Cytotoxic Bagremycins from Mangrove-Derived Streptomyces sp. Q22

May 15, 2017 - Isochromophilones A–F, Cytotoxic Chloroazaphilones from the Marine Mangrove Endophytic Fungus Diaporthe sp. SCSIO 41011. Xiaowei ...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

Cytotoxic Bagremycins from Mangrove-Derived Streptomyces sp. Q22 Lei Chen,† Weiyun Chai,† Wenling Wang,† Tengfei Song,† Xiao-Yuan Lian,*,‡ and Zhizhen Zhang*,† †

Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, People’s Republic of China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: New bagremycins C−E (3−5) and bagrelactone A (6), together with known bagremycins A (1) and B (2), 4hydroxystyrene (7), and 4-hydroxystyrene 4-O-α-D-galactopyranoside (8), were isolated from a mangrove-derived actinomycete, Streptomyces sp. Q22. Structures of these new compounds were elucidated based on their NMR and HRESIMS spectroscopic data as well as chemical degradation. Bagremycin C (3) is a unique analogue with an N-acetyl-(S)-cysteine moiety, while bagrelactone A (6) represents the first example of this type of bagremycin-derived macrolide. Bagremycin C (3) was active against four glioma cell lines, with IC50 values in the range from 2.2 to 6.4 μM, induced apoptosis in human glioma U87MG cells in a dose- and time-dependent manner, and arrested the U87MG cell cycle at the G0/G1 phase.

G

Bagremycins are phenol esters of p-hydroxystyrene and phydroxybenzoic acid. The first two bagremycins (A and B) were originally isolated from Streptomyces sp. Tü 4128, which was obtained from a soil sample collected from Java, Indonesia, in 2001.4 Both bagremycins A and B have been reported to have activity against Arthrobacter aurescens DSM20166 and Streptomyces viridochromogenes Tü 57, with MIC values of 3−30 μg/ mL.4,5 The bagremycins’ biosynthesis in engineered actinomycete Streptomyces sp. Tü 4128 has been recently studied, and it has been found that several genes including bagA, bagB, bagC, and bagI are involved in or may be involved in the biosynthesis of bagremycins.5−7 More recently, bagremycin A was also found in a mycelium extract of Nocardia caishijiensis.8 However, to the best of our knowledge, no other reports on the bagremycins have been reported so far. Herein, we report several new members (3−6) of bagremycins and their activity against glioma cells.

liomas are the most challenging cancers to treat despite recent advances in standard therapy including surgical resection followed by radiation and chemotherapy.1 While chemotherapy has played an important role in treatment of cancers, temozolomide (TMZ) is the only drug that has been alone used for treating gliomas. Moreover, the efficacy of TMZ and other current drugs remains unsatisfactory,1,2 and there is an urgent need to discover lead compounds for development of novel antiglioma drugs. During the course of our ongoing project to discover novel antiglioma agents from marine resources,3 a crude extract prepared from a culture of strain Q22 isolated from a sample of mangrove soil was found to be active against human glioma cells. The 16S rDNA sequence of this strain Q22 completely matched (99% identity for a 1368 bp stretch of sequence) those of several Streptomyces strains including S. f ilipinensis NBRC 12860, S. durhamensis NBRC 13441, and S. durhamensis CSSP538 (Supporting Information, Table S1 and Figure S1). Therefore, this isolated strain was assigned as Streptomyces sp. Q22. In the current study, a large culture of this mangrovederived actinomycete in Gause’s liquid medium resulted in isolation of eight compounds, bagremycins A−E (1−5), bagrelactone A (6), and styrene derivatives (7 and 8). Bagremycins C−E (3−5) and bagrelactone A (6) were found to be new compounds. © 2017 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION A large culture of the isolated marine actinomycete strain Q22 was grown in Gause’s liquid medium, and an 80 L culture of strain Q22 was prepared. A crude extract of this culture was Received: December 9, 2016 Published: May 15, 2017 1450

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456

Journal of Natural Products

Article

Chart 1

Figure 1. Key HMBC correlations of bagremycins C−E (3−5) and bagrelactone A (6).

Table 1. 13C NMR Data of Bagremycins C−E (3−5) and Bagrelactone A (6) (125 MHz, in DMSO-d6) no.

3

4

5

6

no.

