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Antiviral glycosidic bisindole alkaloids from the roots of Isatis indigotica ab

ac

a

a

Yu-Feng Liu , Ming-Hua Chen , Qing-Lan Guo , Sheng Lin , a

a

c

Cheng-Bo Xu , Yue-Ping Jiang , Yu-Huan Li , Jian-Dong Jiang Jian-Gong Shi

ac

&

a

a

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing100050, China

Click for updates

b

Department of Pharmacy, Jining Medical University, Jining272067, China c

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing100050, China Published online: 30 Jun 2015.

To cite this article: Yu-Feng Liu, Ming-Hua Chen, Qing-Lan Guo, Sheng Lin, Cheng-Bo Xu, YuePing Jiang, Yu-Huan Li, Jian-Dong Jiang & Jian-Gong Shi (2015): Antiviral glycosidic bisindole alkaloids from the roots of Isatis indigotica, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1055729 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1055729

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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1055729

Antiviral glycosidic bisindole alkaloids from the roots of Isatis indigotica Yu-Feng Liuab, Ming-Hua Chenac, Qing-Lan Guoa, Sheng Lina, Cheng-Bo Xua, Yue-Ping Jianga, Yu-Huan Lic, Jian-Dong Jiangac and Jian-Gong Shia* a

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State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; bDepartment of Pharmacy, Jining Medical University, Jining 272067, China; c Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China (Received 19 April 2015; final version received 25 May 2015) We dedicate this paper to Prof Xin-Sheng Yao on the occasion of his 80th birthday.

Seven new glycosidic bisindole alkaloids, isatindigobisindolosides A –G (1 – 7), were isolated from an aqueous extract of the Isatis indigotica roots. Their structures including absolute configurations were determined by spectroscopic and chemical methods, together with calculations of electronic circular dichroism (ECD) spectra based on the quantum-mechanical time-dependent density functional theory. In the NMR spectra of 1 –3, it is found that integration of H-2 and intensity of C-2 are affected not only by a substitution group at C-2 but also by solvents. Influences of the glucopyranosyloxy on the calculated ECD spectra of the glycosidic bisindole alkaloids are discussed. Compounds 2, 5, and 6 showed antiviral activity against both the influenza virus A/Hanfang/359/95 (H3N2) and Coxsackie virus B3 with IC50 values of 8.4– 100.0 mM. Keywords: Cruciferae; Isatis indigotica; bisindole alkaloid glucoside; isatindigobisindolosides A– G; Influences of the glucopyranosyloxy on the calculated ECD spectra; antiviral activity

1.

Introduction

The dried roots and leaves of Isatis indigotica Fort. (Cruciferae), named “ban lan gen” and “da qing ye” in Chinese, respectively, are particularly important traditional Chinese medicines used for the treatment of influenza and infection diseases [1]. Several commercialized products containing these herbal drugs or their extracts are recorded in Chinese Pharmacopoeia [2]. Historically and currently, these herbal drugs play a crucial role to prevent and treat influenza during influenza pandemics in China, and a great pharmaceutical demand of the drug *Corresponding author. Email: [email protected] q 2015 Taylor & Francis

materials is fulfilled by cultivation of the plant. Clinical efficacy of “ban lan gen” and “da qing ye” has long attracted attentions of pharmacologists and chemists to search their mechanisms and bioactive chemical constituents. Pharmacological studies showed that extracts of these drugs exhibited antiviral, anti-endotoxic, antinociceptive, anti-inflammatory, and antipyretic effects and cytotoxicity against leukemia cells [3 –5]. Meanwhile, alkaloids, lignans, ceramides, flavonoids, and sulfur-containing metabolites were isolated from the extracts, and the indole alkaloids are considered as the main active

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Y.-F. Liu et al. named isatindigobisindolosides A – G (1 – 7) (Figure 1). According to linkers and linking-positions between the two indolyl moieties, these compounds are categorized as glycosidic derivatives of bis(indol20 /300 -yl)acetonitrile (1 and 2), bis(indol20 /300 -yl)acetamide (3), bis(indol-20 /300 -yl) methane (4 and 7), bis(indol-30 /300 -yl) methane (5), and bis(indol-20 /2 00 -yl) methane (6). Reported herein are the isolation, structure determination, and biological activity of these new isolates.

constituents [6]. However, previous chemical studies were mainly focused on the ethanol and methanol extracts of the drug materials, which is not consistent with the practical utilization of decoctions of the drug materials or the formulations. As part of a program to assess the chemical and biological diversity of traditional Chinese medicines, focusing on the minor components [7 – 13], we carried out on a detailed chemical analysis of an aqueous extract of the roots of I. indigotica. Our previous studies on the aqueous extract led to the isolation of 17 new alkaloids [14] and a pair of indole alkaloid enantiomers containing dihydrothiopyran and 1,2,4-thiadiazole rings [15], as well as 33 constituents firstly isolated from I. indigotica [16]. Some of them exhibited antiviral and hepatocyteprotective activities. A continuation of the investigation on the aqueous extract has led to isolation and characterization of seven new minor bisindole glucosides, HO HO HO

5"' O 3"'

2. Results and discussion Compound 1 was isolated as a white amorphous powder with ½a20 D 2 27.7 (c ¼ 0.17, MeOH). The IR spectrum of 1 exhibited absorption bands assignable to hydroxy and amino (3343 cm21), triple bond (2252 cm21), and aromatic ring (1622, 1597, and 1493 cm21) functional groups. Its molecular formula of C24H23N3O6 was determined by HR-ESIHO HO HO

1 1"' O

CN 3'

OH

2

3"

NH

5'

5"

N 1" H

1' 7'

O

O

CN

OH NH

7"

1 HO HO HO

O

O

2

O 1 NH2

OH

1"' O

OH

O 1"'

O 3'

OH

1

3"

NH N H

3

O

HO HO HO

2

NH

HO HO HO

N H

1 3"

NH

4 HO HO HO

O 1"'

O

NH

NH

5

H N

1

3'

OH

O

3'

N OCH3

O 3"

6 O

OH

N H

O

NH

5a

Figure 1. The structures of compounds 1 – 7.

