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Geranylpyrrol A and Piericidin F from Streptomyces sp. CHQ-64 ΔrdmF Xiaoning Han,† Zengzhi Liu,† Zhenzhen Zhang,† Xiaomin Zhang,† Tianjiao Zhu,† Qianqun Gu,† Wenli Li,† Qian Che,*,† and Dehai Li*,†,‡ †
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, People’s Republic of China S Supporting Information *
ABSTRACT: Two new compounds, geranylpyrrol A (1) and piericidin F (2), were isolated from a reedsmycins nonproducing mutant strain of Streptomyces sp. CHQ-64. Their structures, including absolute configurations, were elucidated by extensive NMR, MS, NOESY, and ECD analyses. Geranylpyrrol A (1) is an unusual naturally occurring 2,3,4trisubstituted pyrrole, and piericidin F (2) showed cytotoxicity against HeLa, NB4, A549, and H1975 cell lines with IC50 values of 0.003, 0.037, 0.56, and 0.49 μM, respectively.
A
ctinomycetes are a rich source of novel compounds for the discovery of new drug leads. Although thousands of bioactive secondary metabolites have already been isolated from different actinomycete strains, those compounds represent only a fraction of the biosynthetic capacity predicted from their genetic information. This may partially be due to the fact that most of the gene clusters encoding the secondary metabolites are silent under the traditional laboratory culture conditions.1 A previous investigation into the secondary metabolites of Streptomyces sp. strain CHQ-64 had led to the isolation of hybrid isoprenoids (indotertines A and B and drimentines F− I)2−4 and skipped-polyol polyene macrolides (reedsmycins A− F).5 To explore the potential biosynthetic ability of this strain, a reedsmycins nonproducing mutant strain ΔrdmF of Streptomyces sp. CHQ-64 was obtained by knocking out the positive regulatory gene rdmF involved in reedsmycins biosynthesis (unpublished data, Prof. W. Li personal communication). Chemical component studies of the ΔrdmF mutant strain led to the isolation of two new alkaloids, named geranylpyrrol A (1) and piericidin F (2) (Figure 1). Geranylpyrrol A (1) is an unusual naturally occurring 2,3,4-trisubstituted pyrrole, and piericidin F (2) showed cytotoxic activities against a panel of cancer cells (HeLa, NB4, H1975, and A549) with IC50 values ranging from 0.003 to 0.56 μM. Herein, we report the isolation, structure elucidation, and bioactivity of the new compounds. The ΔrdmF mutant strain was cultured, and the whole broth (65 L) was extracted with EtOAc. According to the UPLC-MS analysis (Figure S19, Supporting Information), the few peaks that did not exist in the extract of the wild-type strain were focused on. Guided by the MS information, the organic extract © 2017 American Chemical Society and American Society of Pharmacognosy
Figure 1. Key COSY, HMBC, and partial NOESY correlations of 1 and 2.
(17.0 g) was fractionated by repeated column chromatography including silica gel, LH-20, and HPLC ODS column, which led to the isolation of compounds 1 (1.5 mg) and 2 (5.0 mg). Geranylpyrrol A (1) was isolated as a pale yellow powder. The molecular formula was established as C 18 H 26 N 2 O3 according to HRESIMS. The analysis of the 1H/13C NMR and gHSQC data suggested the presence of five methyls (including one methoxy), three methylenes, three aromatic/ olefinic methines, and seven nonprotonated carbons (two carbonyls and five aromatic/olefinic ones). The chemical shifts of C-1−C-9 indicated the presence of a geranyl chain,6 which was further confirmed by the HMBC correlations (Table 1 and Received: January 5, 2017 Published: April 18, 2017 1684
DOI: 10.1021/acs.jnatprod.7b00016 J. Nat. Prod. 2017, 80, 1684−1687
