Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors from - American

Nov 13, 2013 - Korea Bioactive Natural Material Bank, College of Pharmacy and Research ... Food Research Institute, Sungnam 463-746, Republic of Korea...
0 downloads 0 Views 792KB Size
Download PDF
Article pubs.acs.org/jnp

Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors from Morinda citrifolia (Noni) and Their Insulin Mimetic Activity Phi-Hung Nguyen,†,∥ Jun-Li Yang,‡,∥ Mohammad N. Uddin,†,‡ So-Lim Park,§ Seong-Il Lim,§ Da-Woon Jung,⊥ Darren R. Williams,⊥ and Won-Keun Oh*,‡ †

College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea Korea Bioactive Natural Material Bank, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea § Fermentation and Functionality Research Group, Korea Food Research Institute, Sungnam 463-746, Republic of Korea ⊥ New Drug Targets Laboratory, School of Life Sciences, Gwangju Institute of Science and Technology, 1 Oryong-Dong, Buk-Gu, Gwangju 500-712, Republic of Korea ‡

S Supporting Information *

ABSTRACT: As part of our ongoing search for new antidiabetic agents from medicinal plants, we found that a methanol extract of Morinda citrifolia showed potential stimulatory effects on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation of this active extract yielded two new lignans (1 and 2) and three new neolignans (9, 10, and 14), as well as 10 known compounds (3−8, 11−13, and 15). The absolute configurations of compounds 9, 10, and 14 were determined by ECD spectra analysis. Compounds 3, 6, 7, and 15 showed inhibitory effects on PTP1B enzyme with IC50 values of 21.86 ± 0.48, 15.01 ± 0.20, 16.82 ± 0.42, and 4.12 ± 0.09 μM, respectively. Furthermore, compounds 3, 6, 7, and 15 showed strong stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. This study indicated the potential of compounds 3, 6, 7, and 15 as lead molecules for antidiabetic agents.

D

iabetes mellitus has long been a threat to human health.1 The number of people with diabetes is expected to exceed 300 million globally by 2025.2 According to the American Diabetes Association, the number of people with diabetes in the United States was about 22.3 million, about 7% of the population. With the increase of diabetic patients, diabetes-related social expenses in 2012 were estimated to include direct medical costs of $176 billion and productivity reduction of $69 billion in the U.S.3 Although a wide range of antidiabetic agents have been used in the clinic, it is difficult for many patients to achieve targeted levels of glycosylated hemoglobin (HbA1C).4 Thus, the development of new antidiabetic agents is an urgent need, together with adequate therapeutic approaches to control this disease. Protein tyrosine phosphatases (PTPs) participate in intracellular signaling and metabolism by dephosphorylating tyrosine residues. Among a number of PTPs, such as SH2domain-containing phosphotyrosine phosphatase (SHP2), PTP-α, and leukocyte antigen-related tyrosine phosphatase (LAR), PTP1B has critical roles in the insulin receptor (IR) signaling pathway.5 PTP1B overexpression results in insulin© 2013 American Chemical Society and American Society of Pharmacognosy

resistant states, while PTP1B knockout mice display increased insulin sensitivity and show lower weight gain when consuming normal and high-fat diets.6 Leptin, which is secreted from adipose tissue, also controls food intake and weight loss in the hypothalamus. As PTP1B is also the key regulator of the leptin signaling pathway, it is highly expressed in hypothalamic tissues. PTP1B-deficient mice also show decreased leptin levels and hypersensitivity to leptin compared with wild-type littermates on low- and high-fat diets.7 Thus, PTP1B inhibitors could be useful in treating type 2 diabetes as well as obesity.8 The genus Morinda (Rubiaceae) comprises approximately 80 species and is found in many tropical regions. Morinda citrifolia L., also known as noni, has been utilized throughout southern Asia and the Pacific Islands for a long time.9 Its syncarpous fruit can grow 5−10 cm long. This plant has long been used as a traditional medicine for the treatment of diabetes, cholecystitis, menstrual cramps, asthma, lumbago, cancer, dysentery, and Received: July 2, 2013 Published: November 13, 2013 2080

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

Article

many other ailments.10−12 Diverse types of secondary metabolites including iridoid glycosides, triterpenoids, anthraquinones, lignans, neolignans, and polypropanoid derivatives have been discovered.9−13 In this regard and as part of our ongoing efforts to search for new potential antidiabetic agents from medicinal plants, we found that a MeOH extract of M. citrifolia showed stimulatory effects on glucose uptake in vitro using 2-[N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) as a fluorescent-tagged glucose probe in 3T3-L1 adipocyte cells. The 2-NBDG probe has been suggested as a useful reagent for discovering insulin mimetic compounds.14,15 Bioassay-guided fractionation of this active extract yielded two new lignans (1 and 2) and three new neolignans (9, 10, and 14), as well as 10 known compounds (3−8, 11−13, and 15). Furthermore, we evaluated the PTP1B inhibitory activity of compounds 1−15 and their stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Herein we discuss the purification, structure determination, and antidiabetic properties of these isolates.

