(±)-Alternamgin, a Pair of Enantiomeric Polyketides, from the

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

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(±)-Alternamgin, a Pair of Enantiomeric Polyketides, from the Endophytic Fungi Alternaria sp. MG1 Jun-Chen Wu,† Yanan Hou,† Qianhe Xu,† Xiao-Jie Jin,‡ Yaxiong Chen,§ Jianguo Fang,† Burong Hu,§ and Quan-Xiang Wu*,†

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†

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡ College of Pharmacy, Gansu University of Chinese Medicine, Lanzhou 730000, People’s Republic of China § Key Laboratory of Heavy Ion Radiation Biology and Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: A pair of enantiomeric polyketides, (+)- and (−)-alternamgin (1), featuring an unprecedented 6/6/6/6/5/6/6 seven ring backbone, were isolated from the endophytic fungi Alternaria sp. MG1. The relative configuration of 1 was determined using X-ray diffraction, and the absolute configurations of (±)-1 were confirmed by comparing the experimental and calculated ECD data. Plausible biosynthetic pathways for 1 were proposed. Compound (−)-1 exhibited moderate necrosis rates to Hela and HepG2 cells, but (+)-1 only showed similar necrosis rates to HepG2 cells.

enantiomeric polyketides, (+)- and (−)-alternamgin (1) (Figure 1, left), were discovered. Structurally, (±)-1 are

Alternaria Nees, comprising pathogens and saprophytes, is a widely distributed fungal genus, with more than 250 species, in the natural environment. The Alternaria species can produce multifarious toxic metabolites, belonging principally to three different structural groups, dibenzopyrone, perylene, and tetramic acid derivatives. Based on their carcinogenicity, mutagenicity, and teratogenicity, Alternaria mycotoxins can cause serious diseases in humans and animals and significant economic losses to agriculture.1−6 Polyketides, widespread in nature, have been isolated from a variety of sources including bacteria, fungi, and plants. They showed diverse bioactivities, encompassing cytotoxic, antioxidant, antibacterial, antifungal, antimalarial, and immunoregulatory traits. In pathogenic fungi, polyketides are potential virulence factors and immunosuppressants.7,8 Polyketides exhibit a high degree of structural diversity and can be synthesized from simple acyl building blocks.9,10 The investigation of natural products for potential lead compounds in the development of pharmaceuticals is of strong interest because natural products and inspired compounds still imply a significant proportion of currently available pharmaceuticals.11 As part of our continuing efforts in exploring bioactive molecules from fungi,12−16 Alternaria sp. MG1, a phytopathogenic fungus (GenBank accession no. JN102357) isolated from Vitis quinquangularis,17−19 was cultivated in a potato dextrose liquid medium. As a result, a pair of © XXXX American Chemical Society

Figure 1. Chemical structure (left) and key HMBC correlations (from H to C) (right) of 1.

unconventional polyketides, featuring an unprecedented 6/6/ 6/6/5/6/6 ring framework. Herein, we report the details of their structural elucidation, hypothetical biosynthetic pathways, and bioactivity evaluations. Alternamgin (1) was obtained as a red wine colored lump crystal (CH2Cl2/MeOH 3:1). The molecular formula of C29H22O9 was deduced from the (+)-HR-ESI-MS ions at m/ z 515.1342 [M + H]+ (calcd 515.1337), revealing 19 degrees of unsaturation. Absorption bands at 1727, 1675, 1617, and Received: February 4, 2019

A

DOI: 10.1021/acs.orglett.9b00475 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters 1459 cm−1 in the IR spectrum indicate the existence of ketone carbonyl, ester carbonyl, and olefinic groups, and the band at 3346 cm−1 implies the presence of exchangeable protons. The 1 H NMR spectrum (Table 1) of 1 shows proton signals for a

Analysis of the HMBC spectrum (Figure 1, right) aids the construction of the partial structure of 1. According to the HMBC correlations from the methyl (CH3-1) to C-2 (δC 85.2), C-3, and C-19 (δC 89.3) and from H2-3 to C-2, C-4 (δC 198.0), C-5, and C-19, the ketone carbonyl and two oxygenated sp3-hybridized quaternary carbons are connected by the only methylene group (CH2-3) in the structure. The observed HMBC correlations of H-8/C-6, C-10; H-10/C-8, C6; H-21/C-23, C-25; and H-23/C-21, C-25 indicate two 1,2,3,5-tetrasubstituted benzene rings (rings C and E). The HMBC correlations of H-14/C-12, C-16 and H3-27/C-12, C13, C-14 revealed the presence of a pentasubstituted benzene ring (ring D) and the position of the methyl (CH3-27). On the basis of the HMBC correlations of H3-28/C-9 and H3-29/C22, two methoxyls were confirmed, consistent with the chemical shifts. The rings C and D were concatenated by the C-11/C-12 carbon bond determined by HMBC correlations from H-10 and H3-27 to C-12. In the same manner, the link between rings A and E through C-19/C-20 carbon bond was determined by HMBC correlations from H31, H2-3, and H-21 to C-19. The HMBC spectrum of 1 is not informative enough to determine the presence of the deprotonated benzene (ring B), δ-lactone (ring F), and furan (ring G) cores. Due to the crowded quaternary carbons, their positions of rings B, F, and G in the structure were partly resolved by the molecular formula, the degrees of unsaturation, and the chemical shifts.20,21 A single-crystal X-ray diffraction analysis confirms the novel carbon skeleton possessed by 1 (Figure 2). The crystal

