Letter pubs.acs.org/OrgLett
Nonthmicin, a Polyether Polyketide Bearing a Halogen-Modified Tetronate with Neuroprotective and Antiinvasive Activity from Actinomadura sp. Yasuhiro Igarashi,*,† Noriaki Matsuoka,† Yasuko In,‡ Tetsushi Kataura,§ Etsu Tashiro,§ Ikuo Saiki,∥ Yuri Sudoh,⊥ Kannika Duangmal,# and Arinthip Thamchaipenet⊗ †
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1041, Japan § Bioscience and Informatics, Keio University, Yokohama, Kanagawa 223-8522, Japan ∥ Department of Bioscience, Institute of Natural Medicine, Toyama University, 2630 Sugitani, Toyama 930-0194, Japan ⊥ Hyphagenesis, Inc., 2-18-28 Tamagawa Gakuen, Machida, Tokyo 194-0041, Japan # Department of Microbiology, Faculty of Science, and ⊗Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand ‡
S Supporting Information *
ABSTRACT: Nonthmicin (1), a new polyether polyketide bearing a chlorinated tetronic acid, was isolated from the culture extract of a soil-derived Actinomadura strain. The structure of 1 was elucidated by interpretation of NMR and MS spectroscopic data, and the absolute configuration of 1 was proposed on the basis of the crystal structure of its dechloro congener ecteinamycin (2) also isolated from the same strain. Tetronic acids modified by halogenation have never been reported from natural products. Compounds 1 and 2 were found to have neuroprotective activity and antimetastatic properties at submicromolar concentrations in addition to antibacterial activity.
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acterial polyketides are the most prolific source of structurally unique, diverse secondary metabolites, mostly associated with intriguing biological activity.1 Among them, products of type I polyketide synthase (PKS), which utilizes a wide array of malonate-based building blocks in assembling carbon skeletons, are crucially attractive to drug discovery in terms of the structural diversity rather than the products from type II or type III PKSs in which malonates are basically used for chain elongation.2 In many cases, metabolites from bacterial type I PKS, except for polyenes, contain relatively shortconjugated chromophores that have UV absorption bands around 210−330 nm, distinguishable from compounds containing highly conjugated aromatic systems. In our continuing search for structurally novel type I PKS products from bacterial sources by HPLC/UV-based spectroscopic screening,3 two unknown compounds with absorption maxima around 250 and 300 nm were detected in the culture extract of Actinomadura sp. K4S16. UV-guided purification from the 1butanol extract of strain K4S16 led to the isolation of a new polyether polyketide nonthmicin (1) along with its dechloro congener ecteinamycin (2) (Figure 1). Herein, we present the details of the characterization and biological properties of these compounds. After 6 days of production fermentation, the whole culture broth was extracted with 1-butanol. The crude extract was © XXXX American Chemical Society
Figure 1. Structures of nonthmicin (1) and ecteinamycin (2).
fractionated consecutively using silica gel and ODS column chromatographies to give fractions containing the target compounds. From the faster eluted ODS fraction, ecteinamycin (2) was isolated. Compound 2 is described in a patent as an antibiotic from an Actinomadura strain with incomplete assignment of stereochemistry.4 The planar structure of 2 obtained from strain K4S16 was confirmed by NMR and MS analyses, and our NMR spectral data (Table S1) were in good agreement with those described in the patent. In order to establish the relative and absolute configurations of 2, crystallization was attempted, and the crystals from acetoReceived: January 30, 2017
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DOI: 10.1021/acs.orglett.7b00318 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Table 1. 1H and 13C NMR Spectral Data for Nonthmicin (1) Sodium Salt in CDCl3
ne−CH2Cl2 solution were found to be suitable for X-ray crystallographic analysis. The absolute configuration was determined as 8R,9R,11R,12S,13R,14R,15R,17R,18S,21S,22R,25S,26S,36R (CCDC accession no. 1520743; Figure 2).
position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 2. ORTEP drawing of the crystal structure of ecteinamycin (2) sodium salt, illustrating its absolute configuration and coordination to a sodium cation.
