Carbon-13 and proton NMR assignments of a new agathisflavone derivative

Article abstract of DOI:10.1002/mrc.1720

The ¹H and ¹³C NMR spectra of 5-acetyl-7,4′- dimethoxyflavone-(6-8″)-5′-acetyl-7″,4?- dimethoxyflavone, a new agathisflavone derivative, were completely assigned on the basis of 1D and 2D NMR techniques. Copyright

Full text of DOI:10.1002/mrc.1720

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2006; 44: 35–37
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
1
Mario Geraldo de Carvalho,1∗ Mario Sergio do Rocha Gomes, Andre Hilario Fernandes
´
´
1
1
2
´
Pereira, Juliana Feijo de Souza Daniel and Jan Schripsema
1
´
´
Universidade Federal Rural do Rio de Janeiro, Departamento de Quımica, Br 465, Km. 07, CEP: 23980-000; Seropedica-RJ, Brazil
2
Grupo Metaboloˆ mica-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 1H and 13C NMR spectra of 5-acetyl-7,4ꢀ-dimethoxyflavone-(6–8ꢀꢀ)-5ꢀꢀ-acetyl-7ꢀꢀ,4ꢀꢀꢀ-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; 1H NMR; 13C NMR
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 pre-
pare 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 sol-
vents, 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 deriva-
tive (Fig. 1). The product 2 (35.0 mg) was dissolved in 3.0 ml
of an acetic anhydride : pyridine (1 : 1) solution and refluxed
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 Atlaˆnica4 region near Joa˜o Pessoa,
Paraiba state, Brazil. The NMR spectral investigation of the
new agathisflavone derivative permitted the confirmation of
the expected structure, the assignment of the 1H and 13C
chemical shifts and the proposal of a conformer to explain
some signal distortion in the 1H NMR spectra.
EXPERIMENTAL
°
at 60 C overnight. Usual work-up yielded 3 (gum, 30.0 mg).
General procedures
Fourier transform NMR spectra, 13C (BroadBand Decou-
pling (BBD) and Attached Proton Test (APT)), 1H–1H-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
RESULTS AND DISCUSSION
The 1H NMR of 2 (in acetone-d6) showed two low-frequency
singlet signals attributed to the two H-bonded hydroxyl
functionalities (υ 13.27 and 13.24 ppm), four signals of
methoxyl groups (υ 3.93, 3.88, 3.88 and 3.79 ppm) besides
four singlets (υ 6.57, 6.70, 6.81, 6.92 ppm) attributed to H-600,
300, 3 and 8, respectively, and four doublets (υ 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 1H NMR (1D and 2D 1H–1H-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 AA0BB systems [υ 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 [υ 6.50 (H-
300), 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 υ 3.91 (H3C–O-40), 3.84* (H3C–O-700),
1
for H and 100 MHz for 13C. Typical NMR parameters for
1
°
the H NMR were pulse angle of 45 , spectral size of 16 K,
and a spectral width of 15 ppm. Heteronuclear 1H–13C-
COSY (HMQC and HMBC) experiments were modulated
with JCH D 140 Hz and JCH (n D 2 and 3ꢀ D 9 Hz. The
Nuclear Overhauser Effect (NOE)-difference spectroscopy
experiment was recorded on a Bruker AC 200 spectrome-
1
n
1
ter operating at 200 MHz for H using the Bruker program
ŁCorrespondence to: Mario Geraldo de Carvalho, Universidade
´
Federal Rural do Rio de Janeiro, Departamento de Quımica, Br 465,
Km. 07, CEP: 23980-000; Serope´dica-RJ, Brazil.
E-mail: mgeraldo@ufrrj.br
Contract/grant sponsor: CNPq.
Contract/grant sponsor: FAPERJ.
Contract/grant sponsor: CAPES.
Copyright  2005 John Wiley & Sons, Ltd.

