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Oct 13, 2014 - Two novel resin glycosides isolated from Ipomoea cairica with α-glucosidase inhibitory activity. Jie-Hong LI , Jie-Tao PAN , Yong-Qin ...
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Merremins A−G, Resin Glycosides from Merremia hederacea with Multidrug Resistance Reversal Activity Wen-qiong Wang, Wei-bin Song, Xiao-jing Lan, Min Huang, and Li-jiang Xuan* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Five new pentasaccharide resin glycosides, named merremins A−E (1−5), two new pentasaccharide resin glycoside methyl esters, named merremins F and G (6, 7), and four known resin glycosides, murucoidin IV, murucoidin V, stoloniferin IV, and murucoidin XVII, were obtained from the aerial parts of Merremia hederacea. This is the first report of resin glycosides obtained from M. hederacea. In addition, the new compounds can be divided into three types: those possessing an 18membered ring (1−4), compound 5 with a 20-membered ring, and those with an acyclic core (6, 7). Furthermore, the different types of resin glycosides were evaluated for their multidrug resistance reversal activities. Compounds 1, 5, 6, and murucoidin V were noncytotoxic and enhanced the cytotoxicity of vinblastine by 2.3−142.5-fold at 25 μM. Compound 5 and murucoidin V, with 20-membered rings, were more active than compound 1, with an 18-membered ring.

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Herein, the isolation and structure elucidation of seven new pentasaccharide resin glycosides, merremins A−G (1−7), and four known compounds, murucoidin IV, murucoidin V,13 stoloniferin IV,14 and murucoidin XVII,15 from the aerial parts of M. hederacea are described. In addition, the different types of resin glycosides were evaluated for their MDR reversal activities. Compounds 1, 5, 6, and murucoidin V were noncytotoxic and enhanced the cytotoxicity of vinblastine 2.3−142.5-fold at 25 μM. Compound 5 and murucoidin V, with 20-membered rings, were more active than compound 1, with an 18-membered ring.

esin glycosides, which are primarily found in the family Convolvulaceae, are unusual amphipathic metabolites with structures including hydrophobic (fatty acid aglycone) and hydrophilic (oligosaccharide) moieties.1 To date, hundreds of resin glycosides have been isolated from different genera of the family Convolvulaceae, including Ipomoea,2 Calonyction,3 and Pharbitis.4 These resin glycosides exhibit various pharmacological activities, such as cytotoxic,5 multidrug resistance (MDR) reversal,6 ionophoretic,7 and phytogrowth-inhibitory activities.8 Moreover, as novel P-glycoprotein inhibitors, resin glycosides have gained increasing attention due to their intriguing structures. Therefore, the discovery of various structural resin glycosides from the family Convolvulaceae and the investigation of their MDR reversal activities aroused our interest. The genus Merremia (Convolvulaceae) comprises 70 species, which are primarily distributed throughout the warm and tropical regions of Asia and Africa.9 To date, various resin glycosides have been detected in some Merremia spp., including M. mammosa Choisy,10 M. tuberosa L,11 and M. hungaiensis.12 Merremia hederacea, also called “Lilanwang” in China, is a perennial plant with persistent leaves. Traditionally, M. hederacea is widely used for the treatment of pharyngitis and furuncles. Therefore, the rarely reported resin glycosides from M. hederacea encourage a study of the isolation and MDR reversal activity of its resin glycosides. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Air-dried, powdered aerial parts of M. hederacea (5.0 kg) were extracted with 95% EtOH at room temperature. After removal of the organic solvent, the sample was suspended in H2O and sequentially extracted with petroleum ether and EtOAc. The petroleum ether layer was successively separated over silica gel, NH2 gel, Sephadex LH-20, and preparative HPLC to yield merremins A−G (1−7), murucoidin IV, murucoidin V, stoloniferin IV, and murucoidin XVII. The structures of the known compounds were elucidated based on comparisons of their HRESIMS and 1H, and 13C NMR data with reported data. Received: June 13, 2014

