Letter - spectral assignment Received: 12 June 2014
Revised: 2 September 2014
Accepted: 3 September 2014
Published online in Wiley Online Library: 30 September 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4155
Structure determination of two unusual C25 steroids with bicyclo[4.4.1]A/B rings from Penicillium decumbens by NMR spectroscopy Sheng Lin,a,b Ke-Yv Chen,a Peng Fu,a,c Ji Ye,a Yong-Qing Su,a Xian-Wen Yang,a Zhong-Xiao Zhang,a Lei Shan,a Hui-Liang Li,a Yun-Heng Shen,a Run-Hui Liu,a Xi-Ke Xua and Wei-Dong Zhanga* Introduction The genus Penicillium, despite the large numbers of natural products identified, still proves to be rich in structurally unique and biologically active secondary metabolites. A number of fascinating secondary metabolites with antibacterial, antifungal, cytotoxic, and free radical scavenging activities, including citrinadins, scalusamides, perinadine A, and C25 steroids, have been previously isolated from this genus.[1–5] Our recent investigation of a Penicillium decumbens strain from a limestone soil led to the isolation of cyclopenicillone with a unique 2,5-dimethylcyclopent-2-enone carbon skeleton.[6] It demonstrated a dose-dependent inhibition against LPS-induced NO production in RAW264.7 macrophages. As part of our ongoing effort to study the chemical and biological diversity of this strain, we report herein the isolation and structure elucidation of two unusual C25 steroids with bicyclo[4.4.1]A/B rings (Fig. 1), along with 12 known structurally related compounds. To our knowledge, C25 steroids with bicyclo[4.4.1]A/B rings from natural sources are extremely rare. The extensive application of 1D and 2D NMR techniques, high-resolution ESI-MS (HRESIMS) spectrometry, and circular dichroism spectral analysis were performed to characterize their structures and establish the complete 1H and 13C resonance assignments.
Results and discussion
Magn. Reson. Chem. 2015, 53, 223–226
* Correspondence to: Wei-Dong Zhang, School of Pharmacy, Second Military Medical University, Shanghai 200433, China. E-mail: [email protected] a School of Pharmacy, Second Military Medical University, Shanghai 200433, China b State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China c Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Copyright © 2014 John Wiley & Sons, Ltd.
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Compound 1 was obtained as a white, amorphous powder, and exhibited a molecular formula C 25H 36O 5 as evidenced by its positive HRESIMS at m/z 417.2648 ([M + H]+), indicating eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxy (3422 cm 1) and carbonyl units (1649 cm 1). The 1H NMR (600 MHz, CD 3OD) (Table 1) showed resonances for two trisubstituted double bonds at δ H 5.56 (1H, t, J = 6.0 Hz) and 5.79 (1H, s), one trans disubstituted double bond at δ H 5.70 (1H, d, J = 15.6 Hz) and 5.57 (1H, dd, J = 15.6, 6.0 Hz), three oxymethines at δ H 3.26 (1H, m), 4.11 (1H, ddd, J = 13.8, 9.6, 4.2 Hz), and 4.18 (1H, dt, J = 6.0, 6.0 Hz), two tertiary methyls at δ H 0.78 (3H, s) and 1.26 (3H, s), one secondary methyl at δ H 1.16 (3H, d, J = 6.6 Hz), as well as partially overlapping resonances between δ H 1.58 and 2.90 that could be ascribed to aliphatic methylene and methine units. A detailed analysis of the 13C NMR, DEPT, and HSQC data (Table 1) disclosed the existence of three methyls including two sp3 singlet methyls and one
sp3 doulet methyls, two trisubstituted and one trans disubstituted double bonds, seven methines including four sp3 methines and three sp3 methines linked to an oxygen atom, six sp3 methylenes, two sp3 quaternary carbons including one oxygenated sp3 quaternary carbon, and one carbonyl carbon. The remaining four degrees of unsaturations required by the molecular formula indicated that 1 contains four rings. The structure of 1 was finally deduced from a comprehensive 2D NMR spectroscopic data analysis, especially of the 1H–1H COSY and HMBC spectra. Interpretation of the 1H–1H COSY correlations permitted the establishment of spin systems corresponding to the C-1/C-2/C-3/C-4/C-5/C-18, C-9/C-11/C-12, and C-14/C-15/C-16/C-17 portions of 1. HMBC correlations from H-9 to C-1, C-7, and C-18, from H-7 to C-5, C-6, C-8, C-9, C-14, and C-18, from H-5 to C-6, C-7, and C-10, from H 2-18 to C-1, C-4, C-6, and C-9, from H-14 to C-7, C-8, C-9, and C-19, from H 3-19 to C-12, C-13, C-14, and C-17 led to the construction of a unique four-ring system including two seven-membered rings possessing a double bond on the bridgehead in 1. In addition, a side chain could be readily assigned by the COSY correlations from H 3-25 through H-24, H-23 to H-22 and the HMBC correlations from H 3-21 to C-20 and C-22, from H-23 to C-20. Finally, the key correlations of both H 3-21 and H-22 with C-17, and of H 2-16 with C-20 connected this side chain to C-17, completing compound 1 as a C25 steroid with bicyclo [4.4.1]A/B ring.[3,5] 1 H NMR and 13C NMR spectra made it clear that compound 1 closely resembled the co-occurring 24-epi-cyclocitrinol (3).[3,5] The chemical shift of C-15 at δ C 69.8 showed a significant upper frequency shift than that of 3 at δ C 23.7 in the 13C NMR spectrum, and an oxymethine at δ H 4.11 (ddd, J = 13.8, 9.6, 4.2 Hz) instead of an aliphatic methylene at δ H 1.74 and 1.58 (each 1H, m) in 3 appeared in the 1H NMR spectrum of 1, suggesting an hydroxy substitution at C-15. Cross-peaks of the vicinal coupling protons for
