot. 1995, 48, 604−607. (10) Liu, C. M.; Hermann, T. E. J. Biol. Chem. 1978, 253, 5892− 5894. (11) Reed, P. W.; Lardy, H. A. J. Biol. Chem. 1972, 247, 6970−6977. (12) Haslam, R. J.; Davidson, M. M. L.; Desjardins, J. V. Biochem. J. 1978, 176, 83−95. (13) Liu, J. S.; Zhu, Y. L.; Yu, C. M.; Zhou, Y. Z.; Han, Y. Y.; Wu, F. W.; Qi, B. F. Can. J. Chem. 1986, 64, 837−839. (14) Martinek, T.; Riddell, F. G.; Rutherford, T. J.; Sareth, S.; Weller, C. T. Chem. Commun. 1998, 1893−1894. (15) Rodrigues, S. V.; Viana, L. M.; Baumann, W. Anal. Bioanal. Chem. 2006, 385, 895−900. (16) White, K. N.; Amagata, T.; Oliver, A. G.; Tenney, K.; Wenzel, P. J.; Crews, P. J. Org. Chem. 2008, 73, 8719−8722. (17) Molinski, T. F.; Morinaka, B. I. Tetrahedron 2012, 68, 9307− 9343. (18) Shin-ya, K.; Imai, S.; Furihata, K.; Hayakawa, Y.; Kato, Y.; Vanduyne, G. D.; Clardy, J.; Seto, H. J. Antibiot. 1990, 43, 444−447. (19) Alibes, R.; Bourdelande, J. L.; Gregori, A.; Font, J.; Rustullet, A.; Parella, T. J. Carbohydr. Chem. 2003, 22, 501−511. (20) Gregori, A.; Alibes, R.; Bourdelande, J. L.; Font, J. Tetrahedron Lett. 1998, 39, 6963−6966. (21) Amagata, T.; Xiao, J.; Chen, Y. P.; Holsopple, N.; Oliver, A. G.; Gokey, T.; Guliaev, A. B.; Minoura, K. J. Nat. Prod. 2012, 75, 2193− 2199. (22) Bretschneider, G.; Elizalde, J. C.; Perez, F. A. Livest. Sci. 2008, 114, 135−149. (23) Huczynski, A. Bioorg. Med. Chem. Lett. 2012, 22, 7002−7010. (24) Tumova, L.; Pombinho, A. R.; Vojtechova, M.; Stancikova, J.; Gradl, D.; Krausova, M.; Sloncova, E.; Horazna, M.; Kriz, V.; Machonova, O.; Jindrich, J.; Zdrahal, Z.; Bartunek, P.; Korinek, V. Mol. Cancer Ther. 2014, 13, 812−822. (25) Shah, A. M.; Wani, A.; Qazi, P. H.; Rehman, S. U.; Mushtaq, S.; Ar, S. A.; Hussain, A.; Shah, A.; Qazi, A. K.; Makhdoomi, U. S.; Hamid, A.; Kumar, A. Chem.-Biol. Interact. 2016, 256, 198−208. (26) Winter, J. M.; Moffitt, M. C.; Zazopoulos, E.; McAlpine, J. B.; Dorrestein, P. C.; Moore, B. S. J. Biol. Chem. 2007, 282, 16362−16368. (27) Bernhardt, P.; Okino, T.; Winter, J. M.; Miyanaga, A.; Moore, B. S. J. Am. Chem. Soc. 2011, 133, 4268−4270. (28) Kaysser, L.; Bernhardt, P.; Nam, S. J.; Loesgen, S.; Ruby, J. G.; Skewes-Cox, P.; Jensen, P. R.; Fenical, W.; Moore, B. S. J. Am. Chem. Soc. 2012, 134, 11988−11991. (29) Frisch, M. J. T., 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, Jr., J. A.; 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.;

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00996. 16S rRNA sequence of Streptomyces sp. CP26-58, HRESIMS, UV, 1D and 2D NMR spectra for 1−4 and ECD spectra for 3 and 4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 415 338 7713. E-mail: [email protected]. ORCID

R. Scott Lokey: 0000-0001-9891-1248 Taro Amagata: 0000-0002-8375-0098 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided to T.A. from NIGMS (SC2GM088057), California State University Program for Education & Research in Biotechnology (CSUPERB), and Department of Chemistry and Biochemistry, San Francisco State University. S.E.C. and S.R.G. were supported by Beckman Scholarship. Q Exactive analysis work was supported by an NSF MRI grant (CHE-1228656). The cytological profiling experiments were performed in the UCSC Chemical Screening Center, which is supported by QB3, a California initiative in quantitative biosciences. We thank M. Schorn (SIO, UCSD) for assistance with the 16S rRNA sequence analysis.

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DEDICATION Dedicated to Professor Phil Crews, of the University of California, Santa Cruz, for his pioneering work on bioactive natural products.

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REFERENCES

(1) Bickle, M. Anal. Bioanal. Chem. 2010, 398, 219−226. (2) Zanella, F.; Lorens, J. B.; Link, W. Trends Biotechnol. 2010, 28, 237−245. (3) Fraietta, I.; Gasparri, F. Expert Opin. Drug Discovery 2016, 11, 501−514. 690


Journal of Natural Products Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009.

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Naphthablins B and C, Meroterpenoids Identified ... - ACS Publications

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