Sekgranaticin, a SEK34b-Granaticin Hybrid Polyketide from

Jun 26, 2019 - Sekgranaticin, a SEK34b-Granaticin Hybrid Polyketide from Streptomyces sp. 166#. S. 1. Supporting Information. Sekgranaticin, a SEK34b...
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Cite This: J. Org. Chem. 2019, 84, 9087−9092

Sekgranaticin, a SEK34b-Granaticin Hybrid Polyketide from Streptomyces sp. 166# Qianqian Lv,†,∥ Yaqin Fan,†,∥ Ganzheng Tao,‡ Peng Fu,†,§ Jingxin Zhai,‡ Boping Ye,*,‡ and Weiming Zhu*,†,§

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†

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China ‡ School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China § Open Studio for Druggability Research of Marine Natural Products, Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266003, China S Supporting Information *

ABSTRACT: Sekgranaticin (1), a novel hybrid polyketide with a complex 6/6/6/6/6/6/6 7-ring system, was isolated together with granaticins A (2) and B (3) and methyl granaticinate (4) from the culture broth of Streptomyces sp. 166#. Its structure was elucidated by spectroscopic analysis. The absolute configuration was determined on the basis of the calculated 13C NMR and electronic circular dichroism data. Compounds 1−4 exhibited potent cytotoxicity against cancer cell lines MCF-7, A549, P6C, and HCT-116 with IC50 values of 0.02−6.77 μM. The biosynthetic pathway of sekgranaticin (1) was proposed.

1. INTRODUCTION Actinobacteria, a phylum of Gram-positive bacteria, are an important source of natural products with new structures and potent biological activities. They are the origins of many drugs, especially antibiotics.1 Drugs from actinobacteria, represented by streptomycin, rifamycin, abamectin, and so on, have made a great contribution to the fight against diseases.2 Type II aromatic polyketides, a large class of secondary metabolites from actinobacteria, have attracted much attention due to their wide range of biological activities.3 Some of them have been used in the clinic, such as the antibiotic tetracycline and the antitumor drug daunorubicin.4 The enormous potential in antibacterial and antitumor applications of type II aromatic polyketides, especially quinones,5 attracted us to discover new derivatives. We screened actinobacterial strains isolated from extreme environment, such as marine and saline soil, to find new active quinone derivatives. In the process, Streptomycin sp. 166# isolated from clayey cold saline soil was selected because its metabolites showed cytotoxic activity against A549 cells (Table S3) and had UV absorptions similar to those of naphthaquinones.6 Chemical investigation resulted in the isolation of a novel polyketide, sekgranaticin (1), with a rare 6/6/6/6/6/6/6 7-ring system. This compound was postulated to be biogenetically derived from two known natural products, SEK34b and granaticin A (2), which were bridged together by a pyran ring. Granaticins A (2) and B (3),5b,7 as well as methyl granaticinate (4),8 were also identified from the fermentation broth of this strain. All of the compounds 1−4 displayed significant © 2019 American Chemical Society

cytotoxicity against the cancer cell lines MCF-7, A549, P6C, and HCT-116 with IC50 values ranging from 0.02 to 6.77 μM.

2. RESULTS AND DISCUSSION Sekgranaticin (1) was obtained as a yellow-green powder. Its molecular formula was determined as C38H32O15 based on the high-resolution mass spectrometry (HRMS, ESI-Orbitrap) peak at m/z 729.1798 [M + H]+ (Figure S5). The 13C NMR spectrum (Figures S10−S12) showed 38 signals, which were classified by HSQC (Figures S13−S16) as 20 nonprotonated carbons (1 Received: April 14, 2019 Published: June 26, 2019 9087

DOI: 10.1021/acs.joc.9b01022 J. Org. Chem. 2019, 84, 9087−9092

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The Journal of Organic Chemistry Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Sekgranaticin (1) in DMSO-d6 no.