S. Lin et al.
Figure 1. The structures of compounds 1–3.
H-14/H-15/H 2-16/H-17 in the 1H–1H COSY spectrum and longrange correlations from H-15 to C-8, C-13, C-14, C-16, and C-17 in the HMBC spectrum confirmed the OH at C-15. Therefore, compound 1 was established as OH-15 derivative of 24-epi-cyclocitrinol (3). The relative configuration of 1 was clarified on the basis of the ROESY spectrum and the comparison of NMR data with 24-epi-cyclocitrinol (3).[3,5] Compound 1 showed similar NMR chemical shifts, coupling patterns, and NOE correlations to those of 3 except for C/H-15, indicating that they possessed the same relative configuration except for C-15. The OH-15 was determined to be α-oriented by the NOE correlations of H-15 with H 3-19 and H-16β, and H-14 with H-17 and H-16α (Fig. 2). The CD spectrum of 1 showed a negative Cotton effect at 321 (Δε 0.78) nm and a positive Cotton at 246 (Δε + 2.13) nm, which were consistent with those of reported C25 steroid analogues.[5] This suggested that the configuration of the [4.4.1]A/B ring was identical to that of C25 steroids having the same chromophores and for which the configuration was determined by X-ray crystallographic analysis.[3,5] From the previous observations and on biogenetic grounds, compound 1 was determined to be (15S)-15-hydroxy-24-epi-cyclocitrinol. Compound 2 was shown to possess the same molecular formula and exhibited similar NMR spectroscopic features to those of 1. After assignment of all protons to their directly attached carbon atoms via HSQC, 1H–1H COSY correlations were observed between H-14 and H 2-15, H 2-15 and H-16, and H-16 and H-17, suggesting that OH-15 in 1 was replaced in 2 by OH-16. This inference was confirmed by the key HMBC correlation between H-16 and C-13, C-14 and C-20. The presence of NOE correlations of H-16/H 3-19/ H-15β and H-14/H-17/H-15α also revealed the α-orientation of OH-16 (Fig. 2). Thus, on the basis of these data and the similarity between compounds 2 and 1, compound 2 was elucidated as (16R)16-hydroxy-24-epi-cyclocitrinol. The known compounds were identified as 24-epi-cyclocitrinol (3), cyclocitrinol, 12R-hydroxycyclocitrinol, neocyclocitrinols A, B, C, and D, isocyclocitrinol A, isocyclocitrinol B, 22-acetylisocyclocitrinol A, 20-O-methyl-24-cyclocitrinol, and 20-O-methyl-24-epi-cyclocitrinol, by comparison of spectral data with those reported in the literature.[3,5] General experimental procedures
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The UV spectrum was recorded on a Shimadzu UV-1601 (Kyoto, Japan). Optical rotations were acquired with Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Bruker Vector 22 spectrometer with KBr pellets. ESIMS were measured on an Agilent LC/MSD Trap XCT spectrometer (Waters, USA), while HRESIMS on a Q-TOF micro mass spectrometer (Waters, USA). Medium-pressure liquid chromatography was carried out on a Buchi Pump Manager C615 with two C-605 pumps. Materials for CC were silica gel (Huiyou Silical Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-