HO HO HO

1

O

3'

1"' S

OH

HN

7

3"

N H

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Journal of Asian Natural Products Research MS combined with the NMR data (Tables 1 and 2). The NMR spectrum of 1 in DMSOd6 showed resonances attributable to two ortho-disubstituted phenyl rings at dH 7.72 (d, J ¼ 7.5 Hz, H-40 ), 6.97 (t, J ¼ 7.5 Hz, H-50 ), 7.06 (dd, J ¼ 7.5 and 8.0 Hz, H-60 ), and 7.24 (d, J ¼ 8.0 Hz, H-70 ), and 7.51 (d, J ¼ 7.5 Hz, H-400 ), 6.95 (t, J ¼ 7.5 Hz, H500 ), 7.08 (t, J ¼ 7.5 Hz, H-600 ), and 7.38 (d, J ¼ 7.5 Hz, H-700 ); a trisubstituted double bond at dH 7.45 (d, J ¼ 2.5 Hz, H-200 ); and an isolated methine at dH 6.31 (s, H-2). It also displayed diagnostic resonances for a b-glucopyranosyl moiety (Table 1) and two nitrogen-bearing protons at dH 11.00 (s, NH-10 ) and 11.23 (d, J ¼ 2.5 Hz, NH100 ). The 13CNMR (DEPT) spectra showed carbon resonances corresponding to the above units (Table 2) and three additional quaternary carbons [dC 119.5 (C-1), 121.8 (C-20 ), and 133.2 (C-30 )]. The presence of the b-glucopyranosyl unit was confirmed by enzymatic hydrolysis of 1 with snailase. The sugar isolated from the hydrolysate exhibited retention factor (Rf) on TLC, 20 specific rotation f½aD }, and 1H NMR spectroscopic data identical to those of an authentic glucopyranose (Experimental Section and Supporting Information), indicating it is D -glucopyranose. As compared with those of the alkaloids previously reported from I. indigotica [14 – 16], these data indicated that 1 is an unusual alkaloid b-D -glucopyranoside, which was further elucidated by 2D NMR data analysis. The proton resonances and proton-bearing carbon resonances in the NMR spectra were assigned based on the HSQC data. In the 1H – 1H COSY spectrum of 1, homonuclear vicinal coupling correlations of H-40 /H-50 /H-60 / H-70 and H-400 /H-500 /H-600 /H-700 (Figure 2), combined with the chemical shifts and coupling constants of these protons, confirmed the presence of the two orthodisubstituted phenyl rings. In the HMBC spectrum, two- and three-bond correlations from H-40 to C-30 , C-60 , and C70 a; from H-50 to C-30 a and C-70 ; from H-60

3

to C-40 and C-70 a; from H-70 to C-30 a and C-50 ; and from NH-10 to C-20 , C-30 , C-30 a, and C-70 a; together with their chemical shifts, demonstrated that there was a 20 ,30 disubstituted indole moiety in 1. Similarly the occurrence of a 300 -substituted indole moiety in 1 was revealed by the HMBC correlations from H-200 to C-300 , C-300 a, and C-700 a; from H-400 to C-300 , C-600 , and C-700 a; from H-500 to C-300 a and C-700 ; from H-600 to C-400 and C-700 a; from H-700 to C-300 a and C500 ; and from NH-100 to C-200 , C-300 , C-300 a, and C-700 a; in combination with the 1H – 1H COSY cross-peak between H-200 and NH100 . In addition, the HMBC correlation from H-1000 to C-30 indicated that the b-D glucopyranosyl unit was located at C-30 of the 20 ,30 -disubstituted indole moiety. The HMBC correlations from H-2 to C-20 , C200 , and C-300 , together with the molecular composition, suggested that both C-20 and C-300 of the two indole moieties must connect to C-2 of an acetonitrile unit. The presence of the acetonitrile unit was collaborated with the triple-bond absorption (2252 cm21) in the IR spectrum of 1. Accordingly, the planar structure of 1 was elucidated (Figure 2). The circular dichroism (CD) spectrum of 1 displayed typical coupled Cotton effects, negative at 229 nm and positive at 211 nm, arising from the interaction between the 1Bb transition (around lmax 220 nm) moments of the two indole chromophores [17] in the molecule. Based on the CD exciton chirality method [18], the absolute configuration at C-2 of 1 was assigned to be R. This was supported by calculations of electronic circular dichroism (ECD) spectra using the time-dependent density functional theory (TDDFT) method [19]. The calculated ECD spectra of 1 and its epimer (2S)-1 showed almost mirror Cotton effects at the similar maximum wavelengths, and the calculated ECD spectrum of 1 is in agreement with the experimental CD spectrum (Figure 3). Therefore, compound 1 was determined as (2 )-(2R)-2-(3-b- D -glucopyranosyloxy-

6.33 s 10.87 s 7.79 d (8.0) 6.94 dd (8.0, 8.0) 7.05 dd (8.0, 8.0) 7.21 d (8.0) 11.23 d (2.0) 7.44 d (2.0) 7.46 d (7.5) 6.96 dd (7.5, 8.0) 7.08 dd (8.0, 8.0) 7.38 d (8.0) 4.51 d (8.0) 3.35 m 3.27 m 3.27 m 3.17 m 3.59 dd (11.0, 4.5) 3.51 dt (11.0, 6.0)

7.50 s 7.15 s 5.63 s 10.38 s 7.69 d (7.8) 6.91 dd (7.8, 7.8) 6.98 dd (7.8, 7.8) 7.31 d (7.8) 10.94 brs 7.28 d (1.8) 7.59 d (7.8) 6.93 dd (7.8, 7.8) 7.04 dd (7.8, 7.8) 7.32 d (7.8) 4.54 d (7.8) 3.34 m 3.26 t (9.0) 3.22 t (9.0) 3.13 m 3.74 d (11.4) 3.56 m

3

7.53 6.94 7.09 7.56 6.51s 7.34 d (7.5) 6.79 dd (7.5, 7.5) 6.90 dd (7.5, 7.5) 7.12 d (7.5) 4.11 d (8.0) 3.27 t (8.0) 3.17 dd (8.0, 9.0) 3.24 m 2.79 m 3.57 dd (12.5, 2.5) 3.49 dd (12.5, 5.0)

7.46 s 7.61 d (8.0) 6.98 dd (7.5, 8.0) 7.14 dd (7.5, 8.0) 7.37 d (8.0) 4.54 d (8.0) 3.32 m 3.23 m 3.22 m 3.09 m 3.66 dd (11.5, 4.5) 3.51 dt (11.5, 6.0)

d (7.0) dd (7.0, 8.0) dd (8.0, 7.5) d (7.5)

3.51 d (13.0) 3.44 d (13.0)

5

10.40 s 7.69 d (8.0) 6.88 dd (7.0, 8.0) 6.94 dd (7.0, 7.0) 7.15 d (7.0)

4.26 d (16.0) 4.15 d (16.0)

4b

7.63 d (7.5) 6.88 dd (7.5, 7.5) 7.49 dd (7.5, 7.5) 7.04 d (7.5) 4.72 d (8.0) 3.31 m 3.22 m 3.21 m 3.18 m 3.69 dd (11.0, 4.0) 3.53 dt (11.0, 6.0)

12.46 s 7.82 d (7.5) 7.02 dd (8.0, 7.5) 7.22 dd (7.5, 8.0) 7.50 d (7.5) 9.92 s

7.17 brs

6

7.56 d (7.2) 6.87 dd (7.2, 7.8) 7.45 dd (7.8, 7.8) 7.09 d (7.8) 4.70 d (9.6) 3.09 t (9.0) 3.23 m 3.13 t (9.0) 3.24 m 3.71 dd (12.0, 1.5) 3.52 dd (12.0, 6.0)

11.44 s 7.81 d (7.8) 7.16 dd (7.8, 7.8) 7.23dd (7.8, 7.8) 7.40 d (7.8) 9.20 s

7.07 s

7

a NMR data (d) were measured in DMSO-d6 for 1–4, 6, and 7 and in CD3OD for 5 at 500 MHz for 1, 2, and 4–6 and at 600 MHz for 3 and 7. Coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H – 1H COSY, HSQC, and HMBC experiments. b Data of OCH3 in 4: d 4.02 (3H, s).

