Helvolic Acid Derivatives with Antibacterial Activities against

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Helvolic Acid Derivatives with Antibacterial Activities against Streptococcus agalactiae from the Marine-Derived Fungus Aspergillus fumigatus HNMF0047 Fan-Dong Kong,†,⊥ Xiao-Long Huang,‡,⊥ Qing-Yun Ma,† Qing-Yi Xie,† Pei Wang,† Peng-Wei Chen,† Li-Man Zhou,† Jing-Zhe Yuan,† Hao-Fu Dai,† Du-Qiang Luo,*,§ and You-Xing Zhao*,†

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†

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, China ‡ Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou 570228, China § College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China S Supporting Information *

ABSTRACT: Streptococcus agalactiae is a hazardous pathogen that can cause great harm to humans and fish. In the present study, the known fungal metabolite helvolic acid (10), seven new helvolic acid derivatives named 16-O-deacetylhelvolic acid 21,16-lactone (2), 6-O-propionyl6,16-O-dideacetylhelvolic acid 21,16-lactone (3), 1,2-dihydro-6,16-Odideacetylhelvolic acid 21,16-lactone (4), 1,2-dihydro-16-O-deacetylhelvolic acid 21,16-lactone (5), 16-O-propionyl-16-O-deacetylhelvolic acid (6), 6-O-propionyl-6-O-deacetylhelvolic acid (7), and 24-epi6β,16β-diacetoxy-25-hydroxy-3,7-dioxo-29-nordammara-1,17(20)diene-21,24-lactone (9), and two known ones (1 and 8) were isolated from the marine-derived fungus Aspergillus fumigatus HNMF0047 obtained from an unidentified sponge from Wenchang Beach, Hainan Province, China. The structures and the absolute configurations of the new compounds were unambiguously elucidated by spectroscopic data and electronic circular dichroism (ECD) spectroscopic analyses along with quantum ECD calculations. In addition, the spectroscopic data of compound 1 are reported here for the first time, the configuration of C-24 of known compound 8 was revised based on comparison of its ROESY data with its C-24 epimer 9, and the absolute configuration of 8 was also determined for the first time. Compounds 6, 7, and 10 showed stronger antibacterial activity than a tobramycin control against S. agalactiae with MIC values of 16, 2, and 8 μg/mL, respectively. was found, and it soon won clinical acceptance as an antibiotic against Staphylococcus aureus.10 Fusidane-type antibiotics have recently attracted renewed attention for their lack of crossresistance with other commonly used antibiotics. A recent phase II clinical trial of fusidic acid against acute bacterial skin and skin structure infections in the United States showed that it exhibited comparable efficacy to the positive control linezolid.11 Recently, the biosynthetic pathway of helvolic acid was successfully elucidated, which led to the isolation of 21 derivatives.8 Our previous research on secondary metabolites from marine fungi has led to the isolation and identification of a series structurally new and biologically active metabolites.12,13 In the course of our ongoing search, Aspergillus f umigatus HNMF0047 was isolated and identified from an unidentified sponge from the beach of Wenchang, Hainan Province, in

Streptococcus agalactiae, also known as group B streptococcus (GBS), is an important human, livestock, and fish-borne pathogen. This bacterium is distributed throughout the world and can infect a variety of freshwater and marine fish.1,2 Due to high morbidity and mortality, the global economic losses caused by it are very serious each year.3 S. agalactiae can also cause great harm to human health.1,4 It parasitizes the maternal reproductive tract and can cause infant infections. It can also cause postpartum infections, bacteremia, endocarditis, skin and soft tissue infections, and osteomyelitis. Fusidane-type antibiotics, such as helvolic acid5 and fusidic acid,6 represent a class of microbial metabolites from microorganisms with unique tetra- or pentacyclic skeletons. Among them, only streptoseolactone was found from an actinomycete,7 and all of the rest are fungal metabolites.8 The most attractive and representative bioactivity of this class of compounds is their potent activity against Gram-positive bacteria.9 As early as the 1960s, the successful use of fusidic acid in the treatment of staphylococcal infections in humans © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 13, 2018

A

DOI: 10.1021/acs.jnatprod.8b00382 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