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GEL ODS-A (YMC, MA, USA). Semipreparative HPLC was conducted on an octadecylsilane column (Ultimate, 5 μm, 300 × 10 mm) using a photodiode array UV detector. NMR spectral methods 1D and 2D NMR spectra were recorded on a Bruker Avance 600 NMR spectrometers in CD 3OD or DMSO-d 6 using TMS as 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) = 12 003 Hz, fourier transform size (SI) = 65 536, acquisition time (AQ) = 2.73 s, line broadening (LB) = 0.2 Hz, relaxation delay (RD) = 1.0 s, number of dummy scans (DS) = 2, and number of scans (NS) = 16 (1) or 28 (2); 13C NMR spectrum: SF = 150.9 MHz, SW = 45 454.5 Hz, SI = 65 536, AQ = 0.72 s, LB = 2.00 Hz, DS = 0, NS = 3572 (1) or 5019 (2); HSQC: SF = 600.13 MHz, SW 1H = 3597.1 Hz, SW 13C = 230.07 ppm, AQ = 0.28 s, DS = 32, NS = 32 (1) or 16 (2); HMBC: SF = 600.13 MHz, SW 1H = 3597.1 Hz, SW 13 C = 230.07 ppm, AQ = 0.28 s, DS = 32, NS = 32; COSY: SF = 600.13 MHz, SW = 3597.1 Hz, AQ = 0.28 s, DS = 32, NS = 2; ROESY: SF = 600.13 MHz, SW = 3597.1 Hz, AQ = 0.28 s, DS = 32, NS = 16. Fugal material The fungus strain LXY7-1 was isolated from the limestone soil collected from the limestone terrain Lingxiaoyan in Guangdong, China. It was identified according to its morphological characteristics and 18S RNA by Ke-Yv Chen, School of Pharmacy, Second Military Medical University, Shanghai, China. A voucher specimen is deposited in our laboratory at 80°C. Fermentation and extraction P. decumbens LXY7-1 was cultured at 28°C for 6 days while shaking at 165 rpm in 4 × 20 L volumes of the liquid medium (composed of 30 g of sucrose, 1 g of yeast extract, 3 g of NaNO 3, 0.5 g of MgSO 4·7H 2O, 0.01 g of FeSO 4·7H 2O, 1 g of K 2HPO 4, 0.5 g of KCl, per 1 L of water, pH 6.5). The whole broth was filtered through cheesecloth to separate the broth supernatant and mycelia. The former was applied to an Amberlite XAD-16 macroporous adsorbent resin column. Elution of the column with H 2O and MeOH (5 L each) yielded two corresponding residues after removing solvents. The MeOH residue (10.1 g) was suspended in H 2O (150 mL) and then partitioned with EtOAc (6 × 150 mL). The EtOAc extract was evaporated under reduced pressure to yield 2.5 g of residue, which was subjected to Sephadex LH-20 CC eluting with CHCl 3MeOH (1 : 1) as eluent to produce four fractions (A–D). Fraction C was further fractionated by reversed-phase C-18 silica flash chromatography eluting with a step gradient from 20 to 80% MeOH in H 2O, to yield six subfractions (C1–C6). Subfraction C2 was subjected to reversed-phase preparative HPLC (RP 18, 5 μm, 205 nm, MeCNH 2O, 20 : 80) to afford 1 (2.1 mg) and 2 (1.5 mg).
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 223–226
Copyright © 2014 John Wiley & Sons, Ltd.
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δ H (mult, J, Hz)
1 (CD 3OD)
1.26 s 5.70 d (15.6) 5.57 dd (15.6, 6.0) 4.18 dq (6.0, 6.6) 1.16 d (6.6)
1.53 m 2.23 ddd (13.2, 10.8, 4.2) 1.98 dd (10.2, 10.2) 2.54 dd (13.2, 4.8) 2.57 dd (13.2, 7.2) 0.78 s
2.14 d (9.6) 4.11 ddd (9.6, 9.6, 4.2)
1.53 m 1.81 m 1.53 m 2.15 m
2.77 dd (13.2, 5.4)
5.79 s
5.56 dd (7.2, 7.8) 2.17 ddt (13.8, 7.8, 1.8) 2.42 ddd (13.8,7.2, 6.6) 3.26 m 2.78 brd (12.6) 1.60 dd (12.6, 4.2) 2.71 m
13
1
16.2 75.3 29.0 137.8 132.1 69.2 23.8
59.4 28.5
35.4
48.0 63.9 69.8
41.0
49.9 207.5 126.0 159.5 55.7 147.5 28.7
65.3 42.3
123.2 36.9
δ C (mult)
H and C NMR data (δ) for compounds 1–2a
1
13
1.17 s 5.60 d (15.5) 5.48 dd (15.5, 6.0) 4.09 m 1.07 d (6.0)