d, J = 1.9 Hz, H-2′), 6.75 (1H, d, J = 8.2 Hz, H-5′), and 6.65 (1H, dd, J = 8.2, 1.9 Hz, H-6′). The 1H−1H COSY spectrum showed three spin systems: H-5/H-6, H-5′/6′, and H-7′/H-8′/ H-9′ (Figure 1). The HMBC correlations from H-7′ (δH 5.52) to C-1′ (δC 133.9), C-2′ (δC 113.6), and C-6′ (δC 118.4) (Figure 1) established the linkage from C-1′ to C-7′, which was also confirmed by the NOESY correlations from H-7′ (δH 5.52) to H-2′ (δH 6.72) and H-6′ (δH 6.65) (Figure 2). In addition, a broad singlet at δH 7.47 (H-7) showed HMBC correlations (Figure 1) to C-1 (δC 127.0), C-2 (δC 118.0), and C-6 (δC 125.1), as well as to an olefinic carbon at δC 122.5 (C-8), and an ester carbonyl carbon at δC 175.3 (C-9), which strongly suggested an α,β-unsaturated lactone moiety connected to C-1. Further HMBC analysis demonstrated three key correlations from H-7′ (δH 5.52) to C-8 (δC 122.5) and C-9 (δC 175.3) and from H-8′ (δH 3.64) to C-9 (δC 175.3) (Figure 1), which indicated a five-membered 7′,9-lactone ring in compound 1. These observations supported that compound 1 possessed a similar structure to those of phellinsin A17a,b and cis-(4)E-2(3,4-dihydroxyphenyl)-4-[(3,4-dihydroxyphenyl)methylene]tetrahydro-5-oxo-3-furancarboxylic acid.17c The major difference was the occurrence of a hydroxymethylene moiety (δC 62.6; δH 3.89 and 3.63) in 1 instead of the carboxylic group in phellinsin A, implying the replacement of the carboxylic group at C-8′ in phellinsin A by a hydroxymethylene group. The relative configuration of compound 1 was established by a NOESY experiment and comparison of NMR data with literature data. The NOESY correlation (Figure 2) from H-6 (δH 7.03) to H-8′ (δH 3.64) established the E-type Δ7,8 double bond,17a,b which was also confirmed by the similar chemical shifts of C-7 and C-8 between compound 1 [δC 140.8 (C-7), 122.5 (C-8) in methanol-d4] and phellinsin A [δC 140.9 (C-7), 121.6 (C-8) in methanol-d4].17a,b The trans relationship between H-7′ and H-8′ was established via the similar NMR patterns of H-7′ and C-7′ of compound 1 (δH 5.52, d, J = 4.5 Hz; δC 83.4 in methanol-d4) and those of phellinsin A (δH 5.59, d, J = 2.5 Hz; δC 83.7 in methanol-d4),17a,b which was also supported by the different coupling constant of H-7′ between compound 1 (J = 4.5 Hz) and cis-(4)E-2-(3,4-dihydroxyphenyl)-4-[(3,4-dihydroxyphenyl)methylene]tetrahydro-5-oxo-3-furancarboxylic acid (J = 7.6 Hz).17c Thus, compound 1 was elucidated as trans-(3)E-3-(3,4-dihydroxybenzylidene)-5-(3,4dihydroxyphenyl)-4-(hydroxymethyl)dihydrofuran-2(3H)-one. Compound 2 was obtained as a brown, amorphous powder. The molecular formula of 2 was assigned as C18H16O7 by HREIMS at m/z 344.0894 [M]+ (calcd 344.0896) combined with the 13C NMR data (Table 1). The IR absorptions suggested the existence of OH (3274 cm−1), CO (1747 cm−1), and CC (1648 cm−1) functionalities. Its UV spectrum showed absorption bands at λmax 231 and 282 nm, indicating the presence of benzene moieties.8,16 Its 1H and 13C NMR data indicated the existence of two 1,3,4-trisubstituted benzene systems, a pair of oxymethylene protons, two oxymethine signals, and an ester carbonyl carbon (Table 1). Further analysis of the 1H−1H COSY, HMBC (Figure 1), and NOESY (Figure 2) spectra determined that compound 2 possessed the same structure, including the relative configuration, as that of the known lignan (+)-3,4,3′,4′-tetrahydroxy-9,7′α-epoxylignano-7α,9′-lactone, which was also present in noni.8 However, compound 2 gave a negative specific rotation [α]25 D −5.8 (c 0.1, MeOH), opposite that of (+)-3,4,3′,4′-tetrahydroxy-9,7′αepoxylignano-7α,9′-lactone ([α]D25 +7.5 (c 0.2, MeOH)).8

■

RESULTS AND DISCUSSION Two new lignans (1 and 2), three new neolignans (9, 10, and 14), and 10 known compounds (3−8, 11−13, and 15) were purified from a MeOH extract of M. citrifolia by successive chromatographic procedures, including silica gel, RP-C8, MPLC, and HPLC. Compound 1 was obtained as a yellowish and amorphous powder with [α]25 D −5.6 (c 0.1, MeOH). The molecular formula C18H16O7 was determined by a quasi-molecular ion peak at m/z 344.0898 [M]+ (calcd 344.0896) in the HREIMS, combined with its 13C NMR data (Table 1). The IR absorbance bands at 3394, 1747, and 1646 cm−1 indicated the presence of OH, C O, and CC functionalities, respectively. Strong UV bands at λmax 233 and 284 nm suggested the existence of benzene moieties.8,16 The 1H NMR spectrum of compound 1 displayed the existence of two pairs of ABX aromatic protons (Table 1): δH 7.08 (1H, d, J = 1.8 Hz, H-2), 6.83 (1H, d, J = 8.4 Hz, H-5), and 7.03 (1H, dd, J = 8.4, 1.8 Hz, H-6), as well as δH 6.72 (1H, 2081

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

Article

Table 1. NMR Spectroscopic Data (600 MHz, in methanol-d4) for Compounds 1, 2, 9, 10, and 14 1 no. 1 2 3 4 5 6 7 8

δH (J in Hz) 7.08, d (1.8)

6.83, d (8.4) 7.03 dd (8.4, 1.8) 7.47, br s

9

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

OMe

6.72, d (1.9)

6.75, d (8.2) 6.65, dd (8.2, 1.9) 5.52, d (4.5) 3.64, m 3.89, dd (16.8, 9.0) 3.63, dd (16.8, 8.4)

2 δC

9

δH (J in Hz)

δC 132.7, 114.0, 147.1, 146.6, 116.6, 118.6,

10

δH (J in Hz)

δC

C CH 6.77, d (1.2) C C CH 6.78, d (7.8) CH 6.68, dd (7.8, 1.2) 140.8, CH 5.30, d (3.6) 122.5, C 3.27, m

C CH 7.02, d (1.8) C C CH 6.85, d (8.5) CH 6.92, dd (8.5, 1.8) 87.2, CH 4.92, d (8.0) 52.0, CH 4.15, m

129.3, 112.2, 149.4, 148.6, 116.4, 121.9,

175.3, C

73.9, CH2

127.0, 118.0, 146.9, 149.6, 116.9, 125.1,

133.9, 113.6, 146.9, 147.0, 116.6, 118.4,

4.25, dd (9.6, 7.2) 3.97, dd (9.6, 4.2)

