HETEROCYCLES

Paper | Regular issue | Vol. 91, No. 10, 2015, pp. 1926-1936
Received, 9th August, 2015, Accepted, 14th September, 2015, Published online, 24th September, 2015.

■ Four New Compounds from Pouzolzia zeylanica (L.) Benn. var. Microphylla

Chu-Qian Zhong, Shu-Hong Tao, Zhi-Bo Yi, Li-Bing Guo,* Yu-Feng Xie, and Yan-Fen Chen

School of Traditional Chinese Medicine, Guangdong Pharmacutical University, University City, Waihuan Road 280#, Guangzhou, Guangdong, 510006, China

Abstract

Two new stilbenes, pouzolignan D (1) and K (2), and two new norlignans, pouzolignan L (3) and M (4), together with four known flavonoids, rhamnocitrin (5), rhamnetin (6), isorhamnetin (7) and quercetin(8), were isolated from the aerial parts of Pouzolzia zeylanica (L.) Benn. var. microphylla (Wedd.) W. T. Wang. Their structures were elucidated by spectroscopic methods, including UV, IR, HR-ESI-TOF-MS, 1D and 2D NMR experiments.

INTRODUCTION
Pouzolzia zeylanica (L.) Benn. var. microphylla (Wedd.) W. T. Wang (Urticaceae), one of the four medicinal plants in the genus Pouzolzia, is extensively distributed in Japan, India, Malaysia, Indonesia, Australia and South China.1, 2 It has been traditionally used for the treatment of gangrenous ulcers, sores, boils, diarrhea, syphilis, gonorrhea, etc.3-5 A series of compounds including flavonoids, lignans, norlignans and triterpenoids have been isolated from this species and the genus Pouzolzia.6-11 Previously, we reported the pharmacological activities of the extracts from Pouzolzia zeylanica var. microphylla, investigating its anti-inflammatory and analgesic effects,12 therapeutic effects on mouse subcutaneous abscess13 and skin ulcers in rats.14 As part of our continuing study of this plant, two new stilbenes, pouzolignan D (1) and K (2), and two new norlignans, pouzolignan L (3) and M (4), along with four known flavonoids (5-8) (Figure 1) were obtained. Herein, we reported the isolation, structure elucidation of the four new compounds.