3

4

5

6

1 2 3 4 5 6 7 8 9 10

126.9 113.4 140.4 147.3 113.2 118.8 165.8 150.5 122.1 127.1

118.4 121.3 139.7 149.8 113.8 120.2 164.1 150.1 122.1 127.1

119.2 132.1 115.5 162.6 115.5 132.1 164.3 150.4 122.0 127.1

123.5 115.6 134.0 147.5 115.8 118.6 167.4 157.3 115.1 127.9

11 12 13 14 15 16 17 18 19 20

134.8 127.1 122.1 135.8 114.4 36.1 52.3 172.1 169.3 22.3

134.9 127.1 122.1 135.7 114.5

134.7 127.1 122.0 135.7 114.3

129.0 127.9 115.1 75.3 45.9

7.22 (2H, d, J = 8.5 Hz, H-9 and H-13), 7.54 (2H, d, J = 8.5 Hz, H-10 and H-12), 6.74 (1H, dd, J = 17.1, 11.1 Hz, H-14), 5.26 (1H, d, J = 11.1 Hz, H-15a), 5.82 (1H, d, J = 17.1 Hz, H-15b), while the moiety of 3-amino-4-hydroxybenzoic acid resonated at δC 126.9 (C-1), 113.4 (C-2), 140.4 (C-3), 147.3 (C-4), 113.2 (C-5), 118.8 (C-6), 165.8 (C-7) and δH 6.80 (1H, d, J = 8.1 Hz, H-5), 7.07 (1H, d, J = 8.1 Hz, H-6). HMBC correlations as described in Figure 1 further confirmed the structures of the phydroxystyrene and 3-amino-4-hydroxybenzoic acid units. The 13 C NMR data of 3 showed 20 carbon signals, of which 15 were assigned to the two units mentioned above and the remaining five to an N-acetylcysteine unit. This N-acetylcysteine displayed its characteristic NMR signals at δC 172.1 (C-18) and 169.3 (C19) for two carbonyls, δC 52.3 and δH 4.18 (1H, m) for a nitrogenated methine at C-17, δC 36.1 and δH 2.96 (1H, dd, J = 13.0, 8.8 Hz) and 3.13 (1H, dd, J = 13.0, 4.9 Hz) for a methylene at C-16, δC 22.3 and δH 1.80 (3H, s) for a methyl at C-20, and δH 8.25 (1H, d, J = 7.8 Hz) for a NH unit at C-17.

fractionated on Diaion HP-20 and then with ODS column chromatography, following by HPLC purification, to yield four new compounds, bagremycins C−E (3−5) and bagrelactone A (6), together with four known compounds. The known compounds were shown to be bagremycins A (1) and B (2),4 4-hydroxystyrene (7), and 4-hydroxystyrene 4-O-α-Dgalactopyranoside (8)9 based on their NMR spectroscopic data, which was compared to published information. The NMR data of bagremycins A (1) and B (2) are listed in Table S2 (Supporting Information). Compound 3 was obtained as a colorless amorphous powder and has a molecular formula of C20H20N2O6S deduced from negative HRESIMS and 13C NMR data. The structure of 3 is made of three subunits, namely, p-hydroxystyrene, 3-amino-4hydroxybenzoic acid, and N-acetylcysteine. The p-hydroxystyrene unit4 was easily recognized by its characteristic NMR signals at δC 150.5 (C-8), 122.1 (C-9 and C-13), 127.1 (C-10 and C-12), 134.8 (C-11), 135.8 (C-14), 114.4 (C-15) and δH 1451

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456

Journal of Natural Products

Article

combination with the HRESIMS data of 4. The 13C and 1H NMR assignments (Tables 1 and 2) of 4 were made based on HSQC and HMBC correlations (Figure 1). The structure of 4 was also identified as a new compound, bagremycin D. Compound 5 gave a [M − H]− ion at m/z 239.0714 in its negative HRESIMS spectrum, 15 mass units lower than that of bagremycin A (1), implying 5 was a derivative of 1 with the absence of a nitrogen-bearing group. Further NMR analysis indicated that compounds 5 and 1 are different only in the substituent of the NH2 group at the C-3 position. Compared to bagremycin A (1), compound 5 is a simpler bagremycin without an amino substitute at C-3, which exhibited characteristic NMR data for the two moieties p-hydroxystyrene and phydroxybenzoic acid. The 13C and 1H NMR data of 5 as assigned by HSQC and HMBC correlations (Figure 1) are listed in Tables 1 and 2. Therefore, the structure of 5 was assigned as bagremycin E, a new member of the bagremycins. The HRESIMS data of 6 supported a molecular formula of C15H13NO4, 16 mass units higher than that of bagremycin (1), indicating the presence of an additional oxygen-bearing function in 6. The 1H NMR data of 6 showed five peaks in the region of δ 6.5−7.5. The first three peaks, at δ 7.27 (1H, d, J = 2.1 Hz), 6.76 (1H, d, J = 8.3 Hz), and 7.12 (1H, dd, J = 8.3, 2.1 Hz), were obviously related to the 3-amino-4-hydroxybenzoic acid unit and were assigned to H-2, H-5, and H-6, respectively. The remaining two peaks at δ 6.77 (2H, d, J = 8.6 Hz) and 7.22 (2H, d, J = 8.6 Hz) were attributed to H-9, H-13 and H-10, H-12, respectively. The exocyclic double-bond signals at δC 135.8 (C-14) and 114.3 (C-15) and δH 6.73 (1H, dd, J = 17.6, 11.0 Hz, H-14), 5.26 (1H, d, J = 11.0 Hz, H-15a), and 5.80 (1H, d, J = 17.6 Hz, H-15b) (Supporting Information, Table S2) of 1 were replaced by an oxymethine signal at δC 75.3 (C-14) and δH 4.93 (1H, dd, J = 8.5, 2.4 Hz, H-14) and a methylene signal at δC 45.9 (C-15) and δH 3.20 (1H, dd, J = 12.0, 8.5 Hz, H-15a) and 3.41 (1H, d, J = 12.0 Hz, H-15b) in the NMR data of 6. As shown in Figure 1, HMBC correlations of δH 7.22 (H-10 and H-12) with δC 75.3 (C-14), δH 4.93 (H14) with δC 127.9 (C-10 and C-12), 129.0 (C-11), and 45.9 (C15), and δH 3.20 (H-15) with δC 129.0 (C-11) and 75.3 (C-14) determined the positions of the oxymethine at C-14 and the methylene at C-15. Significant HMBC correlations of δH 3.20 and 3.41 (H-15) with δC 134.0 (C-3) demonstrated that the amino group at C-3 was connected to the methylene at C-15 to form a macrolide structure. The preparation of Mosher esters was attempted to determine the absolute configuration of the C-14 position. Treatment of 6 with (S)-(+)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride [(S)-(+)-MTPA-Cl)] or (R)-(−)- MTPA-Cl) gave S-Mosher ester 6S or R-Mosher ester 6R. Unfortunately, no 1H chemical shift difference (ΔδS−R) between 6S and 6R was observed. Therefore, Mosher methodology was not able to assign the configuration at C14, probably because the group of the Mosher regent was very close to its neighbor benzene ring. However, the C-14 configuration was proposed to be S based on comparison of its positive optical rotation value with those of known compounds S-(+)-octopamine (positive value) and R-(−)-octopamine (negative value)12 and its positive Cotton effect in the region of 241−257 nm. It was noted that compound 6 might derive from bagremycin A (1). A possible mechanism for formation of 6 from 1 has been proposed in Figure 2. On the basis of the foregoing analyses, the structure of 6 was determined to be a new bagremycin macrolide, bagrelactone A. To the best of our knowledge, bagrelactone A (6) is the first

