Isatindolignanoside A, a glucosidic indole-lignan conjugate from an aqueous extract of the Isatis indigotica roots

Article abstract of DOI:10.1016/j.cclet.2017.12.001

A glucosidic indole-lignan conjugate with a novel carbon skeleton, named isatindolignanoside A (1), was isolated from an aqueous extract of the Isatis indigotica roots “ban lan gen”. Its structure was elucidated by comprehensive analysis of spectroscopic data, including J- and NOESY correlation-based configurational analysis, circular dichroism (CD) data, enzyme hydrolysis, and theoretical ECD calculation. In a preliminary assay, compound 1 showed antiviral activity against Coxsackie virus B3. This compound is the first example of natural product having a structural feature of conjugation between indole and lignan units, and its biosynthetic pathway is postulated.

Full text of DOI:10.1016/j.cclet.2017.12.001

Accepted Manuscript
Title: Isatindolignanoside A, a glucosidic indole-lignan
conjugate from an aqueous extract of the Isatis indigotica roots
Authors: Lingjie Meng, Qinglan Guo, Minghua Chen,
Jiandong Jiang, Yuhuan Li, Jiangong Shi
PII:
DOI:
Reference:
S1001-8417(17)30517-X
https://doi.org/10.1016/j.cclet.2017.12.001
CCLET 4363
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Chinese Chemical Letters
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Revised date:
Accepted date:
2-10-2017
30-11-2017
1-12-2017
Please cite this article as: Lingjie Meng, Qinglan Guo, Minghua Chen, Jiandong
Jiang, Yuhuan Li, Jiangong Shi, Isatindolignanoside A, a glucosidic indole-lignan
conjugate from an aqueous extract of the Isatis indigotica roots, Chinese Chemical
Letters https://doi.org/10.1016/j.cclet.2017.12.001
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Communication
Isatindolignanoside A, a glucosidic indole-lignan conjugate from an aqueous extract of
the Isatis indigotica roots
Lingjie Meng, Qinglan Guo, Minghua Chen, Jiandong Jiang, Yuhuan Li, Jiangong 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
 Corresponding author.
E-mail address: shijg@imm.ac.cn (J. Shi).
Graphical abstract
A glucosidic indole-lignan conjugate with a novel carbon skeleton, named isatindolignanoside A (1), was isolated from an aqueous extract of the Isatis indigotica
roots “ban lan gen”. Its structure was determined by comprehensive analysis of spectroscopic data, enzyme hydrolysis, and electronic CD calculations. Compound
1 is active against Coxsackie virus B3 and represents the first example of natural product having a structural feature of conjugation between indole and lignan, and
its plausible biosynthetic pathway is postulated.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A glucosidic indole-lignan conjugate with a novel carbon skeleton, named isatindolignanoside A
(1), was isolated from an aqueous extract of the Isatis indigotica roots “ban lan gen”. Its
structure was elucidated by comprehensive analysis of spectroscopic data, including J- and
NOESY correlation-based configurational analysis, circular dichroism (CD) data, enzyme
hydrolysis, and theoretical ECD calculation. In a preliminary assay, compound 1 showed
antiviral activity against Coxsackie virus B3. This compound is the first example of natural
product having a structural feature of conjugation between indole and lignan units, and its
biosynthetic pathway is postulated.
Available online
Keywords:
Isatis indigotica
Cruciferae
Ban lan gen
Indole-lignan conjugate
Isatindolignanoside A
Antiviral activity
The dried roots of Isatis indigotica Fort. (Cruciferae), named “ban lan gen” in Chinese, is among the most popular herbal drugs for
the treatment of colds, especially of the flu during influenza pandemics in China [1]. Many formulations containing extracts of “ban lan
gen” or the raw material are compiled in Chinese Pharmacopoeia [2]. Agricultural cultivation of this plant not only fulfils demands of
medicinal industry but also plays roles of environment protection and increasing local economy in several provinces of China.
Considerable investigations on this herbal medicine indicated that “ban lan gen” extracts had antiviral, anti-endotoxic, anti-
inflammatory, antipyretic, and cytotoxic activities [3-7] and contained alkaloids [8-13], lignans [14,15], flavonoids [16], and epigoitrin
and related analogues [17]. However, the previous chemical studies were mainly carried out on the ethanol or methanol extracts of the
drug material, which differs from a practical application by decocting with water. Therefore, an aqueous decoction of “ban lan gen”
was investigated as part of a program to systematically study the chemical diversity of traditional Chinese medicines and their
biological activities [18-39]. More than 100 compounds including 61 with new structures from the decoction were reported in our
previous papers [39-51]. Antiviral activity against influenza virus A/Hanfang/359/95 (H3N2) or Coxsackie virus B3, protective activity
against dl-galactosamine-induced hepatocyte (WB-F344 cell) damage, and inhibitory activity against the LPS-induced NO production
in mouse peritoneal macrophage [40,41,43-45,47] were found for the isolates with varied structural types. A continuation of the study,
focusing on the minor constituents, has led to characterization of an unusual glucosidic indole-lignan conjugate with a novel carbon
skeleton, named isatindolignanoside A (1) (Fig. 1). This paper describes details of the isolation, structure elucidation, and bioassay of
the new isolate.