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= 7.4 Hz, 1H) in the 1H NMR spectrum. The α-configuration of L-rhamnose was suggested by the chemical shift of C-5 of rhamnose (δC 69.2, 68.8, 68.5) in the 13C NMR spectrum.17 The long-range HMBC correlations between H-1 of β-Fuc (δH 4.75) and C-11(δC 82.9) of the 11-hydroxyhexadecanoyl moiety indicated that β-Fuc was the first hexose unit in the sugar moiety. The sequence of the sugar moiety was determined to be glucosyl-(1→3)-[rhamnosyl-(1→4)]-rhamnosyl-(1→4)-rhamnosyl-(1→2)-fucosyl by their long-range HMBC correlations: H-1 of α-Rha (δH 5.43) to C-2 of β-Fuc (δC 77.4), H-1 of α-Rha′ (δH 5.81) to C-4 of α-Rha (δC 81.7), H-1 of α-Rha″ (δH 6.19) to C-4 of α-Rha′ (δC 79.0), and δH H1 of β-Glc (4.99) to C-3 of α-Rha′ (δC 80.5). In addition, the positions of esterification, i.e., Ac located at OH-3 of β-Fuc, 2Mba′ at OH-4 of α-Rha″, and 2-Mba at OH-2 of α-Rha′, were inferred from the long-range correlations: H-2 of α-Rha′ (δH 6.27) to C-1 of 2-Mba (δC 176.6), δH H-4 of α-Rha″ (5.76) to C-1 of 2-Mba′ (δC 176.7), and H-3 of β-Fuc (δH 5.29) to C-1 of Ac (δC 171.1), respectively. The C-2 (Rha) site of lactonization was corroborated by the correlation between C1 of Jal (δC 173.6) and H-2 of Rha (δH 5.71). Thus, the structure of compound 1 was identified as (11S)-jalapinolic acid 11-O-β-D-glucopyranosyl-(1→3)-O-[4-O-(2S-methylbutyryl)α-L-rhamnopyranosyl-(1→4)]-O-[2-O-(2S-methylbutyryl)]-αL-rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-O(3-O-acetyl)-β-D-fucopyranoside-(1,2′-lactone). The molecular formulas of merremins B−D (2−4) were determined as C61H104O26, C58H98O26, and C63H108O26, respectively, based on the 13C NMR (Table 3) and HRESIMS data. The 1H and 13C NMR spectra indicated that 2−4 contained the same complex sugar moiety and (11S)hydroxyhexadecanoyl group as 1. Additionally, the identities of the simple short fatty acid moieties were determined by EIMS and TLC analyses after alkaline hydrolysis. (2S)Methylbutanoic acid in 2−4, n-decanoic acid (Deca) in 4, and n-octanoic acid (Octa) in 2 were identified. Furthermore, in compounds 2 and 4, the carbonyl resonances for the 11hydroxyhexadecanoyl moieties (δC 174.0 in 2; 173.6 in 4) were identified through correlations (2JCH) with the C-2 diastereotopic methylene protons (δH 2.35, 2.27 in 2; 2.44, 2.30 in 4). The positions of esterification were determined by the HMBC correlations (Figure 1) as follows: OH-4 of Rha″ was acylated by 2-Mba in 2−4, OH-2 of Rha′ was acylated by Octa in 2, OH-2 of Rha′ was acylated by 2-Mba in 3, OH-2 of Rha′ was acylated by Deca in 4, and OH-4 of Fuc was acylated by Ac in 2−4. Moreover, the site of lactonization was corroborated as C2 of Rha according to the correlation between C-1 of Jal and H2 of Rha. Consequently, the structures of 2−4 were determined as shown. Merremin E (5) possessed the molecular formula C51H88O24 based on the HRESIMS and 13C NMR data (Table 3). The comparison of the 1H and 13C NMR data of 5 and murucoidin V indicated that murucoidin V contained an additional 2methybutanoyl unit at OH-4 of Rha″.13 Thus, the structure of 5 was determined to be (11S)-jalapinolic acid 11-O-β-Dglucopyranosyl-(1→3)-O-[α-L-rhamnopyranosyl-(1→4)]-O-[2O-(2S-methylbutyryl)]-α-L-rhamnopyranosyl-(1→4)-O-α-Lrhamnopyranosyl-(1→2)-O-β-D-fucopyranoside-(1,3′-lactone). Merremins F and G (6, 7) had the same molecular formula, C52H92O25, based on the HRESIMS and 13C NMR data (Table 3). These compounds were determined to be methyl esters of resin glycoside acid by the HMBC correlations between C-1 of Jal (δC 176.7 in 6; 174.4 in 7) and the methyl singlet (δH 3.62