6.31 s 11.00 s 7.72 d (7.5) 6.97 t (7.5) 7.06 dd (7.5, 8.0) 7.24 d (8.0) 11.23 d (2.5) 7.45 d (2.5) 7.51 d (7.5) 6.95 t (7.5) 7.08 t (7.5) 7.38 d (7.5) 4.51 d (8.0) 3.35 td (9.0, 4.5) 3.24 td (9.0, 4.5) 3.31 m 3.13 ddd (9.0, 4.5, 2.0) 3.77 ddd (11.5, 4.5, 2.0) 3.59 ddd (11.5, 7.0, 4.5)

1a 1b 2 10 40 50 60 70 100 200 400 500 600 700 1000 2000 3000 4000 5000 6000 a 6000 b

2

H NMR spectral data (d) for isatindigobisindolosides A (1) – G (7)a.

1

1

No.

Table 1.

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4 Y.-F. Liu et al.

Journal of Asian Natural Products Research

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Table 2.

13

5

C NMR spectral data (d) for isatindigobisindolosides A (1) – G (7)a.

No.

1

2

3

4b

5

6

7

1 2 20 30 30 a 40 50 60 70 70 a 200 300 300 a 400 500 600 700 700 a 1000 2000 3000 4000 5000 6000

119.5 24.6 121.8 133.2 120.4 118.2 119.1 122.1 111.6 133.4 123.7 108.1 125.1 118.4 119.1 121.8 111.8 136.4 106.4 73.6 76.5 69.4 76.9 60.6

119.3 24.3 120.9 133.8 120.3 118.5 119.1 122.0 111.6 133.4 123.9 107.7 125.1 118.5 119.1 121.8 111.8 136.5 106.6 73.7 76.6 69.6 77.1 60.7

172.9 38.6 126.5 132.4 120.4 117.4 118.2 120.7 111.6 132.8 123.4 112.5 126.5 118.6 118.3 121.0 111.3 135.8 106.2 73.7 76.4 69.8 77.0 61.1

20.2

34.9

108.4

105.3

127.2 132.8 121.4 117.7 118.3 120.5 111.0 132.6 122.4 109.5 123.4 119.6 119.3 122.2 108.1 132.1 106.4 73.8 76.8 69.9 77.0 61.1

180.6 86.1 129.5 128.6 123.6 131.4 111.4 144.0 125.9 108.2 129.6 120.2 119.9 122.4 112.3 137.8 102.2 75.6 78.3 71.7 78.1 62.9

124.3 139.7 119.7 119.0 119.7 125.0 112.2 133.6 133.4 183.6 120.4 124.5 118.6 136.1 112.0 151.6 105.7 73.9 76.6 69.9 77.2 61.0

129.9 114.1 125.7 120.1 120.4 123.0 111.6 137.6 133.7 185.2 120.9 123.7 119.0 135.4 112.9 153.2 87.2 72.9 77.8 69.7 81.0 60.8

a

Data were measured in DMSO-d6 for 1 –4, 6, and 7 and in CD3OD for 5 at 125 MHz for 1, 2, and 4–6 and at 150 MHz for 3 and 7. The assignments were based on DEPT, 1H– 1H COSY, HSQC, HMQC, and HMBC experiments. b Data of OCH3 in 4: d 65.7. HO HO HO

O

O

HO HO HO

CN

O

OH NH

O

O

NH

N H

O

O

HO HO HO

OH NH

O

NH

O

OH

O

N

NH

OCH3

4 HO HO HO

O

5 O

O

H N

OH NH

6

N H

3

1 HO HO HO

NH2

OH

O

HO HO HO

O OH

S

N H

HN

7

Figure 2. Main 1H – 1H COSY (thick lines) and HMBC (arrows, from 1H to compounds 1 and 3 – 7.

13

C) correlations of

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6

Y.-F. Liu et al.

Figure 3. (Colour online) The experimental CD spectra (full line) of 1 (blue) and 2 (red) and the calculated ECD spectra (dashed line) of 1 (blue) and (2S)-1 (red).

1H-indol-2-yl)-2-(1H-indol-3-yl)acetonitrile and named isatindigobisindoloside A. Compound 2, a white amorphous powder with ½a20 D 2 14.6 (c ¼ 0.26, MeOH), is an isomer of 1 as indicated by spectroscopic data. Comparison of the NMR spectroscopic data of 2 and 1 (Tables 1 and 2) demonstrated that H-10 , H-400 , H-6000 a, and H-6000 b and C-20 in 2 were shielded by DdH 2 0.13, 2 0.05, 2 0.18, and 2 0.08 and DdC 2 0.9, respectively, whereas H-40 and C-30 were deshielded by DdH þ 0.07 and DdC þ 0.6. The other proton and carbon resonances were shifted by DdH # ^ 0.03 and DdC # ^ 0.3 ppm, respectively. Since the protons and carbons with chemical shift changes are mainly around the chiral center C-2, compound 2 was suggested to be the C-2 epimer of 1. This was confirmed by 2D NMR data analysis. In particular, the location of the b-glucopyranosyl in 2 was proved by the HMBC correlation from H1000 to C-30 , while the acetonitrile-bridged connection of the two indole moieties was verified by the HMBC correlations from H-2 to C-20 , C-200 , and C-300 . Furthermore, the CD spectrum of 2 was consistent with the calculated ECD spectrum of (2S)-1

