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Two new sesquarterpenoids from the bark of Cryptomeria japonica
Phytochemistry Letters 22 (2017) 56–60
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Two new sesquarterpenoids from the bark of Cryptomeria japonica a
b,c,d
e
e,1
MARK f
Chi-I Chang , Sheng-Yang Wang , Ming-Der Wu , Ming-Jen Cheng , Horng-Huey Ko , ⁎ Hsun-Shuo Changg,h, Jih-Jung Cheni,j,1, Cheng-Chi Chenk,1, Yueh-Hsiung Kuol,m, a
Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan Department of Forestry, National Chung-Hsing University, Taichung 402, Taiwan Agricultural Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan d Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan e Food Industry Research and Development Institute, Hsinchu 300, Taiwan f Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung 807, Taiwan g School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan h Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan i School of Pharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan j Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan k Department of Chemistry, National Taiwan University, Taipei 106, Taiwan l Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan m Department of Biotechnology, Asia University, Taichung 413, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Cupressaceae Cryptomeria japonica Terpenoid Abietane Sesquarterpenoid
Two new sesquarterpenoids, i.e. ferrugicadinol A (1) and ferrugicryptomeridiol (3), and one known sesquarterpenoid, ferrugicadinol (2) were isolated from the bark of Cryptomeria japonica D. Don. Their structures were identified by extensive spectral analysis and comparison with the data of known analogues.
1. Introduction
Several crude extracts and secondary metabolites of this plant have been reported to exhibit antibacterial (Li et al., 2008), antifungal (Kofujita et al., 2001), cytotoxic (Kofujita et al., 2002), anti-inflammatory (Shyur et al., 2008), anti-androgenic (Tu et al., 2007), and insect antifeedant (Wu et al., 2008), and repellent (Morisawa et al., 2002) properties. As part of our studies on the new chemical ingredients of the bark of C. japonica, we have already reported the isolation of a cytotoxic sesquarterpene (C35), cryptotrione, with an unprecedented skeleton possessing a conjugated abietane and cadinane (Chen et al., 2010) and five abietane-type diterpenoids (Chang et al., 2016). In this report, we describe the isolation and structure elucidation of two new sesquarterpenoids (Fig. 1).
Cryptomeria japonica D. Don belongs to the family Cupressaceae and is the only species existing in the genus Cryptomeria. It is endemic to Japan, known as sugi (Japanese cedar) in Japanese (Gan, 1958); and has been an important plantation coniferous tree species in Taiwan since 1906. C. japonica is a massive evergreen coniferous tree, growing up to 50 m in height. Its wood is aromatic, soft, lightweight but sturdy, waterproof, and reddish-pink in color and has been used as a building material for Japanese-style houses and other wood products. Previous phytochemical investigations of the leaves, heartwood, and barks of C. japonica have resulted in the isolation of diverse terpenoids, including monoterpenoids, sesquiterpenoids, and diterpenoids (Arihara et al., 2004a, 2004b; Chen et al., 2001; Kofujita et al., 2001, 2002; Morita et al., 1995; Nagahama and Tazaki, 1993; Nagahama et al., 1993, 1996a, 1996b, 1998; Narita et al., 2006; Shibuya, 1992; Shieh et al., 1981; Shimizu et al., 1988; Su et al., 1993, 1994a, 1994b, 1995a, 1995b, 1996; Morisawa et al., 2002; Yoshikawa et al., 2006a, 2006b).
2. Results and discussion A methanol extract of the bark of C. japonica was suspended in H2O and then partitioned successively with EtOAc and n-BuOH. The EtOAc fraction was subjected to repeated silica gel column chromatography
⁎ Corresponding authors at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, No. 91, Hsueh-Shih Rd Taichung City, 404 Taiwan. E-mail address: [email protected] (Y.-H. Kuo). 1 Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2017.09.003 Received 24 March 2017; Received in revised form 24 August 2017; Accepted 13 September 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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Fig. 1. Structures of compounds 1-3.
