Guanidine Alkaloids from Plumbago zeylanica - Journal of Natural

The molecular formula was determined to be C11H19N3O2 by HRESIMS. .... Plumbagine E (6) had the molecular formula C15H27N3O5 as determined by ..... 19...
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Guanidine Alkaloids from Plumbago zeylanica Hai-Jian Cong, Shu-Wei Zhang, Yu Shen, Yong Zheng, Yu-Jie Huang, Wen-Qiong Wang, Ying Leng, and Li-Jiang Xuan* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Eleven new guanidine alkaloids, plumbagines A−G (2−8) and plumbagosides A−D (9−12), as well as two known analogues (1, 13), were isolated from the aerial parts of Plumbago zeylanica. Their structures were elucidated by spectroscopic analyses including 1D and 2D NMR, MS, IR, and CD methods. The absolute configuration of 1 was determined by single-crystal X-ray diffraction of its derivative (1a).

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atural products have played a significant role in drug discovery historically. One intriguing paradigm is metformin. On the basis of the identification of galegine, a toxic guanidine hemiterpene from Verbena encelioides (Cav.) and Galega of f icinalis L (French lilac), metformin was developed half a century ago and still is the first choice for treatment of type II diabetes mellitus.1 However guanidine compounds are relatively rare as secondary metabolites, mostly reported from marine organisms. Their bioactivities are versatile and raise the interest of many chemists and biologists.2−6 Cytotoxic compounds nitensidine E and other guanidine alkaloids were isolated from Pterogyne nitens Tul growing in Argentina.7,8 Five hypotensive guanidine compounds, caracasanamides G1, G3, G5, G6, and G7, were isolated from extracts of the leaves of Verbesina caracasana Rob. & Greenman.9−12 Many other biological activities of cyclic guanidines derived from plants have been reported, including the leaf-closing activity of p-coumaroylagmatine from Albizzia ulibrissin Durazz13 and 5-HT7 activity of cimipronidine from Cimicif uga racemosa.14 Plumbago zeylanica L. (Plumbaginaceae), a tropical and subtropical medicinal plant abundant in Southeast Asia, is used as folk medicine for treating rheumatic pain, menostasis, carbuncle, and injury by bumping in China. Previous phytochemical investigations led to the isolation of naphthoquinones,15 triterpenoids,16 steroids,17 coumarins, and plumbagic acid glycosides.18 In our study, 11 novel guanidine derivatives and two analogues were isolated from the hydrophilic extract based on HPLC-TOFMS detection. Plantagoguanidinic acid (1) and plantagoamidinic acid B (13) were first isolated from Plantago asiatica, but their absolute configurations had not been determined previously.19,20 © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A crude aqueous acetone (H2O/acetone = 3:7) extract of airdried P. zeylanica plant material was partitioned between CHCl3 and H2O to obtain the water-soluble fraction. After removal of sugars and salts by MCI eluted with H2O, the aqueous layer was examined by HPLC-TOFMS. The profile of the total ion current showed 13 alkaloid peaks (Figure S1, Supporting Information). The main alkaloids (1−13) were detected on the basis of the results of HPLC-TOFMS. Compounds 1−13 were then isolated, and their structures were unambiguously elucidated by NMR and other spectroscopic data. Received: April 16, 2013

A

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Table 1. 1H NMR and 13C NMR Data (400 and 100 MHz, CD3OD) for 1−4 1 no. 1 2 3a 3b 4 5 6 7 8 2′ 4′ 5′a 5′b

δH (J in Hz) 2.33 1.60 1.60 2.05 5.15

m m m m t (7.1)

1.67 s 1.61 s 4.09 m 3.75 t (9.7) 3.50 dd (9.7, 6.6)

2 δC 181.2 55.4 31.2 27.5 125.7 133.2 26.5 18.4 161.8 59.5 50.1

3

δH (J in Hz) 2.32 1.62 1.54 2.13 5.27

m m m m t (7.4)

4.10 d (12.2) 3.97 d (12.2) 1.76 s 4.09 m 3.74 t (9.6) 3.51 dd (9.6, 6.6)

δC

δH (J in Hz)

181.2 55.0 31.0 26.9 128.8 137.2 61.8 22.2 161.7 59.5 48.9

2.85 2.57 2.13 4.60 5.17

m td (12.5, 8.3) dd (12.5, 6.6) t (8.7) d (9.3)

1.77 s 1.75 s 4.43 q (9.4) 3.82 t (9.6) 3.54 t (9.3)

4 δC 179.4 51.8 42.6 57.3 123.5 139.5 26.4 18.8 159.8 67.8 48.5

δH (J in Hz) 2.61 2.06 1.60 5.03 7.30

m m m m s

1.86 s 4.13 m 3.75 m 3.57 m

δC 180.0 52.1 35.0 82.3 152.2 130.9 176.7 11.0 161.7 59.4 48.5

groups. The 1H and 13C NMR spectra (Table 1) of 2 revealed a guanidine moiety (δC 161.7), one carboxylic moiety (δC 181.2), a trisubstituted double bond [(δH 5.27, δC 128.8) and (δC 137.2, s)], and one methyl, four methylene, and two methine groups. 1H−1H COSY correlations between H-5′/H-4′/H-2/ H-3/H-4/H-5 and HMBC correlations (Figure 2) revealed the

