Cytotoxic Dibohemamines D–F from a Streptomyces Species

Note Cite This: J. Nat. Prod. 2017, 80, 2825-2829

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Cytotoxic Dibohemamines D−F from a Streptomyces Species Bingya Jiang, Wei Zhao, Shufen Li, Hongyu Liu, Liyan Yu, Yixuan Zhang, Hongwei He, and Linzhuan Wu* Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Three dimeric analogues of bohemamines, dibohemamines D−F (1−3), together with dibohemamine A (4), were isolated from Streptomyces sp. CPCC 200497. Their structures were solved using a combination of mass spectrometry, 1D and 2D NMR spectroscopy, and CD. Dibohemamines D and E were new dimeric analogues of bohemamines, and dibohemamine F was a known compound obtained previously by semisynthesis. Dibohemamine F displayed potent cytotoxicity against cancer cell lines A549 and HepG2 with IC50 values of 1.1 and 0.3 μM, respectively. Dibohemamines D and E showed moderate cytotoxicity against cancer cell lines A549 and HepG2.

P

methyl groups (δH 1.17 (d, J = 6.6 Hz), 1.11 (d, J = 6.0 Hz), 1.25 (s), and 1.14 (s)), two pairs of vinyl methyl groups (δH 2.16 (d, J = 1.2 Hz), 2.16 (d, J = 1.2 Hz), 1.93 (d, J = 1.2 Hz), and 1.93 (d, J = 1.2 Hz)), two amide exchangeable protons at δH 9.80 (s) and δH 10.00 (s), five sp3-hybridized methine signals (δH 3.78 (d, J = 6.0 Hz), 3.70 (dd, J = 3.6, 0.6 Hz), and 3.67 (d, J = 3.0 Hz), 3.88 (dd, J = 8.4, 7.2 Hz), 3.92 (t, J = 3.0 Hz)), and two sp2-hybridized methine signals (δH 5.96 (t, J = 1.2 Hz), 5.96 (t, J = 1.2 Hz)). Two sp3-hybridized methylene signals could also be observed (Figure S4). Although the molecular formula (C29H38N4O6) of 1 contained 29 carbon atoms, the 13C NMR data of 1 resolved only 28 carbon signals. However, one of the carbon signals (at δC 118.1) results from two overlapping signals and was much more intense than other methine signals (such as δC 56.1 and δC 64.4 in 1), and there are two close signals at δC 118 in the 13 C NMR data of 2 and 3 (Figures S15, S25). Most of the carbon signals appeared as approximate pairs. Comparing these data with those of bohemamines reported in the literature showed that 1 may be a dimeric analogue of bohemamines.7−9,11 Further examination of the NMR data indicated that 1 had a set of signals very similar to those of bohemamine (designated

yrrolizidine alkaloids are usually considered to be plant secondary metabolites, typically from Boraginaceae, Compositae, and Leguminosae. They are hepatotoxic, genotoxic, or mutagenic toward humans.1−3 Pyrrolizidine-like compounds such as bohemamines have been reported as microbial secondary metabolites from actinomycetes.4−8 Dimers of bohemamines (dibohemamines A−C) with cytotoxic activity toward cancer cell lines were recently identified from a strain of Streptomyces, and additional dimers of bohemamines have been generated by semisynthesis.9 Streptomyces sp. CPCC 200497 was identified by us as a new producer of bohemamines and quinomycins with antibacterial and cytotoxic activities.10,11 The strain was also found to produce four compounds with UV absorption profiles very similar to bohemamines by HPLC analysis of ODS column fractions of an ethyl acetate extract of the strain (Figure S1).11 These compounds showed m/z 539, 539, 537, and 551 ([M + H]+) in the hyphenated MS analysis (Figure S2), approximately double the molecular mass of bohemamines. This paper describes the characterization of these dimeric analogues of bohemamines (including two new compounds) and their in vitro cytotoxicity against two cancer cell lines. Compound 1 was obtained as a white powder with UV absorption at 247, 285, and 346 nm. The molecular formula was determined as C29H38N4O6 using LC-HR-(+)ESIMS. The 1 H NMR data of 1 in DMSO-d6 indicated the presence of four © 2017 American Chemical Society and American Society of Pharmacognosy

Received: February 14, 2017 Published: October 16, 2017 2825

DOI: 10.1021/acs.jnatprod.7b00136 J. Nat. Prod. 2017, 80, 2825−2829

Journal of Natural Products

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Chart 1

Figure 1. Key HMBC and 1H−1H COSY correlations of dibohemamines D−F (1−3).