The significant HMBC correlations (Figure 1) of H2-16 (δ 2.96 and 3.13) with C-2 (δ 113.4) demonstrated that this Nacetylcysteine unit was linked to the C-2 position. The configuration at C-17 in 3 was suggested to be S by a comparison of its negative optical rotation value with those of known compounds N-acetyl-(S)-cysteine (3a) and N-acetyl(R)-cysteine (3b), where a negative value was suggestive of an S-configuration, while a positive value indicated an Rconfiguration.10 In order to confirm this assigned conformation, compound 3 was degraded by palladium-catalyzed reductive cleavage11 to produce N-acetyl-(S)-cysteine (3a), which was further hydrolyzed by hydrochloric acid to give (S)-cysteine (3c) as detected by chiral HPLC analysis using a standard amino acid as reference. On the basis of the foregoing evidence, the structure of 3 was elucidated as a new compound, a unique analogue of bagremycins, that we named bagremycin C. Its 13C and 1H NMR data are listed in Tables 1 and 2. Table 2. 1H NMR Data of Bagremycins C−E (3−5) and Bagrelactone A (6) (500 MHz, in DMSO-d6, J = Hz) no.

3

4

5

6.90, 1H, d (8.2) 7.33, 1H, d (8.2) 7.21, 2H, d (8.8) 7.53, 2H, dd (8.8, 2.5) 7.53, 2H, dd (8.8, 2.5) 7.21, 2H, d (8.8) 6.73, 1H, dd (17.6, 11.0) 5.26, 1H, d (11.0); 5.82, 1H, d (17.6)

7.97, 1H, d (8.8) 6.91, 1H, d (8.8) 6.91, 1H, d (8.8) 7.97, 1H, d (8.8) 7.21, 2H, d (8.7) 7.54, 2H, d (8.7) 7.54, 2H, d (8.7) 7.21, 2H, d (8.7) 6.74, 1H, dd (17.6, 11.0) 5.27, 1H, d (11.0); 5.82, 1H, d (17.6)

2 3 5 6 9 10 12 13 14 15

16

17 20 NH3/17

6.80, 1H, d (8.1) 7.07, 1H, d (8.1) 7.22, 2H, d (8.5) 7.54, 2H, d (8.5) 7.54, 2H, d (8.5) 7.22, 2H, d (8.5) 6.74, 1H, dd (17.1, 11.1) 5.26, 1H, d (11.1); 5.82, 1H, d (17.1) 2.96, 1H. dd (13.0, 8.8); 3.13, 1H, dd (13.0, 4.9) 4.18, 1H, m 1.80, 3H, s 8.25, 1H, d (7.8)

6 7.27, 1H, d (2.1)

6.76, 1H, d (8.3) 7.12, 1H, dd (8.3, 2.1) 6.77, 2H, d (8.6) 7.22, 2H, d (8.6) 7.22, 2H, d (8.6) 6.77, 2H, d (8.6) 4.93, 1H, dd (8.5, 2.4) 3.20, 1H, dd (12.0, 8.5); 3.41, 1H, dd (12.0)