Merremin A (1) was obtained as a white, amorphous powder. The molecular formula was determined to be C58H98O26 based on the 13C NMR data (Table 3) and the HRESIMS ion at m/z 1209.6344 [M + Na] + (calcd 1209.6239). The 1H NMR spectrum of 1 showed 10 methyl groups, five anomeric protons, and long-chain fatty acid signals. The 13C NMR and DEPT spectra of 1 showed 58 carbons, including five anomeric and four carbonyl carbons. The NMR data of 1 could be divided into two parts: resonances in the anomeric region and those representative of the aglycone moieties. The 1H NMR spectrum of 1 showed two methine groups (δH 2.51, 2.35) due to H-2 of the 2methylbutyryl moieties (2-Mba), two primary methyl groups (δH 0.92, 0.73) due to Me-4 of 2-Mba, and two methyl groups (δH 1.19, 0.96) due to Me-2 of 2-Mba in the aglycone region (Table 1). After alkaline hydrolysis, the S absolute configuration of 2-Mba was determined by its specific rotation ([α]23D +15.4).16 The methyl signal at δH 2.00 correlated with the ester carbonyl carbon at δC 171.1 in the HMBC spectrum (Figure 1), suggesting an acetyl group. Furthermore, the 11hydroxyhexadecanoyl moiety (Jal) was suggested by the diagnostic signals of the methyl triplet (δH 0.87), methylene group (δH 2.26 and 2.40), and oxygenated methine at δH 3.79. After alkaline and acid hydrolysis, the resulting 11-hydroxyhexadecanoic acid showed a fragment ion at m/z 201 [M − CH3(CH2)4]+ by the EIMS data, suggesting the 11-OH group of Jal. The absolute configuration was determined to be S by Mosher’s method. In the anomeric region, the 1H−1H COSY data indicated five spin systems, which were attributed to a hexose and four 6deoxyhexose units. The sugars obtained from the acidic hydrolysates were identified as L-rhamnose, D-fucose, and Dglucose through the HPLC analysis and their corresponding optical rotations. The β-configurations of D-glucose and Dfucose were suggested by the coupling constants of the anomeric protons at δH 4.99 (d, J = 7.7 Hz, 1H) and 4.75 (d, J B

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Table 1. 1H NMR (500 MHz) Data of 1−5 (in ppm) (Pyridine-d5)a position

1

2

3

4

5

Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Jal-2 11 16 2-Mba-2 3 4 2-Me 2-Mba′-2 3 4 2-Me Ac-2 Deca-2 10 Octa-2 8

4.75 d (7.4) 4.38 m*b 5.29 dd (10.2, 3.3) 4.23 d (3.3) 3.79 q (6.3) 1.45 d (6.3) 5.43 d (2.0) 5.71 dd (2.0, 3.4) 4.63 dd (9.5, 3.4) 4.13 dd (9.5, 9.5) 4.28 m* 1.60 d (6.3) 5.81 d (1.8) 6.27 dd (3.4, 1.8) 4.26 m* 4.28 m* 4.39 m* 1.65 d (6.1) 6.19 d (1.9) 4.90 br s 4.51 dd (9.4, 3.5) 5.76 dd (9.4, 9.4) 4.36 m* 1.39 d (6.3) 4.99 d (7.7) 3.88−3.92 m 4.03−4.05 m 3.70−4.74 m 3.96−4.00 m 4.42 m*, 4.16 m* 2.40 m*, 2.26 m* 3.79 m* 0.87 t (6.9) 2.35 q (6.9) 1.34 m* 0.73 t (7.4) 0.96 d (7.0) 2.51 q (6.5) 1.49 m* 0.92 t (7.4) 1.19 d (6.9) 2.00 s

4.77 d (7.6) 4.11 d (7.6, 9.5) 4.22−4.25 m 5.54 d (3.6) 3.84 q (6.3) 1.28 d (6.3) 5.59 d (1.8) 5.94−5.96 m 4.96−4.99 m 4.18 dd (9.3, 9.3) 4.40 m* 1.64 d (6.1) 5.95 d (1.7) 6.29 br s 4.75−4.78 m 4.32 m* 4.36 m* 1.66 d (5.5) 6.27 br s 4.97 br s 4.54 dd (9.4, 3.5) 5.79 dd (9.4, 9.4) 4.37 m* 1.40 d (6.2) 5.08 d (7.6) 3.96 d (7.6, 8.8) 4.03−4.08 m 3.75 d (6.5) 4.00 d (5.9) 4.38 m*, 4.08 m* 2.35 m*, 2.27 m* 3.82−3.85 m 0.87 t (9.0) 2.52 q (6.9) 1.49 m* 0.93 t (7.4) 1.20 d (7.0)

4.76 d (7.5) 4.12 d (7.5, 9.9) 4.22 dd (9.9, 3.8) 5.53 d (3.6) 3.86 q (6.3) 1.28 d (6.3) 5.57 d (1.9) 5.95 dd (3.4, 1.9) 4.90−4.94 m 4.16 dd (9.5, 9.5) 4.43 m* 1.63 d (6.3) 5.87 d (1.9) 6.29 br s 4.72 dd (9.4, 3.5) 4.30 dd (9.4, 9.4) 4.36 m* 1.66 d (6.1) 5.87 d (1.9) 4.96 d (2.9) 4.54 dd (9.4, 3.5) 5.79 dd (9.4, 9.4) 4.58 m* 1.41 d (6.3) 5.04 d (7.7) 3.93−4.96 m 4.06 dd (9.1, 9.1) 3.71−3.75 m 3.92−3.95 m 4.36 m*, 4.03 m* 2.54 m*, 2.29 m* 3.84−3.85 m 0.86 t (7.0) 2.39 q (6.9) 1.41 m* 0.77 t (7.5) 1.01 d (6.9) 2.52 q (6.9) 1.48 m* 0.93 t (7.0) 1.20 d (6.9) 1.91 s

