Letter Cite This: Org. Lett. 2018, 20, 6037−6040
pubs.acs.org/OrgLett
Streptoglycerides A−D with a Rare 6/5/5 Tricyclic Ring Skeleton from a Marine Actinomycete Streptomyces species Byeoung-Kyu Choi,†,‡ Shin-Young Park,§ Dong-Kug Choi,§ Bora Shin,∥ Yern-Hyerk Shin,∥ Dong-Chan Oh,∥ Hwa-Sun Lee,‡ Hyi-Seung Lee,‡ Yeon-Ju Lee,‡ Jong Seok Lee,‡ Ji Hoon Lee,‡ and Hee Jae Shin*,†,‡ †
Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon 34113, Republic of Korea Marine Natural Products Chemistry Laboratory, Korea Institute of Ocean Science and Technology, 385 Haeyang-ro, Yeongdo-gu, Busan 49111, Republic of Korea § Department of Applied Life Science, Graduate School of Konkuk University, Chungju 27478, Republic of Korea ∥ Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
Org. Lett. 2018.20:6037-6040. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 10/05/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Four novel secondary metabolites possessing a unique 6/5/5 tricyclic ring system, streptoglycerides A−D (1−4), were isolated from a marine actinomycete Streptomyces sp. derived from a mangrove sample collected on Kosrae Island. Their structures and absolute configurations were elucidated by spectroscopic data and electronic circular dichroism data. Streptoglyceride C (3) showed a weak inhibitory effect on the production of nitric oxide in BV-2 microglia cells.
T
he discovery of new secondary metabolites from marine sources has attracted increasing attention because of their chemical diversity and potential applications as lead compounds for the treatment of human diseases.1−3 Marine actinobacteria are well-known as an important source of bioactive natural products.4,5 Among bioactive secondary metabolites produced by microorganisms, a considerable portion of compounds are produced by Streptomyces species.6 The novel compounds from marine Streptomyces sp. have been researched as a wide range of clinical leads such as antifungals, antivirals, antitumorals, antihypertensives, antibiotics, and immunosuppressives.7 In our continuous research toward the discovery of new lead compounds from marine microorganisms, we encountered a Streptomyces strain isolated from a mangrove sample collected on Kosrae Island. Through a mass-scale culture (60 L) and extensive spectroscopic analysis, four new compounds, streptoglycerides A−D (1−4) possessing a unique ring system (Figure 1), were obtained from the strain. Microglial cells are commonly the macrophage of the brain that produces appropriate responses against brain injury or infection.8 Uncontrolled and excessive activation of microglia often contributes to inflammation-mediated neurodegeneration.9 Therefore, downregulation of microglial activation may lead the development of therapeutic agents for neurodegenerative disorders.10 Thus, it is imperative to search new lead compounds to treat related diseases such as Alzheimer’s © 2018 American Chemical Society
Figure 1. Structures of streptoglycerides A−D (1−4).
disease and Parkinson’s disease.11 Herein we report the isolation, structure elucidation, and bioactivities of 1−4. Streptoglyceride A (1) was obtained as a pale-yellow viscous oil. The molecular formula of 1 was determined to be C15H22O4 (m/z 289.1412 [M + Na]+, calcd 289.1416) by HRESI-MS analysis. The 1H NMR spectrum of 1 (Table 1) showed the presence of a methyl group at δH 1.70, four olefinic protons at δH 5.54 × 2 and δH 6.00 × 2, an oxygenated Received: July 29, 2018 Published: September 27, 2018 6037
