ORGANIC LETTERS

Trichodermatides AD, Novel Polyketides from the Marine-Derived Fungus Trichoderma reesei
Yi Sun,, Li Tian, Jian Huang, Hong-Yu Ma, Zhe Zheng, A-Li Lv, Ken Yasukawa, and Yue-Hu Pei*,
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical UniVersity, Shenyang 110016, P.R. China, College of Pharmacy, Nihon UniVersity, Chiba 274-8555, Japan, and College of Chemical Engineering, Qingdao UniVersity of Science and Technology, Qingdao 266042, P.R. China peiyueh@[Link]
Received November 4, 2007

2008 Vol. 10, No. 3 393-396

ABSTRACT

Four new polyketide derivatives, Trichodermatides AD (14) were isolated from the marine-derived fungus Trichoderma reesei. Trichodermatide A (1) is an unprecedented example of a polyketide with a ketal-containing pentacyclic skeleton. The chemical structures and absolute configurations of compounds 14 were elucidated by extensive spectroscopic methods, especially 2D NMR and CD spectral analysis, and supported by their proposed biosynthesis pathway. The cytotoxicity of 14 was evaluated against A375-S2 human melanoma cell line.

Marine-derived fungi are increasingly of interest as a potential source of new metabolites with a wide range of biological activities.1 Early chemical investigation of the fungus Trichoderma spp. led to the isolation of several bioactive metabolites,2 and terrestrial Trichoderma spp. have demonstrated utility as biocontrol agents.3 Furthermore, the special metabolites of the genus Trichoderma, octaketide derivatives, display significant antibiotic activity.4 In our continuing search for novel and bioactive substances from marine-derived fungi, we isolated four new polyketides, trichodermatides A, B, C, and D (1-4), from the fungus
Shenyang Pharmaceutical University. Nihon University. Qingdao University of Science and Technology. (1) Bugni, T. S.; Ireland, C. M. Nat. Prod. Rep. 2004, 21, 143-146. (2) (a) Garo, E.; Starks, C. M.; Jensen, P. R.; Fenical, W.; Lobkovsky, E;, Clardy, J. J. Nat. Prod. 2003, 66, 423-426. (b) Sperry, S.; Samuels, G. J.; Crews, P. J. Org. Chem. 1998, 63, 10011-10014. (c) Kobayashi, M.; Uehara, H.; Matsunami, K.; Aoki, S.; Kitagawa, I. Tetrahedron Lett. 1993, 34, 7925-7928. (3) Elad, Y. Crop Protect. 2000, 19, 709-714. (4) (a) Ghisalberti, E. L.; Rowland, C. Y. J. Nat. Prod. 1993, 56, 17991804. (b) Almassi, F.; Ghisalberti, E. L.; Narbey, M. J. J. Nat. Prod. 1991, 54, 396-402. (c) Dunlop, R. W.; Simon, A.; Sivasithamparam, K.; Ghisalberti, E. L. J. Nat. Prod. 1989, 52, 67-74.

Trichoderma reesei. Trichodermatide A (1) is an unprecedented polyketide with a ketal-containing pentacyclic skeleton. In this contribution, we describe the structural elucidation, a plausible biogenetic pathway, and cytotoxic evaluation of these four metabolites. The fungus T. reesei was obtained from marine mud in the tideland of Lianyungang, China. Its mycelial mass and fermentation broth were extracted with acetone and EtOAc, respectively. Both crude extracts (14.6 g) were isolated by normal-phase silica gel column and reversed-phase HPLC chromatography, yielding four metabolites Trichodermatide A (1, 3.6 mg), B (2, 4.5 mg), C (3, 2.0 mg), and D (4, 1.5 mg). Trichodermatide A (1)5 was obtained as a colorless oil with the molecular formula C22H32O7 as determined by HREIMS (m/z 408.21465 [M]+; calcd 408.21480), indicating seven degrees of unsaturation. Its UV spectrum (max 257 nm) and IR spectrum (max 1650 and 1620 cm-1) suggested
(5) Trichodermatide A (1), Colorless oil. [R]20D -62.5 (c 0.04, MeOH). UV (MeOH) max 257 nm. CD (MeOH) 296 -0.8601, 253 +1.9531. IR (KBr) max 3450, 2924, 1650, 1620, 1170, 1132 cm-1. 1H and 13C NMR data, see Table 1. EIMS m/z 408.2 [M]+.

