Isatindigodiphindoside, an alkaloid glycoside with a new diphenylpropylindole skeleton from the root of Isatis indigotica

Article abstract of DOI:10.1016/j.cclet.2017.05.019

A novel indole alkaloid glycoside with an unprecedented 2-(diphenylpropyl)indole skeleton, isatindigodiphindoside (1), was isolated from an aqueous extract of the roots of Isatis indigotica. The structure was determined by extensive spectroscopic studies, especially by 2D NMR data analysis combined with enzymatic hydrolysis and ECD calculations. Plausible biosynthetic pathways of compound 1 are also discussed.

Full text of DOI:10.1016/j.cclet.2017.05.019

Accepted Manuscript
Title: Isatindigodiphindoside, an alkaloid glycoside with a
new diphenylpropylindole skeleton from the root of Isatis
indigotica
Authors: Ling-Jie Meng, Qing-Lan Guo, Cheng-Gen Zhu,
Cheng-Bo Xu, Jian-Gong Shi
PII:
S1001-8417(17)30191-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.05.019
CCLET 4089
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
1-4-2017
2-5-2017
17-5-2017
Please cite this article as: Ling-Jie Meng, Qing-Lan Guo, Cheng-Gen Zhu, Cheng-
Bo Xu, Jian-Gong Shi, Isatindigodiphindoside, an alkaloid glycoside with a new
diphenylpropylindole skeleton from the root of Isatis indigotica, Chinese Chemical
Lettershttp://dx.doi.org/10.1016/j.cclet.2017.05.019
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Communication
Isatindigodiphindoside, an alkaloid glycoside with a new diphenylpropylindole
skeleton from the root of Isatis indigotica
Ling-Jie Meng, Qing-Lan Guo, Cheng-Gen Zhu, Cheng-Bo Xu, Jian-Gong Shi
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
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel indole alkaloid glycoside with an unprecedented 2-(diphenylpropyl)indole
skeleton, isatindigodiphindoside (1), was isolated from an aqueous extract of the roots of
Isatis indigotica. The structure was determined by extensive spectroscopic studies,
especially by 2D NMR data analysis combined with enzymatic hydrolysis and ECD
calculations. Plausible biosynthetic pathways of compound 1 are also discussed.
Available online
Graphical abstract
Keywords: Cruciferae
Isatis indigotica
Ban lan gen
Diphenylpropylindole
Alkaloid glycoside
Isatindigodiphindoside
A novel indole alkaloid glycoside with an unprecedented 2-(diphenylpropyl)-
indole skeleton, isatindigodiphindoside (1), was isolated from an aqueous
extract of the roots of Isatis indigotica. The structure was determined by
extensive spectroscopic studies, especially by 2D NMR data analysis
combined with enzymatic hydrolysis and ECD calculations. Plausible
biosynthetic pathways of compound 1 are also discussed.
Isatis indigotica Fort. is widely cultivated to meet demands of
medicinal utilization in China. Its dried roots and leaves, named
“ban lan gen” and “da qing ye” in Chinese, respectively, are
among the most popular herbal drugs in traditional Chinese
medicine for the treatment of influenza [1]. Many formulations
containing “ban lan gen” and/or “da qing ye” are marketed and
recorded in Chinese Pharmacopoeia [2]. In history and at present
these formulations play a crucial role to treat and prevent
influenza during influenza pandemics in China. Clinical efficacy of
these herbal medicines has long attracted attentions of
pharmacologists and chemists to search their mechanisms and
bioactive constituents. Pharmacological studies showed that
extracts of these medicines exhibited a broad spectrum of
activities, including antiviral, anti-endotoxic, antinociceptive,
anti-inflammatory, and antipyretic effects and cytotoxicity
against leukemia cells [3-6]. Meanwhile, different types of
chemical constituents with various biological activities were
isolated from the extracts, such as alkaloids, lignans, and
epigoitrin [7-9] etc., of which indole alkaloids are the main active
constituents. However, previous chemical studies were mainly
carried out on the ethanol and methanol extracts of the drug
materials. This is not in accordance with the practical application
of the herbal medicines by decocting with water. Therefore, as
part of a program to access the chemical diversity of Chinese
traditional medicines and study their biological effects, especially
focusing on minor constituents [10-29], we systematically
 Corresponding author.
