HETEROCYCLES

Short Paper | Regular issue | Vol. 85, No. 11, 2012, pp. 2775-2780
Received, 20th August, 2012, Accepted, 12th September, 2012, Published online, 20th September, 2012.
DOI: 10.3987/COM-12-12571

■ Three New Iridoid Compounds from Swertia cincta Burkill

Jin Yang, Feiyan Huang, Yongfu Zhou, Wei Liu, Ping Yi, and Ganpeng Li*

Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, School of Chemistry and Biotechnology, Yunnan University of Nationalities, Kunming, Jingming South Road, Chenggong New District, Kunming, Yunnan 650500, China

Abstract

Three new iridoid compounds, 3,4,5,6-tetrahydro-8-hydroxy- 6-(hydroxymethyl)pyrano[3,4-c]pyran-1(8H)-one (1), 3,4,5,6-tetrahydro-8-hydroxy-6-methylpyrano[3,4-c]pyran-1(8H)-one (2), 5,6-dihydro-4-(1,3-dihydroxybutan-2-yl)-3-(hydroxymethyl)pyran-2-one (3), named swercinctolides A, B and C, were isolated from the 80% methanol extract of the whole plant of Swertia cincta Burkill, which is a traditional medicine in China. Their structures were defined on the basis of spectral evidences. Compounds 1, 2 and 3 showed TMV (Tobacco Mosaic Virus) inhibitory activity.

Swertia cincta Burkill, a plant of the genus Swertia (Gentianaceae), is mainly distributed in Sichuan, Guizhou and Yunnan of China.1,2 It has been used as traditional medicinal plant to remedy jaundice hepatitis.3 Previous research4-6 suggests that iridoid, flavone, triterpenoid, and xanthone are main constituents of this plant. Our phytochemical investigation on the constituents of this plant has resulted in the isolation of three new iridoid compounds, named swercinctolides A, B and C. Their structures were elucidated by spectroscopic methods and shown in Figure 1. Compounds 1, 2 and 3 showed TMV (Tobacco Mosaic Virus) inhibitory activity compared with the positive control spot.
Compound 1 was obtained as colorless gum. Its molecular formula was deduced to be C9H12O5 by 13C NMR spectra and HRESI-MS, with quasi-molecular ion peaks at m/z 199.0606 (C9H11O5, [M-H]-). The IR spectrum showed the presence of hydroxyl group (3434 cm-1) and C = O bond (1638 cm-1) of α, β-unsaturated ester.