6.20, 1H, brs

The HRESIMS of compound 4 supported a molecular formula of C15H13NO3S. Compared to bagremycin C (3), compound 4 consisted of only two units of p-hydroxystyrene and 2-thiol-3-amino-4-hydroxybenzoic acid. The typical NMR signals of p-hydroxystyrene were observed at δC 150.1 (C-8), 122.1 (C-9 and C-13), 127.1 (C-10 and C-12), 134.9 (C-11), 135.7 (C-14), 114.5 (C-15) and δH 7.21 (2H, d, J = 8.8 Hz, H-9 and H-13), 7.53 (2H, dd, J = 8.8, 2.5 Hz, H-10 and H-12), 6.73 (1H, dd, J = 17.6, 11.0 Hz, H-14), 5.26 (1H, d, J = 11.0 Hz, H15a), 5.82 (1H, d, J = 17.6 Hz, H-15b). The existence of the 2thiol-3-amino-4-hydroxybenzoic acid unit was confirmed by its NMR signals at δC 118.4 (C-1), 121.3 (C-2), 139.7 (C-3), 149.8 (C-4), 113.8 (C-5), 120.2 (C-6), 164.1 (C-7) and δH 6.90 (1H, d, J = 8.2 Hz, H-5), 7.33 (1H, d, J = 8.2 Hz, H-6), in 1452

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456

Journal of Natural Products

Article

Figure 2. Possible mechanism for formation of bagrelactone A (6) from bagremycin A (1).

example of this type of bagremycin-derived macrolide. Its 13C and 1H NMR data were assigned based on the HSQC and HMBC correlations (Figure 1) and are summarized in Tables 1 and 2. The activity of all isolated compounds against four different glioma cell lines, U87MG, U251, SHG44, and C6, was assayed by the sulforhodamine B (SRB) method, a method that measures total cellular protein content for evaluating the antitumor activity of tested compounds. Glioma cells were treated with tested compounds for 72 h at different concentrations. The results (Table 3 and Figure S2) indicated

4 and Figure S3) showed that bagremycin C (3) at both concentrations did not induce apoptosis in U87MG cells at early treatment of 24 h, but significantly induced apoptosis at late treatments of 48 and 72 h. The total apoptotic cells (early and late apoptotic cells) induced by bagremycin C (3) increased by 20.59% (48 h) and 58.32% (72 h) at 2.2 μM and 61.16% (48 h) and 93.00% (72 h) at 4.4 μM, when compared to the control (C, 5.80% for 48 h and 5.24% for 72 h). The positive control DOX (10.0 μM) induced apoptosis in U87MG cells by 10.42% (48 h) and 26.87% (72 h) increase of total apoptotic cells. These data suggest that bagremycin C (3) induced apoptosis in U87MG cells in a dose- and timedependent manner. To determine if an alteration of the cell cycle occurred after treatment of bagremycin C (3), DNA content of treated cells was measured by flow cytometric analysis. U87MG cells were treated with bagremycin C (3, 2.2, 4.4, and 8.8 μM) for 12 h. DOX (0.8 μM) was used as a positive control. The percentages of each phase in the cell cycle are shown in Table 5 and Figure

Table 3. Activity of Bagremycins B and C against Glioma Cells (IC50: μM, mean ± SD, n = 5) compound

U87MG

U251

SHG44

C6

bagremycin B bagremycin C DOX

10.2 ± 0.5 2.2 ± 0.1 0.4 ± 0.0

9.7 ± 1.9 4.3 ± 0.4 3.3 ± 0.7

7.3 ± 0.8 2.4 ± 0.2 1.9 ± 0.0

13.3 ± 2.4 6.4 ± 0.5 0.5 ± 0.1

Table 5. Analysis of Cell Cycle in U87MG Cells Treated by Bagremycin C

bagremycin C (3) was active against four different glioma cell lines, with IC50 values in a range from 2.2 to 6.4 μM, compared to the activity of the positive control doxorubicin (DOX, 0.4 to 3.3 μM). Bagremycin B (2) also showed activity against the four different glioma cell lines with IC50 values of 7.3 to 13.3 μM. Tumor cell apoptosis and cell cycle are important targets for drug development.13,14 Therefore, the cell apoptosis and cell cycle arrest induced by the most active bagremycin (3) were further investigated in U87MG cells, which are more sensitive to bagremycin C. The apoptotic cells induced by bagremycin C (3) were quantified by flow cytometry using annexin V-FITC/ propidium iodide (PI) double staining. U87MG cells were treated with bagremycin C (3) in concentrations of 2.2 and 4.4 μM for 24, 48, and 72 h, stained with annexin-V FITC and PI, and then analyzed by using flow cytometry. The results (Table

treatment (12 h) C DOX (0.8 μM) bagremycin C (2.2 μM) bagremycin C (4.4 μM) bagremycin C (8.8 μM)

G0/G1

S

G2/M

compound(G0/G1) − C(G0/G1)

58.14% 82.88% 63.85%

32.49% 10.84% 27.23%

9.37% 6.28% 8.92%

24.74% 5.71%

74.32%

16.76%

8.92%

16.18%

78.70%

13.77%

7.53%

20.56%

S4. The results showed that the cell population at the G0/G1 phase was enhanced 16.18% and 20.56% after 12 h of exposure to 4.4 and 8.8 μM bagremycin C (3). These changes occurring

