A flavone dimer from Ouratea hexasperma

Article abstract of DOI:10.1016/S0031-9422(99)00106-5

From the leaves of Ouratea hexasperma, the flavone dimer 4',5,7- trihydroxyflavone-(6 →, 8'')-4''',5''-dihydroxy-7''-methoxyflavone was isolated in addition to methyl myoinositol. The structures were established by various analyses including 2D-NMR spectroscopy.

Full text of DOI:10.1016/S0031-9422(99)00106-5

Phytochemistry 51 (1999) 833±838
A ¯avone dimer from Ouratea hexasperma
Isabel C. Moreiraa, Mario G. de Carvalhoa,*, Ana Beatriz F.O. Bastosa,
Raimundo Braz-Filhob
a
Â
Ãncias
Departamento de Quõmica, Instituto de Cie
Â
Setor de Quõmica de Produtos Naturais, LCQUI, Centro de Cie
Â
Exatas, Universidade Federal Rural do Rio de Janeiro, 23851 Seropedica, RJ, Brazil
b
Ãncias
e Tecnologia, Universidade Estadual do Norte Fluminense, 28015-620
Campos, RJ, Brazil
Received 21 April 1998; received in revised form 24 September 1998
Abstract
From the leaves of Ouratea hexasperma, the ¯avone dimer 4',5,7-trihydroxy¯avone-(6 4 80)-41,50-dihydroxy-70-
methoxy¯avone was isolated in addition to methyl myoinositol. The structures were established by various analyses including
2D-NMR spectroscopy. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Ouratea hexasperma; Ochnaceae; Flavone dimer; Methyl myoinositol; Spectral data
1. Introduction
agathis¯avone (3) (Mashima et al., 1970; Geiger,
1994). This new bi¯avone 1 was a potent inhibitor
of cellular growth and also of DNA and protein
synthesis using Sarcoma 180-tumor cells (Grynberg et
al., 1994).
In a previous report, we described the isolation from
Ouratea hexasperma (Ochnaceae) (Moreira, Sobrinho,
de Carvalho, & Braz-Filho, 1994) of the biiso¯avo-
noid, hexaspermone A, from a hexane extract of roots
and hexaspermones B and C and 4',5,7-trimethoxyiso-
¯avone from a methylene chloride extract of stem
bark, together with a mixture of aliphatic esters and a
mixture of aliphatic acids.
In this paper, we report the chemical investigation
of the leaves of this species. The new ¯avone dimer
4',5,7-trihydroxy¯avone-(6 4 80)-41,50-dihydroxy-70-
methoxy¯avone (1, 70-O-methyl agathis¯avone) and
methyl myoinositol (4) were identi®ed in the methanol
extract.
The spectral data, including 2D NMR experiments
1H±1H-COSY and 13C±1H-COSY-nJCH (n=1; n=2
and 3, COLOC) spectra were used to establish the
structures and the unambiguous 1H and 13C-NMR
assignments of 1 and its acetyl (1a) and methyl (1b)
derivatives. The bi¯avone 1 is a monomethyl ether of
agathis¯avone (2) and an isomer of 7-O-methyl-
2. Results and discussion
The carbohydrate methylmyoinositol (4) was ident-
i®ed by analysis of the chemical shifts of monoproto-
nated carbon atoms revealed in the 13C NMR
spectrum and comparison with values described in the
literature (Breitmaier & Voelter, 1987). The 1H and
13C NMR spectra of 4a (see Section 3) and compari-
son with an authentic sample con®rmed the identity of
carbohydrate 4 (Dutra, Alves, Carvalho, & Braz-Filho,
1992; Sanders & Hunter, 1993).
