Article pubs.acs.org/jnp
Polyketides with New Delhi Metallo-β-lactamase 1 Inhibitory Activity from Penicillium sp. Maoluo Gan,†,‡,⊥ Yufeng Liu,‡,§,⊥ Yinlei Bai,† Yan Guan,† Li Li,‡ Rongmei Gao,† Weiying He,† Xuefu You,† Yuhuan Li,† Liyan Yu,† and Chunling Xiao*,† †
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China § Jining Medical College, Jining, Shandong 272067, People’s Republic of China S Supporting Information *
ABSTRACT: Three new polyketide compounds (1−3), a new quinolone alkaloid (4), and seven known polyketide derivatives were identified from the cultures of Penicillium sp. I09F 484, a strain isolated from the rhizosphere soil of the plant Picea asperata from Kanas Lake, Xinjiang, China. Their structures were elucidated by extensive spectroscopic data analysis. The absolute configurations of 1 and 4 were established by quantum chemical time-dependent density functional theory electronic circular dichroism calculation and Marfey’s method, respectively. Compounds 1 and 2 displayed inhibitory activity against New Delhi metallo-β-lactamase 1 with IC50 values of 94.9 and 87.9 μM, respectively.
I
our ongoing search for bioactive compounds from microbial metabolites,10 a fungal strain, Penicillium sp. I09F 484, was isolated from the rhizosphere soil of the plant Picea asperata, collected from Kanas Lake, Xinjiang, China. The crude extract of this strain showed considerable antibacterial activity against a multidrug-resistant enteric bacterium, Morganella morganii 0709, in preliminary screening. Bioassay-guided isolation of the extract resulted in the isolation and characterization of three new polyketide compounds (1−3), a new quinolone alkaloid (4), and seven known polyketides, patulin,11 gentisyl alcohol,11 2-methylhydroquinone, 12 epoformin, 13 epiepoformin, 13 (4S,5S)-4,5-dihydroxy-2-methylcyclohex-2-enone,14 and 3-hydroxybenzyl alcohol.15 Compound 2 was a polyketide alkaloid with an unusual chromeno[3,2-c]pyridine moiety. Compared with other reported MBL inhibitors,5a 1 and 2 showed moderate inhibitory activity against NDM-1, and the antibacterial activity of the crude extracts was mainly due to the presence of the known patulin. Herein we report the isolation, structure elucidation, and biological activities of these compounds.
nfections with carbapenem-resistant Enterobacteriaceae, particularly Klebsiella pneumoniae, are emerging as an important challenge in healthcare settings worldwide.1 The resistance of Enterobacteriaceae to carbapenems is increasing because of the production of various carbapenemases, including metallo-β-lactamases (MBLs).2 In 2008, a novel MBL, called New Delhi metallo-β-lactamase 1 (NDM-1), was identified from a K. pneumoniae clinical isolate and has attracted great attention worldwide since then.3 NDM-1 inactivates all βlactams except aztreonam, conferring upon enteric bacteria nearly complete resistance to all β-lactam antibiotics in clinical use, including the late-generation carbapenems meropenem and imipenem.3a,4 NDM-1 is encoded on a readily transferable plasmid, which facilitates its transmission.4 Of particular concern is the fact that NDM-1 producers are already becoming highly prevalent, and NDM-1 has been observed in clinical isolates of the opportunistic bacterium Acinetobacter baumannii and in strains of Escherichia coli and other Enterobacteriaceae.3b NDM-1 is not inhibited by current β-lactamase inhibitors such as clavulanic acid and tazobactam.3 Although some MBL inhibitors have been reported, none of them are clinically approved, and to date, there are few reports of compounds with inhibitory activity against the newly emerging NDM-1.5 Penicillium species, known for their ability to produce bioactive compounds, are a rich source of polyketide metabolites.6 Some important pharmaceutical agents from Penicillium sp., such as griseofulvin7 and compactin (mevastatin),8 are derived from the polyketide biosynthesis pathway.9 In © 2013 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Compound 1 was isolated as an amorphous powder. Its molecular formula was established as C14H12O7 by analysis of Received: January 31, 2013 Published: August 23, 2013 1535
dx.doi.org/10.1021/np4000944 | J. Nat. Prod. 2013, 76, 1535−1540
