Trichoderpyrone, a Unique Polyketide Hybrid with a Cyclopentenone

Jun 1, 2017 - The 1H, 13C, and HMQC NMR data of 1 (Table 1) revealed the ... 6b, 2.50 d (16.8) .... HepG2, and HeLa, was evaluated, and IC50 values of...
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Trichoderpyrone, a Unique Polyketide Hybrid with a Cyclopentenone−Pyrone Skeleton from the Plant Endophytic Fungus Trichoderma gamsii Lin Chen,†,# Shu-Bin Niu,‡ Li Li,§ Gang Ding,*,† Meng Yu,† Gui-shan Zhang,∥ Meng-hua Wang,† Lu-ying Li,† Tao Zhang,† Hong-Mei Jia,† Hong-wu Zhang,† Hai Shang,† Xing-zhong Liu,⊥ and Zhong-mei Zou*,† †

Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, People’s Republic of China ‡ School of Biological Medicine, Beijing City University, Beijing 450046, People’s Republic of China § State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Beijing 100050, People’s Republic of China ∥ Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China ⊥ State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100090, People’s Republic of China # Zhengzhou Key Laboratory of Medicinal Resources Research, Institute of Nanostructured Functional Materials, Huanghe Science and Technology College, Zhengzhou, Henan 450006, People’s Republic of China S Supporting Information *

ABSTRACT: Trichoderpyrone (1), a unique polyketide with a cyclopentenone−pyrone hybrid skeleton, was isolated from the plant endophytic fungus Trichoderma gamsii. The structure of 1 was determined by detailed analysis of NMR data together with comparison of chemical shift values of similar fragments. The relative and absolute configurations were established by NOESY correlations and CD experiment. Trichoderpyrone (1) displayed weak cytotoxic activities against A549, HepG2, and HeLa cancer cell lines. 1 might originate from a hybrid biosynthetic pathway through two nonreduced (NR) polyketide megasynthetases.

F

double bonds are generally reduced to aliphatic chains by ER (Figure 1). In the majority of documented cases, fungal polyketides were synthesized by a PKS, whereas occasionally a polyketide may be biosynthesized by two PKSs combined.2 We previously reported a series of PKS-NRPS hybrid secondary metabolites, cytochalasans with new ring systems or novel skeletons, from the endophytic fungus Trichoderma gamsii.3,4 During our ongoing investigation to find novel secondary metabolites from this fungus, a new polyketide trichoderpyrone (1) containing a unique cyclopentenone−pyrone hybrid skeleton together with a known lactone compuond (2)5 was isolated. The structural features implied that compound 1 might be biosynthesized by two PKSs. In this Note, the structural elucidation, evaluation of cytotoxicity, and possible biogenetic pathway are presented.

ilamentous fungi contain a large number of gene clusters that produce a wealth of structurally diverse secondary metabolites including polyketides (PKs), nonribosomal peptides (NRPs), polyketides and nonribosomal peptides hybrids (PK-NRPs), terpenes, and indole-alkaloids. The PKs can be further grouped into three subclasses: nonreduced (NR), partially reduced (PR), and highly reduced (HR) polyketides (Figure 1).1 NR-PK synthases (NR-PKSs) usually contain three core domains: β-ketoacyl synthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) sometimes together with methyl transferase (MT) and reductase (R). PR-PKSs contain KS, AT, ACP, ketoreductase (KR), and dehydratase (DH), and HRPKSs contain KS, AT, ACP, KR, DH, and enoyl reductase (ER).2 According to the different combinations of domains mentioned above, the structures of NR polyketides often possess β-keto carbonyl or hydroxy groups at an alternate position, and the β-keto carbonyl or hydroxy groups in the structures of PR polyketides are usually further metabolized to double bonds, whereas in the structures of HR polyketides, the © 2017 American Chemical Society and American Society of Pharmacognosy

Received: March 5, 2017 Published: June 1, 2017 1944

DOI: 10.1021/acs.jnatprod.7b00190 J. Nat. Prod. 2017, 80, 1944−1947

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Figure 1. Core structural features of NR-PKS, PR-PKS, and HR-PKS.

Trichoderpyrone (1) was obtained as an orange oil. Its molecular formula was determined to be C15H17NO5 on the basis of HRESIMS. The 1H, 13C, and HMQC NMR data of 1 (Table 1) revealed the presence of two methyls, one methylene Table 1. NMR Spectroscopic Data of Trichoderpyrone (1) in (DMSO-d6) pos 1′ 2′ 3′ 4′ 4′-OH 5′ 6′ 7′- Me 1 2 3 4 5 5-OH 6a 6b 7 8 9a 9b -NH2 a

δCb, type

Figure 2. Structures of 1, myrothenone A, and 2.

