Introduction

Gliomas remain the most common and high death malignant brain tumors despite advances in therapies including chemotherapy, radiotherapy, and surgical resection1,2,3. Chemotherapy has played a more important role in treatment and prevention of gliomas, because gliomas usually locate at many important brain function areas, which makes the surgical resection extremely difficult. However, so far very few drugs have been approved for treating gliomas including temozolomide (TMZ), carmustine, and lomustine, and only TMZ has been independently used for treatment of gliomas1. Furthermore, the efficacy of TMZ and other current anti-glioma drugs remain unsatisfactory1, 4. Therefore, there is an urgent need to discover lead compounds for development of novel anti-glioma drugs. Bioactive natural products from mangrove-derived actinomycetes are important sources for discovery of drug lead compounds5,6,7. For example, the well-known salinosporamide A actually produced by a mangrove-derived Salinospora strain8 was the first and most advanced marine actinomycete secondary metabolite to be processed for clinical trials for cancer treatment.

During the course of our ongoing project to discover novel anti-glioma agents from marine resources9,10,11,12,13,14,15, ten bacterial strains were isolated from a sample of mangrove soils. An extract prepared from the culture of strain ZQ4BG was found to be the most active against the proliferation of glioma cells with an inhibition of 56.9% for U87MG and 48.9% for U251. This mangrove-derived strain was assigned as Streptomyces sp. ZQ4BG based on its 16S rDNA sequence (Supplementary Information, Fig. S1), which completely (99% identity for a 1404 bp stretch of sequence) matched those of two species of Streptomyces including S. fulvissimus and S. flavofungini (Table S1) in GenBank database. The actinomycete S. flavofungini was previously reported to produce polyene-polyol flavofungins16 and polyol macrolides of desertomycins17.

Polyene-polyol macrolides are usually microbial metabolites whose main structural elements are a continuously conjugated chain of four to seven unsubstituted double bonds connected to a polyol fragment with alternating hydroxy groups. Nystatin, produced by Streptomyces noursei, was the first polyene-polyol macrolide showing antifungal and antibacterial activities18, 19. Amphotericin B, a heptaene-polyol macrolide produced by S. nodosus, is a standard antifungal drug for treating serious deep-seated systemic fungal infections20. Most of the polyene-polyol macrolides discovered since are produced by a variety of terrestrial Streptomyces spp., such as mycoticins from S. ruber 21, faeriefungins from S. griseus var. autotrophicus 19, PK-397 from Streptomyces sp. 87–39722,23,24, nystatins from S. noursei 25, and reedsmycins from Streptomyces sp. CHQ-6426. Recently, polyene-polyol macrolides were also isolated from marine actinomycetes, such as marinisporolides from marine Marinispora sp. CNQ14027 and bahamaolides from marine Streptomyces sp28. Some of these polyene-polyol macrolides mentioned above were found to have significant antifungal, antibacterial, antiviral, and antitumor activities18,19,20,21,22,23,24,25,26,27,28.

A HPLC analysis of the active extract from strain ZQ4BG revealed that this mangrove-derived actinomycete produced rich metabolites with similar UV absorption (λmax) at 365 nm or 330 nm. Further LC/MS analysis of this extract showed a series of [M + Na]+ ions at m/z 673.4, 675.4, 687.4, 689.4, 703.4, and 707.4. The compounds with MS data at 673.4 and 687.4 with UV absorption at 365 nm were match to those of known flavofungins I (1) and II (2), respectively, in AntiBase database29 and references30, 31. The others with m/z 675.4, 689.4, 703.4, and 707.4 with λmax 330 nm did not match any previously reported compounds, prompting us to characterize them further. Therefore, large culture of this isolated mangrove-derived strain ZQ4BG in Gause’s liquid medium resulted in isolation and identification of a series of polyene-polyol macrolides, including new flavofungins III–IX (3–9) and known flavofungins I (1) and II (2) and spectinabilin (10). Flavofungins IV–VIII (4–8) are rare 32-membered polyene-polyol macrolides with a tetrahydrofuran ring, while flavofungin IX (9) is the first example of this type of macrolide with a unique oxepane group. Herein, we report the isolation and culture of strain ZQ4BG, the structural elucidation of these novel flavofungins, and the anti-glioma and antimicrobial active evaluation of these isolated metabolites.

Results and Discussion

An actinomycete strain ZQ4BG which produced rich polyene-polyol macrolides was isolated from mangrove soils and assigned as Streptomyces sp. ZQ4BG based on the result of its 16S rDNA sequence. Culture (total 60 L) of this isolated actinomycete in Gause’s liquid medium resulted in isolation of compounds 1–10 (Fig. 1).

