Letter Cite This: Org. Lett. 2018, 20, 1007−1010
pubs.acs.org/OrgLett
Acaulide, an Osteogenic Macrodiolide from Acaulium sp. H‑JQSF, an Isopod-Associated Fungus Ting Ting Wang,†,§ Ying Jie Wei,‡ Hui Ming Ge,† Rui Hua Jiao,† and Ren Xiang Tan*,†,‡ †
State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, Nanjing University, Nanjing 210046, China ‡ State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China § State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China S Supporting Information *
ABSTRACT: Acaulide (1), a macrodiolide with an unprecedented framework, was characterized along with its shunt productsacaulones A (2) and B (3)from the culture of Acaulium sp. H-JQSF associated with the isopod Armadillidium vulgare. The spiro-linked 14-, 14-, and 6-membered cycles of 1 arise likely from iterative intermolecular Michael addition reactions. Biological evaluation in the prednisolone-induced osteoporotic zebrafish demonstrated that 1 is antiosteoporotic at 0.4 and 2.0 μM.
A
core with spiro-linked 14-, 14-, and 6-membered cycles is likely formed via iterative intermolecular Michael addition reactions (Scheme 1). Acaulide (1) was indicated to have a molecular formula of C34H44O15 by the Na+-liganded molecular ion at m/z 715.2564 (calcd for C34H44O15Na, 715.2572) in its high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The 1 H and 13C NMR spectra of 1 underscored that it possesses two olefins, five methyls, five methylenes, and seven oxymethines, together with three ketone and five ester motifs. Subtracting the 10 double bond equivalents (2 olefins and 8 carbonyls), the molecular formula implied that 1 was tricyclic. Furthermore, the two olefinic bonds were evidenced to be polarized from the magnitude of their 1H and 13C NMR chemical shifts, indicating their conjugation with carbonyls (Table S1). The assumption was confirmed by the 1H−1H COSY spectrum of 1, highlighting that the two double bonds were actually edited in a pair of 4,5-dioxygenated hex-2(E)enoyl motifs (C-1/C-1′ ∼ C-6/C-6′; J2,3 = J2′,3′ = 15.8 Hz). Correlating with its HMBC spectrum, the remaining NMR signals of 1 were assigned to a 2,2-disubstitued 6-methyldihydropyran-2,4-dione (C-1″−C-6″) and two 2-substuted 4-oxooctanoyl moieties (C-7/C-7′ ∼ C-14/C-14′). In view of the HMBC correlations of C-1/1′ with H-3/3′ and H-13/13′, and of C-7/7′ with H-5/5′ and H-9/9′, this substructure information suggested the hybridization of a triketide unit with two macrodiolide molecules similar in structure to clonostachydiol.14 With this in mind, the five ascertained substructures were pieced together by the key HMBC correlation of C-2″ with H-9/9′ (Figure 1). Though no
s the average lifespan of human beings grows, the incidence of osteoporosis has increased remarkably in the past few decades.1 Furthermore, osteoporosis can be induced as well by other disease states2,3 and administration of some chemotherapeutics.4 Accordingly, more drugs of choice for osteoporosis are desired for a personalized optimal treatment of the patients cosuffering from other illnesses because the successive administration of drugs with different modes of osteoprotective action is reasoned to potentially have better outcomes in osteoporosis management.5 Thus, there is an urgent need to identify novel antiosteoporotic molecules that may energize consequently the development of new treatments of osteoporosis. Encouraged by the chemodiversity and biosynthesis of Daldinia eschscholzii, a mantis-associated fungus,6−8 we intuited that a particularly diversified microbial community associated with the crustacean isopod Armadillidium vulgare could be another source of new secondary metabolites.9,10 As a follow-up to our previous effort of searching for osteogenic drug leads,11 we were motivated to characterize new compound(s) with preferred antiosteoporosis properties from the culture of the isopod-associated microbes. Thus, the microbes associated with A. vulgare were isolated, cultured, and extracted with ethyl acetate. The title strain was shown to be metabolically productive and therefore refermented on a 500-L scale, followed by a series of fractionation procedures, to obtain three skeletally undescribed macrodiolides named acaulide (1), acaulone A (2), and acaulone B (3) after the Latin name of the producing fungus. Encouragingly, 1 was demonstrated to be antiosteoporotic in osteoporosis zebrafish, which is appropriate for the in vivo antiosteoporosis evaluation.12,13 Co-characterization of 1−3 from a single fungal strain enabled the biosynthetic proposal, thereby rationalizing that the unique © 2018 American Chemical Society
Received: December 19, 2017 Published: January 27, 2018 1007
DOI: 10.1021/acs.orglett.7b03949 Org. Lett. 2018, 20, 1007−1010
Letter
Organic Letters Scheme 1. Plausible Biosynthetic Pathways toward Acaulide (1) and Acaulones A (2) and B (3)