(Figure 3). Thus, compound 2 was determined as (2 )-(2S)-2-(3-b-D -glucopyranosyloxy-1H-indol-2-yl)-2-(1Hindol-3-yl)acetonitrile and named isatindigobisindoloside B. Compound 3, a white amorphous powder with ½a20 D 2 33.9 (c ¼ 0.11, MeOH), has the molecular formula C24H25N3O7 as indicated from HR-ESIMS. Comparison of the NMR spectroscopic data of 3 and 1 (Tables 1 and 2) revealed that the H-400 and C-2, C-20 , and C-300 resonances in 3 were deshielded by DdH þ 0.08 and DdC þ 14.0, þ 4.7, and þ 4.4, respectively; in contrast, the H-10 , H-100 , and H-200 resonances were shielded by DdH 2 0.62, 2 0.29, and 2 0.17. In addition, the cyano carbon resonance in 1 was replaced by those ascribed to a carbamide group [dH 7.15 and 7.50 (1H each, NH2); dC 172.9 (C-1)] in 3. The IR spectrum of 3 also showed an absorption band (1670 cm21) due to the carbamide, instead of the cyano absorption of 1. These data suggested that 3 was the amide analogue of 1 with substitution of the cynao group by the carbamide group, which was confirmed by 2D NMR experiments and enzymatic hydrolysis. Especially, linkages of the structural

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Journal of Asian Natural Products Research moieties were verified by the correlations from H-2 to C-1, C-20 , C-200 , C-30 , C-300 , and C-300 a; from NH2 to C-1; and from H1000 to C-30 (Figure 2) in the HMBC spectrum of 3. Using the same protocol as described for 1, D -glucopyranose was isolated and identified from the hydrolysate of 3. Similarity of the CD spectra between 3 and 1 indicated the same 2R configuration for the two compounds. The calculated ECD spectra of 3 and (2S)-3 displayed the same order of Cotton effect signs, e.g., positive, negative, and positive (Figure 4), indicating that the b-D glucopyranosyl is critical for the Cotton effects of the two C-2 epimers. The wavelengths of the Cotton effects in the experimental and calculated spectra of 3 were consistent with each other, but differed from those in the calculated ECD spectrum of (2S)-3. This supports that 3 has the (2R)-configuration. Therefore, compound 3 was determined as (2)-(2R)-2-(3b-D -glucopyranosyloxy-1H-indol-2-yl)-2(1H-indol-3-yl)acetamide and named isatindigobisindoloside C. Compound 4 was obtained as a white amorphous powder with ½a20 D 2 56.4 (c ¼ 0.5, MeOH). Its molecular formula C24H26N2O7 was indicated from HRE-

7

SIMS. Comparison of the NMR data between 4 and 1 (Tables 1 and 2) indicated that the acetonitrile unit and the nitrogenbearing proton (H-100 ) in 1 were replaced by an methylene [ dH 4.26 (1H, d, J ¼ 16.0 Hz, H-1a) and 4.15 (1H, d, J ¼ 16.0 Hz, H-1b) and dC 20.2 (C-1)] and a N-methoxy group [dH 4.02 (3H,s) and dC 65.7] in 4, respectively. Based on the differences, 4 was deduced to be a decyano derivative of 1 with the methoxy group at N-100 , and confirmed by 2D NMR data analysis. Particularly, in the HMBC spectrum of 4, the correlations from H2-1 to C-20 , C-200 , C-30 , C-300 , and C-300 a; NH-10 to C-20 , C-30 , C-30 a, and C-70 a; and from H-1000 to C-30 secured connection of the structural moieties. The D -configuration of glucopyranosyl was verified by enzymatic hydrolysis of 4 using the aforementioned method. Thus, compound 4 was determined as (2 )-(3-b-D -glucopyranosyloxy1H-indol-2-yl)(1-methoxy-1H-indol-3-yl) methane and named isatindigobisindoloside D. Compound 5, a white amorphous powder with ½a20 D 2 52.1 (c ¼ 0.37, MeOH), has the molecular formula C23H24N2O7 indicated by HR-ESI-MS and NMR data (Tables 1 and 2).

Figure 4. (Colour online) The experimental CD spectrum (full line) of 3 (blue) and the calculated ECD spectra (dashed line) of 3 (blue) and (2S)-3 (red).

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8

Y.-F. Liu et al.

Comparison of the NMR and IR spectroscopic data of 5 with those of 4 indicated that the two sp2 hybridized quaternary carbons (C-20 and C-30 ) in 4 were replaced by a carbonyl carbon (dC 180.6; nmax 1716 cm21) and an oxygen-bearing sp3 hybridized quaternary carbon (dC 86.1) in 5, respectively, in addition to the absence of the N-methoxy group. The above evidences suggested the presence of a 2oxo-indolin-3-yl in 5 in place of the 1Hindol-2-yl moiety in 4. In the HMBC spectrum of 5, besides the correlations confirming the 1H-indol-3-yl moiety, the correlations from H-40 to C-30 , C-60 , and C70 a; from H-50 to C-30 a and C-70 ; from H-60 to C-40 and C-70 a; from H-70 to C-30 a and C-50 ; and from H-1000 to C-30 ; together with the chemical shifts of these proton and carbon resonances including C-20 , proved the presence of the 3-b-glucopyranosyloxy-2-oxo-indolin-3-yl moiety. In addition, the methylene-bridged linkage between the indolin-3-yl and indol-3-yl moieties in 5 was confirmed by the HMBC correlations from H2-1 to C-20 , C-200 , C-30 , C-300 , C-30 a, and C-300 a. Using the aforementioned protocol, the D -configuration of b-glucopyranosyl in 5 was verified, while the aglycone (5a) was obtained from the

hydrolysate and characterized by its ESIMS and NMR spectroscopic data. Based on similarity between the CD spectrum of 5 and those of 1 and 3, the (30 R)-configuration was assigned for 5. The assignment was further supported by the ECD spectral calculations of 5 and 5a. Although the calculated ECD spectra of 5 and its epimer (30 S)-5 did not show mirror curves, the calculated and experimental spectra of 5 were consistent with each other (Figure 5). The experimental and calculated spectra of 5a are almost identical, and mirrored the calculated ECD spectrum of (30 S)-5a (Figure 6). Therefore, the structure of compound 5 was determined (Figure 1) and named isatindigobisindoloside E. Compound 6 was isolated as a pink amorphous powder with ½a20 D 2 52.9 (c 0.23, MeOH). Its molecular formula C23H22N2O7 was determined by HR-ESIMS combined with the NMR data (Tables 1 and 2). Comparison of the NMR data between 6 and 4 revealed the presence of 1H-oxoindolinylidene and sp2 hybridized methine units in place of the respective 1-methoxy-1H-indol-3-yl and methylene units in 4. This was verified by the 1H – 1H COSY cross peaks of H-400 /H-500 /H-600 /H-

Figure 5. (Colour online) The experimental CD spectrum (full line) of 5 (blue) and the calculated ECD spectra (dashed line) of 5 (blue) and (30 S)-5 (red) (the calculated ECD spectra were shifted by 210 nm).