4.74 d (7.5) 4.09 m* 4.21 dd (9.8, 3.4) 5.51 d (3.4) 3.85 m* 1.26 d (6.5) 5.55 d (2.0) 5.92 d (2.0, 4.0) 4.95 dd (4.0, 9.5) 4.15 dd (9.5, 9.5) 4.41 m* 1.60 d (6.1) 5.90 br s 6.25 d (2.4) 4.74 m* 4.33 m* 4.37 m* 1.63d (6.2) 6.23 br s 4.95 d (3.1) 4.51 dd (9.2, 3.1) 5.75 dd (9.2, 9.2) 4.34 m* 1.38 d (6.1) 5.04 d (7.7) 3.94 m* 4.02 dd (8.9, 8.9) 3.68−3.74 m 3.94 m* 4.35 m*, 4.04 m* 2.44 m*, 2.30 m* 3.84−3.87 m 0.86 t (5.6) 2.48−2.53 m 1.46 m 0.92 t (7.4) 1.18 d (6.9)

4.83 d (7.2) 4.53 dd (7.2, 9.4) 4.22 d (9.4) 3.86−3.90 m 3.82−3.85 m 1.53 d (6.5) 6.36 br s 5.25 br s 5.66 d (11.3) 4.67−4.71 m 4.98−5.01 m 1.60 d (6.2) 5.61 br s 6.02 d (3.2) 4.58 m* 4.17 m* 4.32 m* 1.66 d (6.0) 6.21 br s 4.92 br s 4.42−4.45 m 4.17 m* 4.29 m* 1.53 m 5.09 d (7.4) 3.94−3.95 m 4.16 m* 3.85 m* 3.83 m* 4.45 m*, 4.36 m* 2.54 m*, 2.33 m* 3.84 m* 0.91 t (6.8) 2.45 q (6.4) 1.41 m 0.83 t (7.4) 1.10 d (7.0)

1.91 s

1.90 s 2.25 m* 0.77 t (7.1)

2.44 m* 0.78 t (7.2)

a

Chemical shifts (ppm) are referenced to pyridine-d5 (δH 7.58) at 500 MHz. bChemical shifts marked with an asterisk (*) indicate overlapped signals.

in 6; 3.61 in 7) (Figure 1). According to the 1H and 13C NMR data, 6 and 7 contained the same complex sugar moiety and 11hydroxyhexadecanoyl group as 1, and the positions of esterification were identified by the HMBC correlations: H-4 of Rha″ (δH 5.72) to C-1 of 2-Mba (δC 174.4) in 6 and H-2 of Rha (δH 6.40) to C-1 of 2-Mba (δC 176.7) in 7. Thus, the structure of 6 was established as (11S)-jalapinolic acid methyl ester 11-O-β-D-glucopyranosyl-(1→3)-O-[4-O-(2S-methylbutyryl)-α-L-rhamnopyranosyl-(1→4)]-O-α-L-rhamnopyranosyl(1→4)-O-α-L-rhamnopyranosyl-(1→2)-O-[6-O-acetyl]-β-D-fucopyranose, while 7 was defined as (11S)-jalapinolic acid

methyl ester 11-O-β-D-glucopyranosyl-(1→3)-O-[α-L-rhamnopyranosyl-(1→4)]-O-[2-O-(2S-methylbutyryl)]-α-L-rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-O-[6-O-acetyl]-β-D-fucopyranose. To determine the cytotoxicity and MDR reversal activity of the isolated resin glycosides, their MDR reversal activities in KB/VCR cells using the SRB method were evaluated (Table 4). The cytotoxicity assay showed that the inhibition ratios of 1, 5, 6, and murucoidin V were less than 50% at 25 μM, indicating that these compounds were noncytotoxic at 25 μM, while these compounds enhanced the cytotoxicity of vinblastine by 2.3− C

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Table 2. 1H NMR (500 MHz) Data of 6 and 7 (in ppm) (Pyridine-d5)a position

6

7

Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Jal-2 11 16 OMe 2-Mba-2 3 4 2-Me

