New acetylated flavone C-glycosides from Iris lactea

Article abstract of DOI:10.1016/j.tetlet.2017.04.080

Chemical investigation of the aerial parts of Iris lactea afforded three new flavone C-glycosides including 4?-O-acetyl-embinin (1), 2?,4?-O-diacetyl-embinin (2) and 6″,4?-O-diacetyl-embinin (3) along with the known analogue embinin (4). Their structures

Full text of DOI:10.1016/j.tetlet.2017.04.080

Accepted Manuscript
New Acetylated Flavone C-Glycosides from Iris lactea
Andrei K. Whaley, Weaam Ebrahim, Mona El-Neketi, Elena U. Ancheeva,
Ferhat Can Özkaya, Nina I. Pryakhina, Nadezhda U. Sipkina, Vladimir G.
Luzhanin, Zhen Liu, Peter Proksch
PII:
S0040-4039(17)30534-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.04.080
TETL 48872
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2017
20 April 2017
24 April 2017
Please cite this article as: Whaley, A.K., Ebrahim, W., El-Neketi, M., Ancheeva, E.U., Özkaya, F.C., Pryakhina,
N.I., Sipkina, N.U., Luzhanin, V.G., Liu, Z., Proksch, P., New Acetylated Flavone C-Glycosides from Iris lactea,
Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.04.080
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New Acetylated Flavone C-Glycosides from Iris lactea
Andrei K. Whaleya,b, Weaam Ebrahima,c, Mona El-Neketic, Elena U. Ancheevaa,b, Ferhat Can
Özkayaa, Nina I. Pryakhinab, Nadezhda U. Sipkinab, Vladimir G. Luzhaninb, Zhen Liua,*,
Peter Prokscha,*
a
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-Universität Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
b Saint Petersburg State Chemical Pharmaceutical Academy, Street Prof. Popova 14, 197376 Saint
Petersburg, Russian Federation
c Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
*Corresponding authors.
Tel.: +49 211 81 14163; fax: +49 211 81 11923;
e-mail: zhenfeizi0@sina.com (Z. Liu),
proksch@uni-duesseldorf.de (P. Proksch)
1

ABSTRACT
Chemical investigation of the aerial parts of Iris lactea afforded three new flavone C-
glycosides including 4ꢀ-O-acetyl-embinin (1), 2ꢀ,4ꢀ-O-diacetyl-embinin (2) and 6ꢁ,4ꢀ-O-
diacetyl-embinin (3) along with the known analogue embinin (4). Their structures were
elucidated by 1D and 2D NMR spectroscopic analysis as well as by HRESIMS data. The
sugars were characterized following acid hydrolysis of the respective glycosides and TLC
analysis compared to known standards. Duplicated signals can be observed in the NMR
spectra, indicating the presence of rotamers caused by rotational hindrance around the
glycosyl-flavone C-C linkage. All isolated compounds were tested for their antimicrobial and
cytotoxic activities but found to be inactive.
Keywords: Flavone C-glycosides; rotamers; Iris lactea.
2

Introduction
Iris lactea Pall. is a rhizomatous perennial flowering plant distributed throughout mostly
temperate and some tropical areas of Asia. Early research of plants from the Iris genus
mainly focused on the rhizomes, of which flavonoids were reported to be the major
secondary metabolites.1 Flavone C-glycosides have been found in the aerial parts of Iris
lactea2,3 and other Iris species including I. tectorum4 and I. germanica5. Previous study on I.
lactea suggested the presence of flavone C-glycosides derivatives of embinin, which was first
isolated in 1962 from I. tectorum but the structure was incorrectly prescribed at that time. In
1968, embinin was re-isolated from I. germanica and the structure was correctly elucidated.5
Up to now, three rhamnose-residue acetate ester derivatives of embinin, 5-hydroxyl-7,4′-
1
ꢂ2
dimethoxyflavone-6-C-[O-(α-L-2-acetylrhamnopyranosyl)-1
-β-D-glucopyranoside],
ꢂ2
5-
hydroxyl-7,4′-dimethoxyflavone-6-C-[O-(α-L-3-acetylrhamnopyranosyl)-1
glucopyranoside],3
-β-D-
and
5-hydroxyl-7,4′-dimethoxyflavone-6-C-[O-(α-L-2,3-
diacetylrhamnopyranosyl)-1ꢂ2-β-D-glucopyranoside]2 have been isolated and described in
literature. In this study, we isolated and characterized three new embinin derivatives. The
elucidation of the structures and their bioassay results are reported herein.
Fig. 1. Structures of isolated compounds.
Results and Discussion
Compound 1 was obtained as a brown solid. It exhibited characteristic UV absorption of
3