10.1021/ol702674f CCC: $40.75 Published on Web 12/29/2007

2008 American Chemical Society

the presence of an R,-unsaturated cyclohexenone group. The resonances in the 1H NMR (Table 1) spectrum revealed five

Figure 1. (a) Key HMBC correlations (HfC) and 1H-1H COSY (s) for connecting substructures A-C to the gross structure of 1. (b) Key NOESY correlations of 1.

Table 1.
position 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1H

and 13C NMR Data for Trichodermatide A (1)a,b

C 197.7 70.7 29.4 27.4 167.9 111.7 21.7 38.1 100.0 68.2 26.5 24.8 106.2 29.5

2.85 (ddd, 7.8, 6.0, 2.9) 1.94 (d, 7.8) 3.68 (dt, 8.1, 4.0) 1.85 (m), 1.61 (m) 1.39 (m), 1.23 (m) 1.68 (ddd, 15.2, 3.3, 2.9) 1.83 (ddd, 15.2, 6.0, 3.3)
b 1H

a Data recorded in DMSO-d . 6 at 150 MHz.

was recorded at 600 MHz, and

13C

methylenes at H 1.36 (m, H-17), 1.23 (m, H-18-21), and a methyl at H 0.85 (t, J ) 7.0 Hz, H-22). The corresponding resonances in the 13C NMR spectrum (Table 1) were readily assigned to an aliphatic chain by analysis of the 2D NMR correlations. Comparison of the 13C NMR and DEPT spectra revealed the presence of a methyl, ten methylenes, two methines, four oxymethines, and five quaternary carbons (including a carbonyl group, two olefinic carbons, and two hemiketal or ketal groups). Moreover, the 13C NMR chemical shift of a downfield olefinic carbon (C 167.9, C-5) indicated the presence of an oxygen substituent. The above functionalities accounted for two of the seven degrees of unsaturation in the molecule, revealing a pentacyclic structure for 1. The gross structure of 1 was established with the assistance of HSQC, HMBC, and 1H-1H COSY experiments (Figure 1a). Substructures A-C (Figure 1a) were connected using HMBC and 1H-1H COSY correlations. Substructure A was elucidated by starting with a series of HMBC correlations from H-2 (H 3.91) to C-1 (C 197.7), C-3 (C 29.4), and C-4 (C 27.4); and from H-4 (H 2.45 and 2.32) to C-5 (C 167.9), C-6 (C 111.7). These carbon signals (C-1-C-6) were assigned to the aforementioned R,-unsaturated cyclohex394

