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Bioassay-Guided Isolation of Antibacterial Metabolites from Emericella sp. TJ29 Yan He,†,§ Zhengxi Hu,†,§ Qin Li,†,§ Jinfeng Huang,† Xiao-Nian Li,‡ Hucheng Zhu,† Junjun Liu,† Jianping Wang,*,† Yongbo Xue,*,† and Yonghui Zhang*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China S Supporting Information *

ABSTRACT: Bioassay-guided isolation of metabolites from cultures of the plant-derived fungus Emericella sp. TJ29 yielded three new terpene−polyketide hybrid meroterpenoids, emervaridones A−C (1−3), two new polyketides, varioxiranediols A and B (5 and 6), and three known analogues (4, 7, and 8). The structures and absolute configurations of these new compounds were elucidated by spectroscopic analyses, single-crystal X-ray diffraction, Mo2(OAc)4-induced electronic circular dichroism (ECD) data, and ECD calculations. To date, only one compound (4) bearing the emervaridone-type carbocyclic skeleton has been reported. The structures of emervaridones A−C (1−3) are new members of this type of natural product, and 1 features the first example of an α-directional H-7′ in this structural category. Compounds 1 and 5 were active against five drug-resistant microbial pathogens [methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, extended-spectrum β-lactamase-producing Escherichia coli (ESBL-producing E. coli), Pseudomonas aeruginosa, and Klebsiella pneumoniae] with minimum inhibitory concentration (MIC) values in the micrograms per milliliter range. Notably, the inhibitory effect of emervaridone A (1) against ESBL-producing E. coli was comparable to that of the clinically used antibiotic amikacin, with an MIC value of 2 μg/mL. Compounds 1 and 5, both with low toxicities to mammalian cells, were bacteriostatic and bactericidal, respectively. Importantly, these two compounds may provide novel chemical scaffolds for the discovery of antibacterial agents for drug-resistant microbial pathogens.

T

either microbial products or their analogues.4−6 In this study, an extensive biological screen against five antibiotic-resistant bacteria was conducted to identify novel antibiotics, and the endophytic fungus Emericella sp. TJ29 showed bioactivity against the indicator bacteria.7 The bioassay-guided isolation of metabolites from cultures of this fungal strain yielded five new active compounds (1−3, 5, and 6) and three known analogues (Figure 1). Among these compounds, emervaridone A (1) and varioxiranediol A (5) demonstrated antibacterial activity against ESBL-producing E. coli and P. aeruginosa, in which 1 had minimum inhibitory concentration (MIC) values of 2 and 8

he emergence and spread of antibiotic-resistant bacteria has potentially drastic consequences for human health worldwide. 1 Methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase-producing Escherichia coli (ESBL-producing E. coli), Enterococcus faecalis (E. faecalis), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) express a remarkable array of resistance and virulence factors, which contribute to their prominent roles in hospital-acquired infections in all regions of the world.2 To respond to this emerging crisis, global organizations such as the WHO have urged the scientific community to identify new approaches to combat antibiotic resistance.1,3 Microorganisms are a prolific source of novel antibiotics, and most antibacterial agents in clinical or preclinical trials are © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 25, 2017 Published: September 13, 2017 2399

DOI: 10.1021/acs.jnatprod.7b00077 J. Nat. Prod. 2017, 80, 2399−2405

Journal of Natural Products

Article

Figure 1. Selected 1H−1H COSY and HMBC correlations of compounds 1−3, 5, and 6.

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of Compounds 1−3, 5, and 6 (δ in ppm, J in Hz) 1a

3a

5b

6b

δC

δH

δC

δH

δC

δH

δC

δH

δC

δH

1

152.1

6.58 d (10.3)

151.7

152.8

6.50 br d (10.3)

71.6

4.98 br d (12.3); 5.09 dd (2.5, 12.3)

71.4

4.97 br d (12.3); 5.06 dd (2.7, 12.3)

2

125.9

5.90 d (10.3)

125.8

6.51 br d (10.2) 5.81 br d (10.2)

125.6

5.87 br d (10.3)