identical to that of streptoseolactone, as confirmed by detailed interpretation of the 2D NMR data. The large J-value (12.4 Hz) of H-4/H-5 (Table 1) suggested that H-4 and H-5 adopt a trans-diaxial orientation. ROESY correlations of H3-28/H-5/H3-29/H-13/H-16 and H3-28/H-6 (Figure 2) suggested the cofacial relationship of these protons, and they were arbitrarily assigned as α-oriented. ROESY correlations of H-4/H3-19/H-9/H3-18 (Figure 2) indicated that these protons were β-oriented. The absolute configuration was established by electronic circular dichroism (ECD) quantum chemical calculations in Gaussian 03. 13 The experimental and calculated ECD spectra for (4S,5S,6S,8S,9S,10R,13R,14S,16S)-1 showed agreement (Figure 3), consistent with the absolute configuration of helvolic acid. Based on the above, the structure of compound 1, named 6,16-O-dideacetylhelvolic acid 21,16-lactone, was elucidated. The molecular formula of compound 2 was established as C31H40O6 by HRESIMS, indicating an additional C2H2O unit in comparison with compound 1. The NMR spectra of 2 were closely related to those of 1, with the main differences being the appearance of signals for an additional acetyl group. The location of an acetoxy group at C-6 in 2 was implied by the fact that the H-6 proton signal was deshielded by 1.25 ppm relative to that of compound 1. An HMBC correlation from H-6 to the carbonyl carbon of this acetoxy group confirmed this deduction. Thus, compound 2 was elucidated as 16-Odeacetylhelvolic acid 21,16-lactone. Compound 3 exhibited a prominent protonated molecule peak at m/z 523.3053 [M + H]+ in the HRESIMS spectrum, suggesting a molecular formula of C32H42O6, with an additional methylene group compared to 2. The 13C NMR data of 3 were also quite similar to those of 2, except for the presence of an additional methylene signal (δC 27.6) that correlated to a quartet methylene proton signal (δH 2.37) in the HSQC spectrum, consistent with the presence of a propionyl group in place of the acetyl group in 2. Thus, compound 3 was assigned as 6-O-propionyl-6,16-O-dideacetylhelvolic acid 21,16-lactone. The molecular formula of compound 4 was determined as C29H40O5 by HRESIMS analysis, with a difference of 2 Da more than that of compound 1. The spectroscopic data of 4 were in accordance with those of 1, except that the Δ1 double bond was replaced by a pair of neighboring methylenes, as corroborated by COSY correlations between H2-1 and H2-2 and HMBC correlations from H3-19 to C-1. The relative configuration of 4 was determined to be the same as 1 based on the large J-value (12.5 Hz) of H-4/H-5 resulting from a trans-diaxial coupling, combined with the key cross-peaks of H4/H3-19/H-9/H-18, H3-28/H-6, and H-5/H3-29/H-13/H-16 displayed in the ROESY spectrum (Figure 2). Thus, compound 4 was identified as 1,2-dihydro-6,16-O-dideacetylhelvolic acid 21,16-lactone. Compound 5 exhibited a protonated molecule peak at m/z 511.3051 [M + H]+ in the HRESIMS spectrum, suggesting a molecular formula of C31H42O6, with an additional acetyl group compared to 4. Their NMR data were also quite similar, except for the presence of additional signals due to an acetyl group (δC/H 169.0, 20.9/2.09), as well as a distinctive deshileding of the H-6 signal (δH 5.29) compared to that of 4. These data led to a deduction that one acetoxy group was located at C-6, as confirmed by COSY correlation of H-5 (δH 2.03) with H-6, which correlated with the acetoxy carbonyl carbon (δC 169.0) in the HMBC spectrum. Consequently,

China. Subsequent chemical investigation of the EtOAc extract of the fermentation broth led to the identification of helvolic acid (10)5 and seven new helvolic acid derivatives (2−7 and 9) along with two known ones (114 and 815). Among them, helvolic acid and compounds 6 and 7 showed potent antibacterial activities against S. agalactiae. Herein, the isolation, structure elucidation, and bioactivities of these compounds are described.

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RESULTS AND DISCUSSION Compound 1, an amorphous, white powder, was previously reported as a semisynthetic compound from helvolic acid in a Japanese patent in 1966,14 without any spectroscopic or bioactivity data supplied for reference. It was determined to have a molecular formula of C29H38O5 with 11 degrees of unsaturation, based on its HRESIMS data with a [M + H]+ ion peak at m/z 467.2790. Analysis of the 1H and 13C NMR spectra, with the aid of HSQC data, revealed the presence of two keto carbonyls including an α,β-unsaturated one, one α,βunsaturated lactone carbonyl, six olefinic carbons with three protonated, six sp3 methines with two oxygenated, five sp3 methylenes, six methyls, and three nonprotonated sp3 carbons. The double-bond equivalents of 1 were calculated to be 11. The presence of three olefinic functionalities and three carbonyl groups indicated that compound 1 has a pentacyclic ring system. The above NMR data are similar to those reported for streptoseolactone,7 suggesting that compound 1 also has a steroidal skeleton as in 16-deacetylfusidic acid γ-lactone. The main NMR differences between these compounds are the signals for an α,β-unsaturated ketone carbon and a 1,2disubstituted CC double bond, as well as the lack of signals for an oxymethine and two methylenes in 1 compared to streptoseolactone. In the HMBC spectrum of 1, correlations from both the mutually coupled protons H-1 and H-2 to the ketone carbonyl C-3, from H3-28 to C-3, and from H3-19 to C1 were observed, indicating a CH(1)−CH(2)−CO(3) fragment. The remaining substructure of 1 was found to be B



Article

Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 1−4 in CDCl3 1 position

δC

2 δH (J in Hz)

δC

3 δH (J in Hz)

δC

4 δH (J in Hz)

δC

δH (J in Hz)

1

158.1, CH

7.32, d (10.2)

157.0, CH

7.31, d (10.0)

156.9, CH

7.31, d (10.2)

33.9, CH2

2

127.6, CH

5.83, d (10.2)