enone ring in which a hydroxyl was located at the R-position of C-1. Moreover, proton H-7 (H 2.85) showed CHcorrelations with C-5, C-6, C-8 (C 38.1), and C-9 (C 100.0), connecting C-9 and the olefinic carbon C-5 through an ether linkage to yield a pyran ring on the basis of the chemical shifts of C-5 and C-9. The additional correlations in the HMBC from H-8 (H 1.94), OH-9 (H 6.06) to C-9 suggested the presence of a hemiketal group at C-9. Analysis of the 1 H-1H COSY spectrum indicated the presence of a spin system (H-7/H-8, H-14; and H-15/H-14, H-16) that was assembled into another pyran ring. Further HMBC correlations from H-8 to C-13 (C 106.2) and C-14 (C 29.5), from H-15 (H 4.11) to C-13, C-16 (C 77.5), and the chemical shift of C-13 indicated the presence of a ketal group connected to the pyran ring. Additional COSY correlations showed a spin system (H-10/H-11, H-12) to complete the substructure B, which was also supported by the HMBC correlations from OH-9 to C-10 (C 68.2) and from H-12 to C-8, C-13. Finally, HMBC correlations from H-15 to C-17 (C 35.4) and from H-17 (H 1.36) to C-16, C-18 (C 28.4) connected substructure B with the aliphatic side chain (substructure C) at C-16 to complete the structure of 1. The relative configuration of 1 was determined by NOESY experiment (Figure 1b) and 1H NMR J values. In the 1H NMR, a large coupling constant (J ) 9.7 Hz) was observed for H-2, which required a trans-diaxial relationship between the oxymethine proton (H-2) and H-3R (H 2.06). In addition, a NOESY correlation between H-2 and H-4 (H 2.45) indicated that these two protons were in axial orientations and cis to one another. Correlations between H-16 (H 3.94) and H-14R (H 1.83), and especially between H-16 and H-7 and between H-8 and H-7, H-10 and H-12R (H 1.39), suggested that these protons were on the same side and were assigned as the R-orientation. The correlation of H-8/H-10 indicated that H-8 and H-10 were at the axial positions. Further NOESY correlations of OH-9 with H-11 (H 1.85) suggested that OH-9 was at the axial position and -oriented but on the opposite side of H-7, H-8, and H-10. Thus, the observed NOESY correlations for 1 were fully consistent with its relative configuration.
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Trichodermatide B (2)6 had the molecular formula C16H24O4, as derived from HREIMS data, indicating five degrees of unsaturation. Evidence for the presence of an R,-unsaturated cyclohexenone was obtained from the UV and IR absorptions, which were similar to those of 1. The 13C NMR spectrum of 2 contained signals for four sp2 carbons assigned to two carbonyl groups (C 207.8 and 194.7) and two olefinic carbons (C 176.1 and 110.7); thus, compound 2 was bicyclic. Comparison of the 1H and 13C NMR spectra suggested the presence of two oxymethines (H 3.94, H-2/ C 70.7 and H 5.14, H-9/C 81.7) and an aliphatic chain (H 2.42, 1.45, 1.26 and 0.85, H-11-16/C 42.3, 22.9, 28.8, 31.1, 22.0, and 14.0). The HMBC spectrum of 2 (Figure 2a) established the

Figure 2. (a) Key HMBC correlations for 2. (b) Key NOESY correlations for 2.

5.78 (dt, J ) 15.4, 7.4 Hz, H-11), along with the corresponding carbon signals at C 128.6 (C-10) and 134.6 (C11). The HMBC correlations from H-9 (H 5.29) to C-8 (C 28.2), C-10 and C-11 placed the double bond at C-10 and C-11 and connected the pyran ring with the aliphatic side chain at C-9, as was seen in 2. Additional HMBC correlations from the D2O exchangeable proton (H 5.06, OH-2) to C-1 (C 194.6) and C-2 (C 70.7) confirmed that the oxymethine was R to the carbonyl group. The NOESY cross-peaks of H-2/H-4R (H 2.53) and H-9/H-7 (H 2.83) and the coupling constants (9.4 and 9.5 Hz) of H-2 and H-9 were consistent with the relative configuration of 2 as depicted. The molecular formula of 48 was deduced from HREIMS as C16H24O4. A comparison of the 1H and 13C NMR data of 2 and 4 showed that they differed in the position of the hydroxyl. The presence of the hydroxyl in the NMR of 4 was confirmed by the oxygenated carbon at C 61.7 (C-3) and by the corresponding proton at H 4.36 (tt, J ) 3.9, 3.9 Hz, H-3), which revealed that the hydroxyl was at the -position of the carbonyl group and was axial. The NOESY cross-peak of H-9 (H 5.15) with H-7R (H 2.81) and a large coupling constant of H-9 (J ) 9.8 Hz) confirmed that these two protons were cofacial and were at the axial positions. The absolute configurations of compounds 1-4 were determined on the basis of the CD spectral analysis (Figure 3). The nf* and f* transitions in the CD curves clearly