C CH 6.82, d (1.8) C C CH 6.75, d (8.4) CH 6.70, dd (8.4, 1.8) 83.4, CH 5.16, d (3.6) 51.4, CH 3.57, dd (9.0, 3.6) 62.6, CH2

3.72, dd (12.6, 2.4) 3.50, dd (12.6, 4.1)

133.4, 114.2, 146.9, 146.3, 116.4, 118.4,

C CH 7.56, d (1.8) C C C 7.00, d (8.0) C 7.57, dd (8.0, 1.8) 85.2, CH 54.5, CH

14

δH (J in Hz)

δC

C CH 6.87, d (1.8) C C CH 6.79, d (8.2) CH 6.77, dd (8.2, 1.8) 77.7, CH 4.89, d (7.8) 80.5, CH 4.14, m

129.0, 115.6, 146.9, 147.5, 116.5, 120.5,

62.1, CH2

62.0, CH2

124.7, 119.8, 145.0, 149.0, 117.8, 124.7,

3.73, dd (12.6, 2.4) 3.50, dd (12.6, 4.2)

C CH 7.45, d (1.6) C C CH 7.13, d (8.0) CH 7.49, dd (8.0, 1.6) 169.5, C 9.80, s

δH (J in Hz) C CH 6.82, d (1.8) C C CH 6.76, d (8.4) CH 6.71, dd (8.4, 1.8)

77.6, CH 80.8, CH

132.1, 119.1, 146.0, 151.1, 118.7, 125.5,

5.18, d (3.3) 4.45, ddd (8.4, 3.6, 3.3) 3.54, dd (12.0, 3.6) 3.46, dd (12.0, 8.4)

C CH 7.16, d (1.8) C C CH 6.98, d (8.4) CH 7.12, dd (8.4, 1.8)

192.9, CH 7.48, d (15.9) 6.35, d (15.9)

179.9, C

δC 130.5, 114.6, 146.7, 144.0, 116.4, 119.3,

C CH C C CH CH

77.1, CH 79.5, CH 60.2, CH2

129.2, 118.5, 145.4, 146.2, 119.1, 123.1,

C CH C C CH CH

146.7, CH 117.4, CH 170.5, C

3.89, s

56.6, CH3

Figure 2. NOESY correlations of compounds 1 and 2.

C17H16O7 on the basis of a quasi-molecular ion at m/z 332.0898 [M]+ (calcd 332.0896) in the HREIMS, combined with 13C NMR data (Table 1). Its IR spectrum suggested the existence of OH (3371 cm−1), CO (1720 cm−1), and CC (1603 cm−1) functionalities. The UV spectrum showed absorption bands at λmax 224, 253, and 299 nm, indicating the presence of benzene moieties.18,19 The 1H and 13C NMR data of 9 indicated the occurrence of two 1,3,4-trisubstituted benzene systems [δH 7.02 (1H, d, J = 1.8 Hz, H-2), 6.85 (1H, d, J = 8.5 Hz, H-5), 6.92 (1H, dd, J = 8.5, 1.8 Hz, H-6), 7.56 (1H, d, J = 1.8 Hz, H-2′), 7.00 (1H, d, J = 8.0 Hz, H-5′), and 7.57 (1H, dd, J = 8.0, 1.8 Hz, H-6′); δC 129.3 (C, C-1), 112.2 (CH, C-2), 149.4 (C, C-3), 148.6 (C, C-4), 116.4 (CH, C-5), 121.9 (CH, C-6), 124.7 (C, C-1′), 119.8 (CH, C-2′), 145.0 (C, C-3′), 149.0 (C, C-4′), 117.8 (CH, C-5′), and 124.7 (CH, C-6′)], two oxymethine signals [δH 4.92 (d, J = 8.0 Hz, H-7) and 4.15 (m, H-8); δC 77.7 (C-7) and 80.5 (C-8)], oxymethylene signals [δH 3.72 (dd, J = 12.6, 2.4 Hz, H-9a) and 3.50 (dd, J = 12.6, 4.1 Hz,

Figure 1. 1H−1H COSY () and HMBC (↷) correlations of compounds 1, 2, 9, 10, and 14.

Consequently, compound 2 was determined as the new lignan (−)-3,4,3′,4′-tetrahydroxy-9,7′β-epoxylignano-7β,9′-lactone. Compound 9, obtained as a brown, amorphous powder with [α]25 D +4.7 (c 0.2, MeOH), possessed a molecular formula of 2082

