May 2006 Notes Chem. Pharm. Bull. 54(5) 693—695 (2006) 693 New Constituents from the Heartwood of Picea morrisonicola HAYATA Tzong-Huei LEE,a Ming-Hsi YEH,b Chi-I CHANG,c Ching-Kuo LEE,a Yi-Yuan SHAO,d and Yueh-Hsiung KUO*,b a Graduate Institute of Pharmacognosy Science, Taipei Medical University; Taipei, Taiwan 110, Republic of China: Department of Chemistry, National Taiwan University; Taipei, Taiwan 106, Republic of China: c Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung; Taiwan 912, Republic of China: and d Department of Food Science, Nutrition and Nutraceutical Biotechnology, Shih Chien University; Taipei, Taiwan 104, Republic of China. Received October 5, 2005; accepted January 7, 2006 b Three new constituents, including a p-menthane type monoterpene, trans-p-menthane-7,8,9-triol (1), an aromatic, 2,6-dihydroxy-3,4-dimethylbenzoic acid methyl ester (2), and a lignan, (ⴙ)-morrisonicolanin (3), were isolated from the low polar layer of heartwood extracts of Picea morrisonicola, and their structures were elucidated on the basis of spectroscopic analysis. Of these compounds identified, 1 was obtained as its diacetylated derivative 1a, and 2 was the chemical entity first isolated from a natural source. Key words Picea morrisonicola; Pinaceae; heartwood; monoterpene; lignan; aromatic A total of six species of the genus Picea (Pinaceae), including five transplantation (P. abies, P. glauca, P. glehnii, P. asperata, P. orientalis) and one endemic species (P. morrisonicola), were grown in Taiwan. Among them, cumulative phytochemical examinations have revealed that P. abies,1—3) P. glauca,4) and P. glehnii 5) contained lignans, flavonoids and their glucosides, and diterpenoids. P. morrisonicola HAYATA, a large tree, is distributed at high altitudes of about 2000— 2500 m in the central range, and scattered in ravines and mountain slopes.6) So far its wood has been used only for building materials, but not for any other purpose locally. In our previous papers, bundles of novel compounds, including four abietane-type diterpenes7) and two norditerpenes,8) were isolated and identified from the low polar layer of acetonic extracts of its heartwood. However, many constituents in other fractions were still not fully investigated. Therefore, further studies on different fractions of the same extracts yielded three new components including a monoterpene 1, an aromatic 2, and a lignan 3. The following describes the structural assignments of these compounds. The acetonic extract of the heartwood of P. morrisonicola was concentrated to give a residue which was subjected to partitioning with EtOAc and water. The combined EtOAc soluble layer was then separated sequentially using Si-gravity column chromatography and HPLC to yield three new components, 1—3. Compound 1 was purified as its diacetylated derivative 1a. Compound 1a was obtained as a colorless oil, whose molecular formula was confirmed to be C14H24O5 as deduced from HR-EI-MS and 13C-NMR spectra. Its IR spectrum indicated the presence of a hydroxyl group (3429 cm⫺1) and a carbonyl group (1719 cm⫺1). The 1H-NMR spectrum of 1a showed signals due to one tertiary methyl group at d H 1.09 (3H, s), two methine protons at d H 1.39 (1H, m, H-4a ) and 1.55 (1H, m, H-1b ), one pair of acetoxymethylene protons at d H 3.86 (2H, d, J⫽6.4 Hz, H-7), one pair of AB type acetoxymethylene protons at d H 3.96 (1H, d, J⫽11.2 Hz, H-9) and 4.03 (1H, d, J⫽11.2 Hz, H-9), and four pairs of methylene protons at [d H 0.95 (2H, m, H-2a ,-6a ), 1.85 (2H, m, H-2b , -6b )], [d H 1.15 (1H, m, H-3b ), 1.92 (1H, m, H-3a )], and [d H 1.24 (1H, m, H-5b ), 1.87 (1H, m, H-5a )] as further evidenced by ∗ To whom correspondence should be addressed. e-mail: yhkuo@ntu.edu.tw 13 C-NMR, distortionless enhancement by polarization transfer (DEPT) and 1H-detected heteronuclear multiple quantum coherence (HMQC) spectra. The 1H-NMR spectrum of 1a also revealed two acetyl methyl singlets at d H 2.02 (3H, s) and 2.09 (3H, s), suggesting that there existed two acetoxyl and one hydroxyl (3429 cm⫺1) groups in 1a. Based on the molecular formula C14H24O5, the degree of unsaturation of 1a was three, including two acetyl carbonyls. Thus, the number of rings of 1a should be one. In the heteronuclear multiple bond connectivity (HMBC) spectrum of 1a, the longrange 13C–1H correlations, including one acetyl carbonyl with H2-9, the other acetyl carbonyl with H-7, and one hydroxy-bearing carbon C-8 with H3-10, established the locations of two acetyl groups and one residual hydroxyl group. All the above spectroscopic data, especially the symmetrical H-2 and H-6 signals and nuclear Overhauser and exchange spectroscopy (NOESY) correlations of 1a, suggested that 1a was a p-menthane type skeleton, except that both H3-7 and H3-9 were converted to CH2OAc functionalities, and a hydroxyl group was attached to C-8. In the NOESY spectrum of 1a, mutual correlations were as follows: H2-7/H2-2, -6; H- © 2006 Pharmaceutical Society of Japan 694 Fig. 1. Vol. 54, No. 5 Key NOE Correlations Observed in NOESY Spectrum of 1a Fig. 2. 4a /H-2a , -6a ; H-1/H-3b , -5b , which demonstrated that the acetoxymethylene and 1-acetoxy-2-hydroxyisopropyl are both in equatorial orientation, as shown in Fig. 1. Further evidence of the relative configuration was the distinguishing features in the 1H-NMR data, especially its d H-7. In contrast to the lower field shift of d H-7 (d H 4.03) in axial-oriented CH2OAc of a cis-(4-t-butylcyclohexyl)methyl acetate analogue,9,10) 1a with a relative higher field shift of d H-7 (d H 3.86), closely compatible with that (ca. d H 3.81—3.88) of a trans-(4-t-butylcyclohexyl)methyl acetate analogue,9,10) suggested that its CH2OAc should be equatorial-oriented, the same as its hydroxyl isopropyl group. Accordingly, compound 1a was concluded to be trans-7,9-diacetoxy-p-menthan-7-ol. The molecular formula for 2, C10H12O4, was determined by 13C-NMR and HR-EI-MS data. The IR spectrum of 2 indicated the presence of a hydroxyl group (3409 cm⫺1), a conjugated ester carbonyl with strong hydrogen bonding (1633 cm⫺1), and a benzene ring (1608, 1493 cm⫺1). The 1HNMR spectrum of 2 showed signals for a phenyl proton at d H 6.19 (1H, s), aryl methyl protons at d H 2.08 (3H, s) and 2.43 (3H, s), methoxyl protons at d H 3.98 (3H, s), and two phenolic protons including one normal signal at d H 5.51 (1H, s) and another signal at 12.01 (1H, s) due to strong hydrogen bonding, suggesting the presence of a benzene ring with five substituents. The two methyls, two hydroxys, and one carbonyl methoxy functionalities were determined to be located at C-3, -4, C-2, -6, and C-7, respectively, since correlations between H3-9 and C-2, -3, -4; H3-10 and C-3, -4, -5; OH-6 and C-1, -5, -6; and H3-8 and C-7 were observed in the HMBC spectrum. Two aromatic carbon signals at d C 158.0 and 163.1 in the 13C-NMR spectrum further supported the oxygenation of the benzene ring at C-2 and -6, respectively. All the above assignments were also supported by mutual correlations of H3-9 (d H 2.08) and H3-10 (d H 2.43, 8.0% enhancement), and H-5 and H3-10 (8.3% enhancement), and OH-6 (d H 12.01, 12.8% enhancement) from the NOE difference experiments, as shown in Fig. 2. Thus, the structure of 2 was confirmed to be 