Research article Received: 7 January 2014
Revised: 5 March 2014
Accepted: 5 March 2014
Published online in Wiley Online Library: 2 April 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4065
Structure determination of two new indolediterpenoids from Penicillium sp. CM-7 by NMR spectroscopy Yu-Hong Zhang,a,b Sheng-Dong Huang,a,b Hua-Qi Pan,c Xi-Qing Bian,a,b Zai-Ying Wang,a,b Ai-Hong Hand*,* and Jiao Baia,b* Two new indole-diterpenoids 4b-deoxy-1′-O-acetylpaxilline (1) and 4b-deoxypenijanthine A (2) were isolated from the fermentation broth and the mycelia of the soil fungus Penicillium sp. CM-7, along with three known structurally related compounds, 1′-O-acetylpaxilline (3), paspaline (4) and 3-deoxo-4b-deoxypaxilline (5). The structures of compounds 1 and 2 were elucidated by extensive spectroscopic methods, especially 2D NMR, and their absolute configurations were suggested on the basis of the circular dichroism spectral analysis and the NOESY data. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; 1H NMR; 13C NMR; Indole-diterpenoids; Penicillium sp.
Introduction Indole-diterpenoids are a large, structurally diverse group of fungal secondary metabolites, many of which are potent tremorgenic mammalian mycotoxins.[1] These metabolites have a common core structure comprising a cyclic diterpene skeleton and an indole group.[2,3] The fungi of genus Penicillium are ubiquitous in nature. Previous investigations of these fungi led to the isolation of many indole-diterpenoids secondary metabolites.[4] During our search for new bioactive compounds from fungal sources, two new indole-diterpenoids derivatives, 4b-deoxy-1′O-acetylpaxilline (1) and 4b-deoxypenijanthine A (2) were isolated from Penicillium sp. CM-7, along with 1′-O-acetylpaxilline (3),[5] paspaline (4)[6] and 3-deoxo-4b-deoxypaxilline (5).[7] (Fig. 1) The isolation and the structural elucidation of 1 and 2 are described herein.
Results and Discussion
306
Compound 1 was obtained as a white, amorphous powder. Its HRESIMS exhibited a [M + H]+ at m/z 462.2618 (calcd for C29H36NO4, 462.2639), corresponding to a molecular formula of C29H35NO4, with 13° of unsaturation. Its UV spectrum showed characteristic peaks of an indole chromophore at λmax 229 and 280 nm. The IR spectrum further indicated the presence of an indole group at 3310, 1454, 1102 and 748 cm1, and the presence of an ester carbonyl at 1731 cm1 and a conjugated ketone at 1680 cm1. The 1H NMR (600 MHz, CDCl3) (Table 1) spectrum exhibited a –NH singlet at δ 7.75 (1H, s), four aromatic protons at δ 7.43 (1H, m), 7.30 (1H, m), 7.08 (2H, m), one olefinic proton at δ 5.81 (1H, d, J = 1.3 Hz), two oxymethine protons at δ 4.80 (1H, d, J = 1.7 Hz), 4.24 (1H, brt, J = 9.1 Hz) and five methyl singlets at δ 1.00, 1.06, 1.44, 1.67 and 2.03 (each 3H, s). The 13C NMR (150 MHz, CDCl3) and HSQC (Table 1) spectra revealed 29 carbon signals, including two carbonyls, five methyls, five methylenes, nine methines (including five sp2 carbons, two oxygenated sp3
Magn. Reson. Chem. 2014, 52, 306–309
carbons and two sp3 carbons), and eight quaternary carbons (including five sp2 carbons, one oxygenated sp3 carbons and two sp3 carbons). The structure of 1 was elucidated by the analysis of 2D NMR data including the HSQC, HMBC and 1H–1H COSY spectra. The HMBC correlations observed from H-8 to C-10, C-11a, from H-9 to C-7b, C-11, from H-10 to C-8, C-11a, from H-11 to C-7b, C-9, from NH to C-7a, C-7b, C-11a and C-12a constructed an indole moiety. The HMBC correlations of from H-2 to C-3, C-14a, C-1′ and C-3′, from H-4 to C-2, C-4b and C-14a, from H-5 to C-12c, from H-6 to C-4b and C-12b, from H-7 to C-6, C-6a, C-7a, C-12a and C-12b, from H-13 to C-12c, from H-14 to C-13, from H-2′ to C-2, C-1′ and C-3′, from H-3′ to C-2, C-1′ and C-2′, from 12b-CH3 to C-6a, C-12a, C-12b and C-12c, from 12c-CH3 to C-12b, C-12c and C-13 and the 1H-1H COSY correlations of H-4b and H-5, H-5 and H-6, H-14 and H-14a constructed a diterpenoid unit. All the aforementioned evidences characterized compound 1 as an indole-diterpenoid. 1 H NMR and 13C NMR spectra made it clear that compound 1 closely resembled the known compound 3. The chemical shift
* Correspondence to: Jiao Bai, School of Traditional Chinese Materiel Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. E-mail: [email protected] ** Correspondence to: Ai-Hong Han, College of Chemistry and Life Science, Shenyang Normal University, Shenyang, 110034, China. E-mail: hanhong@ hotmail.com a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China c Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China d College of Chemistry and Life Science, Shenyang Normal University, Shenyang 110034, China
Copyright © 2014 John Wiley & Sons, Ltd.
Indole-diterpenoids from Penicillium sp.
Figure 1. The structures of compounds 1–5.