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Journal of Asian Natural Products Research

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Figure 6. (Colour online) The experimental CD spectrum (full line) of 5a (blue) and the calculated ECD spectra (dashed line) of 5a (blue) and (30 S)-5a (red) (the calculated ECD spectra were shifted by 23 nm).

700 and HMBC correlations from H-1 to C200 , C-30 , and C-300 ; from NH-100 to C-200 , C300 , C-300 a, and C-700 a; from H-400 to C-300 , C-600 , and C-700 a; from H-500 to C-300 a and C-700 ; from H-600 to C-400 and C-700 a; and from H-700 to C-300 a and C-500 ; together with their chemical shifts (Tables 1 and 2). In the NOE difference spectrum of 6, irradiation of H-1 enhanced H-100 and vice versa (Figures S102 and S103), indicating a trans geometry of the double bond between C-1 and C-200 . The D -configuration of glucopyranosyl was verified by enzymatic hydrolysis of 6 using the protocol as described for 1. Thus, compound 6 was determined as (2 )-(E)-(3-bD -glucopyranosyloxy-1H-indol-2-yl)(1H3-oxo-indolin-2-ylidene)methane and named isatindigobisindoloside F. Compound 7, an orange amorphous powder with ½a20 D 2 58.6 (c ¼ 0.09, MeOH), has the molecular formula C23H22N2O6S as indicated by HR-ESIMS. The NMR spectroscopic data of 7 (Tables 1 and 2) demonstrated the presence of bisindole and b-glucopyranosyl moieties similar to those of 6. However, the anomeric carbon of the bglucopyranosyl was resonated at dC 87.2 in the 13C NMR spectrum of 7. This,

combined with the molecular composition, suggests that 7 is a bisindole b-thioglucopyranoside [6], which was confirmed by 2D NMR data analysis. The 1H – 1H COSY cross peaks of H-40 /H-50 /H-60 /H-70 and H400 /H-500 /H-600 /H-700 and the HMBC correlations from H-1 to C-20 , C-30 a, and C-300 ; from H-100 to C-200 , C-300 , C-300 a, and C-700 a; and from H-40 to C-30 , C-60 , and C-70 a; and from H-400 to C-300 , C-600 , and C-700 a; together with their chemical shifts, verified the presence of an aglycone moiety of (1Hindol-3-yl)(1H-3-oxo-indolin-2-ylidene) methane in 7. Additionally, in the HMBC spectrum of 7, correlation from H-1000 to C-20 , in combination with the quaternary nature of C-20 , revealed that the bglucopyranosylthio unit was located at C-20 . Although no enhancement was observed when irradiation of H-1 or H-100 in the NOE difference spectrum of 7, the NOESY spectrum showed the correlation between H-100 with H-40 (Figures S118 and S119). This supports a cis geometry of the double bond between C-1 and C-200 . Because an isolated sample amount of 7 is not enough for hydrlysis, the D configuration of glucopyranosyl in 7 was temporately assigned based on similarity of the specific rotation values between 7

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and the co-occurring indole-3-acetonitrile4-methoxy-2-S-b-D- glucopyranoside [6], of which the indole-derived aglycone moieties have no chirality. The specific rotation of the known compound, which was not presented in the literature [6], was measured to be ½a20 D 2 48.4 (c ¼ 1.0, MeOH) with a sample obtained in this study. Using the aforementioned method, the configuration of b-glucopyranosylthio was proved by acid hydrolysis of the sample and subsequent isolation and identification of D -glucopyranose from the hydrolysate. Thus, compound 7 was determined as (2 )-(Z)-(2-b-D glucopyranosylthio-1H-indol-3-yl)(1H-3oxo-indolin-2-ylidene)methane and named isatindigobisindoloside G. In the NMR spectra of 1 or 2 in DMSO-d6, integration of the H-2 signal is abnormally less than one proton and intensity of the C-2 resonance is too weak to be distinguished from the baseline. However, the exchangeable hydroxy and amino protons have integrations similar to those of the carbon-bearing protons. Additionally, in the 1H NMR spectra of 2 in CD3OD (Figure S42) and 3 in DMSO-d6, integration of H-2 and intensity of C-2 are normal. These phenomena suggest that H-2 in 1 – 3 is not exchangeable under the experimental conditions and that integration of the H-2 signal and intensity of the C-2 resonance in the NMR spectra are affected not only by the substitution group at C-2 but also by the solvents. The ECD spectra calculations of the bisindole glucosides [1, (2S)-1 (2), 3, (2S)3, 5, and (30 S)-5] demonstrate that the ECD spectra are significantly disturbed by the chiral glucopyranosyloxy group on the indole chromophores (Supporting Information). The calculated ECD spectra of 1 and 2 display the mirror Cotton effects with different intensities at the same wavelengths (Figure 3), indicating that only the intensities of the Cotton effects are affected by the glucopyranosyloxy

group at C-30 of the indole chromophore and that the signs of the Cotton effects mainly depend on the C-2 chirality. However, the calculated ECD spectra of 3 and (2S)-3 show the Cotton effects at the different wavelengths, with the different intensities and the same order of the signs, e.g., positive, negative, and positive (Figure 4). This indicates that both the intensities and wavelengths of the Cotton effects are influenced by the glucopyranosyloxy group at C-30 and that the b-D glucopyranosyloxy play a decisive role for the signs of the Cotton effects of 3 and (2S)-3. Meanwhile, it is indicated that the signs of the Cotton effects are independent upon the C-2 chirality in 3 and (2S)-3, which is completely reversed to the cases of 1 and 2. Furthermore, the ECD spectra of 5 and (30 S)-5 show the Cotton effects at the different wavelengths, with both the different intensities and signs (Figure 5), demonstrating that the intensities and wavelengths, as well as the signs of the Cotton effects, are disturbed by the b-D glucopyranosyl at C-30 . Together, the ECD spectra calculations of the bisindole glucosides reveal that the b-D -glucopyranosyl at C-30 has great influences on the intensities, wavelengths, and signs of the Cotton effects. Comparison of the experimental CD spectra with the ECD spectra predicted from TDDFT calculations has become a recent approach increasingly applied for the determination of absolute configurations of natural products [19], and some flexible units including the b-D -glucopyranosyl were occasionally replaced by a methyl group to simplify the computation [20]. The ECD spectra calculations carried out in this study demonstrate that replacement of the b-D -glucopyranosyl by the methyl group may result in an ambiguous assignment of the absolute configurations, such as 3 and (2S)-3. In the preliminary in vitro assays, compounds 2, 5, and 6 showed inhibitory activity against a Coxsackie virus B3 replication [21], with respective IC50