128.0, CH

5.88, d (10.0)

128.1, CH

5.88, d (10.2)

37.4, CH2

3 4 5 6 7 8 9 10 11

202.6, C 40.0, CH 47.2, CH 73.2, CH 213.3, C 52.4, C 41.7, CH 38.5, C 24.0, CH2

12

22.1, CH2

13 14 15

43.6, CH 52.7, C 36.7, CH2

16 17 18 19 20 21 22

81.9, CH 168.1, C 20.7, CH3 28.2, CH3 124.9, C 176.5, C 24.0, CH2

23

27.3, CH2

24 25 26 27 28 29 6a/6b

123.1, CH 133.0, C 25.8, CH3 17.9, CH3 12.6, CH3 17.9, CH3

3.00, dq (12.4, 6.8) 2.15, overlap 4.01, br s

2.61, dd (13.1, 3.2) 1.97, 1.61, 2.16, 1.75, 2.92,

m m overlap m br d (12.5)

2.44, dd (15.6, 11.2) 1.74, overlap 4.91, dd (11.2, 4.1) 0.82, s 1.50, s

2.34, 2.18, 2.18, 2.18, 5.08,

m overlap overlap overlap m

1.65, 1.23, 1.19, 1.57,

s s d (6.8) s

201.5, C 40.4, CH 47.1, CH 73.2, CH 208.5, C 53.1, C 42.0, CH 38.3, C 23.9, CH2 22.1, CH2 43.5, CH 52.4, C 36.4, CH2 81.8, CH 167.6, C 20.4, CH3 27.6, CH3 125.1, C 176.3, C 24.2, CH2 27.3, CH2 123.1, CH 133.2, C 25.8, CH3 17.9, CH3 13.2, CH3 18.4, CH3 169.0, C 20.8, CH3

2.73, m 2.30, br d (12.6) 5.26, br s

2.54, br d (12.5) 1.99, 1.64, 2.20, 1.77, 2.94,


2.47, dd (13.2, 10.6) 1.74, overlap 4.92, br d (10.6) 0.80, s 1.41, s

2.36, 2.20, 2.20, 2.20, 5.09,


1.66, 1.58, 1.26, 1.29,

s s d (6.8) s

201.5, C 40.5, CH 47.2, CH 73.1, CH 208.6, C 53.1, C 42.1, CH 38.3, C 23.9, CH2 22.1, CH2 43.5, CH 52.5, C 36.5, CH2 81.7, CH 167.5, C 20.5, CH3 27.7, CH3 125.1, C 176.2, C 24.3, CH2 27.3, CH2 123.2, CH 133.2, C 25.8, CH3 17.9, CH3 13.2, CH3 18.3, CH3 172.5, C 27.6, CH2 9.2, CH3

2.09, s

compound 5 was determined to be 1,2-dihydro-16-Odeacetylhelvolic acid 21,16-lactone. Based on similar J values, ROESY data, and experimental ECD curves, compounds 2−5 are assigned the same absolute configurations as for compound 1. Compound 6 was assigned the molecular formula C34H46O8 from HRESIMS data, requiring 12 degrees of unsaturation. The 13C NMR spectra for 6 showed 34 carbon signals, which showed great similarity to those of helvolic acid (10), coisolated with 6. The key NMR differences between them were the appearance of additional signals for a methylene, which correlated with a triplet methyl in the COSY spectrum. In the HMBC spectrum, correlations from both protons of this methylene and H-16 to an ester carbonyl were observed, indicating that a propionyloxy rather than an acetoxy group as in helvolic acid was located at C-16. The remaining substructure was determined to be the same as helvolic acid by detailed interpretation of the 2D NMR data (Figure 1). The relative configuration of the stereocenters in the tetracyclic ring

2.73, m 2.31, overlap 5.28, br s

2.55, br d (13.3) 1.99, 1.64, 2.17, 1.75, 2.95,


2.47, dd (13.2, 11.2) 1.75, overlap 4.92, br d (11.2) 0.81, s 1.41, s

2.37, 2.22, 2.22, 2.22, 5.09,

overlap overlap overlap overlap m

1.67, 1.59, 1.26, 1.29,

s s d (6.5) s

213.4, C 41.9, CH 46.3, CH 73.0, CH 213.9, C 52.6, C 41.5, CH 35.3, C 22.8, CH2 22.1, CH2 43.1, CH 52.7, C 36.6, CH2 82.0, CH 168.2 C 20.8, CH3 23.9, CH3 124.8, C 176.5, C 24.2, CH2 27.4, CH2 123.2, CH 133.1, C 25.8, CH3 17.9, CH3 12.5, CH3 17.0, CH3

1.85, 1.70, 2.50, 2.50,

overlap overlap overlap overlap

2.88, overlap 1.88, dd (12.5, 2.2) 3.96, d (2.2)