Figure 2. Key 2D-NOE correlations of dibohemamines D−F (1−3).

NOE correlations were observed between H-8 and H-6, H-8 and H-4, H-8″ and H-6″, H-6″ and H-5″a, and H-5″a and H4″, suggesting the relative configuration of 1 as shown in Figure 2, which is identical to that of bohemamine A and bohemamine C, respectively. The absolute configurations of bohemamines had been determined by Sundararaman et al. using the octant rule for cyclopentenone.7−9,12,13 The absolute configurations of dibohemamine D should be the same as that reported for the bohemamines, as its circular dichroism (CD) spectrum showed the same Cotton effects as bohemamines (Figure S13).7−9 The NMR chemical shifts of dibohemamine D were assigned completely by HSQC, COSY, and HMBC, as indicated in Table 1. Compound 2 was obtained as a white powder with UV absorption at 248, 286, and 350 nm. Its molecular formula was determined as C30H38N4O6 by LC-HR-(+)ESIMS. It is one carbon atom more than 1. The NMR signals of 2 were also in pairs, except the signals for a methyl group (δC/H 16.1/1.24) and a methine group (δC/H 21.6/3.37). The NMR data were similar to and more symmetrical than those of 1, which suggested that compound

as bohemamine A hereafter, a major component of this strain) and a set of signals very similar to those of bohemamine C. The bohemamine A unit in 1 was confirmed by the COSY correlation of H-4/H-9 and the key HMBC correlations of H-8 to C-1/C-6/C-7, H-4 to C-3/C-5/C-6, H-9 to C-4/C-5, 3-NH to C-3/C-1′, H-2′ to C-1′/C-3′/C-4′/C-5′, and H-4′ to C-3′/ C-5′ (Figures 1 and S6−S9). The bohemamine C unit in 1 was also confirmed by the corresponding COSY and HMBC correlations (Figure 1). Both the bohemamine A unit and the bohemamine C unit in 1 lost the signals for the methine group (C-2 and C-2″). At the same time, 13C NMR and DEPT data of 1 indicated three new signals, two (δC 103.3, 103.9) for two nonprotonated olefinic carbons and one (δC 13.6) for a methylene carbon. Thus, the methylene carbon was proposed to serve as the bridge linking the two olefinic carbons (C-2 and C-2″) of the two units. The methylene linkage was verified by the HMBC correlations of H10 to C-1/C-2/C-3/C-1″/C-2″/C-3″ (Figure 1). Compound 1 consists of a skeleton of bohemamine A−methylene−bohemamine C and was designated as dibohemamine D, a new dimeric analogue of bohemamines. 2826