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Journal of Asian Natural Products Research values of 40.5, 33.3, and 8.4 mM, and respective SI values of 2.2, 3.0, and 11.8 (the positive control ribavirin gave an IC50 value of 229.5 mM and a SI value of 6.8). Compounds 2 and 4 – 6 inhibited the influenza virus A/Hanfang/359/95 (H3N2) [21], with IC50 values of 53.3, 12.6, 100.0, and 66.6 mM and SI values of 2.1, 2.7, 2.4, and 3.6, respectively, while the positive control ribavirin gave an IC50 value of 1.4 mM and a SI value of 814.1. In addition, these compounds were also assessed for their inhibitory activity against HIV-1 replication [22], DL -galactosamine (GAlN)-induced hepatocyte (WB-F344 cell) damage [23], and several human cancer cell lines [24], but all were inactive at a concentration of 10 mM. In conclusion, seven new glycosidic bisindle alkaloids, possessing diverse structure features, were isolated as the minor components from the aqueous extract of I. indigotica roots. Compounds 2 and 4 – 6 showed activities against the Coxsackie virus B3 (CVB3) and/or the influenza virus A/Hanfang/359/95 (H3N2). These results, combined with our previous studies [14 – 16], show that diverse compounds contribute toward pharmacological efficacy that supports the traditional uses of I. indigotica roots. The new structures provide an important clue for further studies of biomimetic and total syntheses, chemical transformation, structural modification, and structure – activity relationships, as well as biosynthesis of the diverse indole alkaloids, from this indispensable medicinal plant. 3. Experimental 3.1 General experimental procedures Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were measured on a V-650 spectrometer (JASCO, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR Microscope Transmission) (Thermo Elec-

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tron Corporation, Madison, WI, USA). NMR spectra were obtained at 500 MHz or 600 MHz for 1H, and 125 MHz or 150 MHz for 13C, respectively, on Inova 500 or SYS 600 (Varian Associates Inc., Palo Alto, CA, USA) or Bruker 600 NMR spectrometers (Bruker Corp., Karlsruhe, Germany) in DMSO-d6 or MeOH-d4, with solvent peaks used as references. ESI-MS and HR-ESI-MS data were measured using an AccuToFCS JMS-T100CS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA). Column chromatography (CC) was performed with silica gel (200 – 300 mesh, Qingdao Marine Chemical Inc. Qingdao, China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), or CHP 20P (Mitsubishi Chemical Inc., Tokyo, Japan). HPLC separation was performed on an instrument consisting of an Agilent ChemStation for LC system, an Agilent 1200 pump, and an Agilent 1100 single-wavelength absorbance detector (Agilent Technologies, Ltd.) with a Grace semipreparative column (250 £ 10 mm i.d.) packed with C18 reversed phase silica gel (5 mm) (W.R. Grace & Co., Columbia, MD, USA) or a Chiralpak AD-H column (250 £ 10 mm i.d.) packed with amylose tris(3,5-dimethylphenylcarbamate) coated on 5 mm silica gel (Daicel Chiral Technologies Co. Ltd., Shanghai, China). TLC was carried out with glass precoated silica gel GF254 plates (Qingdao Marine Chemical Inc.). Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH followed by heating. Unless otherwise noted, all chemicals were obtained from commercially available sources and were used without further purification. 3.2

Plant material

The roots of I. indigotica were collected in December 2009 from Anhui Province, People’s Republic of China. Plant identity was verified by Mr Lin Ma (Institute of Materia Medica, Beijing, China). A voucher

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specimen (No. ID-S-2385) was deposited at the herbarium of Natural Medicinal Chemistry, Institute of Materia Medica. 3.3 Extraction and isolation The air-dried and pulvarized plant material (50 kg) was decocted with H2O (150 L 3 £ 1 h). The aqueous extracts were combined and evaporated under reduced pressure to yield a dark-brown residue (32 kg). The residue was dissolved in H2O (122 L), loaded on a macroporous adsorbent resin (HPD-110, 19 kg) column (20 £ 200 cm), and eluted successively with H2O (50 L), 50% EtOH (125 L), and 95% EtOH (100 L) to yield three corresponding fractions A, B, and C. After removing the solvent under reduced pressure, fraction B (0.9 kg) was separated by CC over MCI gel CHP 20P (5 L), with successive elution using H2O (10 L), 30% EtOH (30 L), 50% EtOH (20 L), 95% EtOH (10 L), and Me2CO (8 L), to give fractions B1 –B5. Fraction B3 (165 g) was subjected to CC over silica gel, eluted by a gradient of increasing MeOH (0 – 100%) in EtOAc to afford subfractions B3-1 –B3-16. Subfraction B3-3 (7.5 g) was separated by CC over Sephadex LH-20, eluting with CHCl3 – MeOH (1:1), to yield subfractions B3-31– B3-3-4, of which subfraction B3-3-4 (3.0 g) was further chromatographed over silica gel, eluted by a gradient of increasing MeOH (0 –100%) in CHCl3, to yield subfractions B3-3-4-1 –B3-3-414. Subfraction B3-3-4-12 (481.0 mg) was further fractionated by CC over Sephadex LH-20 (MeOH) to afford B3-3-4-12-1– B3-3-4-12-10. Of these, subfraction B33-4-12-2 (250.0 mg) was purified by RP-HPLC (57% MeOH in H2O, 1.5 ml/min) to afford indole-3-acetonitrile-4-methoxy-2-S-b-D -glucopyranoside (64.0 mg, 0.0013%, tR ¼ 44.0 min). Separation of B3-3-4-12-6 (21.0 mg) using RP-HPLC (42% MeOH in H2O, 2.0 ml/min) yielded a mixture (7.0 mg), which was further separated by HPLC on

a semi-preparative Chiralpak AD-H column, using n-hexane-iPrOH (2:1) as the mobile phase (2.0 ml/min) to yield 1 (4.0 mg, 0.000008%, tR ¼ 37.3 min) and 2 (3.0 mg, 0.000006%, tR ¼ 26.2 min). Subfraction B3-3-4-13 (101.0 mg) was fractionated again by CC over Sephadex LH-20 (MeOH) to afford subfractions B3-3-4-13-1 – B3-3-4-13-7, subsequently purification of subfraction B3-3-4-13-6 by RP-HPLC (70% MeOH in H2O, 1.5 ml/min) to obtain 6 (2.5 mg, 0.000005%, tR ¼ 24 min). Subfraction B3-3-4-14 (413.0 mg) was chromatographed over Sephadex LH-20 (MeOH) to yield subfractions B3-3-4-14-1 –B3-34-14-10. Purification of subfraction B3-34-14-3 (21.0 mg) by RP-HPLC (39% MeOH in H2O, 2.0 ml/min) afforded 5 (4.8 mg, 0.0000096%, tR ¼ 62 min). Subfraction B3-4 (11.0 g) was subjected to CC over Sephadex LH-20 (MeOH) to yield subfractions B3-4-1 – B3-4-6, of which subfraction B3-4-5 (2.5 g) was further fractionated by RP-MPLC, eluted with a gradient of increasing MeOH (20 – 100%) in H2O, to yield subfractions B3-4-5-1 – B3-4-5-30. Further separation of subfraction B3-4-5-13 (405 mg) by CC over Sephadex LH-20 (MeOH) yielded subfractions B3-4-5-13-1 –B3-45-13-9. Purification of subfraction B3-4-5-13-6 (7.1 mg) by RP-HPLC (42% MeOH, 2.0 ml/min) to afford 3 (2.5 mg, 0.000005%, tR ¼ 35 min), and of subfraction B3-4-5-27 (40.0 mg) by RPHPLC (70% MeOH, 1.5 ml/min) to yield 7 (1.5 mg, 0.000003%, tR ¼ 33 min). Fraction C (88.0 g) was subjected to CC over silica gel, with elution using a gradient of increasing acetone concentration (0 – 100%) in petroleum ether, to afford fractions C1 – C11. Fraction C10 (3.5 g) was subjected to CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to give C10-1 – C10-4. Purification of C10-1 (21.1 mg) by RP-HPLC (30% MeCN in H2O, 2.0 ml/min) yielded 4 (12.3 mg, 0.000025%, tR ¼ 45 min).

