ORGANIC LETTERS
Coicenals AD, Four New Diterpenoids with New Chemical Skeletons from the Plant Pathogenic Fungus Bipolaris coicis
2013 Vol. 15, No. 15 3982–3985
Quan-xin Wang,†,‡ Qiu-yue Qi,† Kai Wang,† Li Li,§ Li Bao,† Jun-jie Han,† Miao-miao Liu,† Li-xin Zhang,†,‡ Lei Cai,† and Hong-wei Liu*,† State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 8 Beiertiao, Zhongguancun, Haidian District, Beijing 100190, P. R. China, School of Life Science, University of Science and Technology of China, No. 96 JinzhaiRoad, Hefei 230026, P. R. China, and Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, P. R. China
[email protected] Received June 23, 2013
ABSTRACT
Coicenals AC (13) possessing a previously undescribed 10-(sec-butyl)-6-hydroxy-1,7,9-trimethyl-1,6,7,8,9,9a-hexahydro-1,4-methanobenzo[d]oxepin-2(4H)-ylidene)acetaldehyde skeleton and coicenal D (4) with a new 2-(sec-butyl)-5-hydroxy-1,6,8-trimethyl-2,5,6,7,8,8a-hexahydro-1H4a,1-(epoxymethano)naphthalen-10-ylidene)acetaldehyde skeleton were isolated from the solid culture of the plant pathogenic fungus Bipolaris coicis. The absolute configurations in 1 and 4 were assigned by electronic circular dichroism (ECD) calculations. Compounds 1 and 2 were transformed into 4 and 5 by treatment with acetyl chloride, respectively. Compounds 14 showed moderate inhibitory activity against NO release with IC50 values of 16.34 ( 1.12, 23.55 ( 1.37, 10.82 ( 0.83, and 54.20 ( 2.82 μM, respectively.
Secondary metabolites with various chemical skeletons and interesting biological activities have been reported from the plant pathogenic fungi of Bipolaris sp. Examples include nematocidal and cytotoxic ophiobolins,1 cochlioquinones with inhibitory activity against diacylglycerol acyltransferase and cytotoxicity,2 phytotoxic victoxinine †
Chinese Academy of Sciences. University of Science and Technology of China. § Chinese Academy of Medical Sciences & Peking Union Medical College. (1) Au, T. K.; Chick, W. S. H.; Leung, P. C. Life Sci. 2000, 67, 733– 742. (2) Lee, H. B.; Lim, C. H.; Kwon, H. J.; Kim, Y. K.; Lee, H. S.; Kim, C. J. J. Antibiot. 2003, 56, 967–969. (3) Penarodriguez, L. M.; Chilton, W. S. J. Nat. Prod. 1989, 52, 899– 901. (4) Sugawara, F.; Strobel, G.; Fisher, L. E.; Vanduyne, G. D.; Clardy, J. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 8291–8294. (5) Nakajima, H.; Ishida, T.; Otsuka, Y.; Hamasaki, T.; Ichinoe, M. Phytochemistry 1997, 45, 41–45. (6) Nihashi, Y.; Lim, C. H.; Tanaka, C.; Miyagawa, H.; Ueno, T. Biosci. Biotechnol. Biochem. 2002, 66, 685–688. ‡
10.1021/ol401736z r 2013 American Chemical Society Published on Web 07/22/2013
and prehelminthosporolactone,3 bipolaroxins,4 radicinin,5 and 11-epiterpestacin,6 antimicroalgal bipolal,7 and antioxidant spirostaphylotrichin.8 B. coicis is a phytopathogenic fungus causing serious leaf blight on Coix lachryma-jobi L. An early chemical investigation on its secondary metabolites resulted in the identification of four phytotoxic redicinin derivatives.5 In our ongoing search for new bioactive secondary metabolites from pathogenic fungi, the solid culture of B. coicis fermented on rice was found to contain secondary metabolites with characteristic UV maximal absorption at 280 nm by HPLC-DAD analysis. Chromatographic separation on an EtOAc extract prepared from its solid culture afforded four new diterpenes possessing new chemical (7) Watanabe, N.; Fujita, A.; Ban, N.; Yagi, A.; Etoh, H.; Ina, K.; Sakata, K. J. Nat. Prod. 1995, 58, 463–466. (8) Arunpanichlert, J.; Rukachaisirikul, V.; Tadpetch, K.; Phongpaichit, S.; Hutadilok-Towatana, N.; Supaphon, O.; Sakayaroj, J. Phytochem. Lett. 2012, 5, 604–608.
skeletons (14, Figure 1). Details of the structure elucidation as well as antibacterial and nitric oxide inhibitory activities of compounds 14, and the transformation of compound 1 and 2 into respectively 4 and 5 upon treatment of acyl chloride, are reported herein.