The molecular formula of 1 was determined as
C31H20O10 by analysis of its mass spectrum (m/z 552,
[M]Á+, 100%). Five hydroxyl groups were con®rmed
by formation of penta-acetyl derivative 1a, and one
methoxy group, four singlet signals corresponding to
hydrogens bonded to sp2 carbon atoms and four doub-
lets attributed to two AA'BB' systems in two para-sub-
1
* Corresponding author.
stituted aromatic rings) were observed in the H NMR
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00106-5

834
I.C. Moreira et al. / Phytochemistry 51 (1999) 833±838
Table 1
1H (200 MHz) and 13C (50 MHz) NMR spectral data for bi¯avone 1
(CD3COCD3) and its pentaacetyl 1a (CDCl3) and trimethyl ether 1b
(CDCl3) derivatives. Chemical shifts are in d (ppm) and coupling
constants (J, in parenthesis) in Hza
1
1a
1b
C
dC
dH
dC
dH
dC
dH
2
165.03 ±
183.54 ±
161.29 ±
104.08 ±
163.08 ±
158.18 ±
105.12 ±
123.16 ±
161.81 ±
165.20 ±
183.14 ±
163.43 ±
164.82 ±
101.00 ±
155.50 ±
105.55 ±
123.16 ±
161.81 ±
161.57 ±
176.40 ±
152.72 ±
117.38 ±
155.36 ±
157.06 ±
115.08 ±
128.30 ±
153.23 ±
161.57 ±
176.15 ±
151.38 ±
160.99 ±
106.02 ±
160.99 ±
110.74 ±
128.43 ±
152.89 ±
169.30 ±
168.78 ±
168.70 ±
167.96 ±
167.46 ±
163.96
182.85
159.41
105.50
163.54
157.78
104.41
123.27
162.63
163.96
182.40
162.33
163.36
98.08
154.47
105.17
123.57
162.33
±
4
5
6
7
9
10
1'
4'
20
40
50
70
80
90
100
11
41
AcO
AcO
AcO
AcO
AcO
±
±
±
±
±
±
±
±
±
±
±
±
±
±
13Cx1H-COSY-
1JCH
13Cx1H-COSY-
1JCH
13Cx1H-COSY-
1JCH
spectrum. The 13C NMR spectrum, on the other hand,
showed 27 carbon signals which are consistent with
the superimposition of resonances at dH 129.25 (2CH-
2',6'), 129.08 (2CH-21,61) and 116.75 (2CH-3',5' and
2CH-31,51) correlated to four carbons involved in two
AA'BB' systems revealed by the 1H NMR spectrum
(Table 1). The IR spectrum of 1 showed absorption
bands for a conjugated and H-bonded carbonyl group
(nmax 1654 cm 1), an aromatic ring (nmax 1600, 1570,
1560, 1510 cm 1) and a hydroxyl group (nmax 3370
and 3200 cm 1).
CH
3
104.04 6.65 (s)
94.50 6.80 (s)
108.50 6,60 (s)
109.90 7.48 (s)
104.42 6.63 (s)
89.96 6.65 (s)
8
2',6'
3',5'
30
129.25 7.94(d, 6.8) 127.48 7.92 (d, 8.7) 127.98 7.86 (d, 8.4)
116.75 7.05 (d, 6.8) 122.27 7.27 (d, 8.7) 114.49 6.99 (d, 8.4)
103.56 6.60 (s)
96.03 6.54 (s)
107.72 6.54 (s)
103.87 6.72 (s)
103.53 6.51 (s)
95.47 6.48 (s)
60
21,61
31,51
CH3
129.08 7.62 (d, 7.1) 127.43 7.54 (d, 8.7) 127.73 7.47 (d, 8.3)
116.75 6.83 (d, 7.1) 122.09 7.06 (d, 8.7) 114.36 6.81 (d, 8.3)
MeO-70 56.67 3.88 (s)
56.42 3.82 (s)
56.25 3.85 (s)
55.47 3.89 (s)
56.16 3.83 (s)
55.38 3.79 (s)
MeO-4'
MeO-7
MeO-41
AcO
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
The presence of two monosubstituted 4',5,7-tri-
hydroxy¯avone (C15H9O5) and 4',5-dihydroxy-7-
methoxy¯avone (C16H11O5) moieties in 1 was recog-
±
±