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nuclear magnetic resonance (NMR) and HRAPCIMS data. The infrared (IR) bands at 1503, 1578, 1602, and 1700 cm−1 suggested the presence of an aromatic ring and conjugated carbonyl groups, which was supported by the UV absorptions at 216 and 295 nm. The 1H NMR spectrum exhibited signals that could be attributed to a conjugated double bond proton at δ 8.06 (s) and a pair of meta-coupled aromatic protons (δ 6.74, brs; δ 6.84, brs), indicative of a 1,3,4,5-tetrasubstituted benzene ring. Other signals in the 1H NMR spectrum included one singlet methyl (δ 2.13), two geminal-coupled methylene protons (δ 2.80, dd, J = 15.6, 3.0 Hz; δ 2.57, dd, J = 15.6, 9.0 Hz), and one oxymethine (δ 5.03, dd). The 13C NMR and DEPT data revealed one methyl (δ 30.4), one methylene (δ 50.0), one oxygenated sp3-hybridized methine (δ 62.2), three carboxyl/carbonyl carbons (δ 169.8, 174.1, and 206.6), and eight aromatic/olefinic carbons, three of which were methine carbons. The spectroscopic data were similar to those of chromone derivatives from Penicillium species,16 suggesting that 1 was also a chromone analogue with an aliphatic side chain moiety. This supposition was corroborated by two-dimensional (2D) NMR data analysis (Figures S8 and S9 of the Supporting Information). Cross-peaks in the HMBC spectrum of 1 from H-8 to C-4a, C-6, C-7, and C-8a; from H-6 to C-4a, C-8, and the carboxyl group (C-9); and from H-2 to C-3, C-4, and C-8a indicated that 1 had a 5-carboxyl-7-hydroxychromone nucleus. Additionally, HMBC correlations from H-1′ to C-2′ and C-3′; from H-2′ to C-1′ and C-3′; and from H-4′ to C-2′ and C-3′ suggested the presence of the 1′-hydroxy-3′-butanoyl side chain. The linkage of C-1′ of the side chain to C-3 of the chromone moiety was established by HMBC correlations from H-1′ to C-2, C-3, and C-4 and from H-2′ to C-3. Accordingly, the planar structure of 1 was determined as shown. To determine the absolute configuration of 1, attempts were made to prepare α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) ester derivatives by application of the modified Mosher’s method.17 Unfortunately, the approaches employed did not yield the desired product. More extensive efforts toward this end were hampered by sample limitations. Finally, we decided to use circular dichroism (CD) analysis because of the presence of an α,β-unsaturated carbonyl function adjacent to the asymmetric center in 1. The electronic CD spectrum (ECD) was experimentally recorded, and the theoretical ECD was calculated using the time-dependent density functional theory (TDDFT) method.18 The experimental CD spectrum of 1 exhibited a positive Cotton effect (CE) at 215 nm, two negative CEs near 249 and 286 nm, and a positive CE at 319 nm and was in excellent agreement with the calculated ECD spectrum of 1 with the S configuration at C-1′ (Figure 1). Therefore, the structure of 1 was established as (1′S)-7hydroxy-3-(1′-hydroxy-3′-butanoyl)chromone-5-carboxylic
Figure 1. Experimental and TDDFT-calculated ECD spectra of 1.
acid. Chromone derivatives with a similar aliphatic side chain have been reported,16,19 but the configuration of the chiral carbon in the side chain had not been determined. On the basis of the configuration assignment of 1 described above, PI-3, previously isolated from Penicillium italicum16 with the same negative optical rotation as 1, should also possess the 1′S configuration. Compound 2 was isolated as a brown powder with a molecular formula of C15H11O6N by HRESIMS (m/z 300.0514 [M − H]−) and NMR data. This formula required 11 degrees of unsaturation, two more than compound 1. The NMR data for 2 were similar to those for 1 (Table 1). The main differences were the absence of signals for the 1′-hydroxy-3′butanoyl side chain and the presence of signals for an allylic methyl (δH 2.61) and a methoxyl group (δH 3.78) in the 1H NMR spectrum of 2. Additionally, the downfield shifts of three aromatic protons [H-1 (δH 9.09), H-4 (δH 7.46), and H-6 (δH 7.03)] were observed. In total, 15 carbon resonances were observed in the 13C NMR and DEPT spectra of 2, which were ascribed to one allylic methyl (δC 24.9), one methoxyl (δC 61.6), three sp2 methines [one nitrogen-bearing (δC 149.1)], and 10 sp2 quaternary carbons (including one carboxyl at δC 167.4 and one conjugated carbonyl at δC 173.6). On the basis of the carbon chemical shifts of these resonances, 2 was composed of a carboxyl, a ketone, five CC groups, and a C N bond that accounted for 8 of the 11 degrees of