Curtachalasins A and B, Two Cytochalasans with a Tetracyclic

Letter Cite This: Org. Lett. 2018, 20, 7758−7761

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Curtachalasins A and B, Two Cytochalasans with a Tetracyclic Skeleton from the Endophytic Fungus Xylaria curta E10 Wen-Xuan Wang, Zheng-Hui Li, Tao Feng, Jing Li, Huan Sun, Rong Huang, Qing-Xia Yuan, Hong-Lian Ai,* and Ji-Kai Liu* School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, Hubei 430074, PR China

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ABSTRACT: Two unique cytochalasans, curtachalasins A (1) and B (2), were purified from the endophytic fungus Xylaria curta E10 harbored in the plant Solanum tuberosum. Their structures were determined by extensive spectroscopic methods, Xray crystallographic analysis, and electronic circular dichroism calculations. These two compounds feature an unprecedented pyrolidine/perhydroanthracene (5/6/6/6 tetracyclic skeleton) fused ring system.

C

ytochalasans are a large group of fungal polyketide− nonribosomal peptide products with remarkable biological activities and structural diversity.1 Cytochalasins A and B were first reported in 1967, and they were named for their significant effects on fungal and mammalian cells (Greek: cytos = cell, chalasis = relaxation).2 Cytochalasans can bind to actin filaments and inhibit the polymerization and elongation process leading to the change of cellular morphology.3 Subsequent investigations showed that cytochalasans have a great variety of bioactivities, including cytotoxic, antimicrobial, antiparasitic, phytotoxic, and antiviral.3 The high structural complexity of cytochalasans is mainly due to the variations in size, substitution, or further cyclization of the macrocycle4 as well as the coupling pattern with themselves or other moieties.5 Endophytic fungi are rich resources of bioactive secondary metabolites with a broad range of structural diversity.6 In the process of discovering unique bioactive compounds from untapped endophytic fungi, Xylaria curta E10 was isolated from Solanum tuberosum. Its rice culture afforded two novel cytochalasans curtachalasins A (1) and B (2) (Figure 1).

To the best of our knowledge, 1 and 2 are the first reported tetracyclic 10-phenylcytochalasans with a pyrolidine/perhydroanthracene-fused core structure. Their isolation, structural elucidation, plausible biosynthesis pathway, and preliminary bioassay are reported herein. Curtachalasin A (1) was obtained as colorless powder with the molecular formula C28H37NO7 from its HRESIMS data ([M + Na]+, m/z 522.2460, calcd for 522.2462). The 1H NMR data (Table 1) showed characteristic signals, including single substituted phenyl at δH 7.24 (2H, d, overlapped, H-25/H-29), 7.30 (2H, dd, J = 7.5, 7.5 Hz, H-26/H-28), 7.22 (1H, overlapped, H-27), an olefinic methylene group at δH 5.10 (1H, d, J = 0.9 Hz, Ha-12), 4.95 (1H, d, J = 0.9 Hz; Hb-12), four oxygenated methine groups at δH 4.20 (1H, d, J = 9.6 Hz, H-7), 4.40 (1H, dd, J = 9.6, 9.6 Hz, H-13), 3.94 (1H, d, J = 10.8 Hz, H-19), 3.82 (1H, d, J = 1.8 Hz, H-21), and three methyl groups at δH 0.63 (3H, d, J = 6.8 Hz, H3-11), 2.27 (3H, s, H3-22), 0.74 (3H, d, J = 6.7 Hz, H3-23). The 13C NMR (Table 1), DEPT, and HSQC spectra of 1 presented 28 signals assignable to three methyls, two aliphatic methylenes, 11 aliphatic methines, two aliphatic quaternary carbons, one ketone, one amide, one exocyclic carbon−carbon double bond, and one single substituted phenyl group. The above-mentioned results indicated that compound 1 has six double bonds, which suggested that there are five rings according to the 11 degrees of unsaturation deduced by the molecular formula. The 1H−1H COSY spectrum of 1 displayed two proton spin−spin systems in the core structure, namely H-10/H-3/H4/H-5/H3-11 and H-7/H-8/H-13/H-14(H-15/H-16/H3-23)/ H-20 (H-19)/H-21 (Figure 2). The HMBC correlations from

Figure 1. Structures of compounds 1 and 2.