2,6-dihydroxy-3,4-dimethylbenzoic acid methyl ester. To our knowledge, 2 has ever been synthesized via the methylation of 2,6-dihydroxy-3,4-dimethylbenzoic acid using diazomethane previously.11) However, 2 was found to be new from a natural source. (⫹)-Morrisonicolanin (3) was isolated as an amorphous solid. Its molecular formula C18H16O4 was deduced from 13CNMR data and an [M]⫹ ion at m/z 296.1121 in the HR-EIMS. Analysis of the IR spectrum of 3 suggested that it contained a hydroxyl group (3387 cm⫺1), a conjugated aldehyde (2760, 1660 cm⫺1), and a benzene ring (1594, 1515 cm⫺1). The 1H-NMR spectrum of 3 showed a conjugated transpropenal group [d H 6.68 (1H, dd, J⫽15.8, 7.8 Hz, H-8⬘), 7.40 (1H, d, J⫽15.8 Hz, H-7⬘), 9.62 (1H, d, J⫽7.8 Hz, H-9⬘)], NOE Correlations Observed in NOE Difference Spectra of 2 three ABX type phenyl protons [d H 6.88 (1H, d, J⫽8.5 Hz, H-5⬘), 7.41 (1H, d, J⫽8.5 Hz, H-6⬘), 7.45 (1H, s, H-2⬘)], two methine protons [d H 3.59 (1H, m, H-8), 5.60 (1H, d, J⫽6.3 Hz, H-7)], and a hydroxymethylene group [d H 3.93 (2H, m, H-9)], similar to those of the A and B rings (phenyldihydrobenzofuran) of the neolignan skeleton with two branches,12) and a hydroxymethylene and a trans propenal, attached at C-8 (d C 52.3) and C-1⬘; (d C 126.7), respectively. The remaining signals, four A2X2 type aromatic protons at d H 6.80 (2H, d, J⫽8.4 Hz, H-3, -5) and 7.21 (2H, d, J⫽8.4 Hz, H-2, -6), indicated a 4-substituted phenol moiety for the C ring. The HMBC spectrum of 3 showed phenyl proton signals H-2, -6 (d H 7.21) coupled to C-7 (d C 87.8), which was also coupled to the H2-9 signal (d H 3.93), the H-8 signal (d H 3.59) coupled to C-2⬘ (d C 130.7), the phenyl proton signal H-6⬘ (d H 7.41) coupled to C-7⬘ (d C 153.0), and the H-7⬘ signal (d H 7.40) coupled to the aldehyde carbonyl signal C-9⬘ (d C 193.3). All these correlations also suggested the main part of the structure of 3 is a neolignan aldehyde, as described above. The relative configuration of C-7 and C-8 was determined to be an E form, as evidenced from the observation of cross peaks between H-7 and H2-9, and a lack of correlation between H-7 and H-8 in the NOESY experiment.13) The positive [a ]24 D value [⫹6.7° (c⫽1.4, CHCl3)] and chemical shifts of H-7 (d H 5.60) and H-8 (d H 3.59) in 3 suggested a 7S,8R-configuration, as in the case of (E)-3-[(2S,3R)-2,3-dihydro-2-(4⬘-hydroxy-3⬘-methoxyphenyl)-3-hydroxymethyl7-methoxy-1-benzo[b]furan-5-yl]-2-propenal reported previously.14) Further analysis of all the spectral data allowed the assignment of 3 as (E)-3-[(2S,3R)-2,3-dihydro-2-(4⬘-hydroxyphenyl)-3-hydroxymethyl-1-benzo[b]furan-5-yl]-2propenal, and named as (⫹)-morrisonicolanin. Experimental General Experimental Procedures Melting points were collected using a Yanagimoto micromelting point apparatus. Optical Rotations were measured using a JASCO DIP-180 digital polarimeter at room temperature. IR spectra were recorded on a Perkin-Elmer 781 spectrophotometer. 1H- and 13 C-NMR spectra were recorded on a Bruker AM-300 instrument using tetramethylsilane (TMS) as an internal standard. Chemical shifts are given in d values (ppm), and coupling constants (J) are given in hertz (Hz). Electronimpact mass spectra (EI-MS) were obtained on a Finnigan TSQ-U6C, and JEOL SX-102A mass spectrometer, respectively. Extracts were chromatographed on silica gel (Merck 3374, 70—230 mesh), and further purified with a semipreparative normal phase HPLC column (250⫻10 mm, 7 m m, Li Chrosorb Si 60). Plant Material The heartwood of P. morrisonicola HAYATA was collected from MountTaichung, Taiwan, and was identified by Prof. Shao-Shun Ying, Department of Forest, National