20.98 2.46 (s)
20.98 2.34 (s)
20.88 2.24 (s)
20.69 2.13 (s)
20.36 1.94 (s)
±
±
±
±
±
±
±
±
±
±
±
AcO
AcO
±
±
1
nized by analysis of the H (one- and two-dimensional
±
±
1H±1H-COSY) and 13C (PND and DEPT) NMR spec-
tra of 1 and 1a (pentaacetyl derivative) in combination
with the following additional data: (a) the number of
bonded hydrogens for each carbon signal deduced by
comparative analysis of the PND- and DEPT-13C
NMR spectra of 1 and its 1a and 1b derivatives; (b)
additional data obtained from 1H NMR (one- and
AcO
±
±
AcO-7
HO-5
HO-50
HO-40
HO-41
HO-70
±
±
13.06 (s)
13.04 (s)
13.34 (s)
13.18 (s)
9.15 (br s)
9.15 (br s)
9.10 (br s)
±
±
±
±
±
±
±
±
±
±
±
±
±
a Homonuclear 2D-1H±1H-COSY and heteronuclear 2D-13C±1H-
COSY-nJCH (n=1 (1JCH); n=2 (2JCH) and n=3(3JCH), COLOC
Table 2) were also used in these assignments. Chemical shifts and
coupling constants (J) of hydrogens deduced by 1D 1H NMR spec-
tra.
1
two-dimensional H±1H-COSY) spectra of 1 revealing
the presence of singlet signals attributed to two H-
bonded hydroxyl functionalities (dH 13.34 (HO-5) and
13.18 (HO-50)) and one methoxy group (dH 3.88),

I.C. Moreira et al. / Phytochemistry 51 (1999) 833±838
835
along with four isolated hydrogens attached to sp2
carbon (dH 6.65 (H-3), 6.80 (H-8), 6.60 (H-30) and
6.54 (H-60)) and four doublet signals corresponding to
eight hydrogens bonded to sp2 carbon atoms of two
AA'BB' systems (dH 7.94 (d, J=8.3 Hz, 2H-2', 6'),
7.05 (d, J=8.3 Hz, 2H-3', 5'), 7.62 (d, J=8.0 Hz, 2H-
21, 61), 6.83 (d, J=8.0 Hz, 2H-31, 51)); (c) the pre-
sence of singlet signals (dH 2.46, 2.34, 2.24, 2.13 and
1.94) corresponding to ®ve acetoxy groups in the 1H
NMR spectrum of 1a (Table 1); (d) formation of a tet-
ramethyl ether derivative (1b: dH 3.85 (s), 3.89 (s), 3.83
(s) and 3.79 (s); dC 56.25 (MeO-70), 56.16 (MeO-7),
55.47 (MeO-4') and 55.38 (MeO-40)) by treatment of 1
with CH2N2, selective methylation to obtain 5,50-dihy-
droxylated derivative 1b (dH 13.06 (HO-5) and 13.04
(HO-50)); (e) connections for signals of all hydrogen
and carbon atoms in the heteronuclear 13C±1H corre-
lation via one bond 13C±1H-COSY-1JCH and long-
range couplings 13C±1H-COSY-nJCH (n=2 (2JCH) and
3 (3JCH)) spectra of 1, 1a and 1b (Tables 1 and 2),
along with the intensities of the signals corresponding
to hydrogens 2H-2',6', 2H-3',5', 2H-21,61 and 2H-
31,51 and carbons 2CH-2',6', 2CH-3',5', 2CH-21,61
and 2CH-31,51; f) the existence of major peaks
(Scheme 1) at m/z 521 (32%, M-31 (MeO), C31H20O10
([M]Á+)±MeOÁ=C30H17O9), 283 (8%, C16H11O5, corre-
sponding to 4',5-dihydroxy-methoxy¯avone derived
fragment) and 270 (12%, C15H10O5, attributed to
4',5,7-trihydroxy¯avone derived fragment) in the
EIMS of 1, along with the molecular ion ([M]Á+) at m/
z 552 (100% (basic peak), C31H20O10 4 C16H11O5 (m/z
283)+C15H9O5 (m/z 270-HÁ)).