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

Article

H-9b); δC 62.1 (CH2, C-9)], a carboxylic group (δC 169.5, C7′), and an oxymethyl signal [δH 3.89 (s); δC 56.6]. The carboxylic and oxymethyl groups were attached to C-1′ and C3, respectively, based on HMBC correlations from H-2′ (δH 7.56) and H-6′ (δH 7.57) to the carboxylic carbon at δC 169.5 and from the O-methyl proton at δH 3.89 to C-3 (δC 149.4) (Figure 1). Furthermore, the 1H−1H COSY spectrum showed a spin system H-7/H-8/H2-9, which was connected to C-1 on the basis of HMBC correlations from H-7 (δH 4.92) to C-1 (δC 129.3), C-2 (δC 112.2), and C-6 (δC 121.9). In addition, H2-9 (δH 3.72 and 3.50) showing a clear HMBC correlation to C-3′ (δC 145.0) (Figure 1), combined with the characteristic chemical shifts of C-8 (δC 80.5) and C-3′ (δC 145.0) (Table 1), indicated the linkage pattern C-8−O−C-3′. Although no HMBC correlation from H-7 to C-4′ could be observed, the diagnostic chemical shifts of C-7 (δC 77.7) and C-4′ (δC 149.0), combined with the established molecular formula, C17H16O7, suggested the linkage pattern of C-7−O−C-4′. The above evidence indicated that compound 9 possessed a similar structure to that of arteminorin D,20 with the replacement of the side chain in arteminorin D by a carboxylic group in compound 9. A large coupling constant between H-7 and H-8 (J7,8 = 8.0 Hz) indicated a trans relationship of the two protons.20 The absolute configuration of 9 was determined using the electronic circular dichroism (ECD) exciton chirality method.21 The UV spectrum of 9 showed a strong absorption at 224 nm attributable to the benzene moiety (π → π*). Corresponding to this UV absorption, the ECD spectrum of 9 showed a negative Cotton effect at 225 nm due to the transition interaction between two different benzene moieties in the structure. The above information demonstrated a negative chirality for 9, and the two aforementioned chromophores should be oriented counterclockwise in space (Figure 3A). Thus, the absolute configuration of 9 was determined as (7R, 8R). Compound 9 was elucidated as (7R,8R)-3-methoxy-1′carboxy-4′,7-epoxy-8,3′-oxyneolignan-4,9-diol. Compound 10 was isolated as a yellowish, amorphous powder with [α]25 D +2.5 (c 0.2, MeOH). Its molecular formula was determined as C16H14O6 from a quasi-molecular ion peak at m/z 302.0794 [M]+ (calcd 302.0790) in the HREIMS. Strong UV absorptions at λmax 224, 252, and 300 nm and IR bands at 3361 (OH), 1705 (CO), and 1604 (CC) cm−1 suggested a similar skeleton of 10 compared to 9. This observation was supported by 1H and 13C NMR data of 10 (Table 1): two 1,3,4-trisubstituted benzene systems [δH 6.87 (1H, d, J = 1.8 Hz, H-2), 6.79 (1H, d, J = 8.2 Hz, H-5), 6.77 (1H, dd, J = 8.2, 1.8 Hz, H-6), 7.45 (1H, d, J = 1.6 Hz, H-2′), 7.13 (1H, d, J = 8.0 Hz, H-5′), 7.49 (1H, dd, J = 8.0, 1.6 Hz, H6′); δC 129.0 (C, C-1), 115.6 (CH, C-2), 146.9 (C, C-3), 147.5 (C, C-4), 116.5 (CH, C-5), 120.5 (CH, C-6), 132.1 (C, C-1′), 119.1 (CH, C-2′), 146.0 (C, C-3′), 151.1 (C, C-4′), 118.7 (CH, C-5′), and 125.5 (CH, C-6′)], two oxymethine signals [δH 4.89 (d, J = 7.8 Hz, H-7) and 4.14 (m, H-8); δC 77.6 (C-7) and 80.8 (C-8)], and oxymethylene signals [δH 3.73 (dd, J = 12.6, 2.4 Hz, H-9a) and 3.50 (dd, J = 12.6, 4.2 Hz, H-9b); δC 62.0 (C-9)]. However, the O-methyl signal [δH 3.89 (s); δC 56.6] and the carboxylic group [δC 169.5 (C-7′)] in compound 9 disappeared, and a formyl group [δH 9.80 (s); δC 192.9] was observed in the NMR spectra of 10, suggesting replacement of the O-methyl group by a hydroxy group and of the carboxylic group by a formyl group in the structure of 10. Furthermore, the planar structure of 10 was confirmed by the HMBC experiment as shown in Figure 1. The coupling constant J7,8 =

Figure 3. ECD and UV spectra of compounds 9 (A), 10 (B), and 14 (C). The arrow denotes the electronic transition dipole of the chromophores.

7.8 Hz implied a trans relationship between H-7 and H-8.20 Similar to 9, the absolute configuration of 10 was also determined by the ECD exciton chirality method.21 Corresponding to strong UV absorption at 224 nm, which is attributable to the benzene moiety (π → π*), the ECD spectrum of 10 showed a negative Cotton effect at 226 nm due 2083

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

Article

significant activity with an IC50 value of 4.12 ± 0.09 μM and was used as a positive control in this assay (Table 2). In