Figure 1
figure 1

Structures of compounds (1–10) isolated from Streptomyces sp. ZQ4BG.

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Compounds 1, 2, and 10 were proved to be known compounds of flavofungins I (1) and II (2)30, 31 and spectinabilin (10)32,33,34 based on their HRESIMS and NMR data (Tables 1 and 2) as well as a comparison to the data of references. Flavofungins I (1) and II (2) were 32-membered polyene-polyol macrolides with a conjugated chain of five unsubstituted double bonds connected to a polyol fragment with eight hydroxy groups alternated. Both flavofungins I (1) and II (2) were reported to have antifungal and antiviral activities19, while spectinabilin (10) was a nitro-substituted polyketide with activity to block the binding of androgen to the ligand-binding domain of androgen receptor34.

Table 1 13C-NMR data of compounds 1–9 (125 MHz, in DMSO-d 6)
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Table 2 1H-NMR data of compounds 1–4 (500 MHz, in DMSO-d 6, J = Hz).
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Compounds 3–9 are new members of flavofungins I (1) and II (2), with only four unsubstituted conjugated double bonds and more oxygenated moieties, and named as flavofungins III–IX (3–9). Flavofungin III (3) is an analogue of flavofungin I (1) with two hydroxy groups substituted at C-10 and C-11. Flavofungins IV–IX (4–9) are rare 32-membered polyene-polyol macrolides with a tetrahydrofuran (THF) ring between C-10 and C-13 for flavofungins IV–VIII (4–8) or an oxepane ring between C-10 and C-15 for flavofungin IX (9). It has been found that new flavofungins III–IX (3–9) might derive from known flavofungins I (1) and II (2), suggesting all flavofungins produced by strain ZQ4BG might have same biosynthetic pathway. A hypothetical mechanism for the formation of 3–9 from 1 and 2 is proposed in Fig. S2 (Supplementary Information). The 10,11-epoxide compounds (Fig. S2B) might be formed by enzymatic epoxidation of the double bond at C10 and C11 (Fig. S2A), followed by hydrolysis of these epoxides to give corresponding 10,11-dihydroxylated compounds (Fig. S2C)35, 36. New flavofungins IV-IX (4–9, Fig. S2D and E) might derive from 10,11-dihydroxylated compounds by cyclization at C-10 and C-13 or C-10 and C-15. The exact structures of these new flavofungins were elucidated based on their HRESIMS data and extensive NMR spectroscopic analyses. The stereochemical assignments were made by a combination of NOE information, 1H and 13C NMR chemical shifts, universal NMR database27, 37,38,39, and chemical reactions including preparations of acetonide derivatives27, 28, 40 and Mosher esters.

Flavofungins IV (4), VI (6), and VII (7) had a same molecular formula of C36H58O11 deduced from their HRESIMS ions at m/z [M + Na]+ 689.3870, 689.3872, 689.3867 and their 13C data. Extensive NMR spectroscopic analyses including 1H, 13C, 1H-1H COSY, HSQC, and HMBC concluded that these three compounds shared a same planar structure, which is a 32-membered macrolide with four unsubstituted conjugated double bonds at C-2 to C-9, one double bond at C-28 and C-29, 11 oxymethines at C-10, C-11, C-13, C-15, C-17, C-19, C-21, C-23, C-25, C-27 and C-31, two methyls at C-14 and C-30, one isopropyl at C-31, and a tetrahydrofuran (THF) ring between C-10 and C-13. All double bonds in 4, 6, and 7 were assigned as E geometries based on their trans-coupling constants (≥14.8 Hz, Tables 2 and 3). As mentioned above, flavofungins IV (4), VI (6), and VII (7) might derive from flavofungin I (1) by enzymatic epoxidation and hydrolysis with different configurations at C-10 and C-11. The big differences of the coupling constants of 3 J H9−10 and 3 J H10−11 and the 13C NMR chemical shifts of C-10 and C-11 in 4, 6, and 7 were observed, supporting that the structural differences among 4, 6, and 7 were from their different configurations at C-10 and C-11.