DOI: 10.1021/acs.orglett.8b02411 Org. Lett. 2018, 20, 6037−6040
Letter
Organic Letters Table 1. 1H and
13
C NMR Data for 1−4 in CD3OD 1
1a 1b 2 3 4 5 6a 6b 7 8 9 10 11 12 13a 13b 14 15a 15b 1’ 2’ 3′ 4’ 5′ 6’ 7’ NH2b
2
3
δH (J, Hz)
δC
δH (J, Hz)
δC
3.51, 3.91, 1.74, 4.26, 2.24,
dd (11.0, 4.0) dd (11.0, 2.0) 1.82, m t-like (4.0) d (7.5)
55.0
3.55, dd (11.0, 3.5) 4.01, overlapped 1.85,m 4.29, t (3.5) 2.39, d (7.5)
55.2
1.74, 1.80, 2.16, 5.54, 6.00, 6.00, 5.55, 1.70, 3.80, 3.63,
m m m m dd (14.0, 10.0) dd (14.0, 10.0) m d (6.0) d (9.5) d (9.5)
no.a
4.02, d (9.5) 3.98, d (9.5)
26.5 74.5 51.4 106.9 39.2 25.9 130.8 130.3 131.6 126.0 16.7 77.2 87.5 77.3
5.68, d (15.5) 6.41, 6.12, 6.27, 6.12, 5.76, 1.77, 3.80, 3.66,
dd (15.5, 10.5) dd (14.5, 11.0) dd (14.5, 10.5) dd (14.5, 10.5) m d (7.0) dd (9.0, 1.5) dd(9.5, 1.5)
4.01, overlapped 4.01, overlapped
26.2 74.5 51.6 104.9 132.4 130.1 108.8 134.6 130.3 131.4 16.9 77.8 87.2 77.1
4
δH (J, Hz)
δC
δH (J, Hz)
δC
3.54, 4.00, 1.83, 4.25, 2.53,
dd (11.5, 2.5) dd (12.5, 2.5) m t (4.0) d (7.5)
55.1
3.59, dd (11.0, 3.0) 4.10, t (10.0) 1.87, m 4.32, t-like (4.0) 2.71 d (7.5)
55.2
1.72, 1.80, 2.16, 5.51, 5.96, 5.96, 5.51. 1.68, 4.56, 3.75,
m m dt (9.5, 7.5) m dd (14.0, 10.0) dd (14.0, 10.0) m d (6.5) d (9.0) d (9.0)
4.38, d (9.0) 4.05, d (9.0)
6.54, 7.32, 6.64, 7.94, 8.50,
d (8.5) t (7.0) t (7.5) d (8.0) br s
26.2 73.6 53.0 106.5 39.3 25.9 130.8 130.4 131.6 126.1 16.7 77.7 71.1 77.3 170.7 111.4 149.2 112.5 134.1 115.4 132.4
5.65, d (15.5) 6.38, 6.07, 6.22, 6.07, 5.73, 1.74, 4.59, 3.80,
dd (15.5, dd (14.5, dd (14.5, dd (14.5, m d (6.5) d (9.0) d (9.0)
4.43, d (9.0) 4.02, d (9.0)
6.56, 7.32, 6.62, 7.91, 8.50,
d (8.5) t (7.0) t (7.5) d (8.0) br s
10.5) 11.0) 10.5) 10.5)
25.9 73.7 52.9 104.7 131.8 130.1 130.6 134.8 128.6 131.4 16.9 78.2 70.9 77.4 170.5 111.4 149.1 112.4 134.0 115.3 132.3
a
The assignments were aided by 1H−1H COSY, ROESY, HSQC, and HMBC NMR spectra. bDetected in CDCl3.
methine at δH 4.26, and six characteristic protons at δH 4.02, 3.98, 3.91, 3.80, 3.63, and 3.51 for three oxygenated methylenes. The 13C NMR data and HSQC spectra of 1 indicated the presence of three oxygenated methylenes, three methylenes, six methines, one methyl, and two quaternary carbons (Table 1). Detailed analyses of the 2D NMR (1H−1H COSY, HSQC, and HMBC) data for 1 led to the assignment of the spin systems and their correlations. The 1H−1H COSY correlations of H2-1/H2-2/H-3/H-4 and HMBC correlations from H2-1 and H-3 to the quaternary carbon C-5 suggested the presence of a six-membered ring system (A ring) connected to three oxygen atoms (Figure 2). The presence of an unsaturated
side chain was revealed by a proton spin system of H2-6/H2-7/ H-8/H-9/H-10/H-11/H3-12, as shown in Figure 2. The 3JH,H coupling constants for H-8/H-9 (14.0 Hz) and H-10/H-11 (14.0 Hz) were consistent with an E,E geometry of the conjugated diene in the side chain. In the HMBC spectrum, the correlations from H2-6 to C-5 and H-4 to C-6 suggested that the side chain is connected to the A ring. The HMBC correlations from H2-13 to C-14 and C-15 and from H2-15 to C-13 and C-14 suggested that 1 possesses a glycerol moiety. Afterward, the linkage of the partial structures was deduced from the key HMBC correlations from H-4 to C-13 and C-14 and from H2-15 to C-4 and C-5 (Figure 2). Finally, by detailed analysis of the 2D NMR data combined with the five