correlations from H-2 (H 3.94), OH-2 (H 5.08) to C-1 (C 194.7), C-3 (C 30.1) and from H-3 (H 2.08, 1.78) to C-1, C-4 (C 21.9) and C-5 (C 176.1), which illustrated the presence of the R,-unsaturated cyclohexenone ring, as was seen for 1. A -oxy substituent of an enol ether group was indicated by the low-field resonance of the tetrasubstituted olefinic carbon C-5 and the HMBC correlations. The methylene proton H-7 (H 2.81 and 2.36) showed the same correlations as those of 1, indicating that a pyran ring adjoined with the R,-unsaturated cyclohexenone. Further HMBC correlations from H-9 (H 5.14) to C-10 (C 207.7) and from H-11 (H 2.42) to C-10, C-12 (C 22.9), and C-13 (C 28.8) suggested that the carbonyl group linked the alphatic side chain to the furan ring at C-9. Analysis of NOESY data (Figure 2b) and J values established the relative configuration of 2. Two strong cross-peaks of H-2/H-4R (H 2.48) and H-9/H-7 (H 2.81), along with the large vicinal couplings (9.7 and 9.3 Hz) observed for H-2 and H-9, supported assignment of the axial positions for H-2 and H-9. The molecular formula of trichodermatide C (3)7 was C16H24O3 as deduced from HREIMS data. The NMR spectral data for 3 were similar to those of 2. In the 1H and 13C NMR, the presence of a trans-double bond was revealed by two proton signals at H 5.58 (dd, J ) 15.4, 7.6 Hz, H-10) and
(6) Trichodermatide B (2). Colorless needles (MeOH); mp. 65-68 C; [R]20D +64.0 (c 0.05, MeOH); UV (MeOH) max 255 nm; IR (KBr) max 3443, 2927, 1714, 1651, 1621 cm-1; CD (MeOH) 295 +3.3949, 263 +2.4531; 1H and 13C NMR data, see Supporting information. EIMS m/z 280.2 [M]+; HREIMS m/z 280.16800 [M]+ (calcd for C16H24O4, 280.16744). (7) Trichodermatide C (3). Colorless oil. [R]20D +80.0 (c 0.02, MeOH); UV (MeOH) max 253 nm; CD (MeOH) 298 +1.205; 1H and 13C NMR data, see Supporting information. EIMS m/z 264.2 [M]+; HREIMS m/z 264.17257 [M]+, (calcd for C16H24O3, 264.17253).

Figure 3. CD spectra of 1-4. (a) Standard conformation of cyclohexenone ring showing the positive CE and its application of the octant rule. (b) Opposite conformation of (a) showing the negative CE and its application of the octant rule.

related to the helicity rule of the R,-unsaturated ketone.9 All the CD data for 1-4 were entirely compatible with the view that the hecility of enone moiety established the sign of the Cotton effect (CE) as a dominant factor. Furthermore, the enone helicity was determined by the conformation of cyclohexenone ring.10 Thus, the ring conformation (with
(8) Trichodermatide D (4). Colorless oil. [R]20D +22.5 (c 0.01, MeOH). CD (MeOH) 295 -1.0281, 261 +0.1880. 1H and 13C NMR data, see Supporting information. EIMS m/z 280.1 [M]+; HREIMS m/z 280.16731 [M]+ (calcd for C16H24O4, 280.16744). (9) (a) Kirk, D. N. Tetrahedron 1986, 42, 777-818. (b) Lightner, D. A.; Gurst, J. E. Organic Conformational Analysis and Stereochemistry from Circular Dichroism Spectroscopy; Wiley-VCH: New York, 2000; Chapters 4, 7, and 11. (c) Frelek, J.; Szczepek, W. J.; Weiss, H. P. Tetrahedron: Asymmetry 1995, 6, 1419-1430. (10) (a) Ye, X. L. Stereochemistry; Zhu, X. T., Ed.; Beijing University Press: Beijing, 1999; pp 257-258. (b) Burnett, R. D.; Kirk, D. N. J. Chem. Soc., Perkin I. 1981, 1460-1468. 395