Magn. Reson. Chem. 2014, 52, 306–309
General experimental procedures The UV spectrum was recorded on a Shimadzu UV-1601 (Kyoto, Japan). The IR spectrum was obtained from a Bruker IFS-55 spectrophotometer using KBr pellet. The HR-ESI-MS data were obtained on a Varian QFT-ESI instrument. A JASCO CD-2095 chiral detector was used for CD determination using MeOH as solvent. 1D and 2D NMR spectra were run on a Bruker AVANCE-400/-600 spectrometer. Column chromatography was performed on Si gel G (200–300 mesh; Qingdao Haiyang Chemical Factory, Qingdao, China) and Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA) columns. Thin layer chromatography was carried out using Si gel GF254 (Qingdao Haiyang Chemical Factory) plates. HPLC was performed using Hitachi L-6000 pump, Hitachi L-7400 UV detector and performed with an Octadecylsilyl (ODS) column (10 × 250 mm, 5 μm; YMC-Pack, Japan). NMR spectral methods The NMR spectra were recorded on a Bruker AVANCE-400/-600 instrument, and the chemical shifts are given with TMS as an internal standard. The NMR experiments were carried out at 300 K with the following parameters: 1H NMR spectrum: spectrometer frequency (SF) = 600.13 MHz, spectral width (SW) = 12019.2 Hz, fourier transform size (SI) = 32768, acquisition time (AQ) = 2.73 s, line broadening (LB) = 0.3 Hz, relaxation delay (RD) = 1.0 s, number of dummy scans (DS) = 2 and number of scans (NS) = 8, digitizer resolution (DR) = 18 and digital digitizer resolution (DDR) = 2; 13C NMR spectrum: SF = 100.61 MHz, SW = 24038.5 Hz, SI = 32 768, AQ = 1.36 s, LB = 1.00 Hz, RD = 2.0 s, DS = 4, NS = 4000 (1) or 10240 (2), DR = 22 and DDR = 10; HSQC: SF = 600.13 MHz, SW 1 H = 6009.6 Hz, SW 13C = 30183.3 Hz (1) or 27164.7 (2), AQ = 0.09 s, RD = 1.5 s, DS = 16, NS = 8 (1) or 16 (2), DR = 18 and DDR = 2;
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of C-4b of 1 at δC 42.4 showed a significant lower-frequency shift than that of 3 at δ C 77.4 in the 13C NMR spectrum, and one more signal at δH 2.39 (H-4b) appeared in the 1H NMR spectrum of 1, suggesting a deoxygenated substitution at C-4b. In the HMBC spectrum, H-4 (δ 5.81, d, J = 1.3 Hz) and H-6 (δ 1.83, m) showed long-range correlations with the carbon signal of C-4b, which further confirmed the presence of a deoxygenated carbon at C-4b. Therefore, the structure of compound 1 was established as 4b-deoxy-1′-O-acetylpaxilline. The relative configuration of compound 1 was clarified on the basis of the NOESY spectrum and the comparison of NMR data with the relative compound 3. Compound 1 showed similar NMR chemical shifts and coupling constants to those of 3 except for C/H-4b, suggesting that they possessed the same relative configuration which was supported by NOESY experiment. The NOE correlations of 12c-CH3 with H-6a and H-14β indicated that these protons were all on the same face of the ring system. On the other hand, the NOE correlations of H-2 with H-14a; H-4b with 12b-CH3; H-14a with H-2, H-4b and H-14α showed that these protons were on the opposite face of the ring system. Fan et al. reported that negative Cotton effect at λmax 210– 250 nm in circular dichroism (CD) spectrum corresponded to (6aS,12bS,12cR,4bS) and (6aS,12bS,12cS,4bR) configurations of hexacyclic indolediterpenoids with and without 4b-OH substitutions, respectively.[7] The CD spectrum of compound 1 displayed a strong negative Cotton effect at 238 (Δε 86.6) nm, and the structure of compound 1 was without 4b-OH substitution. Therefore, on the basis of the CD spectral analysis and the NOESY data, the absolute configuration of compound 1 was determined as 2R,4bR,6aS,12bS,12cS and14aS. Compound 2 was obtained as a white, amorphous powder. Its HRESIMS exhibited a [M + H]+ at m/z 404.2572 (calcd for C27H34NO2, 404.2590), corresponding to a molecular formula of C27H33NO2, with 12° of unsaturation. The similar IR and UV absorptions to those of 1 implied that it was an analogue. The 1 H and 13C NMR data (Table 1) for 2 also exhibited characteristic signals as an indole-diterpenoid. The structure of 2 was elucidated by the analysis of 2D NMR data including the HSQC, HMBC and 1H-1H COSY spectra. The HMBC correlations observed from H-4 to C-4b and C-14a, from H-7 to C-7a, C-12a and C-12b, from H-8 to C-10, from H-9 to C-7b, from H-10 to C-11a, from H-11 to C-9, from H-2′ to C-3′, from H-3′ to C-2, C-1′ and C-2′, from
12b-CH3 to C-12a, C-12b, C-12c and 12c-CH3, from 12c-CH3 to C-4b, C-12b and C-13,and the 1H-1H COSY correlations of H-3 and H-4, H-4b and H-5, H-5 and H-6, H-6 and H-6a, H-13 and H-14, H-14 and H-14a established an indole-diterpenoid structure. The 1H NMR and 13C NMR spectra made it clear that compound 2 closely resembled penijanthine A.[8] In the 13C NMR spectrum, the chemical shift of C-4b of 2 at δ 41.3 showed a significant lower-frequency shift than that of penijanthine A at δ 77.7, and one more signal at δH 2.23 (H-4b) appeared in the 1H NMR spectrum of 2, suggesting a deoxygenated substitution at C-4b. In the HMBC spectrum, H-4 (δ 5.72, d, J = 6.0 Hz) and 12c-CH3 (δ 0.95, s) showed long-range correlations with the carbon signal of C-4b, which supported the presence of a deoxygenated carbon at C-4b. Therefore, the structure of compound 2 was established as 4b-deoxypenijanthine A. The relative configuration of compound 2 was clarified on the basis of NOESY spectrum. The NOE correlations of 12c-CH3 with H-6a, H-13β; H-4b with 12b-CH3, H-13α and H-14a; H-14a with H-2, H-4b and H-13α suggested that it possessed the same relative configuration as penijanthine A. Similar to that of compound 1, the CD spectrum of compound 2 also displayed a strong negative Cotton effect at 236 (Δε 26.7) nm. Therefore, on the basis of the CD spectral analysis and the NOESY data, the absolute configuration of compound 2 was determined as 2R,3R,4bR,6aS,12bS,12cS and14aS. The known compounds were identified as 1′-O-acetylpaxilline (3), paspaline (4) and 3-deoxo-4b-deoxypaxilline (5), by comparison of spectral data with those reported in the literature.[5–7]
308 1
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Copyright © 2014 John Wiley & Sons, Ltd.
