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Three new compounds from the stem bark of Juglans mandshurica ab

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Hua Lin , Yu-Wei Zhang , Ying Hua , Yong-Li Bao , Yin Wu , Lued

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Guo Sun , Chun-Lei Yu , Yan-Xin Huang , En-Bo Wang , Hong-Yu f

Jiang & Yu-Xin Li

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National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, ChangChun 130024, China b

Key Laboratory of Polyoxometalate Science of Ministry of Education, Department of Chemistry, Northeast Normal University, Changchun 130024, China c

Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, ChangChun 130112, China d

Institute of Genetics and Cytology, Northeast Normal University, ChangChun 130024, China e

Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, ChangChun 130024, China f

Department of General Internal Medicine, the First Hospital of Jilin University, Changchun 130021, China Published online: 21 Jul 2014.

To cite this article: Hua Lin, Yu-Wei Zhang, Ying Hua, Yong-Li Bao, Yin Wu, Lu-Guo Sun, Chun-Lei Yu, Yan-Xin Huang, En-Bo Wang, Hong-Yu Jiang & Yu-Xin Li (2014) Three new compounds from the stem bark of Juglans mandshurica, Journal of Asian Natural Products Research, 16:8, 819-824, DOI: 10.1080/10286020.2014.923406 To link to this article: http://dx.doi.org/10.1080/10286020.2014.923406

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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 8, 819–824, http://dx.doi.org/10.1080/10286020.2014.923406

Three new compounds from the stem bark of Juglans mandshurica

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Hua Lina,b1, Yu-Wei Zhangc1, Ying Huaa,d, Yong-Li Baoa,e, Yin Wua,d, Lu-Guo Sund,e, Chun-Lei Yud,e, Yan-Xin Huanga,d, En-Bo Wangb*, Hong-Yu Jiangf* and Yu-Xin Lia,d* a National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, ChangChun 130024, China; bKey Laboratory of Polyoxometalate Science of Ministry of Education, Department of Chemistry, Northeast Normal University, Changchun 130024, China; c Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, ChangChun 130112, China; dInstitute of Genetics and Cytology, Northeast Normal University, ChangChun 130024, China; eResearch Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, ChangChun 130024, China; f Department of General Internal Medicine, the First Hospital of Jilin University, Changchun 130021, China

(Received 24 December 2013; final version received 8 May 2014) Three new compounds, 3,6-dihydroxy-4,5-dimethoxy-1,8-naphalic anhydride (1), 3,4,5,6-tetrahydroxy-1,8-naphalic anhydride (2), and methyl (7E,9E)-6,11-dioxononadeca-7,9-dienoate (3), were isolated from the stem bark of Juglans mandshurica. Their structures were elucidated on the basis of spectroscopic evidence, including 1D and 2D NMR, HR-TOF-MS, and by comparison with the literature data. Keywords: Juglandaceae; Juglans mandshurica; naphalic anhydride; fatty acid ester

1.

Introduction

Juglans mandshurica Maxim. (Juglandaceae) is widely distributed in Korea and the northeast of China. Its roots, stembark, and fruits have long been used as folk medicine for the treatment of cancer in China and Korea [1]. Previously, we reported one new anthracene derivative and two new anthraquinones from the stem bark of J. mandshurica [2]. Further phytochemical analysis of this plant has resulted in the isolation of two new naphalic anhydrides, named 3,6dihydroxy-4,5-dimethoxy-1,8-naphalic anhydride (1) and 3,4,5,6-tetrahydroxy1,8-naphalic anhydride (2), along with one new fatty acid ester, methyl (7E,9E)-6,11dioxononadeca-7,9-dienoate (3) (Figure 1). Herein, the isolation and structure elucidation of the three isolated compounds are described.

2.

Results and discussion

Compound 1 was isolated as a white amorphous powder. Its HR-TOF-MS gave an [M þ H] þ ion at m/z 291.0497, consistent with a molecular formula of C14H10O7, corresponding to 10 degrees of unsaturation. The IR spectrum showed the presence of hydroxy (3274 cm – 1) and carbonyl (1725 cm – 1) groups, which was further confirmed by 1D NMR (dH 10.72 and dC 158.4). The 1H NMR spectrum also exhibited two singlet resonances (dH 7.51 and dH 4.04) assignable to an isolated aromatic proton and a methoxy group, respectively. Compared with the 13C NMR spectrum (Table 1), total eight carbon resonances were observed, except for the carbonyl (dC 158.4) and methoxyl (dC 60.9) groups, the remaining carbon resonances (dC 111.4, 111.6, 112.1, 140.2, 141.2, and 152.2) could be assigned to a

*Corresponding authors. Email: [email protected], [email protected], [email protected] jlu.edu.cn q 2014 Taylor & Francis

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O

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O 8a

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OH

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1 R= –OCH3 2 R= –OH O

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OCH3

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Figure 1. Chemical structures of compounds 1 – 3. Table 1.

