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Hinduchelins A−D, Noncytotoxic Catechol Derivatives from Streptoalloteichus hindustanus Fei He,*,†,‡ Hitomi Nakamura,† Shotaro Hoshino,† Joyce Seow Fong Chin,§ Liang Yang,§ Huiping Zhang,⊥ Fumiaki Hayashi,⊥ and Ikuro Abe*,† †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Integrative Microbiology Research Centre, College of Agriculture, South China Agricultural University, Guangzhou 510642, People’s Republic of China § Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551, Singapore ⊥ RIKEN Center for Life Science Technology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokuhama 230-0045, Japan ‡

S Supporting Information *

ABSTRACT: Four new catechol derivatives, hinduchelins A−D (1−4), composed of 2,3- dihydroxybenzoic acid, threonine, and decarboxylated phenylalanine, were isolated from Streptoalloteichus hindustanus. Their structures and absolute configurations were elucidated by interpretation of NMR and HRMS data and quantum chemical ECD calculations. The iron-binding properties of the compounds were evaluated by a pyoverdine production assay in Pseudomonas aeruginosa, and compound 4 showed moderate ability to induce pyoverdine production at 50 μM. None of the compounds were cytotoxic toward HL-20, A549, SMMC-7721, MCF-7, and SW-480 tumor cell lines.

I

were evaluated by a pyoverdine production assay in P. aeruginosa, and compound 4 showed moderate ability to induce pyoverdine production at 50 μM. None of the compounds showed cytotoxicities toward HL-20, A549, SMMC-7721, MCF-7, and SW-480 tumor cell lines. Herein, we report details of the isolation, structure elucidations, and biological activities of these new siderophores. The strain S. hindustanus was cultured in A3M medium at 28 °C on a rotary shaker. The EtOAc extract of the fermentation medium was fractionated on an open silica gel column and by semipreparative HPLC to afford compounds 1−4. Hinduchelin A (1) was isolated as a white, amorphous powder. Its molecular formula was determined to be C21H22N2O5, based on HREISMS, requiring 12 degrees of unsaturation. Analyses of the 1H and 13C NMR and HSQC data (Table 1) revealed eight sp2 nonprotonated carbons (δC 121.4−163.5), eight aromatic methines (δH 7.05−7.48; δC

ron acquisition is essential for almost all bacteria. However, in the presence of oxygen and at physiologic pH, iron forms insoluble ferric hydroxides that cause the nutrient to be unavailable to bacterial cells.1 Small-molecule siderophoremediated transport is one of the most efficient and commonly employed strategies in many microorganisms. Siderophores are small, iron-chelating molecules produced by many bacteria to make insoluble ferric iron available to the microbial cell.2 For example, the main siderophore of Pseudomonas aeruginosa is pyoverdine, which is required for iron acquisition and regulates the expression of several virulence factors.3 Pyoverdine production is induced in response to iron-limited growth conditions and the presence of iron-chelating compounds.4−6 In our continuing search for structurally novel compounds with diverse bioactivities,7−11 we have isolated four unusual catechol siderophores, hinduchelins A−D (1−4), from Streptoalloteichus hindustanus. Catechol is one of the most common iron-binding motifs found in nature. Many catecholderived structurally complex siderophores have been isolated, and their biosynthetic pathways have also been investigated.12−15 The iron-binding properties of compounds 1−4 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 12, 2018

