Cybastacines A and B: Antibiotic Sesterterpenes from a Nostoc sp

Note Cite This: J. Nat. Prod. 2018, 81, 410−413

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Cybastacines A and B: Antibiotic Sesterterpenes from a Nostoc sp. Cyanobacterium Alfredo H. Cabanillas,† Víctor Tena Pérez,† Santiago Maderuelo Corral,‡ Diego Fernando Rosero Valencia,‡ Antera Martel Quintana,§ Montserrat Ortega Doménech,*,‡ and Á ngel Rumbero Sánchez*,† †

Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain Valoralia I más D SLU, Tres Cantos, 28760 Madrid, Spain § Banco Español de Algas, Universidad de las Palmas de Gran Canaria, 35214 Telde, Las Palmas, Canary Islands, Spain ‡

S Supporting Information *

ABSTRACT: Cybastacines A (1) and B (2) were discovered as a novel pentacyclic sesterterpenoid−alkaloid skeleton structure, with a guanidinium group. These molecules were isolated from a Nostoc sp. cyanobacterium collected in the Canary Islands. Their structures were elucidated primarily by a combination of spectroscopic analyses and X-ray diffraction. These compounds showed antibiotic activities against several clinically relevant bacterial strains.

C

and cybastacine B (2), which exhibit an unprecedented pentacyclic guanidinium-bearing sesterterpene skeleton (nostorene). Herein, we report the isolation and structure elucidation of these compounds from the extract of Nostoc sp. (BEA-0956). The strain was selected on the basis of a biological screening assay against pathogenic bacterial species (Supporting Information), exhibiting significant activity against Actinomycetales (Nocardia spp., Mycobacterium spp., and related species), Streptococcus spp., Enterococcus spp., and Staphylococcus spp. The freeze-dried biomass was extracted by repeated maceration with CH2Cl2/MeOH. Further separation and purification of the organic fraction led to the isolation of two new compounds, cybastacine A (1) and cybastacine B (2). Cybastacine A (1), an optically active compound, was obtained as colorless crystals from CH2Cl2/EtOH (7:3). HRESIMS yielded an [M + H]+ peak m/z 414.3478, consistent with the formula C26H44N3O, and implied seven degrees of unsaturation. Analysis of 13C NMR, DEPT, and HSQC data showed the presence of 23 sp3 carbon signals (six CH3, nine CH2, three CH, and five C) and three sp2 carbon signals (Table 1). The 1H NMR and DEPT spectra of 1 revealed an oxygenated methine proton at δH 4.21 (1H; dd, J = 4.5 1.8 Hz; H-11), two methine protons at δH 1.37 (1H; dd, J = 13.0, 2.0 Hz; H-5) and δH 2.74 (1H; dd, J = 11.9, 4.4 Hz; H-18); and six singlet signals for methyl groups at δH 0.87 (3H, s, H-22), 0.89 (3H, s, H-20),

yanobacteria are a group of photosynthetic prokaryotes that likely appeared on Earth about 3 billion years ago. They have colonized all possible environments and are largely present in oceans and freshwater. Besides the fundamental interest in these photosynthetic prokaryotes that fix CO2 and nitrogen, researchers have been fascinated by their ability to produce a wide range of diverse and biologically active secondary metabolites.1 Various compounds have been identified as new potent lead structures for the development of novel pharmaceuticals against infectious diseases, cancer, and other diseases.2−5 Approximately 25% of all cyanobacterial metabolites are derived from the order Nostocales, predominately from the genus Nostoc.6 The major structural classes found in Nostoc spp. are peptides, glycopeptides, and alkaloids. However, terpenes are not commonly found in cyanobacteria.7,8 A few examples are tolypodiol,9 noscomin,10 tasihalide,11 and bacteriohopanepolyols.12 The only guanidine-bearing-sesterterpene compound reported from cyanobacteria is the antimicrobial scytoscalarol from a Scytonema sp.13 In our search for antimicrobial compounds from cyanobacteria, we have isolated cybastacine A (1)

Received: July 24, 2017 Published: February 12, 2018 © 2018 American Chemical Society and American Society of Pharmacognosy