Compound 1 was isolated as a yellowish amorphous powder with [α]2D0 –28.3 (c 0.04, MeOH). Its IR spectrum displayed absorption
bands due to hydroxyl (3395 cm1) and aromatic (1600 cm1 and 1515 cm1) functionalities. The ESIMS of 1 exhibited quasimolecular
ion peaks at m/z 838 [M + Na]+ and 814 [M – H]–. The molecular formula C40H49NO17 was determined by HR-ESIMS at m/z 838.2905
[M + Na]+ (calcd. for C40H49NO17Na: 838.2893), in combination with the NMR data (Table 1). The 1H NMR spectrum of 1 in CD3OD
showed resonances assignable to (a) two meta-para-disubstituted phenyls at δH 7.09 (d, J = 1.8 Hz, H-2), 7.12 (d, J = 8.4 Hz, H-5), and
7.05 (dd, J = 8.4 and 1.8 Hz, H-6) and 6.82 (d, J = 1.8 Hz, H-2), 6.59 (d, J = 7.8 Hz, H-5), 6.80 (dd, J = 7.8 and 1.8 Hz, H-6); (b) an
ortho-substituted phenyl at δH 7.63 (brd, J = 7.8 Hz, H-4), 6.90 (brt, J = 7.8 Hz, H-5), 6.98 (brt, J = 7.8 Hz, H-6), and 7.21 (brd, J =
7.8 Hz, H-7); and (c) two methoxy groups at δH 3.87 (s, 3-OCH3) and 3.72 (s, 3-OCH3). In addition, the spectrum showed resonances
assignable to two -glucopyranosyls, two oxygen-bearing methylenes, and four methines (one oxygen-bearing) between δH 4.89 and
1.99. The -configuration of the glucopyranosyls were indicated by the coupling constant value of the anomeric protons resonated at δH
4.86 (d, J = 7.2 Hz, H-1′′′) and 4.88 (d, J = 7.8 Hz, H-1′′′′), respectively. The 13C NMR and DEPT spectra of 1 showed 40 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 [40-51], these
spectroscopic data suggest that 1 is an unusual alkaloid diglucopyranoside, of which the structure was further elucidated by 2D NMR
data analysis.
The proton-bearing carbon resonances and corresponding proton resonances in the NMR spectra were readily assigned by HSQC
spectroscopic data analysis (Table 1). The presence of the three phenyls were confirmed by cross-peaks of the ortho- and meta-
coupling aromatic protons H-5/H-6/H-2, H-5/H-6/H-2, and H-4/H-5/H-6/H-7 (Fig. 2, thick lines) in the 1H-1H COSY spectrum of
1. Meanwhile, vicinal coupling correlations of H-1/H-2/H-3/H-4/H-5/H2-6 and H-1/H-2/H-3/H-4/H-5/H2-6
1
proved the presence of the two -glucopyranosyls. Additionally, the H-1H COSY spectrum showed the correlations of H-7/H-8/H2-9
and H-7/H-8/H2-9, indicating that there were 7,7,8- and 7,7,8-trisubstituted propyloxy units in 1. In the HMBC spectrum of 1, two-
and three-bond heteronuclear correlations (Fig. 2, arrows) 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; from H-7 to C-1, C-2 and C-6; and from H-1 to C-4; together with the chemical shifts of these proton and carbon resonances;
indicated that C-7 of one propyloxy unit was substituted by a 4--glucopyranosyloxy-3-methoxyphenyl. Similarly substitution at C-7
of the other propyloxy unit by a 4-oxy-3-methoxyphenyl was evidenced by the 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, along with their chemical shift values. The HMBC
correlations from H-7 to C-8 and from both H-7 and H2-9 to C-8 revealed that the two phenylpropyloxy units connected via the C-
8C-8 bond to give a lignan moiety in 1. The HMBC correlations of H-7/C-9 and H2-9/C-7, in combination with the chemical shift
values of C-7 and C-9, indicated an oxygen-bridged linkage between C-7 and C-9 to form a tetrhydrofuran ring. Meanwhile, the
HMBC spectrum of 1 showed correlations from H-4 to C-3, C-3a, C-6, and C-7a; from H-5 to C-3a, C-6, and C-7; from H-6
to C-4 and C-7a; from H-7 to C-3a and C-5; and from H-1 to C-3. These correlations, combined with their chemical shift
values, demonstrated that one end (C-3) of tetra-substituted double bond not only connected to C-3a of the ortho-substituted phenyl
but also was substituted by the remaining -glucopyranosyloxy. In addition, the HMBC correlations of both C-2 and C-3 with H-7
established a connection between C-2 and C-7. Taking into account of unconnected sites C-2, C-7a, C-4, and C-9, especially the
chemical shift values of these carbon resonances, as well as the molecular formula, the two carbons C-2 and C-7a must connected via
the remaining nitrogen atom to construct an indole ring while the carbons C-4 and C-9 must be substituted by two hydroxyls.
Therefore, the planar structure of 1 was deduced to be an unusual lignan-coupled indole alkaloid diglucopyranoside as shown in Fig. 2.
The deduction was supported by comparing the NMR spectroscopic data of 1 with those of the co-occurring lignanoid and indole
derivatives [42].
The configuration of 1 was determined by J-based configurational analysis and NOESY spectrum, together with enzyme hydrolysis
and circular dichroism (CD) data. In the 1H NMR spectrum, the large 3JH-7 ,H-8 coupling constant (12.6 Hz) revealed that free rotation of