127.4

3 4 5 6

203.3 44.5 43.5 18.6

1.63 m 1.85 m

203.1 44.3 41.1 16.8

203.4 44.6 39.3 17.0

2.26 m 1.65 m; 1.85 m

155.6 110.3 130.5 114.6

7

26.1

1.85 m; 2.12 m

26.0

26.3

1.95 m; 2.12 m

145.1

8 9

47.7 60.6

1.65 s

48.0 60.3

47.8 63.1

2.05 br s

84.6 40.1

10 11 12

37.8 65.9 50.1

3.56 s 1.49 br s

37.6 68.1 55.2

37.4 66.9 56.3

2.98 br s 1.65 m; 1.78 m

13 14 15 1′

26.2 21.8 24.5 70.4

2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

54.6 149.5 207.1 50.1 74.1 58.9 174.5 107.5

4.87 s; 5.01 s

55.1 139.5 202.1 50.7 143.1 155.7 166.3 102.4

10′

21.9

1.24 s

20.4

no.

a

2a

1.23 s 1.02 s 1.11 s 4.39 d (9.9); 4.52 d (9.9)

4.02 s 2.72 s

25.8 21.5 24.2 72.5

1.82 m 1.53 m; 1.77 m 1.87 m; 2.04 m 1.76 m

3.64 br s 1.63 m; 1.74 m 1.21 s 0.98 s 1.06 s 4.50 br s

6.69 br s

4.58 br s; 4.65 br s 1.27 s

26.7 21.8 24.4 67.9 53.7 72.4 202.5 51.3 143.9 138.5 167.0 47.3 20.6

1.32 s 1.08 s 1.19 s 4.36 d (9.7); 4.44 d (9.7)

145.5

6.84 m 7.26 t (7.8) 6.85 m

114.2 130.6 110.2 155.5

6.79 d (7.5) 7.26 t (7.8) 6.84 d (8.1)

127.6 82.7 40.2

5.44 m 1.85 m

74.2 75.5 35.9

5.41 m 1.81 dt (8.4, 14.4); 2.11 ddd (3.4, 4.8, 14.4) 3.73 ddd (3.4, 5.7, 8.4) 3.47 m 1.37 m; 1.59 m

72.7 75.9 36.0

3.72 m 3.46 m 1.38 m; 1.55 m

20.1 14.5 55.8

1.38 m; 1.59 m 0.95 t (7.0) 3.82 s

20.1 14.5 55.8

1.37 m; 1.54 m 0.94 t (6.8) 3.83 s

6.84 br s

2.44 br s; 2.72 br s 1.40 s

Recorded in CDCl3. Recorded in CD3OD; “m” = overlapped or multiplet with other signals. b

μg/mL, respectively, and 5 had MICs of 4 μg/mL for both (Table 2). Herein, the isolation, structure elucidation, and bioactivity evaluations of these compounds are presented.

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detailed analysis of its 1H NMR data (Table 1) demonstrated the presence of four methyl singlets at δH 1.02 (H3-14), 1.11 (H3-15), 1.23 (H3-13), and 1.24 (H3-10′); an oxygenated methylene at δH 4.39/4.52 (both d, J = 9.9 Hz, H2-1′); four olefinic protons at δH 6.58 (d, J = 10.3, H-1), 5.90 (d, J = 10.3, H-2), and 4.87/5.01 (both s, H2-9′); and an oxygenated methine at δH 4.02 (s, H-6′). The 13C NMR data (Table 1), together with the DEPT spectrum, revealed the presence of 25

RESULTS AND DISCUSSION

Compound 1 was obtained as a colorless crystal from a mixture of CHCl3−MeOH (10:1). The molecular formula was determined as C25H30O5 by HRESIMS and 13C NMR data. A 2400



Article

Table 2. Antibacterial Activities of Compounds 1−8 (MIC, μg/mL) pathogen

1

2

3

4

5

6

7

8

reff

a

32 32 2 8 32

64 >128 32 32 64

>128 >128 32 64 >128

>128 >128 64 >128 >128

32 32 4 4 16

>128 32 8 32 >128

>128 >128 >128 >128 >128

64 >128 32 32 >128

0.5 (Va) 16 (Ch) 2 (Am) 1 (Ce) 1 (Ce)

MRSA E. faecalisb ESBL-E. colic P. aeruginosd K. pneumoniaee a

MRSA = methicillin-resistant Staphylococcus aureus ATCC43300. bE. faecalis = Enterococcus faecalis ATCC 29212. cESBL-E. coli = ESBL-producing Escherichia coli ATCC 35218. dP. aeruginos = Pseudomonas aeruginosa ATCC 15442. eK. pneumonia = Klebsiella pneumoniae ATCC 700603. fThe activities of reference compounds recommended by the National Committee for Clinical Laboratory Standards (CLSI); Va = vancomycin; Ch = chloramphenicol; Am = amikacin; Ce = ceftriaxone.