Compound 1 was obtained as a white, amorphous powder and gave an alkaloid-positive test when sprayed with Dragendorff’s reagent. The molecular formula was determined to be C11H19N3O2 by HRESIMS. The IR and 1H and 13C NMR spectroscopic data (Table 1) were identical to those of plantagoguanidinic acid,19 whose absolute configuration had not been determined previously. Compound 1 was treated with di-tert-butyl dicarbonate to give compound 1a. A crystal of 1a was obtained from MeOH, and the absolute configuration of 1 was finally determined to be (2R, 4′R) by single-crystal X-ray diffraction of 1a (Figure 1). Compound 1 was also named (2R, 4′R)-plantagoguanidinic acid. Plumbagine A (2), a white, amorphous powder, also gave an alkaloid-positive test. HRESIMS provided the molecular formula C11H19N3O3. The IR spectrum of 2 indicated the presence of OH (3399 cm−1) and carboxyl (1691 cm−1)

Figure 2. Key HMBC (H→C), 1H−1H COSY (), and selected ROESY (↔) correlations of 2.

presence of a terpenoid moiety. The HMBC correlations from H-2, H-3, and H-4′ to C-1 showed that the carboxylic moiety (δC 181.2) was attached to C-2. H-4′ and H-5′ showed HMBC correlations to the C-2′ guanidine carbon, indicating that the guanidine moiety together with the C-4″ and C-5′ formed an imidazoline ring. In the ROESY spectrum of 2, the correlations between Me-8/H-5 and H-7/H-4 supported the Z-doublebond geometry (Figure 2). Compounds 1 and 2 displayed very similar CD curves (Figure 3), suggesting that compound 2 possessed the same absolute configuration as 1. Plumbagine B (3) was isolated as a white, amorphous powder, with the molecular formula C 11 H 17 N 3 O 2 by HRESIMS. Its IR spectrum revealed the presence of OH (3369 cm−1) and carboxyl (1681 cm−1) groups. Comparison of 1D and 2D NMR data of 3 with those of 1 clearly revealed that the structure of 3 was similar to that of 1. The major difference was that the methylene (C-4) in 1 was replaced by methine (C4) in 3. The HMBC correlation between H-4/C-4′ showed that C-4 was connected with N-3″ to form a 4H-pyrrole moiety. Thus, the planar structure was determined. The relative configuration was determined as (2R*, 4S*, 4′R*) through the ROESY correlations between H-5′b/H-2, H-5′a/H-4′, H2/H-5, H-3b/H-5, and H-3a/H-4′ (Figure 4). As compound 3 is presumed to originate from compound 1 in a biogenetic pathway (Figure S2, Supporting Information), the absolute configuration of compound 3 at C-2 and C-4′ should be the

Figure 1. Single-crystal X-ray diffraction of 1a (Cu Kα). B

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from consideration.21 Comparison of the CD spectra (Figure 3) between 1 and 4 supported the (2R, 4′R)-configuration for compound 4. The R-configuration at C-4 of compound 4 was determined by the negative Cotton effect at 219.9 nm (Δε = −0.47) in the CD spcectrum, which was similar to that of (23R)-3-oxo-9β-lanosta-7,24-dien-26,23-olide.22 Hence, the absolute configuration of 4 was determined to be (2R, 4R, 4′R). Plumbagine D (5), a white, amorphous powder, had HRESIMS and NMR spectra consistent with the molecular formula C15H27N3O4. The IR spectrum of 5 indicated the presence of OH (3315 cm−1) and carboxyl (1687 cm−1) groups. The 1H and 13C NMR spectra (Table 2) of 5 revealed signals for a guanidine moiety (δC 160.2), a carboxylic moiety (δC 180.5), a trisubstituted double bond [(δH 5.08, δC 125.2) and (δC 132.8, s)], and two methyl, six methylene, and three methine groups. 1H−1H COSY correlations between H-1″/H2″/H-3″/H-4″ indicated the presence of a butanediol group. The 1H and 13C NMR data (Table 2) were similar to those of compound 1, except for an additional butanediol group. HMBC correlations from H-1″ to C-2′ and C-5′ showed that the butanediol moiety was located at N-1′. The (2R, 4′R)configuration of compound 5 was determined by comparison of the CD spectra between 1 and 5. However, the absolute configuration at C-2″ was not determined. Thus, the structure of 5 was identified as shown. Plumbagine E (6) had the molecular formula C15H27N3O5 as determined by HRESIMS. The 1H and 13C NMR spectra (Table 2) were similar to those of 5, and the only difference was that the methyl (δC 25.9, C-7) in 5 was replaced by an oxymethylene (δC 61.3, C-7) in 6. Combined with HSQC, 1 H−1H COSY, HMBC, ROESY, and CD spectra, the structure was concluded to be as shown. Plumbagine F (7) had the molecular formula C15H25N3O4 by HRESIMS. Comparison of 1D and 2D NMR data of 7 with those of 5 revealed that the structure of 7 was similar to that of 5. The major difference was that the methylene (C-4) in 5 was replaced by a methine (C-4) in 7. The HMBC correlation between H-4/C-4′ showed that C-4 was connected to N-3′. The (2R, 4S, 4′R)-configuration of compound 7 was determined by the ROESY and CD spectra. Plumbagine G (8) had the molecular formula C15H23N3O6. Its IR spectrum revealed absorption bands similar to those of 4. The 1H and 13C NMR spectra (Table 2) were similar to those of 4, except for an additional butanediol group. The 1H−1H COSY correlations between H-1″/H-2″/H-3″/H-4″ showed connections in the butanediol group, and HMBC correlations between H-1″/C-2′ and C-5′ indicated that the butanediol group was attached to N-1′. The absolute configuration at C-2, C-4, and C-4′ was elucidated as (2R, 4R, 4′R) by the ROESY and CD spectra. Therefore, the structure was determined as shown. Plumbagoside A (9) had the molecular formula C20H35N3O8, and its IR spectrum revealed absorption bands similar to those of 5. The 1H and 13C NMR spectra (Table 3) were similar to those of 5, except for resonances of an additional sugar unit. The sugar was identified as α-xylose from the anomeric proton at δH 4.70 (1H, d, J = 3.7 Hz), anomeric carbon at δC 100.3, and some other characteristic NMR resonances. This was confirmed by acid hydrolysis of 9, which yielded D-xylose. The α-D-xylose unit was located at C-4″ due to the HMBC correlation between the anomeric proton at δH 4.70 and C-4″ at δC 65.3 (Figure S3, Supporting Information). Thus, the