4.82 d (7.8) 4.50−4.55 m 4.16 m*b 4.09−4.13 m 4.30 q (6.6) 1.32 d (6.6) 6.27 d (1.5) 4.69 br s 4.18 m* 4.01−4.05 m 3.82 m* 1.54 d (6.3) 5.88 d (2.1) 5.24 br s 4.74 dd (8.6, 3.0) 4.47 m* 4.92 m* 1.63 d (6.1) 6.21 d (1.8) 4.89 br s 4.45 m* 5.72 dd (9.5, 9.5) 4.65 d (6.3) 1.62 d (6.1) 5.24 d (7.7) 3.97−3.99 m 4.33 m* 4.58 m* 3.95−3.96 m 4.57 m*, 4.29 m* 2.32 t (7.5) 3.82 m* 0.93 t (7.3) 3.62 s 2.51 q (6.9) 1.49 m* 0.93 t (7.3) 1.20 d (7.0)

4.81 d (7.8) 4.53−4.55 m 4.18 m* 4.04−4.05 m 3.81 m* 1.53 d (6.4) 6.28 d (1.5) 4.68 br s 4.63 dd (9.6, 3.5) 4.24 dd (9.6, 9.6) 4.90 dd (9.6, 6.2) 1.56 d (6.2) 5.74 d (1.9) 6.40 d (1.4) 4.78 dd (9.6, 3.4) 4.33 dd (9.6, 9.6) 4.42 m* 1.66 d (6.1) 6.26 d (1.7) 4.93 dd (3.6, 1.7) 4.46 dd (9.4, 3.6) 4.28 dd (9.4, 9.4) 4.37 m* 1.65 d (6.1) 5.16 d (7.7) 4.00 m* 4.15 m* 3.87 m* 3.95−3.96 m 4.56 m*, 4.23 m* 2.33 t (7.5) 3.81 m* 0.92 t (6.7) 3.61 s 2.42 q (6.9) 1.40 m* 0.79 t (7.4) 1.04 d (6.9)

out on an Agilent 1100 series HPLC system with a YMC-Pack ODS-A column (250 × 10 mm, 5 μm). Analytical HPLC was performed on an Agilent 1200 series HPLC system with an Agilent ZORBAX NH2 column (150 × 4.6 mm, 5 μm). Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd., People’s Republic of China), C18 reversedphase (RP-18) silica gel (20−45 μm; Fuji Silysia Chemical Ltd., Japan), NH2 silica gel (100−200 mesh; Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 gel (Amersham Biosciences, Sweden) were used for column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd., People’s Republic of China) were used for TLC. Plant Material. Aerial parts of Merremia hederacea were collected in Guangxi, China, in September 2011 and identified by Prof. Heming Yang. A voucher specimen (No. SIMM183) was deposited at the Herbarium of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, People’s Republic of China. Extraction and Isolation. Air-dried, powdered aerial parts of M. hederacea (5.0 kg) were extracted with EtOH−H2O (19:1, v/v, 7 × 20 L) at room temperature, each for 24 h. After removal of the organic solvent, the sample was suspended in H2O and sequentially extracted with petroleum ether and EtOAc. The petroleum layer was concentrated, subjected to silica gel CC, eluted with CHCl3−MeOH mixtures of increasing polarity (from 19/1 to 4/1), to yield fractions A−J. Fraction H was further subjected to NH2 CC, eluted with CHCl3−MeOH mixtures of increasing polarity (from 20/1 to 9/1), to yield fractions H1−H4. Murucoidin XVII and murucoidin V were obtained from subfraction H3 through preparative HPLC (MeCN− H2O, 17/3); murucoidin XVII (80.6 mg) eluted at 16.8 min, and murucoidin V (22.3 mg) at 5.6 min. Compounds 5 (34.5 mg), 6 (4.6 mg), and 7 (10.1 mg) were obtained from subfraction H4 through silica gel CC eluted with CHCl3−MeOH (17/3). Fraction I was further subjected to NH2 CC, eluted with CHCl3−MeOH mixtures of increasing polarity (from 15/1 to 4/1), to yield fractions I1−I3. Compounds 2 (14.3 mg), 3 (13.6 mg), and 4 (52.3 mg) were obtained from subfraction I2 through silica gel CC, eluted with CHCl3−MeOH (10/1). Compound 1 (43.8 mg), murucoidin IV (173.9 mg), and stoloniferin IV (52.0 mg) were obtained from subfraction I3 through silica gel CC, eluted with CHCl3−MeOH (10/1). All compounds were purified by Sephadex LH-20. Merremin A (1): white, amorphous powder; [α]24D −16 (c 0.2, MeOH); 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 1209.6344 [M + Na]+ (calcd for C58H98O26, 1209.6239). Merremin B (2): white, amorphous powder; [α]24D −19 (c 0.2, MeOH); 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 1275.6780 [M + Na]+ (calcd for C61H104O26, 1275.6708). Merremin C (3): white, amorphous powder; [α]24D −18 (c 0.2, MeOH); 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 1209.6341 [M + Na]+ (calcd for C58H98O26, 1209.6239). Merremin D (4): white, amorphous powder; [α]24D −23 (c 0.2, MeOH); 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 1303.7044 [M + Na]+ (calcd for C63H108O26, 1303.7021). Merremin E (5): white, amorphous powder; [α]24D −67 (c 0.4, MeOH); 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 1107.5652 [M + Na]+ (calcd for C51H88O24, 1107.5558). Merremin F (6): white, amorphous powder; [α]24D −48 (c 0.4, MeOH); 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 1139.5856 [M + Na]+ (calcd for C52H92O25, 1139.5820). Merremin G (7): white, amorphous powder; [α]24D −30 (c 0.2, MeOH); 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 1139.5827 [M + Na]+ (calcd for C52H92O25, 1139.5820). Aglycone Identification. Compounds 1−5 (10 mg, each) and the mixture of compounds 6 and 7 (10 mg) were separately dissolved in 5% KOH (10 mL) and refluxed at 95 °C for 4 h. Each of the reaction mixtures was acidified to pH 4, followed by extraction (CHCl3, 3 × 10 mL) and concentration to afford (2S)-methylbutanoic acid (EtOAc− toluene−HCOOH, 7/8/2, Rf = 0.38), n-octanoic acid (petroleum ether−CHCl3, 4/1, Rf = 0.22), and n-decanoic acid (petroleum ether− CHCl3, 4/1, Rf = 0.35), which were detected by TLC analysis with authentic samples. (2S)-Methylbutanoic acid was further purified using an ODS column (MeOH−H2O, 40/60), and the S configuration was