and semipreparative NP-HPLC to afford two new sesquarterpenoids, ferrugicadinol A (1) and ferrugicryptomeridiol (3) and one known sesquarterpenoid, ferrugicadinol (2) (Hsieh et al., 2006) (Fig. 1). The IR spectrum of 1 indicated the presence of the aromatic (1610 and 1507 cm−1) and hydroxy (3409 cm−1) groups. Its HR-EI-MS gave a molecular ion at m/z 506.8120, establishing the molecular formula of 1 as C35H54O2, with nine degrees of unsaturation. The base peak of EI-MS fragmental ions of 1 at m/z 285 [C20H29O] + (Fig. 2) indicated that 1 should be a dimer of diterpene and sesquiterpene. The 1H and 13C NMR data of 1 (Table 1) were similar to those of the known compound, sugikurojin H with a abietane incorporate cadinane skeleton, isolated from the bark of C. japonica (Yoshikawa et al., 2006b). One set of dehydroabietane proton signals including three tertiary-linked methyls [δH 0.88, 0.94, 1.12 (3H each, s, Me-19, Me-18, Me-20)], an isopropyl group attached on the benzene ring [δH 1.22 (3H, d, J = 7.0 Hz, Me16), 1.23 (3H, d, J = 7.0 Hz, Me-17), 3.09 (1H, sept, J = 7.0 Hz, H15)], two singlet phenyl protons [δH 6.53 (1H, s, H-11), 6.83 (1H, s, H14)], and a typical downshifted Hβ-1 signal [δH 2.14 (1H, br d, J = 12.5 Hz)] of dehydroabietane (Yoshikawa et al., 2006b) was observed in the 1H NMR spectrum of 1. The 1H and 13C NMR data of diterpene moiety of 1 (Table 1) closely resembled those of ferruginol (Tezuka et al., 1998) and sugikurojin H (Yoshikawa et al., 2006b), which led to establish the partial structure of 1 as a ferruginol with a substituent at C-7. The relative configurations of sterogenic C-atoms in ferruginol were determined by significant NOE correlations between H5 (δH 1.38)/H-15′ (δH 2.26) and Me-19 (δH 0.88)/Me-20 (δH 1.12) in the nuclear Overhauser enhancement exchange spectroscopy (NOESY) spectrum (Fig. 3). With the aid of 1H–1H COSY, the 1H NMR spectrum of 1 (Table 1) also revealed one set of sesquiterpene signals as follows: one isopropyl group [δH 0.90 (3H, d, J = 7.0 Hz, Me-13′), δH 0.93 (3H, d, J = 7.0 Hz, Me-12′), and 1.55 (1H, m)], one doublet methyl [δH 1.08 (3H, d, J = 7.0 Hz, Me-14′)], one downshifted methine [δH 2.39 (1H, br
Table 1 1 H NMR data for compounds 1 and 3. (CDCl3, δin ppm, J in Hz, 400 MHz for 1H NMR, 100 MHz for 13C NMR). No.
1 δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ a
38.7 19.3 41.7 33.2 45.1 22.1 34.9 131.5 148.8 37.9 110.6 150.7 131.4 127.5 26.9 22.7 22.5 33.7 21.6 24.8 73.1 27.5 24.0 137.3 123.1 45.2 40.9 23.6 28.7 39.3 29.3 21.4 21.1 15.4 47.7
3 δH 1.34 m, 2.14 br d (12.5) 1.57 m, 1.72 m 1.22 m, 1.47 m 1.38 m 1.63 m 2.85 m
6.53 s
6.83 s 3.09 sept (7.0) 1.22 d (7.0) 1.23 d (7.0) 0.94 s 0.88 s 1.12 s 1.74 m, 2.01 m 2.17 m, 2.37 m 5.37 s 2.39 br s 1.51 m 1.68 m, 1.16 m 1.56 m, 1.31 m 1.78 m 1.55 m 0.93 d (7.0) 0.90 d (7.0) 1.08 d (7.0) 2.26 t (15.2), 2.18 m
a
δC
δH
38.7 19.2 42.1 34.0 46.3 21.6 32.7 131.4 149.8 37.0 109.9 150.6 131.2 126.7 27.1 22.7 22.5 33.2 21.2 24.2 58.7 22.5 43.8 72.1 56.5 21.3 49.3 21.0 42.0 38.4 73.0 27.1 27.2 16.2 22.7
1.36 m, 2.17 br d (12.5) 1.66 m, 1.58 m 1.23 m 1.40, m 1.90 br d (14.4), 1.61 m 3.14 br d (8.8)
6.61 s
6.99 s 3.10 sept (6.9) 1.21 d (6.9) 1.21 d (6.9) 0.97 s 0.86 s 1.03 s 1.66 m 1.75 m 1.76 m, 1.30 m 1.33 m 1.50 m, 1.16 m 1.42 m 1.95 br d (12.2), 1.51 m 2.09 br d (11.2), 1.42 m
1.22 s 1.22 s 0.99 s 1.10 s
Coupling constants are presented in Hz.
s, H-6′)], and one olefinic proton [5.37 (s, H-5′)]. Additionally, six of a total of nine degrees of unsaturation was accounted for ferruginol and one was attributable to a double bond, the remaining two degrees of unsaturation hinted that 1 exhibited a bicyclic sesquiterpene moiety. By comparison of 13C NMR data of 1 (Table 1) with cadinane sesquiterpenes, 1-methoxy-4-cadinene (Arihara et al., 2004b) and cubenol (Oyarzun and Garbarino, 1988), the sesquiterpene moiety of 1 was tentatively proposed to be an α-cadinenol derivative. The NOE correlations between H-6′ (δH 2.39)/Me-12′ (δH 0.93) and Me-14′ (δH 1.08), HMBC correlations between Me-14′/C-1′ (δC 73.1) and C-9′ (δC 28.7) (Fig. 3), together with the broad singlet of the olefinic proton (H-5′) (Kuo et al., 2003; He et al., 1997), indicated that α-cadinol derivative exhibited a trans ring junction and a hydroxy group located on C-1′ in
Fig. 2. EI-MS fragmental ion.