E-mail address: shijg@imm.ac.cn (J.-G. Shi).

investigated the aqueous extracts of “ban lan gen” and “da qing
ye”, respectively, since these extracts exhibited significant
difference in chemical constituents based on HPLC-ESIMS
analysis. In our previous papers, we reported more than 100
constituents with highly diverse structure features from the “ban
lan gen” extract [30-38] and four indole alkaloid stereoisomers
from the “da qing ye” extract [39]. Some of these compounds
showed antiviral activity against influenza virus. Further
investigation of the remaining fraction from the “ban lan gen”
extract led to the isolation of an alkaloid glycoside (1), which
possesses an unprecedented carbon skeleton (Fig. 1). Herein, we
report details of the isolation, structure elucidation, postulated
biogenetic pathway of 1.
position δH
δC
position
9a
9b
1
δH
δC
2
130.7
134.5
122.9
119.0
119.5
121.8
111.6
134.5
135.2
113.3
149.0
146.1
116.2
3.76 dd (10.8, 9.6) 65.6
3.68 dd (10.8, 3.6)
134.5
3
3a
4
7.59 d (7.8)
2
6.79 d (1.8)
114.4
148.3
145.7
115.7
5
6.85 t (7.8)
6.93 t (7.8)
7.20 d (7.8)
3
6
4
7
5
6.59 d (8.4)
7a
1
2
3
4
5
6
7
8
6
6.83 dd (8.4, 1.8) 122.3
4.02 d (7.8) 107.6
1
2
3
4
5
6a
6b
7.23 d (1.8)
3.47 dd (9.6, 7.8) 75.3
3.41 dd (9.6, 9.0) 78.1
3.48 t (9.0)
2.99 m
71.4
77.6
6.76 d (7.8)
7.10 dd (7.8, 1.8) 122.3
3.72 dd (11.4, 2.4) 62.5
3.70 dd (11.4, 4.8)
4.68 d (12.0)
3.58 m
45.3
52.5
3-OCH3 3.91 s
56.7
56.4
3-OCH3 3.59 s
1
aNMR data (δ) were measured at 600 MHz for H and at 150 MHz for 13C.
Proton coupling constants (J) in Hz are given in parentheses. The assignments
were based on DEPT, 1H-1H COSY, HSQC, and HMBC experiments.
Fig. 1. Structure of compound 1.
Compound 1 was obtained as a yellowish powder with []2D0 –
58.1 (c 0.07, MeOH). Its IR spectrum showed absorption bands
assignable to hydroxy (3364 cm-1) and aromatic ring (1603 and
1519 cm-1) functionalities. The positive and negative mode ESI-
MS of 1 exhibited quasimolecular ion peaks at m/z 620 [M + Na]+
and 636 [M + K]+ and 596 [M  H] and 632 [M + Cl],
respectively. The molecular formula of C31H35NO11, with 15
degrees of unsaturation, was deduced from HR-ESI-MS at m/z
620.2124 [M + Na]+ (calcd. for C31H35NO11Na, 620.2102) and
NMR spectroscopic data (Table 1). The 1H NMR spectrum of 1 in
CD3OD showed resonances attributable to an ortho-substituted
phenyl at δH 7.59 (d, J = 7.8 Hz, H-4), 6.85 (t, J = 7.8 Hz, H-5),
6.93 (t, J = 7.8 Hz, H-6), and 7.20 (d, J = 7.8 Hz, H-7); two meta-
para-disubstituted phenyls at δH 7.23 (d, J = 1.8 Hz, H-2′), 6.76
(d, J = 7.8 Hz, H-5′), and 7.10 (dd, J = 7.8 and 1.8 Hz, H-6′) and
6.79 (d, J = 1.8 Hz, H-2), 6.59 (d, J = 8.4 Hz, H-5), 6.83 (dd, J
= 8.4 and 1.8 Hz, H-6); and two methoxy groups at δH 3.91 (s, 3′-
OCH3) and 3.59 (s, 3-OCH3). In addition, the spectrum showed
resonances assignable to a -glucopyranosyl-oxy, an oxygen-
bearing methylene, and two methines between δH 4.68 and 2.99,
of which the -configuration of glucopyranosyl-oxy was indicated
by the coupling constant value of anomeric proton resonated at δH
4.02 (d, J = 7.8 Hz, H-1′′′). The 13C NMR and DEPT spectra of 1
showed 29 carbon signals corresponding to the above described
functional units, as well as two additional sp2 hybridized
quaternary carbons attributed to a tetra-substituted double bond.