1.39 m 2.15 m 1.83 dd (10.5, 10.0) 2.45 brs 2.50 m 0.70
1.99 d (9.0) 3.93 m
1.49 m 1.71 m 1.44 m 2.07 m
2.72 dd (12.5, 6.0)
5.70 s
5.52 dd (6.5, 7.0 ) 2.07 brdd (13.5, 7.0) 2.33 (13.5, 7.0, 6.5) 3.11 m 2.62 brd (13.0) 1.51 dd (13.0, 4.0) 2.65 m
δ H (mult, J, Hz)
1 (DMSO-d 6)
15.6 73.1 29.1 136.1 131.0 66.6 24.2
57.6 27.3
34.4
46.2 62.2 67.8
40.0
48.0 204.3 124.9 155.0 53.6 145.7 27.3
63.2 41.5
122.0 36.0
δ C (mult)
1.34 s 5.79 d (15.6) 5.62 dd (15.6, 6.0) 4.19 dq (6.0, 6.6) 1.16 d (6.6)
1.66 d (6.0) 2.53 brd (13.2) 2.57 dd (13,2, 7.2) 0.77 s
2.48 dd (13.2, 4.8) 1.47 dd (13.2, 4.8) 1.91 ddd (13.2, 13.2, 6.6) 4.23 brdd (6.6, 6.0)
1.53 m 1.82 m 1.53 m 2.16 m
2.84 dd (12.6, 5.4)
5.43 s
5.57 dd (7.2, 7.8) 2.17 ddt (13.8, 7.8, 1.8) 2.42 ddd (13.8, 7.2, 6.6) 3.26 m 2.78 brd (12.6) 1.60 dd (12.6, 4.2) 2.71 m
δ H (mult, J, Hz)
2 (CD 3OD)
16.1 75.4 29.2 138.4 131.9 69.2 23.8
71.5 28.5
72.5
49.5 54.3 36.1
41.1
50.0 207.5 125.6 159.5 55.4 147.5 28.5
65.3 42.2
123.2 36.9
δ C (mult)
1.23 s 5.72 d (15.5) 5.53 dd (15.5, 6.0) 4.09 m 1.08 d (6.5)
1.59 d (6.0) 2.46 brs 2.50 m 0.67 s
2.39 dd (12.0, 4.5) 1.33 dd (13.0, 6.0) 1.78 ddd (13.0, 12.5, 6.5) 4.09 m
1.49 m 1.71 m 1.44 m 2.13 brdd (14.0, 5.0)
2.83 dd (12.5, 6.0)
5.31 s
5.54 dd (6.5, 7.0) 2.07 brdd (13.5, 7.0) 2.32 ddd (13.5, 7.0, 6.5) 3.11 m 2.61 brd (13.0) 1.51 dd (13.0, 4.0) 2.65 m
δ H (mult, J, Hz)
2 (DMSO-d 6)
1
1
15.7 73.5 29.8 136.2 131.0 66.8 24.0
70.2 27.2
70.4
47.0 52.5 35.1
40.0
48.1 204.2 124.4 156.9 53.2 145.7 27.2
63.2 41.4
122.0 36.0
δ C (mult)
NMR data (δ) were measured at 600 or 500 MHz for H NMR and at 150 or 125 MHz for C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, H– H COSY, HSQC, and HMBC experiments.
225
Magn. Reson. Chem. 2015, 53, 223–226
a
1 2α 2β 3 4α 4β 5 6 7 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16α 16β 17 18α 18β 19 20 21 22 23 24 25
Position
Table 1.
C25 steroids with bicyclo[4.4.1]A/B rings from Penicillium decumbens
S. Lin et al. Acknowledgements
Figure 2. Key ROESY correlations for compounds 1 and 2.
(15S)-15-hydroxy-24-epi-cyclocitrinol (1) Amorphous powder; [α]20 D : +116.0 (c 0.23, MeOH); UV (MeOH): λ max (log ε) 204 (4.38), 243 (4.23) nm; CD (MeOH) λ max (Δε) 321 ( 0.78), 246 (2.13); IR (KBr): ν max 3422, 2942, 2866, 1649, 1452, 1369 cm 1; 1 H-(CD 3OD or DMSO-d 6, 600 MHz) and 13C NMR (CD 3OD or DMSO-d 6, 150 MHz) data, refer to Table 1; ESIMS (positive): 417 [M + H]+, 439 [M + Na]+; ESIMS (negative): m/z = 415 [M H] , 451 [M + Cl] ; HRESIMS (positive): m/z = 417.2648 [M + H]+ (calcd. for C 25H 37O 5: 417.2641). (16R)-15-hydroxy-24-epi-cyclocitrinol (2) Amorphous powder; [α]20 D : +99.6 (c 0.19, MeOH); UV (MeOH): λ max (log ε) 204 (4.38), 242 (4.21) nm; CD (MeOH) λ max (Δε) 317 ( 0.92), 244 (2.48); IR (KBr): ν max3421, 2868, 1649, 1451, 1366 cm 1; 1H(CD 3OD or DMSO-d 6, 600 MHz) and 13C NMR (CD 3OD or DMSO-d 6, 150 MHz) data, refer to Table 1; ESIMS (positive): 417 [M + H]+, 439 [M + Na]+; ESIMS (negative): m/z = 415 [M H] , 451 [M + Cl] ; HRESIMS (positive): m/z = 439.2466 [M + Na]+ (calcd. for C 25H 37O 5Na: 439.2460).
The work was supported by program NCET Foundation, NSFC (81230090), Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03), and the National Key Technology R&D Program of China (2012BAI29B06).