Chart 1

carbon resonances corresponding to four sp3 methyls, five methylenes (an oxygenated and an olefinic), seven methines (two olefinic and one oxygenated), and nine nonprotonated carbons (two keto, one ester carbonyl, and one olefinic). The presence of three carbonyls (δC 207.1, 203.3, and 174.5) and two double bonds (δC 152.1/125.9 and 149.4/107.5) accounted for five of 11 indices of hydrogen deficiency, requiring six rings in the structure. The 1H and 13C NMR data of 1 showed signals similar to those of the known compound emervaridone (4),8 indicating that both share an emervaridone-type carbocyclic skeleton, except that an oxygenated tertiary carbon (C-9) and a Δ6′,7′ double bond in 4 were replaced by three methines (δC 74.1, C6′; 58.9, C-7′; 60.6, C-9) in 1, which is supported by the 2D NMR data (Figures S13−S16, Supporting Information). This includes 1H−1H COSY correlations of H-9 (δH 1.65)/H-11 (δH 3.56) and H-6′ (δH 4.02)/H-7′ (δH 2.72) and HMBC correlations from H3-10′ (δH 1.24) to C-12, C-4′, C-5′, and C-6′ (δC 74.1) and from H-6′ and H-7′ to C-8′ (δC 174.5). Thus, the planar structure of 1 was determined and further confirmed as shown in Figure 1. Distinguished from the known andilesin A,9 in which H-6′ (δH 4.07, d) and H-7′ (δH 2.93, d) have a cis-relationship with an obvious vicinal coupling constant (∼7 Hz), the singlet of H6′ (δH 4.02, s) with no recognizable vicinal coupling constant (∼0 Hz) to H-7′ (δH 2.72, s) in 1 indicated that H-6′ and H-7′ possessed a unique trans-relationship with a dihedral angle of approximately 90°. This conclusion was supported by the observed NOESY cross-peaks (Figure 2) of H3-14 (δH 1.02)/ H3-13β (δH 1.23)/H-9 (δH 1.65)/H-12b (δH 1.49), H-12a (δH 1.49)/H-6′ (δH 4.02), H-7′ (δH 2.72)/H-1′a (δH 4.39), and H1′b (δH 4.52)/H-5 (δH 1.63)/H3-15 (δH 1.11) and no NOESY correlation between H-5 and H3-13. According to these data, H-9 and H-6′ were determined to be β-oriented, whereas H-7′

Figure 2. Selected NOESY correlations of compounds 1 and 3.

was α-oriented, thereby establishing the relative configuration of 1. To determine its absolute configuration, a time-dependent density functional theory (TDDFT) method at the B3LYP/6311++G** level with polarizable continuum model (PCM) in MeOH was performed for (5R,8S,9S,10R,11R,2′R,5′S,6′R,7′S)1 (Figure S1, Supporting Information), in which the experimental electronic circular dichroism (ECD) spectrum of 1 was identical to the calculated ECD curve of 1, indicating that the absolute configuration of 1 was 5R,8S,9S,10R,11R,2′R,5′S,6′R,7′S. By slow evaporation of a mixture of CHCl3−MeOH (10:1) at room temperature, a suitable crystal of 1 was obtained and subjected to a single-crystal X-ray diffraction experiment with Cu Kα radiation (Figure S2, Supporting Information, CCDC 1525519),10 supporting that the structure, including stereochemistry, was consistent with previous assignments based on the NMR spectral data and ECD calculations. Notably, compound 1 was reported as the second natural compound with an identical carbocyclic skeleton to that of emervaridone8 and represented the first example of an α-directional H-7′ in this structural category. It was given the name emervaridone A. Compound 2 was also isolated as a colorless crystal and gave an HRESIMS ion peak corresponding to a molecular formula of 2401



