(+)- and (−)-Preuisolactone A: A Pair of Caged Norsesquiterpenoidal

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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(+)- and (−)-Preuisolactone A: A Pair of Caged Norsesquiterpenoidal Enantiomers with a Tricyclo[4.4.01,6.02,8]decane Carbon Skeleton from the Endophytic Fungus Preussia isomera Lu-Lin Xu,†,‡,∥ Hai-Li Chen,†,‡,∥ Ping Hai,⊥ Yuan Gao,⊥ Chuan-Dong Xie,‡ Xiao-Long Yang,*,†,‡ and Ikuro Abe*,§ †

School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, People’s Republic of China Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People’s Republic of China § Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan ⊥ Department of Chemical Engineering, Yibin University, Yibin 644000, People’s Republic of China

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ABSTRACT: A pair of enantiomeric norsesquiterpenoids, (+)- and (−)-preuisolactone A (1) [(+)-1 and (−)-1)] featuring an unprecedented tricyclo[4.4.01,6.02,8]decane carbon scaffold were isolated from Preussia isomera. Their structures were determined by spectroscopic and computed methods and X-ray crystallography. Compounds (+)-1 and (−)-1 are two rare naturally occurring sesquiterpenoidal enantiomers. A plausible biosynthetic pathway for 1 is proposed. Additionally, (±)-1 exhibited remarkable antibacterial activity against Micrococcus luteus with an MIC value of 10.2 μM.

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from the stems of Panax notoginseng, gained our attention because of its significant antimicrobial properties. Further bioassay-guided fractionations afforded a pair of enantiomeric norsesquiterpenoids, (+)- and (−)-preuisolactone A (1) [(+)-1 and (−)-1)] (Figure 1). Herein, we describe the details of the isolation, structural elucidation, biological activities, and plausible biosynthetic pathway of 1. (±)-Preuisolactone A (1) was obtained as colorless crystals with a molecular formula of C15H14O7 as evident from the positive HRESIMS (m/z 329.0630 [M + Na]+, calcd for

lant endophytic fungi, the major group of special ecoenvironmental fungi inhabiting living plants without any negative effects, have an excellent capacity for secreting diverse biological secondary metabolites with a variety of unique structures and with potential therapeutic applications.1−5 Fungi of the Preussia genus, belonging to the family Sporormiaceae, have been frequently found as plant endophytes.6,7 Previous phytochemical investigations of some species have afforded many novel bioactive secondary metabolites, such as six novel bicyclic polyketides, preussilides A−F, with antiproliferative activity from Preussia similis,8 two novel polyketides, minimoidiones A and B, as α-glucosidase inhibitors from Preussia minimoides,9 two novel dibenzofurans, preussiafurans A and B, possessing antiplasmodial and cytotoxic activities from Preussia sp.,10 an unusual thiopyranchromenone, preussochromone A, with cytotoxic activity from Preussia af ricana,11 six aromatic bisketals, preussomerins A−F, with antifungal and antibacterial activities from Preussia isomera,12,13 etc. Accordingly, the fascinating properties of the secondary metabolites produced by fungi of the Preussia genus stimulated us to search for additional novel bioactive compounds for new drug development. In the course of our ongoing efforts to explore the unique structurally bioactive compounds from the plant endophytic fungi, one of the isolates, Preussia isomera XL-1326, obtained © XXXX American Chemical Society

Figure 1. Structures of (+)- and (−)-preuisolactone A (1). Received: December 26, 2018

A

DOI: 10.1021/acs.orglett.8b04123 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

C-9, as well as the 1H−1H COSY correlation of H-6 with H-7, facilitated the construction of an 8-methyl-bicyclo[2.2.15,7]heptane moiety (fragment B, rings B and C). The fusing pattern of fragments A and B was determined through C-1 and C-6 to form a tricyclo[4.4.01,6.02,8]decane carbon scaffold, as evident from the key HMBC correlations of H-7 with C-1 and C-5. The remaining two ester carbonyls at δC 174.0 (C-12) and 175.6 (C14) were determined to be attached to C-7 and C-10, respectively, as supported by the HMBC correlations of H-6 with C-14, and H-9 with C-12. The key HMBC correlation of H9 with C-14 allowed us to construct the five-membered lactone (ring E). Analysis of HMBC correlations was not sufficient to determine the remaining five-membered lactone (ring D), due to the lack of a hydrogen atom at C-2. Considering the chemical shift of C-2 as well as the ring tension factor, we finally constructed the remaining five-membered lactone (ring D) by the linkage of C-2 and C-12 via an oxygen atom. Finally, the gross structure of 1 was elucidated, as shown in Figure 1. In the NOESY spectrum (Figure 3), the observed NOE correlations of H3-13 with H-7 and H-9 suggested that H-7, H-9,

