Baeckeins L and M, two novel C-methylated triflavonoids from the roots of Baeckea frutescens L.

Article abstract of DOI:10.1080/14786419.2018.1528590

Baeckea frutescens is a medicinal plant distributing from Southeast Asia to Australia. A pair of novel diastereomeric C-methylated triflavonoids named baeckeins L (1) and M (2) were isolated from the roots of B. frutescens. The structures of these isolates were elucidated by analysis of the 1D (¹H/¹³C) and 2D NMR (HSQC/HMBC/NOESY) and HR-ESI-MS spectroscopic data, and the absolute configurations of chiral carbons (C-2/C-3/C-2°/C-3°) were established by CD spectrometry combined with quantum chemical calculations.

Full text of DOI:10.1080/14786419.2018.1528590

Natural Product Research
Formerly Natural Product Letters
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Baeckeins L and M, two novel C-methylated
triflavonoids from the roots of Baeckea frutescens
L.
Cheng-Jun Jia, Xiao-Min Sun, Xiang-Yun Zhang, Zi-Jiao Wei & Bei-Xi Jia
To cite this article: Cheng-Jun Jia, Xiao-Min Sun, Xiang-Yun Zhang, Zi-Jiao Wei & Bei-Xi Jia
(2018): Baeckeins L and M, two novel C-methylated triflavonoids from the roots of Baeckea
frutescensꢀL., Natural Product Research, DOI: 10.1080/14786419.2018.1528590
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1528590
Baeckeins L and M, two novel C-methylated triflavonoids
from the roots of Baeckea frutescens L.
Cheng-Jun Jiaa, Xiao-Min Sunb, Xiang-Yun Zhangb, Zi-Jiao Weib and Bei-Xi Jiab
aDepartment of Pharmacy, Henan Province Hospital of Traditional Chinese Medicine, Zhengzhou,
b
P. R. China; Department of Pharmacognosy, College of Pharmacy, Zhengzhou University,
Zhengzhou, P. R. China
ABSTRACT
ARTICLE HISTORY
Received 31 July 2018
Accepted 20 September 2018
Baeckea frutescens is
a medicinal plant distributing from
Southeast Asia to Australia. A pair of novel diastereomeric
C-methylated triflavonoids named baeckeins L (1) and M (2) were
isolated from the roots of B. frutescens. The structures of these
isolates were elucidated by analysis of the 1D (1H/13C) and 2D
NMR (HSQC/HMBC/NOESY) and HR-ESI-MS spectroscopic data,
and the absolute configurations of chiral carbons (C-2/C-3/C-2ꢀ/
C-3ꢀ) were established by CD spectrometry combined with
quantum chemical calculations.
KEYWORDS
Baeckea frutescens;
C-methylated triflavonoid;
Baeckeins L and M;
Absolute configuration
1. Introduction
Baeckea frutescens L. (Myrtaceae) distributes from Southeast Asia to Australia, and is
widely used as a folk medicine in the southern part of China. Previous investigations
of B. frutescens revealed that essential oil (Jantan et al. 1998; Tam et al. 2004), chro-
mones (Tsui and Brown 1996a; Satake et al. 1999) and flavonoids (Tsui and Brown
1996b; Makino and Fujimoto 1999; Quang et al. 2008; Kamiya and Satake 2010) were
the mainly active constituents. Recent phytochemical studies on B. frutescens in our
laboratory have reported 11 C-methylated flavonoids and biflavonoids (Jia, Yang, et al.
2011; Jia, Zhou, et al. 2011; Jia et al. 2013; Jia et al. 2014; Jia et al. 2016). As the
CONTACT Jia Bei-Xi
jiabeixi_2003_123@163.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1528590.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
C.-J. JIA ET AL.
Figure 1. Structures of compounds 1 and 2.
literature suggested, C-methylated flavonoids might be distinctive of the Myrtaceae
family and provide the significance of plant chemotaxonomy (Gottlieb et al. 1972;
Mayer 1990; Rao and Rao 1991; Wollenweber et al. 2000). In ongoing search for struc-
turally interesting C-methylated flavonoids, two novel C-methylated triflavonoids
named baeckeins L (1) and M (2) were obtained from the roots of B. frutescens
(Figure 1). In this paper, the detailed isolation and structural elucidation for the
isolates were described.
2. Results and discussion
Baeckein L (1) was obtained as a yellow amorphous solid. HR-ESI-MS in the positive
ion mode gave a quasi-molecular ion at m/z 1105.1901 [M – H]– (calcd 1105.1891
for C54H42O26), which was consistent with the molecular formula C54H42O26. IR
spectrum showed absorption bands for hydroxyl group (3396 cmꢁ1), carbonyl
group (1636 cmꢁ1), and aromatic functionalities (1506 and 1442 cmꢁ1). UV spectrum