References [1] T. Mugishima, M. Tsuda, Y. Kasai, H. Ishiyama, E. Fukushi, J. Kawabata, M. Watabane, L. Akao, J. Kobayashi. J. Org. Chem. 2005, 70, 9430–9435. [2] M. Tsuda, M. Sasaki, T. Mugishima, K. Komatsu, T. Sone, M. Tanaka, Y. Mikami, J. Kobayashi. J. Nat. Prod. 2005, 68, 273–276. [3] M. Sasaki, M. Tsuda, M. Sekiguchi, Y. Mikami, J. Kobayashi. Org. Lett. 2005, 7, 4261–4264. [4] T. Amagata, A. Amagata, K. Tenney, F. A. Valeroote, E. Lobkovsky, J. Clardy, P. Crews. Org. Lett. 2003, 5, 4393–4396. [5] L. Du, T. J. Zhu, Q. Q. Gu, W. M. Zhu. J. Nat. Prod. 2008, 71, 1343–1351. [6] S. Lin, T. Shi, K. Y. Chen, Z. X. Zhang, L. Shan, Y. H. Shen, W. M. Zhu. Chem. Commun. 2011, 47, 10413–10415.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s website.
226 wileyonlinelibrary.com/journal/mrc
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2015, 53, 223–226
Revised: 2 September 2014
Accepted: 3 September 2014
Published online in Wiley Online Library: 30 September 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4155
Structure determination of two unusual C25 steroids with bicyclo[4.4.1]A/B rings from Penicillium decumbens by NMR spectroscopy Sheng Lin,a,b Ke-Yv Chen,a Peng Fu,a,c Ji Ye,a Yong-Qing Su,a Xian-Wen Yang,a Zhong-Xiao Zhang,a Lei Shan,a Hui-Liang Li,a Yun-Heng Shen,a Run-Hui Liu,a Xi-Ke Xua and Wei-Dong Zhanga* Introduction The genus Penicillium, despite the large numbers of natural products identified, still proves to be rich in structurally unique and biologically active secondary metabolites. A number of fascinating secondary metabolites with antibacterial, antifungal, cytotoxic, and free radical scavenging activities, including citrinadins, scalusamides, perinadine A, and C25 steroids, have been previously isolated from this genus.[1–5] Our recent investigation of a Penicillium decumbens strain from a limestone soil led to the isolation of cyclopenicillone with a unique 2,5-dimethylcyclopent-2-enone carbon skeleton.[6] It demonstrated a dose-dependent inhibition against LPS-induced NO production in RAW264.7 macrophages. As part of our ongoing effort to study the chemical and biological diversity of this strain, we report herein the isolation and structure elucidation of two unusual C25 steroids with bicyclo[4.4.1]A/B rings (Fig. 1), along with 12 known structurally related compounds. To our knowledge, C25 steroids with bicyclo[4.4.1]A/B rings from natural sources are extremely rare. The extensive application of 1D and 2D NMR techniques, high-resolution ESI-MS (HRESIMS) spectrometry, and circular dichroism spectral analysis were performed to characterize their structures and establish the complete 1H and 13C resonance assignments.
Results and discussion
Magn. Reson. Chem. 2015, 53, 223–226
* Correspondence to: Wei-Dong Zhang, School of Pharmacy, Second Military Medical University, Shanghai 200433, China. E-mail: [email protected] a School of Pharmacy, Second Military Medical University, Shanghai 200433, China b State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China c Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Copyright © 2014 John Wiley & Sons, Ltd.
223
Compound 1 was obtained as a white, amorphous powder, and exhibited a molecular formula C 25H 36O 5 as evidenced by its positive HRESIMS at m/z 417.2648 ([M + H]+), indicating eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxy (3422 cm 1) and carbonyl units (1649 cm 1). The 1H NMR (600 MHz, CD 3OD) (Table 1) showed resonances for two trisubstituted double bonds at δ H 5.56 (1H, t, J = 6.0 Hz) and 5.79 (1H, s), one trans disubstituted double bond at δ H 5.70 (1H, d, J = 15.6 Hz) and 5.57 (1H, dd, J = 15.6, 6.0 Hz), three oxymethines at δ H 3.26 (1H, m), 4.11 (1H, ddd, J = 13.8, 9.6, 4.2 Hz), and 4.18 (1H, dt, J = 6.0, 6.0 Hz), two tertiary methyls at δ H 0.78 (3H, s) and 1.26 (3H, s), one secondary methyl at δ H 1.16 (3H, d, J = 6.6 Hz), as well as partially overlapping resonances between δ H 1.58 and 2.90 that could be ascribed to aliphatic methylene and methine units. A detailed analysis of the 13C NMR, DEPT, and HSQC data (Table 1) disclosed the existence of three methyls including two sp3 singlet methyls and one
