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Trichodermone, a Spiro-cytochalasan with a Tetracyclic Nucleus (7/5/ 6/5) Skeleton from the Plant Endophytic Fungus Trichoderma gamsii Gang Ding,†,‡ Hailou Wang,† Li Li,‡ Bo Song,† Hong Chen,§ Hongwu Zhang,† Xinzhong Liu,⊥ and Zhongmei Zou*,† †

Institute of Medicinal Plant Development, Beijing, People’s Republic of China State Key Laboratory of Bioactive Substance and Fuction of Natural Medicines, Institute of Materia Medica, Beijing 100050, People’s Republic of China § Medical College of Chinese People’s Armed Police Force, Tianjin, People’s Republic of China ⊥ Institute of Microbiology, Beijing, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Trichodermone (1), the first spiro-cytochalasan with an unprecedented tetracyclic nucleus (7/5/6/5), together with its possible biosynthetic precursor aspochalasin D (2), was isolated from the endophytic fungus Trichoderma gamsii. Compound 2 displayed moderate inhibitory activity against HeLa cells with an IC50 value of 5.72 μM.

C

reactive regions within the precursor and hypothesized intermediates, we assumed there might exist secondary metabolites possessing similar structural features in the fermentation extract. In order to investigate the theoretical minor intermediates, we fermented the endophyte and isolated trichodermone (1), the first spiro-cytochalasan containing a decahydrospiro[benzo[7]annulene-2,3′-pyrrolidine] tetracyclic skeleton from the crude extract. We herein present the structure elucidation, bioactivities, and plausible biogenetic pathway of trichodermone (1). The molecular formula of trichodermone (1) was determined to be C24H33NO5 (9 degrees of unsaturation) on the basis of HRESIMS (m/z 438.2264 [M + Na]+; Δ −1.3 mmu). Analysis of the 1H, 13C, and HMQC NMR data of 1 (Table1) revealed the presence of five methyl groups, six methylene units, five methine units, two quaternary carbons (one oxygenated), two olefinic carbons, and four carbonyl carbons. These data accounted for all the 1H and 13C NMR resonances and required compound 1 to be a tetracyclic secondary metabolite. Interpretation of the 1H−1H COSY NMR data for this compound identified two isolated proton spin-systems, which correspond to C-15−C-20 and C-11−C-5−C-4−C-3−C-10− C-22 (C-23)−C-24 subunits (Figure 1). The remaining

ytochalasans are mycotoxins found in different fungi including Aspergillus, Phomopsis, Penicillium, Zygosporium, Chaetomium, Rosellinia, and Metarrhizium1 and recently also isolated from the plant endophytic fungus Trichoderma gamsii.2 Structurally, this group of fungal toxins contains one isoindole unit fused with one macrocyclic ring. Isotope labeling experiments and genetic investigation revealed that cytochalasans originate from a polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid pathway.1,3,4 Due to the various amino acid precursors including Leu, Phe, Ala, Trp, and Val, together with diverse oxygenated regions in the macrocyclic ring, more than 100 cytochalasan analogues have been reported to date.5 Among these known compounds, several unusual analogues with a unique pentacyclic system such as chaetochalasin A,6 spicochalasin A,7 aspergillin PZ,8 and trichoderones A (3) and B (4)2 were reported. Recently, Overman and co-workers totally synthesized aspergillin PZ.9−11 As part of our ongoing search for new bioactive secondary metabolites from endophytes,12,13 the endophytic fungus Trichoderma gamsii was obtained from the traditional Chinese medicinal plant Panax notoginseng (BurK.) F. H. Chen. Our previous chemical investigation of T. gamsii led to the isolation of two unique pentacyclic cytochalasans, trichoderones A (3) and B (4),2 in which the carbon bond between C-13 and C-19 was hypothetically formed by an intramolecular 1,4-Michael addition reaction from their putative precursor aspochalasin D (2).2,14 Considering the fact that there are different chemically © 2014 American Chemical Society and American Society of Pharmacognosy