NATURAL PRODUCT RESEARCH
3
(kmax 233, 307 and 368 nm) indicated the presence of a conjugated system. And the
positive results for Mg-HCl reaction and Molish reagent suggested 1 to be a flavon-
oid glycoside.
13C-NMR spectrum (Table S1) of compound 1 displayed a group of signals (dC
100.5, 76.7, 75.9, 73.3, 69.7 and 60.7) belonging to a hexosyl unit, and the remaining
48 carbon signals attributing for a triflavonoid structure, among which are three car-
bonyl groups (dC 188.2, 185.4 and 176.3), three aromatic methyl groups (dC 7.4, 6.9
and 6.8), 11 CH (dC 122.1, 119.5, 118.1, 116.4, 116.2, 115.9, 115.6, 115.3, 114.9, 95.5 and
1
94.3) and 31 quaternary carbons. H-NMR spectrum (Table S1) of 1 showed signals for
three sets of typical ABX coupling systems [dH 7.13 (1H, d, J ¼ 8.7 Hz, H-20), 7.82 (1H, d,
J ¼ 2.0 Hz, H-50) and 6.18 (1H, dd, J ¼ 8.7, 2.0 Hz, H-60)], [dH 7.11 (1H, d, J ¼ 8.7 Hz, H-
0
0
0
ꢂ
ꢂ
ꢂ
2 ), 6.93 (1H, d, J ¼ 2.0 Hz, H-5 ) and 7.05 (1H, dd, J ¼ 8.7, 2.0 Hz, H-6 )], and [dH 6.73
(1H, d, J ¼ 8.7 Hz, H-2ꢀ0), 7.14 (1H, d, J ¼ 2.0 Hz, H-5ꢀ0) and 7.01 (1H, dd, J ¼ 8.7, 2.0 Hz,
H-6 )], corresponding to the 3 ,4 -dihydroxy-substituted rings B, B and Bꢀ of
a triflavonoid moiety, and a series of signals in the range dH 5.5-3.0 related to a sugar
moiety. Also two aromatic protons [dH 6.07 (1H, s, H-8) and 5.91 (1H, s, H-8ꢀ)] and
ꢀ0
0
0
ꢂ
ꢂ
three aromatic methyl signals [dH 1.91 (3H, s, Me-6), 2.10 (3H, s, Me-6 ) and 1.84 (3H,
s, Me-6ꢀ)] were presented. The above 1D-NMR data suggested 1 to be a triflavonoid
glycoside, and the aglycone moiety was composed of three molecules of flavonoids
with certain structural characteristics of 6-C-methylquercetin (Ibewuike et al. 1996;
Quang et al. 2008).
Comprehensive analysis of 1D-NMR and HSQC (Figure S4) spectra of 1 made assign-
ꢂ
ꢂ
ꢂ
ments of one flavonoid molecule (the segment II), referring to rings A , B and C ,