Article

8 (δH 5.41)/H2-9 (δH 1.81 and 2.11)/H-10 (δH 3.73)/H-11 (δH 3.47)/H2-12 (δH 1.37 and 1.59) and H2-13 (δH 1.38 and 1.59)/H3-14 (δH 0.95) and an HMBC correlation from H3-14 to C-12 (δC 35.9). Thus, the planimetric map of 5 was determined. A detailed examination of the 1H NMR data of 5 revealed that H-10 (δH 3.73) and H-11 (δH 3.47) showed a diagnostic coupling constant of 5.7 Hz, consistent with the known varioxiranediol (J = 5.5 Hz),8 indicating that the relative configuration of H-10 and H-11 corresponded to an erythro configuration, according to the reported values (threo configuration: J < 5.0 Hz; erythro configuration: J > 5.0 Hz).11 The absolute configuration of the 10,11-diol motif in 5 was ascertained according to the in situ dimolybdenum CD method developed by Snatzke and Frelek.12,13 According to the empirical helicity rule relating the Cotton effect sign of the diagnostic O−C−C-O moiety, the negative CD effect observed at approximately 310 nm (Figure S5, Supporting Information) permitted assignment of the 10R and 11S configurations. To determine the absolute configuration of 5, the calculated ECD spectra (Figure S6, Supporting Information) of (8R,10R,11S)-5 and (8S,10R,11S)-5 were performed at the B3LYP/6-311+ +G** level, and the former was consistent with the experimental curve of 5, suggesting that the absolute structure of 5 is 8R,10R,11S. A side-by-side comparison of the 1D and 2D NMR spectral data (Table 1) of 5 and 6 revealed that these two compounds are a pair of stereogenic isomers, differing only in the location of a methoxy group. In 6, the methoxy group is located at C-6, which was confirmed by 1H−1H COSY cross-peaks (Figure 1) of H-3 (δH 6.79)/H-4/H-5 (δH 6.84) and HMBC correlations from H3-15 (δH 3.83) to C-6 (δC 155.5) and from H-4 (δH 7.26) to C-2 (δC 145.5) and C-6. Furthermore, using Mo2(OAc)4-induced ECD (Figure S5, Supporting Information) and calculated ECD methods (Figure S7, Supporting Information) as for 5, the absolute configuration of 6 was also assigned as 8R,10R,11S. The structures of known compounds 4, 7, and 8 were identified by analysis of the 1H and 13C NMR and HRESIMS data with the literature values and were assigned as emervaridone (4),8 varioxiranediol (7),8 and varioxirane (8).14 Emervaridones A−C (1−3) are new terpene-polyketide hybrid meroterpenoids with a rare emervaridone-type carbocyclic core. Remarkably, emervaridone A (1) has a previously unreported α-directional H-7′, resulting in the exceeding twist of the five-membered lactone motif, which differs significantly from those originating from the heterologous expression of the Emericella variecolor gene cluster in Aspergillus oryzae,9 such as andiconin, anditomin, and andilesins A, C, and D. The cycloaddition,15 redox,16 and Wagner−Meerwein rearrangement reactions were key steps proposed for its biosynthesis (Scheme S1, Supporting Information). Highest antibacterial activities against ESBL-producing E. coli for 1 and P. aeruginosa for 5 were observed (Table 2), with MIC values of 2.0 and 4.0 μg/mL, respectively. The results of time−kill assays17,18 performed for ESBL-E. coli demonstrated that 1 was bacteriostatic and 5 was bactericidal (Figure S8, Supporting Information). Meanwhile, 1 and 5 exhibited little effect on the BEAS-2B cells (Figure S9, Supporting Information). As antibiotic resistance is a growing public health threat, this study might provide novel chemicals for the development of new antibacterial agents.