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Scheme 1. Plausible Biosynthesis Route of 1-4

positive ring chirality) in Figure 3a gave rise to a positive CE for the nf* band, whereas the opposite conformation (Figure 3b) defined a negative CE. Therefore, the absolute configuration of 1 was assigned as 2R, 7R, 8S, 9S, 10S, 13R, 15S, and 16S according to the observed negative Cotton effect at 296 nm ( 296 -0.8601). Moreover, both the C-3 and C-9 of 4 could be assigned as R-configuration, and conformation of its cyclohexenone ring was identified to be that of 1. The CD curves of both 2 and 3 showed similar positive Cotton effects at 295 nm ( 295 +3.395) and 298 nm ( 298 +1.205), indicating the same absolute configuration of 2S, 9S. Polyketide derivatives with different skeletons have been isolated from several fungi and originated via similar biosynthesis pathways.2,11 Scheme 1 shows a plausible biosynthesis pathway for polyketides 1-4 that is based on the linear octaketide-folding chain.12 Initial Claisen condensation between C-5/6 (via an oxygen atom) followed by cyclization from C-5 to C-9 and reduction of the carbonyl groups (except for C-1) would yield octaketide-derived intermediate 5. A subsequent oxidation of 5 at C-10 and hydroxylation at C-2 or C-3 would yield 2, 3, and 4, respectively. When the octaketide-folding chain is condensed with a triketide unit (C-1-C-6), it would produce 1. The cyclizations between the octaketide and triketide would
(11) Brady, S. F.; Wagenaar, M. M.; Singh, M. P.; Janso, J. E.; Clardy, J. Org. Lett. 2000, 2, 4043-4046. (12) (a) Torssell, K. B. G. In Natural Product Chemistry; Sunden, M., Ed.; Swedish Pharmaceutical Society: Sweden, 1997; pp 174-210. (b) Mann, J. In Secondary Metabolism; Huaang, Z. Z., Ed.; Science Press: Beijing, 1983; pp 46-78. (c) Radzom, M.; Zeeck, A.; Antal, N.; Fiedler, H-P. J. Antibiot. 2006, 59, 315-317. 396

proceed by further Claisen condensations. Subsequent steps would involve reduction and hydroxylation to yield 6. Then the nucleophilic OH-9 and OH-10 would attack the carbonyl at C-5 in turn to form the ketal group in 1. The cytotoxicity of compounds 1-4 was evaluated against the A375-S2 melanoma cell line using the MTT assay. 1-4 showed weak cytotoxicity with IC50 values at 102.2, 187.3, 38.8, and 222.0 g/mL, respectively. Trichodermatide A (1) is the first example of a polyketide with a katal-containing pentacyclic skeleton. Octaketide derivatives with the unique bicyclic fusion of an R,unsaturated cyclohexenone and a pyran ring are produced only by fungi of the genus Trichoderma; this skeleton has been reported for about 10 metabolites to date.4,13 The new octaketide derivatives Trichodermatides B-D (2-4) contain some structural features that differ from those of compounds previously reported; they contain a rare keto group or a pair of double bonds at C-10, instead of the hydroxyl. Acknowledgment. This work was supported by 863 HiTech Research and Development Program of China (Grants 2007AA09Z413 and 2007AA09Z435). Supporting Information Available: Experimental details and 1D, 2D NMR, and MS data. This material is available free of charge via the Internet at [Link]
OL702674F
(13) (a) Parker, S. R.; Culter, H. G.; Schreiner, P. R. Biosci. Biotechnol. Biochem. 1995, 59, 1747-1749. (b) Culter, H. G.; H Culter, H. G.; Himmelsbach, D. S.; Yagen, B.; Arrendale, R. F.; Jacyno, J. M.; Cole, P. D.; Cox, R. H. J. Agric. Food. Chem. 1991, 39, 977-980.

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