13C and DEPT NMR experiments differentiated the skeleton carbons of 1 as four methylenes (δC: 66.9, 65.2, 32.1, 29.5), two methines (δC: 88.0, 67.2), and three quaternary (δC: 165.2, 156.0, 125.3) carbons. The three quaternary carbons showed the presence of α, β-unsaturated ester. The HMQC spectra of 1 showed the position assignment of H-atoms, given in Table 1. In the HMBC spectra of 1, H-7 (δH 4.41) exhibited cross-peaks with C-5, C-6, C-10 at δC 156.0, 29.5 and 165.2, and H-6 (δH 2.60 and 2.40) showed correlations with C-4, C-5, C-7, and C-9 at δC 125.3, 156.0, 66.9, and 32.1, respectively. These suggested the presence of 5,6-dihydropyran-2-one. Also, H-3 (δH 5.64) exhibited cross-peaks with C-1, C-4, C-5, and C-10 at δC 67.2, 125.3, 156.0, and 165.2, and H-9 (δH 2.39 and 2.21) showed correlations with C-1, C-4, C-5, C-6 and C-8 at δC 67.2, 125.3, 156.0, 29.5 and 65.2, respectively. And H-8 (δH 3.64) did correlations with C-1 and C-9 at δC 67.2 and 32.1. The above evidences educed the molecule skeleton of 1. Further, H-H COSY experiment showed correlations between H-6 and H-7, H-1 and H-8, H-1 and H-9. Meanwhile, ROESY experiment showed same correlations. From 1H NMR spectrum of 1, the single peak at δH 5.64 (C-3, δC 88.0, tertiary carbon) suggested a hydroxyl was attached to C-3. The peak at δH 3.64 (C-8, δC 65.2, secondary carbon) showed a hydroxyl was attached to C-8. Biogenetically, the H-1 of iridoid skeleton is α-orientation. No ROESY cross-peak between H-1 and H-3 revealed that the relative configuration of H-3 was β-orientation. Thus, the structure of 1 was determined as 3,4,5,6-tetrahydro-8-hydroxy-6-(hydroxymethyl)pyrano[3,4-c]pyran-1(8H)-one, named swercinctolide A.
Compound 2 was obtained as colorless gum. Its molecular formula was deduced to be C9H12O4 by 13C NMR spectra and HRESI-MS, with quasi-molecular ion peaks at m/z 185.0815 ([M＋H]＋) and 207.0633 ([M＋Na]＋). The IR and UV spectra were similar to those of 1.
The 1H and 13C NMR spectra of 2, given in Table 1, resembled those of 1. The 13C and DEPT NMR spectroscopic data showed the presence of one methyl, three methylenes, two methines, and three quaternary carbons. The linkage of C-8 (CH3) to C-1 was confirmed by correlations between H-1 and H-8 in H-H COSY experiment and H-8 (δH 1.22) with C-1 and C-9 at δC 62.6 and 37.7 in HMBC spectra. The ROESY experiment of 2 suggested the stereo configurations of H-1 and H-3 of 2 resembled those of 1. Thus, the structure of 2 was determined as 3,4,5,6-tetrahydro-8-hydroxy-6-methylpyrano[3,4-c]pyran- 1(8H)-one, named swercinctolide B.
Compound 3 was obtained as colorless gum. Its molecular formula was deduced to be C10H16O5 by 13C NMR spectra and HRESI-MS, with quasi-molecular ion peaks at m/z 215.0917 ([M-H]-). The IR spectrum showed the presence of hydroxyl group (3433 cm-1) and C = O bond (1652 cm-1) of α, β-unsaturated ester.
13C and DEPT NMR experiments differentiated the skeleton carbons of 3 as one methyl (δC: 19.3), four methylenes (δC: 67.5, 63.5, 60.2, 27.0), two methines (δC: 71.7, 48.4), and three quaternary (δC: 165.9, 153.5, 125.4) carbons. The three quaternary carbons showed the presence of α, β-unsaturated ester. The HMQC spectrum of 3 showed the position assignment of H-atoms, given in Table 1. In the HMBC spectrum of 3, H-7 (δH 4.45) exhibited cross-peaks with C-5, C-6, C-11 at δC 153.5, 27.0 and 165.9, and H-6 (δH 2.75 and 2.35) showed correlations with C-4, C-5, C-7, and C-9 at δC 125.4, 153.5, 67.5, and 48.4, respectively. These suggested the presence of 5, 6-dihydropyran-2-one. Also, H-10 (δH 1.32) exhibited cross-peaks with C-8 and C-9 at δC 71.7 and 48.4, and H-9 (δH 2.21) showed correlations with C-1, C-4, C-5, C-6, C-8 and C-10 at δC 63.5, 125.4, 153.5, 27.0, 71.7 and 19.3, respectively. And H-3 (δH 3.84, 3.74) did correlations with C-4, C-5 and C-11 at δC 125.4, 153.5 and 165.9. The above information educed the molecule skeleton of 3. Further, H-H COSY experiment showed correlations between H-6 and H-7, H-8 and H-10, H-8 and H-9, H-1 and H-9. From 1H NMR spectrum of 3, the peak at δH 3.83 (C-8, δC 71.7, tertiary carbon) showed a hydroxyl was attached to C-8. The peak at δH 3.84, 3.74 (C-3, δC 60.2, secondary carbon) showed a hydroxyl was attached to C-3. The peak at δH 4.31, 4.21 (C-1, δC 63.5, secondary carbon) showed a hydroxyl was attached to C-1. Thus, the structure of 3 was determined as 5,6-dihydro-4-(1,3-dihydroxybutan-2-yl)-3-(hydroxymethyl)pyran-2-one, named swercinctolide C.
In the leaves of tobacco, the TMV inhibitory activity of 1, 2 and 3 was performed. The solution concentrations of three compounds were all 10 μg/mL. The right part of a leaf was sprayed with 100 μL solution of 1 and the left part was done with the same concentration and volume solution of DMSO. Two hours later, the whole leaf was inoculated with 200 μL solution of TMV (10 μg/mL). After 5 days, the spot numbers of right and left part on leaf were counted up and then the spot inhibitory rate of 1 was calculated according to the spot number [the negative control DMSO gave an inhibitory rate of 0.00%]. In the same way, the spot inhibitory rates of 2 and 3 were calculated. The spot inhibitory rates of 1, 2 and 3 were 65.41%, 46.01% and 42.59%, respectively.