3′ 12b-CH3 12c-CH3 1′-OCOCH3 1′-OCOCH3 NH
14a 1′ 2′
14
7a 7b 8 9 10 11 11a 12a 12b 12c 13
6a 7
6
2 3 4 4a 4b 5
No.
2.03 (3H,s) 7.75 (1H,s)
1.44 (3H,s) 1.06 (3H,s) 1.00 (3H,s)
1.67 (3H,s)
α 1.66 (1H,m) β 1.71 (1H,m) α 2.25 (1H,m) β 1.96 (1H,m) 4.24 (1H,brt,9.1)
7.43 (1H,m) 7.08 (1H,m) 7.08 (1H,m) 7.30 (1H,m)
2.82 (1H,m) 2.41 (1H,dd, 13.2,10.9) 2.75 (1H,dd, 13.2,6.2)
2.39, Overlap α 1.69 (1H,m) β 1.55 (1H,m) 1.83 (2H,m)
5.81 (1H,d,1.5)
4.80 (1H,d,1.5)
δH
22.9, CH3 14.7, CH3 16.4, CH3 170.9, C 22.5, CH3
75.2, CH 82.2, C 24.0, CH3
29.9, CH2
118.7, C 125.1, C 118.7, CH 119.9, CH 121.0, CH 111.7, CH 140.1, C 149.5, C 50.6, C 42.3, C 33.0, CH2
49.1, CH 27.4, CH2
24.2, CH2
80.0, CH 194.5, C 122.4, CH 166.0, C 42.4, CH 25.7, CH2
δC
13
1′-OCOCH3 C-7a,7b,11a,12a
C-2,1′,2′ C-6a,12a,12b,12c C-12b,12c,13
C-2,1′,3’
C-13
C-12c
C-10,11a C-7b,11 C-8,11a C-7b,9
C-6,6a,7a,12a C-6a,7a,12a,12b
C-12c C-4b,12b
C-2,4b,14a
C-3,14a, 1′,3′
HMBC
1
Table 1. Nuclear magnetic resonance data (600 MHz for H, 150 MHz for
H-2,2′ H-4b,5α H-6a,14β
H-2,3′
H-5β H-5α,14a,3′ 12c-CH3 H-2,4b,14α
H-5β,12c-CH3
H-5α, 12b-CH3 H-4b H-6a,13β, 12c-CH3
H-14a,2′,3′
NOE
α β α β
7.75 (1H,s)
5.21 (1H,s) 5.06 (1H,s) 1.81 (3H,s) 1.06 (3H,s) 0.95 (3H,s)
2.05 (1H,m) 1.65 (1H,m) 1.86 (1H,m) 2.23 (1H,m) 4.03 (1H,m)
7.43 (1H,m) 7.08 (1H,m) 7.08 (1H,m) 7.30 (1H,m)
2.23, Overlap α 1.65 (1H,m) β 1.55 (1H,m) 1.80 (1H,m) 1.71 (1H,m) 2.81 (1H,m) 2.40 (1H,dd,13.2,10.7) 2.72 (1H,dd,13.3,6.4)
3.91 (1H,s) 3.99 (1H,m) 5.72 (1H,d,6.0)
δH
C, CDCl3) of compounds 1 and 2 (J in hertz)
20.0, CH3 14.7, CH3 16.1, CH3
76.5, CH 142.1, C 111.6, CH2
29.8, CH2
118.4, C 125.2, C 118.6, CH 119.8, CH 120.8, CH 111.6, CH 140.0, C 150.3, C 50.5, C 41.2, C 33.3, CH2
49.3, CH 27.5, CH2
24.6, CH2
79.1, CH 63.2, CH 119.3, CH 145.8, C 41.3, CH 26.1, CH2
δC
C-3′ C-2,1′,2′ C-12a,12b,12c,12c-CH3 C-4b,12b,13
C-10 C-7b C-11a C-9
C-7a,12a C-7a,12a,12b
C-4b,14a
HMBC
2
H-4b H-6a,13β
H-2,4b,13α
H-5α,13α,14α,14a, 12b-CH3
H-3,6α
NOE
Y.-H. Zhang et al.
