MRC letters Received: 25 September 2013

Revised: 21 October 2013

Accepted: 24 October 2013

Published online in Wiley Online Library: 25 November 2013

(wileyonlinelibrary.com) DOI 10.1002/mrc.4031

Structural elucidation and NMR assignments of two new pyrrolosesquiterpenes from Streptomyces sp. Hd7-21 Dong-Ze Liua* and Bo-Wen Liangb Two new pyrrolosesquiterpenes were isolated from cultures of the soil actinomycete Streptomyces sp. Hd7-21. The structures of these compounds were elucidated by extensive spectroscopic analyses including MS and 1D and 2D NMR data. Their cytotoxic activity against a panel of human cancer cell lines were biologically evaluated. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: 1H NMR;

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C NMR; pyrrolosesquiterpene; actinomycetes; cultures

Introduction Natural products play an important role in drug discovery and have been used for the treatment of diseases for decades.[1] Actinomycetes are widely distributed in nature and are typically useful in the pharmaceutical industry for their seemingly unlimited capacity to produce secondary metabolites with diverse chemical structures and biological activities.[2] It is essential to continue searching for new antibiotics because of the toxicity of some currently used compounds and the emergence of resistant pathogens.[3] Streptomyces produce over 70% of the known antibiotics, and about 70% of all known medicines were isolated from actinomycetes bacteria of which 75% and 60% have been used in medicine and agriculture, respectively.[4–6] Pyrroles have been incorporated into a variety of natural products, yet their appearance among terpenes is rare. To our knowledge, the only previously described pyrroloterpene natural products are pyrrolostatin,[7] glyciapyrroles A–C,[8] and heronapyrroles A C.[9] As part of our ongoing search for biologically active secondary metabolites, streptomyces sp. Hd7-21 from soil sample collected in Daxinganling virgin forest came to our attention as it displayed an interesting chemical profile from our initial LC–MS analysis. This prompted us to investigate the chemical diversity of this strain, which led to the isolation of two new pyrrolosesquiterpenes 1–2 (Figure 1). We herein report the fermentation, isolation, structure elucidation, and the biological activity of these compounds.

Results and Discussion

Magn. Reson. Chem. 2014, 52, 57–59

* Correspondence to: Dong-Ze Liu, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. E-mail: [email protected]. a Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China b Northeast Agricultural University, Harbin 150030, China

Copyright © 2013 John Wiley & Sons, Ltd.

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Compound 1 was assigned the molecular formula of C19H25NO3 by positive HRESIMS (found [M + Na]+ 338.1769, calcd for 338.1732), requiring eight degrees of unsaturation. The 13C and DEPT NMR spectra displayed 19 signals for one ketone carbonyl (δC 179.5 (C-6)), four carbons with chemical shifts and multiplicities that agreed well with a substituted pyrrole (δC 135.1 (C-2), 125.7 (C-5), 115.9 (C-3), 109.8 (C-4)), six olefinic carbons (δC 122.6 (C-7), 146.8 (C-8), 132.4 (C-9), 132.8 (C-10), 124.5 (C-15), 133.5 (C-16)), three aliphatic carbons bearing oxygen (δC 62.3 (C-11), 63.1 (C-12), 68.6 (C-17)), two aliphatic methylene carbons

(δC 37.8 (C-13), 22.7 (C-14)), and three methyl groups (δC 20.6 (C-18), 16.4 (C-19), 13.6 (C-20)). These observations, in combination with molecular formula, revealed that 1 possessed two rings and one OH group. Because four of the carbons could be assigned to the pyrrole, the remaining 15 carbons were postulated to comprise a sesquiterpenoid side chain, which was further confirmed through a series of COSY and HMBC correlations (Figure 2). Cross-peaks between H-10 and H-11, H-9 and H-11, H-14 and H-15, and H13 and H-15 were observed in the 1H,1H COSY spectrum. 13C,1H long-range couplings observed in the HMBC experiments gave correlations from H-20 to C-15, C-16, and C-17, H-17 to C-15 and C-16, H-13 to C-11 and C-12, H-19 to C-11, C-12, and C-13, H-18 to C-7, C-8, and C-9, and H-9 to C-7, C-8, and C-18. Considering the molecular weight and dicyclic feature, it was required that one of two unassigned oxygen atoms must be connected to C-11 and C-12 to make an epoxide ring. By combining all this evidence and data, we were able to assign constitution of the fragment 2,6, 10-trimethyl-5, 6-epoxide-11-hydroxy-undecan-1,3,9-triene. The characterization of the sesquiterpenoid side chain was completed by joining the C-7/C-8 olefin to the ketone carbonyl through an HMBC correlation from H-7 to C-6. A correlation was observed between H-3 and H-7 on the basis of the NOESY experiment, establishing the connectivity between the pyrrole and its sesquiterpene side chain. The geometries of the C-7/C-8 and C-15/C-16 double bonds were determined to be Z and E, respectively, on the basis of NOESY correlations of H-7 to H-18 and H-14 to H-20. The H-9/H-10 coupling constant (J = 16.1 Hz) supported assignment of the E-geometry for the C-9/C-10 olefin.