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Journal of Asian Natural Products Research 3.3.1 Isatindigobisindoloside A (1) White amorphous solid; ½a20 D 2 27.7 (c 0.17, MeOH); UV (MeOH) lmax (log 1) 219 (4.52), 280 (3.88) nm; CD (MeOH) D1211 nm þ 41.03, D1229 nm 2 69.58, D1286 nm þ 6.77; IR nmax 3343, 2923, 2252, 2128, 1700 (sh), 1622, 1597, 1493, 1456, 1424, 1343, 1244, 1176, 1099, 1071, 1023, 898, 825, 749 cm21; 1H NMR (DMSO-d6, 500 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) spectral data, see Table 2; (þ )-ESI-MS: m/z 472 [M þ Na]þ; (2 )-ESI-MS: m/z 448 [M 2 H]2, 484 [M þ Cl]2; (þ)-HR-ESIMS: m/z 472.1479 [M þ Na]þ (calcd for C24H23N3O6Na, 472.1479). 3.3.2

Isatindigobisindoloside B (2)

White amorphous solid; ½a20 D 2 14.6 (c 0.26, MeOH); UV (MeOH) lmax (log 1) 218 (4.24), 280 (3.63) nm; CD (MeOH) D1212 nm 2 32.21, D1229 nm þ 40.03, D1286 nm 2 2.41; IR nmax 3376, 2922, 2853, 2250, 2129, 1700 (sh), 1622, 1595, 1493, 1456, 1344, 1245, 1176, 1100, 1071, 1023, 898, 827, 748 cm21; 1H NMR (DMSO-d6, 500 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) spectral data, see Table 2; (þ )-ESI-MS: m/z 472 [M þ Na] þ; (2 )-ESI-MS: m/z 448 [M 2 H]2, 484 [M þ Cl]2; (þ )-HRESI-MS: m/z 472.1487 [M þ Na]þ (calcd for C24H23N3O6Na, 472.1479). 3.3.3

Isatindigobisindoloside C (3)

White amorphous solid; ½a20 D 2 33.9 (c ¼ 0.11, MeOH); UV (MeOH) lmax (log 1) 222 (4.38), 281 (3.79) nm; CD (MeOH) D1211 nm þ11.34, D1231 nm 221.75, D1293 nm þ1.49; IR nmax 3351, 2919, 2852, 1670, 1619, 1593, 1492, 1458, 1413, 1343, 1319, 1263, 1158, 1101, 1072, 1045, 1022, 930, 897, 822, 748 cm21; 1H NMR (DMSO-d6, 600 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 150 MHz) spectral data, see Table 2; (þ )-ESI-MS: m/z 490 [M þ Na]þ; (2 )-ESI-MS: m/z 466

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[M 2 H]2, 502 [M þ Cl]2; (þ)-HR-ESIMS: m/z 490.1584 [M þ Na]þ (calcd for C24H25N3O7Na, 490.1585). 3.3.4

Isatindigobisindoloside D (4)

White amorphous solid; ½a20 D 2 56.4 (c 0.5, MeOH); UV (MeOH) lmax (log 1) 204 (4.61), 225 (4.63), 288 (3.97), 317 (3.58) nm; CD (MeOH) D1212nm þ 1.82, D1289nm þ 0.43; IR nmax 3401, 2933, 2892, 1618, 1457, 1342, 1241, 1072, 1043, 744 cm21; 1H NMR (DMSO-d6, 500 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) spectral data, see Table 2; (2 )-ESI-MS: m/z 453 [M 2 H]2, 489 [M þ Cl]2; (þ )-HRESI-MS: m/z 477.1657 [M þ Na]þ (calcd for C24H26N2O7 Na, 477.1632). 3.3.5

Isatindigobisindoloside E (5)

White amorphous solid; ½a20 D 2 52.1 (c 0.37, MeOH); UV (MeOH) lmax (log 1) 202 (4.15), 216 (3.98), 261 (3.22), 282 (3.15), 291 (3.08) nm; CD (MeOH) D1210 nm þ 41.39, D1237 nm 2 29.77, D1267 nm þ4.37, D1303 nm þ 1.03; IR nmax 3399, 2922, 2881, 1716, 1622, 1570, 1472, 1458, 1427, 1342, 1291, 1223, 1183, 1153, 1077, 1033, 890, 792, 746 cm21; 1H NMR (CD3OD, 500 MHz) spectral data, see Table 1; 13C NMR (CD3OD, 125 MHz) spectral data, see Table 2; (þ)-ESI-MS: m/z 463 [M þ Na]þ, 479 [M þ K]þ; (2)-ESIMS: m/z 439 [M 2 H]2; (þ)-HR-ESI-MS: m/z 441.1654 [M þ H]þ (calcd for C23H25N2O7, 441.1656), 463.1479 [M þ Na]þ (calcd for C23H24N2O7Na, 463.1476). 3.3.6

Isatindigobisindoloside F (6)

Pink amorphous solid; ½a20 D 2 52.9 (c 0.23, MeOH); UV (MeOH) lmax (log 1) 205 (4.13), 278 (3.75), 334 (3.80), 385 (3.68), 519 (3.88) nm; CD (MeOH) D1224 nm 2 0.98, D1249 nm þ 0.25, D1328 nm þ1.06; IR nmax 3274, 2923, 1678, 1652,

