Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Frondoplysins A and B, Unprecedented Terpene-Alkaloid Bioconjugates from Dysidea frondosa Wei-Hua Jiao,†,⊥ Jing Li,†,‡,⊥ Meng-Meng Zhang,§ Jie Cui,† Yu-Han Gui,† Yun Zhang,∥ Jing-Ya Li,§ Ke-Chun Liu,∥ and Hou-Wen Lin*,†
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†
Research Center for Marine Drugs, State Key Laboratory of Oncogene and Related Genes, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China ‡ Biotech Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai, 201106, China § National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China ∥ Institute of Biology, Qilu University of Technology, Jinan, 250103, China S Supporting Information *
ABSTRACT: The chemical investigation of the marine sponge Dysidea f rondosa discovered a pair of unprecedented bioconjugates that are composed of a meroterpene and an unusual psammaplysin alkaloid. The structures of frondoplysins A (1) and B (2) were characterized by analysis of HRMS and NMR data coupled with single-crystal X-ray diffraction. Frondoplysin A was found to be a potent inhibitor targeting protein-tyrosine phosphatase 1B (PTP1B) with an IC50 value of 0.39 μM.
M
arine sponges have been demonstrated as an exceptionally valuable resource for biologically active natural products.1 Secondary metabolites derived from marine sponges usually embrace rare chemical scaffolds with unique assemblages of functionalities.2,3 These metabolites showed a spectrum of intriguing biological activities. In light of the marvelous potential of sponge metabolites as drug leads, there is a continuing endeavor in discovering new bioactive metabolites from marine sponges.4−6 Their unique chemical scaffolds usually serve as candidates for drug development or new targets for the synthetic or semisynthetic chemists.7−9 Protein-tyrosine phosphatase 1B (PTP1B) has been known as a negative regulator for insulin and leptin receptor signaling for a long time,10,11 and an increasing number of inhibitors targeting PTP1B have been discovered and developed.12−17 Recently, PPT1B was found to be aberrantly expressed in some cancer cells; moreover, it could function as an important oncogene closely related to breast cancer progression.18 In our ongoing discovery program for PTP1B inhibitors from marine sponges,19,20 we analyzed a collection of sponge specimens from the South China Sea using the LCMS method in conjunction with the PTP1B inhibitory screening assay. An active organic extract derived from the sponge Dysidea f rondosa (no. XD1506A) showed very different LCMS profiles from those previously reported. Further chemical investigation of the active extract resulted in the isolation of a pair of unique sponge metabolites, frondoplysins A (1) and B (2). Frondoplysin A (1) was obtained as purple needles. The ESIMS showed a characteristic tetrabrominated ion peak © XXXX American Chemical Society
cluster at m/z 1078/1080/1082/1084/1086 [M + Na]+ (1:4:6:4:1). The molecular formula C42H49O9N3Br4 was established based on the HRMS ion at m/z 1078.0128 [M + Na]+. Analysis of the 13C NMR and HSQC data suggested the presence of 42 carbon signals, including three carbonyls, six double bonds (including an exocyclic one on account of the presence of an exocyclic methylene at δC 103.2), one benzene ring, and four methyl groups (Tables 1 and S1). Four spin systems a−d as depicted in bold bonds were deduced from COSY correlations (Figure 1). The vinyl protons at δH 4.46 and 4.45 (H2-11) were correlated by HMBC to C-3 and C-5. Further, methyl protons H3-12 showed HMBC correlations with C-4, C-5, C-6, and C-10, H3-13 displayed correlations with C-7, C-8, and C-9, while H3-14 exhibited HMBC Received: May 18, 2019
A
DOI: 10.1021/acs.orglett.9b01754 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Frondoplysin A (1) in CDCl3 no.
δC, type
1α 1β 2α 2β 3α 3β 4 5 6α 6β 7 8 9 10 11a 11b 12
22.7, CH2 28.1, CH2 32.9, CH2 159.6, C 40.3, C 36.7, CH2 27.5, CH2 37.0, CH 42.6, C 49.0, CH 103.2, CH2 20.7, CH3
δH (J in Hz) 1.51, 1.88, 1.87, 1.11, 2.10, 2.31,
m m m m m td (15.6, 3.6)
1.38, 1.55, 1.43, 1.21,
m m m m
0.79, 4.46, 4.45, 1.05,
d (11.4) s s s
no.