A

DOI: 10.1021/acs.jnatprod.8b00049 J. Nat. Prod. XXXX, XXX, XXX−XXX

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114.6−128.5), one oxygenated methine (δH 4.99, dd, J = 8.1, 3.2; δC 74.1), one methylene group (δH 3.85 and 3.59; δC 47.4), two methoxyl groups (δH 3.93, δC 56.1; and δH 3.92, δC 61.2), and one methyl group (δH 2.76; δC 11.9). In the 1H−1H COSY spectrum, the correlations between the aromatic protons at δH 7.05 (H-4), 7.16 (H-5), and 7.48 (H-6) and between 7.46 (H2″/H-6″), 7.39 (H-3″/H-5″), and 7.32 (H-4″) indicated two spin coupling systems of aromatic rings. In the HMBC spectrum, the correlations from δH 4.99 (H-7″) to δC 141.9 (C-1″) and 125.9 (C-2″/C-6″), from δH 2.76 (H3-6′) to δC 153.4 (C-5′) and 129.8 (C-4’), and from δH 7.48 (H-5) to δC 156.8 (C-2′) helped us draw the following three partial structures: 1, 2,3-trisubstituted phenyl group A, methyl-bearing unsaturated C3 unit B, and 1-phenylethanol group C (Figure 1). The positions of the two methoxyl groups were determined by the HMBC correlations from δH 3.92 to δC 141.9 (C-1) and from δH 3.93 to δC 153.8 (C-2). The key correlations from H28″ (δ H 3.85 and 3.59) to 15 N (107.5 ppm) in the HMBCGP-15N spectrum and those from H2-8″ to C-10″ (δC 163.5) and from H3-6′ to C-10″ (δC 163.5) in the HMBC spectrum, combined with the molecular formula, the degrees of unsaturation, and the HRESIMS data of 1, suggested the linkage of fragments A, B, and C through an oxazole ring, as shown in Figure 1.

Figure 1. Structure of compound 1 and structural fragments A−C and COSY, key HMBC, HSQMBC, and HMBCGP-15N correlations

The absolute configuration of the hydroxy group on C-7″ could not be determined by Mosher’s method, due to the strongly overlapped 1H NMR signals of the phenyl groups, and the quantum chemical ECD calculation method was used to establish the absolute configuration. The preliminary conformation distribution search was performed with Spartan’16 by a conformation analysis with the MMFF94 force field, followed by geometry optimization using the TDDFT, B3LYP/631G(d,p) calculations. The overall predicted electronic circular dichroism (ECD) spectra of 1 (7″S and 7″R) were subsequently compared with the experimentally derived spectrum (Figure 2), and the structure 7″S matched well with the experimental data in the region of 200−400 nm. The complete structure of 1 was determined.

Table 1. 1H NMR and 13C NMR Data of Compounds 1−4 in CDCl3a 1 no.

δC, mult

1 2 3 4 5 6 2′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 10″ 1-OCH3 2-OCH3

141.9, qC 153.8, qC 114.6, CH 124.4, CH 121.2, CH 121.4, qC 156.8, qC 129. 8, qC 153.4, qC 11.9, CH3 141.9, qC 125.9, CH 128.5, CH 127.8, CH 128.5, CH 125.9, CH 74.1, CH 47.4, CH2 163.5, qC 61.2, CH3 56.1, CH3

2 δH (J in Hz)

7.05, dd (8.1, 1.2) 7.16, t (8.1, 8,1) 7.48, dd (8.1, 1.2)

2.76, s 7.46, 7.39, 7.32, 7.39, 7.46, 4.99, 3.85,

m m m m m dd (8.1, 3.2) m; 3.59, m

3.92, s 3.93, s

δC, mult. 147.8, qC 153.8, qC 114.7, CH 124.4, CH 121.2, CH 121.4, qC 156.8, qC 129.8, qC 153.3, qC 11.8, CH3 124.7, qC 155.4, qC 116.6, CH 128.3, CH 120.1, CH 130.4, CH 31.0, CH2 40.2, CH2 163.6, qC 61.2, CH3 56.1, CH3

4a

3 δH (J in Hz)

7.01, dd (8.0, 1.2) 7.18, overlapped 7.50, dd (8.0, 1.2)

2.77, s

6.93, m 7.17, overlapped 6.86, m 7.12, m 3.01 t (7.7, 7.7) 3.59, m 3.95, s 3.95, s

δC, mult 146.8, qC 148.4, qC 114.2, CH 119.7, CH 117.8, CH 110.8, qC 158.5, qC 128.8, qC 152.4, qC 11.6, CH3 125.3, qC 154.6, qC 115.9, CH 128.2, CH 120.7, CH 130.8, CH 29.6, CH2 41.4, CH2 161.9, qC 56.2, CH3