Received: September 29, 2018 Published: November 30, 2018

© 2018 American Chemical Society

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DOI: 10.1021/acs.orglett.8b03110 Org. Lett. 2018, 20, 7758−7761

Letter

Organic Letters Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data of Compounds 1 and 2 (δ in ppm) 1 (CD3OD) 1

no. 1 2-NH 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25, 29 26, 28 27

H NMR

2 (CDCl3) 13

1

C NMR

H NMR

177.6 3.27 (ddd, 8.0, 6.1, 2.0) 2.76 (dd, 6.1, 2.0) 2.63 (m) 4.20 (d, 9.6) 2.15 (dd, 9.6, 9.6) 2.83 2.67 0.63 5.10 4.95 4.40 1.68 1.84 1.19 1.91

(dd, 13.1, 8.0) (dd, 13.1, 6.1) (d, 6.8) (d, 0.9) (d, 0.9) (dd, 9.6, 9.6) (m) (ddd, 12.9, 3.8, 3.8) (ddd, 12.9, 12.9, 12.9) (ddq, 12.9, 6.7, 3.8)

3.94 2.28 3.82 2.27 0.74

(d, 10.8) (overlapped) (d, 1.8) (s) (d, 6.7)

7.24 (overlapped) 7.30 (dd, 7.5, 7.5) 7.22 (overlapped)

53.4 46.4 31.8 149.4 74.4 41.6 52.9 44.2 11.6 112.1 73.3 39.2 31.0 35.7 84.3 213.3 72.5 41.4 68.4 24.0 14.0 137.6 129.4 128.2 126.4

13

C NMR 175.5

6.34 3.82 2.90 1.76 2.19

(br s) (m) (dd, 4.2, 3.2) (m) (dq, 12.9, 6.6)

2.96 2.73 0.98 1.04

(dd, 13.7, 4.7) (dd, 13.6, 8.0) (d, 7.0) (d, 6.6)

6.85 2.28 1.64 1.41 1.92

(d, 1.9) (overlapped) (ddd, 12.9, 3.3, 3.3) (ddd, 12.9, 12.9, 12.9) (m)

3.85 2.58 3.72 2.30 0.67

(d, 10.8) (br dd, 10.8, 10.8) (br s) (s) (d, 6.7)

7.21 (d, 7.6) 7.36 (dd, 7.6, 7.6) 7.27 (overlapped)

53.2 45.7 35.3 42.9 202.0 132.0 52.5 45.0 15.2 11.9 142.9 33.3 33.6 36.9 84.6 211.5 72.0 41.2 68.3 24.1 14.5 137.2 129.5 128.9 127.2

tion between H-8 and H-14 indicated that H-14 has the same orientation as H-8. Moreover, the relative configuration of the 17-acetyl can be determined to be β by the NOE correlation between H-19 and H3-22. A single crystal of 1 was successfully analyzed by X-ray diffraction with a Flack parameter x value of −0.05 ± 0.017. Therefore, the absolute configuration of compound 1 was determined to be 3S,4R,5S,7S,8S,9R,13S,14R,16S,17S,19R,20R,21R (CCDC 1869563, Figure 3). Compound 2 was obtained as an amorphous powder with the molecular formula C28H35NO6 determined by HRESIMS ([M + Na]+, m/z 504.2354, calcd for 504.2357). The 1H NMR

Figure 2. Key 1H−1H COSY, HMBC, and ROESY correlations of 1.