Table 1. NMR Data of 1 δH (J, Hz)a

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 7-OH 15-OH 24-OH a

δC (DEPT)b

1.51 (s, 3H) 3.31 (d, J = 11.6 Hz, 1H) 3.55 (d, J = 11.6 Hz, 1H)

6.73 (d, J = 1.6 Hz, 1H) 7.81 (d, J = 1.6 Hz, 1H)

7.42 (s, 1H)

5.74 (d, J = 1.6 Hz, 1H) 6.64 (d, J = 1.6 Hz, 1H)

2.97 3.98 3.72 11.87 9.28 11.43

(s, 3H) (s, 3H) (s, 3H) (s, 1H) (brs, 1H) (s, 1H)

20.3 (CH3) 85.2 (C) 50.4 (CH2) 198.0 122.3 114.6 158.5 103.9 161.0 104.2 137.0 124.6 130.9 130.9 153.7 144.4 139.6 139.6 89.3 127.5 107.8 168.0 102.3 165.5 100.9 167.4 24.0 55.8 56.5

(C) (C) (C) (C) (CH) (C) (CH) (C) (C) (C) (CH) (C) (C) (C) (C) (C) (C) (CH) (C) (CH) (C) (C) (C) (CH3) (CH3) (CH3)

Figure 2. X-ray ORTEP drawing of 1.

structure of 1 was demonstrated to be racemic by the space group H-M P-1. The enantiomers (−)-1 and (+)-1 (Figure 3C) were obtained by the chiral HPLC resolution (Figure 3A). The NMR, MS, IR, and UV data of (−)-1 and (+)-1 were identical, the CD curves were completely reversed (Figure 3B), and the optical rotation data were similar but in opposite direction ([α]25 D −46 and +48). The absolute stereochemistry was determined on the basis of the comparison of experimental and the quantum mechanically calculated electric circular dichroism (ECD) data by using the time-dependent density functional theory (TDDFT) at the B3LYP/dgdzvp level in MeOH.22,23 The computed ECD curve of (2S,19S)-1 matches the experimental CD spectrum for (−)-1 (Figure 3B1). The first calculated positive Cotton effect at 209 nm (π- to π*-type molecular orbital) could be assigned to the experimentally observed Cotton effect at 217 nm. The negative 231 nm and positive 296 nm Cotton effects [π- and n- to π*-type (CC and CO double bonds)] 20 were assigned to the experimental absorption bands at 228 and 305 nm, respectively. Given the aforementioned data, the most probable absolute configuration of (−)-1 was elucidated as 2S,19S and its enantiomer as 2R,19R. Compound (±)-1 has been identified as a new type of polyketide that features a rare 6/6/6/6/5/6/6 seven-ring

b

Recorded at 600 MHz in (CD3)2CO. Recorded at 150 MHz in (CD3)2CO.

pentasubstituted benzene ring [δH 7.42 (1H, s, H-14)], two 1,2,3,5-tetrasubstituted benzene rings [δH 5.74 (1H, d, J = 1.6 Hz, H-21), 6.64 (1H, d, J = 1.6 Hz, H-23), 6.73 (1H, d, J = 1.6 Hz, H-8), and 7.81 (1H, d, J = 1.6 Hz, H-10)], and three downfield signals at δH 9.28 (15-OH), 11.43 (24-OH), and 11.87 (7-OH). Additional signals are attributed to two tertiary methyls at δH 1.51 (3H, s, H-1) connected with an sp3hybridized quaternary carbon and δH 2.97 (3H, s, H-27) joined to an aromatic nucleus, two methoxyls [δH 3.72 (3H, s, H-29) and 3.98 (3H, s, H-28)], and a methylene [δH 3.31 (1H, d, J = 11.6 Hz, H-3a) and 3.55 (1H, d, J = 11.6 Hz, H-3b)] connected with an electron-withdrawing group. Its 13C NMR spectrum (Table 1) reveals 29 carbon resonances corresponding to four methyls (two methoxyls), one methylene, five methines, and 19 quaternary carbons [an ester carbonyl, a ketone carbonyl, two oxygenated sp3-hybridized, and 15 olefinic (six oxygenated sp2-hybridized)] as distinguished by the DEPT and HSQC spectra. B