flavones at maxima 213, 273 and 335 nm. The HRESIMS displayed a [M+H]+ peak at m/z
649.2127, corresponding to the molecular formula C31H36O15. The 1H NMR data (Table 1) of
1 showed similar signals to the co-isolated embinin (4),6 including signals for an AA′BB′
system of 4′-substituted B-ring at δH 8.08 (2H, d, J = 8.8 Hz, H-2′ and H-6′) and 7.13 (2H, d,
J = 8.8 Hz, H-3′ and H-5′), a pair of singlet signals for H-3 at δH 6.96 and 6.99, a pair of
singlet signals for H-8 at δH 6.95 and 6.90, a pair of singlet signals for chelated 5-OH at
13.43 and 13.47, a pair of singlet signals for MeO-7 at δH 3.93 and 3.90 as well as a singlet
signal for MeO-4′ at δH 3.86. Detailed analysis of the 2D NMR of 1 confirmed the nature of
5-hydroxyl-7,4′-dimethoxyflavone core structure. The observation of anomeric protons at δH
4.69 (or 4.70) (d, 1H, J = 9.8 Hz, H-1ꢁ) and 5.20 (or 5.15) (d, 1H, J = 1.2 Hz, H-1ꢀ)
suggested the presence of the disaccharide moiety in 1. The chemical shifts of the anomeric
carbons at δC 71.1 and 99.3 (or 99.5) are in accordance with C- and O-glycones, respectively.
1
The doublet methyl signal at δH 0.41 (or 0.51) (d, 3H, J = 6.2 Hz) in the H NMR suggested
the presence of the rhamnosyl residue, which was further confirmed by the hydrolysis results.
The α-L-rhamnosyl residue was established by the small coupling constant (1.2 Hz) of H-1ꢀ
and by comparison with a known standard using TLC analysis after acid hydrolysis of 1. The
other sugar residue was suggested to be β-D-glucopyranosyl based on the J value (9.8 Hz) of
H-1ꢁ, the COSY correlations between the sugar protons and by comparison with data of
embinin (4). In the HMBC spectra, the correlations from H-1ꢁ to C-5, C-6 and C-7 indicated
the β-D-glucopyranosyl moiety to be attached at the C-6 position via C-C bond while the
correlation from H-1ꢀ to C-1ꢁ concluded the connection between α-L-rhamnosyl and β-D-
glucopyranosyl through C(1ꢀ)-O-C(1ꢁ) linkage. Unlike embinin (4), additional signals for an
acetoxy group at δH 1.78 (or 1.65) (s, 3H) and at δC 20.6 (or 20.4) and 169.6 (or 169.5) were
found in NMR spectra of 1. The obvious downfield shift of H-4ꢀ (δH 4.43) in 1 compared to
4

that (δH 2.91) of embinin (4) as well as the HMBC correlation from H-4ꢀ to the carboxy
carbon of the acetoxy group confirmed the location of the additional acetoxy group at C-4ꢀ.
Thus the structure of compound 1 was elucidated as 5-hydroxyl-7,4′-dimethoxyflavone-6-C-
[O-(α-L-4-acetylrhamnopyranosyl)-1ꢂ2-β-D-glucopyranoside]. Compound 1 is 4ꢀ-O-acetyl
analogue of embinin (4), representing a new 6-C-glycosylflavone derivative. Duplicated
signals observed in the NMR spectra of 1 were caused by rotational hindrance at the C(6)-
C(1′) linkage, which is very common for 6-C-glycosylflavones.7,8
Figure 2. COSY and key HMBC correlations of 1.
The molecular formula of compound 2 was determined to be C33H38O16 by HRESIMS
data, containing two additional C, two additional H and one additional O compared to 1. The
UV and NMR data (Table 1) of compound 2 resembled those of 1 except for an additional
acetoxy group (δH 1.99, δC 20.8 and 169.4) and the obvious downfield shift of H-2ꢀ (δH 4.91
or 4.93 in 2 compared to δH 3.60 or 3.62 in 1). These above data suggested compound 2 to be
the 2ꢀ-O-acetyl derivative of 1, which was indicated by the HMBC correlations from H-2ꢀ to
the carboxy carbon of the additional acetoxy group. The remaining structure of 2 was
determined to be the same as 1 after detailed analysis of the 2D NMR spectra of 2. Following
the same procedure as described for 1, acid hydrolysis was carried out for 2, which gave rise
to α-L-rhamnose and embigenin and confirmed the structure of 2 to be 2ꢀ,4ꢀ-O-diacetyl-
embinin as shown.
5