degrees of unsaturation, the planar structure of 1 was elucidated to possess a rare 6/5/5 tricyclic ring system. The unique ring system of 1 was found to have a rigid conformation on the basis of general stereochemical considerations. The relative configuration of 1 was determined by the proton−proton coupling constants and ROESY correlations of 1 and its methylation product. The remarkable ROESY correlations from H-3 to H-4 placed these protons on the same face. The coupling constant (7.5 Hz) for H-3 and H-4 indicated that these two protons are located in a syn arrangement (Figure 3). Additionally, the strong correlations from H-4 to H-6b suggested that H-4 and the side chain in 1 are on the same face of the molecule. Methylation of 1 was conducted to determine the orientation of C-14-OH. To avoid unnecessary byproduct formation, 1 was hydrogenated prior to methylation. The key ROESY correlation from 14-OMe of the methylated derivative of 1 to H-4 revealed that C-14-OMe and
Figure 2. (a) Partial structures and COSY correlations and (b) HMBC correlations for 1 in CD3OD. 6038
DOI: 10.1021/acs.orglett.8b02411 Org. Lett. 2018, 20, 6037−6040
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Organic Letters
the same planar structure as 1 except for the presence of an additional double bond, as indicated by the molecular formula. The E,E,E geometry of the conjugated triene in the side chain was determined by the 3JH,H values (14.5−15.5 Hz). Streptoglyceride C (3) was obtained as a pale-brown amorphous solid. Compound 3 possesses a molecular formula of C22H27NO5 (m/z 384.1813 [M − H]−, calcd 384.1811) as determined by HR-ESI-MS analysis. The 1H NMR data of 3 were similar to those of 1, except for the noticeably downfield shifted H2-13 (δH 3.75, 4.56) and H2-15 (δH 4.05, 4.38). The 1 H and 13C NMR data for 3 showed additional signals for four aromatic protons at δH 6.54, 6.64, 7.32, and 7.94, four sp2 methine carbons at δC 112.5, 115.4, 132.4, and 134.1, and three quaternary carbons at δC 111.4, 149.2, and 170.7. In addition, a proton spin system of H-4′/H-5′/H-6′/H-7′ in the 1 H−1H COSY spectrum and HMBC correlations of H-7′ with C-1′ and C-3′ and H-4′ and H-6′ with C-2′ were observed (Figure 5). Furthermore, the 1H NMR spectrum taken in
Figure 3. Configuration and conformation of 1 established on the basis of a coupling constant (3JH,H) and key ROESY correlations.
H-4 are cofacial (Figures S10 and S11). Thus, there were only two possible structures for 1, with absolute configurations of 1A (3S,4R,5S,14S) and 1B (3R,4S,5R,14R). For the analysis of the absolute configuration of 1, a conformational search for 1 was carried out using MacroModel with the Merck Molecular Force Field (MMFF), an upper energy limit of 4 kJ/mol, and a convergence threshold of 0.001 kJ mol−1 Å−1 on the RMS gradient to minimize the computational complexity and expense. Eight different conformers were obtained for 1A and for 1B, and the energyminimized model of each conformer was calculated by timedependent density functional theory (TD-DFT) at the B3LYP/def2-TZVPP//B3LYP/def-SV(P) level for all atoms with Turbomole 6.5.12 The calculated ECD spectra of the conformers were merged on the basis of their Boltzmann populations (Table S18), and the averaged calculated ECD spectrum of 1A presented a positive Cotton effect at 226 nm (Figure 4 and Tables S1−S8).13 The experimental ECD
Figure 5. Key COSY and HMBC correlations of 3.