Magn. Reson. Chem. 2014, 52, 306–309
Indole-diterpenoids from Penicillium sp. HMBC: SF = 600.13 MHz, SW 1H = 6009.6 Hz, SW 13C = 36220.6 Hz (1) or 27164.7 (2), AQ = 0.09 s, RD = 1.5 s, DS = 16, NS = 108 (1) or 200 (2),DR = 18 and DDR = 2; COSY: SF = 600.13 MHz, SW = 6009.6 Hz, AQ = 0.09 s, RD = 2.0 s, DS = 8, NS = 4, DR = 18 and DDR = 2; NOESY: SF = 600.13 MHz, SW = 6009.6 Hz, AQ = 0.09 s, RD = 2.0 s, DS = 4, NS = 4, DR = 18 and DDR = 2. Fungal material The soil fungal strain CM-7 was separated from the soil sample of Chama at Lashihai City in Yunnan province, China. The fungus was identified as Penicillium sp. (GenBank Accession No. KJ018761) on the basis of morphological and molecular taxonomic methods and was deposited at the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, China. Fermentation and extraction The fresh mycelia of Penicillium sp. CM-7 growing on potato dextrose agar medium at 28 °C for 3 days were inoculated in the liquid medium and cultured statically at same temperature. The liquid medium was composed of mannitol of 20.0 g, D-glucose of 20.0 g, yeast extract of 5.0 g, peptone of 10.0 g, KH2PO4 of 0.5 g, MgSO4 of 0.3 g and corn syrup of 1.0 g, which were dissolved in distilled water of 1000 ml. After 30 days, the fermented broth (40 l) was filtered through cheesecloth to be separated into the supernatant and the mycelia. For the chemical investigation, the fermentation broth was concentrated and successively extracted with ethyl acetate, yielding 40.4 g of extract. The mycelia were extracted with acetone by ultrasonic extraction method, yielding 26.3 g of extract. Isolation and purification The combined extracts (66.7 g) of the fermentation broth and the mycelia were fractioned on a silica gel column eluting with CH2Cl2–MeOH (100 : 0–0 : 100) to yield ten fractions (Fr. 1–10). Fr. 3 (20.9 g) was separated on a silica gel column eluting with petroleum ether-ethyl acetate (100 : 0–1 : 1) to yield three subfractions (Fr. 3.1–3.3). Fr. 3.1 was further separated by Sephadex LH-20 eluting with MeOH to obtain Fr. 3.1.13.1.3. Fr. 3.1.2 was purified by semi-preparative HPLC eluting with 84% MeOH-H2O to yield compounds 1 (2.7 mg, tR 38.2 min) and 2
(1.6 mg, tR 43.5 min). Fr. 3.2 was further separated by Sephadex LH-20 eluting with MeOH to obtain Fr. 3.2.13.2.3. Fr. 3.2.2 was purified by semi-preparative HPLC eluting with 84% MeOHH2O to yield compound 3 (3.4 mg, tR 20.8 min), 4 (2.7 mg, tR 62.0 min), and 5 (4.4 mg, tR 84.4 min). (2R,4bR,6aS,12bS,12cS,14aS)-4b-Deoxy-1′-O-acetylpaxilline (1): white, amorphous powder; ½α20 D 7.2 (c 0.195, MeOH); UV (MeOH) λmax 229 (0.59) and 280 (0.11) nm; CD (c 0.4, MeOH) λmax (Δε) 221 (+29.8), 238 (86.6), 270 (16.5) and 277 (17.1) nm; IR (KBr) νmax 3310, 1731, 1680, 1615, 1454, 1102 and 748 cm1; HRESIMS m/z 462.2618 [M + H]+ (calcd for C29H36NO4, 462.2639); 1H and 13C NMR and 2D NMR data are shown in Table 1. (2R,3R,4bR,6aS,12bS,12cS,14aS)-4b-Deoxypenijanthine A (2): white, amorphous powder; ½α20 D 28.2 (c 0.085, MeOH); UV (MeOH) λmax 228 (0.62) and 280 (0.14); CD (c 0.4, MeOH) λmax (Δε) 215 (4.8), 236 (26.7), 257 (2.4) nm; IR (KBr) νmax 3445, 3336, 1640, 1448, 1100 and 745 cm1; HRESIMS m/z 404.2572 [M + H]+ (calcd for C27H34NO2, 404.2590); 1H and 13C NMR and 2D NMR data are shown in Table 1.
References [1] S. Saikia, M. J. Nicholson, C. Young, E. J. Parker, B. Scott. Mycol. Res. 2008, 112, 184–199. doi:10.1016/j.mycres.2007.06.015. [2] K. M. Byrne, S. K. Smith, J. G. Ondeyka. J. Am. Chem. Soc. 2002, 124, 7055–7060. doi:10.1021/ja017183p. [3] A. E. de Jesus, C. P. Gorst-Allman, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels. J. Chem. Soc. Perkin Trans. I. 1983, 1, 1863–1868. doi:10.1039/P19830001863. [4] H. Sings, S. Singh, in The Alkaloids: Chemistry and Biology (Ed: G. A. Cordell), Academic Press, London, 2003, pp. 51–163. [5] K. Nozawa, Y. Horie, S. Udagawa, K. Kawai, M. Yamazaki. Chem. Pharm. Bull. 1989, 37, 1387–1389. doi:10.1248/cpb.37.1387. [6] S. C. Munday-Finch, A. L. Wilkins, C. O. Miles. Phytochemistry 1996, 41, 327–332. doi:10.1016/0031-9422(95)00515-3. [7] Y. Q. Fan, Y. Wang, P. P. Liu, P. Fu, T. H. Zhu, W. Wang, W. M. Zhu. J. Nat. Prod. 2013, 76, 1328–1336. doi:10.1021/np400304q. [8] T. Itabashi, T. Hosoe, D. Wakana, K. Fukushima, K. Takizawa, T. Yaguchi, K. Okada, G. M. Takaki, K. Kawai. J. Nat. Med. 2009, 63, 96–99. doi:10.1007/s11418-008-0292-6.
