Diplomorphanins A and B: New C-methyl flavonoids from Diplomorpha canescens

Article abstract of DOI:10.1248/cpb.c12-00927

Two new C-methyl flavonoids, diplomorphanins A (1) and B (2) were isolated from the aerial parts of Diplomorpha canescens ( Meisn.) C. A. Meyer along with a known compound, farrerol 7-O-β-dglucopyranoside (3). Structures of these compounds were determined on the basis of spectroscopic data.

Full text of DOI:10.1248/cpb.c12-00927

242
Note
Chem. Pharm. Bull. 61(2) 242–244 (2013)
Vol. 61, No. 2
Diplomorphanins A and B: New C-Methyl Flavonoids from Diplomorpha
canescens
,a,b
Hari Prasad Devkota,a Masato Watanabe,b Takashi Watanabe,c and Shoji Yahara*
a Graduate School of Pharmaceutical Sciences, Kumamoto University; b Medicinal Plants Eco-frontier Center, School
c
of Pharmacy, Kumamoto University; 5–1 Oe-honmachi, Chuo-ku, Kumamoto 862–0973, Japan: and Research
Organization for Regional Alliances, Kochi University of Technology; 185 Miyanokuchi, Tosayamada, Kami, Kochi
782–0003, Japan.
Received October 22, 2012; accepted November 7, 2012; advance publication released online November 14, 2012
Two new C-methyl flavonoids, diplomorphanins A (1) and B (2) were isolated from the aerial parts
of Diplomorpha canescens (Meisn.) C. A. Meyer along with a known compound, farrerol 7-O-β-d-
glucopyranoside (3). Structures of these compounds were determined on the basis of spectroscopic data.
Key words Diplomorpha canescens; Thymelaeaceae; diplomorphanin A; diplomorphanin B
Diplomorpha canescens (Meisn.) C. A. Meyer (Family: C-4′, which revealed that the one glucose moeity was attached
Thymelaeaceae) (Synonym: Wikstroemia canescens Meisn.) to the 4′ position in the B ring. Correlation of anomeric proton
is called as “Sanhijyou” in traditional Chinese medicine1) at δ 4.59 with carbon at 164.1 (C-7) suggested the attachment
and used in antitumor therapy.2) We have previously reported of second glucopyranosyl moiety in C-7 which was also sup-
four new compounds such as (2R,3S)-6,8-di-C-methyldihy- ported by the downfield shift of A ring carbons. The signal
drokaempferol, (2R,3R)-6,8-di-C-methyldihydrokaempferol,3) for C5-OH (δ 12.10) had correlations with carbons at δ 157.9
farrerol 4′-O-β-d-glucopyranoside (4),4) and diplomorphanone (C-5), 111.2 (C-6) and 104.8 (C-10). Key HMBC correlations
A5) along with twenty-five known compounds from the aerial are given in Fig. 2. In the differential NOE spectra, irradiation
parts of D. canescens. In continuing further chemical analy- of C6-CH3 proton (δ 2.09ppm) enhanced the proton signal at
sis, we isolated and identified two new C-methyl flavonoids, δ 4.59 (C7-Glc-C1-H) and 12.10 (C5-OH). Also the irradiation
diplomorphanins A (1) and B (2) along with farrerol 7-O-β-d- of C8-CH3 proton (δ 2.07ppm) enhanced the proton signal at
glucopyranoside (3)6) (Fig. 1) which are reported in this paper. δ 4.59 (C7-Glc-C1-H). The circular dichroism (CD) spectrum of
Diplomorphanin A (1) was obtained as pale yellow amor- 1 showed the positive Cotton effect at 347nm suggesting the
phous powder, [α]D21 −22.2. The high resolution (HR)-FAB- absolute configuration at the C-2 position is S.7) Hence, on the
MS of 1 showed the quasi-molecular ion [M+Na]+ peak at
1
m/z: 647.1979 supporting the formula C29H36O15. The H-NMR
spectrum of 1 (Table 1) showed signals due to two aromatic
methyl groups at δH 2.07 (3H, s) and 2.09 (3H, s), and the
proton resonances at δH 7.45 (2H, d, J=8.5Hz, C2′-H, C6′-H)