1

H (400 MHz) and 13C (100 MHz) spectral data of compounds 1 and 2 (DMSO-d6). 1

Position 1 2 3 4 4a 5 6 7 8 8a C1-CO C8-CO C3-OH C6-OH C4-OCH3 C5-OCH3 a

dC a

112.1 111.4 141.2 140.2 111.6a 140.2 141.2 111.4 112.1a 152.2 158.4 158.4 – – 60.9 60.9

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dH

Position

dC

dH

– 7.51 (1H, s) – – – – – 7.51 (1H, s) – – – – 10.72 (1H, br.s) 10.72 (1H, br.s) 4.04 (3H, s) 4.04 (3H, s)

1 2 3 4 4a 5 6 7 8 8a C1-CO C8-CO C3-OH C6-OH C4-OH C5-OH

112.3 110.2 136.4 139.6 107.6 139.6 136.4 110.2 112.3 148.1 159.1 159.1 – – – –

– 7.46 (1H, s) – – – – – 7.46 (1H, s) – – – – 10.68 (H, br.s) 10.68 (H, br.s) 10.68 (H, br.s) 10.68 (H, br.s)

Data may be exchanged.

five-substituted benzene ring. These foregoing functionalities accounted for 5 out of the total 10 degrees of unsaturation, which combined with the analysis of 1D NMR and HR-TOF-MS, confidently suggesting the presence of an axisymmetric naphthalinic ring with double substituent groups and the formation of an anhydride moiety at a-positions of the naphthalinic ring. The connectivity of hydroxy and methoxy groups in compound 1 was elucidated on the basis of HMBC (Figure 2) and NOESY techniques. The HMBC correlations between H-2/7 (dH

7.51) and C-4/5 (dC 140.2), C-8a (dC 152.2), C-11/9 (dC 158.4) determined the attachment of the isolated aromatic protons to C-2/7, adjacent to the anhydride moiety on the naphthalinic ring. The HMBC spectrum also showed crosspeaks between the 4/5-OCH3 (dH 4.04) and C-4/5 (dC 140.2) indicating the location of the methoxy groups at C-4/5, and unambiguously confirming the location of the hydroxyl groups at C-3/6, which was further confirmed by the absence of NOESY correlation between H-2/7 and 4/5-OCH3. Consequently, the

Journal of Asian Natural Products Research O

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H

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OH OH

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2 O

O O O 3

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Figure 2. Key HMBC correlations of compounds 1 –3.

structure of compound 1 was characterized as 3,6-dihydroxy-4,5-dimethoxy-1,8naphalic anhydride. Compound 2 was also obtained as a white amorphous powder. A molecular formula of C12H6O7, which gave 10 degrees of unsaturation, was determined on the basis of the positive HR-TOF-MS ion at m/z 263.0188 [M þ H]þ. The presence of hydroxy and carbonyl groups was revealed by the bands at 3158 and 1724 cm – 1 in the IR spectrum and was confirmed by signals at dH 10.68 and dC 159.1 in the 1D NMR spectra. Comparison of the 1H and 13C NMR data (Table 1) of 2 with those of 1 suggested that their structures were closely related, except that the methoxy groups at C-4/5 were absent and replaced by additional hydroxy groups in 2. In addition, the HMBC spectrum (Figure 2) showed cross-peaks between H2/7 (dH 7.46) and C-11/9 (dC 159.1), C-4/5 Table 2.

1

(139.5), C-8a (148.1), indicating the isolated aromatic protons at C-2/7. Accordingly, the structure of compound 2 was determined to be 3,4,5,6-tetrahydroxy-1,8-naphalic anhydride. Compound 3 was obtained as a white amorphous powder, and its molecular formula was analyzed as C19H30O4 from its quasi-molecular ion at m/z 345.2035 [M þ Na]þ in the HR-TOF-MS spectrum. The IR spectrum of 3 indicated the presence of ester carbonyl (1736 cm – 1). The 13C NMR spectrum revealed 19 carbon signals that were sorted by a DEPT experiment as a single methyl group at d 13.8, a methoxyl group at d 51.4, 10 methylenes (Table 2), and 2 ketone carbonyl groups at d 200.1 and 199.9, a ester carbonyl group at d 174.2, as well as two pairs of olefinic carbons at d 138.8, 138.8, 136.0, and 135.9. The 1H NMR spectrum of 3 displayed a terminal

H (400 MHz) and 13C (100 MHz) spectral data of compound 3 (CDCl3).

