MAGNETIC RESONANCE IN CHEMISTRY

Magn. Reson. Chem. 2006; 44: 3537

Published online 31 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1720

Carbon-13 and proton NMR assignments of a new

agathisflavone derivative

Mario Geraldo de Carvalho,1 Mario Sergio do Rocha Gomes,1 Andre Hilario

Fernandes
1
1
2
Pereira, Juliana Feijo de Souza Daniel and Jan Schripsema
1
2

Universidade Federal Rural do Rio de Janeiro, Departamento de Qumica, Br 465, Km. 07, CEP: 23980-000; Seropedica-RJ,
Brazil

Grupo Metabolomica-LCQUI-CCT,
Universidade Estadual do Norte Fluminense, Av. Alberto Lamego, 2000, CEP: 28015-620, Campos-RJ, Brazil

Received 7 June 2005; Revised 25 August 2005; Accepted 31 August 2005

The 1 H and 13 C NMR spectra of 5-acetyl-7,4 -dimethoxyflavone-(68 )-5 -acetyl-7 ,4 -dimethoxyflavone,
a new agathisflavone derivative, were completely assigned on the basis of 1D and 2D NMR techniques.
Copyright 2005 John Wiley & Sons, Ltd.

KEYWORDS: biflavone; agathisflavone; 1 H NMR; 13 C NMR

INTRODUCTION
In a previous report, the structure determination of a
new flavone dimer isolated from the leaves of Ouratea
hexasperma (Ochnaceae) was described.1 Continuing the
phytochemical and pharmacological investigation of Ouratea
species,2 5 we have isolated a reasonable amount of
700 -methyl-agathisflavone, an abundant constituent from
O. hexasperma, collected both in the Amazon cerrado, Amapa
state,1 and in the Mata Atlanica4 region near Joao Pessoa,
Paraiba state, Brazil. The NMR spectral investigation of the
new agathisflavone derivative permitted the confirmation of
the expected structure, the assignment of the 1 H and 13 C
chemical shifts and the proposal of a conformer to explain
some signal distortion in the 1 H NMR spectra.

EXPERIMENTAL

NOEDIFF.AU. The samples were placed in 5-mm tubes

using CDCl3 or acetone-d6 as solvent and tetramethylsilane
as internal standard. For the NOEDIFF experiment of 3,
the sample was prepared by bubbling dry nitrogen through
the solution for 30 min in order to ensure the removal of
oxygen. The natural biflavone (1) isolated from the leaves of
O. hexasperma as described in the literature1,3 was used to prepare the 5,500 -diacetyl-40 ,4000 ,7,700 -tetramethyl-agathisflavone
(3) (Fig. 1). This new derivative 3 was prepared by treating a
methanol solution of the natural biflavone (1) (50.0 mg) with
ethereal diazomethane solution. After evaporation of the solvents, the residue was dissolved in acetone and purified by
column chromatography on silica gel. The fraction eluted
with acetone yielded 2 (gum, 40.0 mg), a tetramethyl derivative (Fig. 1). The product 2 (35.0 mg) was dissolved in 3.0 ml
of an acetic anhydride : pyridine (1 : 1) solution and refluxed
at 60 C overnight. Usual work-up yielded 3 (gum, 30.0 mg).

General procedures
Fourier transform NMR spectra, 13 C (BroadBand Decoupling (BBD) and Attached Proton Test (APT)), 1 H 1 H-COSY,
HMQC, HMBC and NOESY, were performed using standard
pulse sequences from the Delta NMR software (version 4.3)
of the JEOL Eclipse C 400 spectrometer operating at 400 MHz
for 1 H and 100 MHz for 13 C. Typical NMR parameters for
the 1 H NMR were pulse angle of 45 , spectral size of 16 K,
and a spectral width of 15 ppm. Heteronuclear 1 H 13 CCOSY (HMQC and HMBC) experiments were modulated
with 1 JCH D 140 Hz and n JCH (n D 2 and 3
D 9 Hz. The
Nuclear Overhauser Effect (NOE)-difference spectroscopy
experiment was recorded on a Bruker AC 200 spectrometer operating at 200 MHz for 1 H using the Bruker program
Correspondence to: Mario Geraldo de Carvalho, Universidade
Federal Rural do Rio de Janeiro, Departamento de Qumica, Br 465,
Km. 07, CEP: 23980-000; Seropedica-RJ, Brazil.
E-mail: mgeraldo@ufrrj.br
Contract/grant sponsor: CNPq.
Contract/grant sponsor: FAPERJ.
Contract/grant sponsor: CAPES.