RESULTS AND DISCUSSION
The aerial parts of P. zeylanica var. microphylla were extracted with petroleum ether and EtOAc, respectively. The resulting EtOAc extract was chromatographed on silica gel, RP-C18, sephadex LH-20 and preparative RP-C18 HPLC to yield compounds 1-8.
Compound 1 was obtained as brown amorphous powder. Its molecular formula was established as C26H28O8 by HR-ESI-TOF-MS ([M-H]-, m/z 467.1729) and had 13 degrees of unsaturation. The IR spectrum showed the presence of hydroxy group (3360 cm-1), aromatic ring (1608, 1520 and 1455 cm-1). The 1H NMR spectrum (Table 1) showed two 1,3,4-trisubstituted phenyl rings signals (ABX system) at δH 7.31 (1H, d, J = 2.0 Hz, H-2), 7.19 (1H, dd, J = 8.5, 2.0 Hz, H-6), 6.98 (1H, d, J = 8.5 Hz, H-5) and 6.78 (1H, d, J = 1.5 Hz, H-2), 6.72 (1H,dd, J = 8.0, 1.5 Hz, H-6), 6.61 (1H, d, J = 8.0 Hz, H-5), one 1,3,5-trisubstituted phenyl ring signal (AB2 system) at δH 6.10 (2H, d, J = 2.0 Hz, H-2, 6), 6.01 (1H, t, J = 2.0 Hz, H-4) and three methoxy groups signals at δH 3.87, 3.83, 3.68 (each 3H, s). Since three phenyl rings accounted for 12 out of 13 degrees of unsaturation, the molecule was tetracyclic. NMR and HSQC spectra showed the presence of four methines signals at δH 2.52 (1H, m, H-4) with δC 57.0 (C-4), δH 3.62 (1H, m, H-3) with δC 53.7 (C-3), δH 4.90 (1H, d, J = 8.5 Hz, H-5) with δC 81.8 (C-5), δH 5.20 (1H, d, J = 7.5 Hz, H-2) with δC 83.4 (C-2), which indicated the existence of a tetrahydrofuran ring, and another methylene signal at δH 3.80 (2H, m, H-6) with δC 61.3 (C-6) were recognized.
The NMR data suggested that compound 1 had the same fundamental skeleton as the known compound cestrumoside.15 The difference between them was 1 had three phenyl rings but cestrumoside had two, and their substituents on the phenyl rings were different. Comparison of the NMR data of 1 with those of cestrumoside showed that C-3 (δC 53.7) was shifted downfield 20.3 ppm, which suggested another phenyl ring attached on C-3 of the tetrahydrofuran ring. And it was confirmed by the HMBC correlations between H-3 and C-1and C-2, 6. The position of the hydroxy and methoxy groups in compound 1 were established by extensive analysis of the HMBC spectra (Figure 2). The relative trans configuration between the methine protons at C-3 and C-4, C-4 and C-5, and cis configurantion between the methine protons at C-2 and C-3, C-2 and C-5 were established by the 2D NOESY spectral analysis. Thus, the structure of 1, named pouzolignan D, was elucidated as 5-(3,4-dimethoxyphenyl)-2-(4-hydroxy-3- methoxyphenyl)-3-(3,5-dihydroxyphenyl)-4-(hydroxymethyl)tetrahydrofuran.
Compound 2 was obtained as a brown tabular crystal (CHCl3-MeOH 1:1) and had a molecular formula of
C30H28O9 established by HR-ESI-TOF-MS ([M+Na]+, m/z 555.1608, [M-H]-, m/z 531.1740), correspongding to 17 degrees of unsaturation. The IR spectrum showed the presence of hydroxy group (3351 cm−1), aromatic ring (1605, 1516 and 1459 cm-1). In the 1H NMR spectrum (Table 1), two sets of aromatic rings signals at δH 6.85 (2H, d, J = 1.5 Hz, H-2, 2), 6.80 (2H, dd, J = 8.5, 1.5 Hz, H-6, 6), 6.76 (2H, d, J = 8.5 Hz, H-5, 5) and 6.11 (4H, d, J = 2.0 Hz, H-2, 6, 2, 6), 6.09 (2H, t, J = 2.0 Hz, H-4, 4) were recognized, revealing a 1,3,4-trisubstituted phenyl ring (ABX system) and a 1,3,5-trisubstituted phenyl ring (AB2 system), respectively, as well as one methoxy group signal at δH 3.80 (6H, s, 3, 3-OMe). Moreover, the 13C NMR and DEPT spectra showed ten olefinic carbon signals at

