Structure determination of baeckeins C and D from the roots of Baeckea frutescens

Article abstract of DOI:10.1002/mrc.2803

Baeckeins C (compound 1) and D (compound 2), a pair of new diastereomeric biflavonoids, were isolated from the roots of Baeckea frutescens. Their structures were elucidated on the basis of spectroscopic analysis, including HR-ESI-MS and oneand two-dimensional NMR spectra (¹H and ¹³C NMR, HSQC, HMBC, and ROESY). The absolute configurations of the 2,3-epoxide moiety for compounds 1 and 2 were determined by circular dichroism spectrometry combined with quantum chemical calculations. Copyright

Full text of DOI:10.1002/mrc.2803

MRC Letters
Received: 5 June 2011
Revised: 25 July 2011
Accepted: 1 August 2011
Published online in Wiley Online Library: 16 October 2011
(wileyonlinelibrary.com) DOI 10.1002/mrc.2803
Structure determination of baeckeins C and D
from the roots of Baeckea frutescens
Bei-Xi Jia,a Yu-Xin Zhou,b Xiao-Qing Chen,a Xiao-Bing Wang,c Jie Yang,a
Mao-Xiang Laid and Qiang Wanga*
Baeckeins C (compound 1) and D (compound 2), a pair of new diastereomeric biflavonoids, were isolated from the roots of
Baeckea frutescens. Their structures were elucidated on the basis of spectroscopic analysis, including HR-ESI-MS and one-
and two-dimensional NMR spectra (1H and 13C NMR, HSQC, HMBC, and ROESY). The absolute configurations of the 2,3-epoxide
moiety for compounds 1 and 2 were determined by circular dichroism spectrometry combined with quantum chemical calcu-
lations. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: NMR; 1H; 13C; two-dimensional NMR; circular dichroism; biflavonoids; baeckein C; baeckein D; Baeckea frutescens; Myrtaceae
119.0, 117.1, 116.6, 115.8, 115.5, 95.7, and 92.7), and 20 quaternary
Introduction
carbons. The 1H NMR spectrum (Table 1) of compound 1 showed
Baeckea frutescens L. (Myrtaceae) is an aromatic low-growing shrub
distributing from Southeast Asia to Australia. The roots of B.
frutescens, known as “Pu-Lao-Zhong,” have been used for treating
rheumatism and snake bites in the southern part of China.[1] Many
active constituents have been isolated from this species, including
essential oil, some sesquiterpenes, phloroglucinols, chromones,
flavones, and their derivatives.[2–10] In our search for structurally
and pharmacologically interesting natural products, a phytochemical
study of B. frutescens was undertaken. As a result, baeckeins C
(compound 1) and D (compound 2), a pair of new diastereomeric
biflavonoids with a 2,3-epoxide ring, were isolated from the roots
of this plant (Fig. 1). Herein, their isolation and structural elucidation
are presented. The one- and two-dimensional NMR techniques and
HR-ESI-MS were extensively applied to characterize their structures
and to establish the 1H and 13C resonance assignments. Furthermore,
the absolute stereochemistry of the 2,3-epoxide moiety for com-
pounds 1 and 2 were determined by circular dichroism (CD) spec-
trometry combined with quantum chemical calculations.
signals for two sets of typical ABX coupling systems [dH 7.03 (1H,
d, J = 8.7Hz, H-2′), 7.19 (1H, d, J = 2.0Hz, H-5′), and 7.10 (1H, dd,
J = 8.7, 2.0 Hz, H-6′)] and [dH 7.28 (1H, d, J = 8.7Hz, H-2*′), 7.82 (1H,
d, J = 2.0Hz, H-5*′), and 7.85 (1H, dd, J = 8.7, 2.0 Hz, H-6*′)],
corresponding to the 3′,4′-dihydroxy-substituted B ring of flavonoids
(rings B and B*). Also, two aromatic protons [dH 6.08 (1H, s, H-8)
and 6.53 (1H, s, H-8*)] and two aromatic methyl signals [dH 1.88
(3H, s, Me-6) and 2.0 (3H, s, Me-6*)] were observed, revealing
the presence of the 6-methyl-substituted A ring of quercetin
(rings A and A*). In addition, a series of signals in the range of
approximately dH 5.5–3.0 related to a sugar moiety were
presented. Taken together, the 1H and 13 C NMR data revealed that
compound 1 was composed of two 6-C-methylquercetin[10,11]
moieties and a hexosyl residue.