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Fig. 3. Key HMBC and NOESY correlations of compounds 1 and 3.
an α orientation. The H-15′ (δH 2.26) showed the correlations with C-4′ (δC 137.3) and C-5′ (δC 123.1) led to identify the structure of sesquiterpene moiety as cadin-4-en-1α-ol with a substituent at C-15′. The methylene C-15′ of cadin-4-en-1α-ol was attached on C-7 of ferruginol in an α-peudo-axial orientation, which was assured by the HMBC correlations (Fig. 3) between H-15′ (δH 2.26)/C-6 (δC 22.1) and C-7 (δC 34.9) and the NOE correlations (Fig. 2) between H-15′/H-5 (δH 1.38) and H-14 (δH 6.83). Based on these observations, the structure of 1 was unambiguously established as ferrugicadinol A (1) (Fig. 1). The IR spectrum of 3 indicated the presence of the aromatic (1613 and 1500 cm−1) and hydroxy (3383 cm−1) groups. Its HR-EI-MS gave a molecular ion at m/z 524.8266, establishing the molecular formula of 3 as C35H56O3, with eight degrees of unsaturation. The EI-MS base peak of 3 at m/z 285 [M- C15H27O2]+ as in 1 hinted that 3 should be also a dimer of diterpene and sesquiterpene. Comparing the NMR data of 3 (Table 1) with those of 1, the 1H and 13C NMR spectra of 3 also displayed the signals for ferruginol with a substituent at C-7 (Tezuka et al., 1998) including three tertiary-linked methyls [δH 0.86, 0.97, 1.03 (3H each, s, Me-19, Me-18, Me-20)], an isopropyl group attached on the benzene ring [δH 1.21 (3H × 2, d, J = 6.9 Hz), 3.10 (1H, sept, J = 6.9 Hz)], two singlet phenyl protons [δH 6.61 (1H, s, H-11), 6.99 (1H, s, H-14)], and a typical downshifted Hβ-1 signal [δH 2.17 (1H, br d, J = 12.5 Hz)] of dehydroabietane (Yoshikawa et al., 2006b). In addition, one set of sesquiterpene signals was observed as follows: four methyl singlets [δH 0.99 (3H, s, Me-14′), 1.10 (3H, s, Me-15′), 1.22 (3H × 2, s, Me-12′, Me-13′)], two oxygenated carbons [δc 72.1 and 73.0]. Six degrees of unsaturation were attributable to ferruginol, and the remaining two degrees of unsaturation hinted the substituent of ferruginol should be a bicyclic sesquiterpene. The 13C NMR signals of sesquiterpene moiety of 3 (Table 1) were similar to those of cryptomeridiol (Mohamed et al., 2011), except for the signals of C-1′, 2′, 10′, and 14′ in ring A. Thus, the structure of the sesquiterpene moiety was tentatively elucidated as cryptomeridiol with a substituent at C-1′. The HMBC correlations (Fig. 3) between Me-12′ (δH 1.22)/C-7′ (δC 49.3), Me-14′ (δH 0.99)/C-9′ (δC 42.0) and C-10′ (δC 38.4), and Me-15′ (δH 1.10)/C-3′ (δC 43.8), C-4′ (δC 72.1) and C-5′ (δC 56.5), along with the NOE correlations (Fig. 3) between H-1′ (δH 1.66)/H-5′ (δH 1.33) and Me-14′/Me-15′ further confirm the above proposal. The linkage between C-1′ of cryptomeridiol and C-7 of ferruginol was assigned by the
HMBC correlations (Fig. 3) between H-7 (δH 3.14)/C-5 (δC 46.3), C-1′ (δC 58.7), and C-10′ (δC 38.4). The broad doublet signal of H-7 exhibited a larger coupling constant (J = 8.8 Hz) and showed the NOE correlation with H-5 (δH 1.40) hinted H-7 was in an α- peudo-axial orientation. A steric congestion from the β-peudo-equatorial substituent at C-7 in 3 resulted in the lower field proton signal of H-14 (δH 6.99) than that of ferruginol (δH 6.81). The NOE correlations (Fig. 3) H-1′/H5′ and H-1′/H-7′ indicated all three protons were inα-axial orientation. In Fig. 4, C-7 of the ferruginol moiety showed a γ-gauche relationship to C-14′ and C-9′ of cryptomeridiol moiety, which caused the higher field 13 C NMR signals of C-14′ and C-9′ than those of cryptomeridiol. In turn, due to the γ-anti-relationship between C-7/C-3′ and C-5′, the downshifted 13C NMR signals of C-3′ and C-5′ were also observed. The above evidences confirmed that the C-7 of ferruginol moiety was located with C-1′ of the cryptomeridiol moiety both inβ-equatorial orientation. Thus, the structure of 3 was unambiguously established as ferrugicryptomeridiol (3) (Fig. 1).
3. Experimental 3.1. General experimental procedures
UV
Optical rotations were measured with a Jasco-DIP-180 polarimeter. spectra were obtained with a Shimadzu UV-1601PC
Fig. 4. The differences of
58
13
C NMR chemical shift.