C25H28O4, which is one oxygen atom and two hydrogen atoms less than 1. The 1H and 13C NMR data (Table 1) closely resembled those of 1, indicating that these two compounds possess identical carbon skeletons and substitution patterns, with the only difference being the presence of a Δ6′,7′ double bond (δC 143.1, C-6′; 155.7, C-7′) in 2 rather than two methines (including one oxygenated) at C-6′ (δC 74.1) and C7′ (δC 58.9), further confirmed by HMBC correlations (Figure 1) from H3-10′ (δH 1.27) to C-12 (δC 55.2), C-4′(δC 202.1), C5′(δC 50.7), and C-6′ (δC 143.1) and from H-6′ (δH 6.69) to C12, C-2′ (δC 55.1), C-7′ (δC 155.7), and C-8′ (δC 166.3). The NOESY data (Figure S16, Supporting Information) show that the relative configuration of 2 was identical to that of 1. The absolute configuration of 2 was ascertained by comparison of its experimental ECD curve with the simulated ECD spectrum (Figure S1, Supporting Information) generated by the TDDFT for (5R,8S,9S,10R,11R,2′R,5′S)-2. Accordingly, the calculated ECD spectrum of 2 showed a good fit with the experimental plot of 2, with the absolute configuration of 5R,8S,9S,10R,11R,2′R,5′S. Furthermore, using repeated recrystallization, an appropriate crystal of 2 was obtained and used for single-crystal X-ray diffraction analysis (Figure S3, Supporting Information, CCDC 1525518), supporting our conclusion of its absolute structure based on the Flack parameter of −0.19(18).10 Therefore, the structure of 2, emervaridone B, was established as shown. Compound 3, obtained as a white powder, was assigned the molecular formula C 25 H28 O5 (12 indices of hydrogen deficiency) based on the HRESIMS analysis, which was 16 mass units greater than that of 1. The 1H and 13C NMR data of 3 (Table 1) were closely related to those of 2, indicating that 3 is a structural analogue of 2, except for the replacement of a Δ3′,9′ double bond (δC 139.5, C-3′; 102.4, C-9′) in 2 by a 3′,9′oxirane moiety in 3, as indicated by its molecular formula and resonances at δC 72.4 (C-3′) and 47.3 (C-9′) and a resonance attributed to H2-9′ at δH 2.44 and 2.72. This conclusion was further supported by the HMBC correlations from H-9 (δH 2.05), H-11 (δH 2.98), H2-1′ (δH 4.36 and 4.44), and H2-9′ (δH 2.44 and 2.72) to C-3′ (δC 72.4). The 3′,9′-oxirane functional group was deduced as β-oriented through the diagnostic NOESY correlations (Figure 2) of H-11 (δH 2.98)/H-9′b (δH 2.72), H-9′a (δH 2.44)/H-1′a (δH 4.44), H-1′b (δH 4.36)/H-7α (δH 1.95), and H-7β (δH 2.12)/H3-13 (δH 1.32). Accordingly, the relative configuration of 3 was determined. To determine its absolute configuration, the ECD spectra (Figure S4, Supporting Information) of 3 [enantiomers: 3 (5R,8S,9S,10R,11R,2′R,3′R,5′S); ent-3 (5S,8R,9R,10S,11S,2′S,3′S,5′R)] were calculated at the B3LYP/6-311++G** level. There is an overall agreement between the experimental ECD and predicted ECD curves of 3. Therefore, the absolute configuration of 3 was established as 5R,8S,9S,10R,11R,2′R,3′R,5′S, and it was named emervaridone C. Compounds 5 and 6 were obtained as white powders with the same molecular formula, C15H22O4, as confirmed by the HRESIMS analysis together with the 13C NMR data. The 1H and 13C NMR data (Table 1) of 5 were comparable to those of varioxiranediol (7),8 the absolute structure of which was verified using single-crystal X-ray diffraction analysis, with the only distinction being that a 12,13-oxirane moiety was absent in the NMR spectra of 5. Instead, resonances for two methylenes at δC 35.9 (C-12) and δC 20.1 (C-13) were observed in the 13C NMR data for 5. This was further supported by the 2D NMR data (Figure 1), incorporating 1H−1H COSY cross-peaks of H2402