As compared with those of compounds previously isolated from I.
indigotica [30-39], these spectroscopic data indicate that 1 is an
unusual alkaloid -glucopyranoside, for which the structure was
further elucidated by 2D NMR spectroscopic analysis.
Fig. 2. The 1H-1H COSY (thick lines) and three-bond HMBC correlations (red
arrows, from 1H to 13C) of 1.
The proton and hydrogen-bearing carbon resonances in the
NMR spectra of 1 were assigned unambiguously by 1H-1H COSY
and HSQC spectroscopic data interpretation. In the 1H-1H COSY
spectrum, the homonuclear coupling correlations (Fig. 2) of H-
4/H-5/H-6/H-7, H-5/H-6/H-2, H-5/H-6/H-2, and H-1/H-
2/H-3/H-4/H-5/H2-6 confirmed the presence of the
phenyl and -glucopyranosyloxy moieties. In addition, the 1H-1H
COSY cross-peaks between H-8 with H-7 and H2-9, together
with their chemical shifts, splitting patterns, and coupling constant
values, revealed that there was a 7,7,8-trisubstituted propyloxy
unit in 1. The HMBC spectrum of 1 showed two- and three-bond
heteronuclear correlations from H-4 to C-3, C-6, and C-7a, from
H-5 to C-3a and C-7, from H-6 to C-4 and C-7a, and from H-7 to
C-3a and C-5. These correlations demonstrated that the sp2
hybridized quaternary carbon (C-3) connected to C-3a of the
ortho-substituted phenyl. The HMBC correlations from H-2 and
H-6 to C-4 and C-7, from H-5 to C-1 and C-3, from 3-OCH3
to C-3, and from H-7 to C-1, C-2 and C-6 indicated that C-7
of the propyloxy unit was substituted by a 4-hydroxy-3-
methoxyphenyl. Similarly a 4-hydroxy-3-methoxyphenyl was
located at C-8 according to the HMBC correlations from H-2 and
H-6 to C-4 and C-8, from H-5 to C-1 and C-3, from 3-OCH3
to C-3, from H-8 to C-1 and C-2, and from H2-9 to C-1. In
addition, the HMBC correlations of both C-2 and C-3 with H-7
Table 1
NMR spectroscopic data of 1 in CD3ODa.

established a connection between C-2 and C-7, while the HMBC
correlation of C-3 with H-1 located -glucopyranosyloxy at C-
3. Considering the molecular formula, the C-2 must connect to C-
7a via the remaining nitrogen atom to afford the gross structure for
1 as shown in Fig. 2. The structure assignment was further proved
by 1D and 2D NMR spectroscopic data analysis of 1 in DMSO-
d6, wherein the exchangeable proton resonances were observed
(Table S1 and Figs. S23–S29 in Supporting information).