and 7.09 (2H, d, J=8.5Hz, C3′-H, C5′-H) were characteristic
of p-substituted phenyl ring. Signals for two anomeric protons
were observed at δ 4.59 (1H, d, J=7.6Hz) and 4.89 (1H, d,
J=7.6Hz). Similarly, three resonances at δ 5.58 (1H, dd, J=2.7,
12.3Hz, C2-H), 3.32 (1H, dd, J=12.3, 16.0Hz, C3-Ha) and 2.87
(1H, dd, J=2.7, 16.0Hz, C3-Hb) were characteristic of C ring
of a flavanone moiety. The 13C-NMR (Table 1) and distortion-
Fig. 1. Structures of Compounds 1–6
less enhancement by polarization transfer (DEPT) spectra
showed signals equivalent to 29 carbons. Among them, seven-
teen carbon signals were assignable to 6,8-di-C-methyl-5,7,4′-
trihydroxyflavanone or farrerol (5)4) and twelve carbons sig-
nals were assignable to two units of β-glucopyranosyl moiety
which was also supported by the acid hydrolysis of 1 affording
5 and d-glucose. The attachments of O-β-d-glucopyranosyl
moieties in C-4′ and C-7 position were confirmed on the
basis of heteronuclear multiple quantum coherence (HMQC)
and heteronuclear multiple bond connectivity (HMBC) cor-
relations and differential nuclear Overhauser effect (NOE)
spectra. In the HMBC spectrum, proton resonances at δ 7.45
(C2′-H, C6′-H) and 7.09 (C3′-H, C5′-H) had long range correla-
tion with carbon resonance at δ 157.3 (C-4′). Similarly, the
signal for an anomeric proton at δ 4.89 had correlations with
The authors declare no conflict of interest.
Fig. 2. Key HMBC Correlations of 1 and 2
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: yaharas1@gpo.kumamoto-u.ac.jp

February 2013
243
basis of these data the structure of 1 was assigned to be far- resonance at δ 159.4 (C-4′). The C6-methyl signal (δ 2.21)
rerol 4′,7-di-O-β-d-glucopyranoside. had correlation with carbons at δ 154.9 (C-5), 113.2 (C-6) and
Diplomorphanin B (2) was obtained as pale yellow amor- 158.5 (C-7). Similarly, C8-methyl signal (δ 2.42) had correla-
phous powder, [α]D21 +2.6. The HR-FAB-MS of 2 showed the tion with carbons at δ 150.0 (C-9), 109.8 (C-8) and 158.5 (C-7).
quasi-molecular ion [M−H]− peak at m/z: 475.1280 supporting The signal for C5-OH had correlations with carbons at δ 154.9
1
the formula C23H24O11. The H-NMR of 2 showed two signals (C-5), 113.2 (C-6) and 106.0 (C-10). The anomeric proton sig-
due to two aromatic methyl groups at δH 2.21 (3H, s) and 2.42 nal at δ 4.66 had correlation with carbon at 158.5 (C-7) which
(3H, s), and the proton resonances at δH 8.11 (2H, d, J=8.7Hz, suggested that the glucopyranosyl moiety was attached at C-7
C2′-H, C6′-H) and 6.97 (2H, d, J=8.7Hz, C3′-H, C5′-H) were position. Key HMBC correlations have been given in Fig. 2.
characteristic of p-substituted phenyl ring. Signal for an In the differential NOE spectra, irradiation of C6-CH3 proton
anomeric proton was observed at δ 4.66 (1H, d, J=7.7Hz). (δ 2.21ppm) enhanced the proton signal at δ 4.66 (Glc-C1-H)
The 13C-NMR (Table 1) and DEPT spectra showed signals and 12.58 (C5-OH) and the irradiation of C8-CH3 proton (δ
equivalent to 23 carbons. Among them, 17 carbon signals 2.42ppm) enhanced the proton signal at δ 4.66 (Glc-C1-H)
were assignable to 6,8-di-C-methylkaempferol (6)8) and the which also supported the above assignments. Hence, on the
six carbon signals at δ 61.1, 71.5, 74.0, 76.3, 77.0 and 104.0 basis of these data the structure of 2 was assigned to be
were assignable to a β-glucopyranosyl moiety which was 6,8-di-C-methylkaempferol 7-O-β-d-glucopyranoside.