sp3 doulet methyls, two trisubstituted and one trans disubstituted double bonds, seven methines including four sp3 methines and three sp3 methines linked to an oxygen atom, six sp3 methylenes, two sp3 quaternary carbons including one oxygenated sp3 quaternary carbon, and one carbonyl carbon. The remaining four degrees of unsaturations required by the molecular formula indicated that 1 contains four rings. The structure of 1 was finally deduced from a comprehensive 2D NMR spectroscopic data analysis, especially of the 1H–1H COSY and HMBC spectra. Interpretation of the 1H–1H COSY correlations permitted the establishment of spin systems corresponding to the C-1/C-2/C-3/C-4/C-5/C-18, C-9/C-11/C-12, and C-14/C-15/C-16/C-17 portions of 1. HMBC correlations from H-9 to C-1, C-7, and C-18, from H-7 to C-5, C-6, C-8, C-9, C-14, and C-18, from H-5 to C-6, C-7, and C-10, from H 2-18 to C-1, C-4, C-6, and C-9, from H-14 to C-7, C-8, C-9, and C-19, from H 3-19 to C-12, C-13, C-14, and C-17 led to the construction of a unique four-ring system including two seven-membered rings possessing a double bond on the bridgehead in 1. In addition, a side chain could be readily assigned by the COSY correlations from H 3-25 through H-24, H-23 to H-22 and the HMBC correlations from H 3-21 to C-20 and C-22, from H-23 to C-20. Finally, the key correlations of both H 3-21 and H-22 with C-17, and of H 2-16 with C-20 connected this side chain to C-17, completing compound 1 as a C25 steroid with bicyclo [4.4.1]A/B ring.[3,5] 1 H NMR and 13C NMR spectra made it clear that compound 1 closely resembled the co-occurring 24-epi-cyclocitrinol (3).[3,5] The chemical shift of C-15 at δ C 69.8 showed a significant upper frequency shift than that of 3 at δ C 23.7 in the 13C NMR spectrum, and an oxymethine at δ H 4.11 (ddd, J = 13.8, 9.6, 4.2 Hz) instead of an aliphatic methylene at δ H 1.74 and 1.58 (each 1H, m) in 3 appeared in the 1H NMR spectrum of 1, suggesting an hydroxy substitution at C-15. Cross-peaks of the vicinal coupling protons for
S. Lin et al.
Figure 1. The structures of compounds 1–3.
H-14/H-15/H 2-16/H-17 in the 1H–1H COSY spectrum and longrange correlations from H-15 to C-8, C-13, C-14, C-16, and C-17 in the HMBC spectrum confirmed the OH at C-15. Therefore, compound 1 was established as OH-15 derivative of 24-epi-cyclocitrinol (3). The relative configuration of 1 was clarified on the basis of the ROESY spectrum and the comparison of NMR data with 24-epi-cyclocitrinol (3).[3,5] Compound 1 showed similar NMR chemical shifts, coupling patterns, and NOE correlations to those of 3 except for C/H-15, indicating that they possessed the same relative configuration except for C-15. The OH-15 was determined to be α-oriented by the NOE correlations of H-15 with H 3-19 and H-16β, and H-14 with H-17 and H-16α (Fig. 2). The CD spectrum of 1 showed a negative Cotton effect at 321 (Δε 0.78) nm and a positive Cotton at 246 (Δε + 2.13) nm, which were consistent with those of reported C25 steroid analogues.[5] This suggested that the configuration of the [4.4.1]A/B ring was identical to that of C25 steroids having the same chromophores and for which the configuration was determined by X-ray crystallographic analysis.[3,5] From the previous observations and on biogenetic grounds, compound 1 was determined to be (15S)-15-hydroxy-24-epi-cyclocitrinol. Compound 2 was shown to possess the same molecular formula and exhibited similar NMR spectroscopic features to those of 1. After assignment of all protons to their directly attached carbon atoms via HSQC, 1H–1H COSY correlations were observed between H-14 and H 2-15, H 2-15 and H-16, and H-16 and H-17, suggesting that OH-15 in 1 was replaced in 2 by OH-16. This inference was confirmed by the key HMBC correlation between H-16 and C-13, C-14 and C-20. The presence of NOE correlations of H-16/H 3-19/ H-15β and H-14/H-17/H-15α also revealed the α-orientation of OH-16 (Fig. 2). Thus, on the basis of these data and the similarity between compounds 2 and 1, compound 2 was elucidated as (16R)16-hydroxy-24-epi-cyclocitrinol. The known compounds were identified as 24-epi-cyclocitrinol (3), cyclocitrinol, 12R-hydroxycyclocitrinol, neocyclocitrinols A, B, C, and D, isocyclocitrinol A, isocyclocitrinol B, 22-acetylisocyclocitrinol A, 20-O-methyl-24-cyclocitrinol, and 20-O-methyl-24-epi-cyclocitrinol, by comparison of spectral data with those reported in the literature.[3,5] General experimental procedures