DOI: 10.1021/acs.jnatprod.7b00136 J. Nat. Prod. 2017, 80, 2825−2829

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at C-2 and C-2″, and this was verified by the 1H−1H COSY correlations of the methyl group (δC/H 16.1, 1.24) to the methine group (δC/H 21.6/3.37) and HMBC correlations of H10/C-1, C-2, C-3, C-1′, C-2′, C-3′ and H-11/C-2, C-2″, C-10 (Figure 1, Figures S14−S19). Thus, compound 2 had a scaffold of bohemamine A−methylmethine−bohemamine A. It was designated as dibohemamine E, another new dimeric analogue of bohemamines. The NOE correlations between H-8 and H-4, H-8 and H-6, H-8″ and H-4″, and H-8″ and H-6″ (Figure 2) indicated that 2 had the same relative configuration as bohemamine. The absolute configuration of dibohemamine E should be the same as dibohemamine D, as its CD spectrum showed the same Cotton effects as dibohemamine D (Figure S23). Compound 3 was considered most likely a regioisomer of dibohemamine D, as it had a molecular formula of C29H38N4O6 according to LC-HR-(+)ESIMS (Figure S2), the same as dibohemamine D. After careful interpretation of 1D and 2D NMR data, in particular the COSY correlations from the H-4″ methine proton to both the methyl protons (H-9″) and the adjacent oxygenated methine proton (H-5″) that coupled further to methylene protons (H-6″), it was concluded that compound 3 contained a scaffold of bohemamine A− methylene−bohemamine B (Figure 1, Figure S24−S29) and was designated as dibohemamine F. Compound 3 had been obtained before by semisynthesis, i.e., in vitro incubation of 2hydroxymethylbohemamine A and bohemamine B in tetrahydrofuran,9 and here it is reported as a natural metabolite. The NMR data of dibohemamine F were assigned completely as indicated in Table 1. The relative and absolute configurations of 3 were also determined as shown in Figure 2 by ROSEY correlations and CD spectrum (Figure S33). Compound 4 showed a very similar UV absorption profile to dibohemamines D−F. Its molecular formula was determined to be C29H36N4O6 according to LC-HR-(+)ESIMS, indicating its structure to be bohemamine A−methylene−bohemamine A, the same as dibohemamine A previously reported.9 The 13C NMR data of 4 gave only partial carbon signals owing to insufficient quantities, but these signals still confirmed the above structure deduction (Figures S34, S35). Thus, compound 4 was dibohemamine A. Catabolism of carbohydrates by bacteria may generate small amounts of formaldehyde and acetaldehyde. Both formaldehyde and acetaldehyde as electrophiles are able to attack C-2 of bohemamines to form dimeric bohemamines by a Baylis−Hillman reaction.9 To further prove this, compound 2 (the dimer of bohemamine A) was synthesized by us with in vitro incubation of bohemamine A with acetaldehyde (Figures S36−S40). Therefore, these dimers (dibohemamines A−F) may appear as metabolites in bohemamines-producing strains such as Streptomyces sp. CPCC 200497 and Streptomyces spinoverrucosus by this route.9,11 Bohemamines were essentially inactive toward cancer cell lines.6 However, some dimers of bohemamines showed moderate or potent cytotoxicity against cancer cell lines.9 An in vitro cytotoxicity assay of dibohemamines D−F and bohemamines A and B against A549 (lung cancer cell line) and HepG2 (liver cancer cell line) was performed, with doxorubicin as positive control. Dibohemamines D and E displayed moderate cytotoxicity against the two cancer cell lines. Dibohemamine F possessed potent cytotoxicity against the two cancer cell lines tested, with IC50 values of 1.1 and 0.3 μM, respectively. Bohemamines A and B were inactive. This

Table 1. 1H and 13C NMR Data for Dibohemamines D−F (1−3)a dibohemamine D (1) position

δC

1 2 3 4

199.4 103.4 166.2 56.1

5

64.4

6

55.8

7 8 9

75.5 20.0 14.1

10

13.4

δH (J in Hz)

3.78 q (6.0) 3.70 dd (3.6, 0.6) 3.67 d (3.0) 1.25 s 1.17 d (6.6) 2.64 (2H, m)

11

dibohemamine E (2) δC 199.3 105.8 165.9 56.0 64.3

56.0 75.2 20.1 14.0 21.6

16.1

1′ 2′ 3′ 4′

163.0 118.1 156.7 27.7

5′

20.4

3-NH 1″ 2″ 3″ 4″

199.4 104.0 163.4 55.0

5″

43.8

6″

73.2

5.96 t (1.2) 1.93 d (1.2) 2.16 d (1.2) 9.80 s

3.88 dd (8.4, 7.2) 2.62 m, 1.75 d (13.8) 3.92 t (3.0)

164.0 117.8 157.4 27.8 20.4

198.9 107.1 165.4 56.2 64.3

56.1

δH (J in Hz)

3.68 d (6.0) 3.72 dd (4.2, 1.2) 3.68 d (6.0) 1.25 s 1.16 d (6.6) 3.37 q (6.6)

dibohemamine F (3) δC 199.4 103.3 166.2 56.0

δH (J in Hz)

3.80 q (6.6)

64.4

3.72 dd (3.6, 0.6)

55.8

3.69 d (3.0)

75.5 19.7 14.0

1.26 s 1.17 d (6.6)

13.6

2.63 d (15.6), 2.72 d (15.6)

1.24 d (6.0) 5.92 s 1.93 d (0.6) 2.16 d (0.6) 9.63 s

3.80 m 3.72 dd (4.2, 1.2) 3.69 d (6.0)

164.0 117.8 157.4 27.7

1.93 d (3.0)

20.4

2.17 d (3.0)

5.92 s

9.93 s 203.2 103.9 166.4 58.7

3.85 q (6.6)

72.3

4.55 m

36.0

1.42 t (14.4) 1.79 dd (6.0, 12.0)