Supporting Information Additional supporting information may be found in the online version of this article at the publishers website.
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Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc
Revised: 5 March 2014
Accepted: 5 March 2014
Published online in Wiley Online Library: 2 April 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4065
Structure determination of two new indolediterpenoids from Penicillium sp. CM-7 by NMR spectroscopy Yu-Hong Zhang,a,b Sheng-Dong Huang,a,b Hua-Qi Pan,c Xi-Qing Bian,a,b Zai-Ying Wang,a,b Ai-Hong Hand*,* and Jiao Baia,b* Two new indole-diterpenoids 4b-deoxy-1′-O-acetylpaxilline (1) and 4b-deoxypenijanthine A (2) were isolated from the fermentation broth and the mycelia of the soil fungus Penicillium sp. CM-7, along with three known structurally related compounds, 1′-O-acetylpaxilline (3), paspaline (4) and 3-deoxo-4b-deoxypaxilline (5). The structures of compounds 1 and 2 were elucidated by extensive spectroscopic methods, especially 2D NMR, and their absolute configurations were suggested on the basis of the circular dichroism spectral analysis and the NOESY data. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; 1H NMR; 13C NMR; Indole-diterpenoids; Penicillium sp.
Introduction Indole-diterpenoids are a large, structurally diverse group of fungal secondary metabolites, many of which are potent tremorgenic mammalian mycotoxins.[1] These metabolites have a common core structure comprising a cyclic diterpene skeleton and an indole group.[2,3] The fungi of genus Penicillium are ubiquitous in nature. Previous investigations of these fungi led to the isolation of many indole-diterpenoids secondary metabolites.[4] During our search for new bioactive compounds from fungal sources, two new indole-diterpenoids derivatives, 4b-deoxy-1′O-acetylpaxilline (1) and 4b-deoxypenijanthine A (2) were isolated from Penicillium sp. CM-7, along with 1′-O-acetylpaxilline (3),[5] paspaline (4)[6] and 3-deoxo-4b-deoxypaxilline (5).[7] (Fig. 1) The isolation and the structural elucidation of 1 and 2 are described herein.
Results and Discussion
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Compound 1 was obtained as a white, amorphous powder. Its HRESIMS exhibited a [M + H]+ at m/z 462.2618 (calcd for C29H36NO4, 462.2639), corresponding to a molecular formula of C29H35NO4, with 13° of unsaturation. Its UV spectrum showed characteristic peaks of an indole chromophore at λmax 229 and 280 nm. The IR spectrum further indicated the presence of an indole group at 3310, 1454, 1102 and 748 cm1, and the presence of an ester carbonyl at 1731 cm1 and a conjugated ketone at 1680 cm1. The 1H NMR (600 MHz, CDCl3) (Table 1) spectrum exhibited a –NH singlet at δ 7.75 (1H, s), four aromatic protons at δ 7.43 (1H, m), 7.30 (1H, m), 7.08 (2H, m), one olefinic proton at δ 5.81 (1H, d, J = 1.3 Hz), two oxymethine protons at δ 4.80 (1H, d, J = 1.7 Hz), 4.24 (1H, brt, J = 9.1 Hz) and five methyl singlets at δ 1.00, 1.06, 1.44, 1.67 and 2.03 (each 3H, s). The 13C NMR (150 MHz, CDCl3) and HSQC (Table 1) spectra revealed 29 carbon signals, including two carbonyls, five methyls, five methylenes, nine methines (including five sp2 carbons, two oxygenated sp3
Magn. Reson. Chem. 2014, 52, 306–309
carbons and two sp3 carbons), and eight quaternary carbons (including five sp2 carbons, one oxygenated sp3 carbons and two sp3 carbons). The structure of 1 was elucidated by the analysis of 2D NMR data including the HSQC, HMBC and 1H–1H COSY spectra. The HMBC correlations observed from H-8 to C-10, C-11a, from H-9 to C-7b, C-11, from H-10 to C-8, C-11a, from H-11 to C-7b, C-9, from NH to C-7a, C-7b, C-11a and C-12a constructed an indole moiety. The HMBC correlations of from H-2 to C-3, C-14a, C-1′ and C-3′, from H-4 to C-2, C-4b and C-14a, from H-5 to C-12c, from H-6 to C-4b and C-12b, from H-7 to C-6, C-6a, C-7a, C-12a and C-12b, from H-13 to C-12c, from H-14 to C-13, from H-2′ to C-2, C-1′ and C-3′, from H-3′ to C-2, C-1′ and C-2′, from 12b-CH3 to C-6a, C-12a, C-12b and C-12c, from 12c-CH3 to C-12b, C-12c and C-13 and the 1H-1H COSY correlations of H-4b and H-5, H-5 and H-6, H-14 and H-14a constructed a diterpenoid unit. All the aforementioned evidences characterized compound 1 as an indole-diterpenoid. 1 H NMR and 13C NMR spectra made it clear that compound 1 closely resembled the known compound 3. The chemical shift
* Correspondence to: Jiao Bai, School of Traditional Chinese Materiel Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. E-mail: [email protected] ** Correspondence to: Ai-Hong Han, College of Chemistry and Life Science, Shenyang Normal University, Shenyang, 110034, China. E-mail: hanhong@ hotmail.com a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China c Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China d College of Chemistry and Life Science, Shenyang Normal University, Shenyang 110034, China
Copyright © 2014 John Wiley & Sons, Ltd.