δC 158.9 (C-3, 5, 3, 5), 148.3 (C-4, 4), 146.6 (C-3, 3), 141.1(C-1, 1), 133.9 (C-1, 1), 119.4 (C-6, 6), 115.5 (C-5, 5), 110.4 (C-2, 2), 107.3 (C-2, 6, 2, 6), 101.8 (C-4, 4), two tertiary methyl carbon signals at δC 88.6 (C-2, 5), 63.3 (C-3, 4) and one methoxy group signal at δC 55.8 (3, 3-OMe).
Furthermore, comparing the molecular formula C30H28O9 and 17 degrees of unsaturation of compound 2 with its fewer 1H NMR and 13C NMR signals, it indicated that this molecule had symmetrical elements and overlapping carbon signals with four phenyl rings and the integration of hydrogen in the 1H NMR spectrum should be doubled. Thus, compound 2 had four phenyl rings which accounted for 16 units of the 17 degrees of unsaturation and the molecule was pentacyclic. Moreover, the 1H NMR, 13C NMR and HSQC spectra revealed four methines signals at δH 3.44 (2H, dd, J = 6.5, 3.0 Hz, H-3, 4) with δC 63.3 (C-3, 4) and δH 5.22 (2H, dd, J = 6.5, 3.0 Hz, H-2, 5) with δC 88.6 (C-2, 5), which suggested the presence of a tetrahydrofuran ring with symmetry substituents. From the above-mentioned analysis and HMBC correlations (Figure 2), it suggested that the four phenyl groups were linked to the four carbons of tetrahydrofuran ring, respectively. It showed 2 was similar to compound 1 and had a symmetrical structure. Confirming evidence was obtained from the HMBC correlations of H-2, 5 with C-3, 4, C-2, 2, C-6, 6 and H-3, 4 with C-1, 1, C-1, 1, C-2, 6, 2, 6. The location of the methoxy group at C-3, 3 were proved by HMBC correlation between methoxy protons (δH 3.80) and C-3, 3 (δc 146.6) and NOESY interactions of methoxy protons with H-2, 2. The relative trans configuration between the methine protons at C-2, 5 and C-3, 4 were established by the weak interactions between H-2, 5 and H-3, 4 in the 1D and 2D NOESY spectra. Moreover, its basic skeleton is similar to the known (2S,3R,4R,5S)-2,3,4,5-tetrakis(4-methoxyphenyl)tetrahydrofuran16 and tricuspidatol-A.17 The 1H NMR data of former compound showed the tetrahydrofuran signals at δH 5.26 (2H, dd, J = 6.3, 2.7 Hz ) and 3.52 (2H, dd, J = 6.3, 2.7 Hz), while the data of tricuspidatol-A at δH 5.25 (2H, dd, J=5.0, 1.5 Hz) and 3.50 (2H, dd, J=5.0, 1.5 Hz), which both resemble the data of compound 2. Thus, on the basis of 2D NOESY of 2, we confirmed the relative stereochemistry structures of 2 in Figure 1. Consequently, the structure of 2, named pouzolignan K, was characterized as 2,5-bis(4-hydroxy-3-methoxyphenyl)-3,4- bis(3,5-dihydroxyphenyl)tetrahydrofuran.
Compound 3 was obtained as white powder, the molecular formula of C27H30O9 was determined from the HR-ESI-TOF-MS ([M+Na]+, m/z 521.1786), suggesting 13 degrees of unsaturation. The UV spectrum showed absorption maxima at 275 and 281 nm in methanol, and the IR spectrum showed absorption bands at 3362, 1720, 1601, 1512 and 1462 cm-1, indicating the presence of hydroxy group, carbonyl group, and aromatic ring.  The 1H NMR and 13C NMR spectrum of 3 (Table 2) along with analysis of the DEPT spectra displayed 27 carbon signals and 30 proton signals. In the 1H-NMR spectrum of 3 (Table 2), two 1, 3, 4-trisubstituted protons (ABX system) at δH 6.94 (1H, d, J = 2.0 Hz, H-2), 6.90 (1H, dd, J = 2.0, 8.0 Hz, H-6'), 6.82 (1H, d, J = 8.0 Hz, H-5), and 6.76 (1H, d, J = 2.0 Hz, H-2), 6.65 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.63 (1H, d, J = 8.0 Hz, H-5), one 1, 3, 5-trisubstituted phenyl protons (AB2 system) at δH 6.17 (2H, d, J = 2.0 Hz, H-2, 6), 6.19 (1H, t, J = 2.0 Hz, H-4), three methine protons at δH 2.84-2.88 (1H, m, H-2), 3.13-3.17 (1H, m, H-3), 3.37 (1H, d, J=12.0 Hz, H-5), two oxymethylene protons at δH 3.31-3.37 (2H, m, H-1), 4.51-4.59 (2H, m, H-4), two methoxyl protons at δH 3.90, 3.78 (each 3H, s), as well as an acetyl methyl protons at δH 1.94 (3H, s) were recognized. DEPT experiment revealed that 3 had ten quaternary carbons, twelve tertiary carbons, two methylenes and three methyl groups (Table 2). In the HMBC spectrum, correlations of H-5 (δH 3.37) with C-1 (δC 137.9), C-6 (δC 121.9), C-1 (δC 137.3), and C-6 (δC 121.1), proved that the two 1, 3, 4-trisubstituted phenyl rings were both linked to C-5.