Position

d (C)

1 2 3 4 5 6 7 8 9 10

174.2 34.0 28.9 29.0 41.0 199.9 135.9 138.8 138.8 136.0

d (H) 2.30 (2H, t, J ¼ 7.6) 1.50– 1.70 (2H, m) 1.50– 1.70 (2H, m) 2.60 (2H, t, J ¼ 7.6) 6.49– 6.50 (1H, 7.15– 7.16 (1H, 7.18– 7.19 (1H, 6.46– 6.47 (1H,

m) m) m) m)

Position

d (C)

11 12 13 14 15 16 17 18 ZOCH3

200.1 42.1 29.0 24.8 23.9 26.1 22.3 13.8 51.4

d (H) 2.59 (2H, t, J ¼ 7.6) 1.50 –1.70 (2H, m) 1.30 –1.40 (8H, m)

0.92 (3H, t, J ¼ 7.2) 3.66 (3H, s)

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methyl group at d 0.92 (3H, t, J ¼ 7.2 Hz, H-18), a methoxyl group at d 3.66 (3H, s), as well as three methylene protons at d 2.60 (2H, t, J ¼ 7.6 Hz, H-5), 2.59 (2H, t, J ¼ 7.6 Hz, H-12), and 2.30 (2H, t, J ¼ 7.6 Hz, H-2), indicating that they were adjacent to carbonyl groups. In addition, four olefinic protons were observed at dH 7.18 –7.19 (1H, m), 7.15– 7.16 (1H, m), 6.49 – 6.50 (1H, m), and 6.46 –6.47 (1H, m), suggesting the presence of two conjugated olefine ketone systems. Because these multiplets were not first order and did not display substantive coupling with the methylene protons, they may be interpreted as the AA0 B0 B system of a conjugated diene– dione. On the basis of the above data, compound 3 was assessed as a long-chain fatty acid ester with two a,b-unsaturated ketone carbonyl groups, and the 1H and 13 C NMR data of 3 were assigned from the HSQC, HMBC, and DEPT spectra (Table 2). The location of these functionalities and the stereochemistry of the double bonds remained to be established. This partial structure assignment for 3 was supported by the UV and IR spectra. For example, the UV spectrum of 3 displayed an absorption maximum at 277 nm, corresponding well with that of octa-3,5-diene-2,7-dione. While the Z,Zand E,Z-isomers absorb at longer wavelengths, the more stable E,E-form displays a lmax at 276 nm [3,4]. In the IR spectrum, two bands attributable to an a,b-unsaturated ketone appeared at nmax 1682 and 1666 cm – 1. The observation of two bands is due to the conformational flexibility of the E-enone moiety [5]. Strong absorption bands at 998 and 990 cm – 1 indicated the E, E-relationship of the double bonds. This was further confirmed by the HMBC experiment which showed the crosspeaks due to 2J and 3J correlations, namely correlations between H-5 at d 2.60 and C-6 at d 200.1, C-7 at d 135.9, H-12 at d 2.59 and C-11 at d 199.9, C-10 at d 136.0, and correlations between double bonds protons

and carbons. In addition, in the HMBC spectrum, correlations between – OCH3 at d 3.66 (3H, s), H-2 at d 2.30 (2H, t, J ¼ 7.6), and C-1 at d 174.2 confirmed the occurrence of ester fragment (Figure 2). Furthermore, The EI-MS of 3 exhibited a molecular ion peak at m/z 322 [M]þ, as well as a hydrocarbonyl ion peak at m/z 99 (C7H15) along with the continual lost fragment peak at m/z 85, 71, 57, 43, which indicated a heptane chain in 3. Thus, the structure of compound 3 was elucidated as methyl (7E,9E)-6,11-dioxononadeca-7,9dienoate. Notably, some polyunsaturated fatty acids, such as (10E,12E,14E)-9,16-dioxooctadeca-10,12,14-trienoic acid [6], (8E,10E)-7,12-dioxo-8,10-octadecadienoic acid [7], and (10E,12E)-9,14-dioxo10,12-octadecadienoic acid [7], whose structures are similar to that of 3, have been obtained from natural resources. 3. Experimental 3.1 General experimental procedures Melting points were determined on Yanaco MP-S3 melting point apparatus and are uncorrected (Yanaco New Science, Inc., Kyoto, Japan). UV spectra were recorded on a Shimadzu UV-2201 spectrometer (Shimadzu Corporation, Kyoto-shi, Japan). IR spectra were recorded on a Bruker Vertex-70 (Bruker Optik BmbH, Ettlingen, Germany) with KBr pellets. NMR spectra were recorded by a Brucker AV 400 NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) with TMS as the internal standard. Chemical shifts were expressed in ppm. HR-TOF-MS were measured on a micrOTOFQ Bruker mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The chromatographic silica gel (300 – 400 mesh) was produced by Qingdao Ocean Chemical Factory (Qingdao, China). Macroporous resin HPD100 was produced by Hebei Cangzhou BaoEn Chemical Factory (Cangzhou, China).