A comprehensive analysis of the one-dimensional NMR and HSQC
spectra of compound 1 made the assignments of segment 1, a 6-C-
methylquercetin unit, referring to rings A*, B*, and C*, which was
confirmed by HMBC correlations (Fig. 2) from Me-6* to [dC = 157.6
(C-5*), 106.3 (C-6*), and 162.4 (C-7*)], from H-8* to [dC = 176.0
(C-4*), 106.3 (C-6*), 162.4 (C-7*), 154.0 (C-9*), and 102.8 (C-10*)], from
H-2*′ to [dC = 44.8 (C-2*), 125.9 (C-1*′), 140.4 (C-3*′), and 122.3 (C-6*′)],
from H-5*′ to [dC = 125.9 (C-1*′), 140.4 (C-3*′), and 141.5 (C-4*′)], and
from H-6*′ to [dC = 144.8 (C-2*), 125.9 (C-1*′), 141.5 (C-4*′), and
Results and Discussion
Baeckein C (compound 1) was obtained as a yellow amorphous
powder and gave positive results for the Mg–HCl reaction and
Molish reagent, indicating it to be a flavonoid glycoside. The HR-
ESI-MS exhibited a [M–H]– ion at m/z 791.1463 (calculated
791.1465), corresponding to the molecular formula C38H32O19, with
23 degrees of unsaturation. The UV spectrum (lmax = 275, 315, and
370 nm) suggested the presence of a conjugated system. The IR
spectrum showed absorption bands for hydroxyl (3377 cm-1) and
carbonyl (1633 cm-1) groups and aromatic functionalities (1599
and 1488 cm-1, respectively).
*
Correspondence to: Qiang Wang, Key Laboratory of Modern Chinese Medi-
cines, Ministry of Education, China Pharmaceutical University, Nanjing
210009, China. E-mail: qwang49@163.com
a Key Laboratory of Modern Chinese Medicines, Ministry of Education, China
Pharmaceutical University, Nanjing 210009, China
b Institute of Chinese Materia Medica, Henan University, Kaifeng 475004, China
The 13 C NMR spectra (Table 1) of compound 1 displayed a group
of signals (dC = 101.6, 77.2, 75.9, 73.2, 69.8, and 60.7) belonging to a
hexosyl unit and 32 carbon signals attributable to a biflavonoid
structure, among which are 2 carbonyl groups (dC = 188.0 and
176.0), 2 aromatic methyl groups (dC = 6.9 and 7.3), 8 CH (dC = 122.3,
c
Department of Natural Medicinal Chemistry, China Pharmaceutical University,
Nanjing 210009, China
d Guangxi Academy of Chinese Medicines and Pharmaceutical Science, Nanning
530022, China
Magn. Reson. Chem. 2011, 49, 757–761
Copyright © 2011 John Wiley & Sons, Ltd.

B.-X. Jia et al.
Figure 1. Structures of compounds 1 and 2.
116.6 (C-5*′)]. Segment 1 was further identified by comparison with
literature values of known compound 6-C-methylquercetin.[10,11]
Inspection of the signals of segment 2 revealed some structural
characteristics of segment 1 and also suggested the presence of
rings A and B. The major differences between segments 1 and 2
were in the ring C. Particularly, the signals assigned to two quater-
nary carbons [dC = 100.0 (C-2) and 90.6 (C-3)] and a carbonyl carbon
[dC = 188.0 (C-4)] were observed, which indicated that the carbon
skeleton of the ring C was converted into a dihydropyrone ring
from a pyrone ring and new substituents were bound to C-2 and
C-3 positions (both of them bearing oxygen atoms).[12] From these
evidence, the presence of a 2,3-epoxide ring was proposed accord-
ing to the molecular weight and the remaining degrees of unsatura-
tion. Segment 2, including rings A, B, and C, was confirmed by HMBC
correlations from Me-6 to [dC = 160.5 (C-5), 105.1 (C-6), and 166.8
(C-7)], from H-8 to [dC = 105.1 (C-6), 166.8 (C-7), 156.3 (C-9), and
99.0 (C-10)], from H-2′ to [dC = 100.0 (C-2), 146.5 (C-4′), and 115.5
(C-6′)], from H-5′ to [dC = 128.1 (C-1′), 146.1 (C-3′), and 146.4 (C-4′)],
and from H-6′ to [dC = 128.1 (C-1′) and 115.8 (C-5′)].