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18a 18b 19 20 21 22 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 8-OH 11-OH 19-OH 20-OH

171.6, C 36.3, CH2 69.7, CH 70.2, CH 43.1, CH 198.1, C 112.1, C 147.0, C 136.1, C 135.5, C 152.7, C 113.7, C 197.8, C 47.7, C 71.8, CH 14.5, CH3 61.4, CH 36.6, CH2 70.1, CH 80.2, C 71.5, CH 16.8, CH3 160.4, C 98.6, C 167.0, C 98.3, CH 165.4, C 36.8, CH2 134.8, C 128.9, CH 133.4, CH 118.3, CH 157.4, C 121.0, C 178.5, C 111.1, CH 165.6, C 19.6, CH3

δH, mult. (J in Hz)

COSY

HMBC

2.66, dd (16.2, 4.4); 2.28, dd (16.1, 8.5) 4.23, dd (8.5, 4.2) 5.09, brs (4.1)a 4.44, brs (4.0)a

3 4, 2 5, 3 4

1, 3 2, 4 5, 6, 14, 3′ 4, 6, 13, 14, 15, 2′

4.12, q (6.6) 1.49, d (6.5) 5.00, brs 2.54, m 1.33, d (14.2) 3.82, d (8.2)

16 15 18 17, 19 17, 19 17, 18, 19-OH

3, 5, 14, 16 14, 15 9, 19, 21

3.65, q (6.2) 0.76, d (6.1)

22 21

10, 19, 20 20, 21

2′, 3′, 5′, 6′

4.52, d (15.8); 4.34, d (16.0)

4′, 5′, 7′, 8′, 12′ 9′ 8′, 10′ 9′

4, 16 5, 3 4, 16

3, 5 19, 21 19-OH 18a, 21

5.58, s

7.29, d (7.4) 7.69, dd (7.4, 8.5) 7.54, d (8.5)

nuclear overhauser effect spectroscop (NOESY)

18a, 19

6′, 9′, 10′, 12′ 7′, 11′ 8′, 11′, 12′

6.15, s

12′, 15′, 16′

2.34, s 11.62, s 11.58, s 5.21, brs 6.05, s

14′, 15′ 7, 8, 9 19

18b 10, 20, 21

a

Half peak width: peak width at half height.

carboxyl carbon, 1 ester carbon, 3 keto carbonyls, 6 oxygenated sp2 carbons, 7 nonoxygenated sp2 carbons, 1 oxygenated sp3 carbon, and 1 nonoxygenated sp3 carbon), 12 methine groups (5 olefinic carbons, 6 oxygenated sp3 carbons, and 1 nonoxygenated sp3 carbon), 3 methylene carbons, and 3 methyl groups (Table 1). In the 1H NMR (Figures S6−S9) and correlation spectroscopy (COSY) (Figures S17−S19) spectra, spin couplings among 7.29 (d, J = 7.4), 7.69 (dd, J = 7.4, 8.5) and 7.54 (d, J = 8.5) revealed the presence of a 1,2,3-trisubstituted benzene ring (Table 1 and Figure 1). Further analysis of its NMR data suggested that sekgranaticin (1) contains a similar moiety (part A in Figure 1) with the known polyketide, SEK34b.9 This fragment could be verified by the COSY correlations of H-8′/H-9′/H-10′, and the key heteronuclear multiple bond correlation (HMBC) (Figures S20−S22)

Figure 1. Key two-dimensional NMR correlations for the structural assignment of 1.

correlations of H-4′ to C-2′/C-3′/C-5′, H2-6′ to C-4′/C-5′/ C-7′/C-12′, H-8′ to C-6′/C-12′, H-9′ to C-7′/C-11′, H-10′ to 9088