Figure 3. CD spectra of compounds 1−4 and 13.

Figure 4. Key HMBC (H→C), 1H−1H COSY (), and selected ROESY (↔) correlations of 3.

same as that of compound 1. Therefore, the absolute configuration of 3 was assigned as (2R, 4S, 4′R). Plumbagine C (4) was obtained as a white, amorphous powder, and its molecular formula was determined to be C11H15N3O4 by HRESIMS. The IR spectrum of 4 indicated the presence of OH (3380 cm−1), carboxyl (1689 cm−1), and α,βunsaturated-γ-lactone (1751 cm−1) groups. The 1H and 13C NMR spectra of 4 (Table 1) revealed a guanidine moiety (δC 161.7), two carbonylic moieties (δC 180.0, 176.7), a trisubstituted double bond [(δH 7.30, δC 152.2) and (δC 130.9, s)], and one methyl, two methylene, and three methine groups. There were 1H−1H COSY correlations between H-5′/ H-4′/H-2/H-3/H-4/H-5. In the HMBC spectrum, the guanidine carbon signal at δC 161.7 showed correlations with protons at δH 3.75 (H-5′a), 3.57 (H-5′b), and 4.13 (H-4′) (Figure 5). The carboxylic carbon at δC 180.0 showed

Figure 5. Structures A and B and key HMBC (H→C) and 1H−1H COSY () of 4.

correlations with protons at δH 2.61 (H-2), 4.13 (H-4′), and 1.60 (H-3). Also, the carbonylic carbon at δC 176.7 had correlations with protons at δH 1.86 (H-8) and 7.30 (H-5). The aforementioned 2D correlations indicated that the structure could be either A or B (Figure 5). The IR adsorption band at 1751 cm−1 (α,β-unsaturated-γ-lactone) excluded structure B C

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Table 2. 1H NMR and 13C NMR Data (400 and 100 MHz, CD3OD) for 5−8 5 no. 1 2 3a 3b 4 5 6 7

δH (J in Hz) 2.28 1.48 1.48 2.04 1.95 5.08

180.5 54.6 30.7

m m m m m t (7.1)

27.1 125.2 132.8 25.9

1.60 s

8 2′ 4′ 5′a 5′b 1″ 2″ 3″

3.96 3.76 3.65 3.24 3.91 1.57

4″

3.67 m

6 δC

1.54 s

17.9 160.2 56.9 53.9

m t (9.7) m m m m

δH (J in Hz) 2.30 1.52 1.52 2.08

26.4

5.23 t (7.2) 4.04 d (12.1) 3.93 d (12.1) 1.71 s

52.1 67.2 37.8 59.5

3.67 m

δH (J in Hz)

180.3 54.1 30.5

m m m m

3.97 3.79 3.64 3.24 3.96 1.61

7 δC

m t (9.7) m m m m

2.8 m 2.5 td (12.4, 8.0) 2.1 dd (12.4, 6.3) 4.63 t (8.5)

128.2 136.6 61.3

5.17 d (9.3)

21.8 160.2 56.8 54.1

1.76 s

52.1 67.2 37.8 59.5

1.78 s

4.40 3.89 3.80 3.36 4.00 1.69 1.65 3.74

q (9.5) t (9.9) t (9.3) m m m m m

8 δC 178.8 52.0 42.5 58.5

δH (J in Hz) 2.57 2.01 1.51 4.95

δC 179.7 51.9 35.0

m m m m

82.3

124.4 138.7 26.4

7.24 s

152.1 130.9 176.7

18.7 159.5 65.3 55.3

1.80 s

11.1 160.7 57.2 54.0

53.3 67.5 38.4

4.00 3.78 3.71 3.24 3.92 1.57

m m m m m m

60.0

3.65 t (5.9)

52.6 67.6 38.2 60.0

Table 3. 1H NMR and 13C NMR Data (400 and 100 MHz, CD3OD) for 9−12 9 no. 1 2 3a 3b 4 5 6 7 8 2′ 4′ 5′a 5′b 1″ 2″ 3″ 4″ 1‴ 2‴ 3‴ 4‴ 5‴

δH (J in Hz) 2.31 1.59 1.47 2.06 1.97 5.10

m m m m m t (7.1)