Furthermore, H-3 (δH 3.13-3.17) with C-2, 6 (δC 109.5) indicated that the 1, 3, 5-trisubstituted phenyl ring was attached to C-3 as well (Figure 2).
The NMR data suggested that compound 3 had the uniform norligan skeleton as pouzolignan F11 which was reported in our earlier phytochemical investigations. Comparing the NMR data of 3 with those of pouzolignan F, C-4 (δC 69.7) was shifted downfield 4.1 ppm and C-3 (δC 46.5) was shifted upfield 2.4 ppm, as well as correlations between H-4 with C-1, C-3, CH3CO in HMBC, thus, the acetoxy group was
attached to C-4. The position of the hydroxy and methoxy groups in compound 3 were established by extensive analysis of the HMBC spectra (Figure 2). Based on the above results, the structure of 3, named pouzolignan L, was estabolished as 1-hydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3- methoxyphenyl)methyl]butyl acetate.
Compound 4 was obtained as white powder, the molecular formula of C28H32O10 was assigned from the HR-ESI-TOF-MS ( [M+Na]+, m/z 551.1889), suggesting 13 degrees of unsaturation. The 1H and 13C NMR spectral data of 4 (Table 2) were similar to those of 3, except for the presence of an additional methoxy group at δH 3.79 (3H, s) and δC 56.9, and the 1, 3, 4, 5-tetrasubstituted phenyl signals at δH 6.48 (2H, s, H-2, 6) with δC 105.9 (C-2, 6). The location of the methoxy group at C-5 was confirmed by the HMBC correlation (Figure 2). Hence, the structure of 4, named pouzolignan M, was identified as 1-hydroxy-3-(3,5-dihydroxyphenyl)-2-[4-hydroxy-3-methoxyphenyl-(4-hydroxy-3,5-dimethoxy-
phenyl)]butyl acetate.
The known compounds were identified as rhamnocitrin (5), rhamnetin (6),18 isorhamnetin (7)19 and quercetin (8),20 by comparing the spectroscopic data with those reported in the literature values.

EXPERIMENTAL
General UV spectra were obtained using a Shimadzu UV-2450 spectrophotometer. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. NMR spectra were run on a Bruker AVANCE Ⅲ 500 spectrometer with TMS as internal standard, and the chemical shifts (δ) were expressed in ppm. HR-ESI-TOF-MS was performed on an API QSTAR time-of-flight spectrometer. TLC was performed on precoated silica gel GF254 plates from Yantai Jiangyou Company. Silica gel 100-200, 200-300 and 300-400 mesh from Qingdao Haiyang Chemical Company, YWG-C18 (50-70 μm) from Tianjin Boruijianhe Chromatography Technology Company and Sephadex LH-20 from Amersham Biosciences were used for column chromatography. Preparactive RP-C18 HPLC was performed by a Shimazu LC-6AD series instrument with Shim-Park RP-C18 column (20×200 mm i.d.).

Plant material The aerial parts of P. zeylanica var. microphylla were collected in Guangzhou, Guangdong province of China on January, 2012 and identified by Professor Ji-Zhu Liu, Guangdong Pharmaceutical University. A voucher specimen (No. 20120113) has been deposited in the Lab of Traditional Chinese Medicine Chemistry, Guangdong Pharmaceutical University.