DOI: 10.1021/acs.joc.9b01022 J. Org. Chem. 2019, 84, 9087−9092

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The Journal of Organic Chemistry C-11′/C-12′, H-14′ to C-12′/C-15′, and H3-16′ to C-14′/C-15′ (Figure 1). The remaining signals (Table 1) were similar to those of granaticin A (2),5b,7 which was also isolated from the culture broth of Streptomyces sp. 166#. Sekgranaticin (1) containing the same fragment (part B in Figure 1) as granaticin A (2) was also evidenced from the COSY correlations of H-17/ H2-18/H-19/19-OH and H-21/H3-22, along with the key HMBC correlations of HO-8 to C-7/C-8/C-9, H-17 to C-9/C21, H-21 to C-10/C-19/C-20, H3-22 to C-20, and HO-20 to C10/C-20 (Figure 1). A double bond (Δ5,14) of granaticin A (2) was replaced by a nonprotonated sp3 carbon (C-14, δC 47.7) and a sp3 methine group (CH-5, δH/C 4.44/43.1) of compound 1. The key HMBC correlations of H2-2 to C-1, H-5 to C-6/C-13/ C-14, H-15 to C-3/C-5, and H3-16 to C-14 revealed that the pyran-fused butanolide (γ-lactone) moiety in 2 was opened to form the corresponding hydroxypyran acetic acid in 1 (Figure 1). The connection between SEK34b moiety and granaticin moiety was confirmed by the HMBC correlations of H-5 to C-2′ and H-4 to C-3′ (Figure 1). The NOESY correlations of H-21/H-18a, H-18a/H-19, H21/H-19, and H-18b/19-OH (Figures 2 and S23) and the

Figure 3. 13C NMR calculation results of two plausible epimers (1 and iso-1) at the B3LYP/6-311++G(2d,p) level. (a) Linear correlation plots of the calculated and experimental 13C values. (b) Relative errors between the calculated and recorded 13C values. (c) DP4 probability of the 13C values of 1.

analysis of the fresh extract with EtOAc indicated that all compounds 1−4 are natural products by Streptomyces sp. 166# (Figure S31). The absolute configuration of 1 could be speculated based on the homologous biosynthetic pathway with granaticin A (2). This deduction was further verified by electronic circular dichroism (ECD) calculations of the simplified model compound I using time-dependent density functional theory method at the B3LYP/6-31G(d,p) level (Figure S4, Tables S8 and S9).12 The measured ECD spectrum of compound 1 matches the calculated ECD curve of the model I (Figure 4). Thus, the absolute configuration of compound 1 was established as (3S,4S,5S,14R,15S,17R,19R,20S,21R)-. The cytotoxicities of compounds 1−4 against four cancer cell lines (MCF-7, A549, P6C, and HCT-116) were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. They displayed a comparable cytotoxic activity to adriamycin against cancer cell lines with the IC50 values ranging from 0.02 to 6.77 μM (Table 2, Figure S33). Among the four compounds, sekgranaticin (1) is the weakest because of the nucleophilic addition to α,β-unsaturated ketone, indicating a quinoid structure (α,β-unsaturated ketone moiety) as a key moiety for the cytotoxicity of this type of compounds.5b,13,14 Compounds 1−4 were also evaluated for their antimicrobial activity against eight bacteria (Staphylococcus aureus subsp. aureus, S. aureus subsp. aureus Rosenbach, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus cereus) and two fungi (Candida glabrata and Candida albicans). Only compounds 2 and 3 exhibited a stronger antibacterial activity than the positive control ciprofloxacin against B. cereus, S. aureus subsp. aureus, and S. aureus subsp. aureus Rosenbach. The corresponding minimum inhibitory concentration (MIC) values of 2, 3, and ciprofloxacin were 3.51, 0.18, and 0.18 μM; 22.40, 0.09, and 0.29 μM; and 37.76, 0.30, and 2.36 μM, respectively (Table 3).