Received: September 12, 2013 Published: January 14, 2014 164

dx.doi.org/10.1021/np4007487 | J. Nat. Prod. 2014, 77, 164−167

Journal of Natural Products

Note

Figure 1. Selective 2D NMR correlations of 1.

peaks from H-4 to C-8 and C-21, H2-18 and H2-20 to C-13, and H-19 to C-8 and C-13 led to the completion of one cyclohex-3-enone fused with the pyrrolidin-2-one at C-9, which established one unique spiro ring system. The key correlation of CH3-25 with C-13, and of C-14 with C-15, led to the connection of C-14 with C-13, C-15, and C-25, forming one cycloheptane moiety fused to the cyclohex-3-enone at C-13 and C-19. Accounting for the chemical shift value of C-8 (δC 124.4), C-13 (δC 178.1), and C-14 (δC 89.0), and unsaturation degrees, the remaining carboxyl group (−COO−, δC 169.7) must be connected with C-8 and C-14, respectively, which established one α,β-unsaturated lactone system to form one furan-2(5H)one ring.15 Thus the planar structure of trichodermone (1) was established as shown in Figure 1. The relative configuration for trichodermone (1) was determined by analysis of coupling constants and NOESY correlations. The coupling constants between H-19 and H-20a and between H-19 and H-20b were 4.5 and 14.0 Hz, respectively, implying that the protons of H-19 and H-20a were on the same side of the cyclohex-3-enone ring, whereas H19 and H-20b were on the opposite side of the same ring system.2 The large coupling constants between H-3 and H-4 (J = 10.8 Hz in acetone-d6) and H-4 and H-5 (J = 8.4 Hz in acetone-d6) implied their trans configuration, respectively. In addition, the relative configurations for C-3, C-4, and C-5 were further supported by NOESY correlations. The cross-peak of H-4 with H2-10 indicated that those protons had the same orientation on the pyrrolidin-2-one ring, whereas the correlation from H-5 to H-3 revealed their closeness and also put the two protons on the opposite side of the same ring. The correlations of CH3-11 to H-20b and CH3-25, respectively, confirmed that those protons were close in space to each other. Thus the relative configuration was determined as depicted in Figure 2. Electronic circular dichroism (ECD) is a powerful method to study the stereochemistry of chiral molecules and has been widely used for absolute configuration assignments of natural

Table 1. NMR Spectroscopic Data of Trichodermone (1) in CDCl3, and in Acetone-d6 pos.

δC,b type

δH,a mult. (J in Hz)

1 2 3

171.9, qC

4

51.5, CH

5.81, brs 4.23, dd (7.0, 8.0) 3.23, m

5

45.3, CH

3.23, m

55.2, CH

6 7 8 9 10

211.1, 169.7, 124.4, 61.4, 29.9,

qC qC qC qC CH2

11 12 13 14 15

17.0, 31.1, 178.1, 89.0, 36.4,

CH3 CH3 qC qC CH2

16

24.9, CH2

17

44.7, CH

18

30.2, CH2

19 20a

35.9, CH 43.0, CH2

20b 21 22 23 24 25

206.8, qC 26.2, CH 23.9, CH3 21.3, CH3 26.5, CH3

δH,c mult. (J in Hz)

4.23, ddt (2.4, 7.8, 10.8) 3.12, dd (8.4, 10.8) 3.22, dd (7.2, 8.4)

key HMBC (H → C#) in CDCl3 C-3, C-4, C-9 C-4, C-5 C-3, C-5, C-6, C9, C-21 C-3, C-4, C-6, C-9

2.13, m; 1.42, m 0.80, d (6.0) 2.21, s

1.30, m; 1.25, m 0.85, d (7.2) 2.20, s

C-3, C-4, C-22, C23, C-24 C-4, C-5, C- 6 C-5, C-6

2.27, m; 1.84, dt (3.0, 16) 1.98, m; 1.68, m 1.41, m; 1.23, m 1.75, m; 1.67, m 3.53, m 2.86, dd (4.5, 16.5) 2.36, dd (14.0, 16.5)