The crystal structure also revealed that 2 coordinates to a sodium cation through three keto carbonyl oxygens at C3, C6, and C16, two cyclic ether oxygens, and the tertiary alcohol oxygen at C25 (Figure 2). Nonthmicin (1) was isolated from a more lipophilic ODS fraction as a colorless amorphous solid. The high-resolution ESITOFMS spectrum indicated a pseudomolecular ion [M − H]− at m/z 741.3622 with a typical isotopic pattern for the presence of one chlorine atom in the molecule (Figure S1).5,6 The molecular formula of 1 was thus determined as C38H58O12Cl (m/z 741.3622; Δ −0.0 mmu, calcd for C38H57O1235Cl), which was corroborated by 1H and 13C NMR data. The IR spectrum displayed the absorption bands for carbonyl groups at 1760 and 1628 cm−1 and hydroxy groups at 3309 cm−1. The UV spectra measured in acidic solution showed absorption bands at 227 and 298 nm, while the absorption maxima were observed at 251 and 300 nm in basic solution, indicative of the presence of a dissociable functionality.5 Analysis of 13C NMR data coupled with interpretation of DEPT135 and HSQC spectral data revealed eight methyl groups, one methoxy group, eight methylenes, four methines, seven oxygenated methines, four fully substituted carbons (three sp3 and one sp2), one olefinic methine, and five oxygenated sp2 quaternary carbons. The 1H NMR spectrum displayed five doublet methyls, two triplet methyls, one methyl singlet, one methoxy group, one olefinic proton, and three exchangeable protons (Table 1). Interpretation of COSY data established 10 small fragments a−j (Figure 3). Fragments a−c were assembled into a tetrahydropyran with two methyl substitutions based on the HMBC correlations from H29 to C10 and C12, H8 to C12, and H12 to C8. A four-carbon fragment d (C32/C31/C15/ C14/14-OH) was connected to C13 by long-range couplings from H14 to C12 and C30 and H30 to C14. Furthermore, H15 showed a correlation to C16 carbonyl carbon, to which were long-range coupled H33 singlet methyl protons. This methyl group displayed HMBC correlations to C18 and a quaternary carbon C17, which was also correlated from an exchangeable proton at δH 5.86. These correlation data established the carbon connectivity from C13 to C18 as well as the presence of a keto group at C16 and a tertiary hydroxy group at C17. The
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 14-OH 17-OH 25-OH
δH mult (J in Hz) 6.27, s
2.45, 3.50, 4.20, 2.24, 1.49, 1.82, 3.63, 1.54, 3.85, 3.64,
dd (11.5, 11.4) dd (11.5, 0.8) dd (11.4, 6.4) m m m m m ddd (10.9, 6.1, 1.3) m
4.46, 1.88, 2.21, 1.78,
dd (10.1, 5.9) m m m
3.56, 1.59, 1.99, 1.48, 1.71,
dd (12.2, 1.8) m m m m
4.01, 1.15, 0.98, 0.93, 0.84, 1.26, 1.64, 0.77, 1.10, 1.45, 1.67, 0.81, 3.08, 1.17, 3.28, 6.28, 5.86, 4.31,
q (6.6) d (6.4) d (6.9) d (7.0) d (7.0) m m t (7.5) s m m t (7.5) q (6.1) d (6.1) s d (6.1) s d (1.9)
δC 98.9 147.8 181.4 169.6 96.4 199.8 35.4 77.7 27.7 36.2 28.8 69.7 36.4 73.7 48.8 222.8 78.6 83.9 24.78a 29.0 88.8 70.2 19.9 24.75a 73.0 74.6 14.1 17.5 11.06b 7.2 21.3 12.6 20.5 30.0 9.1 78.9 11.11b 56.2
HMBC 2, 3
3, 6, 6, 7, 8,
5, 6, 8 8 7, 9, 10, 12 8, 12, 28 12, 29
8, 10, 14, 29 12, 30 12, 13, 15, 16, 30, 31
17 20, 21 17, 18 18, 21, 22 24 21, 22 22
22, 24, 25, 27 25, 26 8, 9, 10 10, 11, 12 12, 13, 14 15, 16 15, 16, 32 15, 31 16, 17, 18 20, 21, 35 20, 21, 35 21, 34 24, 37, 38 25, 36 36 14, 15 16, 17, 33 24, 25
Figure 3. COSY and key HMBC correlations observed for nonthmicin (1) sodium salt.