Especially, in the HMBC spectrum correlations from H-1 to C-2,
C-3, C-3a, and C-7a verified formation of the indole ring in 1.
positive chirality [42-45]. Application of CD exciton chirality
method predicated (7R,8S)-configuration for 1. From the
hydrolysate of 1 with snailase, D-glucose was isolated and
identified by comparison of the retention factor (Rf) on TLC,
specific rotation, and1H NMR spectroscopic data with those of an
authentic sugar sample (Supporting information), while the
aglycone was decomposed into a complex mixture failed to be
separated due to limitation of the sample amount. The absolute
configuration was supported by comparison of the experimental
CD spectrum with the ECD spectrum predicted from quantum
mechanical time dependent density functional theory (TDDFT)
calculations [46]. The theoretically calculated ECD spectra of 1
and the aglycone (1a) were in good agreement with the
experimental CD spectrum (Fig. 5 and Supporting information).
Thus, the structure of compound 1 was determined and designated
as isatindigodiphindoside.
<InlineImage1>
Fig. 5. The experimental CD spectrum of 1 (black) and the calculated ECD
spectra of 1(red full line) and 1a (red dash line).
Compound 1 is characterized by the 2-(diphenylpropyl)-indole
skeleton, which has never been identified in a natural product.
Two plausible biosynthetic pathways for 1 are postulated in
Scheme 1 (shown as a and b, respectively). The biosynthetic
precursor of 1 is proposed to be homovanillic alcohol (2), vanillin
(3), -hydroxypropiovanillone (4), and guaiacol (5), as well as
isatan B (6), which are abundantlyco-occurring in the Isatis plants
[32]. An enzyme-catalyzed Aldol condensation of 2 and 3
generates intermediate (7), which would then undergo a sequential
and/or simultaneous intermolecular nucleophilic addition with 6
to afford 1. Alternatively, Aldol addition of 4 and 6, with
concomitant loss of one molecule of water, affords intermediate 8,
followed by nucleophilic addition with 5 to produce 1. In order to
exclude the possibility of 1 being fortuitously formed by some
type of catalytic effect during the isolation procedure, the putative
precursors 2, 3, and 6 and 4, 5, and 6 were separately refluxed in
acetone or methanol with or without silica gel (the main solvents
and absorbent used in the isolation procedure) for 48 h. HPLC
analysis of the reaction mixtures indicated that 1 was not formed
under the simulated conditions (Figs. S32 and S33 in Supporting
information). This supports that compound 1 is a true natural
product.
Fig. 3. The ROESY correlations (pink dashed double arrows) of 1.
Fig. 4. Newman projection of the stabilized (7R,8S) and (7S,8R)
3
conformations visualizing from C-8 to C-7 based on JH-7 ,H-8 and NOESY


correlation analysis of 1.
The configuration of
1
was deduced from J-based
configurational analysis and ROESY spectrum, combined with
1
circular dichroism (CD) data and enzyme hydrolysis. In the H
NMR spectrum, the magnitude of large 3JH-7 ,H-8 coupling constant

(12.0 Hz) revealed that free rotation of the single bond between C-
7 and C-8 was restricted in solution state and that the two vicinal
protons H-7 and H-8 were anti-oriented in a stabilized
conformation [40]. The ROESY spectrum of 1 showed
correlations between H2-9 with H-2 and H-6, between H-1
with H-2 and H-6 and between H-3 and H-5 (Figs. 3 and S22
in Supporting information). These ROESY correlations
demonstrate that hydroxymethylene (HOCH2-9) and 4-hydroxy-
3-methoxyphenyl, as well as 3-glucopyranosyloxy-1H-indol-2-yl
and 4-hydroxy-3-methoxyphenyl, are gauche to each other in
the stabilized conformation of 1. The lowest-energy 3D
conformation, obtained by Monte Carlo searching with the
MMFF94 molecular mechanics force field using the MOE
softwere [41], was consistent with that assigned by the ROESY
data (Fig. S22 in Supporting information). This revealed (7R,8S)-
or (7S,8R)-configuration for 1 (Fig. 4). The CD spectrum of 1
displayed a positive Cotton effect at 245 ( +6.37) and a negative
Cotton effect at 225 ( –10.03) nm. Configurational analysis of
1 demonstrated that the split Cotton effects would arise from
exciton coupling between * transitions of the substituted
indole and 4-hydroxy-3-methoxyphenyl chromophores with

[18] Y.P. Jiang, Y.F. Liu, Q.L. Guo, J.G.Shi, J. Asian Nat. Prod. Res. 17
(2015) 1166–1179.