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Piericidin F (2) was isolated as a pale yellow oil, and it was determined to have the molecular formula C25H37NO3 on the basis of HRESIMS, which required eight degrees of unsaturation. The NMR spectra show signals for eight methyls (including two oxygenated ones), two methylenes, eight methines (including six aromatic/olefinic ones), and seven aromatic/olefinic carbons. Comparison of its 1H and 13C NMR data (Table 1) with those of Mer-A2026B7,8 indicated that they shared the same skeleton. The only difference was the replacement of the OH-10 in Mer-A2026B by a mythoxyl group in 2, which was confirmed by the HMBC correlation from H3-18 to C-10 (δC 92.7) (Figure 1). The configurations of the piericidin family had been established mainly by total synthesis.8,9 With the flexibility of the side chain and the methylation of the hydroxy group, the determination of the stereochemistry of piericidin F (2) was a challenge. The relative and absolute configurations of 2 were determined based on coupling constant analysis, NOESY experiment, and electronic circular dichroism (ECD) analysis. The geometry of olefins was deduced to be 2E, 5E, 7E, and 11E, respectively, based on the ROESY correlations between H1 and H-17, H-9 and H-16, and H-10 and H-12 (Figure 1). A large coupling constant (8.5 Hz) suggested an anti-relationship between H-9 (δH 2.66) and H-10 (δH 3.18),10 and the 9R*, 10R* relative configuration was supported by the conformational analysis based on the NOEs of H-8/H-10/H-15/H-14/ H-9 and between H-14 and H-18 (Figure 2). To determine the absolute configuration, the solution conformers and ECD spectra of truncated model 2a/2b (Figure 3) were calculated and compared with the experimental
Figure 1). The chemical shifts of C-2′ to C-5′ suggested the presence of a 2,3,4-trisubstituted pyrrole mioety,6 which was further confirmed by the COSY correlations (H-5′/NH-1′) and the HMBC correlations from H-5′ to C-2′ (δC 114.9), C-3′ (δC 126.6), and C-4′ (δC 121.5), from NH-8′ (δH 9.02) to C-2′, C3′, and C-4′, and from H-1′ to C-3′, C-4′, and C-5′. The HMBC correlations also indicated the location of the geranyl group at C-4′. The HMBC correlations of NH-8′ and H-10′ (δH 1.95) to C-9′ (δC 168.4) attached the acetamide group to C-3′. Further HMBC correlation between H-7′ (δH 3.69) and C-6′ (δC 160.8) linked them via an oxygen atom. Finally the planar structure of 1 was established by locating the carbonyl carbon (C-6′) at C-2′ suggested by their chemical shifts as well as the molecular formula. The geometry of the C-2−C-3 double bond was assigned an E configuration on the basis of a NOESY correlation between H-10 (δH 1.55) and H-1.
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for 1 and 2 1a no. 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′
δC 23.8, 123.0, 135.0, 39.6, 26.5, 124.6, 131.2, 25.0, 18.0, 16.1,
CH2 CH C CH2 CH2 CH C CH3 CH3 CH3
114.9, 126.6, 121.5, 120.3, 160.8, 51.2,
C C C CH C CH3
168.4, C 23.3, CH3
2b δH (J in Hz)
no.
2.93, d (7.2) 5.19, t (10.0)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 2′ 3′ 4′c 5′ 6′c 7′ 8′
1.96, m 2.03, m 5.06, t (10.0) 1.63, 1.54, 1.55, 11.42,
s s s s
6.57, d (3.3) 3.69, s 9.02, s 1.95, s
δC 31.5, 118.9, 139.3, 43.0, 124.1, 136.9, 133.2, 135.5, 35.4, 92.7, 133.8, 124.4, 13.0, 10.4, 17.6, 12.8, 16.6, 56.1, 159.8, 92.2, 147.3, 116.4, 147.3, 55.1, 10.1,
CH2 CH C CH2 CH CH C CH CH CH C CH CH3 CH3 CH3 CH3 CH3 CH3 C CH C C C CH3 CH3
δH (J in Hz) 3.37, d (7.0) 5.25, t (6.8) 2.80, d (7.0) 5.48, m 6.09, d (15.5) 5.32, d (8.9) 2.66, m 3.18, d (8.5) 5.41, 1.65, 1.52, 0.78, 1.73, 1.70, 3.11,
m d (6.7) s s s s s
6.01, s
3.80, s 2.02, s
a
The NMR data for 1 in DMSO-d6. bThe NMR data for 2 in CDCl3. cAssignments were made on the basis of DEPT and HMBC/HMQC experimental data. 1685
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to the previously reported isoterpenoid metabolites and skipped-polyol polyene macrolides,2−5 compounds 1 and 2 revealed two additional biosynthetic pathways.