δHa, mult. (J in Hz)

165.8 105.0 167.1 102.4 159.5 19.4 15.7 22.6 111.5 201.1 76.8 42.8 172.5 140.8 113.1

lactone bond must exist between C-2′ and C-6′, which established the 4-hydroxy-6-methyl-2H-pyran-2-one ring system.9,10 Thus, structure 1 possesses two isolated ring system: a 3-aminocyclopent-2-en-1-one ring system and a 4-hydroxy-6methyl-2H-pyran-2-one ring system linked by a methine unit (C-2). To further support the planar structure 1, chemical shift values of similar fragments in different secondary metabolites were analyzed (Figure 4). The 13C NMR data for the 4hydroxy-6-methyl-2H-pyran-2-one ring system in 1 were nearly the same as those fragments found in aszonapyrone B and penicipyrone, whereas the 13C NMR data of the 3-aminocyclopent-2-en-1-one ring in 1 were nearly the same as those of bromomyrothenone B, myrothenone B, and 3-amino-5hydroxy-5-vinyl-2-cyclopenten-1-one (Figure 4).6−10 In addition, the isolation of 2 from the same fungus and of 3-amino-5hydroxy-5-vinyl-2-cyclopenten-1-one from other different Trichoderma sp. supported structure 1.6−8 The relative configuration of 1 was characterized by analysis of NOESY correlations. The correlations of 5-OH with H-6b and 1-Me implied that these two groups were on the same face of the 3aminocyclopent-2-en-1-one ring, whereas the correlations of H6a with H-8 and 7-NH2 and of 7-NH2 with H-2 put these protons on the other side of the corresponding ring system. In addition, the correlations of H-5′ with 4′-OH and H-6′ and of 4′-OH with 1-Me showed that these protons were close in space. The stereochemistry of 1 was determined by analysis of the NMR data and of the CD spectrum. The 13C NMR data, especially the chemical shift values of C-5, C-6, C-8, and C-9 of 1, were nearly the same as those of carbons in bromomyrothenone B (Figure 4), implying that C-5 possessed the same absolute configuration.8 This conclusion was further supported by the CD spectrum of 1. The positive Cotton effect at 280 or so (n → π* transition) was the same as those of (5R)-3-amino5-hydroxy-5-vinyl-2-cyclopenten-1-one, myrothenone B, and bromomyrothenone B, confirming that the stereochemistry of C-5 was the R-configuration.7,8 On account of the fact that the relative configuration of 1 was determined by NOESY correlations, the absolute configuration of 1 was established to be 2(R) and 5(R). The cytotoxic activity of trichoderpyrone (1) against three cancer cell lines, A549, HepG2, and HeLa, was evaluated, and IC50 values of 16.9 ± 1.3, 30.8 ± 0.2, and 33.9 ± 0.7 μM, respectively, were obtained, compared with the positive control etoposide (IC50 values 16.6 ± 1.2, 16.1 ± 0.1, and 15.0 ± 0.2 μM, respectively).

13.16 br s 5.92 br s 2.11 br s 1.32 d (7.2) 4.10 q (7.2)

5.41 br s 2.67 d (16.8) 2.50 d (16.8) 5.77 5.22 5.04 7.39 8.08

dd (16.8, 10.8) dd (16.8, 1.8) dd (10.8, 1.8) br s br s

Recorded at 600 MHz. bRecorded at 150 MHz.

unit, one methine unit, one oxygenated carbon, eight olefinic carbons, and two carbonyl carbons. In addition, there are four free hydroxy or amino protons. These data accounted for all the 1 H and 13C NMR resonances, implying that there might be two rings in structure 1. The 1H−1H COSY correlations established three isolated proton spin-systems corresponding to −C-8−C-9−, −C-1−C2−, and −NH 2 −. The remaining connectivities were determined by detailed analysis of HMBC correlations. The correlations from 5-OH to C-4, C-5, C-6, and C-8 confirmed that the hydroxy group (5-OH) was anchored at C-5, and C-5 was connected with C-4, C-6, and C-8, respectively. The correlations of the methylene unit (−CH2-6) with C-3 and C-7, of −NH2− with C-3, and from H-2 to C-3, C-4, and C-7 established a 3-aminocyclopent-2-en-1-one ring system with a hydroxy group and a terminal alkyne bond located at C-5.6−8 The HMBC correlation of 1-Me with C-2, C-3, and C-3′ confirmed the connection of C-2 with C-1, C-3, and C-3′. In the HMBC spectrum, the cross-peaks of H-2 with C-2′, C-3′, and C-4′, of 4′-OH with C-4′, of H-5′ with C-3′, C-4′, and C6′, and of 7′-Me with C-5′ and C-6′ led to the linkage of −C2−((C-2′)−C-3′−C-4′−C-5′−C-6′−C-7′)− (Figure 3). Considering the unsaturation degrees and chemical shift values, a 1945

DOI: 10.1021/acs.jnatprod.7b00190 J. Nat. Prod. 2017, 80, 1944−1947

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Figure 3. 1H−1H-COSY, HMBC, and NOESY correlations of the structure of 1.