[19] Y.P. Jiang, Q.L. Guo, Y.F. Liu, J.G. Shi, Chin. Chem. Lett. 27 (2016)
55–58.
[20] Y. Jiang, Y. Liu, Q. Guo, et al., Acta Pharm. Sin. B 6 (2016) 46–54.
[21] Z.B. Jiang, B.Y. Jiang, C.G. Zhu, et al., J. Asian Nat. Prod. Res. 16 (2014)
891–900.
[22] Z.B. Jiang, X.H. Meng, B.Y. Jiang, et al., Chin. Chem. Lett. 26 (2015)
653–656.
[23] X.H. Meng, Z.B. Jiang, C.G. Zhu, et al., Chin. Chem. Lett. 27 (2016)
993–1003.
[24] X.H. Meng, Z.B. Jiang, Q.L. Guo, J.G. Shi, Chin. Chem. Lett. 28 (2017)
588–592.
[25] Q. Guo, Y. Wang, S. Lin, et al., Acta Pharm. Sin. B 5 (2015) 350–357.
[26] Q.L. Guo, Y.N. Wang, C.G. Zhu, et al, J. Asian Nat. Prod. Res. 17
(2015) 439–454.
[27] J. He, Z. Luo, L. Huang, et al., Anal. Chem. 87 (2015) 5372–5379.
[28] Q.L. Guo, S. Lin, Y.N. Wang, et al., Chin. Chem. Lett. 27 (2016) 1577–
1581.
[29] Z. Liu, W. Wang, N. Feng, et al., Acta Pharm. Sin. B 6 (2016) 189–197.
[30] M. Chen, S. Lin, L. Li, et al., Org. Lett. 14 (2012) 56685671.
[31] M. Chen, L. Gan, S. Lin, et al., J. Nat. Prod. 75 (2012) 11671176.
[32] X.L. Wang, M.H. Chen, F. Wang, et al., Chin. J. Chin. Mater. Med. 38
(2013) 11721182.
Scheme 1. The plausible biosynthetic pathway of 1.
[33] Y.F. Liu, M.H. Chen, X.L. Wang, et al., Chin. Chem. Lett. 26 (2015)
In conclusion, isatindigodiphindoside (1) was isolated as the
minor component from the aqueous extract of I. indigotica roots.
Although the compound was inactive in the preliminary assay
against the influenza virus, its unique drug-like skeleton is of
interesting for synthetic chemists and biologists. The plausible
biosynthetic pathway associated to the different types of co-
occurring putative precursors provides a clue for further studies
of biomimetic and total synthesis, as well as biosynthesis of the
diverse indole alkaloids from the genus Isatis.
931936.
[34] Y.F. Liu, M.H. Chen, Q.L. Guo, et al., J. Asian Nat. Prod. Res. 17 (2015)
689704.
[35] Y.F. Liu, M.H. Chen, S. Lin, et al., J. Asian Nat. Prod. Res. 18 (2016)
1–12
[36] Y. Liu, X. Wang, M. Chen, et al., Acta Pharm. Sin. B 6 (2016) 141–147.
[37] M.H. Chen, S. Lin, Y.N. Wang, et al., Chin. Chem. Lett. 27 (2016) 643-
648.