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EXPERIMENTAL SECTION
General Experimental Procedures. Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Waters 2487. CD spectra were measured on a JASCO J715 spectropolarimeter. NMR spectra were recorded on Agilent 500 MHz DD2 spectrometers (Agilent, Beijing, China) using tetramethylsilane as an internal standard, and the chemical shifts were recorded in δ values. HRESIMS were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Semipreparative HPLC was performed using an ODS column [HPLC (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 3 mL/min)]. Column chromatography (CC) was performed with silica gel (100−200 mesh, 200−300 mesh, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham Biosciences), respectively. Actinomycetes Material. The Streptomyces sp. CHQ-64 (GenBank No. JQ405211) was isolated from reed rhizosphere soil collected from the mangrove conservation area of Guangdong Province, China. ΔrdmF is a mutant strain of Streptomyces sp. CHQ-64, which is obtained by knocking out the rdmF gene, a regulatory gene for reedsmycins biosynthesis (unpublished data) . Fermentation and Extraction. The spores of ΔrdmF were inoculated into 500 mL Erlenmeyer flasks containing 150 mL of culture medium comprising 1% glucose, 2% dextrin hydrate, 1.5% starch, 1% pharma medium, 0.5% fish meal, 0.2% yeast power, and 0.3% calcium carbonate, pH = 7.0 (in seawater), cultured at 28 °C for 8 days on a rotary shaker at 180 rpm. The whole 65 L of broth was extracted with EtOAc three times to get the crude extract. Purification. The extract (17.0 g) was applied on a VLC column using a stepped gradient elution of MeOH−CH2Cl2, yielding eight subfractions (Fr.1−Fr.8). Fr.6 was applied on a LH-20 column to furnish six subfractions (Fr.6.1−Fr.6.6), and Fr.6.6 was further purified by semipreparative HPLC (80:20 MeOH−H2O, 3 mL/min) to afford compound 1 (1.5 mg). Fr.3 was fractionated on a ODS column and further purified by semipreparative HPLC (50:50 MeCN−H2O, 3 mL/min) to afford compound 2 (5 mg). Geranylpyrrol A (1): pale yellow powder; UV (MeOH) λmax (log ε) 270.5 (0.38) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 319.2018 [M + H]+ (calcd for C18H27N2O3, 319.2016). Piericidin F (2): pale yellow oil; [α]25D −16.7 (c 1.0, CH3OH); ECD (MeOH) λ [nm] (Δε) 200 (23.1), 237 (−13.0); UV (MeOH) λmax (log ε) 231 (0.45) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 398.2698 [M − H]− (calcd for C25H36NO3, 398.2701). Cytotoxicity Assay. Cytotoxicity activities of 1 and 2 were evaluated against HL-60, K562, and NB4 (using the MTT method) and HeLa, A549, MCF-7/ADM, H1975, and HCT-116 (using the SRB method). Adriamycin was used as positive control, and the IC50 values are shown in Table 2. The detailed methodologies for biological testing have been described in a previous report.11 Computation Section. Conformational searches were run by employing the “systematic” procedure implemented in Spartan’1419 using the MMFF. All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian09 program.20 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were
Figure 2. Newman projection and key 1D NOE correlations of the side chain in 2.
Figure 3. Experimental electronic circular dichroism (ECD) spectrum of 2 (black curve) and calculated ECD spectrum of truncated model 2a (red curve) and 2b (blue curve).