2.55, dd (10.3, 3.3) 1.88, 1.52, 2.12, 1.75, 2.90,

m m m m overlap

2.52, overlap 1.73, overlap 4.92, br d (11.0, 4.3) 0.85, s 1.36, s

2.35, 2.21, 2.20, 2.20, 5.09,


1.67, 1.58, 1.11, 1.35,

s s d (6.7) s

2.37, overlap 1.14, overlap

system was determined to be the same as those of compounds 1−5 on the basis of their similar ROESY data (Figure 2) and vicinal coupling constants (Table 2). Further, the ECD spectra of 6 and helvolic acid were quite similar (Figure 4), with a positive Cotton effect (CE) around 234 nm and negative one around 330 nm, leading to the analogous absolute configuration. On the basis of the HRESIMS data, the molecular formula of compound 7 was established to be the same as that of 6. The 1H and 13C NMR spectroscopic data of 7 were nearly identical to those of 6, suggesting that their structures were closely related. However, in the HMBC spectrum of 7, H-6 and H-16 showed correlations to the propionyloxy carbonyl and the acetoxy carbonyl, respectively, rather than the other way around as in compound 6. These spectroscopic features suggested that compound 7 was also a helvolic acid derivative with a propionyloxy unit replacing the corresponding acetoxy substituent at C-6 in helvolic acid. C



Article

Figure 3. Experimental ECD curves for compounds 1−5 and calculated ECD curve for 1.

from the axial protons H-24/H-22a/H-13.15 However, seen from the energy-minimized 3D model with an α-oriented H-24 proton (Figure 5, model for compound 9), the observation of an NOE correlation between the axial protons H-22a and H-13 was scarcely possible due to the distance between them. Further, in the ROESY spectrum (Figure 5) of 8, correlations between axial protons H-22a (δH 2.94) and H-24 (δH 3.92) and H-12a (δH 1.86) suggested a cofacial relationship of H-24 and H-12a, opposite that reported. Although several methods were attempted to crystallize 8 to confirm the orientation of H24, they all failed. On the basis of the HRESIMS ion at m/z 607.2881 [M + Na]+, the molecular formula of 9 was established as C33H44O9, matching that of 8. The NMR data of 9 showed a great similarity to those of 8, with the main differences observed in the 13C NMR data being that C-21 was slightly shielded by 3.1 ppm, while C-17, C-20, and C-22 were slightly deshielded by 3.0, 1.5, and 1.9 ppm, respectively (Table 2). Based on the

Figure 1. Key COSY (bold lines) and HMBC (→) correlations of compounds 1, 5, 6, and 9.

Comparison of the NMR data (Table S1, Supporting Information) of 8 with those reported revealed that it was the known compound 6β,16β-diacetoxy-25-hydroxy-3,7-dioxo-29nordammara-1,17(20)-diene-21,24-lactone,15 which was further confirmed by analysis of its 2D NMR spectra (Supporting Information). The relative configurations of all of the stereocenters except C-24 were assigned to be the same as those of 6 and 7 based on analysis of the ROESY data (Figure 5). As the stereogenic carbon C-24 was far from the others in the structure, H-24 of compound 8 was assigned to be αoriented in the previous report based on NOESY correlations

Figure 2. Key ROESY correlations of compounds 1 and 4−6. D



Article

Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 5−7 and 9 in CDCl3 5 position

δC

1

33.2, CH2

2

36.7, CH2

3 4 5 6 7 8 9 10 11

212.6, C 42.2, CH 46.2, CH 73.0, CH 208.7 C 52.7, C 41.6, CH 35.4, C 22.6, CH2

12

22.2, CH2

13 14 15

43.1, CH 53.1, C 36.4, CH2

16 17 18 19 20 21 22

81.8, CH 167.7, C 20.6, CH3 23.8, CH3 125.0, C 176.3, C 24.3, CH2

23

27.4, CH2

24 25 26 27 28 29 6a

123.2, CH 133.1, C 25.8, CH3 17.9, CH3 13.4, CH3 17.6, CH3 169.0, C 20.9, CH3

6 δH (J in Hz)

1.93, 1.70, 2.50, 2.50,

overlap overlap overlap overlap

2.54, overlap 2.03, br d (12.1) 5.29, br s

2.54, overlap 1.90, 1.53, 2.18, 1.77, 2.92,


2.55, overlap 1.70, overlap 4.92, dd (11.2, 4.5) 0.81, s 1.28, s

2.35, 2.21, 2.20, 2.20, 5.10,


1.67, 1.59, 1.16, 1.41,

s s d (7.0) s

2.09, s

δC

7 δH (J in Hz)

9 δH (J in Hz)

δC

δH (J in Hz)

157.4, CH

7.31, d (10.2)

157.4, CH

7.31, d (10.0)

157.1, CH

7.29, d (10.1)

128.0, CH

5.86, d (10.2)

128.0, CH

5.86, d (10.0)

128.1, CH

5.87, d (10.1)

201.5, C 40.5, CH 47.3, CH 73.9, CH 208.9, C 52.8, C 41.8, CH 38.3, C 24.1, CH2 25.9, CH2 49.6, CH 46.7, C 40.9, CH2 73.3, CH 147.8, C 18.0, CH3 27.7, CH3 130.3, C 173.7, C 28.8, CH2 28.5, CH2 122.9, CH 133.1, C 25.9, CH3 17.9, CH3 13.3, CH3 18.5, CH3 169.1, C 20.9, CH3