DOI: 10.1021/acs.orglett.9b00475 Org. Lett. XXXX, XXX, XXX−XXX

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opening and reducing reactions from 3, and the third KIM c (cis- more stable than trans-conformation, by model building via Chem3D energy minimization calculations) was produced by the reduction of the semiquinone in 5 and the isomerization of the enol−keto and the allyl alcohol. In pathway A, d was obtained after oxidative coupling26 of KIMs a and b. The key electrocyclization27,28 occurred to produce e. After enol−keto tautomerism and an oxidation, f was obtained. The novel polyketide 1 was formed after a ring opening of the epoxide and an esterification. In pathway B, g was generated by a Diels−Alder reaction29 from the KIMs a and c and via an additional aromatization reaction. Finally, 1 was formed after a dehydration reaction. Compounds (−)-1 and (+)-1 were evaluated for their cytotoxic activities against two human cancer cell lines (Hela and HepG2). Both compounds displayed low cytotoxicity to either Hela or HepG2 cells, with the half maximal inhibitory concentration (IC50) values over 20 μM (Figure S10). The apoptosis of Hela and HepG2 cells induced by (−)-1 and (+)-1 were tested. They did not exhibit significant activity. The necrosis rates (20 μM) (Figure 4) of (−)-1 and (+)-1 to HepG2 cells were uniform, but that of (+)-1 to Hela cells was not found in the experiment, different from (−)-1. We also studied the protection of the H2O2- and 6-hydroxydopamine (6-OHDA)-induced damage of PC12 cells by (±)-1 according to our well-established experimental models.30,31 Treatment of the cells with H2O2 and 6-OHDA reduced the cell viability to ∼65% and ∼45%, respectively. However, no significant protection was observed upon pretreatment of the cells with (±)-1 at nontoxic concentrations (10 μM or 20 μM, Figure S11), indicating that both compounds had little effect on the H2O2- and 6-OHDA-induced death of PC12 cells. They did also not show outstanding result in the micronucleus assay. Polyketides have been identified as pharmacologically interesting molecules with extensive biological activity. Compound (±)-1, representing a novel 6/6/6/6/5/6/6 heptacyclic core with dibenzopyrone functionality and

Figure 3. (A) Chiral HPLC chromatogram of 1, (B) calculated and experimental ECD spectra of (−)-1 and (+)-1, and (C) the structures of (−)-1 and (+)-1.

backbone. Two possible biogenetic pathways are proposed in Scheme 1. Four naturally occurring polyketides, alternariol (2),24 generated from acetyl coenzyme A (CoA) and malonyl CoA by polyketide synthase (PKS) along with the cyclization reaction, alternariol 5-O-methyl ether (3),24 altenusin (4),25 and dehydroaltenusin (5),25 were also obtained from this species and are perhaps the parent compounds for novel polyketides. First, a key intermediate molecule (KIM) a was formed by the reaction of a decarboxylation from 4. Simultaneously, the second KIM b was generated via ringScheme 1. Proposed Biosynthetic Pathways of 1

C

DOI: 10.1021/acs.orglett.9b00475 Org. Lett. XXXX, XXX, XXX−XXX

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potential cytotoxicity, were isolated from the phytopathogenic fungus Alternaria sp. MG1. These compounds enrich the structural diversity of polyketides and may attract broad interest of synthetic chemists and biosynthetic chemists.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00475. Experimental details and copies of HR-ESI-MS, IR, UV, and 1D and 2D NMR spectra (PDF) Accession Codes

CCDC 1894352 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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Figure 4. Apoptosis induction effects of the (−)-1 and (+)-1. After treatment with DMSO or compounds for 48 h, cell apoptosis was determined by Annexin V-FITC/PI staining. Cells in the lower right quadrant indicate early apoptotic cells, and cells in the upper right quadrant indicate late apoptotic cells. Cells in the upper left quadrant indicate necrosis cells. The concentrations are 0 μM in (A), (C), and (E) and 20 μM in (B), (D), and (F). The necrosis rates are 7.3% (A), 15.7% (B); 4.9% (C), 14.5% (D); 7.0% (E), and 21.8% (F).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jianguo Fang: 0000-0002-2884-3363 Quan-Xiang Wu: 0000-0002-1895-5417 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Program for National Natural Science Foundation (No. 21672087). We thank Prof. Juling Shi (College of Food Science and Engineering, Northwest A&F University, P.R. China) for presenting the fungi and Prof. Yan-Long Yang (State Key Laboratory of Applied Organic Chemistry, Lanzhou University, P.R. China) for designing the proposed biosynthetic pathways. D

DOI: 10.1021/acs.orglett.9b00475 Org. Lett. XXXX, XXX, XXX−XXX