solution ECD spectrum of 2. The Merck molecular force field (MMFF) conformational search of truncated model 2a/2b followed by density functional theory (DFT) optimization at the B3LYP/6-31G(d) level afforded three slightly different conformers above 3.6% population with 84.5%, 5.6%, and 6.3% populations. The Boltzmann-weighted ECD spectra of (9R,10R)-2a gave the best agreement (Figure 3), which led to the determination of the 9R, 10R absolute configuration of 2. Compounds 1 and 2 were evaluated in vitro for their cytotoxicity against HeLa, A549, H1975, MCF-7/ADM, and HTC-116 cells by the SRB method,11 and the NB4, K562, and HL-60 cells by the MTT method.11 Compound 2 showed cytotoxicity against the HeLa, NB4, A549, and H1975 cell lines with IC50 values of 0.003, 0.037, 0.56, and 0.49 μM (Table 2), while 1 was inactive (IC50 > 30.0 μM). In conclusion, two new alkaloids were discovered from the ΔrdmF mutant strain. Geranylpyrrol A (1) is an unusual naturally occurring 2,3,4-trisubstituted pyrrole, and piericidin F (2) shows cytotoxicity against the HeLa cell line with an IC50 of 3 nM. The monopyrrole family secondary metabolites are rare, with 17 naturally occurring members reported,6,12−17 in which pyrrolostatin was the only one possessing a geranyl group. Geranylpyrrol A (1) represents a product from mixed biosynthesis involving terpenoid precursors and an α-pyrrole component.13,14 The piericidin antibiotics have been shown to be biosynthesized by a modular polyketide synthase (PKS) pathway, and an ATP-dependent amidotransferase, PieD, plays a key role in forming the α-pyridone ring moiety.18 In contrast
Table 2. Cytotoxicity of 1 and 2 against Eight Human Cancer Cell Lines IC50 (μM) compound
HeLa
NB4
A549
H1975
HL-60
MCF-7/ADM
K562
HCT-116
1 2 adriamycin
>30.0 0.003 ± 0.0006 0.6
>30.0 0.037 ± 0.001 0.4
>30.0 0.56 ± 0.03 0.2
>30.0 0.49 ± 0.02 0.2
>30.0 >30.0 0.02
>30.0 >30.0 25.2
>30.0 >30.0 0.3
>30.0 >30.0 0.2
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performed on the three lowest-energy conformations for (9R,10R)-2a and the three lowest-energy conformations for (9S,10S)-2b (>5% population) using 30 excited states and using a polarizable continuum model (PCM) for MeOH. ECD spectra were generated using the program SpecDis21 by applying a Gaussian band shape with 0.4 eV width for 2a/2b from dipole length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 96.4% of the weights. The calculated spectra were shifted by 18 nm to facilitate comparison to the experimental data.
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(11) Du, L.; Zhu, T. J.; Liu, H. B.; Fang, Y. C.; Zhu, W. M.; Gu, Q. Q. J. Nat. Prod. 2008, 71, 1837−1842. (12) Raju, R.; Piggott, A. M.; Diaz, L. X. B.; Khalil, Z.; Capon, R. J. Org. Lett. 2010, 12, 5158−5161. (13) Macherla, V. R.; Liu, J.; Bellows, C.; Teisan, S.; Nicholson, B.; Lam, K. S.; Potts, B. C. M. J. Nat. Prod. 2005, 68, 780−783. (14) Kwon, H. C.; Espindola, A. P. D. M.; Park, J.; Prieto-Davó, A.; Rose, M.; Jensen, P. R.; Fenical, W. J. Nat. Prod. 2010, 73, 2047−2052. (15) Liu, D. Z.; Liang, B. W. J. Antibiot. 2014, 67, 415−417. (16) Liu, D. Z.; Liang, B. W. Magn. Reson. Chem. 2014, 52, 57−59. (17) Liu, D. Z.; Liang, B. W.; Li, X. F. Chem. Biodiversity 2015, 12, 153−156. (18) Chen, Y. L.; Zhang, W. J.; Zhu, Y. G.; Zhang, Q. B.; Tian, X. P.; Zhang, S.; Zhang, C. S. Org. Lett. 2014, 16, 736−739. (19) Spartan′14; Wavefunction Inc.: Irvine, CA, 2013. (20) Frisch, M. J.; Trucks, 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, J. A., Jr.; 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.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009. (21) Bruhn, T.; Hemberger, Y.; Schaumlöffel, A.; Bringmann, G. SpecDis, Version 1.53; University of Wuerzburg: Germany, 2011.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00016. MS and NMR spectra for compounds 1 and 2 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail:
[email protected] (Q. Che). *E-mail:
[email protected] (D. Li). ORCID
Qian Che: 0000-0003-0610-1593 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 (21402180, 21372208, and 21542001), the Shandong Provincial Natural Science Fund (ZR2014BQ014), AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (2015ASTP-ES09), the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02), NSFC-Shandong Joint Fund for Marine Science Research Centers (U1606403), Shandong Province Key Research and Development Program (2016GSF201204), Fundamental Research Funds for the Central Universities (201564026), and the Basic Scientific Research Fund for Young Teachers of University (201413013).
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