Table 3 1H-NMR data of compounds 5–9 (500 MHz, in DMSO-d 6, J = Hz)
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The relative configurations of C-15, C-17, C-19, C-21, C-23, C-25, and C-27 in 4 were initially suggested by comparison of the 13C NMR data from 4 with the Kishi’s 13C Universal NMR database of model compounds (a–d, Fig. 2)27, 37,38,39. As shown in Table 1 and Fig. 2, the 13C NMR chemical shifts of C-17 (δ 68.4), C-19 (δ 66.3), C-21 (δ 62.8), C-23 (δ 62.9), and C-25 (δ 65.8) in 4 were close to the C-5 values at δ 67.8 (syn/syn triol), δ 66.0 (anti/syn triol), δ 63.9 (anti/anti triol), δ 63.9 (anti/anti triol), and δ 65.9 (syn/anti triol), respectively, in the Kishi Database. Thus, the relative configurations of the 1,3-diols were assigned as 15,17-syn, 17,19-syn, 19,21-anti, 21,23-anti, 23,25-anti, and 25,27-syn. The relative configurations of these 1,3-diols were also supported by the 13C NMR chemical shifts of the dimethyl and acetal carbons in the triacetonide derivative 4a (Fig. 3) prepared from flavofungin IV (4). Usually, two distinctive NMR chemical shifts around δ C 20 and 30 for dimethyl and δ C 98 for acetal suggest a 1,3-syn diol, while a 1,3-anti diol is indicated by two identical signals near δ C 25 for dimethyl and δ C 100 for acetal27, 28, 40. The 13C NMR chemical shifts (Table S3) of the acetonide methyl and acetal carbons in 4a were observed at δ 19.9 (C-40), 30.2 (C-41), and 98.0 (C-39) for 17,19-syn, δ 24.5 (C-43), 24.6 (C-44), and 100.1 (C-42) for 21,23-anti, and δ 19.9 (C-47), 30.3 (C-46), and 98.1 (C-45) for 25,27-syn (Fig. 3). NOE correlations (Fig. 4B) of H-40 (δ 1.35) with H-17 (δ 3.99) and H-19 (δ 3.86), H-43 (δ 1.25) with H-23 (δ 3.74), H-44 (δ 1.20) with H-21 (δ 3.80), and H-47 (δ 1.31) with H-25 (δ 3.82) and H-27 (δ 4.24) further confirmed the configurations of C-17, C-19, C-21, C-23, C-25, and C-27. The absolute stereochemistry of C-11 and C-15 was next determined by application of the Modified Mosher ester NMR method27, 28. Treatment of 4a with (S)-(+)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride [(S)-(+)-MTPA-Cl] or (R)-(−)-MTPA-Cl gave di-S-Mosher ester 4S or di-R-Mosher ester 4R. The 1H NMR chemical shift differences (Δδ S-R , Fig. 4E, Table S6) between 4S and 4R in negative values for H-8 to H-10 and positive value for H-12 were observed, suggesting a 11R configuration, while negativeΔδ S-R values for Me-14 and H-14 and positiveΔδ S-R values for H-16 and H-17 indicated a 15R configuration. Thus, the absolute configurations of 17R, 19R, 21S, 23S, 25R, and 27R were assigned based on a 15R configuration and the relative configurations of these 1,3-diols. NOE correlations of Me-14 with H-13 and H-15 observed in NOESY spectra of both 4 and 4a determined that these protons had a same β-orientation, suggesting 13S and 14R conformations. The 13C NMR chemical shifts for C-30 to C-34 and the 1H NMR chemical shifts for H-30 to H-34 in compound 4 and flavofungin I (1) were superimposable, suggesting the two compounds had same 30S and 31S configurations. A small coupling constant (3 J H30−31, 2.2 Hz) and a large coupling constant (3 J H31−32, 10.0 Hz), as well as NOE correlation of H-31 (δ 4.65, dd, J = 10.0, 2.2 Hz) with H-30 (δ 2.54, m) and no NOE between H-31 and H-32 (δ 1.83, m), also supported the 30S and 31S configurations in 4.

Figure 2
figure 2

13C NMR chemical shifts of the central carbon in 1,3,5-triols (a–d) and the relevant portion of flavofungins III–V (3–5).

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Figure 3
figure 3

Acetonide derivatives 3a–6a, 8a, 9a, and 9b of flavofungins III–VI (3–6), VIII (8), and IX (9).

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Figure 4
figure 4

(a) Overview of configurational assignments of polyol chains using 13C NMR chemical shifts, HMBC, and NOE correlations of 1,3-diol acetonides. (b–d) Key NOE correlations of triacetonide derivatives 4a, 6a, and 9a. (e,f) Δδ S-R values for the Mosher diesters 4S, 4R, 9S, and 9R.