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The UV spectrum was recorded on a Shimadzu UV-1601 (Kyoto, Japan). Optical rotations were acquired with Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Bruker Vector 22 spectrometer with KBr pellets. ESIMS were measured on an Agilent LC/MSD Trap XCT spectrometer (Waters, USA), while HRESIMS on a Q-TOF micro mass spectrometer (Waters, USA). Medium-pressure liquid chromatography was carried out on a Buchi Pump Manager C615 with two C-605 pumps. Materials for CC were silica gel (Huiyou Silical Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-
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GEL ODS-A (YMC, MA, USA). Semipreparative HPLC was conducted on an octadecylsilane column (Ultimate, 5 μm, 300 × 10 mm) using a photodiode array UV detector. NMR spectral methods 1D and 2D NMR spectra were recorded on a Bruker Avance 600 NMR spectrometers in CD 3OD or DMSO-d 6 using TMS as 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) = 12 003 Hz, fourier transform size (SI) = 65 536, acquisition time (AQ) = 2.73 s, line broadening (LB) = 0.2 Hz, relaxation delay (RD) = 1.0 s, number of dummy scans (DS) = 2, and number of scans (NS) = 16 (1) or 28 (2); 13C NMR spectrum: SF = 150.9 MHz, SW = 45 454.5 Hz, SI = 65 536, AQ = 0.72 s, LB = 2.00 Hz, DS = 0, NS = 3572 (1) or 5019 (2); HSQC: SF = 600.13 MHz, SW 1H = 3597.1 Hz, SW 13C = 230.07 ppm, AQ = 0.28 s, DS = 32, NS = 32 (1) or 16 (2); HMBC: SF = 600.13 MHz, SW 1H = 3597.1 Hz, SW 13 C = 230.07 ppm, AQ = 0.28 s, DS = 32, NS = 32; COSY: SF = 600.13 MHz, SW = 3597.1 Hz, AQ = 0.28 s, DS = 32, NS = 2; ROESY: SF = 600.13 MHz, SW = 3597.1 Hz, AQ = 0.28 s, DS = 32, NS = 16. Fugal material The fungus strain LXY7-1 was isolated from the limestone soil collected from the limestone terrain Lingxiaoyan in Guangdong, China. It was identified according to its morphological characteristics and 18S RNA by Ke-Yv Chen, School of Pharmacy, Second Military Medical University, Shanghai, China. A voucher specimen is deposited in our laboratory at 80°C. Fermentation and extraction P. decumbens LXY7-1 was cultured at 28°C for 6 days while shaking at 165 rpm in 4 × 20 L volumes of the liquid medium (composed of 30 g of sucrose, 1 g of yeast extract, 3 g of NaNO 3, 0.5 g of MgSO 4·7H 2O, 0.01 g of FeSO 4·7H 2O, 1 g of K 2HPO 4, 0.5 g of KCl, per 1 L of water, pH 6.5). The whole broth was filtered through cheesecloth to separate the broth supernatant and mycelia. The former was applied to an Amberlite XAD-16 macroporous adsorbent resin column. Elution of the column with H 2O and MeOH (5 L each) yielded two corresponding residues after removing solvents. The MeOH residue (10.1 g) was suspended in H 2O (150 mL) and then partitioned with EtOAc (6 × 150 mL). The EtOAc extract was evaporated under reduced pressure to yield 2.5 g of residue, which was subjected to Sephadex LH-20 CC eluting with CHCl 3MeOH (1 : 1) as eluent to produce four fractions (A–D). Fraction C was further fractionated by reversed-phase C-18 silica flash chromatography eluting with a step gradient from 20 to 80% MeOH in H 2O, to yield six subfractions (C1–C6). Subfraction C2 was subjected to reversed-phase preparative HPLC (RP 18, 5 μm, 205 nm, MeCNH 2O, 20 : 80) to afford 1 (2.1 mg) and 2 (1.5 mg).
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Copyright © 2014 John Wiley & Sons, Ltd.
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δ H (mult, J, Hz)