Figure 1. Structures of compounds 15.
The molecular formula of coicenal A (1) was established to be C20H30O3 (6 degrees of unsaturation) on the basis of HRESIMS (found: m/z 319.2279 [MþH]þ, calcd: m/z 319.2268). Analysis of its NMR data (Table 1) revealed an aldehyde group (δC/δH 190.6/9.83), four olefinic carbons with two protonated, five methyls [δH 0.86 (3H, t, J = 6.8 Hz), 1.02 (6H, d, J = 6.5 Hz), 1.41 (3H, s), and 1.15 (3H, d, J = 6.0 Hz) ], two methylenes, seven methines with two oxygenated, and one quaternary carbon. The 1H1H COSY NMR data of 1 showed four isolated spin systems of H-3H-4, H-5H-6H2-7(H3-17)H-8H-8a(H3-18), H-2H-13H2-14(H3-16)H3-15 and H-10H-11, which in combination with the HMBC correlations from H-2 to C-1, C-3, C-4, C-8a, C-13, C-14, and C-16, from H-3 to C-1, C-2, C-4, C-4a, and C-13, from H-4 to C-2, C-3, C-5, and C-8a, from H-5 to C-4, C-4a, C-6, C-7, and C-17, and HMBC cross peaks from five methyls H3-12, H3-15, H3-16, H3-17, and H3-18, as well as the chemical shift consideration of C-3 (δC 78.0) and C-5 (δC 76.8) established the secbutyl-trimethyl-octahydronaphthalene-diol structural moiety. The lack of 1H1H COSY and the coupling constant of nearly 0 Hz between H-2 and H-3 implied a dihedral angle of 90°. Further HMBC cross peaks from H-2, H-3, H-8a, and H3-12 to C-9, from H-10 to C-1, C-9, and C-11, and from H-11 to C-10, together with the oxygenated nature of C-3 and C-9 (δC 186.8) and the 1H1H COSY of H-10 to H-11, confirmed the planar structure of 1. The larger coupling constants of H-5 (JH‑5,H‑6 = 10.8 Hz) and H-8a (JH‑8a,H‑8 = 10.3 Hz) supported the trans configuration between H-5 and H-6 and between H-8 and H-8a. NOESY correlations of H-10 with H-8a and H-13; H-8a with H-5, H3-17, and H3-18; and H-2 with H-3 and H-8 indicated that H-5, H-8a, H3-17, H3-18, the sec-butyl moiety, and the acrylaldehyde moiety were on the same face of the octahydronaphthalene ring (Figure 2). The absolute configuration of 1 was determined by comparison of the experimental and simulated electronic circular Org. Lett., Vol. 15, No. 15, 2013
dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT).9,10 Since the conformationally flexible side chain had an insignificant effect on the CD spectrum of 1, a simplified structure 6 was used for ECD calculations (Figure 3). Considering the relative configuration previously mentioned, two stereoisomers, (1R, 2R, 3S, 5R, 6S, 8R, 8aS)-6a and (1S, 2S, 3R, 5S, 6R, 8S, 8aR)-6b, were deduced to represent the configurations in the octahydronaphthalene moiety of 1. A conformational analysis was carried out for 6a and 6b by the Molecular Operating Environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational search followed by reoptimization using TDDFT at the B3LYP/ 6-31G(d) basis set level afforded the lowest-energy conformers for enantiomers 6a and 6b (Figure S39, Supporting Information (SI)). The overall calculated ECD spectra of 6a and 6b were then generated by Boltzmann weighting of the conformers. The experimental CD spectrum of 1 (positive Cotton effect at 260 nm and negative Cotton effect at 218 nm) was nearly identical to the calculated ECD spectrum of (1R, 2R, 3S, 5R, 6S, 8R, 8aS)-6a (Figure 3). The energy-minimized conformer of 6a showed a dihedral angle of 83.4° between H-2 and H-3 (Figure S39, SI), corresponding to a JH‑2,H‑3 value of nearly 0 Hz as that observed in its 1H NMR. The configuration at C-13 was left unsolved in the current data analysis. Therefore, 1 was deduced to have the 1R, 2R, 3S, 5R, 6S, 8R, 8aS absolute configuration.
Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for 1 and 4 in CDCl3 1 pos.