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DOI: 10.1021/acs.jnatprod.7b00638 J. Nat. Prod. 2018, 81, 410−413

Journal of Natural Products

Note

Table 1. NMR Spectroscopic Data (1H 500 MHz, 13C 125 MHz, CD3OD) for Cybastacine A (1) and Cybastacine B (2) cysbastacine A (1) position

δC, type

1ax 1eq 2ax 2eq 3ax 3eq 4 5 6ax 6eq 7ax 7eq 8 9 10 11 12ax 12eq 13 14 15ax 15eq 16ax 16eq 17 18 19 20 21 22 23 24 25ax 25eq 1′

38.3, CH2 20.1, CH2 42.9, CH2 34.2, C 51.1, CH 20.8, CH2 27.6, CH2 136.0, C 141.0, C 40.2, C 63.2, CH 40.6, CH2 36.6, C 44.8, C 28.1, CH2 36.8, CH2 53.3, 41.3, 33.7, 22.3, 22.2, 23.0, 20.7, 23.4, 38.9,

C CH CH3 CH3 CH3 CH3 CH3 CH3 CH2

154.3, C

cybastacine B (2)

δH, mult. (J in Hz) 1.66, 1.83, 1.63, 1.48, 1.43, 1.24, 1.37, 1.55, 1.83, 2.01, 2.31,

m m m m m ddd (13.2, 13.2, 4.3) dd (13.0, 2.0) m m ddd (17.3, 11.8, 7.3) dd br (17.3, 6.2)

4.21, dd (4.5, 1.8) 1.66, dd (15.2, 4.5) 1.78, d br (15.2)

1.83, 1.51, 1.36, 1.63,

m m m m

2.74, 0.92, 0.89, 1.02, 0.87, 0.89, 1.30, 3.19, 3.75,

dd (11.9, 4.4) s s s s s s dd (11.9, 11.6) dd (11.6, 4.4)

position

δC, type

1ax 1eq 2ax 2eq 3ax 3eq 4 5 6ax 6eq 7

39.3, CH2

8 9 10 11 12

141.3, C 58.1, CH 38.1, C 65.3, CH 42.3, CH2

13 14 15

19.9, CH2 43.5, CH2 34.0, C 51.8, CH 26.0, CH2 124.8, CH

δH, mult. (J in Hz)

ROE

1.28, 1.78, 1.63, 1.52, 1.42, 1.24,

m m m m m m

H-9 H-11 H-19, 21

1.41, 2.25, 2.01, 5.78,

dd (12.2, 5.5) m m dd (5.0, 2.6)

H-9,18,20 H-19, 21 H-20 H-6ax, 6eq, 15, 22

H-19

2.23, m

H-1ax, 5

3.93, ddd, (4.5, 4.0, 4.0) 1.65, d (4.0)

H-1eq, 12, 21 H-11,22,23,25eq

37.3, C 44.0, C 34.3, CH2

1.81, m

H-7, 22, 24

16

35.9, CH2

1.60, m

H-18, 24

17 18 19 20 21 22 23 24 25ax 25eq 1′

53.5, 44.1, 33.2, 21.6, 14.5, 28.1, 20.8, 23.2, 39.0,

2.21, 0.87, 0.90, 0.78, 0.97, 0.81, 1.30, 3.23, 3.82,

H-5, 16, 25eq H-2ax, 3eq, 6ax, 21 H-5, 6eq H-2ax, 6ax, 11, 19 H-7, 12, 15, 23 H-12, 22, 24, 25ax H-16−23, 25ax H-23, 24 H-12, 18

C CH CH3 CH3 CH3 CH3 CH3 CH3 CH2

dd (11.9, 4.4) s s s s s s dd (11.9, 11.9) dd (11.9, 4.4)