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212-133019). We thank Ms Shu-Yun Sun for the MS measurements in the Instrumentation Center of the College of Science, National Taiwan University. We are also grateful to the National Center for high-performance computing for computer time and facilities.
spectrophotometer. Infrared (IR) spectra were recorded on a PerkinElmer-983G FT-IR spectrophotometer. 1H and 13C NMR and 2D NMR spectra were recorded on a Varian-Unity-Plus-400 spectrometer with tetramethylsilane (TMS) as the internal standard. EI-MS and HR-EI-MS were recorded on a Jeol-JMS-HX300 mass spectrometer. Column chromatography (CC) was carried out with Silica gel (230–400 mesh; Merck & Co., Inc.). Thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (silica gel 60 F254; Merck & Co., Inc.). The spots on TLC were detected by spraying with 5% H2SO4 and then heating at 100 °C. Semi-preparative HPLC was performed using a normal phase column (Purospher STAR Si, 5 μm, 250 × 10 mm; Merck & Co., Inc.) on a LDC Analytical-III system.
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3.2. Plant material The bark of C. japonica D. Don was collected in Sitou, Taiwan in June, 2000. The plant material was identified by Dr. Yen-Hsueh Tseng, Department of Forestry, National Chung-Hsing University (NCHU). A voucher specimen (TCF13443) has been deposited at the Herbarium of the Department of Forestry, NCHU, Taiwan. 3.3. Extraction and isolation The air-dried bark of C. japonica (16.0 kg) was extracted by maceration with MeOH (100 L) three times (7 days each time) at room temperature. The combined MeOH extract was concentrated under reduced pressure to afford a crude extract (480 g), which was suspended in H2O (1 L), and then partitioned between H2O and EtOAc (1 L) for three times. The EtOAc soluble fraction (430 g) was subjected to a silica gel (4.0 kg) column and eluted with n-hexane–EtOAc and EtOAc–MeOH mixtures to give 11 fractions. Fr. 4 from n-hexane–EtOAc (4:1) elution (92.4 g), was further purified through a silica gel column (7 × 60 cm), eluted with a gradient mixture of CH2Cl2–EtOAc (100:1 to 0:1) to obtain sixteen fractions, 4A–4P. Further purification of subfraction 4E (3.3 g) by HPLC afforded 1 (3.0 mg, tR = 35.2 min) using nhexane–EtOAc (4:1). Further purification of subfraction 4F (2.3 g) by HPLC gave 2 (51.9 mg, tR = 37.5 min) using n-hexane–EtOAc (4:1). Fr. 5 from n-hexane–EtOAc (7:3) elution (21.6 g) was further purified over a silica gel column (5 × 45 cm), eluted with n-hexane–CH2Cl2－EtOAc (8:8:1 to 0:1:1) to yield fifteen fractions, 5A–5O. Further purification of subfraction 5E (2.5 g) by HPLC gave 3 (2.8 mg, tR = 46.1 min) using nhexane–EtOAc (7:3). 3.3.1. Ferrugicadinol A (1) Gum; [α] 25 D = −14.9°(c 0.5, CHCl3); IR (dry film) νmax 3409, 1639, 1610, 1507, 1460, 1414, 1367, 1241, 1169,1016, 744 cm−1; UV (MeOH) λmax (log ε) 222 (4.04), 279 (3.57) nm; 1H and 13C NMR data, see Table 1; EI-MS (%) m/z 506 (2) [M]+, 488 ([M − H2O]+, 4), 473 ([M+ − H2O − CH3]+, 1), 298 (3), 285 (100), 243 (4), 229 (5), 215 (4), 201 (6), 189 (9). HR-EI-MS [M]+ m/z 506.8120 (calcd for C35H54O2 506.8104). 3.3.2. Ferrugicryptomeridiol (3) Gum; [α] 25 D = +12.6°(c 0.5, CHCl3); IR νmax 3383, 1613, 1500, 1460, 1420, 1387, 1162, 910, 731 cm−1; UV (MeOH) λmax (log ε) 220 (3.28), 283 (2.82) nm; 1H and 13C NMR data, see Table 1; EI-MS (%) m/ z 524 (2) [M]+, 506 ([M − H2O]+, 3), 488 ([M − 2H2O]+, 3), 285 (100), 243 (5), 229 (5), 215 (5), 201 (13), 189 (19). HR-EI-MS [M]+ m/ z 524.8266 (calcd for C35H56O3 524.8256). Acknowledgements This work was kindly supported by a grant from China Medical University (CMU) under the Aim for Top University Plan of the Ministry of Education, Taiwan, and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B59
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Wu, B., Kashiwagi, T., Kuroda, I., Chen, X.H., Tebayashi, S.I., Kim, C.S., 2008. Antifeedants against Locusta migratoria from the japanese cedar, Cryptomeria japonica II. Biosci. Biotechnol. Biochem. 72, 611–614. Yoshikawa, K., Suzuki, K., Umeyama, A., Arihara, S., 2006a. Abietane diterpenoids from the barks of Cryptomeria japonica. Chem. Pharm. Bull. 54, 574–578. Yoshikawa, K., Tanaka, T., Umeyama, A., Arihara, S., 2006b. Three abietane diterpenes and two diterpenes incorporated sesquiterpenes from the bark of Cryptomeria japonica. Chem. Pharm. Bull. 54, 315–319.