2.26, m; 1.76, m 1.98, m; 1.76, m 2.15, m; 1.48, m 1.84, m; 1.30, m 3.46, m 2.78, dd (5.4, 16.8) 2.61, dd (13.2, 16.8)

13, 14, 16, 25

1.53, m

1.83, m

0.93, d (8.0) 0.92, d (8.0) 1.64, s

0.93, d (6.6) 0.92, d (6.6) 1.69, s

C-3, C-10, C-23, C-24 C-10, C-22, C-24 C-10, C-22, C-23 C-13, C-14, C-15

C-15, C-18 C-15, C-16, C-18 C-16, C-17, C-19 C-8, C-13, C-21 C-9, C-13, C-14, C-18, C-19

a c

Recorded at 500 MHz in CDCl3. bRecorded at 125 MHz CDCl3. Recorded at 600 MHz in acetone-d6.

connection was determined by HMBC correlations. The correlations from CH3-12 and CH3-11 to C-5 and C-6 indicated that C-6 was connected to both C-5 and C-12. Those of H-4 with C-1 and C-9, and the free amino-proton (−NH−) with C-3, C-4, and C-9, established one pyrrolidin-2one unit with one isobutyl and one 3-oxobutan-2-yl attached at C-3 and C-4, respectively. In the HMBC spectra, the cross-

Figure 2. Selective NOESY correlations of 1. 165



Note

products.16,17 Thus, the absolute configuration of 1 was tentatively established by a combination of ECD experiments and quantum-chemical calculations adopting time-dependent density functional theory (TDDFT).17 The calculated ECD spectrum of compound 1 was red-shifted 30 nm according to the UV correction and matched well with the experimental ECD spectrum of 1 (Figure 3). Thus, the stereochemistry for 1 was determined to be 3S, 4R, 5S, 8Z, 9R, 14R, and 19S, which was consistent with its biogenetic origin.

carbon (C-13), to construct a cyclohept[cd]isobenzonfuran2(3H)-one ring system, not encountered in any other terrestrial sources.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a Beckman Coulter DU 800 spectrometer. IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. 1H and 13 C NMR data were acquired with a Bruker 500 spectrometer using solvent signals (CDCl3; δH 7.26/δC 77.6; acetone-d6; δH 2.05/δC 29.8, 206.0; pyridine-d5; δH 8.73, 7.58, 7.21/δC 149.9, 135.5, 123.5) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were acquired using an LTQ Orbitrap XL mass spectrometer. Fungal Material. The culture of Trichoderma gamsii was isolated from the traditional Chinese medicinal plant Panax notoginseng. The isolate was identified based on sequence (GenbBank accession no. JF964996) analysis of the ITS region of the rDNA and assigned the accession no. SQP 79-1 in X.L.’s culture collection at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. The fungus was cultured on slants of potato dextrose agar at 25 °C for 10 days. Agar plugs were used to inoculate 250 mL Erlenmeyer flasks, each containing 40 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5 before sterilization. Flask cultures were incubated at 25 °C on a rotary shaker at 170 rpm for five days as seed culture. Fermentation was carried out in Fernbach flasks (500 mL) each containing 80 g of rice. Distilled H2O (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 lb/in.−2 for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of seed culture and cultivated at 25 °C for 40 days. Extraction and Isolation. The fermented material was extracted with ethyl acetate (20 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (100.0 g), which was fractionated by silica gel column chromatography (CC) (10 × 100 cm) using CH2Cl2−CH3OH gradient elution. The fraction (3 g) eluted with 100:2 CH2Cl2−CH3OH was separated by column chromatography, and further purification of this subfraction by RPHPLC (Shimadzu LC-6AD; YMC-Pack ODS-A column; 5 μm; 250 × 10 mm; 2 mL/min, 65% MeOH in H2O) afforded

Figure 3. Calculated and experimental CD spectra of 1.