154.4, C

0.89 (3H, s, H-23), 0.92 (3H, s, H-19), 1.02 (3H, s, H-21), and 1.30 (3H, s, H-24). The 13C NMR spectrum (Table 1) contained 26 carbon signals, three of which appeared above 100 ppm. Two of these were assigned to an endocyclic double bond (C-8 δC 136.0 and C-9 δC 141.0). The carbon signal at δC 154.3 ppm (C-1′) was correlated with a guanidinium group on the basis of the chemical shift as well as consideration of the formula.14 Two of the seven degrees of unsaturation were assigned for the guanidinium group and the endocyclic double bond. It suggested a pentacyclic sesterterpene with a six-membered guanidinium heterocycle, cybastacine A (1). The planar structure of 1 was deduced by a combination of 1D and 2D NMR experiments (Table 1, Figure 1). The absolute configuration was fixed by an X-ray diffraction analysis. The correct configuration of cybastacine A is shown in Figure 2. We can observe that methyl groups C-21, C-22, C-23, and C-24 and the C-25 methylene are all in the β-plane. The hydroxy group at C-11 is in the α-plane; therefore the absolute configuration is 5S, 10S, 11R, 13R, 14S, 17R, and 18S. Rings A/B and D/E exhibited trans-fused configurations. Rings C/D presented a cis-fused configuration. X-ray diffraction showed nitrate as the counterion of the guanidinium functional group. This can be explained by the fact that the three N−C bonds

Figure 1. Planar structure of 1 with supporting 2D NMR correlations.

in the guanidinium moiety displayed the typical sp2 hybridization bond length of 1.33 Å, resulting in positive charge delocalization.15 Cybastacine B (2) was obtained as a white solid and was revealed to have the molecular formula C26H44N3O from the HRESIMS [M + H]+ ion at m/z 414.3472 and 1H and 13 C NMR spectroscopic data. The general features of the 1H and 13 C NMR spectra (Table 1) of cybastacine B (2) exhibited a close similarity of the carbon chemical shifts to those of 1, with the exception of the C-7 (δC 124.8) and C-9 (δC 58.1) carbon signals and the proton resonance at δH 5.78 (1H; dd, J = 5.0, 2.6 Hz; H-7). These changes indicated the presence of the 411



Note

Table 2. Cybastacines A (1) and B (2) MIC Valuesa control

Figure 2. X-ray structure of cybastacine A (1).

double bond at C-7 and C-8, instead of C-8 and C-9 in 1. The relative configuration was determined by analysis of the 2D ROESY spectrum (Figure 3 and Table 1). ROESY correlations

cybastacine A (1)

cybastacine B (2)

imipenem

vancomycin

clinical strain (n = 5)

MIC 50 (μg/mL)

MIC 50 (μg/mL)

MIC 50 (μg/mL)

MIC 50 (μg/mL)

M. abscesuss N. carnea N. cyriacigeorgica T. pulmonis S. pyogenes E. faecalis E. faecium S. aureus S. epidermidis

≤16 ≤16 ≤16

≤4 ≤4 ≤4

≤8 ≤8 ≤8

≤8 ≤32 ≤32 ≤32 ≤32 ≤32

≤2 ≤4 ≤4 ≤4 ≤4 ≤4

≤8 ≤16 ≤8 ≤8 ≤8 ≤8

a

n = 5 different strains/species. Control not tested. MIC data are shown as average values.

In summary, we have obtained two new sesterterpenoid alkaloids, cybastacine A (1) and cybastacine B (2), from the CH2Cl2/ MeOH extract of a Nostoc sp. Compounds 1 and 2 showed moderate in vitro antibiotic activities. Sesterterpenes are rare among microbial secondary metabolites, with only one report of a previous alkaloid−sesterterpene found in cyanobacteria.13 The discovery of 1 and 2 represents a significant addition to the novel chemical structures active against resistant bacterial strains.16

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Figure 3. Key ROESY correlations of cybastacine B (2).

EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotation was determined on a PerkinElmer 241 polarimeter. Infrared spectrometry was performed on an Agilent Cary 630 FTIR. NMR experiments were performed on a Bruker Avance DRX 500 spectrometer operating at 500 MHz (1H) or 125 MHz (13C). The deuterated solvent was methanol-d4 as specified. Spectra were calibrated by assignment of the residual solvent peak to δH 3.31 and δC 49.0 for methanol-d4. HREIMS analyses were performed using a QSTAR XL quadrupole TOF mass spectrometer. MS samples were prepared in MeOH. X-ray crystallographic data for the structure determination of cybastacine A were recorded at 298(2) K on a Bruker D8 Venture (Cu Kα). Molecular graphics were produced with Mercury 3.7. TLC was performed using Merck silica gel 60-F254 plates. Developed chromatograms were visualized by UV absorbance (254 nm) or through application of heat to a plate stained with phosphomolybdic acid (H3PMo12O40). Manual flash chromatography was performed with flash-grade silica gel (60 μm) and the indicated eluent in accordance with standard techniques. Biological Material. Nostoc sp. strain (BEA-0956) was isolated from a sample collected in a cave wall in Montañoń Negro, Gran Canaria (Canary Islands, Spain), available from Banco Español de Algas (Canary Islands, Spain), and grown under axenic conditions by Valoralia I más D. The 16S rRNA gene sequence was deposited in GenBank (accession no. KY346979). The cyanobacteria were cultivated in 2 L flasks containing BG11 medium. The cultures were grown by bubbling with air and incubated at 23 ± 1 °C. The culture was illuminated only with environmental light. The cyanobacterial cultures were harvested after 28 days; the biomass of cyanobacteria was harvested by centrifugation and freeze-dried. Extraction and Isolation. The freeze-dried biomass (34.66 g) was extracted by repeated maceration with CH2Cl2/MeOH (1:1) to yield 2.24 g of extract. The extract showed significant antibacterial activities. A portion of the organic extract (2.10 g) was subjected to antimicrobial bioassay-guided silica gel column chromatography, using a stepwise gradient of CH2Cl2/MeOH to produce eight fractions (A−H). Fractions F and G, which eluted with CH2Cl2/MeOH (10:1), were found to be the most active fractions against microbes. These fractions

observed from H3-21 to H3-19 and H-11, as well as from H3-23 to H3-22 and H3-24, indicated all these groups to be in the β-plane. This led to the determination of the relative configuration as 10R*, 11R*, 13R*, 14S*, and 17R*. Correlations between H-5 and both H3-20 and H-9, as well as between H-18 and H-5, indicated all of these groups to be in the α-plane and determined the relative configuration as 5S*, 9R*, 10S*, and 18S*. The ring junctions A/B and C/D were trans and the D/E junction was cis. This led to a relative configuration of cybastacine B (2) of 5S*, 9R*, 10S*, 11R*, 13R*, 14S*, 17R*, and 18S*. Disk diffusion methods for antimicrobial susceptibility testing of the extract of this Nostoc sp. (2 mg) showed inhibitions (10−15 mm) for Actinomycetales (Nocardia spp., T. pulmonis, Mycobacterium spp.), Enterococcus spp., Streptococcus spp., and Staphylococcus spp. Active fractions (500 μg) improved this inhibition in bacteria tested (15−22 mm). In view of these results, a more accurate antibacterial activity assay was performed, based on detecting antibiotic activity with the two compounds. The in vitro activities of cybastacines A (1) and B (2) against clinically isolated strains, expressed in terms of MIC values, are listed in Table 2. Cybastacine A (1) exhibited weak antibiotic activity against the species tested, with MIC values (≤16 to 32 μg/mL) that were not comparable to reference antimicrobial MICs, except for T. pulmonis, with a similar MIC (8 μg/mL) to imipenem. However, cybastacine B (2) exhibited strong activity against clinical isolates of Nocardia spp., M. abscessus, Enterococcus spp., and Staphylococcus spp., with MICs of ≤4 μg/mL, and with a surprising MIC of ≤2 μg/mL against T. pulmonis. Interestingly, two strains of coagulase-negative Staphylococcus aureus tested in this study showed a lower MIC for 2 compared to their MICs for vancomycin. All bacterial strains were highly susceptible to cybastacine B (Table 2). 412