to the transition interaction between two different benzene moieties in the structure (Figure 3B). Thus, the negative chirality determined the absolute configuration of 10 being (7R, 8R). Consequently, compound 10 was determined as (7R,8R)3,4,9-trihydroxy-4′,7-epoxy-8,3′-oxyneolignan-1′-al. Compound 14, a yellow, amorphous powder with [α]25 D −26.8 (c 0.1, MeOH), was analyzed for the molecular formula C18H16O7 on the basis of a quasi-molecular ion at m/z 344.0896 [M]+ (calcd 344.0896) in the HREIMS. The UV bands at λmax 224, 253, and 310 nm and IR absorptions at 3275 (OH), 1688 (CO), and 1607 (CC) cm−1 suggested a similar carbon skeleton of 14 to those of 9 and 10. This observation was also supported by the 1H and 13C NMR data (Table 1), which showed two pairs of ABX aromatic protons [δH 6.82 (1H, d, J = 1.8 Hz, H-2), 6.76 (1H, d, J = 8.4 Hz, H5), 6.71 (1H, dd, J = 8.4, 1.8 Hz, H-6), 7.16 (1H, d, J = 1.8 Hz, H-2′), 6.98 (1H, d, J = 8.4 Hz, H-5′), 7.12 (1H, dd, J = 8.4, 1.8 Hz, H-6′); δC 130.5 (C, C-1), 114.6 (CH, C-2), 146.7 (C, C-3), 144.0 (C, C-4), 116.4 (CH, C-5), 119.3 (CH, C-6), 129.2 (C, C-1′), 118.5 (CH, C-2′), 145.4 (C, C-3′), 146.2 (C, C-4′), 119.1 (CH, C-5′), and 123.1 (CH, C-6′)], two oxymethine signals [δH 5.18 (d, J = 3.3 Hz, H-7) and 4.45 (ddd, J = 8.4, 3.6, 3.3 Hz, H-8); δC 77.1 (C-7) and 79.5 (C-8)], and oxymethylene signals [δH 3.54 (dd, J = 12.0, 3.6 Hz, H-9a) and 3.46 (dd, J = 12.0, 8.4 Hz, H-9b); δC 60.2 (C-9)]. Further analysis showed that the major difference in NMR spectra of compounds 10 and 14 was the disappearance of the formyl group and introduction of one olefinic bond [δH 7.48 (d, J = 15.9 Hz) and 6.35 (d, J = 15.9 Hz); δC 146.7 (CH) and 117.4 (CH)] and a carboxylic group [δC 170.5 (C)] in 14. The above observations indicated the existence of an α,β-unsaturated carboxylic group connected to C-1′ in 14, which was further confirmed by HMBC correlations from H-7′ [δH 7.48 (d, J = 15.9 Hz)] to C-1′ (δC 129.2), C-2′ (δC 118.5), and C-6′ (δC 123.1) (Figure 1). The coupling constants J7,8 = 3.3 Hz and J7′,8′ = 15.9 Hz indicated the cis relationship between H-7 and H-8 and E-type Δ7′,8′ double bond.20 Our efforts to determine the absolute configuration of compound 14 by the ECD exciton chirality method failed due to no clear Cotton effects corresponding to the strong UV absorption at 224 nm (Figure 3C). However, compound 14 showed a similar ECD spectrum to those of cis-(2R,3S)-3-methyl-2-phenyl-l,4-benzodioxane and eusiderin C,22 which established the absolute configuration of 14 as (7R, 8S). Therefore, compound 14 was elucidated as (7′E),(7R,8S)-3,4,9-trihydroxy-4′,7-epoxy-8,3′-oxyneolignan-7′en-8′-oic acid. Based on NMR, MS, and optical rotation data and comparison with literature values, the known compounds were elucidated as episesamin 2,6-dicatechol (3),16 (−)-3,3′bisdemethylpinoresinol (4),8 (−)-pinoresinol (5),8,23 lirioresinol B (6),24 lirioresinol B dimethyl ether (7),25 americanin A (8),9 arteminorin D (11),20 rel-(7α,8β)-3-methoxy-4′,7-epoxy8,3′-oxyneolignan-4,9,9′-triol (12),26 americanoic acid A (13),19 and ursolic acid (15).27 Herein we also established the 1H and 13C NMR data of compound 3 (Supporting Information) for the first time based on 1H−1H COSY, HSQC, and HMBC techniques. Protein tyrosine phosphatase 1B (PTP1B) has been established as an outstanding drug target for the treatment of diabetes.28−30 Several inhibitors of PTP1B have been developed for the treatment of type 2 diabetes such as ertiprotafib.31 In this regard, we measured the inhibitory effects of compounds 1−15 on the PTP1B enzyme. Ursolic acid (15) showed

Table 2. Inhibitory Effects of Compounds 1−15 on PTP1B Enzyme compound

inhibitory effect (IC50, μM)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15d

NAb NA 21.86 ± 0.48 >30 18.69 ± 0.28 15.01 ± 0.20 16.82 ± 0.42 NA 19.56 ± 0.37 NTc >30 >30 >30 >30 4.12 ± 0.09

a Values are expressed as mean ± SD of three replicates. Seven concentration points were set for establishment of the PTP1B inhibition curve to calculate IC50 values. bNA: not active. Compounds showed no activity at 50 μM. cNT: not tested. dCompound 15 was used as the positive control.

addition, compounds 3, 5−7, and 9 showed moderate inhibitory effects on the PTP1B enzyme with IC50 values ranging from 15.01 ± 0.20 to 21.86 ± 0.48 μM, while compounds 4 and 11−14 showed weak activity (Table 2). We further evaluated the stimulatory effects of compounds 1−15 on glucose uptake in vitro using 2-NBDG in 3T3-L1 adipocyte cells.32 2-NBDG has been reported as a useful fluorescenttagged glucose probe for discovering insulin mimetic compounds.14,15 Screening indicated significant stimulatory effects of compounds 3, 6, 7, and 15 on 2-NBDG uptake in 3T3-L1 cells (Figure 4). Compared with that of DMSO, stimulatory effects of rosiglitazone (positive control) and compounds 3, 6, 7, and 15 were around 1.70 ± 0.19, 1.52 ± 0.08, 1.46 ± 0.08, 1.45 ± 0.16, and 1.82 ± 0.12 times, respectively. Further evaluation of compounds 3, 6, 7, and 15 at concentrations of 20, 10, and 5 μM demonstrated their dosedependent stimulatory effects on 2-NBDG uptake in 3T3-L1 cells (Supporting Information). Similar structure−activity relationships were observed in PTP1B and 2-NBDG assays. Ursolic acid and 7,9′:9,7′-diepoxylignans have stronger inhibitory effects on the PTP1B enzyme than 4′,7-epoxy-8,3′oxyneolignans, and they also demonstrated stronger stimulatory effects on 2-NBDG uptake. Furthermore, in the 7,9′:9,7′diepoxylignan group, isomers 3 and 4 showed significantly different activity in both assays, which may be due to the difference in the relative configuration at C-7′. Compared with 4, compounds 5−7 possessed more O-methyl groups and showed stronger activity than 4, which implied that the Omethyl groups positively influence the inhibitory effects on the PTP1B enzyme and stimulatory effects on 2-NBDG uptake. The worldwide prevalence of diabetes mellitus is increasing at a rapid rate. As insulin resistance is an important cause in the pathogenesis of type-2 diabetes, reconstitution of the insulin sensitivity is one of the key strategies for the treatment of type2 diabetes.33 However, some thiazolidinediones as a repre2084