[38] Y. Liu, M. Chen, Q. Guo, et al., Acta Pharm. Sin. B 7 (2017) 179–184.
[39] D.W. Li, Q.L. Guo, X.H. Meng, et al., Chin. Chem. Lett. 27 (2016) 1745–
1750.
[40] X. Wei, N.M. Henriksen, J.J. Skalicky, et al., J. Org. Chem. 76 (2011)
Acknowledgment
5515–5523.
[41] Molecular Operating Environment software package 2008.10;
Chemical Computing Group Inc., www.chemcomp.com.
[42] N. Harada, K. Nakanishi, J. Am. Chem. Soc. 91 (1969) 39893991.
[43] M. Koreeda, N. Harada, K. Nakanishi, J. Am. Chem. Soc. 96 (1974)
266268.
[44] K. Nakanishi, N. Berova, The exciton chirality method, in: K. Nakanishi,
N. Berova, R.W. Woody (Eds.), Circular Dichroism Principles and
Applications, Wiley-VCH, New York, 1994, pp. 361398.
[45] H.U. Humpf, N. Berova, K. Nakanishi, J. Org. Chem. 60 (1995)
35393542.
Financial support from the National Natural Science
Foundation of China (Nos. 81373287, 81630094 and 30825044)
is acknowledged.
References
[1]
Jiangsu New Medical College, Dictionary of Traditional Chinese
Medicine, Shanghai Science and Technology Publishing House,
Shanghai, 1986.
[2]
Chinese Pharmacopoeia Commission, Pharmacopoeia of People’s
Republic of China, Part 1, China Medical Science Press, Beijing, 2015.
[46] X.C. Li, D. Ferreira, Y. Ding, Curr. Org. Chem. 14 (2010) 16781697.
[3] Y.L. Ho, Y.S. Chang, Phytomedicine 9 (2002) 419424.
[4] J.G. Fang, J. Tang, Z.Q. Yang, et al., Chin. Tradit. Herb. Drugs 36
(2005) 242244.
[5]
J.G. Fang, Y.H. Liu, W.Q. Wang, et al., Acta. Pharmacol. Sin. 26 (2005)
593597.
[6]
S.L. Hsuan, S.C. Chang, S.Y. Wang, et al., J. Ethnopharm. 123 (2009)
6167.
[7]
[8]
L. Zuo, J.B. Li, J. Xu, et al., Chin. J. Chin. Mater. Med. 32 (2007) 688691.
Z. Xie, Y. Shi, Z. Wang, R. Wang, Y. Li, J. Agric. Food Chem. 59 (2011)
1246712472.
[9] L. Yang, G. Wang, M. Wang, et al., Fitoterapia 95 (2014) 175181.
[10] W.D. Xu, Y. Tian, Q.L. Guo, Y.C. Yang, J.G. Shi, Chin. Chem. Lett. 25
(2014) 1531–1534.
[11] Y. Tian, Q.L. Guo, W.D. Xu, et al., Org. Lett. 16 (2014) 3950–3953.
[12] W.X. Song, Y.C. Yang, J.G. Shi, Chin. Chem. Lett. 25 (2014) 1215–1219.
[13] Z.B. Jiang, W.X. Song, J.G. Shi, Chin. Chem. Lett. 26 (2015) 69–72.
[14] Y. Yu, Z. Jiang, W. Song, et al., Acta Pharm. Sin. B 5 (2015) 210–214.
[15] W.X. Song, Q.L. Guo, Y.C. Yang, J.G. Shi, Chin. Chem. Lett. 26 (2015)
517–521.
[16] Y. Jiang, Y. Liu, Q. Guo, et al., Acta Pharm. Sin. B 5 (2015) 215–222.
[17] Y.P. Jiang, Y.F. Liu, Q.L. Guo, et al., J. Asian Nat. Prod. Res. 17 (2015)
601–614.