RESULTS AND DISCUSSION

The 1 H NMR of 2 (in acetone-d6 ) showed two low-frequency
singlet signals attributed to the two H-bonded hydroxyl
functionalities ( 13.27 and 13.24 ppm), four signals of
methoxyl groups ( 3.93, 3.88, 3.88 and 3.79 ppm) besides
four singlets ( 6.57, 6.70, 6.81, 6.92 ppm) attributed to H-600 ,
300 , 3 and 8, respectively, and four doublets ( 6.98, 7.16, 7.69,
8.11 ppm) attributed to the pairs of protons 3000 ,5000 , 30 ,50 , 2000 ,6000
and 20 ,60 , respectively. These signals confirmed the structure
of the product previously described in the literature.1
The 1 H NMR (1D and 2D 1 H 1 H-COSY) spectra of 3
did not show any signals of H-bonded protons, as expected,
but showed four doublets (J D 8.8 Hz) of eight hydrogens
bonded to sp2 carbon atoms of two AA0 BB systems [ 6.84
(H-3000 ,5000 ), 7.45* (H-2000 ,6000 , broad), 7.05 (H-30 ,50 ), 7.89 ppm
(H-20 ,60 )] and four signals of isolated hydrogens [ 6.50 (H300 ), 6.61 (H-3), 6.73 (H-600 ) and 7.03 ppm (H-8)]. The methoxyl
groups were identified by four singlets including two
broadened signals (*), at 3.91 (H3 CO-40 ), 3.84* (H3 CO-700 ),

Copyright 2005 John Wiley & Sons, Ltd.

36

M. G. de Carvalho et al.

of AA0 BB0 systems with CH 127.8 (C-20 ,60 ), 127.7 (C-2000 ,6000 ),
114.5 (C-30 ,50 ), 114.4 ppm (C-3000 ,5000 ) and of H-3, 300 ,600 and
8 with CH 107.4 (C-3), 106.4 (C-300 ), 103.7 (C-600 ) and
97.0 ppm (C-8), respectively. This spectrum showed signals
of methylic carbons at CH3 56.4 (H3 C O-700 ), 56.3 (H3 CO-7),
55.5 (H3 CO-4000 ), 55.4 (H3 CO-40 ), 20.9 (H3 CCOO-5) and
21.2 ppm (H3 CCOO-500 ), which were attributed to the cross
peaks revealed in the HMQC experiment with H 3.84, 3.82,
3.78, 3.91, 2.15 and 2.49 ppm, respectively. The signals due
to 2 JCH observed in the HMBC experiment of the methyl
group at 2.15 (br s) and 2.49 ppm (s) with CO 168.7 and
169.6 ppm, respectively, confirmed the acetyl groups. The
chemical shifts of the quaternary carbons of the flavone
moieties of 3 were established by 1 H 13 C COSY (HMBC)
spectral analysis. The cross peaks of H-3 with C-2, C-4 (2 JCH ),
C-10, and C-10 (3 JCH ), H-300 with C-200 , C-400 (2 JCH ), C-1000 , and
C-1000 (3 JCH ), H-600 with C-500 , C-700 (2 JCH ), C-1000 , and C-800
(3 JCH ), and H-8 with C-9, C-7 (2 JCH ), C-10, and C-6 (3 JCH ) were
used to attribute the chemical shifts of C-800 with 107.1,
C-1000 with 111.0, C-10 with 111.3, C-6 with 107.1, C-10 ,1000
with 123.7 and C-500 with 150.9, C-9 with 158.8, C-700 with

Figure 1. Structures of agathisflavone and its derivatives.

2,6
2,6

3,5,8
3,5
6
3 3

MeO-4/H-3,5
1

6.0

MeO-7/H-6

Hx1H-COSY

MeO-7/H-8
MeO-4/H-3,5
6

7.0

JH/H

3,5
3/2,6
3/2,6 NOE

8.0
8.0

7.0

6.0

Table 1. 1 H (400 MHz) and 13 C (100 MHz) NMR spectral data

of biflavone derivative 3 in CDCl3 ; chemical shifts in (ppm)a

5.0

4.0

3.0

2.0 1

Figure 2. Section of the NOESY spectrum of 3; at 400 MHz in

CDCl3 .

Figure 3. Conformers of 3.