Table 4. Quantification of Total Apoptotic Cells (Early and Late Apoptotic Cells) Induced by Bagremycin C in U87MG Cells compound − C treatment

24 h

48 h

72 h

24 h

48 h

72 h

C DOX (10 μM) bagremycin C (2.2 μM) bagremycin C (4.4 μM)

3.04% 6.21% 4.00% 6.40%

5.80% 16.22% 26.39% 66.96%

5.24% 32.11% 63.56% 98.24%

3.17% 0.96% 3.36%

10.42% 20.59% 61.16%

26.87% 58.32% 93.00%

1453

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456

Journal of Natural Products

Article

rotary shaker (180 rpm). A total of 320 Erlenmeyer flasks (80 L fermentation) were prepared for this study. Extraction and Isolation. The culture broth (80 L) of the isolated mangrove Streptomyces sp. Q22 was filtered to give mycelia and filtrate. The filtrate was applied to a column of Diaion HP-20 eluting with water and 100% MeOH. The MeOH eluent was dried in vacuo to give part A. The mycelia were extracted with MeOH three times. The combined MeOH extract was concentrated under reduced pressure to afford part B. A mixture of part A and part B was fractionated by a Diaion HP-20 column eluting with 50%, 70%, and 90% MeOH to give fractions 50M, 70M, and 90M. Fraction 70M was separated by column chromatography of ODS successively eluting with 50%, 80%, and 100% MeOH to yield fractions 70MA, 70MB, and 70MC, respectively. Fraction 70MB was further separated by HPLC using a Zorbax SBC18 column (250 × 9.4 mm) with an isocratic mobile phase of MeOH and 0.1% HOAc in H2O (75:25) at a flow rate of 1.0 mL/min and UV detection of 256 nm to give compounds 1 (25.6 mg, tR 17.7 min), 2 (10.3 mg, tR 20.4 min), 3 (10.8 mg, tR 15.4 min), 4 (1.8 mg, tR 14.1 min), 5 (1.2 mg, tR 27.0 min), 6 (10.6 mg, tR 12.2 min), and 7 (3.5 mg, tR 15.8 min). Compound 8 (6.3 mg, tR 14.2 min) was obtained from fraction 50M by HPLC purification using the same HPLC conditions for the separation of compounds 1−7, but a different isocratic mobile phase (MeOH/0.1% HOAc in H2O, 65:35). Bagremycin C (3): colorless, amorphous powder; [α]25D −7.67 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 205 (4.89), 250 (4.76), 320 (3.89) nm; IR (KBr) νmax 3398, 2946, 2837, 2352, 1722, 1655, 1603, 1552, 1505, 1387, 1294, 1196, 1118, 1025 cm−1; 13C NMR data, see Table 1; 1H NMR data, see Table 2; HRESIMS m/z 415.0970 [M − H]− (calcd for C20H19N2O6S, 415.0964). Bagremycin D (4): colorless, amorphous powder; UV (MeOH) λmax (log ε) 214 (3.18), 306 (2.50) nm; IR (KBr) νmax 2957, 2858, 2363, 1727, 1603, 1510, 1448, 1376, 1267, 1200, 1113, 1020 cm−1; 13C NMR data, see Table 1; 1H NMR data, see Table 2; HRESIMS m/z 286.0543 [M − H]− (calcd for C15H12NO3S, 286.0538) and [M + Na]+ 310.0513 (calcd for C15H13NO3SNa, 310.0514). Bagremycin E (5): colorless, amorphous powder; UV (MeOH) λmax (log ε) 208 (4.80), 262 (3.89) nm; IR (KBr) νmax 2957, 2858, 2363, 1727, 1603, 1510, 1448, 1376, 1267, 1200, 1113, 1020 cm−1; 13C NMR data, see Table 1; 1H NMR data, see Table 2; HRESIMS m/z 239.0714 [M − H]− (calcd for C15H11O3, 239.0708). Bagrelactone A (6): colorless, amorphous powder; [α]25D +3.30 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 218 (3.32), 226 (3.27), 357 (2.98) nm; ECD (CH3OH, c 0.45 mM, 0.1 cm), Δε +0.62 (250 nm); IR (KBr) νmax 3415, 1674, 1608, 1581, 1490, 1373, 1278, 1220, 1174, 1090, 906, 825, 764 cm−1; 13C NMR data, see Table 1,; 1H NMR data, see Table 2; HRESIMS m/z 272.0922 [M + H]+ (calcd for C15H14NO4, 272.0923). Palladium-Catalyzed Reductive Cleavage of Bagremycin C (3). Bagremycin C (3, 3.0 mg) was dissolved in 500 μL of THF and then treated with 2.1 equiv of Et3SiH and 3 mol % PdCl2.11 The mixtures were shaken with 100 rpm at room temperature for 30 min. The reaction mixtures were dried under reduced pressure to give a residue (sample A) for further acid hydrolysis. Acid Hydrolysis of Sample A. Sample A was dissolved in 1.0 mL of 6 N HCl and heated at 110 °C in an evacuated glass ampule for 24 h. The hydrolysate was concentrated in vacuo to give a residue. This residue was dissolved in 0.2 mL of water to produce sample B for chiral HPLC analysis. Chiral HPLC analysis. Each of sample B and the authentic Dcysteine and L-cysteine was analyzed using a Chirex 3126 (D)penicillamine column (150 × 4.6 nm, Phenomenex) with detection at 254 nm and temperature at 22 °C. The 1 mM aqueous CuSO4 was used as mobile phase with flow rate of 1.0 mL/min. The free amino acid in the acid hydrolysates of sample B was shown to be (S)-cysteine by comparison of its retention time (tR) with those of the authentic amino acids (S)-cysteine (tR 31.3 min) and (R)-cysteine (tR 44.5 min). MTPA Esterification of 6. Compound 6 (2.0 mg) was dissolved in a mixture of anhydrous pyridine (1 mL) and dimethylaminopyridine (10 mg). The mixtures were stirred at room temperature for 15 min, and then either (S)- or (R)-α-methoxy-α-(trifluoromethyl)-