a

Chemical shifts (ppm) referenced to pyridine-d5 (δH 7.58) at 500 MHz. bChemical shifts marked with an asterisk (*) indicate overlapped signals.

142.5-fold when incorporated at 25 μM. Murucoidin V was one of the most active compounds. Compound 5 and murucoidin V, with 20-membered rings, were more active than compound 1, with an 18-membered ring. In addition, the acyclic resin glycoside acid methyl ester (6) exhibited similar activity to 1, suggesting that the macrocyclic structure of resin glycosides was not essential for the anti-MDR activity. This was inconsistent with a previous report that the biological activity was associated with the macrocyclic structure of resin glycoside.18

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. NMR experiments were recorded in pyridine-d5 on Bruker AM-400 and Bruker AM-500 spectrometers referenced to solvent peaks (δH 7.58; δC 135.9). EIMS analyses were performed on a Thermo-DFS mass spectrometer. HRESIMS analyses were performed on an Agilent 6224 TOF mass spectrometer with an ESI interface. Semipreparative HPLC was carried D

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Table 3. 13C NMR (125 MHz) Data of 1−7 (in ppm) (Pyridine-d5)a

a

position

1

2

3

4

5

6

7

Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Jal-1 2 11 16 OMe 2-Mba-1 2 4 2-Me 2-Mba′-1 2 4 2-Me Ac-1 2 Deca-1 2 10 Octa-1 2 8

104.4 77.4 76.3 70.5 70.9 17.5 99.0 73.7 70.0 81.7 69.2 19.2 100.4 73.4 80.5 79.0 68.8 19.4 103.7 72.8 70.1 75.5 68.5 18.4 105.8 75.5 78.8 78.3 71.6 63.2 173.6 34.5 82.9 14.7

104.7 79.8 71.3 74.9 69.4 17.4 99.1 73.9 69.7 81.2 69.4 19.2 100.2 73.4 80.5 78.8 68.5 19.5 103.6 72.8 70.6 75.5 68.8 18.4 105.8 71.7 78.8 78.5 75.5 63.2 174.0 34.8 83.2 14.7

104.7 79.8 71.8 74.9 69.4 17.3 99.1 73.8 69.7 81.3 68.8 19.3 100.2 73.3 80.5 79.1 68.5 19.5 103.7 72.8 70.6 75.5 69.4 18.4 105.8 71.8 78.8 78.4 75.5 63.2 173.7 34.6 83.2 14.7

104.7 79.7 71.2 74.8 69.3 17.4 99.0 73.8 69.6 81.3 69.3 19.2 100.1 73.4 80.4 78.7 68.8 19.4 103.5 72.7 70.6 75.4 68.5 18.0 105.7 71.6 78.8 78.4 75.4 63.2 173.6 34.6 83.2 14.6

101.8 75.5 76.9 78.1 71.6 18.8 100.4 71.0 78.3 75.5 68.3 19.0 99.3 72.6 80.7 78.6 68.6 19.5 103.8 72.7 71.6 79.3 70.3 17.6 105.4 73.8 80.0 78.5 78.5 62.8 174.8 34.7 82.6 14.8