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were subjected to additional bioactivity-guided fractionation using a silica gel chromatography column eluting with CH2Cl2/MeOH (10:1) to afford two pure antibiotic compounds, cybastacines A (1) (445 mg, 21.20%) and B (2) (15 mg, 0.72%). Cybastacine A (1): colorless crystals from CH2Cl2/EtOH (7:3), mp >320 °C (dec); [α]25D +45 (c 0.1, MeOH); IR (neat) νmax 3243.51, 2936.52, 1671.89, 1618.28, 1393.70 cm−1; 1H NMR and 13C NMR, Table 1; HRESIMS m/z 414.3478 [M + H]+ (calcd for C26H44N3O, 414.3479). Cybastacine B (2): white solid; [α]25D +32 (c 0.1, MeOH); IR (neat) νmax 3244.12, 2935.94, 1670.64, 1618.53, 1391.30 cm−1; 1 H NMR and 13C NMR, Table 1; HRESIMS m/z 414.3472 [M + H]+ (calcd for C26H44N3O, 414.3479). Crystallographic Data. Cybastacine A (1): C26H44N3O, 414.35. A clear colorless prismatic-like specimen of approximate dimensions 0.120 mm × 0.120 mm × 0.140 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured in a Bruker D8 Venture diffractometer equipped with a Cu high-brilliance microfocus sealed tube, λ(Cu Kα) = 1.541 78 Å. The integration of the data using a monoclinic unit cell yielded a total of 18 557 reflections to a maximum θ angle of 66.59° (0.84 Å resolution), of which 4394 were independent (average redundancy 4.223, completeness = 99.1%, Rint = 4.42%, Rsig = 3.64%), and 3890 (88.53%) were greater than 2σ(F2). The final cell constants of a = 7.5796(3) Å, b = 11.3035(5) Å, c = 14.7799(7) Å, β = 92.183(2)°, and volume = 1265.36(10) Å3 are based upon the refinement of the XYZ-centroids of reflections above 20 σ(I). The structure was solved and refined using the Bruker SHELXTL software package, using the space group P21, with Z = 2. The final anisotropic full-matrix least-squares refinement on F2 with 314 variables converged at R1 = 4.29% for the observed data and wR2 = 12.12% for all data. The goodness-of-fit was 1.021. The largest peak in the final difference electron density synthesis was 0.208 e−/Å3, and the largest hole was −0.163 e−/Å3 with an RMS deviation of 0.051 e−/Å3. On the basis of the final model, the calculated density was 1.251 g/cm3 and F(000), 520 e−. Crystallographic data for cybastacine A have been deposited with the Cambridge Crystallographic Data Centre (CCDC 1819365). Antibacterial Susceptibility Tests. The descriptions (internal references and the source of isolation) of all strains used for biological evaluation are detailed in the Supporting Information. A bacterial cell suspension in sterile saline was prepared from a culture of 24−72 h, depending on bacterial species, in Mueller−Hinton agar with 5% sheep blood. Each suspension was adjusted to a fixed size inoculum of (1−5) × 108 CFU/mL.17 The Kirby−Bauer agar disk diffusion method was utilized to determine the sensitivity or resistance of pathogenic bacteria against the Nostoc extract, subfractions, and cybastacines A and B. After 18 to 72 h of incubation at 37 °C, with or without CO2, under aerobic or anaerobic conditions, depending on the bacterial species, halos of growth inhibition were obtained and evaluated. The absence of growth around the disks is an indirect measure of the ability of this compound to inhibit an organism.18 Minimum inhibitory concentrations (MICs) were used to determine the sensitivity or resistance of pathogenic bacteria against cybastacines A and B, following a standardized methodology.19 MIC results were analyzed following “The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters”, Version 7.0, 2017; http://www.eucast.org.

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Note

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Á ngel Rumbero Sánchez: 0000-0003-2713-6023 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. J. A. Sáez-Nieto, N. Garrido, and M. A. Fernández, from the National Center for Microbiology, Health Institute Carlos III, Majadahonda, Madrid, Spain, for their technical assistance with antibacterial tests. Also we are especially grateful to the NMR and X-ray departments from SIdI of Universidad Autónoma Madrid and D. ChosquecilloLazarte from IACT-CSIC, Armilla, Granada.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00638. Experimental details including IR, HRESIMS, and NMR (1H, 13C, COSY, HMBC, HSQC, HSQC-TOCSY, and ROESY) spectra of compounds 1 and 2, as well as strains for biological testing (PDF) X-ray data for compound 1 (CIF) 413


Cybastacines A and B: Antibiotic Sesterterpenes from a Nostoc sp

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