δC, type
13 14 15a 15b 16 17 18 19 20 21 22 23 24 25 26 27 28
16.9, CH3 17.6, CH3 35.1, CH2 142.3, C 183.4, C 146.5, C 98.5, CH 185.6, C 139.4, CH 49.5, CH2 70.3, CH 139.9, C 130.1, CH 118.7, C 152.7, C 118.7, C
δH (J in Hz) 0.94, 0.85, 2.54, 2.37,
d (6.6) s d (13.8) d (13.8)
5.43, d (1.8) 6.33, s 3.28, m 4.87, m 7.53, s
no.
δC, type
29 30 31 32 33 34 35 36 37a 37b 38 39 40 41 42 18-NH 32-NH
130.1, CH 71.1, CH2 29.2, CH2 37.2, CH2 159.0, C 155.7, C 79.5, CH 122.2, C 37.1, CH2 103.4, C 148.8, C 105.5, C 145.5, CH 59.1, CH3
δH (J in Hz) 7.53, 4.10, 2.13, 3.74,
s t (6.0) t (6.0) q (6.6)
5.14, s 3.38, d (15.6) 3.13, d (15.6)
7.02, 3.69, 6.00, 7.16,
s s t (7.2) t (5.4)
152.7 (C-27). These HMBC correlations coupled with the two spin systems c and d suggested the presence of a moloka’iamine substructure. Interestingly, the olefinic proton at δH 7.02 (H-41) showed long-ranged correlations with C-36, C-39, and C-40, meanwhile the geminal protons at δH 3.38 and 3.13 (H237) displayed diagnostic HMBC cross-peaks with C-35, C-36, C-38, and C-39, which established an unusual dibrominated 1,6-dioxa-2-azaspiro[4.6]undeca-2,7,9-triene (spirooxepinisoxazoline) ring system.21 Furthermore, the observed HMBC cross-peaks between the methylene H2-32 and C-33 suggested the fragment d attached to the spiro ring system by an amide bond, which finally furnished the planar structure of 1. The relative configuration of the bicyclic sesquiterpene moiety was assigned by J-based configuration analysis coupled with NOESY experiment. The large coupling constant of H-10 (J = 11.4 Hz) suggested the axial orientations of H-1β, H-10, and H3-12 and thus the trans fusion of rings A/B, which was supported by the observed NOESY cross-peaks of H-6α/H-8, H-6β/H3-12, H-8/H-10, H3-12/H3-14, and H3-13/H3-14 (Figure 1). The relative configuration of the spirooxepinisoxazoline could not be established by the current NOESY experimental data, which might be the same as that of psammaplysin A based on comparison of the NMR data.21 Frondoplysin A has three isolated chiral clusters, C-5, C-8, C-9, and C-10, C-23, as well as C-35 and C-36, which make it challenging to unambiguously assign its complete absolute configurations. Fortunately, we obtained needle crystals for 1 in MeOH/CH2Cl2 (2:1); the X-ray crystallographic analysis data unambiguously assigned the absolute configuration of 1 as 5S,8S,9R,10S,23R,35R,36R (Figure 2). Frondoplysin B (2), isomeric with 1, has the same molecular formula of C42H49O9N3Br4 as assigned by the 13C NMR data and HRMS ion at m/z 1078.0101 [M + Na]+. The NMR data (Table S2) of 2 highly resembled those of 1 except for the disappearance of the exomethylene protons at δH 4.46 and 4.45 and the appearance of an olefinic singlet at δH 5.15, indicating the exocyclic double bond Δ4,11 in 1 was transferred to Δ3,4 in 2. This assignment was supported by COSY cross-peaks of H22/H-3 as well as long-ranged HMBC correlations of H3-11 to C-3, C-4, and C-5 (Figure 1). Additionally, HMBC cross-peaks of H2-22/C-19 and 19-NH/C-18 suggested the psammaplysin
Figure 1. Key 2D NMR correlations for frondoplysins A (1) and B (2).