Indole-diterpenoids from Penicillium sp.
Figure 1. The structures of compounds 1–5.
Magn. Reson. Chem. 2014, 52, 306–309
General experimental procedures The UV spectrum was recorded on a Shimadzu UV-1601 (Kyoto, Japan). The IR spectrum was obtained from a Bruker IFS-55 spectrophotometer using KBr pellet. The HR-ESI-MS data were obtained on a Varian QFT-ESI instrument. A JASCO CD-2095 chiral detector was used for CD determination using MeOH as solvent. 1D and 2D NMR spectra were run on a Bruker AVANCE-400/-600 spectrometer. Column chromatography was performed on Si gel G (200–300 mesh; Qingdao Haiyang Chemical Factory, Qingdao, China) and Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA) columns. Thin layer chromatography was carried out using Si gel GF254 (Qingdao Haiyang Chemical Factory) plates. HPLC was performed using Hitachi L-6000 pump, Hitachi L-7400 UV detector and performed with an Octadecylsilyl (ODS) column (10 × 250 mm, 5 μm; YMC-Pack, Japan). NMR spectral methods The NMR spectra were recorded on a Bruker AVANCE-400/-600 instrument, and the chemical shifts are given with TMS as an internal standard. The NMR experiments were carried out at 300 K with the following parameters: 1H NMR spectrum: spectrometer frequency (SF) = 600.13 MHz, spectral width (SW) = 12019.2 Hz, fourier transform size (SI) = 32768, acquisition time (AQ) = 2.73 s, line broadening (LB) = 0.3 Hz, relaxation delay (RD) = 1.0 s, number of dummy scans (DS) = 2 and number of scans (NS) = 8, digitizer resolution (DR) = 18 and digital digitizer resolution (DDR) = 2; 13C NMR spectrum: SF = 100.61 MHz, SW = 24038.5 Hz, SI = 32 768, AQ = 1.36 s, LB = 1.00 Hz, RD = 2.0 s, DS = 4, NS = 4000 (1) or 10240 (2), DR = 22 and DDR = 10; HSQC: SF = 600.13 MHz, SW 1 H = 6009.6 Hz, SW 13C = 30183.3 Hz (1) or 27164.7 (2), AQ = 0.09 s, RD = 1.5 s, DS = 16, NS = 8 (1) or 16 (2), DR = 18 and DDR = 2;
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of C-4b of 1 at δC 42.4 showed a significant lower-frequency shift than that of 3 at δ C 77.4 in the 13C NMR spectrum, and one more signal at δH 2.39 (H-4b) appeared in the 1H NMR spectrum of 1, suggesting a deoxygenated substitution at C-4b. In the HMBC spectrum, H-4 (δ 5.81, d, J = 1.3 Hz) and H-6 (δ 1.83, m) showed long-range correlations with the carbon signal of C-4b, which further confirmed the presence of a deoxygenated carbon at C-4b. Therefore, the structure of compound 1 was established as 4b-deoxy-1′-O-acetylpaxilline. The relative configuration of compound 1 was clarified on the basis of the NOESY spectrum and the comparison of NMR data with the relative compound 3. Compound 1 showed similar NMR chemical shifts and coupling constants to those of 3 except for C/H-4b, suggesting that they possessed the same relative configuration which was supported by NOESY experiment. The NOE correlations of 12c-CH3 with H-6a and H-14β indicated that these protons were all on the same face of the ring system. On the other hand, the NOE correlations of H-2 with H-14a; H-4b with 12b-CH3; H-14a with H-2, H-4b and H-14α showed that these protons were on the opposite face of the ring system. Fan et al. reported that negative Cotton effect at λmax 210– 250 nm in circular dichroism (CD) spectrum corresponded to (6aS,12bS,12cR,4bS) and (6aS,12bS,12cS,4bR) configurations of hexacyclic indolediterpenoids with and without 4b-OH substitutions, respectively.[7] The CD spectrum of compound 1 displayed a strong negative Cotton effect at 238 (Δε 86.6) nm, and the structure of compound 1 was without 4b-OH substitution. Therefore, on the basis of the CD spectral analysis and the NOESY data, the absolute configuration of compound 1 was determined as 2R,4bR,6aS,12bS,12cS and14aS. Compound 2 was obtained as a white, amorphous powder. Its HRESIMS exhibited a [M + H]+ at m/z 404.2572 (calcd for C27H34NO2, 404.2590), corresponding to a molecular formula of C27H33NO2, with 12° of unsaturation. The similar IR and UV absorptions to those of 1 implied that it was an analogue. The 1 H and 13C NMR data (Table 1) for 2 also exhibited characteristic signals as an indole-diterpenoid. The structure of 2 was elucidated by the analysis of 2D NMR data including the HSQC, HMBC and 1H-1H COSY spectra. The HMBC correlations observed from H-4 to C-4b and C-14a, from H-7 to C-7a, C-12a and C-12b, from H-8 to C-10, from H-9 to C-7b, from H-10 to C-11a, from H-11 to C-9, from H-2′ to C-3′, from H-3′ to C-2, C-1′ and C-2′, from