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As mentioned above, compounds 4, 6, and 7 are stereoisomers and their structural differences were from their different configurations at C-10 and C-11. Because the 11R configuration in 4 had already been assigned, the conformations of C-10 and C-11 in compounds 4, 6, and 7 could be established based on their different coupling constants of 3 J H9−10 and 3 J H10−11 and different 13C NMR chemical shifts for C-10 and C-11 as well as NOEs. First, a large coupling constant (3 J H9−10, 6.6 or 7.5 Hz) in 4 and 4a and a small coupling constant (3 J H10−11, 2.9 Hz) in 4a (Tables 2 and S4) indicated an α-orientation for both H-10 and H-11, suggesting a 10R configuration at C-10 in 4, while a β-orientation for both H-10 and H-11 in 6 was indicated by small coupling constants of 3 J H9−10 (3.5 or 5.7 Hz) in 6 and 6a and 3 J H10−11 (3.9 Hz) in 6a (Tables 3 and S5). Second, the 13C NMR chemical shifts (Table 1) at δ 81.8 (C-10) and δ 72.0 (C-11) in 6 were quite different from their counterparts at δ 85.9 (C-10) and δ 74.7 (C-11) in 4. Furthermore, H-9 (δ 5.79) and H-11 (δ 3.89) exhibited NOE correlation in NOESY spectrum of 4a (Fig. 4B), no NOE between H-9 (δ 5.93) and H-11 (δ 4.20) was observed in NOESY spectrum of 6a. Altogether, the data demonstrated that compounds 4 and 6 had different configurations at C-10 and C-11, which were 10R, 11R for 4 and 10S, 11S for 6. By comparison to 4 and 6, a small 3 J H9−10 (3.5 Hz) coupling constant, NOE correlation of H-9 (δ 6.01) with H-11 (δ 3.88) and no NOE between H-10 (δ 4.10) and H-11, as well as 13C NMR chemical shifts at δ 82.6 (C-10) and δ 75.8 (C-11) suggested that 7 had 10S and 11R configurations. Based on the foregoing evidences, the structures of compounds 4, 6, and 7 were elucidated as novel polyene-polyol macrolides, named as flavofungin IV (4), VI (6), and VII (7). Their 13C and 1H NMR data were fully assigned by interpretation of 1H, 13C, 1H-1H COSY, HSQC, HMBC, and NOESY spectra and are listed in Tables 1–3.

Compound 5 gave a [M + Na]+ ion at m/z 703.4022, 14 mass units higher than that of 4, implying the presence of an additional -CH2 moiety in 5. Detailed analyses of extensive NMR spectra of 5 and its triacetonide derivative 5a concluded that 5 was an analogue of 4, with a –CH2CH3 moiety at C-34, instead of the –CH3 group at C-34 in 4. The 13C and 1H NMR data (Tables 1, 3, S3 and S4) of 5 and 5a were assigned based on 1H, 13C, 1H-1H COSY, HSQC, HMBC, and NOESY spectroscopic analyses. The structure of 5 was assigned as a new polyene-polyol macrolide, named as flavofungin V.

Compound 8 had a molecular formula of C35H56O11 deduced from its [M + Na]+ ion at m/z 675.3714, 14 mass units lower than that of 4. Compared to 4, the NMR spectra of 8 showed only three methyls. Further 13C and 1H NMR assignments (Tables 1, 3, S3 and S5) of 8 and its triacetonide derivative 8a confirmed that 8 was very close to 4, with the absence of a methyl at C-14. The structure of 8 was thus elucidated as a new polyene-polyol macrolide, named as flavofungin VIII.

The NMR spectra of 9 showed 36 carbons for a carbonyl, ten olefinic methines, 11 oxymethines, three methines, seven methenes, and four methyls, which were same to those of 4, 6, and 7. Extensive NMR analyses confirmed that their structural differences were the tetrahydrofuran (THF) ring in 4, 6, and 7 replaced by an oxepane ring in 9. Treatment of 9 with 2,2-dimethoxypropane afforded triacetonide 9a and tetraacetonide 9b. Interpretation of the 13C NMR chemical shifts (Table S3) of the acetonide methyl and acetal carbons in 9a and 9b resulted in the configurational assignments of 11,13-syn, 17,19-syn, 21,23-anti, and 25,27-syn 1,3-diols. Triacetonide 9a was further treated with either (S)-(+)-MTPA-Cl or (R)-(−)-MTPA- Cl to produce (S)-(+)- and (R)-(−)-MTPA esters 9S and 9R, respectively. The analysis of Δδ S-R values allowed for the assignment of 11R (Fig. 4F, Table S6). Unfortunately, the results from Δδ S-R values failed to assignment the configuration at C-13, probably because the two groups of Mosher agent were too close. In the NOESY spectrum of 9a (Fig. 4D), strong NOE correlations of H-10 (δ 3.57) with H-15 (δ 3.38), H-13 (δ 3.90) with H-11 (δ 3.31) and Me-14 (δ 0.78), and no NOE between H-10 and H-11, as well as a large coupling constant (3 J H10−11, 8.8 Hz) (Table S5) suggested an α-orientation for H-10 and H-15 and an β-orientation for H-11, H-13, and Me, -14. Therefore, the configurations of C-10, C-13, C-14, and C-15 were assigned as 10S, 13S, 14R, and 15R. Based on the foregoing evidences, the structure of 9 was determined as novel polyene-polyol macrolide, named as flavofungin IX.