δC
1 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16 17 18
52.7 52.4 78.0 119.0 148.2 76.8 40.8 44.7 36.2 57.7 186.8 101.6 190.6 20.5 32.7 25.0 12.2 18.7 18.7 23.6
δH, mult (J in Hz) 2.18, d (3.4) 4.78, d (6.0) 6.22, d, (6.0) 3.48, dd (10.8, 1.3) 1.35, m 1.09, m; 1.69, m 1.75, m 1.82, d (10.3) 5.16, d (8.0) 9.83, d (8.0) 1.41, s 1.58, m 1.02, m; 1.35, m 0.86, t (6.8) 1.02, d (6.5) 1.02, d (6.5) 1.15, d (6.0)
4 δC 51.3 51.1 132.9 126.2 79.0 87.6 35.9 44.1 30.6 60.3 183.3 103.7 189.9 22.4 34.1 27.2 13.2 21.7 18.5 20.4
δH, mult (J in Hz) 2.26, m 5.87, dd (9.7, 2.3) 6.14, dd (9.7, 2.3) 3.46, d (10.4) 1.55, m 0.91, m; 1.66, m 1.87, m 1.39, d (10.8) 5.17, d (8.2) 9.91, d (8.2) 1.34, s 1.55, m 0.79, m; 1.41, m 0.79, t (6.8) 1.03, d (6.7) 1.07, d (6.5) 1.03, d (6.7)
(9) Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 2717–2727. (10) Ren, J. W.; Zhang, F.; Liu, X. Y.; Li, L.; Liu, G.; Liu, X. Z.; Che, Y. S. Org. Lett. 2012, 14, 6226–6229. 3983
Figure 2. Selected HMBC (HfC), 1H1H COSY (bold lines), and NOESY (dash line) correlations of 1 and 4.
Coicenal B (2) was assigned the molecular formula of C24H34O6 by HRESIMS (found: m/z 419.2425 [MþH]þ, calcd: m/z 419.2428). The NMR data of 2 were similar to those of 1, except for the presence of a succinyl moiety [δH 2.70 (2H, m), 2.70 (2H, m); δC 28.8, 29.0, 171.0, 176.7] in 2 (Table S1, SI). The 1H1H COSY between H2-20 and H2-21 as well as the HMBC correlations from H2-20 and H2-21 to C-19 and C-22 further confirmed the succinyl moiety (Figure S1, SI). The HMBC cross peak from H-5 to C-19 indicated the attachment of the succinyl moiety at C-5 via an ester bond, which was also supported by the downfield shift of H-5 (1, δH 3.48; 2, δH 4.79). The relative configuration of 2 was determined by NOESY correlations of H-10 with H-8a and H-13; H-8a with H-5, H3-17, and H3-18; and H-2 with H-3 and H-8 (Figure S1, SI). The CD spectrum of 2 was nearly identical with that of 1, displaying a positive Cotton effect at 259 nm and a negative Cotton effect at 217 nm (Figure S15, SI). Thus, compound 2 was determined to have a 1R, 2R, 3S, 5R, 6S, 8R, 8aS absolute configuration. HRESIMS of 3 showed signals at m/z 433.2587 [MþH]þ, indicating the molecular formula of C25H36O6, which differs from that of 2 by a methyl substituent. The NMR data of 3 closely resemble those of 2, except for a methoxy group (δC/δH 52.0/3.69) (Table S1, SI). The methoxy group was located at C-22 by its HMBC correlation to C-22 (Figure S1, SI). In the CD spectrum of 3, a positive Cotton effect at 259 nm and a negative Cotton effect at 220 nm were observed (Figure S23, SI), which indicated the same absolute configuration between 2 and 3. Thus, compound 3 was identified as a methyl ester of 2 and named as coicenal C. The molecular formula of coicenal D (4) was established to be C20H30O3 (6 degrees of unsaturation) on the basis of HRESIMS (found: m/z 319.2266 [MþH]þ, calcd: m/z 319.2268). Analysis of its NMR data (Table 1) revealed an 3984
Figure 3. Experimental CD spectrum of 1 and 4 in n-hexane and the calculated ECD spectra of 6a, 6b, 7a, and 7b. Structures 6a and 6b, 7a and 7b represent two possible stereoisomers of 6 and 7, respectively.