in the cell cycle indicated that bagremycin C (3) might block the U87MG cell cycle at the G0/G1 phase. Compounds 1−8 were inactive against growth of methicillinresistant Staphylococcus aureus, Escherichia coli, and Candida albicans.

■

EXPERIMENTAL SECTION

General Experimental Procedures. UV and optical rotation were measured on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., China) and a JASCO DIP-370 digital polarimeter (JASCO, Japan), respectively. An AVATAR 370 FT-IR spectrometer (Thermo Nicolet, USA) and a JASCO J 715 spectropolarimeter (JASCO, Japan) were used to record IR and CD spectra, respectively. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer. NMR spectra were acquired on a Bruker 500 spectrometer using standard pulse programs and acquisition parameters. Chemical shifts were expressed in δ (ppm) and referred to the NMR solvent DMSO-d6. Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18-Prep, Nacalai Tesque Inc., Japan) and Diaion HP-20 (Mitsubishi Chemical, Japan) were used for column chromatography. HPLC separation was performed on an Agilent 1260 HPLC system using an Agilent column (Zorbax SB-C18, 250 × 9.4 mm, 5 μm). All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and Et3SiH and PdCl2 were ordered from Energy Chemical (Shanghai, China). Human glioma U251 (XB-0439), U87MG (JDS-2568), and SHG44 (RXJ150) cells and rat glioma C6 (XB-003) cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans were gifts from Drs. Zhongjun Ma, Pinmei Wang, and Bin Wu, respectively. Gause’s agar was purchased from Guangdong Huankai Microbial Science and Technology Co. Ltd. (Guangzhou, China). Doxorubicin (>98.0%) was ordered from SigmaAldrich, and gentamicin (99.6%), and amphotericin B (>95.0%) were from Meilune Biotechnology Co. Ltd. (Dalian, China). Isolation and Taxonomic Identity of Mangrove Streptomyces sp. Q22. Strain Q22 was derived from a sample of mangrove soils, which were obtained from the Qiao Mangrove Forest in Zhuhai City, Guangdong, China, in October 2013. Briefly, the soils were diluted and then spread on the surface of Gause’s solid medium. After 5 days of incubation at room temperature, the grayish-white bacteria colonies were picked with sterile needles and transferred to a Gause’s agar plate. After another 5 days of growth at room temperature, the single colony (strain Q22) that grew well was transferred onto Gause’s agar slants to prepare working stocks and stored at 4 °C until use. Strain Q22 was identified using 16S rDNA sequence analysis 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 Q22 has been deposited in GenBank (accession number KX585211). A voucher strain (Streptomyces sp. Q22) was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhejiang University, China. Preparation of Crude Extract for Bioactivity Assay. The isolated strain was cultured in Gause’s solid medium with a small Petri dish (90 mm, 25 mL of 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 a crude extract. This crude extract was redissolved in DMSO to prepare a crude sample (1.0 mg/mL). The sulforhodamine B assay was used to evaluate the activity of the crude sample against glioma U87MG and U251 cells. Large Culture of Strain Streptomyces sp. Q22. Colonies of the strain Q22 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 then inoculated into a 500 mL Erlenmeyer flask, which contained 250 mL of liquid Gause’s agar media. The flask was incubated at 28 °C for 10 days on a 1454