2.77, dq (13.1, 6.2) 2.26, br d (13.2) 5.23, br s

2.61, dd (13.5, 3.0) 1.96, 1.58, 2.42, 1.82, 2.58,

m m m m br d (13.0)

0.92, s 1.45, s

2.50, 2.42, 2.14, 2.08, 5.11,

m m m m t (7.3)

1.69, 1.61, 1.28, 1.18,

s s d (6.8) s

173.7, C 27.5, CH2 9.2, CH3

201.6, C 40.6, CH 47.4, CH 73.8, CH 209.1, C 52.8, C 41.8, CH 38.3, C 24.1, CH2 26.0, CH2

2.24, dd (15.4, 8.6) 1.91, br d (15.4) 5.90, br d (8.4)

49.5, CH 46.7, C 40.8, CH2 73.6, CH 147.6, C 18.1, CH3 27.7, CH3 130.4, C 173.4, C 28.7, CH2 28.5, CH2 122.9, CH 133.1, C 25.9, CH3 17.9, CH3 13.3, CH3 18.5, CH3

2.77, dq (13.2, 6.7) 2.27, br d (13.2) 5.25, br s

2.60, dd (13.5, 3.0) 1.96, 1.58, 2.42, 1.82, 2.58,

m m m m br d (12.8)

2.24, dd (15.2, 8.6) 1.91, br d (15.2) 5.88, br d (8.6) 0.92, s 1.44, s

2.50, 2.42, 2.14, 2.08, 5.10,

m m m m t (7.3)

1.69, 1.61, 1.28, 1.18,

s s d (6.8) s

2.11, s

16a 6b/16b

δC

170.4, C 20.7, CH3 172.6, C 27.7, CH2 9.2, CH3

2.23, q (7.6) 1.08, t (7.6)

1.95, s

201.4, C 40.6, CH 47.4, CH 73.8, CH 208.6, C 52.8, C 41.7, CH 38.3, C 24.1, CH2 26.6, CH2 51.5, CH 47.0, C 41.0, CH2 73.6, CH 154.7, C 18.6, CH3 27.7, CH3 125.4, C 164.9, C 24.8, CH2 24.1, CH2 85.3, CH 71.9, C 25.8, CH3 24.3, CH3 13.3, CH3 18.4, CH3 169.0, C 20.9, CH3 169.9, C 20.9, CH3

2.77, dq (12.6, 6.9) 2.28, dd (12.6, 1.0) 5.25, d (1.0)

2.65, dd (12.8, 2.5) 1.61, 1.96, 2.50, 1.85, 2.63,

m m m m m

2.18, dd (7.8, 14.4) 20.6, br d (14.4) 5.98, br d (7.6) 1.00, s 1.45, s

3.11, 2.43, 2.07, 1.75, 4.10,

m m m m dd (3.9, 10.9)

1.28, 1.22, 1.29, 1.18,

s s d (6.9) s

2.12, s 2.12, s

2.38, q (7.6) 1.16, t (7.6)

were in good agreement with those of 6 and 7 (Figure 4), indicating the (4S,5S,6S,8S,9S,10R,13R,14S,16S,24R)-configuration for 8 and the (4S,5S,6S,8S,9S,10R,13R,14S,16S,24S)configuration for 9. In order to confirm that the helvolic acid lactones occur naturally in the fungus rather than being artifacts derived from hydrolyzed or partially hydrolyzed helvolic acid derivatives during the extraction or isolation process, hydrolysis experiments of helvolic acid (10) in MeOH under neutral conditions with normal-phase silica gel present, in acidic (pH 2 and 4) and alkaline conditions (pH 11), were performed (Supporting Information), respectively. The samples were incubated for 24 h at room temperature. HPLC analysis of the reaction products (Figure S78, Supporting Information) showed that the 6- and 12-acetoxy groups of 10 were stable and compounds 1 and 2

above, 9 was deduced to be a stereoisomer of 8 and was most likely an epimer at C-24. Analysis of 2D NMR data (Figure 1) confirmed the planar structure of 9. Analysis of the ROESY spectrum (Figure 5) revealed that the configurations for all stereocenters except for C-24 were the same as those of 8, as well as the Δ17(20) double bond. Thus, the difference between the two compounds could only be the configuration of C-24 relative to those of the other stereocenters. Furthermore, unlike the case of 8, there was no correlation between the two axial protons H-22a (δH 2.43) and H-12a (δH 1.85) in the ROESY spectrum, and instead, a strong correlation between the equatorial proton H-22b (δH 3.11) and H-12a was observed. These data indicated that H-24 was on the face opposite H-12a in 9 and further supported the above H-24 orientation reassignment for 8. The ECD spectra for 8 and 9 E



Article

Table 3. Inhibitory Effects of Compounds 1−10 on S. aureus and S. agalactiae pathogenic bacteria (MIC, μg/mL)

Figure 4. Experimental ECD curves for compounds 6−10.