Figure 2. Key NOE correlations of compound 1.

similar coupling constant (Tables 1 and S1) to those of granaticin A (2) suggested the same relative configuration of the left unit of part B of 1 to granaticin A (Figure 1). Meanwhile, the NOESY correlations of H-5/H-4, H-3/H3-16, and H3-16/H-5, and H-3/H-4 (Figure 2) revealed the relative configuration of the right unit of part B as shown in Figure 1, which was further verified by the small half peak widths (hpw) of H-4 (4.1 Hz) and H-5 (4.0 Hz) (Table 1). However, no NOESY correlations supported the relative configuration between the left and the right units of part B. To solve this problem, δC values of two plausible epimers, 1 and iso-1 (Figures 3 and S1), were calculated at the B3LYP/6311++G(2d,p) level (Figures S2 and S3, Tables S4−S7).10 The DP4 probability analysis (Figure 3 and Table S2)11 allowed the determination of the relative configuration of sekgranaticin as 1 instead of iso-1. This deduction could be evidenced from the possible biosynthetic pathway of 1 (Scheme 1), which was postulated to be biogenetically derived from two known microbial polyketides, granaticin A (2) and SEK34b. Granaticin A (2) undergoes a Michael addition with the carbanion of SEK34b by a nucleophilic attack from the front of the ring plane with less steric hindrance, followed by an enol−keto tautomerization and a ring-opening of γ-lactone ring to yield the intermediate 1a. The carboxy anion 1a is subjected to another enol−keto tautomerization to form the second intermediate 1b, which further undergoes an intramolecular Michael addition followed by protonation and enol−keto tautomerization to yield compound 1 with a trans-dihydroisochromene moiety. Thus, compound 4 might be an artificial product of 2 with MeOH in isolation by an easier cleavage of the acyloxy bond of the γ-lactone ring. However, the LC−MS 9089

DOI: 10.1021/acs.joc.9b01022 J. Org. Chem. 2019, 84, 9087−9092

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The Journal of Organic Chemistry Scheme 1. Plausible Biosynthetic Pathway of 1

Figure 4. Experimental ECD curve of compound 1, and the calculated ECD spectra of I and ent-I.

Table 2. IC50 Values of Compounds 1−4 against Cancer Cell Lines (μM) MCF-7 A549 P6C HCT-116

1

2

3

4

adriamycin

1.26 ± 0.010 4.84 ± 0.020 4.45 ± 0.100 6.77 ± 0.160

0.28 ± 0.002 1.55 ± 0.002 2.12 ± 0.086 1.55 ± 0.001

0.37 ± 0.042 3.57 ± 0.037 0.28 ± 0.003 0.01 ± 0.001

0.23 ± 0.006 1.39 ± 0.010 0.88 ± 0.012 0.02 ± 0.001

1.00 ± 0.030 0.63 ± 0.040 0.63 ± 0.030 0.21 ± 0.060

3. EXPERIMENTAL SECTION

Table 3. MIC Values of Compounds 2 and 3 against Pathogenic Bacteria (μM) compounds

B. cereus

S. aureus subsp. aureus

S. aureus subsp. aureus Rosenbach

2 3 ciprofloxacin

3.51 22.40 37.76

0.18 0.09 0.30

0.18 0.29 2.36

3.1. General Experimental Procedures. A JASCO P-1020 digital polarimeter was used to record the optical rotations. UV data were obtained on a Beckman DU 640 spectrophotometer. ECD spectra were recorded on a JASCO J-815 spectrometer. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer in KBr disks. NMR spectra were recorded on a Varian System 500 spectrometer, and the corresponding residual solvent signals (δH/C 2.50/39.52 for DMSOd6) were used to reference the chemical shifts. HRESIMS spectra were recorded on a Finnigan LTQ Orbitrap ThermoFisher (scientific) mass spectrometer. Semipreparative high-performance liquid chromatography (HPLC) was performed using an octadecylsilyl (ODS) column (YMC-pak ODS-A, 10 × 250 mm2, 5 μm, 4.0 mL/min) or (COSMOSIL π-NAP, 10 × 250 mm2, 5 μm, 4.0 mL/min) on Waters 1525. Thin-layer chromatography was performed on plates precoated with silica gel GF254 (10−40 μm). Column chromatography were performed over silica gel (200−300 mesh, Qingdao Marine Chemical