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(MeOH) λmax (Δε) = 200 (−7.2), 220 (−2.2), and 245 (+3.4) nm; for 1 H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS [M + H]+ m/z 411.2189 (calcd for C25H31O5, 411.2171). Emervaridone B (2): colorless crystals; [α]20 D −25.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 238 (3.70) nm; IR (KBr) νmax = 3436, 2972, 2844, 1755, 1641, 1452, 1379, and 1014 cm−1; CD (MeOH) λmax (Δε) = 203 (+8.5), 265 (+4.3), and 310 (−10.1) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS [M + H]+ m/z 393.2063 (calcd for C25H29O4, 393.2066). Emervaridone C (3): white powder; [α]20 D −40.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 236 (3.70) nm; IR (KBr) νmax = 3430, 2950, 2839, 1760, 1707, 1667, 1451, 1382, and 1020 cm−1; CD (MeOH) λmax (Δε) = 200 (−0.83), 251 (+7.3), and 310 (−3.8) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS ion peak at [M + H]+ m/z 409.2016 (calcd for C25H29O5, 409.2015). Varioxiranediol A (5): white powder; [α]20 D +29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 203 (3.8) and 220 (0.9) nm; IR (KBr) νmax = 3492, 3432, 2959, 2866, 1752, 1602, 1487, 1432, 1074, 1039, and 767 cm−1; CD (MeOH) λmax (Δε) = 207 (−0.83), 236 (+7.3), and 300 (−3.1) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS m/z 289.1411 [M + Na]+ (calcd for C15H22O4Na, 289.1416). Varioxiranediol B (6): white powder; [α]20 D +35 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 203 (3.6) and 220 (0.8) nm; IR (KBr) νmax= 3292, 2959, 2928, 2868, 1600, 1485, 1361, 1268, 1049, 928, and 767 cm−1; CD (MeOH) λmax (Δε) = 207 (−0.83), 236 (+7.3), and 300 (−3.1) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS m/z 289.1411 [M + Na]+ (calcd for C15H22O4Na, 289.1416). Crystallographic data for emervaridone A (1): C25H30O5, M = 410.49, a = 11.4327(2) Å, b = 12.1836(2) Å, c = 14.6589(3) Å, α = 90°, β = 90°, γ = 90°, V = 2041.86(6) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.742 mm−1, 11 295 reflections measured, 3493 independent reflections (Rint = 0.0388). The final R1 values were 0.0330 (I > 2σ(I)). The final wR(F2) values were 0.0879 (I > 2σ(I)). The final R1 values were 0.0332 (all data). The final wR(F2) values were 0.0881 (all data). The goodness of fit on F2 was 1.082. Flack parameter = 0.06(7). Crystallographic data for emervaridone B (2): C25H28O4, M = 392.47, a = 12.2820(2) Å, b = 12.44920(10) Å, c = 13.3615(2) Å, α = 90°, β = 103.8280(10)°, γ = 90°, V = 1983.78(5) Å3, T = 100(2) K, space group P21, Z = 4, μ(Cu Kα) = 0.702 mm−1, 15 299 reflections measured, 6501 independent reflections (Rint = 0.0855). The final R1 values were 0.0681 (I > 2σ(I)). The final wR(F2) values were 0.1795 (I > 2σ(I)). The final R1 values were 0.0868 (all data). The final wR(F2) values were 0.1923 (all data). The goodness of fit on F2 was 1.035. Flack parameter = −0.19(18). The crystallographic data for 1 (deposition no. CCDC 1525519) and 2 (deposition no. CCDC 1525518) have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK (fax: + 44-1223336-033; or e-mail: [email protected]). ECD Calculations. The theoretical calculations of new compounds 1−3, 5, and 6 were performed using Gaussian 09.19 Conformational analysis was initially conducted by using Maestro in Schrödinger 2010 conformational searching, together with the OPLS_2005 molecular mechanics methods. The optimized conformation geometries and thermodynamic parameters of all conformations were provided. The OPLS_2005 conformers were optimized at the B3LYP/6-31G(d) level. The theoretical calculation of ECD was performed using TDDFT at the B3LYP/6-311++G** level in MeOH with the PCM model. The calculated ECD curves were generated using SpecDis 1.51. Rvel.20 Absolute Configurations of 10,11-Diols for 5 and 6. A 1.0 mg amount of compound 5 and 1.1 mg of Mo2(OAc)4 were dissolved in dry DMSO and then recorded immediately for the first induced ECD spectra. The additional induced ECD spectra were recorded every 10 min until reaching the stationary state. The absolute configuration of