Table 1. 1H and 13C NMR data for compounds 1 and 2 (d in ppm, J in Hz)
Position
Compound 1
Compound 2
dH
dC
dH
dC
2
100.0
90.6
99.8
90.5
3
4
188.0
160.5
105.1
186.7
160.3
105.1
7.0
5
6
CH3-6
7
1.88 s
6.08 s
6.9 1.83 s
166.8
169.0
95.7
8
95.7 5.93 s
156.3
9
156.2
98.2
10
1′
99.0
128.1
128.4
118.9
146.0
146.4
115.7
115.4
144.8
136.6
176.0
157.5
106.2
7.3
2′
7.03 d (8.7)
119.0 7.03 d (8.4)
146.1
3′
4′
146.5
5′
7.19 d (2.0)
115.8 7.18 d (2.0)
115.5 7.08 dd (8.4, 2.0)
144.8
A C–O–C linkage between segments 1 and 2 was deduced from
1
the above spectral analysis. For segment 1, all H and 13 C reso-
6′
7.10 dd (8.7, 2.0)
2*
3*
4*
5*
6*
CH3-6*
7*
8*
9*
10*
1*′
2*′
3*′
4*′
5*′
6*′
Glc-100
200
300
400
500
600
nance assignments were established, and the carbon signals of
C-3*′ (dC = 140.4) and C-4*′ (dC = 141.5) were shifted dC = 5.0–7.5
low frequency against those of 6-C-methylquercetin, suggesting
one available binding position for segment 2. Elucidated in the same
manner, the 2,3-epoxide ring provided another available position for a
connection between two segments, which was supported by related
literature values of known compound 1,3,11a-trihydroxy-9-(3,5,
7-trihydroxy-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-
4-on-2-yl)-5a-(3,4-dihydroxyphenyl)-5,6,11-hexahydo-5,6,11-
trioxanaphthacene-12-one.[13] A comparative evaluation of
the chemical shifts of quaternary carbons C-2 (dC = 100.0)
and C-3 (dC = 90.6) revealed that the C–O–C linkage should
be bound to C-3, which was confirmed by an HMBC corre-
lation from H-2′ to C-2. As a result, the (C-3)–O–(C-4*′) ether
linkage was determined to be the only possible option for a
linkage between segments 1 and 2 of compound 1. Further-
more, the unusual 2,3-epoxide moiety and the (C-3)–O–(C-4*′)
bond were verified by the NMR data presented by Chervia-
kovsky et al.[14] for the product named as product B.
136.7
176.0
157.6
106.3
2.00 s
6.53 s
7.3 2.00 s
162.4
162.4
92.6
92.7 6.53 s
154.0
153.9
102.7
125.8
117.0
140.4
141.5
116.5
122.2
101.5
73.2
102.8
125.9
7.28 d (8.7)
117.1 7.25 d (8.7)
140.4
141.5
7.82 d (2.0)
7.85 dd (8.7, 2.0)
4.75 d (7.0)
3.28 m
116.6 7.79 d (2.0)
122.3 7.83 dd (8.7, 2.0)
101.6 4.73 d (6.9)
73.2 3.27 m
77.2 3.31 m
69.8 3.14 m
75.9 3.26 m
60.7 3.45 dd (11.1, 5.1)
3.69 dd (11.0, 2.0)
3.33 m
77.1
3.17 m
69.7
The D-glucose was obtained from the acid hydrolysis experiment
of compound 1 and was identified by thin layer chromatograph
3.27 m
75.8
3
(TLC) and gas chromatograph (GC) analysis.[15,16] The large JH-1,H-2
3.47 dd (11.2, 5.0)
3.71 dd (11.3, 2.0)
60.6
coupling constant of the anomeric proton [dH 4.75 (1H, d,
J=7.0Hz, H-100)] suggested a b-configuration for the glucose unit,
and the location of the glucose residue should be at C-4′ (segment
1H NMR was recorded at 500 MHz and 13C at 125 MHz (in DMSO-d6).