the single bond between C-7 and C-8 was restricted in solution and that the two vicinal protons H-7 and H-8 were anti-oriented in a
stabilized conformation of 1 (Figs. 3 and 4). In the NOESY spectrum, correlations between H-7 with H-6 and H-8, indicated that these
protons were cofacial on one side of the tetrahydro-furan ring (Fig. 3). The NOESY correlations between H-8 and H2-9 demonstrated
that these protons were oriented on the other side of the tetrahydrofuran ring. Especially the NOESY correlations between both H-2
and H-6 with H-8 indicated that the C-1C-7 and C-8C-8 bonds were gauche-oriented as shown in the Newman projection viewing
from C-7 to C-8 (Fig. 4). Thus, the relative configuration of 1 was established, which was consistent with the lowest-energy 3D
conformation obtained by Monte Carlo searching with the MMFF94 molecular mechanics force field using the MOE software [52]
(Fig. 3).
Enzymatic hydrolysis of 1 liberated D-glucose, which was isolated from the hydrolysate and identified by comparison of TLC and
the 1H NMR spectroscopic and specific rotation data with those of an authentic D-glucose sample, while the aglycone was decomposed
into a complex mixture which failed to be separated due to limitation of the sample amount (Figs. S26 and S27 in Supporting
information). The circular dichroism (CD) spectrum of 1 showed a negative Cotton effect at max 237.5 (Δε 24.09) and a positive
Cotton effect at max 293.0 (Δε +6.66) nm. As compared with the CD data of the lignanoids and indole derivatives isolated from the
same extract [40-51], the Cotton effects arose from the * transitions of three chromophores in 1. Because of transition overlapping
of the chromophores and because of the absence of characteristic exciton coupling Cotton effects, the available CD rules may not be
applicable for determination of the absolute configuration. Meanwhile, our efforts to crystallize 1 in a variety of solvent systems failed