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456

Journal of Natural Products

Article

phenylacetyl chloride (MTPA-Cl, 45 μL) was added. The reaction was terminated with MeOH (1 mL) after 1 h, and then the reaction mixtures were dried under reduced pressure to give a residue. (S)MTPA ester (6S, 1.3 mg, tR 64.2 min) or (R)-MTPA ester (6R, 1.2 mg, tR 62.0 min) was obtained from the residue by HPLC purification (column: Zorbax SB-C18, 250 × 9.4 mm, 5 μM; mobile phase: MeOH/H2O, 78/22; flow rate: 1.0 mL/min; UV detection: 240 nm). (S)-MTPA ester (6S): colorless, amorphous powder; 1H NMR (DMSO-d6, 500 MHz) δ 7.61 (2H, dd, J = 8.6, 1.1 Hz, H-10, 12), 7.55−7.57 (5H, m, H-MTPA), 7.30 (1H, d, J = 2.1 Hz, H-2), 7.29 (2H, dd, J = 8.6, 1.2 Hz, H-9, 13), 7.16 (1H, dd, J = 8.4, 2.2 Hz, H-6), 6.86 (1H, d, J = 8.4 Hz, H-5), 6.38 (1H, d, J = 2.0 Hz, NH-3), 5.17 (1H, dd, J = 8.1, 2.3 Hz, H-14), 3.53 (1H, m, H-15a), 3.23 (1H, m, H15b), 3.78 (3H, s, OCH3-4), 3.66 (3H, s, OCH3-MTPA); HRESIMS m/z [M + Na]+ 524.1290 (calcd for C26H22F3NO6Na, 524.1297). (R)-MTPA ester (6R): colorless, amorphous powder; 1H NMR (DMSO-d6, 500 MHz) δ 7.61 (2H, dd, J = 8.6, 1.1 Hz, H-10, 12), 7.55−7.57 (5H, m, H-MTPA), 7.30 (1H, d, J = 2.1 Hz, H-2), 7.29 (2H, dd, J = 8.6, 1.2 Hz, H-9, 13), 7.16 (1H, dd, J = 8.4, 2.1 Hz, H-6), 6.86 (1H, d, J = 8.4 Hz, H-5), 6.38 (1H, d, J = 2.0 Hz, NH-3), 5.17 (1H, dd, J = 8.1, 2.3 Hz, H-14), 3.53 (1H, m, H-15a), 3.23 (1H, m, H15b), 3.78 (3H, s, OCH3-4), 3.66 (3H, s, OCH3-MTPA); HRESIMS m/z [M + Na]+ 524.1292 (calcd for C26H22F3NO6Na, 524.1297). Culture of Glioma Cells. Rat glioma C6 and human glioma U251 cells were cultured in DMEM (Dulbecco’s modified Eagle medium, Gibco) with 10% FBS (fetal bovine serum, PAA Laboratories Inc.), human glioma U87MG cells in MEM (minimum essential medium, Gibco), and human glioma SHG44 in RPMI-1640 medium (Roswell Park Memorial Institution 1640 Medium, Gibco). All cells were incubated at 37 °C in a humidified incubator with 5% CO2. Cells after the third generation were used for the experiment. Sulforhodamine B Assay. The SRB assay3a,b was applied to determine the activity of all isolated compounds against glioma U87MG, U251, SHG44, and C6 cells. Doxorubicin (a chemotherapeutic drug)15 was used as a positive control. Briefly, glioma cells were plated in a 96-well plate and then treated with different concentrations of each tested compound after 24 h of cell adhesion. After 72 h of treatment, cells were fixed with 50 μL of 50% cold trichloroacetic acid solution at 4 °C for 1 h, washed with distilled water five times, and then dried at room temperature. The dried cells were stained with 50 μL of 0.4% SRB for 10 min and rinsed with a 1% acetic acid solution five times. After drying, dye was dissolved in a 10 mM Tris buffer and measured at 515 nm on a microplate reader (BioTech) to give optical density (OD) value. The cell viability (%) was calculated from the formula TOD/COD × 100% (TOD: OD value of tested compound; COD: OD value of control), and IC50 value was obtained based on the cell viability (%) by logistic calculation using SPSS software. Annexin V-FITC/PI Double Staining Assay. The quantification of apoptotic cells induced by bagremycin C (3) was done using an annexin V-FITC/PI double staining assay using an annexin V apoptosis detection kit.3a Briefly, human glioma U87MG cells were treated with bagremycin C (2.2, 4.4, and 8.8 μM) for 24, 48, and 72 h, respectively, and then 1 × 106 cells were harvested. The harvested cells were washed in cold phosphate-buffered saline (PBS) buffer and resuspended in 100 μL of 1× binding buffer mixed with 5 μL of annexin V-FITC and 1 μL of 100 μg/mL PI working solution. The stained cells were incubated at room temperature for 15 min, and then 400 μL of 1× binding buffer was added. Fluorescence was analyzed by flow cytometry at the fluorescence emission at 530 and 575 nm using 488 nm excitation. Cell Cycle Assay. Cell cycle arrest by bagremycin C (3) was analyzed by propidium iodide DNA staining using flow cytometry.3a Briefly, human glioma U87MG cells were treated with bagremycin C (2.2, 4.4, and 8.8 μM) for 12 h. After treatment, cells were harvested, prepared as a single cell in ice-cold PBS, and then fixed with 70% icecold ethanol at 4 °C overnight. The fixed cells were harvested by centrifugation (1900 rpm, 7 min), washed with PBS twice, resuspended in PBS with RNase A (50 U/mL), incubated at 37 °C