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

Article

Figure 4. Stimulatory effects of compounds 1−15 on 2-NBDG uptake in 3T3-L1 adipocyte cells (Rosi: rosiglitazone). The tested compounds (1− 15), DMSO (vehicle), and rosiglitazone (positive control, 400 μg/mL) were used to treat the cells for 90 min. Compounds 1, 3, 6, and 9 were used at a concentration of 20 μM, while other compounds were used at 40 μM. partitioned with EtOAc (4 × 1.5 L). The EtOAc fraction was concentrated to give a dark residue (94.6 g). Part of this residue (90 g) was applied to a silica gel open column (10 × 60 cm) with a gradient of n-hexane/acetone (20:1 to 1:20) to yield compound 15 (1 g) and six other fractions (MC-1−MC-6) according to their TLC patterns. Fraction MC-5 (15 g) was fractionated using an MPLC system (VSP1200 ceramic pump (EYELA, Tokyo, Japan) with a plastic column (6.0 × 20 cm) filled with RP-C8 (75 μm particle size), eluted with MeOH/H2O (1:10 to 10:1, each 2.5 L), to give five subfractions (MC5.1−MC-5.5). Subfraction MC-5.2 was further purified on a Gilson HPLC system [mobile phase: MeOH in H2O containing 0.1% HCO2H (0−35 min: 48% MeOH); flow rate: 2 mL/min; UV detection at 205 and 254 nm], resulting in the isolation of compounds 4 (155.0 mg; tR = 20.1 min) and 6 (30.0 mg; tR = 29.2 min). Subfraction MC-5.3 was also purified by HPLC [mobile phase: MeCN in H2O containing 0.1% HCO2H (0−45 min: 24% MeCN); flow rate: 2 mL/min; UV detection at 205 and 254 nm], yielding compounds 3 (4.0 mg; tR = 25.9 min), 8 (1.2 mg; tR = 35.5 min), 9 (2.9 mg; tR = 34.8 min), and 10 (1.5 mg; tR = 37.5 min). Subfraction MC-5.4 was purified by HPLC [mobile phase: MeOH in H2O containing 0.1% HCO2H (0−35 min: 50% MeOH); flow rate: 2 mL/min; UV detection at 205 and 254 nm], giving compounds 5 (3.7 mg; tR = 20.1 min) and 11 (16.0 mg; tR = 30.0 min). With the same prodedure, compound 7 (7.8 mg; tR = 22.2 min) was purified from subfraction MC-5.5 by HPLC [mobile phase: MeOH in H2O containing 0.1% HCO2H (0−30 min: 60% MeOH); flow rate: 2 mL/min; UV detection at 205 and 254 nm]. Fraction MC-6 (20 g) was further fractionated by MPLC systems with a plastic column (6.0 × 20 cm) filled with RP-C8, eluted with MeOH/H2O (1:15 to 1:1, each 3 L), affording compound 12 (20 mg) and five other subfractions (MC6.1−MC-6.5). Subfraction MC-6.3 was purified by HPLC [mobile phase: MeCN in H2O containing 0.1% HCO2H (0−50 min: 20% MeCN); flow rate: 2 mL/min; UV detection at 205 and 254 nm], affording compounds 1 (2.3 mg; tR = 27.4 min) and 2 (20.8 mg; tR = 37.6 min). Similarly, compounds 13 (14.0 mg; tR = 28.5 min) and 14 (4.5 mg; tR = 22.2 min) were purified from subfraction MC-6.4 by HPLC [mobile phase: MeCN in H2O containing 0.1% HCO2H (0−40 min: 26% MeCN); flow rate: 2 mL/min; UV detection at 205 and 254 nm]. trans-(3)E-3-(3,4-Dihydroxybenzylidene)-5-(3,4-dihydroxyphenyl)-4-(hydroxymethyl)dihydrofuran-2(3H)-one (1): yellowish, amorphous powder; [α]25 D −5.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 233 (4.10), 284 (3.60) nm; IR (KBr) νmax 3394, 2919, 1747, 1646, 1508, 1033, 677 cm−1; 1H and 13C NMR data, Table 1; HREIMS m/z 344.0898 [M]+ (calcd for C18H16O7, 344.0896).

sentative class of insulin sensitizers have been withdrawn from the market due to severe side effects, which include edema, weight gain, and the increased risk of heart failure.31 Therefore, there is an urgent demand to discover new antidiabetic agents acting as insulin mimics and/or insulin sensitizers. M. citrifolia (noni) has been utilized as a folk remedy in treating diabetes for a long time. In this study, lignans 3, 6, 7, and ursolic acid (15) isolated from noni not only showed significant (15) or moderate (3, 6, and 7) inhibitory effects on the PTP1B enzyme but also showed potential stimulatory effects on 2-NBDG uptake, which suggested the potential of compounds 3, 6, 7, and 15 as insulin mimetics for developing antidiabetic agents; their antidiabetic properties may be related to their inhibitory effects on the PTP1B enzyme. Furthermore, this study shows that lignans and ursolic acid may be the compounds responsible for the antidiabetic properties of noni.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on an Autopol IV A7040-12 automatic polarimeter using a 100 mm glass microcell. UV spectra were measured with an Optizen 3220UV spectrophotometer (Mekasys Co. Ltd., Daejon, Korea). The ECD spectra were obtained on a JASCO J-710 spectropolarimeter with MeOH (ECD/ORD spectropolarimeter). IR spectra were recorded in a Nicolet 6700 FT-IR (Thermo Electron Corp. USA). 1D and 2D NMR spectra were measured on a Varian Unity Inova 600 MHz spectrometer (Korea Basic Science Institute, Gwangju Center, Korea). HREIMS data were obtained from a Micromass QTOF2 mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography was conducted with silica gel (63−200 μm particle size) from Merck. RP-C18 F254 and silica gel 60 F254 plates were used for checking TLC pattern. HPLC was conducted by using a Gilson System with an Optima Pak C18 column (10 μm particle size, 10 × 250 mm, RS Tech, Korea) and a UV detector. Analytical grade solvents were used for extraction and isolation. Plant Material. The dried powder of M. citrifolia was purchased from Babsaewoo Company in Suwon City, Republic of Korea, on May 25, 2012. The sample was botanically identified by one of the authors (W.-K. Oh), and a voucher specimen (SNU-25-2012) has been deposited at College of Pharmacy, Seoul National University, Korea. Extraction and Isolation. The M. citrifolia powder (1.58 kg) was extracted with MeOH (3 × 5 L, 5 h each) at room temperature using sonication. After removing the solvent under reduced pressure, the dried extract (155 g) was suspended in H2O (1.5 L) and then 2085