12b-CH3 to C-12a, C-12b, C-12c and 12c-CH3, from 12c-CH3 to C-4b, C-12b and C-13,and the 1H-1H COSY correlations of H-3 and H-4, H-4b and H-5, H-5 and H-6, H-6 and H-6a, H-13 and H-14, H-14 and H-14a established an indole-diterpenoid structure. The 1H NMR and 13C NMR spectra made it clear that compound 2 closely resembled penijanthine A.[8] In the 13C NMR spectrum, the chemical shift of C-4b of 2 at δ 41.3 showed a significant lower-frequency shift than that of penijanthine A at δ 77.7, and one more signal at δH 2.23 (H-4b) appeared in the 1H NMR spectrum of 2, suggesting a deoxygenated substitution at C-4b. In the HMBC spectrum, H-4 (δ 5.72, d, J = 6.0 Hz) and 12c-CH3 (δ 0.95, s) showed long-range correlations with the carbon signal of C-4b, which supported the presence of a deoxygenated carbon at C-4b. Therefore, the structure of compound 2 was established as 4b-deoxypenijanthine A. The relative configuration of compound 2 was clarified on the basis of NOESY spectrum. The NOE correlations of 12c-CH3 with H-6a, H-13β; H-4b with 12b-CH3, H-13α and H-14a; H-14a with H-2, H-4b and H-13α suggested that it possessed the same relative configuration as penijanthine A. Similar to that of compound 1, the CD spectrum of compound 2 also displayed a strong negative Cotton effect at 236 (Δε 26.7) nm. Therefore, on the basis of the CD spectral analysis and the NOESY data, the absolute configuration of compound 2 was determined as 2R,3R,4bR,6aS,12bS,12cS and14aS. The known compounds were identified as 1′-O-acetylpaxilline (3), paspaline (4) and 3-deoxo-4b-deoxypaxilline (5), by comparison of spectral data with those reported in the literature.[5–7]
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3′ 12b-CH3 12c-CH3 1′-OCOCH3 1′-OCOCH3 NH
14a 1′ 2′
14
7a 7b 8 9 10 11 11a 12a 12b 12c 13
6a 7
6
2 3 4 4a 4b 5
No.
2.03 (3H,s) 7.75 (1H,s)
1.44 (3H,s) 1.06 (3H,s) 1.00 (3H,s)
1.67 (3H,s)
α 1.66 (1H,m) β 1.71 (1H,m) α 2.25 (1H,m) β 1.96 (1H,m) 4.24 (1H,brt,9.1)
7.43 (1H,m) 7.08 (1H,m) 7.08 (1H,m) 7.30 (1H,m)
2.82 (1H,m) 2.41 (1H,dd, 13.2,10.9) 2.75 (1H,dd, 13.2,6.2)
2.39, Overlap α 1.69 (1H,m) β 1.55 (1H,m) 1.83 (2H,m)
5.81 (1H,d,1.5)
4.80 (1H,d,1.5)
δH
22.9, CH3 14.7, CH3 16.4, CH3 170.9, C 22.5, CH3
75.2, CH 82.2, C 24.0, CH3
29.9, CH2
118.7, C 125.1, C 118.7, CH 119.9, CH 121.0, CH 111.7, CH 140.1, C 149.5, C 50.6, C 42.3, C 33.0, CH2
49.1, CH 27.4, CH2
24.2, CH2
80.0, CH 194.5, C 122.4, CH 166.0, C 42.4, CH 25.7, CH2
δC
13
1′-OCOCH3 C-7a,7b,11a,12a
C-2,1′,2′ C-6a,12a,12b,12c C-12b,12c,13
C-2,1′,3’
C-13
C-12c
C-10,11a C-7b,11 C-8,11a C-7b,9
C-6,6a,7a,12a C-6a,7a,12a,12b
C-12c C-4b,12b
C-2,4b,14a
C-3,14a, 1′,3′
HMBC
1
Table 1. Nuclear magnetic resonance data (600 MHz for H, 150 MHz for
H-2,2′ H-4b,5α H-6a,14β
H-2,3′
H-5β H-5α,14a,3′ 12c-CH3 H-2,4b,14α
H-5β,12c-CH3
H-5α, 12b-CH3 H-4b H-6a,13β, 12c-CH3
H-14a,2′,3′
NOE
α β α β
7.75 (1H,s)
5.21 (1H,s) 5.06 (1H,s) 1.81 (3H,s) 1.06 (3H,s) 0.95 (3H,s)
2.05 (1H,m) 1.65 (1H,m) 1.86 (1H,m) 2.23 (1H,m) 4.03 (1H,m)
7.43 (1H,m) 7.08 (1H,m) 7.08 (1H,m) 7.30 (1H,m)
2.23, Overlap α 1.65 (1H,m) β 1.55 (1H,m) 1.80 (1H,m) 1.71 (1H,m) 2.81 (1H,m) 2.40 (1H,dd,13.2,10.7) 2.72 (1H,dd,13.3,6.4)
3.91 (1H,s) 3.99 (1H,m) 5.72 (1H,d,6.0)
δH
C, CDCl3) of compounds 1 and 2 (J in hertz)
20.0, CH3 14.7, CH3 16.1, CH3
76.5, CH 142.1, C 111.6, CH2
29.8, CH2
118.4, C 125.2, C 118.6, CH 119.8, CH 120.8, CH 111.6, CH 140.0, C 150.3, C 50.5, C 41.2, C 33.3, CH2
49.3, CH 27.5, CH2
24.6, CH2
79.1, CH 63.2, CH 119.3, CH 145.8, C 41.3, CH 26.1, CH2
δC
C-3′ C-2,1′,2′ C-12a,12b,12c,12c-CH3 C-4b,12b,13
C-10 C-7b C-11a C-9
C-7a,12a C-7a,12a,12b
C-4b,14a
HMBC
2
H-4b H-6a,13β
H-2,4b,13α
H-5α,13α,14α,14a, 12b-CH3
H-3,6α
NOE
Y.-H. Zhang et al.