for 30 min, and finally stained with PI in the dark at 4 °C for 30 min. Cell cycle distribution was studied using a FACScan flow cytometer. Antimicrobial Assay. The antimicrobial activity of isolated compounds against the growth of methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans was evaluated by the micro broth dilution method.3e Gentamicin (an antibiotic against both Gram-positive and Gramnegative bacteria) and amphotericin B (an antifungal drug) were used as positive controls. Briefly, 96-well plates were used to make dilutions of the tested compounds. The stock solution of tested compounds was diluted with 50% DMSO to make a series of concentrations, and the final volume was 200 μL. After that 2 μL from 108 cf/mL of culture was added, and the plates were incubated at 37 °C for 12 h overnight. The next day, plates were observed by eye, and those dilutions that were completely clear along with the next dilutions were plated over nutrient agar plates. The agar plates were incubated at 37 °C for 12 h, and the results were recorded the next day. The dilution that inhibited the growth of bacteria was assigned as MIC.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01136. NMR and HRESIMS spectra of isolated bagremycins as well as other supporting data (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-13675859706. Fax: +86-571-88208432. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhizhen Zhang: 0000-0002-8290-3507 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 81273428 and No. 81274137). We appreciate Mrs. Jianyang Pan (Pharmaceutical Informatics Institute of Zhejiang University) for performing the NMR spectrometry. The authors also thank Prof. B.-R. Lin at Institute of Plant Protection of Guangdong Academy of Agricultural Science and Prof. G.-X. Zhou at College of Pharmacy of Jinan University for their help with sample collection.

■

REFERENCES

(1) Patil, S. A.; Hosni-Ahmed, A.; Jones, T. S.; Patil, R.; Pfeffer, L. M.; Miller, D. D. Expert Opin. Drug Discovery 2013, 8, 1135−1151. (2) Chamberlain, M. C. Expert Rev. Neurother. 2010, 10, 1537−1544. (3) (a) Xin, W.; Ye, X.; Yu, S.; Lian, X. Y.; Zhang, Z. Mar. Drugs 2012, 10, 2388−2402. (b) Yu, S.; Ye, X.; Chen, L.; Lian, X. Y.; Zhang, Z. Steroids 2014, 88, 19−25. (c) Yu, S.; Ye, X.; Huang, H.; Peng, R.; Su, Z.; Lian, X. Y.; Zhang, Z. Planta Med. 2015, 81, 152−159. (d) Chen, L.; Liang, L.; Song, T.; Anjum, K.; Wang, W.; Yu, S.; Huang, H.; Lian, X. Y.; Zhang, Z. Bioorg. Med. Chem. Lett. 2015, 25, 2629− 2633. (e) Ye, X.; Anjum, K.; Song, T.; Wang, W.; Yu, S.; Huang, H.; Lian, X. Y.; Zhang, Z. Nat. Prod. Res. 2016, 30, 1156−1161. (f) Liang, Y.; Xie, X.; Chen, L.; Yan, S.; Ye, X.; Anjum, K.; Huang, H.; Lian, X. Y.; Zhang, Z. Mar. Drugs 2016, 14 (1), 10. (g) Zhang, X.; Ye, X.; Chai, W.; Lian, X. Y.; Zhang, Z. Mar. Drugs 2016, 14 (10), 181. (4) Bertasso, M.; Holzenkampfer, M.; Zeeck, A.; Dall’Antonia, F.; Fiedler, H. P. J. Antibiot. 2001, 54, 730−736. 1455

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456

Journal of Natural Products

Article

(5) Zhu, Y.; Liao, S.; Ye, J.; Zhang, H. Biotechnol. Lett. 2012, 34, 269− 274. (6) Zhu, Y.; Xu, D.; Liao, S.; Ye, J.; Zhang, H. Ann. Microbiol. 2013, 63, 167−172. (7) Liu, F.; Ye, J.; Zhang, H.; Xu, D.; Zhang, Y.; Zhu, Y. Microbiol. Res. 2015, 173, 18−24. (8) Tanvir, R.; Sajid, I.; Hasnain, S.; Kulik, A.; Grond, S. Microbiol. Res. 2016, 185, 22−35. (9) Socolsky, C.; Fascio, M. L.; D’Accorso, N. B.; Salvatore, A.; Willink, E.; Asakawa, Y.; Bardon, A. J. Chem. Ecol. 2008, 34, 539−548. (10) Ohta, G.; Nagase, O.; Hosokawa, Y.; Tagawa, H.; Shimizu, M. Chem. Pharm. Bull. 1967, 15, 644−647. (11) Matsumura, T.; Niwa, T.; Nakada, M. Tetrahedron Lett. 2012, 53, 4313−4316. (12) Lundell, K.; Katainen, E.; Kiviniemi, A.; Kanerva, L. T. Tetrahedron: Asymmetry 2004, 15, 3723−3729. (13) Kamal, A.; Faazil, S.; Malik, M. S. Expert Opin. Ther. Pat. 2014, 24, 339−354. (14) De Falco, M.; De Luca, A. Curr. Pharm. Des. 2010, 16, 1417− 1426. (15) Tacara, O.; Sriamornsak, P.; Dass, C. R. J. Pharm. Pharmacol. 2013, 65, 157−170.

1456

DOI: 10.1021/acs.jnatprod.6b01136 J. Nat. Prod. 2017, 80, 1450−1456