5367–5370. Su, W.C., Fang, J.M., Cheng, Y.S., 1996. Diterpenoids from leaves of Cryptomeria japonica. Phytochemistry 41, 255–261. Tezuka, Y., Kasimu, R., Li, J.X., Basnet, P., Tanaka, K., Namba, T., Kadota, S., 1998. Constituent of roots of Salvia deserta schang. (Xinjiang-Danshen). Chem. Pharm. Bull. 46, 107–112. Tu, W.C., Wang, S.Y., Chien, S.C., Lin, F.M., Chen, L.R., Chiu, C.Y., Hsiao, P.W., 2007. Diterpenes from Cryptomeria japonica inhibit androgen receptor transcriptional activity in prostate cancer cells. Planta Med. 73, 1407–1409.
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Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Two new sesquarterpenoids from the bark of Cryptomeria japonica a
b,c,d
e
e,1
MARK f
Chi-I Chang , Sheng-Yang Wang , Ming-Der Wu , Ming-Jen Cheng , Horng-Huey Ko , ⁎ Hsun-Shuo Changg,h, Jih-Jung Cheni,j,1, Cheng-Chi Chenk,1, Yueh-Hsiung Kuol,m, a
Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan Department of Forestry, National Chung-Hsing University, Taichung 402, Taiwan Agricultural Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan d Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan e Food Industry Research and Development Institute, Hsinchu 300, Taiwan f Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung 807, Taiwan g School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan h Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan i School of Pharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan j Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan k Department of Chemistry, National Taiwan University, Taipei 106, Taiwan l Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan m Department of Biotechnology, Asia University, Taichung 413, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Cupressaceae Cryptomeria japonica Terpenoid Abietane Sesquarterpenoid
Two new sesquarterpenoids, i.e. ferrugicadinol A (1) and ferrugicryptomeridiol (3), and one known sesquarterpenoid, ferrugicadinol (2) were isolated from the bark of Cryptomeria japonica D. Don. Their structures were identified by extensive spectral analysis and comparison with the data of known analogues.
1. Introduction
Several crude extracts and secondary metabolites of this plant have been reported to exhibit antibacterial (Li et al., 2008), antifungal (Kofujita et al., 2001), cytotoxic (Kofujita et al., 2002), anti-inflammatory (Shyur et al., 2008), anti-androgenic (Tu et al., 2007), and insect antifeedant (Wu et al., 2008), and repellent (Morisawa et al., 2002) properties. As part of our studies on the new chemical ingredients of the bark of C. japonica, we have already reported the isolation of a cytotoxic sesquarterpene (C35), cryptotrione, with an unprecedented skeleton possessing a conjugated abietane and cadinane (Chen et al., 2010) and five abietane-type diterpenoids (Chang et al., 2016). In this report, we describe the isolation and structure elucidation of two new sesquarterpenoids (Fig. 1).
Cryptomeria japonica D. Don belongs to the family Cupressaceae and is the only species existing in the genus Cryptomeria. It is endemic to Japan, known as sugi (Japanese cedar) in Japanese (Gan, 1958); and has been an important plantation coniferous tree species in Taiwan since 1906. C. japonica is a massive evergreen coniferous tree, growing up to 50 m in height. Its wood is aromatic, soft, lightweight but sturdy, waterproof, and reddish-pink in color and has been used as a building material for Japanese-style houses and other wood products. Previous phytochemical investigations of the leaves, heartwood, and barks of C. japonica have resulted in the isolation of diverse terpenoids, including monoterpenoids, sesquiterpenoids, and diterpenoids (Arihara et al., 2004a, 2004b; Chen et al., 2001; Kofujita et al., 2001, 2002; Morita et al., 1995; Nagahama and Tazaki, 1993; Nagahama et al., 1993, 1996a, 1996b, 1998; Narita et al., 2006; Shibuya, 1992; Shieh et al., 1981; Shimizu et al., 1988; Su et al., 1993, 1994a, 1994b, 1995a, 1995b, 1996; Morisawa et al., 2002; Yoshikawa et al., 2006a, 2006b).
2. Results and discussion A methanol extract of the bark of C. japonica was suspended in H2O and then partitioned successively with EtOAc and n-BuOH. The EtOAc fraction was subjected to repeated silica gel column chromatography
⁎ Corresponding authors at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, No. 91, Hsueh-Shih Rd Taichung City, 404 Taiwan. E-mail address: [email protected] (Y.-H. Kuo). 1 Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2017.09.003 Received 24 March 2017; Received in revised form 24 August 2017; Accepted 13 September 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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Fig. 1. Structures of compounds 1-3.