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

■

(−)-3,4,3′,4′-Tetrahydroxy-9,7′β-epoxylignano-7β,9′-lactone (2): brown, amorphous powder; [α]25 D −5.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (4.11), 282 (3.71) nm; IR (KBr) νmax 3274, 2967, 1747, 1648, 1288, 828 cm−1; 1H and 13C NMR data, Table 1; HREIMS m/z 344.0894 [M]+ (calcd for C18H16O7, 344.0896). (7R,8R)-3-Methoxy-1′-carboxy-4′,7-epoxy-8,3′-oxyneolignan-4,9diol (9): brown, amorphous powder; [α]25 D +4.7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 224 (4.30), 253 (4.12), 299 (4.01) nm; ECD (MeOH, Δε) λ 222 (−1.24), 229 (−1.55) nm; IR (KBr) νmax 3371, 2935, 1720, 1603, 1280, 687 cm−1; 1H and 13C NMR data, Table 1; HREIMS m/z 332.0898 [M]+ (calcd for C17H16O7, 332.0896). (7R,8R)-3,4,9-Trihydroxy-4′,7-epoxy-8,3′-oxyneolignan-1′-al (10): yellowish, amorphous powder; [α]25 D +2.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 224 (4.26), 252 (4.11), 300 (4.02) nm; ECD (MeOH, Δε) λmax 223 (−0.67), 230 (−0.85); IR (KBr) νmax 3361, 2921, 1705, 1604, 1015, 691 cm−1; 1H and 13C NMR data, Table 1; HREIMS m/z 302.0794 [M]+ (calcd for C16H14O6, 302.0790). (7′E),(7R,8S)-3,4,9-Trihydroxy-4′,7-epoxy-8,3′-oxyneolignan-7′en-8′-oic acid (14): yellow, amorphous powder; [α]25 D −26.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (4.25), 253 (4.13), 310 (4.10) nm; IR (KBr) νmax 3275, 2922, 1688, 1607, 1012, 816 cm−1; 1H and 13 C NMR data, Table 1; HREIMS m/z 344.0896 [M]+ (calcd for C18H16O7, 344.0896). PTP1B Inhibition Assay. PTP1B enzyme activity was measured by the catalyzed amount of p-nitrophenyl phosphate by enzyme as previously described.34 Briefly, 0.05−0.1 μg of PTP1B (BIOMOL International L.P., Plymouth Meeting, PA, USA) and 4 mM p-NPP in a buffer containing 1 mM dithiothreitol, 0.1 M NaCl, 1 mM EDTA, and 50 mM citrate (pH 6.0), with or without test compounds, were added as 100 μL of a final volume to each of the 96 well. After the reaction mixture was incubated at 37 °C for 30 min, 10 M NaOH was added to quench the reaction. PTP1B enzyme activity was determined by the amount of produced p-nitrophenol at 405 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control, which did not contain PTP1B enzyme. Cell Culture and Induction of 3T3-L1 Adipocytes. 3T3-L1 preadipocytes were maintained for 48 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with pen/strep and 10% calf serum in an atmosphere of 5% CO2 at 37 °C. To induce adipogenesis, the media was changed to DMEM supplemented with 1 μg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 2 μg/mL dexamethasone, 10% fetal bovine serum (FBS), 1 μM rosiglitazone, and pen/strep and further cultured for 48 h. Every 2 days thereafter, the cells were incubated with fresh DMEM supplemented with 1 μM rosiglitazone, 10% FBS, 1 μg/mL insulin, and pen/strep. 3T3-L1 adipocytes at 7 days after the induction of differentiation were used for experiments. Adipocyte-Based Measurement of 2-NBDG Uptake. 2-NBDG uptake in 3T3-L1 adipocytes was measured as previously described with some modifications.32 Briefly, differentiated 3T3-L1 adipocytes (3 × 104 cells/well) were seeded in a 24-well tissue culture plate (BD Falcon, NJ, USA) and cultured in glucose-free culture media supplemented with 10% FBS and pen/strep for treating 2-NBDG with or without compounds of interest. The culture media was replaced with low-glucose, serum-free DMEM for 3 h. After that the media was changed with DMEM containing test compound, and rosiglitazone served as a positive control at a concentration of 400 μg/ mL. The media was changed with DMEM containing 50 μM 2-NBDG after 30 min. Adipocytes after 1 h were washed twice using prewarmed phosphate-buffered saline (PBS) and lysed by treating with 70 μL of 0.1 M K3PO4 and 1% Triton X-100 in PBS for 10 min in the dark. Thirty microliters of DMSO was added, and the thus formed solution was mixed by using a multichannel pipet. The NBDG signal was recorded on a VICTOR X3 multilabel plate reader (PerkinElmer, MA, USA) at 450 nm excitation and 535 nm emission. Statistical Analysis. Data are represented as a mean of triplicate assays ± SD. For statistical analysis of the data for single comparison, the significance between means was determined by t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control.

Article

ASSOCIATED CONTENT

S Supporting Information *

1D (1H and 13C) and 2D (HSQC and HMBC) NMR spectra for compounds 1, 2, 9, and 10, 1H and 13C NMR spectra for compound 14, and figure of the dose-dependent stimulatory effects of compounds 3, 6, 7, and 15 on 2-NBDG uptake in 3T3-L1 cells, as well as 1H and 13C NMR data of compound 3, are available free of charge via the Internet at http://pubs.acs. org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: +82-02-880-7872. E-mail: [email protected]. Author Contributions ∥

P.-H. Nguyen and J.-L. Yang contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported in part by a grant from the National Research Foundation of Korea (NRF) (2012R1A2A2A01009417) and by Korea Institute for Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET), Korea Ministry of Agriculture, Food and Rural Affairs.