also supported by the acid hydrolysis of 2 affording 6 and d-
glucose. In the HMBC spectrum, proton resonances for C3′-H,
C5′-H and C2′-H, C6′-H had long range correlation with carbon
Experimental
General Experimental Procedures Optical rotations
Table 1. NMR Spectroscopic Data for 1–3 and 6 in DMSO-d6
1
3
2
6
Position
δH, mult. (J in
δH, mult. (J in
δH, mult. (J in
δH, mult. (J in
δC
δC
δC
δC
Hz)
Hz)
Hz)
Hz)
2
3
77.8
42.2
5.58, dd (2.7,
12.3)
2.87, dd (2.7,
16.0)
78.0
42.2
5.49, m
147.5
136.0
146.5
135.5
2.78, dd (2.9,
17.2)
3.32, dd (12.3,
16.0)
3.30, m
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
198.4
157.9
111.2
164.1
110.2
157.2
104.8
132.1
127.8
116.2
157.3
116.2
127.7
8.7
198.6
157.9
111.1
164.4
110.1
157.4
104.8
129.0
128.0
115.2
157.6
115.2
128.0
8.7
176.5
154.9
113.2
158.5
109.8
150.0
106.0
121.8
129.5
115.5
159.4
115.5
129.5
8.9
176.1
155.0
106.3
159.1
101.4
151.4
102.9
122.0
129.4
115.5
159.4
115.5
129.4
8.0
7.45, d (8.5)
7.09, d (8.5)
7.32, d (8.4)
6.80, d (8.4)
8.11, d (8.7)
6.97, d (8.7)
8.07, d (8.8)
6.95, d (8.8)
7.09, d (8.5)
7.45, d (8.5)
2.09, s
2.07, s
12.10, s
4.89, d (7.6)
3.22–3.41
3.22–3.41
3.22–3.41
3.22–3.41
3.70, brd
(10.0)
6.80, d (8.4)
7.32, d (8.4)
2.08, s
2.05, s
12.12, s
6.97, d (8.7)
8.11, d (8.7)
2.21, s
2.42, s
12.58, s
6.95, d (8.8)
8.07, d (8.8)
2.07, s
2.27, s
12.61, s
6′
6-CH3
8-CH3
C5-OH
4′-O-Glc-1
4′-O-Glc-2
4′-O-Glc-3
4′-O-Glc-4
4′-O-Glc-5
4′-O-Glc-6
9.3
9.3
9.2
8.2
100.3
73.2
77.0
69.7
76.3
60.7
3.22–3.41
4.59, d (7.6)
3.22–3.41
3.22–3.41
3.22–3.41
3.22–3.41
3.61, brd
(10.9)
7-O-Glc-1
7-O-Glc-2
7-O-Glc-3
7-O-Glc-4
7-O-Glc-5
7-O-Glc-6
104.2
74.1
77.0
69.8
76.6
61.0
104.1
74.0
77.0
69.8
76.3
61.0
4.57, d (7.3)
3.22, m
3.31, m
3.17, m
3.31, m
3.61, brd
(11.3)
104.0
74.0
77.0
71.5
76.3
61.1
4.66, d (7.7)
3.27, m
3.37, m
3.17, m
3.37, m
3.62, brd
(11.4)
3.22–3.41
3.43, m
3.42, m

244
Vol. 61, No. 2
were measured with a JASCO DIP-1000KUY polarimeter.
Farrerol 7-O-β-d-Glucopyranoside (3): A pale yellow amor-
1
1H-, 13C-NMR spectra were measured on a JEOL α-500 spec- phous power; [α]D21 +7.2 (c=0.57, MeOH); H-NMR (DMSO-
trometer. Chemical shifts are given in ppm with reference d6, 500MHz) and 13C-NMR (DMSO-d6, 125MHz), Table 1;
to tetramethylsilane (TMS). Mass spectra were recorded on CD (MeOH, c=0.024): Δε (nm) −12.9 (288), +3.7 (349).