In summary, we identified a new SEK34b-granaticin hybrid polyketide (1) produced by Streptomyces strain 166#. This compound has a complex 6/6/6/6/6/6/6 7-ring skeleton and exhibited moderate cytotoxicity against MCF-7, A549, P6C, and HCT-116 cancer cell lines without an antimicrobal activity. The discovery of sekgranaticin (1) will provide new insight into the biosynthetic study of polyketide. 9090

DOI: 10.1021/acs.joc.9b01022 J. Org. Chem. 2019, 84, 9087−9092

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The Journal of Organic Chemistry

MTT assay, A549, MCF-7, P6C, and HCT-116 cell lines were grown in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin−streptomycin solution under a humidified atmosphere of 5% CO2 and 95% air at 37 °C. One hundred microliters of cell suspension at a density of 3 × 104 cell/mL was plated in 96-well microtiter plates, allowed to attach overnight, and then exposed to compounds for 72 h with varying concentrations from 10.0 to 0.15 μM by a consecutive 2fold dilution (Figure S34). The MTT solution (20 μL, 5 mg/mL in RPMI-1640 medium) was then added to each well and incubated for 4 h. Old medium containing MTT was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. Adriamycin was used as the positive control. 3.6. Antimicrobial Assay. The antimicrobial activities of compounds 1−4 against fungi and bacteria were determined using the filter paper disk method.16 The pathogenic bacteria (S. aureus subsp. aureus ATCC6538, P. aeruginosa ATCC10145, E. coli ATCC11775, E. aerogenes ATCC 13048, and B. subtilis ATCC 6051) and fungi (C. glabrata ATCC2001 and C. albicans ATCC10231) were purchased from the China General Microbiological Culture Collection Center. The bacteria (B. cereus ATCC14579 and S. aureus subsp. aureus Rosenbach ATCC 43300) were purchased from the Guangdong Microbial Culture Collection Center. The pathogenic bacteria and fungi were cultivated in LB and YPD agar plates at 37 °C, respectively. The testing methanol (MeOH) solutions (1 mg/mL) of 1−4 and positive control (ciprofloxacin hydrochloride for bacteria and ketoconazole for fungi) were diluted into 0.1 mg/mL with MeOH. Then, 10 μL of the testing solutions was separately added to the paper disk (5 mm diameter). After evaporation to dryness, the drug paper disks were added into the cultural plates and incubated at 28 °C for 12 h. Inhibition zones were then recorded as millimeter in diameter. Only those compounds with inhibitory zone diameters larger than 14 mm were further tested for the minimum inhibitory concentration (MIC) using liquid cultures in 96-well plates.16,17 Thus, compounds 2 and 3 and ciprofloxacin (positive control) were respectively prepared by a serial 2-fold dilution method from 100 to 0.049 μg/mL with fresh corresponding liquid media. The bacteria were inoculated in the LB liquid medium. Each well contains 100 μL of bacterial suspension and 100 μL of the testing solution. The final bacterial inoculum was prepared to give approximately 5 × 105 CFU/mL. The medium (100 μL) equipped with 100 μL of bacterial suspension was used as the corresponding negative controls, and the medium (200 μL) was used as blank controls. Each experiment was carried out in three parallel wells. All plates were incubated is stationary state for 15 h at 28 °C. The minimum inhibitory concentration (MIC) was determined as the lowest concentration that inhibited the visible growth of pathogenic bacteria, and the results are listed in Table 3.