and semipreparative NP-HPLC to afford two new sesquarterpenoids, ferrugicadinol A (1) and ferrugicryptomeridiol (3) and one known sesquarterpenoid, ferrugicadinol (2) (Hsieh et al., 2006) (Fig. 1). The IR spectrum of 1 indicated the presence of the aromatic (1610 and 1507 cm−1) and hydroxy (3409 cm−1) groups. Its HR-EI-MS gave a molecular ion at m/z 506.8120, establishing the molecular formula of 1 as C35H54O2, with nine degrees of unsaturation. The base peak of EI-MS fragmental ions of 1 at m/z 285 [C20H29O] + (Fig. 2) indicated that 1 should be a dimer of diterpene and sesquiterpene. The 1H and 13C NMR data of 1 (Table 1) were similar to those of the known compound, sugikurojin H with a abietane incorporate cadinane skeleton, isolated from the bark of C. japonica (Yoshikawa et al., 2006b). One set of dehydroabietane proton signals including three tertiary-linked methyls [δH 0.88, 0.94, 1.12 (3H each, s, Me-19, Me-18, Me-20)], an isopropyl group attached on the benzene ring [δH 1.22 (3H, d, J = 7.0 Hz, Me16), 1.23 (3H, d, J = 7.0 Hz, Me-17), 3.09 (1H, sept, J = 7.0 Hz, H15)], two singlet phenyl protons [δH 6.53 (1H, s, H-11), 6.83 (1H, s, H14)], and a typical downshifted Hβ-1 signal [δH 2.14 (1H, br d, J = 12.5 Hz)] of dehydroabietane (Yoshikawa et al., 2006b) was observed in the 1H NMR spectrum of 1. The 1H and 13C NMR data of diterpene moiety of 1 (Table 1) closely resembled those of ferruginol (Tezuka et al., 1998) and sugikurojin H (Yoshikawa et al., 2006b), which led to establish the partial structure of 1 as a ferruginol with a substituent at C-7. The relative configurations of sterogenic C-atoms in ferruginol were determined by significant NOE correlations between H5 (δH 1.38)/H-15′ (δH 2.26) and Me-19 (δH 0.88)/Me-20 (δH 1.12) in the nuclear Overhauser enhancement exchange spectroscopy (NOESY) spectrum (Fig. 3). With the aid of 1H–1H COSY, the 1H NMR spectrum of 1 (Table 1) also revealed one set of sesquiterpene signals as follows: one isopropyl group [δH 0.90 (3H, d, J = 7.0 Hz, Me-13′), δH 0.93 (3H, d, J = 7.0 Hz, Me-12′), and 1.55 (1H, m)], one doublet methyl [δH 1.08 (3H, d, J = 7.0 Hz, Me-14′)], one downshifted methine [δH 2.39 (1H, br
Table 1 1 H NMR data for compounds 1 and 3. (CDCl3, δin ppm, J in Hz, 400 MHz for 1H NMR, 100 MHz for 13C NMR). No.
1 δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ a
38.7 19.3 41.7 33.2 45.1 22.1 34.9 131.5 148.8 37.9 110.6 150.7 131.4 127.5 26.9 22.7 22.5 33.7 21.6 24.8 73.1 27.5 24.0 137.3 123.1 45.2 40.9 23.6 28.7 39.3 29.3 21.4 21.1 15.4 47.7
3 δH 1.34 m, 2.14 br d (12.5) 1.57 m, 1.72 m 1.22 m, 1.47 m 1.38 m 1.63 m 2.85 m
6.53 s
6.83 s 3.09 sept (7.0) 1.22 d (7.0) 1.23 d (7.0) 0.94 s 0.88 s 1.12 s 1.74 m, 2.01 m 2.17 m, 2.37 m 5.37 s 2.39 br s 1.51 m 1.68 m, 1.16 m 1.56 m, 1.31 m 1.78 m 1.55 m 0.93 d (7.0) 0.90 d (7.0) 1.08 d (7.0) 2.26 t (15.2), 2.18 m
a
δC
δH
38.7 19.2 42.1 34.0 46.3 21.6 32.7 131.4 149.8 37.0 109.9 150.6 131.2 126.7 27.1 22.7 22.5 33.2 21.2 24.2 58.7 22.5 43.8 72.1 56.5 21.3 49.3 21.0 42.0 38.4 73.0 27.1 27.2 16.2 22.7
1.36 m, 2.17 br d (12.5) 1.66 m, 1.58 m 1.23 m 1.40, m 1.90 br d (14.4), 1.61 m 3.14 br d (8.8)
6.61 s
6.99 s 3.10 sept (6.9) 1.21 d (6.9) 1.21 d (6.9) 0.97 s 0.86 s 1.03 s 1.66 m 1.75 m 1.76 m, 1.30 m 1.33 m 1.50 m, 1.16 m 1.42 m 1.95 br d (12.2), 1.51 m 2.09 br d (11.2), 1.42 m
1.22 s 1.22 s 0.99 s 1.10 s
Coupling constants are presented in Hz.
s, H-6′)], and one olefinic proton [5.37 (s, H-5′)]. Additionally, six of a total of nine degrees of unsaturation was accounted for ferruginol and one was attributable to a double bond, the remaining two degrees of unsaturation hinted that 1 exhibited a bicyclic sesquiterpene moiety. By comparison of 13C NMR data of 1 (Table 1) with cadinane sesquiterpenes, 1-methoxy-4-cadinene (Arihara et al., 2004b) and cubenol (Oyarzun and Garbarino, 1988), the sesquiterpene moiety of 1 was tentatively proposed to be an α-cadinenol derivative. The NOE correlations between H-6′ (δH 2.39)/Me-12′ (δH 0.93) and Me-14′ (δH 1.08), HMBC correlations between Me-14′/C-1′ (δC 73.1) and C-9′ (δC 28.7) (Fig. 3), together with the broad singlet of the olefinic proton (H-5′) (Kuo et al., 2003; He et al., 1997), indicated that α-cadinol derivative exhibited a trans ring junction and a hydroxy group located on C-1′ in
Fig. 2. EI-MS fragmental ion.