Extraction and Isolation The air-dried and powdered aerial parts of P. zeylanica var. microphylla (5 kg) were extracted with petroleum ether (150 L) under reflux for two times (1 h per time). After the solvent on the plants was volatized completely, the plants were extracted with EtOAc (150 L) under reflux for three times (1.5 h per time), which was evaporated under reduced pressure to yield a residue (80 g). The EtOAc extract was subjected to silica gel (100-200 mesh) column chromatography, eluted with petroleum ether-EtOAc (50:1→1:1), EtOAc, CH2Cl2-MeOH (1:1), MeOH to yield six fractions, Fr.A-F. Fr.D (13.4 g) was resubjected by silica gel (200-300 mesh) column chromatography, eluted with CHCl3-MeOH (50:1→5:1) to yield Fr.D1-D5. Fr.D1 was separated by Sephadex LH-20, eluted with CHCl3-MeOH (1:1) and purified by RP-C18 column (50-70 μm), using 70% MeOH as the eluent to afford 2 (7.5 mg). Fr.D2 was subjected to sephadex LH-20, eluted with CHCl3-MeOH (1:1) to yield four fractions, and the second fraction (0.3 g) was purified by RP-C18 column (50-70 μm), eluted with 70% MeOH to afford 1 (17 mg). Fr.E (4 g) was resubjected to silica gel (200-300 mesh) column chromatography, eluted with CH2Cl2- MeOH (25:1→8:1) to yield Fr.E1-E4. Fr.E2 was separated repeatedly by silica gel (300-400 mesh) column chromatography, eluted with CH2Cl2-MeOH (17:1) and finally by preparative RP-C18 HPLC, eluted with MeOH-H2O-HCO2H (23:1:0.5) to afford 3 (14 mg). Fr.E3 was further fractionated repeately by silica gel (300-400 mesh) column chromatography, eluted with CH2Cl2-MeOH (14:1), then was separated by Sephadex LH-20, eluted with CHCl3-MeOH (3:2) to afford 4 (14 mg). Fr.F (14 g) was submitted to silica gel (200-300 mesh) column chromatography, eluted with CHCl3-MeOH (20:1→1:1) to afford Fr.F1-F6. Fr.F2 (0.5 g), Fr.F4 (1.0 g) and Fr.F5 (0.3 g) were subjected to Sephadex LH-20, eluted with MeOH to yield compounds 5 (15 mg), 6 (15 mg), 7 (18 mg) and 8 (25 mg), respectively.

Pouzolignan D (1) : Brown amorphous powder; [α]20 D+34.0 (c 0.1, MeOH); IR (KBr) νmax 3360, 2940, 2840, 1608, 1520, 1455, 1347, 1270, 1150, 820 cm-1; UV (MeOH) λmax nm (log ε) 274 (4.02); HR-ESI-TOF-MS m/z 467.1729 [M-H]- (calcd 467.1733 for C26H27O8-), 1H and 13C NMR data (see Table 1).
Pouzolignan K (2): Brown tabular crystals (CHCl3-MeOH 1:1); [α]20 D+71.4 (c 0.1, MeOH); IR (KBr) νmax 3351, 1605, 1516, 1459, 1435, 1273, 1238, 1156, 1124, 1031, 1001 and 921 cm-1; UV (MeOH) λmax nm (log ε) 280 (4.13); HR-ESI-TOF-MS m/z 555.1608 [M+Na]+, 531.1740 [M-H]- (calcd 555.1611 for C30H28NaO9+, 531.1743 for C30H27O9-), 1H and 13C NMR data (see Table 1).
Pouzolignan L (3): white powder; [α]20 D+29.7 (c 0.1, MeOH); UV (MeOH) λmax nm  (log ε) 275 (3.25); IR (KBr) νmax 3362, 2942, 1720, 1601, 1512, 1462, 1365, 1128 and 849 cm-1, HR-ESI-TOF-MS m/z 521.1786 [M+Na]+ (calcd 521.1788 for C27H30NaO9+), 1H and 13C NMR data (see Table 2).
Pouzolignan M (4): white powder; [α]20 D+36.1 (c 0.1, MeOH), UV (MeOH) λmax nm  (log ε) 275(3.34); IR (KBr) νmax 3362, 2941, 1721, 1602, 1514, 1462, 1366, 1126 and 849 cm-1; HR-ESI-TOF-MS m/z 551.1889 [M+Na]+ (calcd 551.1893 for C28H32NaO10+) 1H and 13C NMR data (see Table 2).
ACKNOWLEDGEMENTS
This work was financially supported by the National Natural Science Foundation (NNSF) of China (NO. 81073045) and the Chinese Medicine Administration Bureau of Guangdong, China (NO. 20141162).

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