1 (CD 3OD)
1.26 s 5.70 d (15.6) 5.57 dd (15.6, 6.0) 4.18 dq (6.0, 6.6) 1.16 d (6.6)
1.53 m 2.23 ddd (13.2, 10.8, 4.2) 1.98 dd (10.2, 10.2) 2.54 dd (13.2, 4.8) 2.57 dd (13.2, 7.2) 0.78 s
2.14 d (9.6) 4.11 ddd (9.6, 9.6, 4.2)
1.53 m 1.81 m 1.53 m 2.15 m
2.77 dd (13.2, 5.4)
5.79 s
5.56 dd (7.2, 7.8) 2.17 ddt (13.8, 7.8, 1.8) 2.42 ddd (13.8,7.2, 6.6) 3.26 m 2.78 brd (12.6) 1.60 dd (12.6, 4.2) 2.71 m
13
1
16.2 75.3 29.0 137.8 132.1 69.2 23.8
59.4 28.5
35.4
48.0 63.9 69.8
41.0
49.9 207.5 126.0 159.5 55.7 147.5 28.7
65.3 42.3
123.2 36.9
δ C (mult)
H and C NMR data (δ) for compounds 1–2a
1
13
1.17 s 5.60 d (15.5) 5.48 dd (15.5, 6.0) 4.09 m 1.07 d (6.0)
1.39 m 2.15 m 1.83 dd (10.5, 10.0) 2.45 brs 2.50 m 0.70
1.99 d (9.0) 3.93 m
1.49 m 1.71 m 1.44 m 2.07 m
2.72 dd (12.5, 6.0)
5.70 s
5.52 dd (6.5, 7.0 ) 2.07 brdd (13.5, 7.0) 2.33 (13.5, 7.0, 6.5) 3.11 m 2.62 brd (13.0) 1.51 dd (13.0, 4.0) 2.65 m
δ H (mult, J, Hz)
1 (DMSO-d 6)
15.6 73.1 29.1 136.1 131.0 66.6 24.2
57.6 27.3
34.4
46.2 62.2 67.8
40.0
48.0 204.3 124.9 155.0 53.6 145.7 27.3
63.2 41.5
122.0 36.0
δ C (mult)
1.34 s 5.79 d (15.6) 5.62 dd (15.6, 6.0) 4.19 dq (6.0, 6.6) 1.16 d (6.6)
1.66 d (6.0) 2.53 brd (13.2) 2.57 dd (13,2, 7.2) 0.77 s
2.48 dd (13.2, 4.8) 1.47 dd (13.2, 4.8) 1.91 ddd (13.2, 13.2, 6.6) 4.23 brdd (6.6, 6.0)
1.53 m 1.82 m 1.53 m 2.16 m
2.84 dd (12.6, 5.4)
5.43 s
5.57 dd (7.2, 7.8) 2.17 ddt (13.8, 7.8, 1.8) 2.42 ddd (13.8, 7.2, 6.6) 3.26 m 2.78 brd (12.6) 1.60 dd (12.6, 4.2) 2.71 m
δ H (mult, J, Hz)
2 (CD 3OD)
16.1 75.4 29.2 138.4 131.9 69.2 23.8
71.5 28.5
72.5
49.5 54.3 36.1
41.1
50.0 207.5 125.6 159.5 55.4 147.5 28.5
65.3 42.2
123.2 36.9
δ C (mult)
1.23 s 5.72 d (15.5) 5.53 dd (15.5, 6.0) 4.09 m 1.08 d (6.5)
1.59 d (6.0) 2.46 brs 2.50 m 0.67 s
2.39 dd (12.0, 4.5) 1.33 dd (13.0, 6.0) 1.78 ddd (13.0, 12.5, 6.5) 4.09 m
1.49 m 1.71 m 1.44 m 2.13 brdd (14.0, 5.0)
2.83 dd (12.5, 6.0)
5.31 s
5.54 dd (6.5, 7.0) 2.07 brdd (13.5, 7.0) 2.32 ddd (13.5, 7.0, 6.5) 3.11 m 2.61 brd (13.0) 1.51 dd (13.0, 4.0) 2.65 m
δ H (mult, J, Hz)
2 (DMSO-d 6)
1
1
15.7 73.5 29.8 136.2 131.0 66.8 24.0
70.2 27.2
70.4
47.0 52.5 35.1
40.0
48.1 204.2 124.4 156.9 53.2 145.7 27.2
63.2 41.4
122.0 36.0
δ C (mult)
NMR data (δ) were measured at 600 or 500 MHz for H NMR and at 150 or 125 MHz for C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, H– H COSY, HSQC, and HMBC experiments.
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a
1 2α 2β 3 4α 4β 5 6 7 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16α 16β 17 18α 18β 19 20 21 22 23 24 25
Position
Table 1.
C25 steroids with bicyclo[4.4.1]A/B rings from Penicillium decumbens
S. Lin et al. Acknowledgements
Figure 2. Key ROESY correlations for compounds 1 and 2.
(15S)-15-hydroxy-24-epi-cyclocitrinol (1) Amorphous powder; [α]20 D : +116.0 (c 0.23, MeOH); UV (MeOH): λ max (log ε) 204 (4.38), 243 (4.23) nm; CD (MeOH) λ max (Δε) 321 ( 0.78), 246 (2.13); IR (KBr): ν max 3422, 2942, 2866, 1649, 1452, 1369 cm 1; 1 H-(CD 3OD or DMSO-d 6, 600 MHz) and 13C NMR (CD 3OD or DMSO-d 6, 150 MHz) data, refer to Table 1; ESIMS (positive): 417 [M + H]+, 439 [M + Na]+; ESIMS (negative): m/z = 415 [M H] , 451 [M + Cl] ; HRESIMS (positive): m/z = 417.2648 [M + H]+ (calcd. for C 25H 37O 5: 417.2641). (16R)-15-hydroxy-24-epi-cyclocitrinol (2) Amorphous powder; [α]20 D : +99.6 (c 0.19, MeOH); UV (MeOH): λ max (log ε) 204 (4.38), 242 (4.21) nm; CD (MeOH) λ max (Δε) 317 ( 0.92), 244 (2.48); IR (KBr): ν max3421, 2868, 1649, 1451, 1366 cm 1; 1H(CD 3OD or DMSO-d 6, 600 MHz) and 13C NMR (CD 3OD or DMSO-d 6, 150 MHz) data, refer to Table 1; ESIMS (positive): 417 [M + H]+, 439 [M + Na]+; ESIMS (negative): m/z = 415 [M H] , 451 [M + Cl] ; HRESIMS (positive): m/z = 439.2466 [M + Na]+ (calcd. for C 25H 37O 5Na: 439.2460).
The work was supported by program NCET Foundation, NSFC (81230090), Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03), and the National Key Technology R&D Program of China (2012BAI29B06).
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s website.
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