EXPERIMENTAL
General experimental procedures. Optical rotations and UV data were measured on a JASCO-20 polarimeter and UV-2401PC spectrometer. MS were measured on a VG Auto Spec-3000 spectrometer. HR-ESI-MS data were measured using a JMS-T100CS AccuTOF LC/MS spectrometer. 1H-NMR and 13C-NMR spectra were recorded on Bruker AM-400 instruments with TMS as internal standard. IR spectra were measured on a Bruker Tensor 27 spectrometer with KBr pellets. Column chromatography was performed with ZORBAX Eclips XDB-C18 (10 μm, Agilent, America) and Lichroprep RP-18 gel (40-63 μm; Merk, Darmstadt, Germany), Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd. Uppsala, Sweden), Macroporous resin and MCI gel (75-150 μm; Mitsubishi Chemical Corporation, Japan).
Plant material. Whole herb of Swertia cincta Burkill was collected from Xinping County, Yunnan Province, People’s Republic of China, in October 2009. The plant was identified by Dr. Lipang Yang (Yunnan University of Traditional Chinese Medicine). A voucher specimen (NO. 0601007) is deposited at the Herbarium of Key Laboratory of Ethnic Medicine Resource Chemistry, Yunnan University of Nationalities.
Extraction and Isolation. The dried whole plants of Swertia cincta Burkill (4.5 kg) were extracted four times with MeOH (80%) under ultrasound calefaction (4 h each time). After filtering and evaporating the solvents in vacuo a crude extract (880 g) was obtained. The extract was suspended in water and extracted six times with CH2Cl2. The solvents were evaporated to afford CH2Cl2 extract (368 g) and a residue (510 g) from water. After eliminating coloring matter in the residue with MCI gel column chromatography (CC), the left residue (485 g) was performed on macroporous resin column, and then eluted with water, water-MeOH (3:7) and pure MeOH. The residue of water elution was 156 g, after evaporating water. The residue was chromatographed on a reverse silica gel column (Agilent Eclips XDB-C18, 21.2×250 mm) and eluted with MeOH-water (3:7) to afford four fractions. Fraction 3 was purified with Sephadex LH-20 CC (MeOH) and Lichroprep RP-18 gel (MeCN-H2O 1:9), and 1 (6.2 mg) and 2 (7.0 mg) were obtained. Fraction 2 was chromatographed over Lichroprep RP-18 gel column and eluted with MeCN-H2O (1:9) to yield 3 (6.2 mg).
Compound 1: C9H12O5, colorless gum; [α]19.1D +65.1 (c 0.025, MeOH); UV (MeOH) λmax (log ε) 202 (3.69) nm; IR (KBr): νmax 3434, 2925, 2359, 1638, 1095, 536 cm-1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) spectral data see Table 1; negative-mode ESI-MS m/z 199 [M-H]-; HR-ESI-MS m/z 199.0606 [M-H]- (calcd for C9H11O5, 199.0603).
Compound 2: C9H12O4, colorless gum; [α]19.1D +16.0 (c 0.020, MeOH); UV (MeOH) λmax (log ε) 202 (3.69) nm; IR (KBr): νmax 3429, 2924, 2364, 1628, 1394, 1085, 533 cm-1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) spectral data see Table 1; positive-mode HR-ESI-MS m/z 185.0815 [M+H]+ (calcd for C9H13O4, 185.0814), 207.0633 [M+Na]+ (calcd for C9H12O4Na, 207.0636).
Compound 3: C10H16O5, colorless gum; [α]19.1D +60.7 (c 0.017, MeOH); UV (MeOH) λmax (log ε) 202 (3.67) nm; IR (KBr): νmax 3433, 2926, 2357, 1652, 1093, 672, 538 cm-1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) spectral data see Table 1; negative-mode ESI-MS m/z 215 ([M-H]-); HR-ESI-MS m/z 215.0917 [M-H]- (calcd for C10H15O5, 215.0915).

ACKNOWLEDGEMENTS
This project was supported financially by NSFC (NO. 21262048), and the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), and the Key Project of Chinese Ministry of Education (NO. 208132).

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