δH (J in Hz)

7.03, dd (8.0, 1.2) 6.97, t (8.0, 8.0) 7.45, dd (8.0, 1.2)

2.76, s

6.95, 7.15, 6.89, 7.15, 3.02, 3.64,

m m m overlapped t (6.7, 6.7) m

3.98, s

δC, mult 147.0, qC 148.6, qC 114.5, CH 119.7, CH 117.9, CH 110.8, qC 158.6, qC 128.9, qC 152.4, qC 11.9, CH3 138.7, qC 128.8, CH 128.8, CH 126.8, CH 128.8, CH 128.8, CH 35.9, CH2 40.5, CH2 161.1, qC 56.3, CH3

δH (J in Hz)

6.99, dd (8.2, 1.3) 6.93, t (8.2, 8.2) 7.53, dd (8.2, 1.3)

2.74, s 7.25, 7.35, 7.24, 7.35, 7.25, 2.93, 3.70,

overlapped m overlapped m overlapped t (7.2, 7.2) dd (13.6, 6.8)

3.93, s

a

Compound 4 was recorded on a JEOL ECX-500 spectrometer, and compounds 1−3 were recorded on a Bruker AVANCE III HD 900 spectrometer (900 MHz). B

DOI: 10.1021/acs.jnatprod.8b00049 J. Nat. Prod. XXXX, XXX, XXX−XXX

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were measured on a U-2910 spectrometer (Hitachi). The NMR spectra of 1−3 were recorded on a Bruker AVANCE III HD 900 spectrometer (900 MHz) in CDCl3. The NMR spectra of 4 were recorded on a JEOL ECX-500 spectrometer. All of the HRMS data were obtained from LC-MS analysis of the samples, using a compact micrOTOF-MS (Bruker) attached to an LC-20AD UHPLC system (Shimadzu). Column chromatography (CC) was performed on Sephadex LH-20 (Pharmacia, USA) and ODS (60−80 μm, YMC) matrices. TLC was performed on silica gel plates (SGF254, 0.2 mm, Merck, Germany). Analytical and semipreparative HPLC runs were performed on a Shimadzu LC20-AD HPLC system, using YMC C18 columns (4.6 × 250 mm, 5 μm and 10.0 × 250 mm, 5 μm). Strain Material and Cultivation. The strain Streptoalloteichus hindustanus NBRC 15115 was obtained from the Biological Research Center, National Institute of Technology and Evaluation (NITE, NBRC), Japan. The strain S. hindustanus NBRC 15115 was grown on ISP2 agar plates (glucose 4 g, malt extract 10 g, yeast extract 4 g, and agar 20 g in 1 L of distilled water, pH 7.3) at 28 °C for 3 days, and then its spores were inoculated aseptically into 500 mL baffled Erlenmeyer flasks containing 100 mL of sterile seed medium (ISP2 medium) and cultivated for 2 days at 28 °C on a rotary shaker (160 rpm). Subsequently, 1 mL of seed culture was transferred to 500 mL baffled Erlenmeyer flasks (total number, 25), containing 100 mL of A3M medium, composed of 2% starch, 0.5% glucose, 2% glycerol, 1.5% pharma media, 0.3% yeast extract, and 1% HP-20 resin (pH 7.0), and cultured on a rotary shaker (160 rpm) at 28 °C for 7 days. Extraction and Purification. The fermentation broth (total volume, 2.5 L) was centrifuged (6000 rpm, 20 min), and the supernatant was extracted with EtOAc (3 L × 3). The mycelia were extracted with acetone (200 mL × 2). All extracts were evaporated under reduced pressure and combined to give 1.8 g of crude extract for further purification. The crude extract (1.8 g) was subjected to silica gel CC, using a stepped gradient elution of CHCl3−CH3OH (100:0, 98:2, 95:5, 90:10, 80:20, 50:50, 0:100, v/v) to give seven fractions (Fr. 1 to Fr. 7). Compounds 1−4 were all obtained from Fr. 2 (475 mg) after repeated semipreparative HPLC runs with gradient elution of ACN/H2O (60:40−95:5, 20 min) to obtain the compounds 1 (2.1 mg, tR = 11.0 min), 2 (1.0 mg, tR = 12.1 min), 3 (0.8 mg, tR = 12.7 min), and 4 (6.5 mg, tR = 18.2 min). Hinduchelin A (1): [α]25 D −2.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (3.49) nm; CD (0.35 mM, MeOH) λmax (Δε) 220 (1.65) nm; HRESIMS m/z 383.1604 [M + H]+ (calcd for C21H23N2O5, 383.1601); 1H and 13C NMR data, see Table 1. Hinduchelin B (2): [α]25 D 0.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.57), 273 (3.08) nm; HRESIMS m/z 383.1596 [M + H]+ (calcd for C21H23N2O5, 383.1601); 1H and 13C NMR data, see Table 1. Hinduchelin C (3): [α]25 D −0.