Factory) and Sephadex LH-20 (Amersham Biosciences). Vacuumliquid chromatography (VLC) was carried out over silica gel H (Qingdao Marine Chemical Factory). 3.2. Collection and Phylogenetic Analysis. Streptomyces sp. 166# was isolated from clayey cold saline soil collected at an altitude of 4547 m in Nima County (N31°52′, E87°1′), Naqu District, Tibet Autonomous Region, China. The sample stamped onto the agar media (20 g/L soluble starch, 1 g/L KNO3, 0.5 g/L K2HPO4·3H2O, 0.5 g/L MgSO4·7H2O, 0.01 g/L FeSO4, 0.5 g/L NaCl, in tap water). Actinomycete colonies were selected and streaked to purity using the same agar media. It was characterized as Streptomyces sp. according to its 16S rRNA gene sequences (GenBank accession no. KX101078) by its phylogenetic tree analysis (Figure S32). Streptomyces sp. 166# was preserved in Ye’s Laboratory, China Pharmaceutical University. 3.3. Cultivation and Extraction. Streptomyces sp. 166# was seeded in 150 mL of fermentation medium in a 500 mL conical flask, containing 3 g of soluble starch, 150 mg of KNO3, 75 mg of K2HPO4· 3H2O, 75 mg of MgSO4·7H2O, 1.5 mg of FeSO4, 75 mg of NaCl, and 150 mL tap water, and shaken for 7 days (28 °C, 180 rpm). The whole culture broth (120 L) was extracted exhaustively with EtOAc, yielding 5.0 g of extract. 3.4. Purification. The crude extract (5.0 g) was separated into 12 fractions (Fr.1−Fr.12) on a silica gel VLC column, eluting with a step gradient of petroleum ether−CH2Cl2 (1:0, 1:1 and 0:1) and then of CH2Cl2−MeOH (100:1, 80:1, 70:1, 60:1, 40:1, 20:1, 15:1, 10:1, and 1:1). Fraction 12 (1.06 g) was separated into five subfractions (Fr.12.1−Fr.12.5) on Sephadex LH-20, eluting with MeOH−CH2Cl2 (1:1). Fr.12.3 (7.6 mg) was purified by HPLC on an ODS column using the solvent system of 35% MeCN/H2O (0.1% trifluoroacetic acid (TFA)) to yield compound 1 (2.8 mg, tR 12.1 min) (Figure S27). Fraction 6 (1.19 g) was separated into nine subfractions (Fr.6.1− Fr.6.9) on Sephadex LH-20, eluting with MeOH. Fr.6.9 (94.0 mg) was purified by HPLC on an π-NAP column using the solvent system of 35% MeCN/H2O (0.1% TFA) to yield compound 2 (66.1 mg, tR 14.4 min) (Figures S24 and S28). Fraction 9 (468.2 mg) was separated into nine subfractions (Fr.9.1−Fr.9.9) on Sephadex LH-20, eluting with MeOH. Fr.9.8 (40.0 mg) was purified by Sephadex LH-20 (MeOH) to yield compound 3 (10.3 mg, tR 13.1 min) (Figures S25 and S29). Fr.5 (6.5 mg) was purified by Sephadex LH-20 (MeOH) to yield compound 4 (1.89 mg, tR 13.0 min) (Figures S26 and S30). 3.4.1. Sekgranaticin (1). Yellow-green powder; [α]D20 + 134.4 (c 0.025, MeOH); UV (MeOH) λmax (log ε) 297 (4.03), 409 (3.39) nm; ECD (0.3 mM, MeOH) λmax (Δε) 215 (+31.4), 246 (−1.9), 270 (+5.2), 311 (−4.1) nm; IR (KBr) νmax 3400, 2922, 2854, 1682, 1648, 1559, 1540, 1455, 1421, 1387, 1262, 1208, 1128, 1027, 803 cm−1; 1H and 13C NMR data, see Table 1; HRMS (ESI-Orbitrap) m/z: [M + H]+ Calcd for C38H33O15, 729.1814; Found 729.1798. 