D.-Z. Liu and B.-W. Liang bacterium was grown on modified ISP4 medium agar plates consisting of 1.5% agar, 1% starch, 0.1% K2HPO4, 0.1% MgSO4•7H2O, 0.1% peptone, 0.05% yeast extract, 0.2% (NH4) 2SO4, 0.2% CaCO3, and trace element solution (pH 7.0). A spore suspension and mycelium was inoculated into each of the 250-ml Erlenmeyer flasks containing 50 ml of liquid modified ISP-4 medium. The flasks were incubated at 28 °C on a rotary shaker (200 rpm) for 2 days. Each seed culture (50 ml) was transferred into a 1000-ml Erlenmeyer flask containing 200 ml of modified ISP-4 medium. The flasks were incubated on a rotary shaker (200 rpm) at 28 °C for 2 weeks on rotary shakers (200 rpm).

Figure 1. Structures of compounds 1–2.

Extraction and isolation

Figure 2. Key COSY and HMBC correlations for 1.

The molecular formula of 2 was determined as C19H23NO3 on the basis of the HRESIMS (found [M + Na]+ 336.1570, calcd for 336.1576) and NMR data. Analysis of and comparison of the NMR data for 2 with the data for 1 clearly showed that the two compounds were similar in structure. The key difference was that the hydroxymethylene moiety (δC 68.6, C-17) in 1 was replaced by the aldehyde group (δC 195.1, C-17) in 2. This assignment was confirmed by the HMBC correlations of H-17 (δH 9.41, s) to C-15, C-16, and C-20. The geometries of the C-7/C-8, C-9/C-10, and C-15/C-16 double bonds were the same as that of 1 on the basis of NOESY correlations and proton–proton coupling constants. Compounds 1–2 were biologically evaluated for in vitro cytotoxicity against three human cancer cell lines (A549, HCT-8, and Hep G2) by using an Methylthiazoletetrazolium (MTT) method with camptothecin as the positive control. Compound 1 showed selective and moderate cytotoxicity against the HCT-8 and Hep G2 cell lines with IC50 values of 19.0 and 16.8 μM, respectively. Compound 2 showed cytotoxic activity against the A549, HCT-8, and Hep G2 cell lines with IC50 values of 8, 25.1, and 19.4 μM, respectively.

Experimental Section General experimental procedures NMR experiments were performed at a 500 MHz Bruker Avance spectrometer with TMS as internal standard. Mass spectra were recorded on a VG Auto Spec-3000 (VG, Manchester,. England) or an API QSTAR Pulsar 1 (PE Sciex Instruments, Boston, MA, USA) spectrometer. IR spectra were recorded on a Bruker Tensor 27 spectrometer (Bruker Optics, Ettlingen, Germany) with KBr pellets. Optical rotations were measured on a Horiba SEPA-300 polarimeter (Horiba, Kyoto, Japan). Column chromatography was performed on silica gel (200–300 mesh; Qingdao Marine Chemicals) and Sephadex LH-20 (Amersham Biosciences, Sweden). TLC analysis was carried out at silica gel GF254 precoated plates (0.20-0.25 mm; Qingdao Marine Chemicals) with detection by heating silica gel plates sprayed with 10% H2SO4 in ethanol. Biological material and cultivation

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The strain Hd7-21, isolated from soil sample collected in Daxinganling virgin forest, Heilongjiang, China, was identified as Streptomyces sp. by complete 16S rRNA gene sequence. This