Magn. Reson. Chem. 2014, 52, 306–309
Indole-diterpenoids from Penicillium sp. HMBC: SF = 600.13 MHz, SW 1H = 6009.6 Hz, SW 13C = 36220.6 Hz (1) or 27164.7 (2), AQ = 0.09 s, RD = 1.5 s, DS = 16, NS = 108 (1) or 200 (2),DR = 18 and DDR = 2; COSY: SF = 600.13 MHz, SW = 6009.6 Hz, AQ = 0.09 s, RD = 2.0 s, DS = 8, NS = 4, DR = 18 and DDR = 2; NOESY: SF = 600.13 MHz, SW = 6009.6 Hz, AQ = 0.09 s, RD = 2.0 s, DS = 4, NS = 4, DR = 18 and DDR = 2. Fungal material The soil fungal strain CM-7 was separated from the soil sample of Chama at Lashihai City in Yunnan province, China. The fungus was identified as Penicillium sp. (GenBank Accession No. KJ018761) on the basis of morphological and molecular taxonomic methods and was deposited at the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, China. Fermentation and extraction The fresh mycelia of Penicillium sp. CM-7 growing on potato dextrose agar medium at 28 °C for 3 days were inoculated in the liquid medium and cultured statically at same temperature. The liquid medium was composed of mannitol of 20.0 g, D-glucose of 20.0 g, yeast extract of 5.0 g, peptone of 10.0 g, KH2PO4 of 0.5 g, MgSO4 of 0.3 g and corn syrup of 1.0 g, which were dissolved in distilled water of 1000 ml. After 30 days, the fermented broth (40 l) was filtered through cheesecloth to be separated into the supernatant and the mycelia. For the chemical investigation, the fermentation broth was concentrated and successively extracted with ethyl acetate, yielding 40.4 g of extract. The mycelia were extracted with acetone by ultrasonic extraction method, yielding 26.3 g of extract. Isolation and purification The combined extracts (66.7 g) of the fermentation broth and the mycelia were fractioned on a silica gel column eluting with CH2Cl2–MeOH (100 : 0–0 : 100) to yield ten fractions (Fr. 1–10). Fr. 3 (20.9 g) was separated on a silica gel column eluting with petroleum ether-ethyl acetate (100 : 0–1 : 1) to yield three subfractions (Fr. 3.1–3.3). Fr. 3.1 was further separated by Sephadex LH-20 eluting with MeOH to obtain Fr. 3.1.13.1.3. Fr. 3.1.2 was purified by semi-preparative HPLC eluting with 84% MeOH-H2O to yield compounds 1 (2.7 mg, tR 38.2 min) and 2
(1.6 mg, tR 43.5 min). Fr. 3.2 was further separated by Sephadex LH-20 eluting with MeOH to obtain Fr. 3.2.13.2.3. Fr. 3.2.2 was purified by semi-preparative HPLC eluting with 84% MeOHH2O to yield compound 3 (3.4 mg, tR 20.8 min), 4 (2.7 mg, tR 62.0 min), and 5 (4.4 mg, tR 84.4 min). (2R,4bR,6aS,12bS,12cS,14aS)-4b-Deoxy-1′-O-acetylpaxilline (1): white, amorphous powder; ½α20 D 7.2 (c 0.195, MeOH); UV (MeOH) λmax 229 (0.59) and 280 (0.11) nm; CD (c 0.4, MeOH) λmax (Δε) 221 (+29.8), 238 (86.6), 270 (16.5) and 277 (17.1) nm; IR (KBr) νmax 3310, 1731, 1680, 1615, 1454, 1102 and 748 cm1; HRESIMS m/z 462.2618 [M + H]+ (calcd for C29H36NO4, 462.2639); 1H and 13C NMR and 2D NMR data are shown in Table 1. (2R,3R,4bR,6aS,12bS,12cS,14aS)-4b-Deoxypenijanthine A (2): white, amorphous powder; ½α20 D 28.2 (c 0.085, MeOH); UV (MeOH) λmax 228 (0.62) and 280 (0.14); CD (c 0.4, MeOH) λmax (Δε) 215 (4.8), 236 (26.7), 257 (2.4) nm; IR (KBr) νmax 3445, 3336, 1640, 1448, 1100 and 745 cm1; HRESIMS m/z 404.2572 [M + H]+ (calcd for C27H34NO2, 404.2590); 1H and 13C NMR and 2D NMR data are shown in Table 1.
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Supporting Information Additional supporting information may be found in the online version of this article at the publishers website.
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