The connection (6 4 80) of the two ¯avone moieties
was established by 13C±1H-COSY-nJCH [n=2 (2JCH
)
and 3 (3JCH)) spectra of 1, 1a and 1b, which revealed
cross-peaks showing heteronuclear long-range coup-
lings Table 2 of C-9 (dC 157.06 (1a) and 157.78 (1b))
2
and H-8 (dH 7.48 (1a) and 6.65 (1b), JCH) and C-6
(dC 104.08 (1) and 105.50 (1b)) and both HO-5 (dH
3
13.34 (1) and 13.06 (1b), JCH) and H-8 (dH 7.48 (1a)
and 6.65 (1b), JCH) (Tables 1 and 2). This deduction
was con®rmed by additional NMR data used in the
3
Table 2
Heteronuclear long range coupling observed in the 13C±1H-COSY-nJCH (n=2 and 3) 2D NMR spectra of bi¯avone 1 (CD3COCD3) and its pen-
taacetyl 1a (CDCl3) and tetramethyl ether 1b (CDCl3) derivativesa
1
1a
1b
13Cx1H-COSY-nJCH
13Cx1H-COSY-nJCH
13Cx1H-COSY-nJCH
2JCH
3JCH
2JCH
3JCH
2JCH
3JCH
C
2
H-3
H-3
H-8
2H-2',6'
'H-8
H-3
2H-2',6'
4
5
HO-5
HO-5
6
HO-5
HO-5, H-8
MeO-7
7
H-8
H-8
9
10
H-6, HO-5
H-3, H-8
2H-3',5
HO-5, H-8
2H-3',5'
1'
4'
20
2H-3',5'
H-30
2H-2',6'
2H-21,61
2H-2',6',MeO-4'
2H-21,61
H-30
H-30
40
50
H-60,HO-50
H-60
H-60
H-60
HO-50,H-60
70
80
MeO-70
H-60
MeO-70
H-60
MeO-70
H-60
90
100
11
41
AcO
AcO
AcO
AcO
AcO
H-60,HO-50
H-30, H-60
2H-31,50
'2H-20,60
HO-50,H-60
2H-31,51
H-21,61,MeO-41
2H-30,500
2.46
2.34
2.24
2.13
1.94
CH
60
HO-50
HO-50
a Homonuclear 2D-1H±1H-COSY and 13C±1H-COSY-1JCH data were also used in these assignments.

836
I.C. Moreira et al. / Phytochemistry 51 (1999) 833±838
Scheme 1. Proposed mass spectral fragmentation mechanism for 1 (only shows major peaks).
location of the methoxy group at carbon atom C-70
(vide infra).
(dC 105.55 (1), 110.74 (1a) and 105.17 (1b)) with the
three hydrogens H-60 (dH 6.54 (1), 6.72 (1a) and 6.48
3
The location of the methoxy group at carbon C-70
and not C-7 as in 7-O-methylagathis¯avone (3)
(Mashima et al., 1970; Geiger, 1994), was deduced on
the basis of the unambiguous assignment of the chemi-
cal shifts of the directly bound (1JCH) hydrogen H-60
(dH 6.54 (1), 6.72 (1a) and 6.48 (1b)) and C-60 (dC
96.03 (1), 103.87 (1a) and 95.47 (1b)) by cross-peaks
observed in the 13C±1H-COSY-1JCH spectra of 1, 1a
and 1b in combination with the following data
obtained by 13C±1H-COSY-nJCH (n=2 (2JCH) and 3
(3JCH), COLOC) spectra of 1, 1a and 1b Table 2: (a)
spin±spin interaction of C-50 (dC 163.43 (1), 151.38
(1a) and 162.33 (1b)) and both hydrogens H-60 (dH
6.54 (1), 6.72 (1a) and 6.48 (1b)) and HO-50 (dH 13.18
(1b), JCH), H-30 (dH 6.60 (1), 6.54 (1a) and 6.51 (1b),
3
3JCH) and HO-50 (dH 13.18 (1) and 13.04 (1b), JCH);
(c) carbon C-70 (dC 164.82 (1) and 160.99 (1a)) and
2
both H-60 (dH 6.54 (1) and 6.72 (1a), JCH) and MeO-
3
70 (dH 3.88 (1), 3.82 (1a) and 3.85 (1b), JCH) (Tables 1
and 2); (d) results obtained from NOE di€erence spec-
tra of 1a and 1b performed with irradiations at MeO-
70 (dH 3.82 (1a) and 3.85 (1b)) revealing signal
enhancements at dH 6.72 (H-60, NOE=10% (1a)) and
6.48 (H-60, NOE=9% (1b)), along with other data
summarized in Table 3.