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Magn. Reson. Chem. 2011, 49, 757–761

Structure determination of baeckeins C and D
Figure 2. Key HMBC and ROESY correlations for compound 1.
2), which was supported by the HMBC cross peak from H-100 to C-4′
and the NOE correlations (Fig. 2) between H-100 and aromatic protons
of ring B (H-2′, H-5′, and H-6′). Moreover, the b-D-glucose moiety was
further confirmed by the related data in the literature.[10]
The stereochemical assignment of epoxide rings in complex
natural structures is not an easy task, especially when the
oxiranes are tetrasubstituted.[17] The CD spectrum (Fig. 3A) of
compound 1 showed positive Cotton effects at 307 and 378 nm
and negative Cotton effects at 281 and 332 nm, but these spec-
tral data (including NMR) were not sufficient enough to deter-
mine the absolute stereochemistry of the 2,3-epoxide moiety. In
this case, quantum chemical CD calculations were used.[18] The
geometry was built on a three-dimensional structure of com-
pound 1 with configurations (2S and 3S). The conformational
analysis was analyzed using the semiempirical PM3 method, as
implemented in the Q-Chem program package, starting from
preoptimized geometries generated by the MM2 force field in Chem
three-dimensional software. The electronic circular dichrism (ECD)
spectrum was computed in the gas phase using time-dependent
density-functional theory (TD-DFT) at the B3LYP/6-31 G (d) level.
The shapes of the computed CD (Fig. 3B) were in good agreement
with those of the experimental CD spectrum, which allowed the as-
signment of the absolute configuration of compound 1 as depicted.
On the basis of the results, the structure of baeckein C (compound 1)
was unambiguously established as (1aS,7aS)-4,6-dihydroxy-
Figure 3. (A) Experimental CD spectra and (B, C) calculated CD spectra of
compounds 1 and 2.
1a-(3-hydroxy-4-((2R,3R,4S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)
tetrahydro-2H-pyran-2-yloxy)phenyl)-7a-(2-hydroxy-4-
(3,5,7-trihydroxy-6-methyl-4-oxo-4H-chromen-2-yl)phenoxy)-
5-methyl-1aH-oxireno[2,3-b]chromen-7(7aH)-one.
The HR-ESI-MS and 13 C NMR analysis of baeckein D (compound
2) indicated that it possessed the molecular formula of C38H32O19,
which was the same as that of compound 1. The 1H and 13 C NMR
data (Table 1) as well as the two-dimensional correlations (Fig. 4)
disclosed that both the skeleton and the functional groups pre-
sented in compound 2 were similar to those of compound 1,
Magn. Reson. Chem. 2011, 49, 757–761
Copyright © 2011 John Wiley & Sons, Ltd.
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B.-X. Jia et al.
NMR spectra
NMR spectra were recorded at room temperature on a Bruker
1
AV-500 spectrometer equipped with a QNP 5-mm probe. H, 13C
NMR spectra (500 and 125 MHz, respectively), and two-dimensional
NMR experiments (HSQC, HMBC, and ROESY) were recorded at
303 K using standard Bruker pulse programs. Approximately
3.0- to 5.0-mg samples were dissolved in 0.5ml DMSO-d6, which
was used as the internal lock. The chemical shifts were given in d
(ppm) scale and referenced to residual solvent peaks of DMSO-d6
1
at dH 2.50 for H NMR and dC = 39.51 for 13C NMR. Coupling con-
stants (J) were given in hertz.