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1623, 1588, 1533, 1491, 1468, 1334, 1296, 1244, 1202, 1134, 1103, 1076, 1027, 949, 882, 828, 802, 748, 708, 687 cm21; 1H NMR (DMSO-d6, 500 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) spectral data, see Table 2; (2)-ESI-MS: m/z 437 [M 2 H]2, 473 [M þ Cl]2; (þ)-HRESI-MS: m/z 439.1500 [M þ H]þ (calcd for C23H23N2O7, 439.1500), 461.1319 [M þ Na]þ (calcd for C23H22N2O7Na, 461.1319). 3.3.7 Isatindigobisindoloside G (7) Orange amorphous solid; ½a20 D 2 58.6 (c 0.09, MeOH); UV (MeOH) lmax (log 1) 205 (4.29), 268 (3.89), 395 (3.52), 495 (3.63) nm; CD (MeOH) D1238 nm 2 0.74, D1270 nm þ 0.76; IR nmax 3359, 3201, 2955, 2921, 2851, 1734, 1660, 1630, 1589, 1468, 1424, 1377, 1318, 1290, 1247, 1225, 1199, 1164, 1135, 1099, 1051, 749, 721, 707 cm21; 1H NMR (DMSO-d6, 600 MHz) spectral data, see Table 1; 13C NMR (DMSO-d6, 150 MHz) spectral data, see Table 2; (þ)-ESI-MS: m/z 477 [M þ Na]þ; (2)-ESI-MS: m/z 453 [M 2 H]2; (þ)-HRESI-MS: m/z 455.1269 [M þ H]þ (calcd for C23H23N2O6S, 455.1271), 477.1094 [M þ Na]þ (calcd for C23H22N2O6SNa, 477.1091). 3.3.8 Enzymatic hydrolysis of 1, 3 – 6, and acidic hydrolysis of indole-3acetonitrile-4-methoxy-2-S-b-D glucopyranoside Compounds 1 and 3–6 (1.2–2.0 mg) were separately hydrolyzed in H2O (3 ml) with snailase (3.0 mg, CODE S0100, Beijing Biodee Biotech Co., Ltd., Beijing, China) at 378C for 24 h. Indole-3-acetonitrile-4-methoxy-2-S-b-D -glucopyranoside (9.0 mg) was hydrolyzed in 2N HCl (3 ml) at 808C for 6 h. The hydrolysate was evaporated under reduced pressure, then chromatographed over silica gel eluting with CH3CN–H2O (8:1), to yield sugar. The sugar (0.3– 2.0 mg) from the hydrolysates of 1, 3–6,

and indole-3-acetonitrile-4-methoxy-2-Sb-D -glucopyranoside gave retention factor (Rf < 0.38) on TLC (EtOAc-MeOHAcOH-H2O, 12:3:3:2), with ½a20 D values of þ 44.1 – þ 47.2 (c ¼ 0.02 2 0.11, H2O), and 1H NMR (D2O) data, consistent with those of an authentic D -glucose. The aglycones of 1–4, 6, and indole-3-acetonitrile-4-methoxy-2-S-b-D -glucopyranoside were decomposed into complex mixtures under the hydrolysis condition. The aglycone 5a was obtained from the hydrolysate of 5 as a white powder, ½a20 D 2 31.7 (c 0.10, MeOH); CD (MeOH) D1208 nm þ 13.95, D1238 nm (12.44, D1267 nm þ 2.30, D1295 nm þ0.94; 1 H NMR (DMSO-d6, 500 MHz) d: 10.66 (1H, s, NH), 9.95 (1H, s, NH), 7.37 (1H, d, J ¼ 8.0 Hz, H-40 ), 7.26 (1H, d, J ¼ 7.0 Hz, H-400 ), 7.19 (1H, d, J ¼ 8.0 Hz, H-700 ), 7.07 (1H, t, J ¼ 7.5 Hz, H-60 ), 6.94 (1H, t, J ¼ 7.5 Hz, H-50 ), 6.89 (1H, t, J ¼ 7.5 Hz, H-600 ), 6.83 (1H, t, J ¼ 7.5 Hz, H-500 ), 6.55 (1H, d, J ¼ 8.0 Hz, H-70 ), 6.54 (1H, s, H200 ), 6.05 (1H, s, OH), 3.25 (2H, s, H-1); 13C NMR (DMSO-d6, 125 MHz) d: 179.3 (C-20 ), 142.0 (C-70 a), 135.4 (C-700 a), 132.1 (C-60 ), 128.7 (C-300 a), 127.6 (C-30 a), 124.3 (C-40 ), 123.9 (C-200 ), 121.1 (C-50 ), 120.5 (C-600 ), 118.7 (C-400 ), 118.0 (C-500 ), 111.0 (C-700 ), 109.1 (C-70 ), 107.5 (C-300 ), 76.4 (C-30 ), 33.2 (C-1); (þ)-ESI-MS: m/z 279 [M þ H]þ, 301 [M þ Na]þ. 3.3.9

ECD calculation of 1– 3, 5, and 5a

Conformational analysis was conducted by Monte Carlo searching with the MMFF94 molecular mechanics force field using the Spartan 10 software for 1, (2S)-1 (2), 3, (2S)-3, and 5a, and the molecular operating environment software for 5 and (30 S)5. The lowest-energy conformers having relative energies within 2 kcal/mol [1, (2S)-1 (2), 3, (2S)-3, and 5a] or 5 kcal/ mol [5 and (30 S)-5] were optimized with the Gaussian 09 program. Subsequently, the conformers were re-optimized using DFT at the B3LYP/6-31(G (d, p) level,

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Journal of Asian Natural Products Research with the solvent effects considered using the dielectric constant of MeOH (1 ¼ 32.6) via conductor-like polarizable continuum model. The energies, oscillator strengths, and rotational strengths of the excitations were calculated using the TDDFT methodology at the B3LYP/6311((G (2d, 2p) level in vacuum. The reoptimized conformers showed relative Gibbs free energies (DG) under 2 kcal/ mol were used for ECD spectra simulation. The ECD spectra were simulated by the Gaussian function (s ¼ 0.28 eV). To obtain the final spectra, the simulated spectra of the lowest energy conformers were averaged on the basis of the Boltzmann distribution theory and their relative Gibbs free energy (DG). All quantum computations were performed using Gaussian 09 program package on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. Supplemental data The supplemental data for this article can be accessed at http://dx.doi.org/10.1080/ 10286020.2015.1055729 Acknowledgements We thank Chinese Academy of Medical Sciences and Peking Union Medical College High Performance Computing Platform for supporting the calculation of the ECD spectra.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding Financial support from the National Natural Sciences Foundation of China [NNSFC; grant number 81373287, 30825044, and 21132009], the Program for Changjiang Scholars and Innovative Research Team in University [PCSIRT, grant number IRT1007], and the National Science and Technology Project of

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China [grant numbers 2012ZX09301002-002 and 2011ZX0 9307-002-01] is acknowledged.

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