176.6 41.6 11.8 16.9 176.7 41.9 12.2 17.4 171.1 21.2

176.7 41.9 12.2 17.3

176.7 41.6 11.8 17.0 176.7 41.9 12.2 17.4 171.5 21.1

176.6 41.8 12.1 17.3

176.5 41.6 11.9 17.0

101.5 75.6 77.0 72.1 68.2 18.3 101.9 73.1 78.9 78.2 71.6 17.6 104.0 72.4 83.2 79.6 68.0 19.4 103.5 73.1 70.6 75.5 68.8 19.2 106.1 73.9 79.0 83.1 75.5 63.3 176.7 34.6 83.1 14.9 51.6 174.4 41.9 12.2 17.4

101.5 75.4 77.0 71.8 71.0 17.6 101.8 73.1 72.6 82.6 67.7 18.9 100.3 73.7 81.1 78.8 68.7 19.3 103.9 73.1 72.9 78.7 71.0 19.0 106.2 75.6 78.2 78.9 74.3 63.4 174.4 34.6 82.6 14.8 51.6 176.7 41.6 11.8 17.0

171.5 21.1

171.4 21.0 173.9 34.8 14.5

173.7 34.6 17.3

Chemical shifts (ppm) are referenced to pyridine-d5 (δC 135.9) at 125 MHz.

confirmed by the specific rotation ([α]23D +15.4), whereas the noctanoic acid from alkaline hydrolysates of 2 and the n-decanoic acid from the alkaline hydrolysates of 4 were separated using silica gel chromatography (petroleum ether−CHCl3 from 4/1 to 0/1). In addition, the aqueous phase via CHCl3 extraction was then extracted with n-BuOH (3 × 10 mL) and concentrated to yield a colorless solid. The residue in 1 N H2SO4 (10 mL) was heated at 95 °C for 4 h. The reaction mixture was extracted with CHCl3 (3 × 10 mL), which was

further concentrated and purified to afford 11-hydroxyhexadecanoic acid. 11-Hydroxyhexadecanoic acid was methylated with diazomethane. The methyl ester of 11-hydroxyhexadecanoic acid was further separately treated with (S)-MPTA chloride and (R)-MPTA chloride in pyridine-d5 and allowed to stand for 8 h at room temperature. The S configuration of 11-hydroxyhexadecanoic acid was determined by the chemical shift difference (Δδ = δS − δR, ΔδOMe = +0.002, Δδ16H = −0.035). E

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Figure 1. Key HMBC and 1H−1H COSY correlations of merremins A−G (1−7). (2S)-Methylbutanoic acid: [α]23D +15.4 (c 0.1, MeOH); EIMS m/z 103 [M + H]+ (1), 87 (24), 74 (100), 57 (44). Octanoic acid: EIMS m/z 145 [M + H]+ (1), 127 (2), 115 (10), 73 (72), 60 (100). Decanoic acid: EIMS m/z 172 [M]+ (3), 143 (7), 129 (40), 115 (12), 87 (16), 73 (100), 60 (92). (11S)-Hydroxyhexadecanoic acid: EIMS m/z [M − 17]+ 255 (8), 201 (16), 183 (100), 101 (16), 84 (25), 55 (26); HRESIMS m/z 295.2244 [M + Na]+ (calcd for C16H32O3, 295.2244). 11-(S-MPTA)-hydroxyhexadecanoic acid methyl ester: 1H NMR (in pyridine-d5, 400 MHz) δ 0.785 (3H, t, J = 6.6 Hz, Me-16), 5.266 (1H, m, H-11), 3.674 (3H, s, OMe). 11-(R-MPTA)-hydroxyhexadecanoic acid methyl ester: 1H NMR (in pyridine-d5, 400 MHz) δ 0.820 (3H, t, J = 6.6 Hz, Me-16), 5.267 (1H, m, H-11), 3.672 (3H, s, OMe). Sugar Analysis. Compound 1 (20.0 mg) was hydrolyzed by alkali and acid, as described in the Aglycone Identification section. The aqueous phase was extracted with n-BuOH (3 × 10 mL) after acid hydrolysis and concentrated to yield a colorless solid. The residue was

dissolved in H2O and directly analyzed by HPLC with authentic samples (MeCN−H2O, 95/5): D-glucose eluted at 21.1 min, Lrhamnose at 6.5 min, and D-fucose at 10.0 min. Each of these eluates was individually collected, concentrated, and dissolved in H2O. The eluates were identified as D-glucose, [α]23D +52 (c 0.1, H2O), Lrhamnose, [α]23D +8 (c 0.1, H2O), and D-fucose, [α]23D +78 (c 0.1, H2O), through comparisons of their specific rotations with those of the corresponding authentic samples. MDR Reversal Assays. The MDR reversal activities of the test compounds against the KB/VCR cell lines were measured using a sulforhodamine B (SRB) assay as previously described.19 Briefly, cells were plated in 96-well culture plates for 24 h and treated with serial dilutions of vinblastine (Sigma) ranging from 0.125 to 1 μM, with or without 25 μM of the samples. After incubation for 72 h under a humidified atmosphere of 5% CO2 at 37 °C, cells were fixed with 10% trichloroacetic acid and incubated at 4 °C for 1 h. After washing with distilled H2O and air drying, the plates were stained for 15 min with 100 μL of 0.4% SRB in 1% glacial HOAc. The plates were washed with 1% HOAc and air-dried. For reading of the plates, the protein-bound F