H2-10 to C-24 and C-25/29 indicated that the phenyl group substitutes at position 10. According to the HMBC correlations from H-4 to C-9, H-11 to C-6, H-12 to C-5 and C-7, H-8 to C-9, as well as H-21 to C-4, C-8, and C-9, the fusing pattern of rings A, B, and C can be determined. By combining the HMBC correlations from H3-22 to C-17 and C18, H3-23 to C-17, as well as H-19 to C-17, the structure of ring D was elucidated as shown in Figure 2. Because the B, C, and D rings are typical cyclohexane structures, the J coupling constants between vicinal protons are useful to determine their orientations. The coupling constants of H-7 (J = 9.6 Hz), H-13 (J = 9.6, 9.6), H-16 (J = 12.9, 6.7, 3.8), H-19 (J = 10.8), and H-21 (J = 1.8) suggested that they are axial, axial, axial, axial, and equatorial bonds, respectively. Furthermore, the relative configuration of 1 in rings A and B was deduced as shown in Figure 2 by the NOE correlations of H-3/H3-11, H2-10/H-21, and H-4/H-21. The NOE correla-

Figure 3. X-ray structure of 1. 7759

DOI: 10.1021/acs.orglett.8b03110 Org. Lett. 2018, 20, 7758−7761

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Organic Letters and 13C NMR spectra of 2 showed signals similar to those of 1, including the signals of phenyl group and the D ring (from position 16 to 29, Table 1). However, compound 2 lacks the signals of exocyclic carbon−carbon double bond but has the signals of one more doublet methyl group at δH 1.04 (3H, d, J = 6.6, H3-12), δC 11.9 (C-12), and one more α,β-unsaturated ketone at δH 6.85 (1H, d, J = 1.9, H-13), δC 202.0 (C-7), 132.0 (C-8), 142.9 (C-13). The 1H−1H COSY spectrum of 2 displayed two major spin−spin systems, namely H-10/H-3/H4/H-5(H-11)/H-6/H-12 and H-13/H-14(H-15/H-16/H23)/H-20(H-19)/H-21. The planar structure of 2 was established by the key HMBC correlations from H-10 to C24 and C-25/29, H-12 to C-5, C-6 and C-7, H-13 to C-7 and C-8, H-19 to C-17, H-21 to C-8 and C-9, H3-22 to C-17 and C-18, as well as H3-23 to C-17 (Figure 4), which shares the same scaffold as 1.

level of theory). Therefore, the relative configuration of 17-OH was deduced to be α. To assign the absolute configuration of 2, ECD calculation (TDDFT) was performed on the mPW1PW91/6-311G(d) level of theory. The calculated ECD curve of the enantiomer with absolute configuration 3S,4R,5S,6R,9R,14S,16S,17S,19R,20R,21R is in good accordance with the experimental spectrum (Figure 5). Therefore, the absolute configuration of 2 was unambiguously assigned.

Figure 5. Calculated and experimental ECD of 2.

The carbon skeleton shared by compounds 1 and 2 might be derived from the known compound 19,20-epoxycytochalasin D,7 which was produced by X. curta E10 with a higher amount (around 100 mg per kilogram rice medium). The biosynthesis of compounds 1 and 2 may subsequently involve oxidation, condensation, rearrangement, reduction, and hydrolysis to generate the pyrolidine/perhydroanthracene fused ring system (Scheme 1).

Figure 4. Key 1H−1H COSY, HMBC, and ROESY correlations of 2.