with their molecular weights relating reasonably to 4ketoclonostachydiol. Monitored by LC−MS analysis, the subsequent fractionation efforts led to the characterization of metabolites 2−5 from the fungal culture. Acaulone A (2) was shown to possess a molecular formula of C19H26O7 by the Na+-liganded molecular ion at m/z 389.1576 in its HR-ESI-MS spectrum (calcd for C19H26O7Na, 389.1571). Interpretation of the 1D and 2D NMR spectra of 2 indicated that it harbored a 14-membered macrodiolide moiety identical with the macrocycles of 1 (Scheme 1). In the 1H NMR spectrum of 2, a pair of mutually coupled double doublets at δH 2.52 (J = 17.6, 6.3 Hz) and 3.12 (J = 17.6, 7.8 Hz) could arise from a ketone- and methine-isolated methylene group.6 This observation, along with a coupling sequence from H-3′ through H-5′ (Figure S1), suggested the presence of a 1-substituted (E)-pent-3-en-2-one residue, which might anchor on the 14membered ring through the C-8/C-1′ bond linkage. The singlecrystal X-ray diffraction (Cu Kα) of 2 confirmed the proposed structure and revealed its (4R,5S,8R,13S)-configuration (Figure 2A) with the Flack parameter 0.05(7).15 Acaulone B (3) was obtained as colorless prism. Its molecular formula C19H24O7 was deduced by its HR-ESI-MS spectrum ([M + Na]+, m/z 387.1414; calcd for C19H24O7Na, 387.1421). The 1D and 2D NMR spectra of 3 indicated the coexistence of 14-membered macrodiolide and 1,4-disubstituted pent-3-en-2-one substructures. The HMBC spectrum of 3 displayed the correlations of C-7 with H-1′ and of C-9 with H3′ and H-5′. The observation could only be explained by assuming that C-8 and C-9 linked to C-1′ and C-4′, respectively, to generate a 3-methyl-2-cyclohexenone (Figure S1).17,18 The (4R,5S,8S,9R,13S)-configuration of 3 was clarified by its single-crystal X-ray diffraction (Cu Kα) (Figure 2B) with a Flack parameter of 0.02(18).15
Figure 1. Key 1H−1H COSY, HMBC, and NOESY correlations of 1 (left); X-ray structure of 1 (right).
HMBC correlation was discerned between C-1″ and H-5″, the triketide-derived 6-methyldihydropyran-2,4-dione moiety was evident from the chemical shift magnitude of H-5″ (δH 4.79), the 1H−1H COSY pinpointed coupling sequence from H-4″ through H-6″, and the HMBC correlations of C-3″ with H-5″, and of C-2″ with H-4″. Surprisingly, the carbon between the two carbonyls (C-2″) in the δ-lactone ring serves as the tricyclic spiro linker connecting to the two 14-membered macrodiolide moieties, which give different sets of 1H and 13C NMR signals owing to the C-5″ chirality (Table S1). The structure of 1 was confirmed by its single crystal X-ray diffraction (Cu Kα), underscoring simultaneously that it has a (4R,5S,8R,13S,4′R,5′S,8′R,13′S,5″S)-configuration (Figure 1) with the Flack parameter −0.24(15).15 Structural scrutiny of 1 suggested that it could be formed from 6-methyldihydropyran-2,4-dione by incorporating twice an analogue of 4-ketoclonostachydiol, a representative fungal 14-membered macrodiolide stereochemically finalized by total synthesis.16 To help propose the biosynthetic pathway of 1, our attention was directed to the isolation of the fungal metabolites 1008
DOI: 10.1021/acs.orglett.7b03949 Org. Lett. 2018, 20, 1007−1010
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Organic Letters
tion.19,20 This might allow stereochemical ambiguity which is desired to be clarified by robust approaches like total synthesis16 and X-ray crystallography.22 After clarifying the structural feature of this collection of macrodiolides, we were able to hypothesize the active roles played by 10-ketoacaudiol (5) and the triketides II and IV in the fungal assembly of 1−3 (Scheme 1). The 2-hexenoyl- and octanoyl-based biosynthons resulted from the decarboxylative Claisen condensations of acetyl starter unit with two and three malonyl extension units, respectively. Heterodimerization of the tri- and tetraketide moieties via an ester linkage gave intermediate I, which yielded 4 upon macrolactonization. The 10-hydroxy oxidation of 4 afforded 5 as a key player in the diversification of this array of fungal macrodiolides. Likely, Michael addition of the saturated δ-lactone (IV) with 5 gave intermediate V, which formed 1 via the second-round of intermolecular Michael addition with 5. The δ-lactone motif of V might undergo intramolecular dehydrative saponification to yield 2 presumably via VI. Probably in parallel, the decarboxylative Clasisen condensation of 5 with triketide intermediate II could trigger the formation of 3 likely via VII (Scheme 1). Bioassay on the prednisolone-induced osteoporotic zebrafish model (see the Supporting Information) showed that 1 alleviates the skull loss of the osteoporosis zebrafish at 0.4 and 2.0 μM (Figure 4). However, 4 and 5 were found to be inactive at 10.0 μM in the assay, and unfortunately, 2 and 3 were too scarce to be bioassayed. It is noteworthy that this zebrafish model is appropriate for the in vivo antiosteoporotic evaluation of both small molecules12 and biomacromolecules,13 thereby convincing the osteogenic action of 1. Although likely
Figure 2. X-ray structures of 2 (A), 3 (B), and 4 (C).