unsaturation. This suggested that 2 possessed a fused chromeno-pyridine tricyclic ring structure.20 This suggestion was reflected in the UV chromophore of 2, which showed a significant bathochromic shift (absorption maximum at 330 nm) as compared to 1, and was further confirmed by 2D NMR data analysis (Figures S15−S17 of the Supporting Information). In the HMBC spectrum of 2, H-6 displayed intense 2JCH or 3JCH correlations to C-5a, C-7, C-8, and C-9a and weak 4JCH correlations to C-9 and the carbonyl (C-10). Furthermore, the methoxy protons were correlated to C-8 (δ 142.7). These correlations, combined with the chemical shifts of these carbons and protons, unequivocally established a 7-hydroxy-8-methoxylchromone-9-carboxylic acid moiety for 2. The lack of ROESY correlations between the methoxyl and H-6 confirmed that the methoxyl group was substituted at C-8, instead of C-7. In addition, HMBC correlations of H-1/C-3, C-4a, C-10, and C10a; H-4/C-4a, C-10 and C-10a; and H3-12/C-3 and C-4 demonstrated that the chromone moiety was fused with a 31536
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Table 1. NMR Spectroscopic Data for Compounds 1−4 in DMSO-d6a 1 no. 1 2 3 4 4a 5 5a 6
a
δC 152.5 126.1 174.1 112.0 137.4 113.1
7 8 8a 9 9a 10 10a 11 12 OMe 1′ 2′
162.2 102.3 157.7 169.8
3′
206.6
4′
30.4
62.2 50.0
δH mult. [J (Hz)]
2
3
4
δC
δH mult. [J (Hz)]
δC
149.1
9.09 s
64.6 56.8 55.7 66.3
3.64 2.94 3.07 3.76
26.3
1.38 m
32.2
1.28 ddd (12.6, 6.0, 2.4) 1.23 ddd (12.6, 12.6, 10.2) 0.82 d (6.6)
8.06 s 164.2 111.1 160.9
6.74 brs
158.4 104.7
6.84 brs
153.6 142.7 129.5 111.2 173.6 115.2 167.4 24.9 61.6
7.46 s
7.03 s
17.0
δH mult. [J (Hz)] ddd (10.2, 6.0, 4.8) brd (4.8) dd (2.4, 2.4) ddd (5.4, 2.4, 2.4)
δC
δH mult. [J (Hz)]
161.2 119.7 146.1 116.1 126.0
7.77 d (7.8)
122.0
7.18 t (7.8)
130.9 115.6 139.2 165.6
7.53 t (7.8) 7.35 d (7.8)
6.45 s
2.61 s 3.78 s 172.4b 49.2
5.03 dd (9.0, 3.0) 2.80 dd (15.6, 3.0) 2.57 dd (15.6, 9.0)
36.2
4.72 dd (7.8, 6.0) 2.83 dd (16.8, 6.0) 2.66 dd (16.8, 7.8)
171.9b
2.13 s b
The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments. Exchangeable. Abbreviation: mult., multiple.
methylpyridine ring via C-4a and C-10a to form a chromeno[3,2-c]pyridine ring. Consequently, 2 was established as 7hydroxy-8-methoxy-3-methyl-10-oxo-10H-chromeno[3,2-c]pyridine-9-carboxylic acid. Compound 2, and SB236049 isolated from the fungus Chaetomium f unicola,20 represent the only examples of alkaloids possessing the unique chromeno[3,2-c]pyridine nucleus. Compounds 1 and 2 share the same carbon skeleton with the well-known polyketide metabolites fulvic acid,21 polivione,22 and lapidosin23 isolated from Penicillium species. The close resemblance between these compounds, in the carbon skeleton and oxygenation pattern, indicated that they are probably generated by a common biosynthesis strategy from a heptaketide precursor (5) as suggested by Staunton et al (Scheme 1).24 Compound 3 had the molecular formula C7H12O3, as indicated by HRESIMS. The NMR data (Table 1 and Experimental Section) in DMSO-d6 and a pyridine-d5/D2O mixture (5:1) were very similar to those of ampelomin B,25 suggesting that 3 is a stereoisomer of the reported compound with the same planar structure. This was confirmed by 1H−1H COSY, HSQC, and HMBC data analyses (Figures S24−S26 of the Supporting Information). However, detailed 1H NMR coupling constant analysis and the NOESY experiment revealed an equatorial hydroxyl group at C-1 in 3 rather than the axial hydroxyl found in ampelomin B. The large coupling constant between H-1 and H-6ax (3JH‑1/H‑6ax = 10.2 and 10.8 Hz in DMSO-d6 and a 5:1 pyridine-d5/D2O mixture, respectively) and between H-5 and H-6ax (3JH‑5/H‑6ax = 12.6 and 11.4 Hz in DMSO-d6 and a 5:1 pyridine-d5/D2O mixture, respectively) revealed their axial relationships, thus also indicating an equatorial orientation for OH-1. This was further verified by the obvious correlation of H-1 and H-5 and the lack of
Scheme 1. Plausible Biosynthetic Pathways for 1 and 2
correlation between H-5 and OH-1 in the NOESY spectrum of 3 (Figure S27 of the Supporting Information). Thus, compound 3 was determined to be an epimer of ampelomin B with the opposite configuration at C-1, and its relative 1537
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values of 94.9 and 87.9 μM, respectively (the positive controls L-captopril and EDTA gave IC50 values of 107.6 and