Figure 4. Comparison of 13C NMR chemical shift values of 1 with those of other compounds containing similar fragments.

Figure 5. Postulated biosynthesis of 1.

Many pyrone-derived secondary metabolites originating from a polyketide biosynthetic pathway have been isolated from different fungi.11,12 3-Amino-5-hydroxy-5-vinylcyclopent-2-en1-one and its analogues were purified from other Trichoderma sp., whereas their biosynthetic pathways have not been confirmed.6−8 The metabolite 1 possesses a cyclopentenone− pyrone hybrid skeleton, which has not been encountered in Nature previously and implying an unusual biogenetic pathway. From the structural features of 1, we propose that trichoderpyrone (1) originates from a polyketide biosynthetic

pathway through two PK megasynthases. The possible biogenetic pathway is shown in Figure 5.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 241 polarimeter, and UV data were recorded on a ThermoGenesys-10S UV−vis spectrometer. IR data were recorded using a Nicolet IS5FT-IR spectrophotometer. CD spectra were performed on a JASCO J-810 spectrometer. 1H and 13C NMR data were acquired with a Bruker 600 spectrometer using solvent signals (DMSO-d6; δH 2.49/δC 39.5) as references. The 1946

DOI: 10.1021/acs.jnatprod.7b00190 J. Nat. Prod. 2017, 80, 1944−1947

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HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS were obtained using a TOF-ESIMS (Waters Synapt G2, USA). Semipreparative HPLC separation was performed on a Lumtech instrument with a YMC-Pack ODS-A column (5 μm, 250 × 10 mm). Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) and silica gel (200−300 mesh) (Qingdao Marine Chemical Plant, Qingdao, China) were used. Extraction and Isolation. The culture of T. gamsii was isolated from the traditional Chinese medicinal plant Panax notoginseng (BurK.) F.H. Chen. The isolate was identified on the basis of the sequence (Genbank Accession No. JF964996) obtained by the analysis of the internal transcribed spacer region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 25 °C for 10 days. Agar plugs were used to inoculate Fernbach flasks (500 mL), each containing 80 g of rice, and incubated at 25 °C for 40 days. The fermented material was extracted with ethyl acetate (5 L four times). The solution was concentrated to dryness under vacuum to afford a crude extract (40.0 g), which was fractionated by silica gel column chromatography (10 × 100 cm) using CH2Cl2−MeOH gradient elution. The fraction (100 mg) eluted with CH2Cl2−MeOH (100:4) was separated by Sephadex LH-20 (CH2Cl2−MeOH) column chromatography to afford three fractions (D1−D3). Fraction D2 (40 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL min−1, 55% MeOH in H2O for 30 min) to afford trichoderpyrone (1, 15 mg, tR 25.2 min). Fraction D3 (35 mg) was further separated by silica gel column chromatography (0.5 × 15 cm) using CH2Cl2−MeOH gradient elution to afford (5R,6R)-5-(1-hydroxyethyl)dihydro-2-furanone (2, 2 mg). Trichoderpyrone (1): orange oil; [α]25D +15.0 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 243 (3.60), 200 (3.72) nm; CD (c 0.5 M, methanol) λ (Δε) 278 (6.66), 308 (−1.07), 339 (0.57); IR (neat) νmax 3427, 2924, 2854, 1623, 1410, 1121, 744, 596 cm−1; for 1H NMR and 13 C NMR data see Table 1; positive HRESIMS m/z 314.1005 [calcd for C15H17NO5 Na (M + Na), 314.1004]. MTT Assay.13 The cytotoxic activities of the isolated compounds were evaluated against the A549, HepG2, and HeLa cell lines by the MTT colorimetric method with etoposide as positive control. The IC50 values of the positive control etoposide were 16.6 ± 1.2, 16.1 ± 0.1, and 15.0 ± 0.2 μM, respectively.

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REFERENCES

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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.jnatprod.7b00190. 1 H, 13C, and 2D NMR spectra of 1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: 86-10-57833290. Fax: 8610-57833290. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (31570340, for D.G.), Beijing Nature Science Foundation (7174284), the Chinese National S&T Special Project on Major New Drug Innovation (2011ZX09307-002-01), and the Fundamental Research Funds for the Central Research Institutes for Public Welfare (D.G.), and the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Agricultural Sciences (No. 502-17). 1947

DOI: 10.1021/acs.jnatprod.7b00190 J. Nat. Prod. 2017, 80, 1944−1947