7″ 8″ 9″

81.7 23.4 20.5

1‴ 2‴

164.0 118.1

3‴ 4‴

157.0 27.7

5‴

20.4

3″-NH

1.14 s 1.11 d (6.0) 5.96 t (1.2)

1.93 d (1.2) 2.16 d (1.2) 10.00

75.2 20.1 14.2 163.8 117.8 157.0 27.8 20.4

1.25 s 1.16 d (6.0) 5.95 t (1.2) 1.91 d (1.2) 2.16 d (1.2) 10.44

71.9 26.0 10.1

1.20 s 0.74 d (6.6)

163.0 118.0

5.99 s

157.0 27.7

1.93 d (3.0)

20.4

2.17 d (3.0) 10.30

a1

H and 13C NMR spectra data (δ) were obtained at 600 and 125 MHz, respectively, on a VNS-600 spectrometer or Bruker 600 spectrometer and measured in DMSO-d6 at room temperature.

2 was also a dimeric analogue of bohemamines containing two identical bohemamine units. A careful analysis of 1D and 2D NMR data, particularly the COSY correlations of H-9/H-4/H-5/H-6 and H-9″/H-4″/H5″/H-6″, confirmed that compound 2 included two sets of NMR signals produced by a pair of bohemamine A units. The two identical units were connected by a methylmethine bridge 2827

DOI: 10.1021/acs.jnatprod.7b00136 J. Nat. Prod. 2017, 80, 2825−2829

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NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, see Table 1; ESI(+)HRMS m/z 539.2866 [M + H]+ (calcd for C29H39N4O6, 539.2864, ppm = 0.3). Dibohemamine E (2): white, amorphous solid; [α]20 D −60.1 (c 0.013, MeOH); UV (MeOH) λmax (log ε) 248 (3.37), 285 (3.11), and 250 (3.09) nm; CD (MeOH) λmax 349 (Δε +29.1), 287 (Δε −91.6), 247 (Δε +31.3); IR (KBr) νmax 3285, 3187, 3196, 2975, 2926, 1711, 1638, 1562, 1511, 1451, 1420, 1377, 1227, 1191, 1132, 856, 731, 711, 633 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSOd6, 125 MHz) data, see Table 1; ESI(+)HRMS m/z 551.2864 [M + H]+ (calcd for C30H39N4O6, 551.2864, ppm = 0.04). Dibohemamine F (3): white, amorphous solid; [α]20 D −25.5 (c 0.036, MeOH); UV (MeOH) λmax (log ε) 247 (3.21), 284 (2.94), and 346 (2.91) nm; CD (MeOH) λmax 349.7 (Δε +33.1), 287.5 (Δε −121.2), 246 (Δε +50.3); IR (KBr) νmax 3232, 2921, 2851, 1712, 1641, 1451, 1381, 1299, 1225, 1127, 1075, 850, 801, 722, 629 cm−1; 1 H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, see Table 1; ESI(+)HRMS m/z 539.2865 [M + H]+ (calcd for C29H39N4O6, 539.2864, ppm = 0.1). Dibohemamine A (4): white, amorphous solid; ESI(+)HRMS m/ z 537.2705 [M + H]+ (calcd for C29H37N4O6, 537.2708, ppm = −0.5). Cytotoxicity Assay. Dibohemamines D−F (1−3) were evaluated for cytotoxicity against A549 (lung cancer cell line) and HepG2 (liver cancer cell line) by the SRB assay. All compounds were dissolved in DMSO, with doxorubicin serving as a positive control in the assay.

result, together with previously published data, seems to support that dimerization of bohemamines results in active compounds.6,9 It needs to be determined whether dimers of bohemamines possess hepatotoxic, genotoxic, or mutagenic effects toward animals or humans, just as their monomers do. Table 2. Cytotoxicity of Dibohemamines D−F (1−3) and Bohemamines A and B against Cancer Cell Lines cytotoxicity (IC50, μM)

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compound

A549

HepG2

dibohemamine D (1) dibohemamine E (2) dibohemamine F (3) bohemamine A bohemamine B doxorubicin