Journal of Asian Natural Products Research 3.2 Plant material The stem bark of Juglans mandshurica was collected from Ji’an County, Jilin Province of China, and was identified by Prof. Shaobo Fan (Jilin Agricultural University). A voucher specimen (No. 2012010) was deposited in the National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University.

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3.3

Extraction and isolation

The air-dried and powdered stem bark of J. mandshurica (6 kg) was extracted with 95% EtOH (3 £ 40 l) at room temperature. The resulting EtOH extract was concentrated in vacuo, suspended in H2O, and partitioned successively with petroleum ether, CHCl3, EtOAc, and n-BuOH. A part of the CHCl3 extract (95 g) was fractionated into 22 fractions by silica gel column chromatography [petroleum ether –EtOAc (100:0 to 0:100), followed by EtOAc – MeOH (2:1, 1:1, 1:2, and 0:2)]. Fraction 3 [petroleum ether – EtOAc (95:5)] was recrystallized using petroleum ether – EtOAc to yield compound 3 (12 mg). A part of the EtOAc extract (120 g) was loaded on a reduced pressure silica gel column and eluted with a CHCl3 – MeOH gradient solvent system to provide 10 main fractions (F1 – F10). F2 [CHCl3 – MeOH (95:5)] was rechromatographed on Sephadex LH-20 column eluting with MeOH to obtain 1 (15 mg). F6 [CHCl3 – MeOH (85:15)] was rechromatographed over Sephadex LH-20 column using MeOH and purified by polyamide column with MeOH:H2O (2:1) to furnish 2 (62 mg). 3.3.1 3,6-Dihydroxy-4,5-dimethoxy-1,8naphalic anhydride (1) White amorphous powder (methanol); m. p. 124 –1268C; UV (MeOH) lmax (log 1): 383 (3.36), 239 (3.14) nm; IR (KBr) nmax: 3274, 3070, 3016, 2950, 2856, 1725, 1611, 1578, 1489, 1364, 1213, 1172, 915,

823

760 cm – 1; for 1H and 13C NMR spectral data, see Table 1; HR-TOF-MS: m/z 291.0497 [M þ H]þ (calcd for C14H11O7, 291.0499).

3.3.2 3,4,5,6-Tetrahydroxy-1,8-naphalic anhydride (2) White amorphous powder (methanol); m. p. 140– 1428C; UV (MeOH) lmax (log 1): 375 (3.35), 239 (3.20) nm; IR (KBr) nmax: 3158, 1724, 1669, 1622, 1585, 1490, 1375, 1348, 1185, 1106, 919, 751, 685 cm – 1; for 1 H and 13C NMR spectral data, see Table 1; HR-TOF-MS: m/z 263.0188 [M þ H]þ (calcd for C12H7O7, 263.0186).

3.3.3 Methyl (7E,9E)-6,11dioxononadeca-7,9-dienoate (3) White amorphous powder (petroleum ether – EtOAc); m.p. 84 – 868C; UV (MeOH) lmax (log 1): 245 (2.78), 305 (2.65), 220 (2.38) nm; IR (KBr) nmax: 3048, 2953, 2935, 2869, 1736, 1682, 1584, 1224, 1167, 1113, 1030, 884, 725 cm – 1; for 1H and 13C NMR spectral data, see Table 2; EI-MS: m/z 322 (Mþ), 99, 85, 71, 57, 43. HR-TOF-MS: m/z 345.2035 [M þ Na] þ (calcd for C19H 30O 4Na, 345.2042).

Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Nos. 31170324 and 31070318), the National Key New Drug Creation and Manufacturing Program (No. 2011ZX09401-305-03), the Fundamental Research Funds for the Central Universities, the Natural Science Foundation of Jilin Province (No. 20101503), the Grant of Jilin Province Science & Technology Committee (Nos 20100911, 20102203, 20110938, 20110711 and 20130102100JC), the Scientific and Technical Project of Administration of Traditional Chinese Medicine of Jilin Province (No 2011-zol17, YJS-0216 and YJS-0217), as well as the Grant of Jilin Province Human Resources and Social Security Department (No. 2011250).

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Note 1.

These authors contributed equally to this work.

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Three new compounds from the stem bark of Juglans mandshurica.

Three new compounds, 3,6-dihydroxy-4,5-dimethoxy-1,8-naphalic anhydride (1), 3,4,5,6-tetrahydroxy-1,8-naphalic anhydride (2), and methyl (7E,9E)-6,11-...
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