For 1H NMR, 8–16 transients were acquired using spectral widths
of 7861 and 8620 Hz for compounds 1 and 2 and 90ꢁ pulses
(14.80 ms at ꢂ1.00 dB). For 13C NMR, 400–20 000 transients were
acquired using spectral widths of 30303 Hz and 90ꢁ pulses
(9.50 ms at 2.00dB). Data points (32 K) were used for the processing
with an exponential function (LB = 0.20 Hz for 1H NMR, LB= 2.00 Hz
for 13C NMR).
The two-dimensional spectra used 1024 ꢀ 256 (HSQC),
1024 ꢀ 512 (HMBC), and 1024 ꢀ 128 (ROESY) data point ma-
trices, which were zero filled to 2048 ꢀ 1024, 2048 ꢀ 1024,
and 1024 ꢀ 1024, respectively. A relaxation delay of 2.0 s
was used in all experiments. HSQC data were acquired with
16 transients per t1 increment, and spectral widths
of 12570 Hz/2750 Hz (compound 1) and 27500 Hz/4000 Hz
(compound 2) were used for 13C and 1H, respectively. The
HMBC experiment was performed with 32 transients per t1
increment. Spectral widths of 12570 Hz/2750 Hz (compound
1) and 30050 Hz/4000 Hz (compound 2) were used for 13C
and 1H, respectively. For ROESY, a mixing time of 800 ms
and a spectral width of 4006 Hz for both dimensions were
used, and the number of transients collected for each time
increment in the indirect dimension was eight.
Figure 4. Key HMBC and ROESY correlations for compound 2.
suggesting that compound 2 was a diastereomer of compound 1.
This deduction was verified by the CD spectrum of compound 2
(Fig. 3A), which displayed positive Cotton effects at 280 and
332 nm and negative Cotton effects at 307 and 377 nm, nearly a
mirror image of the CD spectrum of compound 1. Simultaneously,
a TD-DFT calculation of the ECD data for compound 2 was also car-
ried out following similar procedures that compound 1 used. In ad-
dition, the shapes of the computed CD and experimental CD of
compound 2 are almost the same (Fig. 3C). Accordingly, the abso-
lute configuration of the 2,3- epoxide ring for compound 2 was
Plant material
The roots of B. frutescens were collected from Nanning, Guangxi
Province, China, and identified by Q. Wang, China Pharmaceutical
University. A voucher sample (No. GS001) is deposited in the
Department of Chinese Materia Medica Analysis, China Pharma-
ceutical University, Nanjing, China.
1
assigned as (2R and 3R), and the full assignments of H and 13C
NMR data were achieved in combination with HMBC, HSQC, and
ROESY experiments.
Extraction and isolation
The dried roots of B. frutescens (10 kg) were extracted three times
with 90% ethanol and concentrated in vacuum to give residue
(600 g), which was suspended in water and partitioned with
petroleum ether, ethyl acetate, and n-butanol successively. The
ethyl acetate soluble part (200 g) was first subjected to chromatog-
raphy over a silica gel column and eluted with gradient solvent
CHCl3/MeOH (1 : 0–1 : 1) to yield eight fractions on the basis of
TLC analyses. Fraction 6 was then submitted to a Sephadex LH-20
column eluted with CHCl3/MeOH (1 : 1) to afford six subfractions
(F6.1–6.6), of which subfraction F6.3 was repeatedly chromato-
graphed over ODS columns eluted with MeOH/H2O (60 : 40) to
yield compound 1 (20 mg) and compound 2 (5mg).
Experimental
General procedures
Column chromatography was carried out using silica gel (SiO2;
Qingdao Haiyang Chemical Industry, 200–300 mesh), ODS
(40–63 mm, Fuji), and Sephadex LH-20 (20–100 mm, Pharmacia). Opti-
cal rotations were measured with a JASCO P-1020 polarimeter. UV
and CD spectra were determined on a JASCO J-810 CD spectrometer.
IR spectra (KBr discs) were recorded with Nicolet Impact-410 spec-
trometer. MS spectra were obtained on an Agilent LC/MSD TOF.
GC analysis was carried out on a Agilent 6890 Plus gas chromatogra-
Baeckein C (compound 1)
phy using
a
DB-5 capillary column (30 m ꢀ 0.25 mm, i.d.);
detection, FID; detector temperature, 280 ꢁC; injection temperature,
250 ꢁC; initial temperature of 180 ꢁC was maintained for 5 min and
then raised to 260 ꢁC at the rate of 8 ꢁC/min; carrier gas, He.