Table 1. 1H and 13C NMR Data of Compound 1–3.
1 (Ma)a,c
δH (J in Hz)
1 (Mi)a,c
δH (J in Hz)
2 (Ma)b,c
δH (J in Hz)
2 (Mi)b,c
δH (J in Hz)
3 (Ma)b,c
δH (J in Hz)
3 (Mi)b,c
δH (J in Hz)
position
d
d
d
d
δC
δC
δC
δC
163.6
δC
163.2
δC
163.3
2-C
3-CH
4-C
4a-C
5-C
6-C
163.4
103.6 6.96, s
182.3
104.8
160.2
110.0
163.6
90.5
157.0
122.5
163.3
103.8
182.0
104.2
159.1
109.8
164.7
91.4
157.1
122.6
128.3
114.7
162.5
71.1
163.4
103.6
182.3
104.8
160.2
109.7
163.2
90.6
157.0
122.4
128.3
114.7
162.5
70.9
6.99, s
6.98, s
103.8 7.02, s
181.9
103.5 6.98, s
182.2
103.6 7.01, s
181.7
104.1
159.1
109.6
164.6
91.4
157.1
122.5
104.6
160.2
109.5
163.0
90.4
156.9
122.3
103.9
159.1
109.4
164.4
91.5
156.9
122.5
7-C
8-CH
8a-C
1′-C
6.95, s
6.90, s
6.99, s
6.93, s
6.97, s
6.92, s
2′, 6′-CH
3′, 5′-CH′ 114.7 7.13, d (8.8)
4′-C
1ꢁ-CH
2ꢁ-CH
3ꢁ-CH
4ꢁ-CH
5ꢁ-CH
6ꢁ-CH2
128.3 8.08, d (8.8)
8.08, d (8.8)
7.13, d (8.8)
8.09, d (8.8)
7.13, d (8.8)
128.3 8.09, d (8.8)
114.7 7.13, d (8.8)
162.5
128.3 8.09, d (8.8)
114.7 7.14, d (8.8)
162.5
128.3 8.09, d (8.8)
114.7 7.14, d (8.8)
162.5
162.5
71.1
73.5
79.8
70.8
81.6
61.7
4.69, d (9.8)
4.31, m
3.38, m
3.11, m
3.15, m
4.70, d (9.8)
4.10, m
3.39, m
3.11, m
3.15, m
4.72, d (9.7)
4.32, m
3.43, m
3.09, m
3.17, m
70.8
74.6
79.6
70.6
81.6
61.6
4.74, d (9.7)
4.11, m
3.43, m
3.09, m
3.17, m
70.8
73.1
79.3
70.1
77.7
64.3
4.71, d (9.4)
4.29, m
3.41, m
3.17, m
3.38, m
70.8
74.3
79.3
70.1
77.7
64.3
4.73, d (9.4)
4.08, m
3.41, m
3.17, m
3.38, m
74.7
79.9
70.8
81.6
73.2
79.7
70.6
81.6
3.73, m
61.7
3.73, m
61.6
3.72, m
3.72, m
4.40, m
4.40, m
3.39, m
3.39, m
3.37, m
3.37, m
3.88, m
3.88, m
1ꢀ-CH
2ꢀ-CH
3ꢀ-CH
99.3
70.4
67.8
5.20, d (1.2)
3.60, m
99.5
70.4
67.6
5.15, d (1.2)
3.62, m
96.3
71.8
65.7
5.25, d (1.4)
4.91, m
3.36, m
96.5
71.8
65.5
5.18, d (1.4)
4.93, m
3.48, m
99.0
70.2
67.7
5.20, d (1.3)
3.61, m
3.19, m
99.3
70.2
67.4
5.14, d (1.3)
3.61, m
3.30, m
3.21,
dd
3.31,
dd
(9.7, 3.0)
4.43, m
2.17, m
0.41, d (6.2)
13.43, s
3.93, s
(9.7, 3.0)
4.43, m
2.17, m
0.51, d (6.2)
13.47, s
3.90, s
4ꢀ-CH
5ꢀ-CH
6ꢀ-CH3
5-OH
7-OMe
4′-OMe
6ꢁ-OAc
73.8
65.4
17.1
73.9
65.2
17.8
73.4
65.3
17.0
4.36, m
2.29, m
0.49, d (6.3)
13.46, s
3.95, s
73.5
65.2
17.6
4.37, m
2.27, m
0.58, d (6.2)
13.52, s
3.89, s
73.8
65.4
17.1
4.42, m
2.15, m
0.40, d (6.2)
13.43, s
3.94, s
73.8
65.2
17.7
4.42, m
2.11, m
0.50, d (6.2)
13.50, s
3.90, s
56.7
55.6
56.4
55.6
56.7
55.6
56.4
55.6
56.6
55.6
20.7,
170.4
56.4
55.6
20.7,
170.4
3.86, s
3.86, s
3.87, s
3.87, s
3.87, s
1.99, s
3.87, s
2.00, s
2ꢀ-OAc
4ꢀ-OAc
20.8,
169.4
20.4,
169.5
1.99, s
1.76, s
20.8,
169.4
20.2,
169.4
1.99, s
20.6,
169.6
1.78, s
20.4,
169.5
1.65, s
1.66, s
20.6,
169.5
1.78, s
20.4,
1.65, s
169.5
a Recorded at 600 MHz for 1H and 150 MHz for 13C in DMSO-d6. Recorded at 700 MHz for H and 175 MHz for 13C in DMSO-d6. Ma and Mi denote
b
1
c
the major and minor rotamer of each flavone, respectively. d Data extracted from HSQC and HMBC spectra.
6