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with a PerkinElmer 341 polarimeter. UV, CD, and FT-IR spectra were acquired by using a Varian Cary 50, a JASCO-810 CD spectrometer, and a Bruker Vertex 70 instrument, respectively. 1D and 2D NMR data were recorded on a Bruker AM-400 spectrometer, and the 1H and 13C NMR chemical shifts were referenced to the solvent or solvent impurity peaks for CDCl3 (δH 7.26 and δC 77.16) and CD3OD (δH 3.31 and δC 49.0). High-resolution electrospray ionization mass spectrometry (HRESIMS) was performed in the positive ion mode with a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer. The singlecrystal X-ray diffraction experiments were carried out with a Bruker APEX DUO diffractometer using graphite-monochromated Cu Kα radiation. Semipreparative HPLC was carried out on a Dionex Ultimate 3000 with a UV detector using an RP-C18 column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18). Column chromatography (CC) was performed using silica gel (100−200 and 200−300 mesh), RP-C18 gel (50 μm, Merck Co. Ltd., Germany), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Thin-layer chromatography (TLC) was performed with silica gel 60 F254 and RP-C18 F254 plates. Fractions were monitored by TLC, and spots were visualized by spraying heated silica gel plates with 10% H2SO4 in EtOH. Fungus. The fungus Emericella sp. TJ29 was isolated from the root of the plant Hypericum perforatum collected from the Shennongjia areas of Hubei Province, China. The isolated fungus was identified by one of the authors (J.W.) based on morphology and sequence analysis of the ITS region of the rDNA (GenBank accession no. KY346979). A voucher fungal strain was deposited in the culture collection of Tongji Medical College, Huazhong University of Science and Technology. Antibacterial Screening in the Fungal Metabolites for Further Study. Five drug-resistant microbial pathogens (MRSA ATCC 43300, E. faecalis ATCC 29212, ESBL-producing E. coli ATCC 35218, P. aeruginosa ATCC 15442, and K. pneumoniae ATCC 700603) were used to screen the ethyl acetate extract at a fixed concentration (100 μg/mL) in vitro. The obtained sample exhibiting potential antibacterial activities was further fractionated by RP-C18 silica gel CC eluted with MeOH−H2O (20%, 40%, 60%, 80%, and 100%) to afford five fractions, which were tested using the broth microdilution method to determine the MICs. The results showed that the extract (60% MeOH in H2O) exhibited the best antibacterial activity against ESBLproducing E. coli with an MIC value of 32 μg/mL. Fermentation, Extraction, and Isolation. The Emericella sp. TJ29 strain was cultured on potato dextrose agar (PDA) at 28 °C for 7 days in stationary phase to prepare the seed culture. Then the agar plugs were cut into small pieces and inoculated into 40 Erlenmeyer flasks (1 L), previously sterilized by autoclaving, each containing 200 g of rice and 200 mL of distilled water. All flasks were incubated at 28 °C for 30 days. Following incubation, the growth of cells was stopped by adding 250 mL of EtOAc to each flask, and the culture was homogenized. The suspension was extracted repeatedly with EtOAc (4 × 8 L), and the organic solvent was evaporated to dryness under vacuum to yield the crude extract (80.6 g), which was chromatographed on RP-C18 silica gel CC eluted with MeOH−H2O (20%, 40%, 60%, 80%, and 100%) to afford five fractions (Fr.1−Fr.5). Fr.3 (14 g) was further fractionated on a silica gel column eluted with CH2Cl2− MeOH (20:1−0:1) to get six major fractions (Fr.3.1−Fr.3.6) based on TLC analysis. Fr.3.2 (2.1 g) was further separated by Sephadex LH-20 (MeOH) and semipreparative HPLC (MeCN−H2O, 50%) to afford compounds 1 (15.5 mg; tR 12.5 min; 2 mL/min) and 2 (20.2 mg; tR 10.5 min; 2 mL/min). Fr.3.3 (2.8 g) was further separated by repeated semipreparative HPLC (MeCN−H2O, 52%; 2 mL/min), and two peaks eluting at 15, 17, and 20 min were identified as compounds 3 (22.4 mg), 4 (19.8 mg), and 8 (28.5 mg), respectively. Fr.3.4 (1.5 g) was purified by repeated semipreparative HPLC (MeOH−H2O, 60%; 2 mL/min) to yield compounds 5 (12.5 mg; tR 17.2 min), 6 (8.1 mg; tR 23.0 min), and 7 (18.8 mg; tR 15.0 min). Emervaridone A (1): colorless crystals; [α]20 D −22.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 236 (3.64) nm; IR (KBr) νmax = 3449, 2966, 1763, 1708, 1665, 1623, 1440, 1372, and 1243 cm−1; CD 2403