Compound 3 gave a [M + Na]+ ion at m/z 707.3972, correspondence to a molecular formula C36H60O12. The general features of the NMR spectra of 3 were very similar to those of 1, indicating they were structurally related compounds. The main differences were found at the positions of C-10 and C-11, where two olefinic methines of 1 were replaced by two oxymethines in 3. The UV absorption maximum at 330 nm for 3 (365 nm for 1) also supported a smaller conjugated system in 3. The configurational assignments of 11S, 13S, 14R, 15R, 17R, 19R, 21S, 23S, 25R, 27R, 30S, and 31S in 3 were achieved using the same protocol as for compounds 4–9, including 13C Universal NMR database (Fig. 2), 13C NMR chemical shifts (Table S3) of the acetonide methyl and acetal carbons in tetraacetonide derivative 3a, NOE information (Fig. 4A), and the same biosynthetic pathway these macrolides might have. The orientations of αH-10 and βH-11 in 3 were indicated by NOE correlations of H-11 (δ 3.70) with H-9 (δ 5.71) and H-13 (δ 4.20), and no NOE between H-11 and H-10 (δ 3.83) in 3a, as well as the large coupling constant (δ 3.79, t, 7.3 Hz, H-10) for 3 J H9−10 and 3 J H10−11 (Table 2) in 3, suggesting 10R conformation for C-10. Therefore, the structure of 3 was assigned as a new polyene-polyol macrolide, named as flavofungin III.

All isolated compounds (1–10) were evaluated by sulforhodamine B (SRB) assay for their activity in inhibiting proliferation of glioma cells U251, U87MG, SHG44, and C6. Doxorubicin (DOX) was used as a positive control. The results (Supplementary Information, Table S2) indicated that flavofungins I (1) and II (2) and spectinabilin (10) showed different sensitivities to glioma cells. Flavofungin II (2) and spectinabilin (10) showed moderate activity against the proliferation of four tested glioma cell lines with IC50 values of 15.67–56.67 μM for 2 and 10.86–42.75 μM for 10. The anti-glioma property of these two compounds has not been reported previously. Flavofungin I (1) showed weak activity with an IC50 value of 45.91–87.45 μM. Unfortunately, new flavofungins III–IX (3–9) were inactive. Therefore, flavofungin II (2) and spectinabilin (10) are the main components responsible for the anti-glioma activity in the crude extract. The cytotoxicity (CC50) of the two active compounds 2 and 10 towards normal human astrocyte (HA) and human foreskin fibroblast (HFF-1) cells were also assayed. The results (Table S2) indicated both compounds showed a higher selectivity index (CC50/IC50) for HA with 4.6–16.5 for 2 and 3.4–13.3 for 10, when compared to the selectivity index for HFF-1 with 1.8–6.4 for 2 and 1.3–5.1 for 10.

Compounds (1–10) were also tested for their antimicrobial activity against methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans by micro broth dilution method. Flavofungins I (1) and II (2) and spectinabilin (10) showed activity in suppressing the growth of C. albicans with MIC value of 12.5 μg/mL. The control drug amphotericin B had antifungal activity with MIC value of 3.125 μg/mL.

Methods

General Experimental Procedures

This section was supplied as online Supplementary Information.

Strain Isolation

Strains were isolated from a sample of mangrove soils, which were collected from the Qi’ao Mangrove Forest in Zhuhai City, Guangdong, China, in October, 2013. Briefly, the dried soils (1.0 g) were diluted with sterile water to make 0.001 g/mL suspension and 200 μL suspension were covered on the surface of Gause’s solid medium and then incubated at 28 °C for 10 days. The single colony was picked with sterile needles and transferred to a Gause’s-agar plate. After another 10 days of growth at 28 °C, the single colony that grew well was transferred onto Gause’s agar slants and stored at 4 °C until use. A total of ten strains were obtained from this mangrove sample in this study.