Metabolite 4 was demonstrated to have a molecular formula of C14H20O6 by its HR-ESI-MS spectrum ([M + Na]+, m/z 307.1160; calcd for C14H20O6Na, 307.1152). The 1H and 13C NMR spectra of 4 indicated the presence of two olefins and two carbonyl motifs that account in total for four of its five degrees of unsaturation. The 1D NMR spectra of 4 demonstrated that it possesses an identical planar structure with clonostachydiol, a 14-membered fungal macrodiolide reported previously.14,19,20 Our single-crystal X-ray diffraction of 4 (Cu Kα) underpinned its (4R,5S,10S,13S)-configuration (Figure 2C) with the Flack parameter 0.17(9).15 Thus, 4 is an enant iomer o f clon ost ach ydiol th at p ossesses a (4S,5R,10R,13R)-configuration.19,20 To avoid ambiguity, we have named 4 acaudiol. The MS and 1H and 13C NMR spectra of 5 indicated that it was a 10-oxidized derivative of 4. The (4R,5S,13S)-configuration of 5 was demonstrated by its CD spectrum close to that of 4 (Figure 3) and the Dess−Martin oxidation of 5 into 4
Figure 3. CD spectra of 4 (blue) and 5 (red). Figure 4. Evaluation of antiosteoporosis activity of acaulide (1) in zebrafish. (A) Difference in the skull mineralization of zebrafish treated with DMSO (as a blank/positive control), prednisolone (an osteoporosis-affording drug, as a negative control), and prednisolone combined with 1 at 0.4, 2.0, and 10.0 μM, respectively. Columns showed means of mineralized area and integrated optical density (IOD) of the zebrafish skulls in three independent experiments; bars represent the mean ± SEM; *p < 0.05, **p < 0.01 versus the prednisolone-treated group. (B) After 3 days postfertilization (3 dpf), the zebrafish were separately treated for 5 days with DMSO, prednisolone, and the prednisolone−acaulide (1) mixtures dosed above, followed by euthanization and staining with Alizarin Red. Annotated for clarity were the stained opercular bone (op), ceratobranchial 5 (cb5), anterior tip of the notochord (no), and cleithrum (cl), parasphenoid (ps), and otolith (o, stained brown, not bone) in the skull of the DMSO-treated zebrafish.