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Fig. 3. Key HMBC and NOESY correlations of compounds 1 and 3.
an α orientation. The H-15′ (δH 2.26) showed the correlations with C-4′ (δC 137.3) and C-5′ (δC 123.1) led to identify the structure of sesquiterpene moiety as cadin-4-en-1α-ol with a substituent at C-15′. The methylene C-15′ of cadin-4-en-1α-ol was attached on C-7 of ferruginol in an α-peudo-axial orientation, which was assured by the HMBC correlations (Fig. 3) between H-15′ (δH 2.26)/C-6 (δC 22.1) and C-7 (δC 34.9) and the NOE correlations (Fig. 2) between H-15′/H-5 (δH 1.38) and H-14 (δH 6.83). Based on these observations, the structure of 1 was unambiguously established as ferrugicadinol A (1) (Fig. 1). The IR spectrum of 3 indicated the presence of the aromatic (1613 and 1500 cm−1) and hydroxy (3383 cm−1) groups. Its HR-EI-MS gave a molecular ion at m/z 524.8266, establishing the molecular formula of 3 as C35H56O3, with eight degrees of unsaturation. The EI-MS base peak of 3 at m/z 285 [M- C15H27O2]+ as in 1 hinted that 3 should be also a dimer of diterpene and sesquiterpene. Comparing the NMR data of 3 (Table 1) with those of 1, the 1H and 13C NMR spectra of 3 also displayed the signals for ferruginol with a substituent at C-7 (Tezuka et al., 1998) including three tertiary-linked methyls [δH 0.86, 0.97, 1.03 (3H each, s, Me-19, Me-18, Me-20)], an isopropyl group attached on the benzene ring [δH 1.21 (3H × 2, d, J = 6.9 Hz), 3.10 (1H, sept, J = 6.9 Hz)], two singlet phenyl protons [δH 6.61 (1H, s, H-11), 6.99 (1H, s, H-14)], and a typical downshifted Hβ-1 signal [δH 2.17 (1H, br d, J = 12.5 Hz)] of dehydroabietane (Yoshikawa et al., 2006b). In addition, one set of sesquiterpene signals was observed as follows: four methyl singlets [δH 0.99 (3H, s, Me-14′), 1.10 (3H, s, Me-15′), 1.22 (3H × 2, s, Me-12′, Me-13′)], two oxygenated carbons [δc 72.1 and 73.0]. Six degrees of unsaturation were attributable to ferruginol, and the remaining two degrees of unsaturation hinted the substituent of ferruginol should be a bicyclic sesquiterpene. The 13C NMR signals of sesquiterpene moiety of 3 (Table 1) were similar to those of cryptomeridiol (Mohamed et al., 2011), except for the signals of C-1′, 2′, 10′, and 14′ in ring A. Thus, the structure of the sesquiterpene moiety was tentatively elucidated as cryptomeridiol with a substituent at C-1′. The HMBC correlations (Fig. 3) between Me-12′ (δH 1.22)/C-7′ (δC 49.3), Me-14′ (δH 0.99)/C-9′ (δC 42.0) and C-10′ (δC 38.4), and Me-15′ (δH 1.10)/C-3′ (δC 43.8), C-4′ (δC 72.1) and C-5′ (δC 56.5), along with the NOE correlations (Fig. 3) between H-1′ (δH 1.66)/H-5′ (δH 1.33) and Me-14′/Me-15′ further confirm the above proposal. The linkage between C-1′ of cryptomeridiol and C-7 of ferruginol was assigned by the
HMBC correlations (Fig. 3) between H-7 (δH 3.14)/C-5 (δC 46.3), C-1′ (δC 58.7), and C-10′ (δC 38.4). The broad doublet signal of H-7 exhibited a larger coupling constant (J = 8.8 Hz) and showed the NOE correlation with H-5 (δH 1.40) hinted H-7 was in an α- peudo-axial orientation. A steric congestion from the β-peudo-equatorial substituent at C-7 in 3 resulted in the lower field proton signal of H-14 (δH 6.99) than that of ferruginol (δH 6.81). The NOE correlations (Fig. 3) H-1′/H5′ and H-1′/H-7′ indicated all three protons were inα-axial orientation. In Fig. 4, C-7 of the ferruginol moiety showed a γ-gauche relationship to C-14′ and C-9′ of cryptomeridiol moiety, which caused the higher field 13 C NMR signals of C-14′ and C-9′ than those of cryptomeridiol. In turn, due to the γ-anti-relationship between C-7/C-3′ and C-5′, the downshifted 13C NMR signals of C-3′ and C-5′ were also observed. The above evidences confirmed that the C-7 of ferruginol moiety was located with C-1′ of the cryptomeridiol moiety both inβ-equatorial orientation. Thus, the structure of 3 was unambiguously established as ferrugicryptomeridiol (3) (Fig. 1).
3. Experimental 3.1. General experimental procedures
UV
Optical rotations were measured with a Jasco-DIP-180 polarimeter. spectra were obtained with a Shimadzu UV-1601PC
Fig. 4. The differences of
58
13
C NMR chemical shift.