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 211 (4.05), 275 (3.74), 312 (3.22) nm; HRESIMS m/z 369.1450 [M + H]+ (calcd for C20H21N2O5, 369.1445); 1H and 13C NMR data, see Table 1. Hinduchelin D (4): [α]25 D +0.5 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 239 (3.95), 290 3.85), 316 (3.49) nm; HRESIMS m/z 353.1504 [M + H]+ (calcd for C20H21N2O4, 353.1496); 1H and 13C NMR data, see Table 1. Calculated ECD Spectra. The conformer of 1 was calculated using Spartan’16 by a conformational analysis with the MMFF94 force field, followed by geometry optimization using the TDDFT, B3LYP/631G(d,p) calculations.16 The solvent effect was introduced through the polarizable continuum model (PCM). The Boltzmann distribution based on the free energy was then calculated, and five conformers greater than 1% were chosen. The calculated ECD spectrum was generated from the Boltzmann weighting of each conformer in a methanol solution. Finally, the experimental data recorded in the methanol solution were compared with the calculated data. Pyoverdine Quantification Assay. P. aeruginosa PAO1 strain was cultured overnight in Luria Bertani (LB) broth at 37 °C with shaking. Overnight bacteria culture was diluted to OD600 = 0.02 in ABTGC media. Compound 4 was prepared to 200 μM in a 200 μL volume of ABTGC in the first row of a 96-well plate. A 100 μL amount of the

Figure 2. Experimental ECD spectrum of 1 and the calculated ECD spectra of 1 (7″S) and 1 (7″R).

Hinduchelin B (2) was assigned the molecular formula C21H22N2O5, as deduced from the positive HRESIMS. The 1H and 13C NMR data of 2 showed similarities with those of 1 (Table 1), indicating nine sp2 nonprotonated carbons (δC 121.4−163.6), seven aromatic methines (δH 6.86−7.50; δC 116.6−130.4), two methylene groups (δH 3.01, and δC 31.0; δH 3.59, and δC 40.2), two methoxyl groups (δH 3.95, δC 56.1; δH 3.95, δC 61.2), and one methyl group (δH 2.77; δC 11.8). Further comparison of the 1H and 13C NMR data of 2 with those of 1 revealed that one methine group (δH 4.99, H-15; δC 74.1, C-15) had disappeared in 2 and was replaced by a methylene group (δH 3.01; δC 31.0). This was confirmed by the COSY correlations between H2-7″ (δH 3.01) and H2-8″ (δH 3.59). Furthermore, one hydroxy group located on C-2″ (δC 155.4) was deduced from the HMBC correlations from H2-7″ to C-2″, C-1″ (δC 124.7), and C-6″ (δC 130.4) and from the COSY correlations of H-3″ (δH 6.93)/H-4″ (δH 7.17)/H-5″ (δH 6.86)/H-6″ (δH 7.12). Hinduchelin C (3) had the molecular formula C20H20N2O5, as deduced from HRESIMS. The 1H and 13C NMR spectra of 3 were very similar to those of 2 and differed only in the absence of one methoxyl group. In the HMBC spectrum, the methoxyl group (δH 3.98; δC 56.2) placed at C-2 was confirmed by the HMBC correlation from δH 3.98 to δC 148.4 (C-2). The molecular formula of hinduchelin D (4) was established as C20H20N2O4, on the basis of HRESIMS analysis. The 1H and 13 C NMR data of 4 were similar to those of 3 and differed only in the absence of one hydroxy group. The structure was further elucidated and assigned by analyses of its COSY, HSQC, and HMBC correlations, as shown in Figure 1. A literature search revealed that the siderophore catechol group showed iron-chelating activities due to its phenolic hydroxy group(s). In our research, the phenolic hydroxy groups in 1−4 were methylated and showed moderate or weak activities in iron-chelating assays. In P. aeruginosa, 4 showed some moderate iron-chelating ability to induce pyoverdine production at 50 μM. None of the compounds were cytotoxic toward the HL-20, A549, SMMC-7721, MCF-7, and SW-480 tumor cell lines at 50 μM.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an MCP 300 polarimeter (Anton Paar), and UV spectra C