CDCl3 showed a broad singlet proton at δH 8.50, revealing the presence of an amino group (Figure S28). The position of the amino group at C-3′ was determined by the HMBC correlation between NH2 and C-4’ (Figure S29). The downfield-shifted signal of C-3′ (δC 149.2) and molecular formula of 3 also supported the position and presence of the amino group, respectively. Thus, the presence of the 2-aminobenzoate moiety was confirmed. Finally, the planar structure of 3 was determined by attachment of the 2-aminobenzoate to 14-OH of 1, as confirmed by the ROESY correlations between H-4′ and H2-13 as well as H2-15 (Figure S30) and supported by the downfield-shifted signals of H2-13 and H2-15 compared with those of 1. The geometry of the double bonds at C-8 and C-10 was assigned as E,E on the basis of the large coupling constant (14.0 Hz). Streptoglyceride D (4) was obtained as a pale-brown amorphous solid. Compound 4 possesses a molecular formula of C22H25NO5 (m/z 382.1656 [M − H]−, calcd 382.1654) as deduced by HR-ESI-MS analysis, with two mass units less than 3. The 1H NMR data for 4 were similar to those of 3, differing only by the presence of two additional olefin signals, as in 2. Signals for the C-6 (δC 39.3) and C-7 (δC 25.9) saturated carbons in 3 were replaced by two unsaturated carbons at δC 130.1 and 132.4, respectively. The presence of three disubstituted olefins in the side chain was confirmed by the 1 H−1H COSY and HMBC correlations. Almost the same chemical shifts and 2D NMR correlations in 4 compared with 3 suggested that 4 has the same backbone as 3 with an additional double bond. The geometry of the conjugated triene
Figure 4. Comparison of the experimental ECD spectrum of 1 (black) with the averaged calculated ECD spectra of 1A (for the 3S,4R,5S,14S configuration, red) and 1B (for the 3R,4S,5R,14R configuration, blue).
spectrum of 1 also showed a positive Cotton effect at 230 nm. Therefore, the absolute configuration of 1 was determined to be 3S,4R,5S,14S, as shown in Figure 4. Streptoglyceride B (2) was also obtained as a pale-yellow viscous oil. The molecular formula was determined to be C15H20O4 (m/z 287.1262 [M + Na]+, calcd 287.1259) on the basis of HR-ESI-MS analysis, with two mass units less than 1. The 1H NMR data for 2 were similar to those of 1, differing only by the presence of two additional olefin signals and the absence of two methylenes. Signals for the C-6 and C-7 saturated carbons were replaced by two unsaturated carbons at δC 130.1 and 132.4, respectively. The presence of one more double bond in the side chain than in 1 was confirmed by the 1 H−1H COSY and HMBC correlations. Nearly the same chemical shifts and 2D NMR correlations supported that 2 had 6039
DOI: 10.1021/acs.orglett.8b02411 Org. Lett. 2018, 20, 6037−6040
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Organic Letters ORCID
at C-6, C-8, and C-10 in 4 was assigned as E,E,E on the basis of the large coupling constants (14.5−15.5 Hz). Comparison of the optical rotation values and chemical shifts of compounds 1−4 suggested that they have the same backbone and absolute configuration of 3S,4R,5S,14S. Compounds 1−4 were evaluated for their ability to inhibit NO production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and for their cytotoxicity. As shown in Figure 6, 3 showed a weak inhibitory effect on the production of NO. However, streptoglyceride D (4) displayed strong cytotoxicity at the treated concentrations.
Dong-Chan Oh: 0000-0001-6405-5535 Hee Jae Shin: 0000-0002-5952-5325 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors express gratitude to Y.H.K., Korea Basic Science Institute, Ochang, Korea, for providing mass data. This research was supported in part by the Korea Institute of Ocean Science and Technology (Grant PE9969J to H.J.S.).
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Figure 6. Effects of 1−4 on LPS-stimulated NO production and cell viability in BV-2 microglia cells. BV-2 cells were treated with LPS at concentrations of 12.5, 25, 50, and 100 μM for 1−3 and 1, 2, 4, and 8 μM for 4. Viability was evaluated using the MTT assay. The results are displayed as percentages of the control sample.
In summary, we discovered four novel metabolites from a marine actinomycete Streptomyces species that possess an unusual tricyclic ring and in two cases an additional 2aminobenzoate moiety (3 and 4). The available evidence convincingly demonstrates that neuroinflammation is a crucial factor in the onset and progression of neurodegeneration and neuronal loss in neurodegenerative diseases.14 The NO production inhibitory effects of 3 in LPS-stimulated BV-2 microglial cells may contribute to treatment of neuroinflammatory diseases. It is noteworthy that 4 showed stronger cytotoxicity than 3, suggesting that the presence of one more olefinic bond has a vital influence on the biological activity. To the best of our knowledge, this is the first report to describe a rare 6/5/5 tricyclic ring system consisting of a glycerol moiety from marine organisms. This study presents unique anti-neuroinflammatory natural products for further chemical and pharmacological investigation.
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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.orglett.8b02411. Experimental and spectroscopic data for 1−4 (PDF)
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DOI: 10.1021/acs.orglett.8b02411 Org. Lett. 2018, 20, 6037−6040