1.63 s 1.57 s 3.95 3.85 3.68 3.27 3.99 1.78 1.66 3.80 3.50 4.70 3.35 3.52 3.42 3.48 3.44

m m dd (9.8, 6.3) m m m m m m d (3.7) dd (9.3, 3.7) m m m m

10 δC 180.4 54.6 30.7 27.1 125.2 132.8 25.9 17.9 160.1 56.9 53.9 52.1 67.2 34.7 65.3 100.3 73.4 75.2 71.5 63.1

11

δH (J in Hz) 2.31 1.59 1.49 2.10

m m m m

5.25 t (7.2) 4.06 d (12.1) 3.95 d (12.1) 1.73 s 3.99 3.81 3.69 3.27 4.00 1.77 1.66 3.84 3.51 4.70 3.36 3.53 3.41 3.49 3.45

m m dd (9.6, 6.4) m m m m m m d (3.6) dd (9.4, 3.6) m m m m

δC 180.4 54.2 30.5 26.5

δH (J in Hz) 2.76 2.48 2.06 4.55

m td (12.4, 7.9) dd (12.4, 6.3) t (8.3)

128.3 136.6 61.3

5.11 d (9.3)

21.7 160.1 56.9 53.9

1.68 s

52.1 67.2 34.7 65.4 100.3 73.4 75.2 71.5 63.1

structure of compound 9 was identified as plumbagine D α-Dxyloside. Plumbagoside B (10) had the molecular formula C20H35N3O9 as determined by HRESIMS. The 1H and 13C NMR spectra (Table 3) were similar to those of 9; the difference was that the methyl (δC 25.9, C-7) in 9 was replaced

1.70 s

4.32 3.80 3.72 3.25 3.96 1.75 1.64 3.85 3.49 4.69 3.32 3.51 3.41 3.43 3.36

q (9.3) m t (9.4) m m m m m m d (3.5) m m m m m

12 δC 178.8 52.0 42.5 58.4

δH (J in Hz) 2.61 2.02 1.55 4.97

m m m d (9.5)

δC 179.1 51.4 34.6 81.8

124.4 138.7 26.4

7.26 s

151.7 130.4 176.2

18.8 159.3 65.2 55.2

1.82 s

10.5 160.1 56.8 53.4

53.2 67.4 35.4 65.9 100.8 73.9 75.6 71.9 63.6

4.05 m 3.81d (9.8) 3.73 dd (9.8, 6.1) 3.26 m 3.99 m 1.78 m 1.63 m 3.85 m 3.50 m 4.69 d (3.7) 3.34 dd (9.5, 3.6) 3.53 m 3.41 m 3.48 d (7.8) 3.44 d (7.8)

52.1 67.1 34.7 65.3 100.3 73.4 75.2 71.5 63.1

by an oxymethylene (δC 61.3, C-7) in 10. The relative configuration was determined by the ROESY spectrum. Thus, compound 10 was determined to be plumbagine E α-Dxyloside. Plumbagoside C (11), C20H33N3O8, had 1H and 13C NMR spectra (Table 3) similar to those of 7, except for an additional D

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pentose group. The pentose was identified as α-D-xylose. The linkage point of the α-D-xylose unit to the aglycone was established through the HMBC spectrum, and the (2R, 4S, 4′R)-configuration was determined from ROESY and CD spectra. Compound 11 was named plumbagine F α-D-xyloside. Plumbagoside D (12), C20H31N3O10, had IR absorptions similar to those of 8. The 1H and 13C NMR spectra of compound 12 (Table 3) showed five carbon resonances assignable to a xylose unit, which was identified the same way as in 9. Comparison of the 1H and 13C NMR data with those of compound 8 indicated that compound 12 was similar to 8, except for a xylose unit. The linkage point of the α-D-xylose unit to the aglycone was established by the HMBC spectrum. The (2R, 4R, 4′R) configuration was determined by ROESY and CD spectra. Therefore, the structure of 9 was plumbagine G αD-xyloside. Compound 13 was obtained as a white, amorphous powder. The molecular formula was determined to be C11H21N3O3 by HRESIMS. The IR and 1H and 13C NMR spectra were identical to those of plantagoamidinic acid B,20 whose absolute configuration had not been determined. Compounds 1 and 13 displayed very similar CD curves (Figure 3). Thus, the absolute configuration of compound 13 was determined as (2R, 4′R)-plantagoamidinic acid B. The biogenetic pathway of compounds 1−4 was postulated as proposed in Figure S2 (Supporting Information). A monoterpene fragment attached to guanidine to form a monoterpene guanidine and further cyclization produced a pentahydroimidazole unit (compound 1). After oxidation and cyclization, an unprecedented natural 2-imino-1,3diazabicyclo[3.3.0]octane skeleton (compound 3) is assumed. Compound 4 could also be produced by oxidation and esterification from compound 1. Thus, the absolute configurations of these compounds at C-2 and C-4′ were the same. Compounds 1−13 were evaluated against the PANC-1 (human pancreatic carcinoma) and MDA-MB-231 (human breast carcinoma) cell lines. However, none of these compounds exhibited significant cytotoxicity (IC50 >10 μM). By contrast, the IC50 of paclitaxel for these two cell lines was less than 1 μM.