Preparation of Crude Extract for Bioactive Assay

Each isolated strain was cultured in Gause’s solid medium with a small Petri dish (90 mm, 25 mL medium) at room temperature for 10 days. The solid culture was cut into small pieces (about 0.6 × 0.6 cm) and then percolated with MeOH three times (50 mL, each). The combined MeOH solution was dried under reduced pressure to give crude extract. This crude extract was redissolved in DMSO to prepare crude sample (1.0 mg/mL). Sulforhodamine B (SRB) assay was used to evaluate the activity of the crude sample against the proliferation of glioma U87MG and U251 cells. The crude extract prepared from the culture of strain ZQ4BG was found to be the most active and the strain ZQ4BG was thus selected for this study.

Taxonomic identity of Streptomyces sp. ZQ4BG

The 16S rDNA sequence analysis of strain ZQ4BG was conducted by Majorbio (Shanghai, China) and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database. The 16S rDNA sequence of strain ZQ4BG has been deposited in GenBank (accession number: KX601187). The voucher strain of Streptomyces sp. ZQ4BG was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhejiang University, China.

Large culture of strain Streptomyces sp. ZQ4BG

Colonies of the strain ZQ4BG growing on Gause’s-agar slants were inoculated into a 500 mL Erlenmeyer flask containing 200 mL of Gause’s liquid medium and then incubated at 28 °C for 5 days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (5 mL) was inoculated into a 500 mL Erlenmeyer flask, which contains 200 mL of Gause’s liquid medium. All flasks were incubated at 28 °C for 12 days on a rotary shaker (180 rpm). A total of 60 L cultures was prepared for this study.

Extraction and Isolation of compounds 1–10

The 60 L cultures were filtered to give filtrate and mycelia. The filtrate was applied to a column of Diaion HP-20 eluting with water and then 100% MeOH. The MeOH elution was concentrated in vacuo to give Part A. The mycelia were extracted with MeOH five times and the combined MeOH solution was concentrated in vacuo to afford Part B. The combination of Part A and Part B was suspended in water and then partitioned by EtOAc three times to afford a crude extract (30.0 g) after removal of the solvent EtOAc. This crude extract was fractionated on a brown column of ODS eluting successively with 70% MeOH, 80% MeOH, 90% MeOH, and 100% MeOH to give fractions A–J based on the results of TLC and LC/MS analyses. Fraction H was separated by HPLC using an Agilent Zorbax SB-C18 column (mobile phase: MeOH/H2O, 85/15; flow rate: 1.0 mL/min) to give compounds 1 (33 mg, tR 24.3 min) and 2 (31 mg, tR 28.8 min). By using the same column and flow rate for 1 and 2 but different mobile phase, 3 (10 mg, tR 70.1 min, CH3CN/H2O, 30/70) and 8 (18 mg, tR 84.0 min, CH3CN/H2O, 30/70) from fraction C, 4 (80 mg, tR 70.1 min, MeOH/H2O, 65/35) and 7 (6 mg, t R 67.1 min, MeOH/H2O, 65/35) from fraction D, and 10 (200 mg, tR 60.5 min, MeOH/H2O, 85/15) from fraction I, were obtained. While fraction B was separated by a preparative HPLC using a Sepax Amethyst C18-H column (mobile phase: CH3CN/H2O, 30/70; flow rate: 10.0 mL/min) to give 9 (57 mg, tR 37.5 min) and an impure compound, which was further purified by HPLC using an Agilent Zorbax SB-C18 column (mobile phase: CH3CN/H2O, 30/70) to furnish 6 (27 mg, tR 53.4 min). Similarly, fraction E was separated by a preparative HPLC using the same column and flow rate for 9 to afford 5 (30 mg, tR 57.5 min MeOH/H2O, 65/35).

Flavofungin I (1): Yellow amorphous powder; molecular formula C36H58O10; UV (MeOH) λmax (log ε) 365 (4.66) nm; 13C NMR data, see Table 1, 1H NMR data, see Table 2; HRESIMS m/z [M + Na]+ 673.3919 (calcd for C36H58NaO10, 673.3928).

Flavofungin II (2): Yellow amorphous powder; molecular formula C37H60O10; UV (MeOH) λmax (log ε) 364 (4.37) nm; 13C NMR data, see Table 1, 1H NMR data, see Table 2; HRESIMS m/z [M + Na]+ 687.4079 (calcd for C37H60NaO10, 687.4084).