correlations to C-8, C-9, C-10, and C-15. Taken together, these data established the linkage of two spin systems a and b and indicated the existence of a bicyclic sesquiterpene substructure. The observation of two unsaturated ketones at δC 183.4 (C-17) and δC 185.6 (C-20), the two weakly coupled vinyl protons at δH 5.43 (H-19) and δH 6.33 (H-21) with coupling constants of 1.8 Hz, and the HMBC cross-peaks of H19/C-17 implied a meta-disubstituted p-benzoquinone unit. Moreover, two geminal protons at δH 2.54 and 2.37 (H2-15) exhibited HMBC cross-peaks with C-8, C-9, C-10, and C-14 in the sesquiterpene moiety and C-16, C-17, and C-21 in the quinone unit, which linked the two substructures by the carbon bond C-15−C-16. In spin system c, nitrogenated methylene at δH 3.28 (H2-22) showed HMBC correlations with olefinic carbon δC 146.5 (C-18) in the quinone unit, which positioned the N-methylene at C-18. Two overlapped aromatic protons at δH 7.53 (H-25 and H-29) showed HMBC correlations with an oxygenated methine at δC 70.3 (C-23), two pairs of symmetric aromatic carbons at δC 130.1 (C-25 and C-29) and δC 118.7 (C-26 and C-28), and one oxygenated aromatic carbon δC B
DOI: 10.1021/acs.orglett.9b01754 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
potent antioxidative activity over five times stronger than that vitamin C (Figure 4).
Figure 2. X-ray structure of frondoplysin A (1).
moiety was placed at C-19 in 2 instead of at C-18 in 1. The relative configuration of 2 was determined to be the same as that of 1 on account of identical NOESY correlations (Figure 1). Interestingly, the similar Cotton effects in the CD spectra of the two compounds indicated that they shared the same absolute configurations (Figure 3).
Figure 4. In vivo antioxidant activity of frondoplysin A (1) in zebrafish embryos (n = 5). (a) In vivo visualization of zebrafish skin fluorescence treatment. (b) FS number statistic results of all groups (in the white frame). Data were derived from the five independent experiments and represented as mean ± SD, **p < 0.01 compared to the model group. ##p < 0.01 compared to the control group.
In conclusion, two unusual terpene-alkaloid bioconjugates were discovered and characterized from the marine sponge Dysidea f rondosa. Frondoplysin A (1) exhibited potent in vitro PTP1B inhibitory activity and in vivo antioxidant activity in transgenic zebrafish. This finding provides a potent marine sponge-derived PTP1B inhibitor for further chemical and biological studies.
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Figure 3. Experimental CD spectra for 1 and 2.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01754.
The marine environment has provided a number of natural occurring meroterpenoids where the hydroquinone/quinone unit is substituted by amine, amino acids, and even nucleoside.22−24 However, frondoplysins A (1) and B (2) are a pair of unprecedented bioconjugates of a meroterpene and a complex psammaplysin alkaloid. The putative biogeneis of frondoplysins represents at least three distinct biosynthetic pathways: terpene, acetate, and alkaloid (Scheme S1). It seems that nature constructs frondoplysins in a convergent manner by first utilizing the common pathway to the meroterpenoids, avarone, and neovarone, as well as the alkaloid psammaplysin B and then taking the quinone and the amine to link them together by their intrinsic reactivities. The PTP1B inhibitory activity of the two unusual bioconjugates was evaluated. Frondoplysins A (1) and B (2) showed potent inhibitory activity with IC50 values of 0.39 ± 0.04 and 0.65 ± 0.03 μM, using oleanolic acid as a positive control (IC50 3.7 ± 0.03 μM). In comparison with the PTP1B inhibitors in development, frondoplysin A is more potent than thiazolidinediones (IC50 5.0 μM)16 and similar to benzofuran and benzothiophene biphenyls (IC50 0.36 μM).17 Detailed enzymetic kinetic study revealed that frondoplysin A was a mixed PTP1B inhibitor with an unknown mechanism (Figure S2). Further in vivo activity evaluation of frondoplysin A (1) in transgenic fluorescent zebrafish showed that frondoplysin A showed no cytotoxicity at the concentration of 64 μM but
Detailed experimental procedures and 1H, 13C, and 2D NMR data for compounds 1 and 2 (PDF) Accession Codes
CCDC 1915341 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Wei-Hua Jiao: 0000-0003-4835-4775 Hou-Wen Lin: 0000-0002-7097-0876 Author Contributions ⊥
W.-H.J. and J.L. contributed equally to this work.
Notes
The authors declare no competing financial interest. C
DOI: 10.1021/acs.orglett.9b01754 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
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ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (No. 2018YFC0310901) and National Natural Science Foundation of China (Nos. 41576130, U1605221, 81602982, and 81703624).
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DOI: 10.1021/acs.orglett.9b01754 Org. Lett. XXXX, XXX, XXX−XXX