The molecular formula of compound 3 was determined to be the same as for 2. Both
compounds also shared similar UV absorption and NMR spectra. The hydrolysis of 3
afforded α-L-rhamnose and embigenin, suggesting 3 to be an O-diacetyl derivative of
embinin (4) as well. However, the NMR data of the rhamnosyl residue in 3 were more
compatible to those of 1 and the signals of the oxygenated methylene (CH2-6ꢁ) in the
glucosyl unit were obviously downfield shifted. The above findings suggested the two
acetoxy groups to be located at the C-6ꢁ and C-4ꢀ position. This assumption was further
confirmed by key HMBC correlations from H2-6ꢁ and H-4ꢀ to the caboxy carbons of the two
acetoxy groups, respectively.
All isolated compounds (1–4) were examined for their antimicrobial and cytotoxic
activites but none of them exhibited a pronounced effect at a dose of 20 µg/mL. O-glycosyl
flavones are known as one of the major components in genus Iris, which were mainly isolated
from leaves and flowers.9 There are few reports about C-glycosylflavonoids from this genus.
Their contribution to flower colour of the plant was described.10 C-glycosylflavonoids are
found to be minor constituent in the extract and their roles in plant physiology need further
research. Inspection of their NMR data revealed the duplication of the signals, indicating the
presence of rotamers. This is caused by the rotational hindrance at the C(6)-C(1′) linkage,
which can not be found in mono-6-C-substituted derivatives.7
Acknowledgments
P.P. wants to thank the Manchot Foundation for support.
Supporting Information
Supplementary data (UV, MS and NMR spectra of 1–3 as well as experimental section)
associated with this article can be found, in the online version, at
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7

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8

9

Highlights
ꢀ
ꢀ
ꢀ
Three new flavone C-glycosides were isolated from the aerial parts of Iris lactea.
The sugars were characterized following acid hydrolysis and TLC analysis.
Duplicated signals caused by rotational hindrance were observed in the NMR spectra.
10