Yellow amorphous powder, [a]3D0 ꢂ100ꢁ (c = 0.10, MeOH). UV
(MeOH) nm lmax (log e): 272 (2.95), 313 (3.20), and 375 (2.80); IR
(KBr) nmax: 3377, 2921, 2354, 1633, 1599, 1488, 1435, 1274, 1198,
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Copyright © 2011 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2011, 49, 757–761

Structure determination of baeckeins C and D
1160, 1075, 1010, 948, 896, 808, 731, and 623; CD (c = 0.10, MeOH):
281 nm ( Δe -5.50), 307 nm ( Δe +4.32), 332nm ( Δe -4.38), and
378 nm ( Δe +2.51); 1H- and 13C-NMR data, see Table 1; ESI-
MS m/z 791 [M–H]–, HR-ESI-MS m/z 791.1463 [M–H]– (calcu-
lated for C38H31O19, 791.1465).
of Natural Medicinal Chemistry, China Pharmaceutical University),
and Mr. L. Feng (Simcere Pharmaceutical Group) for providing
assistance in technology and equipment.
References
Baeckein D (compound 2)
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Ben Cao, Shanghai Science and Technology Press, Shanghai, 1999.
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Yellow amorphous powder, [a]3D0 ꢂ37.5ꢁ (c = 0.11, MeOH). UV
(MeOH) nm lmax (log e): 272 (2.93), 312 (3.20), and 374 (2.81); IR
(KBr) nmax: 3408, 2919, 2352, 1633, 1599, 1504, 1435, 1273, 1199,
1145, 1076, 1011, 949, 895, 808, 732, and 624; CD (c = 0.11, MeOH):
280 nm ( Δe +4.60), 307 nm ( Δe -3.60), 332 nm ( Δe +3.65), and
378 nm ( Δe -2.11); 1H- and 13C-NMR data, see Table 1; ESI-MS m/z
791 [M–H]–, HR-ESI-MS m/z 791.1463 [M–H]– (calculated for
C38H31O19, 791.1465).
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Acid hydrolysis and GC analysis
Each compound (2mg) was hydrolyzed with 10% HCl–dioxane (1 :
1, 5 ml) at 80 ꢁC for 4 h. After extraction with ethyl acetate (5mlꢀ 3),
the sugar component in the aqueous layer was evaporated under
reduced pressure and then examined by silica gel TLC with
CHCl3–MeOH–H2O (8 : 5 : 1) by comparison with the authentic sam-
ple.[15] Furthermore, the sugar residue was dissolved in 1 ml of dry
pyridine, and 2 mg of L-cysteine methyl ester hydrochloride was
added, followed by heating at 60 ꢁC for 2 h. The reaction mixture
was concentrated to dryness with N2 gas, and then trimethylsilyl
imidazole was added to the residue, followed by stirring at 60 ꢁC
for 1 h. Finally, the resulted solution was extracted with cyclohex-
ane and H2O, and the combined organic phase was analyzed by
GC.[16] The standard D-glucose (Sigma, USA) was subjected to the
same reaction and GC analysis under the same conditions.
Computational
The Q-Chem program package and the Chem three-dimensional
software were used for calculations. Conformation searching was
performed at MM2 molecular mechanics force field. All ground-
state geometries were optimized at the B3LYP/6-31 G(d) level,
and harmonic vibrational frequencies were calculated to confirm
the minima. TD-DFT calculations for excitation energy and rota-
tory strength R were carried out at the same level in the gas
phase. The ECD spectra were then simulated by overlapping
Gaussian functions for each transition.
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Acknowledgements
This work was supported by grants from a project funded by the
Priority Academic Program Development of Jiangsu Higher Edu-
cation Institutions. The authors appreciate the kind help of Dr K.
Zan (State Key Laboratory of Natural and Biomimetic Drugs,
Peking University Health Science Center), Dr J. Luo (Department
Magn. Reson. Chem. 2011, 49, 757–761
Copyright © 2011 John Wiley & Sons, Ltd.
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