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temperature. The combined extracts were concentrated to give a crude extract, which was partitioned between CHCl3 and H2O to obtain the water-soluble fraction. The water-soluble fraction was subjected to a column of MCI and eluted with H2O, 20%, 40%, 60%, and 100% MeOH successively. Seven major fractions (A−G) were obtained. Fraction B was chromatographed on a C18 column eluted with MeOH/H2O (10−20%) to give fractions (B1, B2). Fraction B1 was passed over a MCI column with MeOH/H2O (10−30%) to afford 13 (60 mg) and 2 (20 mg). Fraction B2 was separated by RP-HPLC (YMC 5 μm, 250 × 10 mm, 17% MeOH, 3.0 mL/min, eluting at 7.5− 8.1 min, UV 210 nm) to yield compound 12 (10 mg). Fraction C was subjected to passage over a MCI column with MeOH/H2O (10−30%) to give fractions (C1, C2). Fraction C2 was chromatographed on a C18 column with MeOH/H2O (10−30%) to afford 4 (11 mg) and 8 (13 mg). Fraction D was subjected to a LH-20 column (MeOH) to give fractions (D1, D2). Fraction D1 was separated by RP-HPLC (YMC 5 μm, 250 × 10 mm, 17% MeOH, 3.0 mL/min, eluting at 14−15 min, UV 210 nm) to yield compound 10 (21 mg). Compound 6 (20 mg) was obtained from fraction D2 through a MCI column (MeOH 20− 60%). Fraction E was chromatographed on a C18 column eluted with MeOH/H2O (10−30%) to give 7 (18 mg) and fraction E2. Fraction E2 was separated by RP-HPLC (YMC 5 μm, 250 × 10 mm, 20% MeOH, 3.0 mL/min, UV 210 nm) to yield 3 (15 mg) and 11 (20 mg). Fraction F was passed over a C18 column with MeOH/H2O (10− 30%) to give two subfractions (F1, F2). Fraction F1 was subjected to a LH-20 column (MeOH) to afford 5 (150 mg). Fraction F2 was separated by RP-HPLC (YMC 5 μm, 250 × 10 mm, 21% MeOH) to yield compound 9 (20 mg). Compound 1 (200 mg) was obtained from fraction G by passage through C18 (MeOH, 10−30%) and LH-20 (MeOH) columns. LC-TOF MS Analysis. The aqueous layer (1 mg) was desalted through a MCI column (H2O, MeOH), and then dissolved in H2O. HPLC was performed using an Agilent HPLC system. Chromatographic separation was performed on a Zorbax Eclipse XDB C-18 (5 μm 4.6 × 250 mm, Agilent, USA). Mobile phase A was water containing 0.5% formic acid. Mobile phase B was MeOH. The column temperature was 30 °C. The HPLC flow rate was 1.0 mL/min. A mobile phase gradient was used with the percentage of B in A varying as follows: initial concentration, 5% B; 20 min, 25% B; 40 min, 30% B; 50 min, 60% B. MS experiments were performed on a TOF mass spectrometer with an ESI interface (Agilent, USA). The positive ESI conditions were as follows: gas temperature, 350 °C; drying gas, 9 L/ min, nebulizer, 40 psig; capillary, 3500. Plantagoguanidinic acid (1): white, amorphous powder; [α]20 D +52.5 (c 0.2, MeOH); CD (MeOH) λmax nm (Δε) 201.5 (+8.97); IR (KBr) νmax 3392, 2927, 1689, 1587, 1448, 1398, 1105 cm−1; 1H and 13 C NMR (CD3OD) see Table 1; ESIMS (pos.) m/z 226 [M + H]+; ESIMS (neg.) m/z 224 [M − H]−; HRESIMS m/z 226.1554 [M + H]+ (calcd for C11H20N3O2, 226.1556). Preparation of Compound 1a. To a solution of compound 1 (100 mg) in DMF (5 mL) and pyridine (2 mL) was slowly added dropwise (Boc)2O (100 mg); then the mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated to remove most of the solvents, and then the residue was purified by LH20 column chromatography to give compound 1a (55 mg) as a white solid. Compound 1a: mp 153−154 °C; 13C NMR (100 MHz, CD3OD) δ 180.0 (C-1), 158.2 (C-2′), 151.9 (C-9), 133.4 (C-6), 125.0 (C-5), 86.4 (C-10), 61.9 (C-4′), 49.9 (C-2), 42.9 (C-5′), 30.2 (C-3), 28.9 (3C, C11, C-12, and C-13), 27.0 (C-4), 26.0 (C-8), 17.9 (C-7); 1H NMR (400 MHz, CD3OD) δ 5.13 (1H, td, J = 6.6, 1.1 Hz, H-5), 4.46 (1H, ddd, J = 8.8, 3.0, 1.8 Hz, H-4′), 3.91 (1H, dd, J = 10.0, 1.8 Hz, H-5′a), 3.61 (1H, t, J = 9.5 Hz, H-5′b), 2.79 (1H, dt, J = 10.1, 4.1 Hz, H-2), 2.05 (2H, m, H-4), 1.74 (1H, m, H-3a), 1.68 (3H, s, H-8), 1.60 (3H, s, H-7), 1.58 (9H, s, H-11, H-12, and H-13), 1.21 (1H, m, H-3b); HRESIMS m/z 326.2075 [M + H]+ (calcd for C16H28N3O4, 326.2074). Crystal Data of 1a: C16H27N3O4·CH3OH, M = 357.45 g mol−1, monoclinic, space group C2, a = 19.4525(4) Å, b = 6.8143(2) Å, c = 14.9925(4) Å, α = 90°, β = 90.9140(10)°, γ = 90°, V = 1987.08(8) Å3,