to determine the absolute configuration by single crystal X-ray diffraction analysis. Subsequently, the theoretical electronic CD (ECD)
spectra of 1, its diastereoisomer (1), and the enantiomeric aglycones (1a and 1a) were calculated by using a method based on quantum
mechanical time dependent density functional theory (TDDFT) [53]. Extensive comparison of the calculated ECD spectra of 1, 1, 1a,
and 1a revealed that the -glucopyranosyls on the chromophores of 1 and 1 dominated intensities, wavelengths, and signs of the
Cotton effects in a long wavelength region ( 250 nm) of the calculated ECD spectra (Supporting information). However, in a low
wavelength region (200~245 nm), the Cotton effect curves of 1 and 1 were distinguishable. As compared, in the distinctive region the
experimental CD curve is in well agreement with the calculated curve of 1 but obviously different from that of 1, though in the long
wavelength region (> 250 nm) the two theoretical Cotton effects are replaced by only one relative weak Cotton effect in the
experimental CD spectrum (Fig. 5). This suggests the 7R,7ʹR,8S,8ʹR configuration for 1. Therefore, the structure of compound 1 was
determined and named isatindolignanoside A, which has the systematical name (–)-(7R,7ʹR,8S,8ʹR)-4--D-glucopyranosyloxy-3,3ʹ-
dimethoxy-7ʹ-(3--D-glucopyranosyloxy-1H-indol-2-yl)-7,9ʹ-epoxylignane-4ʹ,9-diol according to IUPAC commented nomenclature
of lignans and neolignans [54].
Isatindolignanoside A (1) possesses a novel skeleton of indole-lignan conjugation, which represents the first example of its kind.
Based on the unique structural feature, the plausible biosynthetic pathway for 1 is postulated in Scheme 1. The biosynthetic precursor
of 1 is proposed to be isatan B (2) and ()-pinoresinol (3), since 2 and 3 as well as their derivatives abundantly co-occur in this plant
[39-51], which are metabolites biosynthesized from L-phenylanaline (L-tyrosine) and L-tryptophan, respectively. Because the C-8
configuration in 1 was reversed comparing to corresponding chiral center in the precursor 3, a C-8/C-7 conjugated intermediate 4 was
proposed to be formed in the biosynthetic pathway. The intermediate 4 could be generated from 3 either via (a) an enzyme-catalyzed
sequential hydrolysis and dehydration process or (b) an oxidative opening of one tetrahydrofuran ring followed by intramolecular
double bond rearrangement. The intermediate 4 undergoes a nucleophilic addition with 2 to afford 1. Glucosidation may take place
during these processes. In order to exclude possible formation of 1 during the isolation procedure, 2 and (–)-pinoresinol mono- or di-
glucosides (5 or 6, Figs. S28 and S29 in Supporting information) was refluxed in H2O 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. This supports that compound 1 is a natural product.
In the preliminary antiviral assay [40-51], compound 1 was active against Coxsackie virus B3 with IC50 and SI values of 25.910-6
mol/L and >3.9, respectively, the positive controls pleconaril and ribavirin gave IC50 values of 0.00110-6 mol/L and 517.410-6 mol/L
and SI values of 17122.2 and 3.9, respectively. However, 1 was inactive against influenza virus A/Hanfang/359/95 (H3N2) at a
concentration of 10-5 mol/L.
In conclusion, isatindolignanoside A (1) with antiviral activity against Coxsackie virus B3 was isolated as the minor constituent
from the aqueous extract of “ban lan gen”. This result, together with our previous studies [40-51], continuously proves that the multiple
chemical constituents with the diverse structures have contributions towards clinical efficacy that supports the conventional
applications of “ban lan gen”. Particularly the structure of 1 possesses the novel carbon skeleton connecting between indole and lignan
parent structures via a carbon-carbon bond, and represents the first indole-lignan conjugating natural product. The structural novelty
not only enriches the diversity of the bioactive chemical constituents of “ban lan gen”, but also challenges synthetic and biosynthetic
chemists to obtain enough amount of the sample for further biological evaluation.
Acknowledgment
Financial support from the National Natural Science Foundation of China (Nos. 81373287, 81630094, and 30825044) is
acknowledged.