Thus, all these data were used to establish the
structure of the new ¯avone dimer as 4',5,7-trihydrox-
y¯avone-(6 4 80)-41,50-dihydroxy-70-methoxy¯avone
(1).
2
(1) and 13.04 (1b), JCH); (b) coupling of carbon C-100

I.C. Moreira et al. / Phytochemistry 51 (1999) 833±838
837
Table 3
1H{1H}-NOE di€erence spectral data (CDCl3) for pentaacetyl 1a and
tetra-methyl ether 1b derivatives
residues A (23.4 g) and B (286.1 g), respectively.
Residue A (23.4 g) was ®ltered on a CaCO3 column to
eliminate chlorophyll, eluted with hexane and the sol-
vent was evaporated under vacuum giving residue C
(6.7 g). The latter (C) was submitted to column chro-
matography (200 g, silica gel) eluted with n-hexane
and n-hexane±CHCl3 mixtures of gradually increasing
polarity) and 101 frs of 100 ml each were collected.
These fractions were analyzed by TLC and the com-
pounds of interest were subsequently combined and re-
crystallized in methanol. Fractions 1±12 yielded a mix-
ture of alkanes; frs 17±24 yielded a mixture of ali-
phatic esters and fr 37±79 produced a ppt which was
washed with n-hexane to a€ord a mixture of triter-
penoids (283.2 mg). Part of residue B (70 g) was parti-
Compounds
Irradiated
NOE enhancement
1H
dH
1H
dH
%
1a
1b
AcO-7
MeO-70
30
MeO-41
MeO-7
MeO-70
MeO-4'
1.94
3.82
6.54
3.79
3.83
3.85
3.89
8
7.48
6.72
7.54
6.81
6.65
6.48
6.99
3
10
20
11
9
60
21,61
31,51
8
60
3',5'
9
15
The homonuclear 1H±1H-COSY and heteronuclear
13C±1H-COSY-nJCH (n=1; n=2 and 3, COLOC) 2D
shift-correlated and 1H±{1H}-NOE spectra were also
used to assign the chemical shifts of all hydrogen and
carbon atoms of 1, 1a and 1b (Tables 1±3). These
assignments were facilitated by application of the
usual shift parameters (Breitmaier & Voelter, 1987;
Dutra et al., 1992; Sanders & Hunter, 1993; Gunther,
1994).
tioned with CHCl3 to produce
a CHCl3-soluble
portion named residue D. This residue (D, 28.6 g) was
chromatographed on a silica gel column and eluted
with CHCl3 (frs 1±50, 100 ml each one) and EtOAc
(frs 51 to 80, 100 ml each one); fr 53 was recrystallized
from EtOAc to a€ord 1 (364.3 mg); frs 54±70 yielded
additional quantity of 1 (466.0 mg). Frs 71±80 (120
mg) were chromatographed on silica gel (chloroform:
methanol, 9:1) and 40 frs of 50 ml each one were col-
The analysis of the mass spectra resulted in the
proposed fragmentation for the major fragments 1 as
shown in Scheme 1.
lected; frs 3±14 (43.0 mg) were analyzed by H and 13C
1
NMR spectra. Acetylation of frs 15±40 (30.0 mg) in
pyridine (2 ml) and Ac2O (2 ml) at room temperature
overnight, following work-up and ®ltration through a
silica gel column, gave pure 4a (m.p. 218±2198C, 26.0
mg).