aldehyde group (δC/δH 189.9/9.91), four olefinic carbons with three protonated, five methyls [δH 0.79 (3H, t, J = 6.8 Hz), 1.03 (6H, d, J = 6.7 Hz), 1.07 (3H, d, J = 6.5 Hz), and 1.34 (3H, s)], two methylenes, six methines (one oxymethine), and two quaternary carbons with one oxygenated. The planar structure of 4 was deduced from the 2D NMR data. The 1H1H COSY NMR data of 4 showed three isolated spin systems of H-5H-6 H-7(H3-17)H-8H-8a(H3-18), H-4H-3H-2H-13 H-14(H3-16)H3-15, and H-10H-11, which together with the HMBC correlations from H-3 and H-4 to C-4a, from H-5 to C-4, C-4a, and C-17, from H-8a to C-1, C-2, C-4, C-5, C-4a, and C-12, and from H3-12 to C-1, C-2, C-8a, and C-9 confirmed the 2-(sec-butyl)-5-hydroxy-1,6,8trimethyl-2,5,6,7,8,8a-hexahydro-1H-4a,1-(epoxymethano)naphthalen-10-ylidene)acetaldehyde skeleton of 4. The relative configuration of 4 was assigned from NOESY correlations of H-10 with H-8a and H-13; H-8a with H-5, H3-17, and H3-18; and H-2 with H-3 and H-8 as depicted in Figure 2. The absolute configuration of 4 was determined as described for 1 by comparison of its Org. Lett., Vol. 15, No. 15, 2013
experimental and ECD spectra. Similarly, a simplified structure 7 was used for ECD calculations (Figure 3). Considering the relative configuration determined, two possible stereoisomers, (1S, 2S, 4aR, 5R, 6S, 8R, 8aR)-7a and (1R, 2R, 4aS, 5S, 6R, 8S, 8aS)-7b were deduced. The conformational analysis afforded the lowest-energy conformers for enantiomers 7a and 7b (Figure S41, SI). The experimental CD spectrum of 4 was nearly identical to the calculated ECD spectrum of (1S, 2S, 4aR, 5R, 6S, 8R, 8aR)-7a (Figure 3). Accordingly, 4 was deduced to have the 1S, 2S, 4aR, 5R, 6S, 8R, 8aR absolute configuration. In our attempt to determine the absolute configuration of 1 using the modified Mosher method, a new rearranged product 1a instead of the corresponding (S)- and (R)-MTPA esters was obtained. Analysis of the MS, 1H NMR spectrum, and HPLC retention time of 1a indicated that it has the same chemical structure as that of 4. To understand the possible reaction mechanism involved in such a rearrangement, compound 1 was treated with the reagent of acetyl chloride, benzoyl chloride, and acetic acid in the same manner as described in Mosher’s method, respectively. The transformation of 1 into 4 was observed in the presence of acetyl chloride and benzoyl chloride (Figure S43, SI). However, such a transformation was not occurred upon treatment of acetic acid. Furthermore, when treated with the acetyl chloride, compound 2 can be transformed into the corresponding rearrangement product 5. On the basis of the above results, it can be concluded that the acyl cation played a very important role in the transformation of 1 into 4. The possible reaction mechanism was described in Scheme 1. In the presence of an acyl cation, an intermediate product 8 was formed from the reaction of the electrophilic carbon (acyl cation) with the nucleophilic oxygen atom at C-11 that was generated from the shift of electrons from the double bond between C-4 and C-4a to the oxygen atom in the carbonyl group. The intermediate product 8 was quickly transformed into 4 by the formation of a new carbonoxygen bond between C-4a and the oxygen atom at C-9. Nitric oxide (NO) plays an important role in the inflammatory process. Natrual products inhibiting NO release could be used as therapeutic agents in inflammatory diseases.11 Compounds 14 were tested for inhibition (11) Lee, H. J.; Kim, N. Y.; Jang, M. K.; Son, H. J.; Kim, K. M.; Sohn, D. H.; Lee, S. H.; Ryu, J. H. Planta Med. 1999, 65, 104–108.
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Scheme 1. Possible Mechanism for the Transformation of 1 and 2 into Respectively 4 and 5 by Treatment with Acetyl Chloride
against NO production in LPS-induced RAW264.7 macrophages. Compounds 14 showed moderate inhibitory activity against NO release from macrophages with IC50 values of 16.34 ( 1.12, 23.55 ( 1.37, 10.82 ( 0.83, and 54.20 ( 2.82 μM, respectively. In an antibacterial bioassay, compound 3 inhibited the growth of Bacillus subtilis and Staphylococcus aureus with MIC values of 62.5 and 125 μM, respectively. Compounds 1, 2, and 4 were inactive against the growth of B. subtilis and S. aureus at the concentration of 200 μM. Compounds 14 represented new types of fungal diterpenoids. Compounds 13 possess a previously undescribed 10-(sec-butyl)-6-hydroxy-1,7,9-trimethyl-1,6, 7,8,9,9a-hexahydro-1,4-methanobenzo[d]oxepin-2(4H)ylidene)acetaldehyde skeleton, and 4 has a new 2-(secbutyl)-5-hydroxy-1,6,8-trimethyl-2,5,6,7,8,8a-hexahydro1H-4a,1-(epoxymethano)naphthalen-10-ylidene)acetaldehyde skeleton. The biosynthetic pathway for these new fungal diterpenes deserves further investigation. Acknowledgment. Financial support from the National Natural Science Foundation (Grant No. 21072219) and the Ministry of Science and Technology of China (2009CB522302 and 2012ZX09301002-003) is gratefully acknowledged. Supporting Information Available. Experimental section, characterization data, NMR and MS data of 14, and ECD calculations for 1 and 4 were provided. This material is available free of charge via the Internet at http://pubs.acs.org. The authors declare no competing financial interest.
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