Flavofungin III (3): Yellow amorphous powder; molecular formula C36H60O12; UV (MeOH) λmax (log ε) 330 (4.76) nm; IR (KBr)ν max 3384, 2925, 1690, 1620, 1596, 1429, 1355, 1309, 1123, 1011 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 2; HRESIMS m/z [M + Na]+ 707.3972 (calcd for C36H60NaO12, 707.3982).

Flavofungin IV (4): Yellow amorphous powder; molecular formula C36H58O11; UV (MeOH) λmax (log ε) 330 (4.59) nm; IR (KBr)ν max 3385, 2937, 1704, 1621, 1596, 1417, 1370, 1296, 1125, 1009 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 2; HRESIMS m/z [M + Na]+ 689.3870 (calcd for C36H58NaO11, 689.3877).

Flavofungin V (5): Yellow amorphous powder; molecular formula C37H60O11; UV (MeOH) λmax (log ε) 331 (4.28) nm; IR (KBr)ν max 3383, 2934, 1703, 1620, 1596, 1425, 1369, 1290, 1125, 1008 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 3; HRESIMS m/z [M + Na]+ 703.4022 (calcd for C37H60NaO11, 703.4033).

Flavofungin VI (6): Yellow amorphous powder; molecular formula C36H58O11; UV (MeOH) λmax (log ε) 335 (4.64) nm; IR (KBr)ν max 3384, 2935, 1684, 1621, 1595, 1435, 1371, 1300, 1125, 1011 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 3; HRESIMS m/z [M + Na]+ 689.3872 (calcd for C36H58NaO11, 689.3877).

Flavofungin VII (7): Yellow amorphous powder; molecular formula C36H58O11; UV (MeOH) λmax (log ε) 335 (3.94) nm; IR (KBr)ν max 3375, 2926, 1695, 1622, 1598, 1422, 1371, 1300, 1128, 1009 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 3; HRESIMS m/z [M + Na]+ 689.3867 (calcd for C36H58NaO11, 689.3877).

Flavofungin VIII (8): Yellow amorphous powder; molecular formula C35H56O11; UV (MeOH) λmax (log ε) 330 (5.02) nm; IR (KBr)ν max 3374, 2936, 1704, 1620, 1596, 1423, 1371, 1296, 1124, 1009 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 3; HRESIMS m/z [M + Na]+ 675.3714 (calcd for C35H56NaO11, 675.3720).

Flavofungin IX (9): Yellow amorphous powder; molecular formula C36H58O11; UV (MeOH) λmax (log ε) 328 (4.45) nm; IR (KBr)ν max 3385, 2937, 1699, 1622, 1597, 1410, 1319, 1297, 1125, 1011 cm−1; 13C NMR data, see Table 1, 1H NMR data, see Table 3; HRESIMS m/z [M + Na]+ 689.3864 (calcd for C36H58NaO11, 689.3877).

Preparation of acetonide derivatives of flavofungins III–VI (3–6), VIII (8), and IX (9)

Pyridinium-p-toluenesulfonate (10 mg) and 2,2-dimethoxypropane (8.0 mL) were added to a solution of each flavofungin (3–6, 8, 9, 9–55 mg) in anhydrous CH2Cl2 (4.0 mL) and MeOH (400 μL) at room temperature. The mixture was stirred in dark under nitrogen for 12 h and then quenched with 5% aqueous NaHCO3. The reaction product was extracted with CH2Cl2 to give crude extract, which was fractionated by an ODS column eluting with 70% and 90% MeOH to give fractions 70 M and 90 M. Fraction 90 M was further purified by HPLC using an Agilent column (Zorbax SB-C18 250 × 9.4 mm, 5μm) to afford pure acetonide derivatives 3a (3.0 mg, tR 72.5 min, CH3CN/H2O, 85/15), 4a (15.0 mg, tR 59.9 min, CH3CN/H2O, 80/20), 5a (7.0 mg, tR 36.6 min, MeOH/H2O, 92/8), 6a (6.0 mg, tR 35.4 min, CH3CN/H2O, 85/15), 8a (3.0 mg, tR 57.4 min, CH3CN/H2O, 75/25), 9a (9.0 mg, tR 38.5 min, MeOH/H2O, 92/8), and 9b (20.0 mg, tR 32.7 min, MeOH/H2O, 100/0).

Compound  3a: Yellow amorphous powder; molecular formula C48H76O12; 13C NMR data, see Table S3, 1H NMR data, see Table S4; HRESIMS m/z [M + Na]+ 867.5227 (calcd for C48H76NaO12, 867.5234).