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an SGW X-4 melting point instrument and are uncorrected. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV and IR spectra were recorded on Shimadzu UV2450 and Perkin-Elmer 577 spectrophotometers, respectively. CD spectra were recorded on a JASCO J-810 spectrometer. NMR spectra were taken on a Varian Mercury NMR spectrometer operating at 400 MHz for 1H and 100 MHz for 13C. LR and HRESIMS were measured using a Finnigan LCQ-DECA and Agilent G6224A TOF spectrometer, respectively. Thin-layer chromatography (TLC): precoated silica gel GF254 plates (Yantai, People’s Republic of China). Column chromatography (CC): Sephadex LH-20 (20−80 μm; Amersham Pharmacia Biotech AB), Chromatorex C18-OPN (20−45 μm; Fuji Silysia Chemical Ltd.), MCI gel CHP-20P (75−150 μm, Mitsubishi Chemical Industries Co., Ltd.). Plant Material. The aerial parts of P. zeylanica were collected from Nanning, Guangxi Province, People’s Republic of China, in September 2009, and authenticated by Prof. He-Ming Yang. A voucher specimen (No. SIMMPZ123) is deposited at the Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, People’s Republic of China. Extraction and Isolation. The dried P. zeylanica plant material (8.0 kg) was extracted three times with 70% aqueous acetone at room E

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T = 140(2) K, Z = 4, D = 1.195 mg/m3, crystal size 0.30 × 0.04 × 0.04 mm3, λ(Cu Kα) = 1.54178 Å, F(000) = 776. Final R indices, R1 = 0.0446, wR2 = 0.1221 [I > 2σ(I)], R1 = 0.0480 (all data), wR2 = 0.1261 (all data). The absolute structure parameter was 0.0(2). Crystallographic data have been deposited in the Cambridge Crystallographic Data Center as entry 929620. A copy of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, U.K. [fax, +44(0)-1223-336033; e-mail, [email protected]]. Plumbagine A (2): white, amorphous powder; [α]20 D +61.5 (c 0.2, MeOH); CD (MeOH) λmax nm (Δε) 200.5 (+8.15); IR (KBr) νmax 3399, 2940, 1691, 1585, 1448, 1398, 1103, 1004 cm−1; 1H and 13C NMR (CD3OD) see Table 1; ESIMS (pos.) m/z 242 [M + H]+; ESIMS (neg.) m/z 240 [M − H]−; HRESIMS m/z 242.1498 [M + H]+ (calcd for C11H20N3O3, 242.1499). Plumbagine B (3): white, amorphous powder; [α]20 D +85 (c 0.2, MeOH); CD (MeOH) λmax nm (Δε) 209.5 (+25.27); IR (KBr) νmax 3369, 2917, 1681, 1581, 1446, 1400, 1301, 1087 cm−1; 1H and 13C NMR (CD3OD) see Table 1; ESIMS (pos.) m/z 224 [M + H]+; ESIMS (neg.) m/z 222 [M − H]−; HRESIMS m/z 224.1396 [M + H]+ (calcd for C11H18N3O2, 224.1394). Plumbagine C (4): white, amorphous powder; [α]20 D +35 (c 0.1, H2O); UV (H2O) (log ε) λmax 203 (4.06); CD (H2O) λmax nm (Δε) 196.9 (+3.39), 219.9 (−0.47), 234.6 (+0.12); IR (KBr) νmax 3380, 2927, 1751, 1689, 1585, 1396, 1058 cm−1; 1H and 13C NMR (CD3OD) see Table 1. ESIMS (pos.) m/z 254 [M + H]+; ESIMS (neg.) m/z 252 [M − H]−; HRESIMS m/z 254.1137 [M + H]+ (calcd for C11H16N3O4, 254.1135). Plumbagine D (5): white, amorphous powder; [α]20 D +46.3 (c 0.3, MeOH); CD (MeOH) λmax nm (Δε) 205.5 (+6.43); IR (KBr) νmax 3315, 2927, 1687, 1587, 1448, 1398, 1056 cm−1; 1H and 13C NMR (CD3OD) see Table 2; ESIMS (pos.) m/z 314 [M + H]+; ESIMS (neg.) m/z 312 [M − H]−; HRESIMS m/z 314.2077 [M + H]+ (calcd for C15H28N3O4, 314.2074). Plumbagine E (6): white, amorphous powder; [α]20 D +31.0 (c 0.1, MeOH); CD (MeOH) λmax nm (Δε) 205.5 (+5.40); IR (KBr) νmax 3380, 2927, 1681, 1585, 1448, 1400, 1052, 1002 cm−1; 1H and 13C NMR (CD3OD) see Table 2; ESIMS (pos.) m/z 330 [M + H]+; ESIMS (neg.) m/z 328 [M − H]−; HRESIMS m/z 330.2024 [M + H]+ (calcd for C15H28N3O5, 330.2023). Plumbagine F (7): white, amorphous powder; [α]20 D +67.3 (c 0.15, MeOH); CD (MeOH) λmax nm (Δε) 214.0 (+19.42); IR (KBr) νmax 3376, 2933, 1675, 1581, 1448, 1384, 1280, 1058 cm−1; 1H and 13C NMR (CD3OD) see Table 2; ESIMS (pos.) m/z 312 [M + H]+; ESIMS (neg.) m/z 310 [M − H]−; HRESIMS m/z 312.1917 [M + H]+ (calcd for C15H26N3O4, 312.1918). Plumbagine G (8): white, amorphous powder; [α]20 D +25 (c 0.1, H2O); UV (H2O) (log ε) λmax 205 (4.37); CD (H2O) λmax nm (Δε) 204.9 (+1.40), 223.3 (−0.71), 234.6 (+0.17); IR (KBr) νmax 3380, 2927, 1747, 1683, 1585, 1398, 1058 cm−1; 1H and 13C NMR (CD3OD) see Table 2; ESIMS (pos.) m/z 342 [M + H]+; ESIMS (neg.) m/z 340 [M − H]−; HRESIMS m/z 342.1659 [M + H]+ (calcd for C15H24N3O6, 342.1660). Plumbagoside A (9): white, amorphous powder; [α]20 D +109.0 (c 0.2, MeOH); CD (MeOH) λmax nm (Δε) 205.5 (+1.55); IR (KBr) νmax 3394, 2927, 1683, 1585, 1448, 1400, 1043 cm−1; 1H and 13C NMR (CD3OD) see Table 3; HRESIMS m/z 446.2493 [M + H]+ (calcd for C20H36N3O8, 446.2497). Plumbagoside B (10): white, amorphous powder; [α]20 D +75.0 (c 0.2, MeOH); CD (MeOH) λmax nm (Δε) 205.5 (+0.98); IR (KBr) νmax 3388, 2927, 1683, 1583, 1402, 1041 cm−1; 1H and 13C NMR (CD3OD) see Table 3; ESIMS (pos.) m/z 462 [M + H]+; ESIMS (neg.) m/z 460 [M − H]−; HRESIMS m/z 462.2450 [M + H]+ (calcd for C20H36N3O9, 462.2446). Plumbagoside C (11): white, amorphous powder, [α]20 D +106.7 (c 0.15, MeOH); CD (MeOH) λmax nm (Δε) 214.0 (+22.36); IR (KBr) νmax 3384, 2933, 1673, 1581, 1448, 1402, 1307, 1043 cm−1; 1H and 13C NMR (CD3OD) see Table 3; ESIMS (pos.) m/z 444 [M + H]+; ESIMS (neg.) m/z 442 [M − H]−; HRESIMS m/z 444.2342 [M + H]+ (calcd for C20H34N3O8, 444.2340).