JEOL JMS 700 MStation mass spectrometer. Column chroma-
Acid Hydrolysis of 1 Acid hydrolysis was performed by
tography was carried out with silica gel 60 (0.040–0.063mm, heating a solution of compound 1 (8.0mg) in 2M HCl (2mL)
Merck), MCI gel CHP20P (75–150µm, Mitsubishi Chemical in a sealed tube at 70°C for 3h. The aglycone (5, 4.0mg) was
Industries Co., Ltd.), Sephadex LH-20 (Amersham Pharma- extracted with EtOAc and the structure was confirmed as
cia Biotech) and Chromatorex ODS (30–50µm, Fuji Silysia farrerol4) by comparing its NMR data and co-TLC with an
Chemical Co., Ltd.). TLC was performed on a precoated sil- authentic sample. d-Glucose (3.3mg) was obtained from the
ica gel 60 F254 (0.2mm, aluminum sheet, Merck). CD spectra aq. layer and confirmed by comparing its optical rotation, [α]D21
were recorded on JASCO J-810 spectropolarimeter.
Plant Material Fresh aerial parts of D. canescens were
+75.0 (c=0.33, H2O), and co-TLC with an authentic sample.
Acid Hydrolysis of 2 Acid hydrolysis was performed by
collected in August, 2007 from Daman, Nepal and shade dried heating a solution of compound 2 (6.0mg) in 2M HCl–dioxane
for one month. The specimen was identified by Mr. Kuber (1:1, 1mL) in a sealed tube at 80°C for 3h. The mixture was
Jung Malla, Scientic Officer, Department of Plant Resources, evaporated and applied to silica gel column (CHCl3–MeOH–
Thapathali, Kathmandu, Nepal. The voucher specimen has H2O=7:3:0.5) to obtain aglycone (6, 2.5mg) and d-glucose
been deposited at the Kochi Prefectural Makino Botanical (1.3mg). The aglycone 6 was confirmed as 6,8-di-C-methyl
Garden, Kochi, Japan.
kaempferol8) by comparing its NMR data and d-glucose was
Extraction and Isolation The detailed extraction and confirmed by comparing its optical rotation, [α]D21 +73.0
fractionation of aerial parts of D. canescens was described in (c=0.13, H2O), and co-TLC with an authentic sample.
previous papers.3,4) Fraction 2 (16.3g) was subjected to Sepha-
dex LH-20 column (MeOH) and then ODS (30% MeOH) to
Acknowledgements We are grateful to Ms. Teruo Tanaka
afford 1 (11mg). Fraction 3 (10.9g) was separated into MeOH and Mr. Toshiyuki Iriguchi of Institute of Resource Develop-
soluble (3-1) and MeOH insoluble (3-2) parts. MeOH soluble ment and Analysis, Kumamoto University for measurement
part (8.5g) was then applied on Sephadex LH-20 column of NMR and Mass spectra, respectively. We would like to
(MeOH) to give seven subfraction. Subfraction 3-1-4 was thank the Ministry of Education, Culture, Sports, Science and
again applied on ODS column (30–50% MeOH) to give fur- Technology (MEXT) of Japan for the scholarship to one of the
ther 12 subfractions. Among them, subfraction 3-1-4-5 was authors, Mr. Hari Prasad Devkota.
applied on silica gel column eluting with CHCl3–MeOH–H2O
(8:2:0.1) to obtain 1 (39mg). Subfraction 3-1-5 was obtained
as compound 2 (20mg). Fraction 4 (10g) was subjected to
Sephadex LH-20 column (MeOH) to obtain 8 subfractions.
Among them, subfraction 4-3 (1.9g) was subjected to ODS
column (30% MeOH) to obtain 3 (569mg). Fraction 5 (2.5g)
was subjected to Sephadex LH-20 column (MeOH) and ODS
column (45% MeOH) to afford 3 (45mg).
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Diplomorphanin A (1): A pale yellow amorphous powder;
1
[α]D21 −22.2 (c=0.66, MeOH); H-NMR (DMSO-d6, 500MHz)
and 13C-NMR (DMSO-d6, 125MHz), Table 1; HR-FAB-MS
m/z: 647.1979 [M+Na]+ (Calcd for C29H36O15Na, 647.1952); CD
(MeOH, c=0.024): Δε (nm) −15.9 (283), +4.4 (347).
Diplomorphanin B (2): A pale yellow amorphous powder;
1
[α]D21 +2.6 (c=0.50, pyridine); H-NMR (DMSO-d6, 500MHz)
and 13C-NMR (DMSO-d6, 125MHz), Table 1; HR-FAB-MS
m/z: 475.1280 [M−H]− (Calcd for C23H23O11, 475.1240).