The relative configuration of 2 was deduced by the ROESY spectrum and proton−proton J coupling constants. The NOE correlations of H2-10/H-21 and H-4/H-21 indicated that H10, H-4, and H-21 are on the same side with β orientation. Correspondingly, the NOE correlations of H-3 and H3-11 suggested that they have α orientation. The splitting pattern of H-6 (2.19, dq, J = 12.9, 6.6 Hz) showed that the coupling constant between H-6 and H-5 was 12.9 Hz, indicating H-6 and H-5 are both axial bonds. Therefore, H-6 has an α orientation related to H3-11. The NOE correlations of H3-11/ H-6 and H3-12/H-5 also proved this assignation. Thus, the relative configuration of the substituent groups on the A ring can be determined as shown in Figure 4. The coupling constant of H-21 was relatively small and did not result in visible splitting of the resonance peak, suggesting H-21 is in the equatorial position. The visible spin−spin coupling and splitting of H-20 (2.58, 1H, br dd, J = 10.8, 10.8 Hz) was induced by the vicinal protons H-19 (3.85, 1H, d, J = 10.8 Hz) and H-14 (2.28, 1H, overlapped), which indicated the H-20, H-19, and H-14 are all axial bonds. The coupling constants of Hax-15 (1.41, 1H, ddd) were 12.9, 12.9, and 12.9 Hz. Besides the geminal coupling with Heq-15, the other two coupling constants of Hax-15 were induced by H-14 and H-16, respectively. Thus, the coupling constant value suggested that H-16 is in the axial position. Therefore, the relative configurations of H-14, H-16, H-19, H-20, and H-21 were deduced to be β, β, β, α, and α, respectively. A NOE correlation between H3-23 and an active proton at δH 3.95 (1H, br s) was observed. This proton was assigned to be 17-OH, considering the distances between H3-23 and other hydroxyl protons are beyond the maximum distance of NOE (distances between H3-23/17-OH, H3-23/19-OH, and H3-23/ 21-OH are 2.655, 4.872, and 7.301 Å respectively, in the lowest energy conformer of 2 optimized on B3PW91-D3/6-311G(d)

Scheme 1. Proposed Biosynthesis Pathway of Compounds 1 and 2 from 19,20-Epoxycytochalasin D

Following the established bioassay methods,8 compounds 1 and 2 were tested for cytotoxic activities (against five human cancer cell lines, HL-60, A-549, SMMC-7721, MCF-7, and SW480), antibacterial activities (against Escherichia coli, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa), and antifungal activities (against Candida albicans, Epidermophyton f loccosum, Trichophyton rubrum, and Microsporum gypseum). Compounds 1 and 2 showed weak antifungal activity against M. gypseum (70.3 ± 0.4% and 68.4 ± 0.7% inhibitory percentage, respectively, at concentration of 200 μM). In summary, curtachalasins A (1) and B (2) are a new family of cytochalasans with a 5/6/6/6-fused tetracyclic skeleton. This unique core structure further enriches the structural diversity of cytochalasans. Their potential bioactivities and 7760

DOI: 10.1021/acs.orglett.8b03110 Org. Lett. 2018, 20, 7758−7761

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Organic Letters

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ecological roles in the endophytic system are worth unveiling in future research.

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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.8b03110. Experimental procedures; 1D and 2D NMR, MS, IR, and ECD spectra for compounds 1 and 2 (PDF) Accession Codes

CCDC 1869563 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 Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Tao Feng: 0000-0002-1977-9857 Hong-Lian Ai: 0000-0002-6832-0970 Ji-Kai Liu: 0000-0001-6279-7893 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 31801789, 31560010, 81773590, 81561148013, 21502239, and 81803395), National Key R&D Plan (No. 2017YFC1704007), Hubei Provincial Natural Science Foundation of China (No. 2018CFB222), Key Projects of Technological Innovation of Hubei Province (No. 2016ACA138), and the Fundamental Research Funds for the Central Universities, South-Central University for Nationalities (Nos. CZP18005, CZT18013, and CZT18014). We are grateful for the HRMS and NMR measurements provided by the Analytical & Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities. We are thankful to Kunming Institute of Botany, Chinese Academy of Sciences, for performing the quantum chemistry calculations on their high-performance computing cluster.

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DOI: 10.1021/acs.orglett.8b03110 Org. Lett. 2018, 20, 7758−7761