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DOI: 10.1021/acs.orglett.7b00318 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Nonthmicin (1) and ecteinamycin (2) were evaluated in several bioassays available in our laboratories. Parkinson’s disease (PD) is a common neurodegenerative disease caused by progressive degeneration of dopaminergic neurons. 1-Methyl-4phenylpyridinium (MPP+), an inhibitor of mitochondrial complex I, is widely used to induce neuronal cell death in the acquired Parkinson’s disease (PD) model.13 As shown in Figure 5, 1 and 2 suppressed MPP+-induced cell death at 0.1 μM. We
remaining part of 1 was assigned to include two cyclic ether substructures. Three fragments g−i were joined into a tetrahydropyran ring on the basis of HMBC correlations from H27 to C25, H26 to C24, and H24 and H26 to C22. A methoxy-substituted ethyl group was connected to C25 by a long-range correlation from the doublet methyl H37 to C25. Fragments e and f and this tetrahydrofuran ring were coupled together at the oxygenated quaternary carbon C21 by HMBC correlations from H20 and H34 to C21 and C22. Two oxygenated carbons C18 and C21 were linked by the ether bridge in consideration of the unsaturation degree, since the remaining six sp2 quaternary carbons and a chlorine atom were assigned to constitute the tetronic acid as described below. HMBC correlations from the methylene H7 to C3, C5, and C6, and from an olefinic methine H1 to C2 and C3 established the carbon connectivity from C1 to C7. A carbonyl carbon C4 (δC 169.6) could be connected directly to C5 and through a lactone oxygen to C2 by comparing the chemical shift with known natural products, agglomerin,7 tetronomycin,8 and SF2487,9 all of which possess a tetronic acid substituted by exo-methylene group. Carbons from C3 to C6 showed high similarity to the corresponding carbons of the above-mentioned compounds. The chemical shift of C1 was 9.6 ppm downfield shifted and that of C2 was 5.4 ppm upfield shifted, compared to the same carbons of 2. This shifting trend is in good accordance with the reported chemical shift values of halogenated alkenes.6 Therefore, the chlorine atom was placed at C1 to complete the planar structure of 1. The double-bond geometry around the C1−C2 axis of 1 was determined by analyzing long-range 1H−13C coupling constants (Figure 4). It is known that 3JCH values in 1H−CC−13C spin
Figure 5. Suppression of MPP+-induced cell death in PC12D cells by 1 and 2.