Taiwan University. A voucher specimen (No. 226237) has been deposited at the Herbarium of the Department of Botany, National Taiwan University, Taipei, Taiwan. Extraction and Isolation The air dried heartwood of P. morrisonicola (7.5 kg) was extracted with 90l Me2CO three times (7 d each) at room temperature. The combined extracts were evaporated under a vacuum to give a black residue, which was suspended in 8 l water and then partitioned with May 2006 EtOAc (2 l⫻3). The ethyl acetate layer (85.5 g) was then chromatographed by a Si-column using mixtures of n-hexane and EtOAc as eluents. HPLC of low polar fractions on a normal-phase column with 30% and 60% EtOAc in n-hexane as eluents afforded 2 (10.5 mg) and 3 (9.0 mg), respectively. Relative high polar fractions were dissolved in pyridine and Ac2O, and the mixture was left overnight at room temperature. Then, the reaction mixture was added dropwise to ice water under stirring, and the resultant suspension was extracted with EtOAc. After the usual work-up, the extract was further purified by normal-phase HPLC with 30% EtOAc in n-hexane to yield the pure diacetate derivative 1a (5.2 mg). trans-7,9-Diacetoxy-p-menthan-7-ol (1a): Colorless oil; [a ]24 D ⫹3.2° (c⫽0.19, CHCl3); IR n max (dry film): 3429, 1719, 1381, 1248, 1036 cm⫺1; 1 H-NMR (CDCl3) d : 0.95 (2H, m, H-2a , -6a ), 1.09 (3H, s, H-10), 1.15 (1H, m, H-3b ), 1.24 (1H, m, H-5b ), 1.39 (1H, m, H-4a ), 1.55 (1H, m, H-1b ), 1.85 (2H, m, H-2b , -6b ), 1.87 (1H, m, H-5a ), 1.92 (1H, m, H-3a ), 2.02, 2.09 (3H, s, OCOCH3⫻2), 3.86 (2H, d, J⫽6.4 Hz, H-7), 3.96 (1H, d, J⫽11.2 Hz, H-9), 4.03 (1H, d, J⫽11.2 Hz, H-9); 13C-NMR (CDCl3) d : 20.8 (C-10), 20.8 (OCOCH3⫻2), 25.6 (C-3), 26.6 (C-5), 29.4 (C-6), 29.5 (C-2), 36.9 (C-1), 44.9 (C-4), 69.3 (C-7), 70.0 (C-9), 73.3 (C-8), 171.2, 171.3 (OCOCH3⫻2); EI-MS (70 eV) (rel. int. %) m/z 272 (M⫹, 18), 229 (35), 165 (32), 131 (42), 119 (63), 105 (100); HR-EI-MS m/z 272.1620 (M⫹, Calcd for C14H24O5 272.1624). 2,6-Dihydroxy-3,4-dimethylbenzoic acid methyl ester (2): Slight yellow solid; mp 107—109 °C; UV l max (MeOH) nm (log e ): 258 (3.8), 321 (3.2); IR n max (KBr): 3409, 1633, 1608, 1493 cm⫺1; 1H-NMR (CDCl3) d : 2.08 (3H, s, H3-9), 2.43 (3H, s, H3-10), 3.98 (3H, s, H3-8), 5.51 (1H, s, OH-2), 6.19 (1H, s, H-5), 12.01 (1H, s, OH-6); 13C-NMR (CDCl3) d : 108.5 (C-1), 158.0 (C-2), 105.2 (C-3), 140.1 (C-4), 110.5 (C-5), 163.1 (C-6), 172.5 (C-7), 51.7 (C-8), 7.6 (C-9), 24.0 (C-10); EI-MS m/z (rel. int. %) 196 (M⫹, 58), 164 (100), 136 (95), 84 (42), 79 (38), 55 (25), 51 (30), 145 (42); HR-EI-MS m/z 196.0751 (M⫹, Calcd for C10H12O4 196.0736). (⫹)-Morrisonicolanin (3): Amorphous solid; [a ]24 ⫹6.7° (c⫽1.4, D CHCl3); UV l max (MeOH) nm (log e ): 230 (4.0), 330 (3.6); IR n max (dry film): 3387, 2760, 1660, 1594, 1515 cm⫺1; 1H-NMR (CDCl3) d : 3.59 (1H, m, H-8), 3.93 (2H, m, H-9), 5.60 (1H, d, J⫽6.3 Hz, H-7), 6.68 (1H, dd, J⫽15.8, 7.8 Hz, H-8⬘), 6.80 (2H, d, J⫽8.4 Hz, H-3, -5), 6.88 (1H, d, J⫽8.5 Hz, H-5⬘), 7.21 (2H, d, J⫽8.4 Hz, H-2, -6), 7.40 (1H, d, J⫽15.8 Hz, 695 H-7⬘), 7.41 (1H, d, J⫽8.5 Hz, H-6⬘), 7.45 (1H, s, H-2⬘) 9.62 (1H, d, J⫽7.8 Hz, H-9⬘); 13C-NMR (CDCl3) d : 52.3 (C-8), 63.5 (C-9), 87.8 (C-7), 109.6 (C-5⬘), 115.1 (C-3, -5), 124.7 (C-6⬘), 125.5 (C-8⬘), 126.7 (C-1⬘), 127.0 (C-2, -6), 129.0 (C-3⬘), 130.7 (C-2⬘), 131.7 (C-1), 153.0 (C-7⬘), 156.6 (C-4), 162.6 (C-4⬘), 193.3 (C-9⬘); EI-MS m/z (rel. int. %) 296 (M⫹, 1), 165 (25), 149 (60), 105 (53), 95 (70), 81 (68), 69 (80), 55 (100), 46 (30); HR-EI-MS m/z 296.1121 (M⫹, Calcd for C18H16O4 296.1094). Acknowledgments This study was supported by grants to Y.-H. 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