were not formed in MeOH under neutral conditions with normal-phase silica gel present or under acidic conditions even at pH 2. These data suggested that the isolated helvolic acid lactones are not likely artifacts. Moreover, even under an alkaline condition (pH 11), only the 6-acetoxy group was hydrolyzed and 6-deacetoxyhelvolic acid was obtained and identified (Table S2), which further indicated that hydrolysis of the 16-acetoxy and subsequent lactone formation do not readily occur during the isolation process. The antibacterial activities of all the isolated compounds against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Streptococcus agalactiae were evaluated using the 2-fold dilution assay.8 Compounds 6, 7, 9, and 10 showed antibacterial activity against S. agalactiae with MIC values of 16, 2, 64, and 8 μg/mL (Table 3), respectively (with tobramycin as the positive control, MIC 32 μg/mL), while compounds 6, 7, and 10 also showed antibacterial activity against S. aureus with MIC values of 16, 8, and 16 μg/mL, respectively (tobramycin as the positive control, MIC 1 μg/ mL). All of the compounds were inactive against E. coli and B. subtilis at 128 μg/mL. On the basis of the structural differences between compounds 6, 7, and 10 and compounds 1−5, we could conclude that the presence of a C-21/C-16 lactone ring significantly reduced the antibacterial activity. Remarkably, compound 7, with a propionyloxy replacing the corresponding acetoxy substituent at C-6 in helvolic acid, exhibited stronger antibacterial activity against S. agalactiae than the antibacterial drugs helvolic acid and tobramycin.

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compound

S. agalactiae

S. aureus

1 2 3 4 5 6 7 8 9 10 tobramycin

>128 >128 >128 >128 >128 16 2 >128 64 8 32

>128 >128 >128 >128 >128 16 8 >128 >128 16 1

with tetramethylsilane as an internal standard. ESIMS, HRESIMS, and HREIMS data were recorded with a Micromass Autospec-UltimaTOF, API QSTAR Pulsar 1, or Waters Autospec Premier spectrometer. Semipreparative HPLC was carried out using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). Thinlayer chromatography (TLC) and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham Biosciences), respectively. Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory). The ODS column (30 × 300 mm) used was from Beijing Synthware Glass Factory. The sea salt used was made from the evaporation of seawater collected in Laizhou Bay, China (Weifang HaiHua Yu Feng Chemical Factory). Fungal Material and Fermentation. The fungus HNMF0047 was isolated from an unidentified sponge (HNMF00) from the beach of Wenchang, Hainan Province, China. After grinding, the sample (1 g) was diluted to 10−2 g/mL with sterile H2O, 100 μL of which was deposited on a PDA (200 g of potato, 20 g of glucose, 20 g of agar per liter of seawater collected in Haikou Bay, China) plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and 18S rRNA gene sequences (GenBank accession no. MH101462, Supporting Information). A reference culture of Aspergillus f umigatus HNMF0047 maintained at −80 °C is deposited in our laboratory. The isolate was cultured on slants of PDA medium at 28 °C for 5 days. Plugs of agar supporting mycelium growth were cut and transferred aseptically to 100 × 1000 mL Erlenmeyer flasks each containing 300 mL of liquid medium (20 g of mannitol, 20 g of maltose, 10 g of glucose, 10 g of monosodium glutamate, 3 g of yeast extract, 0.5 g of corn meal, 0.5 g of KH2PO4, 0.3 g of MgSO4, 33 g of sea salt per liter of tap water, pH 7.0). The flasks were incubated at room temperature under static conditions for 30 days. Extraction and Isolation. The cultures (30 L) were filtered through cheesecloth to separate the mycelial mass from the aqueous layer. The filtrate was then extracted three times by shaking with 3fold volumes of EtOAc, while the mycelium was extracted with

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter, and UV spectra were measured on a Beckman DU 640 spectrophotometer. ECD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were obtained on a Nicolet Nexus 470 spectrophotometer as KBr discs. NMR spectra were recorded on a Bruker AV-500 spectrometer

Figure 5. Key ROESY correlations of compounds 8 and 9 (some protons have been omitted for clarity). F



Article

6β,16β-Diacetoxy-25-hydroxy-3,7-dioxo-29-nordammara1,17(20)-dien-21,24-lactone (8): colorless powder; [α]25D −23 (c 0.1, MeOH); [α]25D −17 (MeOH);15 ECD (0.1 mM, MeOH) λmax 329 (−1.15), 249 (+0.12), 207 (−1.97) nm. 24-epi-6β,16β-Diacetoxy-25-hydroxy-3,7-dioxo-29-nordammara-1,17(20)-diene-21,24-lactone (9): colorless powder; [α]25D −27 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 234 (4.21); ECD (0.34 mM, MeOH) λmax 330 (−1.06), 243 (+0.16), 208 (−1.67) nm; IR (KBr) νmax (cm−1) 3446, 2920, 1741, 1665, 1376, 1221 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 607.2881 [M + Na]+ (calcd for C33H44O9Na, 607.2878). Helvolic acid (10): colorless powder; [α]25D −41 (c 0.1, CHCl3); [α]25D −57 (CHCl3) in the literature;5 ECD (0.35 mM, MeOH) λmax 330 (−0.58), 243 (+0.58), 208 (−1.78) nm.