C15H14O7Na 329.0632), requiring 9 degrees of unsaturation. The IR spectra exhibited typical absorptions suggesting the presence of hydroxy (3289 cm−1), γ-lactone (1795 cm−1), conjugated carbonyl (1640 cm−1), and olefinic (1581 cm−1) functionalities. An analysis of the 1H NMR spectroscopic data (Table 1) of 1 displayed the characteristic signals, assigned to Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data for (±)-Preuisolactone A (1) in Methanol-d4 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

δH (J in Hz)

5.48, d (1.8) 3.03, brs 2.85, brs 4.34, s 1.09, s 1.37, s 3.88, s

δC 63.1, C 93.2, C 188.9, C 100.1, CH 184.3, C 49.5, CH 52.6, CH 60.5, C 82.5, CH 85.6, C 10.5, CH3 174.0, C 13.9, CH3 175.6, C 58.6, CH3

three singlet methyl groups including one oxygenated at δH 1.09 (H-11), 1.37 (H-13), and 3.88 (H-15), one olefinic proton at δH 5.48 (d, H-4), and three methine groups including one oxygenated at δH 3.03 (brs., H-6), 2.85 (brs., H-7), and 4.34 (s, H-9). In agreement with its molecular formula, 15 resolved Catom resonances were observed in the 13C NMR spectrum, which were categorized by DEPT and HSQC experiments as four sp2 quaternary carbons (δC 188.9, 184.3, 175.6, and 174.0), one protonated olefinic carbon (δC 100.1), three methyl groups including one oxygenated (δC 58.6, 13.9, and 10.5), three sp3 methines including one oxygenated (δC 82.5, 52.6, and 49.5), and four sp3 quaternary carbons including two oxygenated (δC 93.2, 85.6, 63.1, and 60.5). The above unsaturated functionalities accounted for four of the nine indices of hydrogen deficiency, illustrating that compound 1 had a pentacyclic ring system (rings A−E). In the HMBC spectrum (Figure 2), the observed correlations of H-4 with C-2, C-3, and C-6, H-6 with C-2 and C-4, H-11 with

Figure 3. NOESY correlations and X-ray crystallographic structure of (±)-preuisolactone A (1).

and H3-13 should be in the same orientation, while H-6 and H311 should be opposite, as evident by the NOE correlations of H6 with H3-11. Due to the existence of four contiguous quaternary carbons in 1, the relative configurations of C-1, C-2, C-8, and C10 could not be directly determined by the NOESY experiment. Fortunately, a single crystal was successfully obtained from a solvent mixture of toluene/MeOH/CH2Cl2 (4:2:1, v/v/v), after many attempts using different solvents. The single-crystal X-ray crystallographic analysis (MoKα radiation) (Figure 3) confirmed the above proposed structure, and the relative configuration of 1 was determined to be 1R*,2R*,6R*,7R*,8S*,9R*,10S*. However, the X-ray structure performed with Cu Kα radiation (Table S5 and Figure S11) revealed the presence of a racemic mixture. Further chiral HPLC analysis indicated that compound 1 contained two enantiomers (+)-1 and (−)-1) with a ratio of about 1:1.05 (Figure S12). We tried to separate this partially racemic mixture (±)-1 by chiral HPLC to afford (−)-1 (0.25 mg) and (+)-1 (0.16 mg). Unfortunately, we only obtained the experimental CD spectra of (−)-1 but failed for (+)-1 due to trace sample amount. The calculated ECD spectra of (−)-1 was in good agreement with its experimental data (Figure 4), suggesting that the absolute configuration in (−)-1 is 1S,2S,6S,7S,8R,9S,10R. The specific 23 rotations ([α]24 D +69 for (+)-1 and [α]D −80 for (−)-1)] were opposite. Furthermore, the specific rotation calculations of (+)-1 (+53.85) and (−)-1 (−53.85) supports that the absolute configurations of (+)-1 and (−)-1 are 1R,2R,6R,7R,8S,9R,10S and 1S,2S,6S,7S,8R,9S,10R, respectively. Finally, compounds

Figure 2. Structural fragments A−B of (±)-preuisolactone A (1) and key HMBC and 1H−1H COSY correlations.