Plumbagoside D (12): white, amorphous powder, [α]20 D +53 (c 0.1, H2O); UV (H2O) (log ε) λmax 205 (4.01); CD (H2O) λmax nm (Δε) 219.6 (−1.40), 239.1 (+0.26); IR (KBr) νmax 3384, 2927, 1743, 1681, 1585, 1400, 1041 cm−1; 1H and 13C NMR (CD3OD) see Table 3; HRESIMS m/z 474.2080 [M + H]+ (calcd for C20H32N3O10, 474.2082). Plantagoamidinic acid B (13): [α]20 D +52 (c 0.55, MeOH); CD (MeOH) λmax nm (Δε) 199.0 (+10.13); IR (KBr) νmax 3330, 3226, 2969, 1693, 1581, 1400, 1303 cm−1; 13C NMR (100 MHz, CD3OD) δ 181.0 (C-1), 161.3 (C-2′), 71.40 (C-6), 58.9 (C-4′), 55.4 (C-2), 48.4 (C-5′), 44.9 (C-5), 31.1 (C-3), 29.2 (C-7), 29.2 (C-8), 23.3 (C-4); 1H NMR (400 MHz, CD3OD) δ 2.33 (1H, m, H-2), 1.57 (2H, m, H-3), 1.41 (2H, m, H-4), 1.51 (2H, m, H-5), 1.16 (s, 6H, H-7 and H-8), 4.08 (1H, m, H-4′), 3.75 (1H, t, J = 9.7 Hz, H-5′a), 3.49 (1H, dd, J = 9.6, 6.7 Hz, H-5′b); HRESIMS m/z 244.1657 [M + H]+ (calcd for C11H22N3O3, 244.1656). Acidic Hydrolysis of Compounds 9−12. Compound 9 (11 mg) was hydrolyzed with 2 M HCl in H2O and heated for 4 h in an 85 °C water-bath. After cooling, the reaction mixture was neutralized with 10% Na2CO3 and extracted with CHCl3 twice. The aqueous layer was desalted (Sephadex LH-20, MeOH) to afford a sugar residue (2.5 mg). The sugar was confirmed to be D-xylose by comparison with an authentic sample on TLC (EtOAc/MeOH/H2O/HOAc, 13:3:3:4, Rf 0.39) and by measurement of its optical rotation ([α]20 D +18.5 c 0.12, H2O). The constituent sugars of compounds 10−12 were identified by the same method as 9. Cytotoxicity Assays. The cytotoxicity of the test compounds against the PANC-1 (human pancreatic carcinoma) and MDA-MB231 (human breast carcinoma) cell lines was measured using a sulforhodamine B (SRB) assay as described in the literature.23 Paclitaxel was used as positive control. Briefly, cells were plated in 96well culture plates for 24 h and then treated with serial dilutions of the compounds, with a maximum concentration of 100 μM. After being incubated 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 water and air-drying, the plates were stained for 15 min with 100 μL of 0.4% SRB (Sigma) in 1% glacial acetic acid. The plates were washed with 1% acetic acid and airdried. For reading of the plates, the protein-bound 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, MWG-Biothech, Inc., Sunnyvale, CA, USA). All tests were performed in triplicate, and results are expressed as IC50 values.