which were confirmed by HMBC correlations (Figure S1) from CH3-6 (dH 2.10) to [dC
ꢂ
0
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
161.0 (C-5 ), 102.9 (C-6 ) and 163.4 (C-7 )0], from H-2 (dH 7.11) to [dC 146.0 (C-2 0),
0
0
0
0
ꢂ
ꢂ
ꢂ
ꢂ
129.3 (C-1 ), 140.0 (C-3 ) and 140.5 (C-4 )], from H-5 (dH 6.93) to [dC 129.3 (C-1 ),
0
0
ꢂ
0
ꢂ
ꢂ
ꢂ
140.0 (C-3 ), 140.5 (C-4 ) and 122.1 (C-6 )], and from H-6 (dH 7.05) to [dC 146.0
(C-2 ) and 115.3 (C-2 )]. Compared with literature values of 6-C-methylquercetin
0
ꢂ
ꢂ
(Ibewuike et al. 1996;0 Quang et al. 2008),0 the segment II was further identified. The
ꢂ
ꢂ
carbon signals of C-3 (dC 140.4) and C-4 (dC 141.5) shifting dC 5.0-7.5 low-frequency
against those of 6-C-methylquercetin, suggested one available binding position for the
ꢂ
ꢂ
segment III, and the absence of an H-8 signal indicated a substituted C-8 , revealing
another binding position for the segment I.
In the segment III, except the signals assignable to rings A and B of a 6-C-methyl
quercetin molecule, the distinctive carbon signals [dC 188.2 (C-4ꢀ), 100.1 (C-2ꢀ), and
90.4 (C-3ꢀ)] suggested a dihydroflavanol structure and a 2ꢀ,3ꢀ-epoxide moiety, and the
carbon of C-3ꢀ provided a possible binding position for the segment II. Between seg-
ꢀ
0
ꢂ
ments II and III, the ether linkage (C-3 )-O-(C-4 ) was elucidated. The segment III,
refering to rings Aꢀ, Bꢀ, and Cꢀ, and the 2ꢀ,3ꢀ-epoxide moiety, was confirmed by
HMBC correlations (Figure S1) from CH3-6ꢀ (dH 1.84) to [dC 160.4 (C-5ꢀ), 104.8 (C-6ꢀ),
and 165.9 (C-7ꢀ)], from H-8ꢀ (dH 5.91) to [dC 104.8 (C-6ꢀ), 165.9 (C-7ꢀ), 156.5 (C-9ꢀ), and
99.3 (C-10ꢀ)], from H-2ꢀ0 (dH 6.73) to [dC 100.1 (C-2ꢀ), 146.8 (C-4ꢀ0), and 119.5 (C-6ꢀ0)],
from H-5ꢀ0 (dH 7.14) to [dC 124.6 (C-1ꢀ0), 146.6 (C-3ꢀ0), and 146.8 (C-4ꢀ0)], and from H-6ꢀ0
(dH 7.01) to [dC 124.6 (C-1ꢀ0) and 115.6 (C-5ꢀ0)], and was verified by related NMR data
of reported baeckeins C, D and E (Jia, Zhou, et al. 2011; Jia et al. 2013).