3.4.2. Granaticin A (2). Red powder; [α]D20 + 211.0 (c 0.063, MeOH); UV (MeOH) λmax (log ε) 223 (4.30), 280 (3.69), 500 (3.47), 523 (3.58), 617 (3.35) nm; ECD (0.3 mM, MeOH) λmax (Δε) 206.5 (+14.1), 221.5 (−28.5), 231.5 (+6.3), 279.0 (−7.6), 322.5 (+8.5), 378.5 (−4.7) nm; IR (KBr) νmax 3439, 2928, 1774, 1613, 1580, 1418, 1272, 1204, 1147, 1088, 1036, 1011 cm−1; 1H and 13C NMR data, see Table S1; MS (ESI-TOF) m/z: [M − H]− 443.7. 3.4.3. Granaticin B (3). Blue powder; [α]D20 + 674.5 (c 0.010, MeOH); UV (MeOH) λmax (log ε) 223 (4.25), 290 (3.41), 528 (3.50), 562 (3.56), 608(3.38) nm; ECD (0.2 mM, MeOH) λmax (Δε) 208 (+33.5), 229 (−53.2), 276 (−8.1), 322 (+8.5), 378 (−6.5) nm; IR (KBr) νmax 3444, 2928, 2362, 1771, 1573, 1419, 1268, 1204, 1146, 1112, 1080, 1017, 991, 1027, 825 cm−1; 1H and 13C NMR data, see Table S1; MS (ESI-TOF) m/z: [M − H]− 557.8. 3.4.4. Methyl Granaticinate (4). Blue powder; [α]D20 + 560.3 (c 0.010, MeOH); UV (MeOH) λmax (log ε) 223 (4.35), 285 (3.50), 528 (3.50), 565 (3.36), 609(3.42) nm; IR (KBr) νmax 3382, 2926, 2853, 1732, 1583, 1417, 1385, 1271, 1085, 1026, 922 cm−1; 1H and 13C NMR data, see Table S1; MS (ESI-TOF) m/z: [M − H]− 475.7. 3.5. Cytotoxicity Assay. Compounds 1−4 were evaluated for cytotoxicity against MCF-7 (human breast adenocarcinoma cell line), A549 (lung cancer cell line), P6C (human colorectal cancer stem cells), and HCT-116 (colon carcinoma cell line) by the MTT assay.15 In the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.9b01022. Theory and calculation of HRESIMS and 1H, 13C, HSQC, H-1H COSY, HMBC, and NOESY NMR spectra for compound 1; 1H NMR spectra for compounds 2−4; HPLC profiles for compounds 1−4; LC−MS and the cytotoxic activity for the EtOAc extracts of Streptomyces sp. 166#, phylogenetic tree of Streptomyces sp. 166#; the proliferations of compounds 1−4 on tumor cell lines (PDF)

1

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (B.Y.). *E-mail: [email protected]. Tel/Fax: +86-53282031268. (W.Z.). 9091

DOI: 10.1021/acs.joc.9b01022 J. Org. Chem. 2019, 84, 9087−9092

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The Journal of Organic Chemistry ORCID

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Peng Fu: 0000-0002-7768-4004 Weiming Zhu: 0000-0002-7591-3264 Author Contributions ∥

Q.L. and Y.F. contributed equally to this work.

Author Contributions

Authors from OUC are responsible for isolation, identification, and cytotoxic assay of the compounds, and those from CPU are responsible for the isolation, identification, and fermentation of Streptomyces sp. 166#. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 41876172, 41806086, U1501221, and U1606403), the State Key Basic Research Program of China (No. 2018YFC0311000), and the Fundamental Research Funds for the Central Universities (No. 201841006).

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DOI: 10.1021/acs.joc.9b01022 J. Org. Chem. 2019, 84, 9087−9092