(Figure S2). We have named 5 10-ketoacaudiol. Compound 5 was characterized herein as a natural product, and its enantiomer was detected as 10-oxoclonostachydiol afforded upon the chemical oxidation of clonostachydiol.21 The macrodiolide configuration of 1−5 is constant and comparable to that of 4-ketoclonostachydiol whose chirality was revised through total synthesis.16 This is reasonable since 1−5 are constructed from the biosynthetic precursors originated from a single fungal strain (Scheme 1). However, the C-5 and C-13 chirality of 1−5 does not match that of other fungal macrodiolides such as clonostachydiol. To our knowledge, this macrodiolide was assigned stereochemically by correlating to the earlier biogenetic consideration/assump1009
DOI: 10.1021/acs.orglett.7b03949 Org. Lett. 2018, 20, 1007−1010
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Organic Letters
(7) Zhang, A. H.; Liu, W.; Jiang, N.; Wang, X. L.; Wang, G.; Xu, Q.; Tan, R. X. Org. Lett. 2017, 19, 2142−2145. (8) Zhang, A. H.; Tan, R.; Jiang, N.; Yusupu, K.; Wang, G.; Wang, X. L.; Tan, R. X. Org. Lett. 2016, 18, 5488−5491. (9) Verdon, J.; Thevenot, P. C.; Rodier, M. H.; Landon, C.; Depayras, S.; Noel, C.; Camera, S. L.; Moumen, B.; Greve, P.; Bouchon, D.; Berjeaud, J. M.; Varnier, C. B. Front. Microbiol. 2016, 7, 1472−1486. (10) Le Clec’h, W.; Raimond, M.; Guillot, S.; Bouchon, D.; Sicard, M. Environ. Microbiol. 2013, 15, 2922−2936. (11) Wang, S. F.; Jiang, Q.; Ye, Y. H.; Li, Y.; Tan, R. X. Bioorg. Med. Chem. 2005, 13, 4880−4890. (12) de Vrieze, E.; Zethof, J.; Merker, S. S.; Flik, G.; Metz, J. R. Bone 2015, 74, 106−113. (13) van Dijk, F.; Zillikens, M. C.; Micha, D.; Riessland, M.; Marcelis, C. L. M.; Smulders, C. E. D.; Milbradt, J.; Franken, A. A.; Harsevoort, A. J.; Lichtenbelt, K. D.; Pruijs, H. E.; Rubio-Gozalbo, M. E.; Zwertbroek, R.; Moutaouakil, Y.; Egthuijsen, J.; Hammerschmidt, M.; Bijman, R.; Semeins, C. M.; Bakker, A. D.; Everts, V.; Klein-Nulend, J.; Obando, N. C.; Hofman, A.; Meerman, G. J.; Verkerk, A. J. M. H.; Uitterlinden, A. G.; Maugeri, A.; Sistermans, E. A.; Waisfisz, Q.; Heijboer, H. M.; Wirth, B.; Simon, M. E. H.; Pals, G. N. Engl. J. Med. 2013, 369, 1529−1536. (14) Grabley, S.; Hammann, P.; Thiericke, R.; Wink, J. J. Antibiot. 1993, 46, 343−345. (15) Flack, H. D.; Bernardinelli, G. Acta Crystallogr., Sect. A: Found. Crystallogr. 1999, 55, 908−915. (16) Han, J.; Su, Y. P.; Jiang, T.; Xu, Y. F.; Huo, X.; She, X. G.; Pan, X. F. J. Org. Chem. 2009, 74, 3930−3932. (17) Yuan, X. Q.; Dong, S. P.; Liu, Z.; Wu, G. B.; Zou, C. C.; Ye, J. X. Org. Lett. 2017, 19, 2322−2325. (18) Taher, A.; Lee, K. C.; Han, H. J.; Kim, D. W. Org. Lett. 2017, 19, 3342−3345. (19) Rama Rao, A. V.; Murthy, V. S.; Sharma, G. V. M Tetrahedron Lett. 1995, 36, 139−142. (20) Rama Rao, A. V.; Murthy, V. S.; Sharma, G. V. M Tetrahedron Lett. 1995, 36 (36), 143−146. (21) Lang, G.; Mitova, M. I.; Ellis, G.; van der Sar, S.; Phipps, R. K.; Blunt, J. W.; Cummings, N. J.; Cole, A. L. J.; Munro, M. H. G. J. Nat. Prod. 2006, 69, 621−624. (22) Flack, H. D.; Bernardinelli, G. Chirality 2008, 20, 681−690.
to be true, the osteogenic effect of 1 in mammals needs more biological work to ascertain. In conclusion, we have characterized acaulide (1) and acaulones A (2) and B (3) as skeletally unprecedented secondary metabolites from Acaulium sp. associated with A. vulgare, with 1 displaying a potent antiosteoporotic activity in the osteoporosis zebrafish. With identification of shunt (2 and 3) and transient products (4 and 5), we were able to rationalize the formation of the unique 14/14/6 ring system of 1, which may be essential for its antiosteoporotic action. The work provides acaulide as an innovative molecule useful for the development of acaulide-based/derived new osteogenic therapeutic(s) and the motivation/inspiration for further studies investigating the mode of action of acaulide and the sample supply related issue via chemical synthesis and/or synthetic biology approaches.
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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.7b03949. General methods; detailed fractionation procedure; LC− MS, 1D and 2D NMR data for compounds 1−3 (PDF) Accession Codes
CCDC 1565576, 1573201, and 1811786−1811787 contain 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]. uk, 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
Hui Ming Ge: 0000-0002-0468-808X Ren Xiang Tan: 0000-0001-6532-6261 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research was cofinanced by the NSFC (Frant Nos. 8153008 9, 814 21091, 2167210 1, 8157 3833, and 21661140001). We are indebted to Prof. L. D. Guo at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, for the fungal identification.
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REFERENCES
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DOI: 10.1021/acs.orglett.7b03949 Org. Lett. 2018, 20, 1007−1010