3. Experimental
3.2. 4',5,7-Trihydroxy¯avone-(6 4 80)-41,50-dihydroxy-
70-methoxy¯avone (1)
M.p.'s are uncorr. NMR spectra in CD3COCD3 (1)
or CDCl3 (1a and 1b) soln were recorded at 200 MHz
for 1H and 50.3 MHz for 13C on a Bruker AC-200
spectrometer using TMS as int. standard or by refer-
ence to solvent signals CHCl3 at dH 7.24 and 13CDCl3
at dC 77.00; EIMS: direct inlet at 70 eV on a VG Auto
Spec-300 spectrometer; CC: silica gel (Merck and
Aldrich 0.05±0.20 mm); TLC: silica gel H or G (Merck
and Aldrich) with visualization by UV (254 and 366
nm) and exposure to iodine vapour. TLC was used to
analyze the frs collected from CC.
M.p. 227±2298C (EtOAc). [a ]D:
2.3 (c 0.5,
ketone
max
H3CCOCH3). UV l
nm (log e): 330 (3.94), 270
1
(3.96), 220 (4.04), 206 (4.11). IR umax (KBr) cm
:
3370, 3200 (OH), 1654 (C.O), 1600, 1570, 1560, 1510
(aromatic rings). 1H and 13C NMR spectra: Tables 1
and 2. EIMS 70 eV: Scheme 1.
3.3. 4',5,7-Tri-O-acetyl¯avone-(6 480)-41,50-di-O-
acetyl-70-methoxy¯avone (1a)
3.1. Plant material
M.p. 186±1888C (MeOH). [a]D: 6.2 (c 0.5, CHCl3).
CHCl3
max
Ouratea hexasperma (St. Hil) Bail (Ochnaceae) was
collected in Amapa State, Brazil and authenticated by
botanist Benedito Vitor Rabelo. A voucher specimen
(No. 01519) is deposited at the Herbario Amapaense
HAMAB of the Divisao de Botanica, Museu Angelo
Moreira da Costa Lima (IEPA), Macapa-AP, Brazil.
UV l
nm (log e): 313 (3.28), 265 (3.26), 240
(3.17). H and 13C NMR spectra: Tables 1 and 2. H
1
1
NMR NOE di€erence spectra: Table 3.
3.4. 5-Hydroxy-4',7-dimethoxy¯avone-(6 4 80)-50-
hydroxy-41,70-dimethoxy¯avone (1b)
3.1.1. Extraction and isolation of leave constituents
Dried and powdered leaves (1.5 kg) were succes-
sively percolated with n-hexane and methanol at room
temp. The solvents were removed under vacuum giving
M.p. 170±1728C (MeOH). [a]D: 1.2 (c 0.5, CHCl3).
CHCl3
max
UV l
nm (log e): 325 (4.17), 275 (14.19), 240
(3.97). H and 13C NMR spectra: Tables 1 and 2. H
1
1
NMR NOE di€erence spectra: Table 3.

838
I.C. Moreira et al. / Phytochemistry 51 (1999) 833±838
3.5. Penta-O-acetyl-methyl myoinositol (4a)
References
M.p. 218±2198C (OEtAc). 1H NMR (200 MHz,
CDCl3): d 5.70(t, 2.8 Hz), 5.40, 5.30 and 5.10 (t, 10.1
Hz, 1H each one) 4.90 (dd, 10.1 and 2.8 Hz), 3.37 (dd,
10.1 and 2.8 Hz), 3.30 (s, 3H), 2.15 (s, 3H), 2.01 (s,
3H) and 1.97 (s, 9H); 13C NMR (50.3 MHz, CDCl3):
dC: 169.9±169.9, dCH: 77.0, 70.9, 70.7, 69.3, 69.1, 66,0
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University Press.
Acknowledgements
The authors are grateful to CNPq, CAPES and
PADCT/FINEP for ®nancial support and to CNPq
for research and graduate fellowships.
Gunther, H. (1994). NMR spectroscopy: basic principles, concepts,
and applications in chemistry ( (2nd ed.).). New York: John Wiley
and Sons.