4
C.-J. JIA ET AL.
Inspection of the signals for segment I indicated the presence of a 5,7-dihydroxy-6-
methyl-substituted ring A and a 30,40-dihydroxy-substituted ring B. Characteristic chem-
ical shifts for one carbonyl carbon [dC 185.4 (C-4)] and two quaternary carbons [dC
104.7 (C-2) and 91.9 (C-3)] revealed a dihydroisoflavanol structure, and carbons of C-2
and C-3 supplied two available binding positions for the segment II. Between seg-
ꢂ
ꢂ
ments I and II, optimal connections of C-2–C-8 and C-3–O–C-7 formed a furan ring
D. The segment I, including rings A, B, C and D, was confirmed by HMBC correlations
(Figure S1) from CH3-6 (dH 1.91) to [dC 161.5 (C-5), 104.8 (C-6), 167.1 (C-7), 94.3 (C-8)
and 98.6 (C-10)], from H-8 (dH 6.07) to [dC 185.4 (C-4), 104.8 (C-6), 167.1 (C-7), 159.3
(C-9) and 98.6 (C-10)], from H-20 (dH 7.13) to [dC 124.3 (C-10), 144.5 (C-30), 145.7 (C-40),
115.9 (C-50) and 118.1 (C-60)], from H-50 (dH 7.82) to [dC 91.9 (C-3), 124.3 (C-10), 144.5
(C-30), 145.7 (C-40) and 118.1 (C-60)], and from H-60 (dH 6.18) to [dC 91.9 (C-3), 145.7
(C-40) and 115.9 (C-50)], which was further identified by literature values of baeckeins
J and K (Jia et al. 2016).
The sugar moiety was identified as D-glucose by TLC and GC analysis (Tang et al.
3
2005; Gao et al. 2008) . The large JH-1,H-2 coupling constant of the anomeric proton
[dH 4.93 (1H, d, J ¼ 7.0 Hz, H-100)] suggested a b-configuration for the glucose unit, and
the HMBC cross-peak (Figure S1) from H-100 to C-4ꢀ0 and NOE correlations (Figure S6)
between H-100 and aromatic protons of ring B (H-2ꢀ0, H-5ꢀ0, and H-6ꢀ0) indicated the
location of the glucose residue should be at C-4ꢀ0. And the b-D-glucose moiety was
further confirmed by related data in the literature (Quang et al. 2008).
In the aglycone structure of compound 1, referring to segments I, II and III, the qua-
ternary carbons C-2, C-3, C-2ꢀ and C-3ꢀ provided four chiral centers. However, our
NOESY experiment (not giving useful NOE increments) and CD spectrum (with positive
Cotton effects (CEs) at 236, 283, 336 and 375 nm, and negative CEs at 213 and
308 nm) (Figure S2A) were not sufficient enough to determine their absolute configu-
rations. In this case, quantum chemical CD calculations were employed (Jia, Zhou,
et al. 2011; Jia et al. 2013; Jia et al. 2014; Jia et al. 2016). All 16 geometries were opti-
ꢂ
mized by M06-2X functional (Zhao and Truhlar 2008) with 6-311G basis-set (Krishnan
et al. 1980), and xB97XD functional (Chai and Head-Gordon 2008) with 6-31G basis-
ꢂ
set (Hariharan and Pople 1973) was employed to conduct the TD-DFT calculations.
Based on the resulting electronic excitation energies and rotatory strengths, Multiwfn
3.3.8 (Lu and Chen 2012) in combination with Origin software were used to obtain
ECD spectra. The calculated CD spectrum (Figure S2B) for the configuration (2R, 3R,
2ꢀS, 3ꢀS) displayed diagnostic negative and positive CEs, which exhibited good agree-
ments with the experimental CD, and allowed the assignments of absolute configura-
tions of 1 as (2R, 3R, 2ꢀS, 3ꢀS). Based on the above results, the structure of baeckein L
(1) was unambiguously established.
Baeckein M (2) was also obtained as a yellow amorphous powder and possessed
the molecular formula C54H42O26, as established by HR-ESI-MS (m/z 1107.2028
1
[M þ H]þ, calcd 1107.2037 for C54H43O26). The H, 13C and 2D-NMR (Table S1, Figure
S1) for compound 2 were very similar to those of 1, and careful analysis of these spec-
tral data suggested that both the carbon skeleton and functional groups presented in
2 were much the same as those of 1. And the CD spectrum of 2 displayed positive
CEs at 212 and 307 nm and negative CEs at 235, 283, 333 and 374 nm, nearly a mirror

NATURAL PRODUCT RESEARCH
5
image of that of 1 (Figure S2A), which disclosed a pair of diastereomers. Also the
absolute stereochemistry of chiral centers C-2, C-3, C-2ꢀ and C-3ꢀ in compound 2 was
assigned as (2S, 3S, 2ꢀR, 3ꢀR) by the similar TD-DFT calculations that 1 used
(Figure S2C).
Acknowledgements
We appreciate the kind help from Mr. Mao-Xiang Lai (Guangxi Academy of Chinese Medicines
and Pharmaceutical Science) for collecting the plant materials.
Funding
This work was supported by the [National Natural Science Foundation of China] under Grant
[NO. 81303162], and the [China Postdoctoral Science Foundation] under Grant [NO.
2018M630841].
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