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Table 4. Results of Modulating MDRa Activities in KB/VCR Cells of Compounds 1−6, Murucoidin IV, Murucoidin V, Stoloniferin IV, and Murucoidin XVII

(6) Figueroa-Gonzál, G.; Jacobo-Herrera, N.; Zentella-Dehesa, A.; Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 93−97. (7) Kitagawa, I.; Ohashi, K.; Kawanishi, H.; Shibiya, H.; Shinkai, K.; Akedo, H. Chem. Pharm. Bull. 1989, 37, 1679−1681. (8) Pereda-Miranda, R.; Mata, R. J. Nat. Prod. 1993, 56, 571−582. (9) Jenett-Siems, K.; Weigl, R.; Böhm, A.; Mann, P.; Tofern-Reblin, B.; Ott, S. C.; Ghomian, A.; Kaloga, M.; Siems, K.; Witte, L.; Hilker, M.; Müller, F.; Eich, E. Phytochemistry 2005, 66, 1448−1464. (10) Kitagawa, I.; Shibuya, H.; Yokokawa, Y.; Baek, N. I.; Ohashi, K.; Yoshikawa, M.; Nitta, A.; Wiriadinata, H. Chem. Pharm. Bull. 1988, 36, 1618−1621. (11) Ono, M.; Nakagawa, K.; Kawasaki, T.; Miyahara, K. Chem. Pharm. Bull. 1993, 41, 1925−1932. (12) Noda, N.; Tsuji, K.; Miyahara, K.; Yang, C. R. Chem. Pharm. Bull. 1994, 42, 2011−2016. (13) Chérigo, L.; Pereda-Miranda, R. J. Nat. Prod. 2006, 69, 595− 599. (14) Noda, N.; Takahashi, N.; Kawasaki, T.; Miyahara, K.; Yang, C. R. Phytochemistry 1994, 36, 365−371. (15) Corona-Castañeda, B.; ChéRigo, L.; Fragoso-Serrano, M.; Gibbons, S.; Pereda-Miranda, R. Phytochemistry 2013, 95, 277−283. (16) Yin, Y. Q.; Kong, L. Y. J. Agric. Food Chem. 2008, 56, 2363− 2368. (17) Yu, B. W.; Luo, J. G.; Wang, J. S.; Zhang, D. M.; Yu, S. S.; Kong, L. Y. J. Nat. Prod. 2011, 74, 620−628. (18) Kinghorn, A. D.; Falk, H.; Kobayashi, J. In Progress in the Chemistry of Organic Natural Products; Budzikiewicz, H.; PeredaMiranda, R.; Rosas-Ramírez, D.; Castań eda-Gómez, J., Eds.; Sprigerwien: New York, 2010; Vol. 92, pp 77−153. (19) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112.

vinblastine + sampleb sample

RFc value

inhibition ratio % (25 μM)

IC50 value (μM)

0.91 93.76 97.80 96.57 −0.08 0.23 97.50 7.72 96.67 91.10

0.253

2.3

0.036 0.230

15.8 2.5

0.004

142.5

1 2 3 4 5 6 murucoidin IV murucoidin V stoloniferin IV murucoidin XVII vinblastine a

0.57 b

MDR: multidrug resistance. Serial dilutions ranging from 0.125 to 1 μM of vinblastine in the presence or absence of 25 μM sample. cRF: IC50 of VCR alone/IC50 of VCR in the presence of sample.

dye was dissolved in 150 μL of 10 mM Tris base. The absorbance was measured at 510 nm on a microplate spectrophotometer (Molecular Devices SpectraMax 340, Sunnyvale, CA, USA). The results were expressed as IC50 values.

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13C, COSY, HMQC, and HMBC spectra of compounds 1− 7 (Figures S1−S35); 1H NMR spectra of 11-(S-MPTA)hydroxyhexadecanoic acid methyl ester and 11-(R-MPTA)hydroxyhexadecanoic acid methyl ester (Figures S36, S37); EIMS and HRESIMS spectra of 11-hydroxyhexadecanoic acid (Figures S38, S39). These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel and Fax: 86-2120231968. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge grants from the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program”, China (No. 2009ZX09301-001), the National Natural Sciences Foundation of China (No. 81473111), and the China Postdoctoral Science Foundation (No. Y412021031).

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REFERENCES

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