DOI: 10.1021/acs.jnatprod.8b00049 J. Nat. Prod. XXXX, XXX, XXX−XXX

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ABTGC medium was added to subsequent rows. The solution of compound 4 was serially diluted by pipetting 100 μL of the 200 μM solution of 4 (first row) to the subsequent row (second row), and this was repeated for the following rows. Then, 100 μL of the diluted culture was added to all the wells. Final working concentrations of the compound to treat the cells (final OD600 = 0.01) were as follows: 50, 25, 12.5, 6.25, 3.125, 1.6, 0.8, 0.4, and 0.2 μM. The pyoverdine production was measured using a microplate reader (Tecan Infinite 2000). OD600 was used to normalize the pyoverdine production, and the data were tabulated as relative fluorescence units. The experiment was done in triplicate (three biological replicates).5

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(10) Hoshino, S.; Okada, M.; Wakimoto, T.; Zhang, H.; Hayashi, F.; Onaka, H.; Abe, I. J. Nat. Prod. 2015, 78, 3011−3017. (11) Hoshino, S.; Wakimoto, T.; Zhang, H.; Hayashi, F.; Okada, M.; Abe, I. Bioorg. Med. Chem. Lett. 2015, 25, 3953−3955. (12) Zane, H. K.; Butler, A. J. Nat. Prod. 2013, 76, 648−654. (13) Kishimito, S.; Nishimura, S.; Hattori, A.; Tsujimoto, M.; Hatano, M.; Igarashi, M.; Kakeya, H. Org. Lett. 2014, 16, 6108−6111. (14) Seyedsayamdost, M. R.; Cleto, S.; Carr, G.; Vlamskis, H.; Vieira, M. J.; Kolter, R.; Clardy, J. J. Am. Chem. Soc. 2012, 134, 13550−13553. (15) Seyedsayamdost, M. R.; Traxler, M. F.; Zheng, S. L.; Kolter, R.; Clardy, J. J. Am. Chem. Soc. 2011, 133, 11434−11437. (16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford, CT, 2016.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00049. 1D and 2D NMR spectra for compounds 1−4, pyoverdine production assay experiment, cytotoxicity assay experiment, and details for ECD calculations (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: +81-3-5841-4740. Fax: +81-3-5841-4744. ORCID

Liang Yang: 0000-0002-9225-1995 Ikuro Abe: 0000-0002-3640-888X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (JSPS KAKENHI Grant Nos. JP16H06443, JP16K13084, and JP15H01836), JST/NSFC Strategic International Collaborative Research Program, the Kobayashi International Scholarship Foundation, the National Basic Research Program of China (2015CB150600), and the National Natural Science Foundation of China (31470236).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00049 J. Nat. Prod. XXXX, XXX, XXX−XXX