Article

the 10,11-diol for compound 5 was demonstrated from the observed Cotton effects at around 310 nm in their induced ECD spectra. Moreover, compound 6 was treated with the same procedures as those of compound 5. Biological Assay Protocols: Strains, Media, and Antibiotics. The test strains were obtained from the ATCC: methicillin-resistant Staphylococcus aureus (MRSA) ATCC43300; Staphylococcus epidermidis ATCC 29887; ESBL-producing Escherichia coli ATCC 35218; Pseudomonas aeruginosa ATCC 15542; Klebsiella pneumoniae ATCC 700603. The reference compounds for the tests were recommended by the National Committee for Clinical Laboratory Standards:21 vancomycin (Sigma, cat # 861987); chloramphenicol (Sigma, cat # 1107300); amikacin (Sigma, cat # 1019508); ceftriaxone (Sigma, cat # 1098184). All the investigated compounds 1−8 were ≥95% pure (HPLC, wavelength = 210 nm). All compounds were dissolved in DMSO as 20 mg/mL stock solutions. Determination of Minimum Inhibitory Concentrations. Determination of the MICs was performed according to our previously reported broth microdilution method.7 In brief, the inoculum was standardized to approximately 5 × 105 CFU/mL. The plates were incubated at 37 °C for 16 h, and MIC values were recorded as the lowest concentration of antibiotic at which no visible growth of bacteria was observed. Each experiment was performed three times. Time−Kill Curves of Compounds 1 and 5. ESBL-producing E. coli was grown in Mueller-Hinton broth (MHB) overnight, and this culture was used to inoculate fresh MHB (5 × 105 CFU/mL). Inoculated media was aliquoted (3 mL) into culture tubes, and varied concentrations of compounds 1 and 5 were added; untreated inoculated media served as the control. Tubes were incubated at 37 °C with shaking. Samples were taken at 2, 4, 6, 8, 12, and 24 h time points, serially diluted in fresh MHB, and plated on tryptic soy agar. Plates were incubated at 37 °C overnight, and the number of colonies was enumerated. To confirm the results, the time−kill assays for each experiment were performed at least three times; the data are represented as mean data or standard deviation (SD). Cytotoxicity Assays. The normal human lung epithelial cells (BEAS-2B cells) were cultured in RPMI-1640 or Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) in a humidified atmosphere containing 5% CO2 at 37 °C. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA).22 Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before addition of the test compound, both with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well and the incubation continued at 37 °C for 4 h. The cells were lysed with 100 μL of 20% sodium dodecyl sulfate−50% dimethylformamide after removal of 100 μL of medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated with Reed and Muench’s method.23 Statistical Analysis. Statistical analysis of the data was carried out with Graph Pad Prism 4.0 software. The data were expressed as the means ± SD. Values were analyzed with SPSS version 12.0 software by one-way analysis of variance (ANOVA), and p < 0.05 was considered statistically significant.

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1D and 2D NMR, HRESIMS, UV, and IR spectra of compounds 1−3, 5, and 6 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: 86-27-83692892. Fax: 86-27-83692762. E-mail: [email protected] (J. Wang). *Tel: 86-27-82609207. E-mail: [email protected] (Y. Xue). *Tel: 86-27-83692892. Fax: 86-27-83692762. E-mail: [email protected] (Y. Zhang). ORCID

Yan He: 0000-0003-2836-1767 Zhengxi Hu: 0000-0002-1247-5615 Junjun Liu: 0000-0001-9953-8633 Yongbo Xue: 0000-0001-9133-6439 Yonghui Zhang: 0000-0002-7222-2142 Author Contributions §

Y. He, Z. Hu, and Q. Li contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is financially supported by the Program for New Century Excellent Talents in University, State Education Ministry of China (NCET-2008-0224), and the National Natural Science Foundation of China (Nos. 81573316, 31370372, 31270395, 81573316, 31570361, 31670354, 21502057, and 81641129).

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00077. 2404



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(16) He, Y.; Hu, Z.; Sun, W.; Li, Q.; Li, X. N.; Zhu, H.; Huang, J.; Liu, J.; Wang, J.; Xue, Y.; Zhang, Y. J. Org. Chem. 2017, 82, 3125− 3131. (17) Su, Z.; Yeagley, A. A.; Su, R.; Peng, L.; Melander, C. ChemMedChem 2012, 7, 2030−2039. (18) Harris, T. L.; Worthington, R. J.; Melander, C. Angew. Chem., Int. Ed. 2012, 51, 11254−11257. (19) 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.; Keith, T.; 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 B.01; Gaussian, Inc.: Wallingford, CT, 2010. (20) Glendening, E. D.; Reed, A. E.; Carpenter, E. J.; Weinhold, F. NBO Version 3.1; Gaussian, Inc.: Wallingford, CT, 2009. (21) CLSI Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline, document M26-A, Clinical and Laboratory Standards Institute, Wayne, PA, 1999. (22) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991, 83, 757−766. (23) Reed, L. J.; Muench, H. Am. J. Epidemiol. 1938, 27, 493−497.

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