acetone. After removing acetone by evaporation under vacuum, the obtained aqueous acetone solution was extracted three times with equal volumes of EtOAc. The combined EtOAc extracts were dried under vacuum to produce 20.5 g of extract. The EtOAc extract was subjected to a silica gel VLC column, eluting with a stepwise gradient of 0%, 10%, 15%, 20%, 40%, 60%, 80%, and 100% EtOAc in petroleum ether (v/v), to give six fractions (Fr. 1−6). Fraction 3 (903 mg) was applied to octadecyl silane (ODS) silica gel with gradient elution of MeOH−H2O (1:5, 2:3, 3:2, 4:1, 1:0) to yield four subfractions (Fr. 4-1−Fr. 4-4). Fr. 4-3 (64 mg) was subjected to semipreparative HPLC (YMC-pack ODS-A, 5 μm; 10 × 250 mm; 70% MeCN−H2O; 4 mL/min) to give compound 1 (tR 8.1 min; 5.4 mg). Fr. 4-3 (103 mg) was subjected to semipreparative HPLC (YMC-pack ODS-A, 5 μm; 10 × 250 mm; 75% MeCN−H2O; 4 mL/ min) to give compounds 2 (tR 8.0 min; 10.9 mg), 5 (tR 8.8 min; 7.6 mg), and 3 (tR 10.0 min; 3.4 mg). Fraction 5 (1.4 g) was applied to ODS silica gel with gradient elution of MeOH−H2O (1:5, 2:3, 3:2, 4:1, 1:0) to yield five subfractions (Fr. 5-1−Fr. 5-5). Fr. 5-3 (53 mg) was subjected to semipreparative HPLC (YMC-pack ODS-A, 5 μm; 10 × 250 mm; 70% MeCN−H2O; 4 mL/min) to give compounds 10 (tR 5.4 min; 9.4 mg), 6 (tR 7.9 min; 2.3 mg), 7 (tR 8.7 min; 2.1 mg), and 4 (tR 9.2 min; 4.7 mg). Fr. 5-2 (205 mg) was subjected to Sephadex LH-20 chromatography with CHCl3−MeOH (1:1) to afford three subfractions (Fr. 5-2-1−Fr. 5-2-3). Fr. 5-2-2 was subjected to semipreparative HPLC (YMC-pack ODS-A, 5 μm; 10 × 250 mm; 65% MeCN−H2O; 4 mL/min) to give compounds 8 (tR 9.4 min; 6.7 mg) and 9 (tR 11.1 min; 1.8 mg). 6,16-O-Dideacetylhelvolic acid 21,16-lactone (1): colorless powder; [α]25D −61 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.43) nm; ECD (0.40 mM, MeOH) λmax 330 (−0.38), 248 (−1.39), 218 (+2.81) nm; IR (KBr) νmax (cm−1) 3414, 2930, 1744, 1669, 1452, 1381, 1257; 1H and 13C NMR data, Table 1; HRESIMS m/z 467.2790 [M + H]+ (calcd for C29H39O5, 467.2792). 16-O-Deacetylhelvolic acid 21,16-lactone (2): colorless powder; [α]25D −82 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.21) nm; ECD (0.39 mM, MeOH) λmax 328 (−0.63), 247 (−1.28), 218 (+2.51) nm; IR (KBr) νmax (cm−1) 2931, 1749, 1674, 1452, 1377, 1214; 1H and 13C NMR data, Table 1; HRESIMS m/z 509.2894 [M + H]+ (calcd for C31H41O6, 509.2898). 6-O-Propionyl-6,16-O-dideacetylhelvolic acid 21,16-lactone (3): colorless powder; [α]25D −142 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (2.99) nm; ECD (0.19 mM, MeOH) λmax 330 (−0.85), 248 (−1.29), 218 (+2.10) nm; IR (KBr) νmax (cm−1) 2943, 1753, 1674, 1453, 1377, 1217; 1H and 13C NMR data, Table 1; HRESIMS m/z 523.3053 [M + H]+ (calcd for C32H43O6, 523.3054). 1,2-Dihydro-6,16-O-dideacetylhelvolic acid 21,16-lactone (4): colorless powder; [α]25D −30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (2.95) nm; ECD (0.43 mM, MeOH) λmax 328 (−0.26), 249 (−1.17), 218 (+3.43) nm; IR (KBr) νmax (cm−1) 3414, 2934, 1744, 1703, 1451, 1386, 1261; 1H and 13C NMR data, Table 1; HRESIMS m/z 469.2947 [M + H]+ (calcd for C29H41O5, 469.2949). 1,2-Dihydro-16-O-deacetylhelvolic acid 21,16-lactone (5): colorless powder; [α]25D −47 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (2.91); ECD (0.39 mM, MeOH) λmax 328 (−0.41), 248 (−0.63), 218 (+1.72) nm; IR (KBr) νmax (cm−1) 2931, 1748, 1708, 1454, 1385, 1214; 1H and 13C NMR data, Table 2; HRESIMS m/z 511.3051 [M + H]+ (calcd for C31H43O6, 511.3054). 16-O-Propionyl-16-O-deacetylhelvolic acid (6): colorless powder; [α]25D −99 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.74) nm; ECD (0.34 mM, MeOH) λmax 330 (−0.33), 234 (+0.27), 209 (−0.72) nm; IR (KBr) νmax (cm−1) 3447, 2920, 1742, 1703, 1655, 1452, 1377, 1214; 1H and 13C NMR data, Table 2; HRESIMS m/z 581.3103 [M + H]+ (calcd for C34H45O8, 581.3109). 6-O-Propionyl-6-O-deacetylhelvolic acid (7): colorless powder; [α]25D −63 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.69) nm; ECD (0.34 mM, MeOH) λmax 327 (−0.43), 241 (+0.45), 208 (−1.31) nm; IR (KBr) νmax (cm−1) 3448, 2922, 1740, 1702, 1660, 1451, 1375, 1244; 1H and 13C NMR data, Table 2; 581.3104 [M + H]+ (calcd for C34H45O8, 581.3109).