Most cytochalasans are cytotoxic against cancer cell lines. Trichodermone (1) and aspochalasin D (2) were evaluated against HeLa cells. Compound 2 displayed moderate inhibitory activity with an IC50 value of 5.72 μM. The possible biogenetic pathway of trichodermone (1) is proposed as shown in Figure 4. To date, more than 100 cytochalasans have been isolated from different fungi.5 Trichodermone (1) differs from other known cytochalasans. The unique decahydro-1H-benzo[7]annulene unit fused with the pyrrolidin-2-one moiety at C-9 establishes an unprecedented decahydrospiro[benzo[7]annulene-2,3′-pyrrolidine] carbon skeleton. In addition, the cycloheptane, cyclohex-3enone, and furan-2(5H)-one rings together share one olefinic

Figure 4. Possible biosynthetic pathways for compounds 1−4. 166



Note

trichodermone (1, 3.6 mg, tR 10.0 min). The other fraction (125 mg) eluted with 100:8 CH2Cl2−CH3OH was separated by Sephadex LH20 CC, and then purification by RPHPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 70% MeOH in H2O for 5 min, and followed by 70−85% for 60 min) afforded aspochalasin D (2; 10.0 mg, tR 35.9 min). Trichodermone (1): amophous powder, [α]D −326.5 (c 0.1, CH3OH); UV (CH3OH) λmax 220 (log ε 0.74), 243 (log ε 0.34) nm; IR (neat) νmax 3168, 2956, 2935, 1742 (brs), 1700, 1392 cm−1; 1 H, 13C NMR, and HMBC data, see Table 1; HRESIMS obsd m/z 438.2264 [M + Na]+, calcd for C24H33NO3Na, 438.2251. Quantum-Chemical Calculation. All quantum-chemical calculations have been performed on the (3S, 4R, 5S, 9R, 14R, 19S) configuration of 1 by the Gaussian 09 program package.18 The flexible isopentyl group was replaced by a methyl group to improve the computation efficacy. A systematic conformational analysis was carried out using the MMFF94 force field via the MOE software package.19 The obtained conformers were further optimized and verified as the true minima of the potential energy surface at the B3LYP/6-31G(d,p) basis set level using the TDDFT method. The conductor-like polarizable continuum model (CPCM) with the dielectric constant of methanol (ε = 32.6) was adopted to take solvent effects into consideration. The 30 lowest electronic transitions were calculated, and dipole velocity (Rvel) representations were converted to a Gaussian-type curve with a half-bandwidth of 0.4 eV. The ECD spectrum was red-shifted 30 nm and compared with the experimental ECD data of 1. MTT Assay. The cytotoxic activities of the isolated compounds were evaluated against the HeLa cell line obtained by the MTT colorimetric method with VP-16 and paclitaxel as positive controls (IC50 value 5.7 and 2.5 nM, respectively).

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13C, and 2D NMR spectra of trichodermone (1). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Chinese National S&T Special Project on Major New Drug Innovation (2011ZX09307-002-01 and 2013ZX09508104), the Open Funding Project of the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Program for Innovative Research Team in IMPLAD (PIRTI), PUMC Youth Fund, and the Fundamental Research Funds for the Central Universities.

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REFERENCES

(1) Qiao, K.; Chooi, Y.; Tang, Y. Metab. Eng. 2011, 13, 723−732. (2) Ding, G.; Wang, H.; Li, L.; Chen, A.; Chen, L.; Chen, H.; Zhang, H.; Liu, X.; Zou, Z. Eur. J. Org. Chem. 2012, 2516−2519. (3) Binder, M.; Kiechel, J. R.; Tamn, C. Helv. Chim. Acta 1970, 53, 1797−1812. (4) Schuemann, J.; Hertweck, C. J. Am. Chem. Soc. 2007, 129, 9564− 9565. (5) Scherlach, K.; Boettger, D.; Remme, N.; Hertweck, C. Nat. Prod. Rep. 2010, 27, 869−886. 167


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