36
M. G. de Carvalho et al.
of AA0BB0 systems with υCH 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 υCH 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 υCH3 56.4 (H3C– O-700), 56.3 (H3C–O-7),
55.5 (H3C–O-4000), 55.4 (H3C–O-40), 20.9 (H3CCOO-5) and
21.2 ppm (H3CCOO-500), which were attributed to the cross
peaks revealed in the HMQC experiment with υH 3.84, 3.82,
3.78, 3.91, 2.15 and 2.49 ppm, respectively. The signals due
2
to JCH observed in the HMBC experiment of the methyl
group at υ 2.15 (br s) and 2.49 ppm (s) with υCO 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 1H–13C COSY (HMBC)
spectral analysis. The cross peaks of H-3 with C-2, C-4 (2JCH),
C-10, and C-10 (3JCH), H-300 with C-200, C-400 (2JCH), C-1000, and
C-1000 (3JCH), H-600 with C-500, C-700 (2JCH), C-1000, and C-800
(3JCH), and H-8 with C-9, C-7 (2JCH), C-10, and C-6 (3JCH) were
used to attribute the chemical shifts of C-800 with υ 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.
3’,5’,8
3’’’,5’’’
∗
2’,6’
2’’’,6’’’
∗
6’’
3 3’’
∗
MeO-4’/H-3’,5’
MeO-7’’/H-6’’
MeO-7/H-8
1Hx1H-COSY
6.0
7.0
8.0
MeO-4’’’/H-3’’’,5’’’
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR spectral data
of biflavone derivative 3 in CDCl3; chemical shifts in υ (ppm)a
6’’
3JH/H
3’,5’
3’’/2’’’,6’’’
∗
HMQC (1JCH
)
HMBC
(3JH–Cꢀ
NOE
3/2’,6’
υH (mult,
J in Hz)
8.0
7.0
6.0
5.0
4.0
3.0
2.0 1
13C/1H
υCH
(2JH–Cꢀ
C-2,C-4
Figure 2. Section of the NOESY spectrum of 3; at 400 MHz in
CDCl3.
3
107.4
106.4
103.7
97.0
6.61 (s)
C-10,C-10
C-1000,C-1000
C-1000,C-800
C-10,C-6
C-40
300
6.50 (s)
6.73 (s)
7.03 (s)
C-200,C-400
C-500,C-700
C-9,C-7
C-30, 50
600
8
20,60
127.8b 7.89 (d, 8,8)
2000,6000
127.7b 7.45 (br d, 8)e C-3000,5000
C-4000
30,50
114.5c 7.05 (d, 8,8)
114.4c 6.84 (d, 8,8)
55.4d 3.91 (s)
C-40
C-4000
C-10
3000,5000
C-1000
H3CO-40
H3CO-4000
H3CO-7
H3CO-700
H3CCO2-5
H3CCO2-5
H3CCO2-500
C-40
55.5d 3.78 (s)
C-4000
56.3
56.4
20.9
3.82 (br s)e
3.84 (br s)e
2.15 (br s)e
C-7
C-700
Figure 3. Conformers of 3.
H3CCO2-5
H3CCO2-500
168.7
21.2
H3CCO2-500 169.6
3.82* (H3C–O-7) and 3.78 ppm (H3C–O-4000). The 1Hf Hg-
1
2.49 (s)
NOE-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 υ 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 1H signal assignments. The
broadening of the H-2000,6000, H3CO-7 and 700 signals assigned
by (*), besides the signal of H3CCOO-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 13C NMR spectra (BBD and APT) was
used to identify the number of bonded hydrogens for each
carbon signal. The heteronuclear correlations via one bond
(1H–13C COSY, HMQC) showed cross peaks of hydrogens
Chemical shifts of quaternary carbons of the flavone skeleton
C
υC
C
υC
C
υC
2
161.9
162.2
176.5
176.9
155.7
150.9
6
7
107.1
161.7
161.0
107.1
158.8
158.8
10
1000
10
111.3b
111.0b
123.7
123.7
162.4
162.2
200
4
700
800
9
400
5
500
1000
40
900
4000
a Homonuclear 2D 1H–1H COSY spectra were also used in these
assignments.
b–d Values may be interchanged.
e Deformed/broadened signals.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 35–37

Carbon-13 and proton NMR assignments
37
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 H3C–O
with υC 161.0 (C-700), 161.7 ppm (C-7) and with υ 162.4 and
162.2 ppm were used to confirm the chemical shifts of the
carbons that support these groups defining the υC of 40 and 4000
as 162.4 and 162.2 ppm, respectively. The HMBC spectrum
did not show cross peaks with the signal of υC 155.7 ppm,
corroborating that C-5 carries the protected acetyl group.
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Acknowledgements
The authors are grateful to CNPq, FAPERJ and CAPES for a research
fellowship and for financial support.
5. Grynberg NF, de Carvalho MG, Velandia JR, de Oliveira MCC,
Moreira IC, Braz-Filho R, Echevarria A. Braz. J. Med. Biol. Res.
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Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 35–37