C-1, C-2, and C-6, and H-15 with C-5 suggested the presence of a 3-methoxy-5-methylcyclohex-2-en-1-one substructure (fragment A, ring A). Furthermore, the observed key HMBC correlations from H-6 to C-8 and C-10, from H-7 to C-1 and C9, from H-9 to C-1 and C-7, and from H-13 to C-2, C-7, C-8, and B

DOI: 10.1021/acs.orglett.8b04123 Org. Lett. XXXX, XXX, XXX−XXX

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biosynthetic pathway (Scheme 1), compound 1 originates from the terpenoid pathway. First, the key tricyclic intermediate, the presilphiperfolan-8-yl cation (I-3), which was previously reported to play a vital role in the formation of many polycyclic sesquiterpenes in plant and fungi,14−18 is derived from farnesyl pyrophosphate (FPP) via cyclization and rearrangement reactions. Second, the intermediate I-3 takes place migration and rearrangement reactions to form intermediate I-5 and followed by two steps of oxidation reaction to form bicyclic intermediate I-7. Then the key aldol reaction occurs in I-7 to construct the new tricyclic intermediate II-1, which is further transformed into the intermediate II-4 after oxidation and esterification reactions. Subsequently, one carbon is degraded in II-4 via a decarboxylation reaction to form the intermediate II-5. Finally, compound 1 is formed from II-5 after migration, oxidation, and methylation reactions. To the best our knowledge, enantiomeric sesterterpenoids are rare naturally occurring.19 There are only a few examples to be reported, for example, (±)-wistarin from Ircinia species,20,21 (±)-germacrene D from Solidago canadensis,22−24 (±)-furodysinin from Dysidea

Figure 4. Experimental and calculated CD spectra of (+)-1 and (−)-1.

(+)-1 and (−)-1 were characterized and named (+)-preuisolactone A and (−)-preuisolactone A. Structurally, compounds (+)-1 and (−)-1 are two enantiomeric norsesterterpenoids with an unprecedented 6/5/5/5/5 pentacyclic scaffold consisting of a caged tricyclo[4.4.01,6.02,8]decane carbon skeleton and two γ-lactone rings. In the proposed Scheme 1. Proposed Biosynthetic Pathway for 1

C

DOI: 10.1021/acs.orglett.8b04123 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters herbaceae,25,26 and (±)-phaeocaulins A−D from Curcuma phaeocaulis.27 However, the biosynthetic formation mechanism of enantiomeric sesterterpenoids remains poorly understood. Interestingly, two enantioslective germacrene D synthases have been recently discovered by König’s group from Solidago canadensis.23,24 The presence of the above two cyclases (designated Sc11 and Sc19) not only explains the biosynthesis of enantiomeric germacrene D within S. Canadensis but also provides us the direct evidence to support the possibility that two different enantioslective cyclases might also exist in P. isomera and be involved in the biosynthesis of (±)-preuisolactone A. However, this hypothesis needs further investigation at genetic levels. Compound 1 was tested for its antifungal activities against eight pathogenic fungi (Sclerotinia sclerotiorun, Helminthosporium maydis, Verticillium dahliae Kleb, Phytophthora parasitica, Gibberella saubinetii, Alternaria alternata, Colletotrichum acutatum Simmonds, and Botrytis cinereal Pers.) as well as for its antibacterial activities toward eight pathogenic bacteria (Micrococcus lysodeikticus, Micrococcus luteus, Bacillus megaterium, Salmonella paratyphi B, methicillin-resistant Staphylococcus aureu, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium). The results (Tables S1 and S2) revealed that compound 1 showed antibacterial activities against M. luteus and B. megaterium with MIC values of 10.2 and 163.4 μM, respectively. Furthermore, compound 1 also showed moderate antifungal activity against A. alternata with an MIC value of 163.4 μM. Compound 1 was also evaluated for its cytotoxicity, and the results (Table S3) indicated that 1 showed no cytotoxic activity against the A549, Huh7, MGC803, HCT116, and LN229 human cancer cell lines (IC50 > 100 μM). In conclusion, a pair of norsesquiterpenoidal enantiomers, (+)- and (−)-preuisolactone A (1), with an unprecedented caged tricyclo[4.4.01,6.02,8]decane carbon skeleton, were isolated from P. isomera XL-1326. Compound (±)-1 showed remarkable antibacterial activity against M. luteus with an MIC value of 10.2 μM. These findings might inspire further investigations of its chemical and pharmacological properties.

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Author Contributions ∥

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was financially supported by the National Natural Science Foundation of China (No. 81872755). REFERENCES