3.82* (H3 CO-7) and 3.78 ppm (H3 CO-4000 ). The 1 Hf1 HgNOE-difference spectra performed with irradiation at H-300
(showed an NOE at the H-2000 ,6000 signal), H-3 (showed NOE
at H-20 ,60 ) and at the methoxyl groups at 3.91 and 3.78 ppm
(showed NOE at H-30 , 50 and 3000 ,5000 , respectively) were used
to confirm those assignments. The assigned signals in the
NOESY expectrum (Fig. 2) confirmed those experiments
and were also used in the 1 H signal assignments. The
broadening of the H-2000 ,6000 , H3 CO-7 and 700 signals assigned
by (*), besides the signal of H3 CCOO-5 (br s, Fig. 2), can
be justified by magnetic anisotropy of the groups according
to the conformer (Fig. 3) because of the restricted rotation
about the bond connecting both the flavone moieties. The
analysis of the 13 C NMR spectra (BBD and APT) was
used to identify the number of bonded hydrogens for each
carbon signal. The heteronuclear correlations via one bond
(1 H 13 C COSY, HMQC) showed cross peaks of hydrogens

Copyright 2005 John Wiley & Sons, Ltd.

HMQC (1 JCH )
13

C/1 H

H (mult,
J in Hz)

CH

3
300
600
8
20 ,60
2000 ,6000
30 ,50
3000 ,5000
H3 CO-40
H3 CO-4000
H3 CO-7
H3 CO-700
H3 CCO2 -5
H3 CCO2 -5
H3 CCO2 -500
H3 CCO2 -500

107.4
106.4
103.7
97.0
127.8b
127.7b
114.5c
114.4c
55.4d
55.5d
56.3
56.4
20.9
168.7
21.2
169.6

HMBC
( 2 JH C

6.61 (s)
6.50 (s)
6.73 (s)
7.03 (s)
7.89 (d, 8,8)
7.45 (br d, 8)e
7.05 (d, 8,8)
6.84 (d, 8,8)
3.91 (s)
3.78 (s)
3.82 (br s)e
3.84 (br s)e
2.15 (br s)e

C-2,C-4
C-200 ,C-400
C-500 ,C-700
C-9,C-7
C-30 , 50
C-3000 ,5000
C-40
C-4000

2.49 (s)

H3 CCO2 -500

(3 JH C

C-10,C-10
C-1000 ,C-1000
C-1000 ,C-800
C-10,C-6
C-40
C-4000
C-10
C-1000
C-40
C-4000
C-7
C-700

H3 CCO2 -5

Chemical shifts of quaternary carbons of the flavone skeleton

C

2
200
4
400
5
500

161.9
162.2
176.5
176.9
155.7
150.9

6
7
700
800
9
900

107.1
161.7
161.0
107.1
158.8
158.8

10
1000
10
1000
40
4000

111.3b
111.0b
123.7
123.7
162.4
162.2

Homonuclear 2D 1 H 1 H COSY spectra were also used in these

assignments.
bd
Values may be interchanged.
e
Deformed/broadened signals.

Magn. Reson. Chem. 2006; 44: 3537

Carbon-13 and proton NMR assignments

161.0, C-2 with 161.9, C-200 with 162.2, C-4 with 176.5 and
C-400 with 176.9 ppm (Table 1). The cross peaks of H3 CO
with C 161.0 (C-700 ), 161.7 ppm (C-7) and with 162.4 and
162.2 ppm were used to confirm the chemical shifts of the
carbons that support these groups defining the C of 40 and 4000
as 162.4 and 162.2 ppm, respectively. The HMBC spectrum
did not show cross peaks with the signal of C 155.7 ppm,
corroborating that C-5 carries the protected acetyl group.

Acknowledgements
The authors are grateful to CNPq, FAPERJ and CAPES for a research
fellowship and for financial support.

Copyright 2005 John Wiley & Sons, Ltd.

REFERENCES
1. Moreira IC, de Carvalho MG, Bastos ABFO, Braz-Filho R.
Phytochemistry 1999; 51: 833.
2. Velandia JR, de Carvalho MG, Braz-Filho R, Werle AA.
Phytochem. Anal. 2002; 13: 283.
3. Daniel JFDS, de Carvalho MG, Cardoso RDS, Agra MDF,
Eberlin MN. J. Braz. Chem. Soc. 2005; 16: 634.
4. de Carvalho MG, Velandia JR, de Oliveira MCC, Echevarria A,
Braz-Filho R, Grynberg NF. In Phytochemical and Pharmacology
II of the Series Recent Progress in Medicinal Plants. vol. 8,
Majumdar DK, Govil JN, Singh VK (eds). SCI Tech Publishing
LLC: Houston, 2002; 77.
5. Grynberg NF, de Carvalho MG, Velandia JR, de Oliveira MCC,
Moreira IC, Braz-Filho R, Echevarria A. Braz. J. Med. Biol. Res.
2002; 15: 819.

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