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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.jnatprod.8b00382.

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HRESIMS, IR, and 2D NMR spectra of compounds 1− 9, the 18S rRNA gene sequence of Aspergillus f umigatus HNMF0047, and the quantum calculation details (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax (D.-Q. Luo): +86-159-31287995. E-mail: [email protected]. *Tel/Fax (Y.-X. Zhao): +86-898-66989095. E-mail: [email protected]. ORCID

You-Xing Zhao: 0000-0002-8107-2510 Author Contributions ⊥

F.-D. Kong and X.-L. Huang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Hainan Province (417256), Natural Science Foundation of China (41606088, 81741157), China Agriculture Research System (CARS-21), Financial Fund of the Ministry of Agriculture and Rural Affairs, P. R. of China (NFZX2018), and Central Public-Interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (17CXTD-15, 1630052016008).

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REFERENCES

(1) Delannoy, C. M.; Crumlish, M.; Fontaine, M. C.; Pollock, J.; Foster, G.; Dagleish, M. P.; Turnbull, J. F.; Zadoks, R. N. BMC Microbiol. 2013, 13, 41−50. (2) Li, L.; Wang, R.; Liang, W.; Huang, T.; Huang, Y.; Luo, F.; Lei, A.; Chen, M.; Gan, X. Fish Shellfish Immunol. 2015, 45, 955−963. (3) Harrison, L. H.; Elliot, J. A.; Dwyer, D. M.; Libonati, J. P.; Ferrieri, P.; Billmann, L.; Anne, S. J. Infect. Dis. 1998, 177, 998−1002. (4) Kim, B. J.; Hancock, B. M.; Del Cid, N.; Bermudez, A.; Traver, D.; Doran, K. S. Microb. Pathog. 2015, 79, 57−60. (5) Chain, E.; Florey, H. W.; Jennings, M. A.; Williams, T. I. Br. J. Exp. Pathol. 1943, 24, 108−119. (6) Godtfredsen, W. O.; Jahnsen, S.; Lorck, H.; Roholt, K.; Tybring, L. Nature 1962, 193, 987. (7) Jiao, R.; Xu, H.; Cui, J. T.; Ge, H.; Tan, R. J. Appl. Microbiol. 2013, 114, 1046−1053.

G



Article

(8) Lv, J. M.; Hu, D.; Gao, H.; Kushiro, T.; Awakawa, T.; Chen, G. D.; Wang, C. X.; Abe, I.; Yao, X. S. Nat. Commun. 2017, 8, 1644− 1653. (9) Von, D. W.; Godtfredsen, W. O.; Rasmussen, P. R. Adv. Appl. Microbiol. 1979, 25, 95−146. (10) Fernandes, P. Cold Spring Harbor Perspect. Med. 2016, 6, a025437. (11) Craft, J. C.; Moriarty, S. R.; Clark, K.; Scott, D.; Degenhardt, T. P.; Still, J. G.; Corey, J. R.; Das, A.; Fernandes, P. Clin. Infect. Dis. 2011, 52, S520−S526. (12) Kong, F. D.; Zhang, R. S.; Ma, Q. Y.; Xie, Q. Y.; Wang, P.; Chen, P. W.; Zhou, L. M.; Dai, H. F.; Luo, D. Q.; Zhao, Y. X. Fitoterapia 2017, 122, 1−6. (13) Kong, F. D.; Ma, Q. Y.; Huang, S. Z.; Wang, P.; Wang, J. F.; Zhou, L. M.; Yuan, J. Z.; Dai, H. F.; Zhao, Y. X. J. Nat. Prod. 2017, 80, 1039−1047. (14) Tsuda, K.; Okuda, S.; Nakayama, Y.; Kunishima, A.; Tabara, K. Jpn. Tokkyo Koho. JP 41022178 B 19661224, 1966. (15) Ssh, A.; Zhuravleva, O. I.; Antonov, A. S.; Kalinovsky, A. I.; Pivkin, M. V.; Menchinskaya, E. S.; Aminin, D. L. Nat. Prod. Commun. 2012, 7, 497−500.

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Helvolic Acid Derivatives with Antibacterial Activities against

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