(1) Martinez-Klimova, E.; Rodríguez-Peña, K.; Sánchez, S. Biochem. Pharmacol. 2017, 134, 1−17. (2) Gouda, S.; Das, G.; Sen, S. K.; Shin, H. S.; Patra, J. K. Front. Microbiol. 2016, 7, 1538−1545. (3) Nisa, H.; Kamili, A. N.; Nawchoo, I. A.; Shafi, S.; Shameem, N.; Bandh, S. A. Microb. Pathog. 2015, 82, 50−59. (4) Venugopalan, A.; Srivastava, S. Biotechnol. Adv. 2015, 33, 873− 887. (5) Alvin, A.; Miller, K. I.; Neilan, B. A. Microbiol. Res. 2014, 169, 483− 495. (6) Khan, A. L.; Asaf, S.; Khan, A. R.; Al-Harrasi, A.; Al-Rawahi, A.; Lee, I. J. J. Biotechnol. 2016, 225, 44−45. (7) Gonzalez-Menendez, V.; Martin, J.; Siles, J. A.; Gonzalez-Tejero, M. R.; Reyes, F.; Platas, G.; Tormo, J. R.; Genilloud, O. Mycol. Prog. 2017, 16, 713−728. (8) Noumeur, S. R.; Helaly, S. E.; Jansen, R.; Gereke, M.; Stradal, T. E. B.; Harzallah, D.; Stadler, M. J. Nat. Prod. 2017, 80, 1531−1540. (9) Rangel-Grimaldo, M.; Rivero-Cruz, I.; Madariaga-Mazón, A.; Figueroa, M.; Mata, R. J. Nat. Prod. 2017, 80, 582−587. (10) Talontsi, F. M.; Lamshöft, M.; Douanla-Meli, C.; Kouam, S. F.; Spiteller, M. Fitoterapia 2014, 93, 233−238. (11) Zhang, F.; Li, L.; Niu, S. B.; Si, Y. K.; Guo, L. D.; Jiang, X. J.; Che, Y. S. J. Nat. Prod. 2012, 75, 230−237. (12) Weber, H. A.; Baenziger, N. C.; Gloer, J. B. J. Am. Chem. Soc. 1990, 112, 6718−6719. (13) Weber, H. A.; Gloer, J. B. J. Org. Chem. 1991, 56, 4355−4360. (14) Pinedo, C.; Wang, C. M.; Pradier, J. M.; Dalmais, B.; Choquer, M.; Le Pècheur, P.; Morgant, G.; Collado, I. G.; Cane, D. E.; Viaud, M. ACS Chem. Biol. 2008, 3, 791−801. (15) Tani, H.; Koshino, H.; Sakuno, E.; Cutler, H. G.; Nakajima, H. J. Nat. Prod. 2006, 69, 722−725. (16) Coates, R. M.; Ho, J. Z.; Klobus, M.; Zhu, L. J. Org. Chem. 1998, 63, 9166−9176. (17) Coates, R. M.; Ho, Z.; Klobus, M.; Wilson, S. R. J. Am. Chem. Soc. 1996, 118, 9249−9254. (18) Fitjer, L.; Majewski, M.; Monzò-Oltra, H. Tetrahedron 1995, 51, 8835−8852. (19) Finefield, J. M.; Sherman, D. H.; Kreitman, M.; Williams, R. M. Angew. Chem., Int. Ed. 2012, 51, 4802−4836. (20) Gregson, R. P.; Ouvrier, D. J. Nat. Prod. 1982, 45, 412−414. (21) Fontana, A.; Fakhr, I.; Mollo, E.; Cimino, G. Tetrahedron: Asymmetry 1999, 10, 3869−3872. (22) Degenhardt, J.; Köllner, T. G.; Gershenzon, J. Phytochemistry 2009, 70, 1621−1637. (23) Prosser, I.; Altug, I. G.; Phillips, A. L.; Kö nig, W. A.; Bouwmeester, H. J.; Beale, M. H. Arch. Biochem. Biophys. 2004, 432, 136−144. (24) Schmidt, C. O.; Bouwmeester, H. J.; Franke, S.; Kçnig, W. A. Chirality 1999, 11, 353−362. (25) Searle, P. A.; Jamal, N. M.; Lee, G. M.; Molinski, T. F. Tetrahedron 1994, 50, 3879−3888. (26) Horton, P.; Inman, W. D.; Crews, P. J. Nat. Prod. 1990, 53, 143− 151. (27) Xia, G. Y.; Sun, D. J.; Ma, J. H.; Liu, Y.; Zhao, F.; Donkor, P. O.; Ding, L. Q.; Chen, L. X.; Qiu, F. Sci. Rep. 2017, 7, 43576−43583.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b04123. Experimental section, biological activities, computational section, and spectroscopic data of 1 (PDF) Accession Codes

CCDC 1885762 and 1892020 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], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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L.-L.X. and H.-L.C. contributed equally to this work.

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Corresponding Authors

*Tel: +86 023 65678450. E-mail: [email protected]. *Tel: 03-3818-2532. E-mail: [email protected]. ORCID

Xiao-Long Yang: 0000-0002-3838-4084 Ikuro Abe: 0000-0002-3640-888X D

DOI: 10.1021/acs.orglett.8b04123 Org. Lett. XXXX, XXX, XXX−XXX