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Fig. 1. Structure of compound 1.

Fig. 2. The 1H-1H COSY (thick lines) and three-bond HMBC correlations (red arrows, from 1H to 13C) of 1.

Fig. 3. The NOESY correlations (pink dashed double arrows) of 1.

Fig. 4. Newman projection of a conformation visualizing from C-7 to C-8 based on 3JH-7,H-8 and NOESY correlation analysis of 1.

Calculated ECD spectrum of 1
Calculated ECD spectrum of 1'
Experimental CD spectrum of 1
25
20
15
10
5
0

-5
-10
-15
-20
-25
-30
-35
200 220 240 260 280 300 320 340 360
Wavelength (nm)
Fig. 5. The overlaid experimental CD (black) and calculated ECD spectra of 1 (red dash) and 1 (blue dot).

Scheme 1. The plausible biosynthetic pathway of 1.

Table 1
NMR spectroscopic data for compound 1a.
Position δH (J in Hz)
δC
Position δH (J in Hz)
δC
1
138.6
111.7
150.8
147.4
117.6
2″
3″
3″a
4″
5″
6″
7″
7″a
131.2
133.9
121.8
118.4
119.8
122.2
112.6
135.1
2
3
4
5
6
7
8
7.09 d (1.8)
7.63 brd (7.8)
6.90 brt (7.8)
6.98 brt (7.8)
7.21 brd (7.8)
7.12 d (8.4)
7.05 d (8.4, 1.8) 120.3
4.72 d (6.0)
1.99 m
85.7
55.6
3.48 dd (10.8,
4.8)
9a
64.1
1′′′
4.86 d (7.2)
102.8
3.40 dd (10.8,
5.4)
9b
2′′′
3.45 dd (9.0, 7.2)
74.9
OCH3-3
1′
2′
3′
4′
5′
6′
7′
3.87 s
57.0
3′′′
4′′′
5′′′
3.42 t (9.0)
3.34 t (9.0)
3.40 m
77.9
71.5
78.2
62.6
134.9
113.4
148.8
146.1
116.2
6.82 d (1.8)
6′′′a
6′′′b
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′a
6′′′′b
3.84 dd (12.0, 1.8)
3.63 dd (12.0, 6.0)
4.88 d (7.8)
3.52 brt (7.8)
3.42 dd (9.0, 7.8)
3.43 t (9.0)
3.10 m
3.68 dd (12.6, 2.4)
3.58 dd (12.6, 4.8)
6.59 d (7.8)
6.80 dd (7.8, 1.8) 121.9
4.49 d (12.6)
2.98 m
3.99 brd (9.0)
3.82 dd (9.0, 6.0)
106.2
75.8
78.1
71.3
78.5
62.2
45.3
47.0
72.8
8′
9′a
9′b
OCH3-3′ 3.72 s
56.5
aNMR data (δ) were measured in CD3OD at 600 MHz for 1H and at 150 MHz for 13C. The assignments were based on DEPT, 1H-1H COSY, HSQC, and HMBC
experiments.