20.2 39.2 1.1 290.2 501.6 1.4

7.3 10.8 0.3 29.7 180.7 0.5

EXPERIMENTAL SECTION

General Experimental Procedures. Analytical HPLC was conducted on an Agilent HPLC system with 1200 Quat-Pump and PAD detector. Preparative HPLC was accomplished on a Shimadzu LC-20AT with PAD detector. Column chromatography (CC) was performed using YMC*GEL ODS-A-HG (12 nm, S-50 μm, AAG12S50, YMC Co., LTD, Japan) or Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Preparative TLC was performed with HSGF254 high-performance silica gel preparative plates (Yantai Chemical Industry Research Institute, Yantai, China). MS and HRMS were analyzed by LTQ XL and Orbitrap XL from Thermo Fisher Scientific, and QTOF/MS from AB SCIEX. The 1D and 2D NMR were assayed in DMSO-d6 using a VNS-600 or Bruker-600 spectrometer (600 MHz for 1H and 150 MHz for 13C), with solvent peaks used as references. Optical rotations were measured using a Rudolph Research Autopol III automatic polarimeter. UV spectra were obtained using a Cary 300 spectrometer, and CD spectra were recorded using a JASCO J-815. IR spectra were recorded using a Nicolet 5700 FTIR microscope (transmission). Fermentation. Fresh spores of Streptomyces sp. CPCC 200497 were spread on 4000 plates of ISP2 medium (80 L; medium composition: yeast extract 0.4%, malt extract 1.0%, glucose 0.4%, and agar 1.5%) and incubated at 28 °C for 8 d to develop mycelia lawns. Extraction and Isolation of Dibohemamines D−F and C. The plate cultures were pooled and extracted three times with equal volume of ethyl acetate (EtOAc). The EtOAc extracts were combined and evaporated under reduced pressure to yield a dark brown residue (10 g). The residue was dissolved in H2O (100 mL), loaded on a YMC*GEL ODS-A-HG column (36 × 460 cm), and eluted with H2O (2 L) and then 10−100% EtOH (10% up for each stepwise gradient, 2 L for each step) to give 11 fractions (F1−11; fraction F2 could be used for bohemamine A purification as it contained bohemamine A as the main component, Figure S41). Fraction F6 was applied onto a Sephadex LH-20 column and eluted by a mixture of chloroform− methanol (1:1) to yield fractions F6-1, F6-2, F6-3, and F6-4. Fraction F6-4 (190.5 mg) was then loaded on HSGF254 high-performance silica gel preparative plates and developed with a mixture of CHCl3− MeOH (1:1), which yielded four fractions, F6-4-1, F6-4-2, F6-4-3, and F6-4-4 (Figure S3). Dibohemamine D (1, 0.4 mg), dibohemamine E (2, 1.2 mg), dibohemamine F (3, 1.4 mg), and dibohemamine A (4, 0.1 mg) were obtained from fraction F6-4-2, F6-4-4, F6-4-1, and F6-43, respectively, by the final polishing of reversed-phase preparative HPLC (Agilent ZorBax SB-C18, 5 μm, 9.4 × 250 mm). Dibohemamine D (1): white, amorphous solid; [α]20 D −33.6 (c 0.011, MeOH); UV (MeOH) λmax (log ε) 247 (3.15), 285 (2.87), and 346 (2.84) nm; CD (MeOH) λmax 345 (Δε +23.3), 286 (Δε −80.0), 246 (Δε +29.7); IR (KBr) νmax 3214, 2919, 1712, 1640, 1562, 1506, 1452, 1378, 1294, 1225, 1198, 1131, 1078, 855, 721, 632 cm−1; 1H

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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.7b00136. TLC and HPLC analysis of fraction F6-4 (from EtOAc extract) of Streptomyces sp. CPCC 200497; MS and NMR spectra of dibohemamines D−F and dibohemamine A; CD spectra for dibohemamines D and E (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-10-63165283. Fax: 86-10-63017302. ORCID

Bingya Jiang: 0000-0002-7415-3179 Liyan Yu: 0000-0002-8861-9806 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Nuclear Magnetic Resonance Center (Institute of Materia Medica, CAMS & PUMC) and Analytical Center of Drugs (Institute of Medicinal Biotechnology, CAMS & PUMC) for the NMR, MS, and CD analyses. This work was supported by the National Natural Science Foundation of China (81302676 and 81321004), CAMS Initiative for Innovative Medicine (CAMS-I2M-3-012), National Infrastructure of Microbial Resources (No. NIMR-20163), National Mega-project for Innovative Drugs (2012ZX09301002-001-016, 2012ZX09301002-003), Fundamental Research Funds for the Central Universities (2012N09), and PUMC Youth Fund (3332013090).

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