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After fermentation, the culture (20 l) was centrifuged to yield supernatant and a mycelial cake. The supernatant was extracted with equal volumes of ethyl acetate three times, and then, samples were evaporated to dryness. The mycelial cake was extracted with 1.5 l of acetone three times, and the solvent was then evaporated to dryness. The two organic extracts were finally combined to give 5.6 g of residue, which was subjected to column chromatography over silica gel (200–300 mesh) using gradient elution with a CHCl3 MeOH mixture (from 1 : 0 to 0 : 1) to afford fractions A–G. Fraction B was subjected to further column chromatography over silica gel, eluting with a gradient of EtOAc in petroleum ether (from 5 : 1 to 0 : 1), to give fractions C1–C5. Subfraction C2 was purified by Sephadex LH-20 column eluting with MeOH and repeated chromatography over silica gel using CHCl3–MeOH (100 : 1–30 : 1) as the eluent to yield compounds 1 (8.6 mg) and 2 (9.7 mg). Cytotoxicity assay The cell growth inhibitory activities of compounds 1–2 against the human cell lines (A549, HCT-8, and Hep G2) were determined using the previously published MTT method.[10] Briefly, the cancer cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37 °C. Then, 100 μl of cell suspension was plated in 96-well plates to a final concentration of 2 × 103 cells per well and incubated for 12 h. Following incubation, 50 μl of the test compound solutions (in DMSO) at various concentrations was added to each well. After the exposure to compounds 1–2 for 48 h, 50 μl of MTT solution (1 mg/ml in sodium perborate ) was added to each well, and the plates were incubated for 4 h at 37 °C. Then, 200 μl of DMSO was added in each well. The absorbance caused by formazan crystallization was read at 550 nm using a microplate reader (model 550, BioRad). The calculation of cell viability used the following formula: cell viability (%) = (the average A550 nm of the treated group/the average A550 nm of the untreated group) × 100. (1) Colorless powder; [α]25 D + 16.5 (c 0.50, CH3OH); UV (CH3OH ) λmax (log ε) 330(14000), 290(6600) nm; IR (KBr) νmax 3430,3260, 2970, 1640, 1610, 1572, 1450, 1400, 1310 cm 1; 1 H and 13C NMR spectroscopic data (Table 1); (+) ESIMS m/z 388 [M + Na]+; (+) HRESIMS m/z 388.1769 [M + Na]+ (calcd for C19H25NO3Na, 338.1732). (2) Colorless powder; [α]25 D + 16.3 (c 0.50, CH3OH); UV (CH3OH ) λmax (log ε) 330(14000), 290(6600) nm; IR (KBr) νmax 3432, 3260, 2970, 2711,1690, 1608, 1570, 1450, 1400 cm 1; 1H and

Copyright © 2013 John Wiley & Sons, Ltd.

Magn. Reson. Chem. 2014, 52, 57–59

Structural elucidation and NMR assignments of two new pyrrolosesquiterpenes from a Streptomyces sp. Table 1.

1

H (500 MHz) and

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C (125 MHz) NMR data for compounds 1 and 2 (DMSO-d6)

No.

1 δH

1-NH 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2 δC

δH

11.77 (br s) 7.03 (br m) 6.20 (dt, 3.8, 2.2) 7.09 (m) 6.78 (br s) 7.95 (d, 16.0) 6.03 (dd, 16.1, 7.6) 3.38 ( d, 7.6) 1.64 (m) 1.49 (m) 2.08 (m) 5.38 (br t, 7.2) 3.99(s) 2.05 (s) 1.26 (s) 1.62(s)

δC

11.77 (br s) 135.1 (C) 115.9 (CH) 109.8 (CH) 125.7 (CH) 179.5 (C) 122.6 (CH) 146.8 (C) 132.4 (CH) 132.8 (CH) 62.3 (CH) 63.1 (C) 37.8 (CH2)

7.03 (br m) 6.20 (dt, 3.8, 2.2) 7.09 (m) 6.78 (br s) 7.95 (d, 16.0) 6.03 (dd, 16.1, 7.6) 3.38 ( d, 7.6) 1.71(m) 1.57 (m) 2.42(m) 6.44 (br t, 7.2)

22.7 (CH2) 124.5 (CH) 133.5 (C) 68.6 (CH2) 20.6 (CH3) 16.4 (CH3) 13.6 (CH3)

9.41 (s) 2.06 (s) 1.26 (s) 1.74 (s)

135.1 (C) 115.9 (CH) 109.8 (CH) 125.7 (CH) 179.5 (C) 122.6 (CH) 146.8 (C) 132.4 (CH) 132.8 (CH) 62.3 (CH) 63.1 (C) 37.3 (CH2) 24.1 (CH2) 152.6 (CH) 138.4 (C) 195.1 (C) 21.4 (CH3) 22.6 (CH3) 9.2 (CH3)

Chemical shifts (δ) are in ppm and J in Hz.

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C NMR spectroscopic data, (Table 1); (+) ESIMS m/z 336 [M + Na]+; (+) HRESIMS m/z 336.1570 [M + Na]+ (calcd for C19H23NO3Na, 336.1576).

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