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ASSOCIATED CONTENT

S Supporting Information *

LC-TOFMS chromatogram of the aqueous layer, postulated biosynthesis pathway for compounds 1−4, key HMBC (H→C) and 1H−1H COSY () correlations of 5−12, selected ROESY (↔) correlations of 6, 7, 10, and 11, 1D and 2D NMR spectra of compounds 2−12, CD spectra of compounds 5−12, and Xray crystallographic data (CIF file) of 1a 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]. (L. J. Xuan). Tel & Fax: +8621-20231968. 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) F

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and the National Natural Sciences Foundation of China (No. 30901851).

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REFERENCES

(1) Bailey, C. J.; Day, C. Pract. Diab. Int. 2004, 21, 115−117. (2) Berlinck, R. G. S. Nat. Prod. Rep. 1996, 13, 377−409. (3) Berlinck, R. G. S. Nat. Prod. Rep. 1999, 16, 339−365. (4) Berlinck, R. G. S. Nat. Prod. Rep. 2002, 19, 617−649. (5) Berlinck, R. G. S.; Kossuga, M. H. Nat. Prod. Rep. 2005, 22, 516− 550. (6) Berlinck, R. G. S.; Burtoloso, A. C. B.; Trindade-Silva, A. E.; Romminger, S.; Morais, R. P.; Bandeira, K.; Mizuno, C. M. Nat. Prod. Rep. 2010, 27, 1871−1907. (7) Bolzani, V. S.; Gunatilaka, A. A. L.; Kingston, D. G. I. J. Nat. Prod. 1995, 58, 1683−1688. (8) Regasini, L. O.; Castro, I. G.; Silva, D. H. S.; Furlan, M.; Barreiro, E. J.; Ferreira, P. M. P.; Pessoa, C.; Lotufo, L. V. C.; Moraes, M. O.; Young, M. C.; Bolzani, V. S. J. Nat. Prod. 2009, 72, 473−476. (9) Delle Monache, G.; Botta, B.; Delle Monache, F.; Espinal, R.; De Bonnevaux, S. C.; De Luca, C.; Botta, M.; Corelli, F.; Carmignani, M. J. Med. Chem. 1993, 36, 2956−2963. (10) Delle Monache, G.; Volpe, A. R.; Delle Monache, F.; Vitali, A.; Botta, B.; Espinal, R.; De Bonnevaux, S. C.; De Luca, C.; Botta, M.; Corelli, F.; Carmignani, M. Bioorg. Med. Chem. Lett. 1999, 9, 3249− 3254. (11) Carmignani, M.; Volpe, A. R.; Delle Monache, F.; Botta, B.; Espinal, R.; De Bonnevaux, S. C.; De Luca, C.; Botta, M.; Corelli, F.; Tafi, A.; Ripanti, G.; Delle Monache, G. J. Med. Chem. 1999, 42, 3116−3125. (12) Carmignani, M.; Volpe, A. R.; Botta, B.; Espinal, R.; De Bonnevaux, S. C.; De Luca, C.; Botta, M.; Corelli, F.; Tafi, A.; Sacco, R.; Delle Monache, G. J. Med. Chem. 2001, 44, 2950−2958. (13) Stoessl, A. Phytochemistry 1965, 4, 973−976. (14) Fabricant, D. S.; Nikolic, D.; Lankin, D. C.; Chen, S. N.; Jaki, B. U.; Krunic, A.; Breemen, R. B.; Fong, H. H. S.; Farnsworth, N. R.; Pauli, G. F. J. Nat. Prod. 2005, 68, 1266−1270. (15) Gunaherath, G. M. K. B.; Gunatilaka, A. A. L.; Thomson, R. H. J. Chem. Soc., Perkin Trans. 1 1988, 407−410. (16) Gupta, A.; Rai, R.; Siddiqui, I. R.; Singh, J. Fitoterapia 1998, 69, 420−422. (17) Nguyen, A. T.; Malonne, H.; Duez, P.; Vanhaelen Fastre, R.; Vanhaelen, M.; Fontaine, J. Fitoterapia 2004, 75, 500−504. (18) Lin, L. C.; Yang, L. L.; Chou, C. J. Phytochemistry 2003, 62, 619−622. (19) Goda, Y.; Kawahara, N.; Kiuchi, F.; Hirakura, K.; Kikuchi, Y.; Nishimura, H.; Takao, M.; Marumoto, M.; Kitazaki, H. J. Nat. Med. 2009, 63, 58−60. (20) Wang, Z.; Geng, F.; Sun, Q.; Yang, L.; Qi, M.; Liao, L.; Ji, G.; Dou, W.; Zheng, X. Patent CN 102050787 A, 2011. (21) Tanaka, R.; Aoki, H.; Wada, S.-i.; Matsunaga, S. J. Nat. Prod. 1999, 62, 198−200. (22) Tanaka, R.; Wada, S. I.; Aoki, H.; Matsunaga, S.; Yamori, T. Helv. Chim. Acta 2004, 87, 240−249. (23) 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.

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