next tested them for autophagy induction because protein aggregation observed in PD patients’ brain is related to the impairment of autophagy, an intracellular bulk degradation system.14 Autophagy flux can be traced using the mCherryGFP-LC3 tandem fluorescent construct (tf-LC3) by assessing colocalization of GFP and mCherry.15 In this construct, red puncta (mCherry-positive-GFP-negative) indicates autolysosome, namely the end point of autophagy. According to the fluorescence-imaging experiments using inducible tf-LC3 expressing PC12 cells, 24 h treatment of 0.1 μM 1 and 2 significantly increased red puncta area, indicating the activation of autophagy flux by these compounds (Figure 6). As 1 and 2 suppressed MPP+-induced cell death and activated autophagy at the same concentrations, their protective effect against MPP+induced neuronal cell death is likely due to their autophagyenhancing ability. Additionally, biological activity of 1 and 2 was investigated in tumor-cell invasion assay and antimicrobial testing. Compounds 1 and 2 inhibited the invasion of murine carcinoma colon 26-L5 cells into the reconstituted extracellular matrix Matrigel with an IC50 of 0.017 and 0.15 μM, respectively, while at these concentrations both compounds showed no cytotoxicity (IC50 1: 1.7 μM, 2: 4.4 μM) (Figure S21). The antiinvasive potency of 1 is comparable to campechic acid A, the most potent inhibitor found in our previous screening.3c As described in the patent, 2 showed strong antimicrobial activity against Gram-positive bacteria. Interestingly, 1 was 2- to 8-fold more potent than 2 (Table 2). In summary, nonthmicin (1), a new polyether polyketide bearing a chlorinated tetronic acid, was isolated from the culture extract of a soil-derived Actinomadura strain along with ecteinamycin (2) through UV-based metabolite analysis. The absolute configuration of 2 was established by X-ray crystallographic analysis, and that of 1 was proposed on the basis of the crystal structure of 2. The most unique structural feature of 1 is the chlorinated tetronic acid. Halogen-modified tetronic acids have never been reported from natural products. Both 1 and 2 displayed neuroprotective properties and antiinvasive activity as
Figure 4. Configuration analysis for C1−C2 double bond in 1 based on long-range 1H−13C coupling constants.
system are larger for trans-double bonds than for cis-double bonds, which can be rationalized by the Carplus equation regarding to the dihedral angles.6,10 In the case of 2, the 3JCH value between the exo-methylene proton at δH 4.78 and C3 was 7.3 Hz and that between another exo-methylene proton at δH 5.17 and C3 was 2.8 Hz. Thus, the former proton could be assigned to be placed trans to C3 and the latter cis to C3. Meanwhile, the 3JCH value for H1 and C3 of 1 was 2.0 Hz. This rather small coupling constant indicated the cis relationship of H1 to C3, and therefore, the trans relationship of the chlorine atom to C3, establishing the Z-configuration for C1/C2.11 The 3 JH1,C3 value is decreased from 2.8 to 2.0 Hz (29% decrease) by chlorine substitution at C1. A similar tendency is observed for the 3JCH values in propene and 1-chloropropene (26% decrease).6,12 Crystals of 1 could not been obtained; however, the absolute configurations of asymmetric centers in 1 were tentatively assigned as same as those for 2 in consideration of biogenesis and chemical shift similarity, which was also corroborated by CD comparison (Figure S4). C
DOI: 10.1021/acs.orglett.7b00318 Org. Lett. XXXX, XXX, XXX−XXX
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ACKNOWLEDGMENTS We acknowledge Dr. Masaya Imoto at Keio University for his assistance and discussions regarding neuroprotective assay.
Figure 6. Enhancement of autophagy flux by 1 and 2. Fluorescence images of differentiated PC12/tet-on/mCherry-GFP-LC3 cells treated with 0.1 μM 1 and 2. Red puncta (mCherry-positive-GFP-negative) was significantly increased by treatment with 1 and 2.
Table 2. Antimicrobial Activity of Nonthmicin (1) and Ecteinamycin (2) MIC (μg/mL) test organisms Kocuria rhizophila ATCC 9341 Bacillus cereus NBRC 15305 Staphylococcus aureus IFO 12732 Enterococcus faecalis NBRC 100480 Escherichia coli NIH-JC2 Candida albicans NBRC 1594
1
2
0.005 0.0013 0.005 0.0025 >10 >10
0.01 0.01 0.01 0.01 >10 >10
well as potent antimicrobial activity against Gram-positive bacteria.
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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.orglett.7b00318. Experimental procedures, analytical data, and MS, UV, IR, CD, and 1D and 2D NMR spectra for 1 and 2 (PDF) Single-crystal X-ray data for 2 (CIF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yasuhiro Igarashi: 0000-0001-5114-1389 Notes
The authors declare no competing financial interest. D
DOI: 10.1021/acs.orglett.7b00318 Org. Lett. XXXX, XXX, XXX−XXX