■

REFERENCES

(1) Zimmet, P.; Alberti, K. G. M. M.; Shaw, J. Nature 2001, 414, 782−787. (2) Marx, J. Science 2002, 296, 686−689. (3) American Diabetes Association.. Diabetes Care 2013, 36, 1033− 1046. (4) Washburn, W. N. J. Med. Chem. 2009, 52, 1785−1794. (5) Bettaieb, A.; Matsuo, K.; Matsuo, I.; Wang, S.; Melhem, R.; Koromilas, A. E.; Haj, F. G. PLoS One 2012, 7, e34412: 1−10. (6) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, C. C.; Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544−1548. (7) Zabolotny, J. M.; Bence-Hanulec, K. K.; Stricker-Krongrad, A.; Haj, F.; Wang, Y.; Minokoshi, Y.; Kim, Y. B.; Elmquist, J. K.; Tartaglia, L. A.; Kahn, B. B.; Neel, B. G. Dev. Cell 2002, 2, 489−495. (8) Ramachandran, C.; Kennedy, B. P. Curr. Top. Med. Chem. 2003, 3, 749−757. (9) Deng, S.; Palu, A. K.; West, B. J.; Su, C. X.; Zhou, B. N.; Jensen, J. C. J. Nat. Prod. 2007, 70, 859−862. (10) Su, B. N.; Pawlus, A. D.; Jung, H. A.; Keller, W. J.; McLaughlin, J. L.; Kinghorn, A. D. J. Nat. Prod. 2005, 68, 592−595. (11) Akihisa, T.; Matsumoto, K.; Tokuda, H.; Yasukawa, K.; Seino, K.; Nakamoto, K.; Kuninaga, H.; Suzuki, T.; Kimura, Y. J. Nat. Prod. 2007, 70, 754−757. (12) Deng, Y.; Chin, Y. W.; Chai, H.; Keller, W. J.; Kinghorn, A. D. J. Nat. Prod. 2007, 70, 2049−2052. (13) Pawlus, A. D.; Su, B. N.; Keller, W. J.; Kinghorn, A. D. J. Nat. Prod. 2005, 68, 1720−1722. (14) Kim, W. H.; Lee, J.; Jung, D. W.; Williams, D. R. Sensors (Basel) 2012, 12, 5005−5027. (15) Lee, J.; Jung, D. W.; Kim, W. H.; Um, J. I.; Yim, S. H.; Oh, W. K.; Williams, D. R. ACS Chem. Biol. 2013, 8, 1803−1814. (16) Miyake, Y.; Fukumoto, S.; Okada, M.; Salaida, K.; Nakamura, Y.; Osawa, T. J. Agric. Food Chem. 2005, 53, 22−27. (17) (a) Hwang, E. I.; Yun, B. S.; Kim, Y. K.; Kwon, B. M.; Kim, H. G.; Lee, H. B.; Jeong, W. J.; Kim, S. U. J. Antibiot. 2000, 53, 903−911. (b) Kim, E.; Lee, H. K.; Hwang, E.-I.; Kim, S.-U.; Lee, W. S.; Lee, S.; 2086

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087

Journal of Natural Products

Article

Jung, S.-H. Synth. Commun. 2005, 35, 1231−1238. (c) Wan, X.; Liu, H.; Huang, X.; Luo, J.; Kong, L. Can. J. Chem. 2008, 86, 821−830. (18) Schwarz, B.; Hofmann, T. J. Agric. Food Chem. 2009, 57, 3729− 3737. (19) Kamiya, K.; Tanaka, Y.; Endang, H.; Umar, M.; Satake, T. J. Agric. Food Chem. 2004, 52, 5843−5848. (20) He, Z. Z.; Yan, J. F.; Song, Z. J.; Ye, F.; Liao, X.; Peng, S. L.; Ding, L. S. J. Nat. Prod. 2009, 72, 1198−1201. (21) Yang, J. L.; Shi, Y. P. Phytochemistry 2012, 76, 124−132. (22) Arnoldi, A.; Merlini, L. J. Chem. Soc., Perkin Trans. 1 1985, 2555−2557. (23) Abdel-Kader, M. S.; Wisse, J.; Evans, R.; Werff, H.; Kingston, D. G. I. J. Nat. Prod. 1997, 60, 1294−1297. (24) Michel, S.; Tillequin, F.; Koch, M.; Assi, L. A. J. Nat. Prod. 1982, 45, 489−494. (25) Pan, J. X.; Hensens, O. D.; Zink, D. L.; Chang, M. N.; Hwang, S. B. Phytochemistry 1987, 26, 1377−1379. (26) Seca, A. M. L.; Silva, A. M. S. Phytochem. Lett. 2010, 3, 126−128. (27) Lee, K. H.; Lin, Y. M.; Wu, T. S.; Zhang, D. C.; Yamagishi, T.; Hayashi, T.; Hall, I. H.; Chang, J. J.; Wu, R. Y.; Yang, T. H. Planta Med. 1988, 53, 308−311. (28) Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373− 381. (29) Tiganis, T. FEBS J. 2013, 280, 445−458. (30) Teng, B. S.; Wang, C. D.; Yang, H. J.; Wu, J. S.; Zhang, D.; Zheng, M.; Fan, Z. H.; Pan, D.; Zhou, P. J. Agric. Food Chem. 2011, 59, 6492−6500. (31) Shrestha, S.; Bhattarai, B. R.; Cho, H.; Choi, J. K.; Cho, H. Bioorg. Med. Chem. Lett. 2007, 17, 2728−2730. (32) (a) Zou, C.; Wang, Y.; Shen, Z. J. Biochem. Biophys. Methods 2005, 64, 207−215. (b) Nitin, N.; Carlson, A. L.; Muldoon, T.; ElNaggar, A. K.; Gillenwater, A.; Richards-Kortum, R. Int. J. Cancer 2009, 124, 2634−2642. (c) Jung, D. W.; Ha, H. H.; Zheng, X.; Chang, Y. T.; Williams, D. R. Mol. BioSyst. 2011, 7, 346−358. (d) Millon, S. R.; Ostrander, J. H.; Brown, J. Q.; Raheja, A.; Seewaldt, V. L.; Ramanujam, N. Breast Cancer Res. Treat. 2011, 126, 55−62. (33) Barnett, A. H. Vasc. Health Risk Manag. 2006, 2, 59−67. (34) Cui, L.; Na, M.; Oh, H.; Bae, E. Y.; Jeong, D. G.; Ryu, S. E.; Kim, S.; Kim, B. Y.; Oh, W. K.; Ahn, J. S. Bioorg. Med. Chem. Lett. 2006, 16, 1426−1429.

2087

dx.doi.org/10.1021/np400533h | J. Nat. Prod. 2013, 76, 2080−2087