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212-133019). We thank Ms Shu-Yun Sun for the MS measurements in the Instrumentation Center of the College of Science, National Taiwan University. We are also grateful to the National Center for high-performance computing for computer time and facilities.
spectrophotometer. Infrared (IR) spectra were recorded on a PerkinElmer-983G FT-IR spectrophotometer. 1H and 13C NMR and 2D NMR spectra were recorded on a Varian-Unity-Plus-400 spectrometer with tetramethylsilane (TMS) as the internal standard. EI-MS and HR-EI-MS were recorded on a Jeol-JMS-HX300 mass spectrometer. Column chromatography (CC) was carried out with Silica gel (230–400 mesh; Merck & Co., Inc.). Thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (silica gel 60 F254; Merck & Co., Inc.). The spots on TLC were detected by spraying with 5% H2SO4 and then heating at 100 °C. Semi-preparative HPLC was performed using a normal phase column (Purospher STAR Si, 5 μm, 250 × 10 mm; Merck & Co., Inc.) on a LDC Analytical-III system.
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3.2. Plant material The bark of C. japonica D. Don was collected in Sitou, Taiwan in June, 2000. The plant material was identified by Dr. Yen-Hsueh Tseng, Department of Forestry, National Chung-Hsing University (NCHU). A voucher specimen (TCF13443) has been deposited at the Herbarium of the Department of Forestry, NCHU, Taiwan. 3.3. Extraction and isolation The air-dried bark of C. japonica (16.0 kg) was extracted by maceration with MeOH (100 L) three times (7 days each time) at room temperature. The combined MeOH extract was concentrated under reduced pressure to afford a crude extract (480 g), which was suspended in H2O (1 L), and then partitioned between H2O and EtOAc (1 L) for three times. The EtOAc soluble fraction (430 g) was subjected to a silica gel (4.0 kg) column and eluted with n-hexane–EtOAc and EtOAc–MeOH mixtures to give 11 fractions. Fr. 4 from n-hexane–EtOAc (4:1) elution (92.4 g), was further purified through a silica gel column (7 × 60 cm), eluted with a gradient mixture of CH2Cl2–EtOAc (100:1 to 0:1) to obtain sixteen fractions, 4A–4P. Further purification of subfraction 4E (3.3 g) by HPLC afforded 1 (3.0 mg, tR = 35.2 min) using nhexane–EtOAc (4:1). Further purification of subfraction 4F (2.3 g) by HPLC gave 2 (51.9 mg, tR = 37.5 min) using n-hexane–EtOAc (4:1). Fr. 5 from n-hexane–EtOAc (7:3) elution (21.6 g) was further purified over a silica gel column (5 × 45 cm), eluted with n-hexane–CH2Cl2－EtOAc (8:8:1 to 0:1:1) to yield fifteen fractions, 5A–5O. Further purification of subfraction 5E (2.5 g) by HPLC gave 3 (2.8 mg, tR = 46.1 min) using nhexane–EtOAc (7:3). 3.3.1. Ferrugicadinol A (1) Gum; [α] 25 D = −14.9°(c 0.5, CHCl3); IR (dry film) νmax 3409, 1639, 1610, 1507, 1460, 1414, 1367, 1241, 1169,1016, 744 cm−1; UV (MeOH) λmax (log ε) 222 (4.04), 279 (3.57) nm; 1H and 13C NMR data, see Table 1; EI-MS (%) m/z 506 (2) [M]+, 488 ([M − H2O]+, 4), 473 ([M+ − H2O − CH3]+, 1), 298 (3), 285 (100), 243 (4), 229 (5), 215 (4), 201 (6), 189 (9). HR-EI-MS [M]+ m/z 506.8120 (calcd for C35H54O2 506.8104). 3.3.2. Ferrugicryptomeridiol (3) Gum; [α] 25 D = +12.6°(c 0.5, CHCl3); IR νmax 3383, 1613, 1500, 1460, 1420, 1387, 1162, 910, 731 cm−1; UV (MeOH) λmax (log ε) 220 (3.28), 283 (2.82) nm; 1H and 13C NMR data, see Table 1; EI-MS (%) m/ z 524 (2) [M]+, 506 ([M − H2O]+, 3), 488 ([M − 2H2O]+, 3), 285 (100), 243 (5), 229 (5), 215 (5), 201 (13), 189 (19). HR-EI-MS [M]+ m/ z 524.8266 (calcd for C35H56O3 524.8256). Acknowledgements This work was kindly supported by a grant from China Medical University (CMU) under the Aim for Top University Plan of the Ministry of Education, Taiwan, and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B59
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5367–5370. Su, W.C., Fang, J.M., Cheng, Y.S., 1996. Diterpenoids from leaves of Cryptomeria japonica. Phytochemistry 41, 255–261. Tezuka, Y., Kasimu, R., Li, J.X., Basnet, P., Tanaka, K., Namba, T., Kadota, S., 1998. Constituent of roots of Salvia deserta schang. (Xinjiang-Danshen). Chem. Pharm. Bull. 46, 107–112. Tu, W.C., Wang, S.Y., Chien, S.C., Lin, F.M., Chen, L.R., Chiu, C.Y., Hsiao, P.W., 2007. Diterpenes from Cryptomeria japonica inhibit androgen receptor transcriptional activity in prostate cancer cells. Planta Med. 73, 1407–1409.
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