Compound  4a: Yellow amorphous powder; molecular formula C45H70O11; 13C NMR data, see Table S3, 1H NMR data, see Table S4; HRESIMS m/z [M + Na]+ 809.4796 (calcd for C45H70NaO11, 809.4816).

Compound  5a: Yellow amorphous powder; molecular formula C46H72O11; 13C NMR data, see Table S3, 1H NMR data, see Table S4; HRESIMS m/z [M + Na]+ 823.4972 (calcd for C46H72NaO12, 823.4972).

Compound  6a: Yellow amorphous powder; molecular formula C45H70O11; 13C NMR data, see Table S3, 1H NMR data, see Table S5; HRESIMS m/z [M + Na]+ 809.4819 (calcd for C45H70NaO11, 809.4816).

Compound  8a: Yellow amorphous powder; molecular formula C44H68O11; 13C NMR data, see Table S3, 1H NMR data, see Table S5; HRESIMS m/z [M + Na]+ 795.4659 (calcd for C44H68NaO11, 795.4659).

Compound  9a: Yellow amorphous powder; molecular formula C45H70O11; 13C NMR data, see Table S3, 1H NMR data, see Table S5; HRESIMS m/z [M + Na]+ 809.4810 (calcd for C45H70NaO11, 809.4816).

Compound  9b: Yellow amorphous powder; molecular formula C48H74O11; 13C NMR data, see Table S3, 1H NMR data, see Table S5; HRESIMS m/z [M + Na]+ 849.5126 (calcd for C48H74NaO11, 849.5129).

MTPA esterification of acetonide derivatives 4a and 9a

Dried acetonide derivative 4a (4.0 mg) was divided into two equal portions. Anhydrous pyridine (1 mL) and 4-dimethylaminopyridine (2 mg) were added to each portion. The mixtures were stirred at room temperature for 10 min and then added either (S)-(+)-MTPA-Cl or (R)-(−)-MTPA-Cl (45 μL). The reaction mixtures were terminated with MeOH (1 mL) after 1 h and then dried under reduced pressure to give a residue. (S)-(+)-MTPA ester (4S, 1.2 mg, tR 35.3 min) or (R)- (−)-MTPA ester (4R, 1.1 mg, tR 35.9 min) was obtained from the residue by HPLC purification using an Agilent Zorbax SB-C18 column (250 × 9.4 mm, 5μm; mobile phase: CH3CN/H2O, 85/15; flow rate: 1.0 mL/min). In the same way, MTPA esterification of acetonide derivative 9a (8.0 mg) gave (S)-(+)-MTPA ester (9S, 3.0 mg, tR 26.2 min, MeOH/H2O, 90/10) and (R)-(−)-MTPA ester (9R, 3.0 mg, tR 26.7 min).

(S)-(+)-MTPA ester  4S: Yellow amorphous powder; molecular formula C56H72F6O15; 1H NMR data, see Table S6; HRESIMS m/z [M + Na]+ 1121.4673 (calcd for C56H72F6NaO15, 1121.4673).

(R)-(−)-MTPA ester  4R: Yellow amorphous powder; molecular formula C56H72F6O15; 1H NMR data, see Table S6; HRESIMS m/z [M + Na]+ 1121.4653 (calcd for C56H72F6NaO15, 1121.4673).

(S)-(+)-MTPA ester  9S: Yellow amorphous powder; molecular formula C56H72F6O15; 1H NMR data, see Table S6; HRESIMS m/z [M + Na]+ 1121.4683 (calcd for C56H72F6NaO15, 1121.4673).

(R)-(−)-MTPA ester  9R: Yellow amorphous powder; molecular formula C56H72F6O15; 1H NMR data, see Table S6; HRESIMS m/z [M + Na]+ 1121.4677 (calcd for C56H72F6NaO15, 1121.4673).

Anti-glioma bioactive assay

Sulforhodamine B (SRB) assay as described in previous publications9, 10 was used to evaluate the activity of all isolated compounds against the proliferation of glioma U87MG, U251, SHG44, and C6 cells as well as normal human astrocytes (HA) and human foreskin fibroblast (HFF-1). Doxorubicin (DOX, a chemotherapeutic drug)41 was used as a positive control (CON).

Antimicrobial assay

The antimicrobial activity of the isolated compounds against growth of methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans was assayed by the micro broth dilution method as described in previous study13. Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and amphotericin B (an antifungal drug) were used as positive controls.