Synthesis of methoxybenzoflavones and assignments of their NMR data

Article abstract of DOI:10.1002/mrc.3790

A phytotoxic root exudate from Acroptilon repens was identified as 7,8-benzoflavone, an inhibitor of cytochrome P450 1A2 and activator of cytochrome P450 3A4. The synthetic 5,6-benzoflavone also is a potent phytotoxin. Six 7,8-benzoflavones and eight 5,6-benzoflavones were synthesized in this study. The NMR data for a few of these compounds have been previously reported; however, the NMR data for most of them have not been reported. For reference purposes, the complete NMR data for the 14 benzoflavones are described.

Full text of DOI:10.1002/mrc.3790

MRC Letter
Received: 27 July 2011
Revised: 21 December 2011
Accepted: 3 January 2012
Published online in Wiley Online Library: 8 February 2012
(wileyonlinelibrary.com) DOI 10.1002/mrc.3790
Synthesis of methoxybenzoflavones and
assignments of their NMR data
Doseok Hwang,a Geunhyeong Jo,a Jiye Hyun,a Sung Dae Lee,b Dongsoo Kohc*
and Yoongho Lima*
A phytotoxic root exudate from Acroptilon repens was identified as 7,8-benzoflavone, an inhibitor of cytochrome P450 1A2 and
activator of cytochrome P450 3A4. The synthetic 5,6-benzoflavone also is a potent phytotoxin. Six 7,8-benzoflavones and eight
5,6-benzoflavones were synthesized in this study. The NMR data for a few of these compounds have been previously reported;
however, the NMR data for most of them have not been reported. For reference purposes, the complete NMR data for the 14 ben-
zoflavones are described. Copyright © 2012 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article.
1
Keywords: NMR; H NMR; 13C NMR; 2D NMR; 5,6-benzoflavones; 7,8-benzoflavones
catalytic amount of iodine for 3 h, and the reaction mixture was
Introduction
poured into ice water.[5] The resulting solid was filtered and washed
7,8-Benzoflavone (a-naphthoflavone) is an in vitro inhibitor of
cytochrome P450 1A2 (CYP1A2) as well as an in vitro activator of
CYP3A4.[1] It was recently identified as one of the most potent
inhibitors of the breast cancer resistance protein.[2] 7,8-Benzoflavone
is a known phytotoxin that can inhibit the seed germination of many
plant species.[3] Root exudates from Acroptilon repens (Russian
knapweed) were found to be phytotoxic, and the phytotoxin in the
exudate was identified to be 7,8-benzoflavone. 5,6-Benzoflavone
(b-naphthoflavone) is known as a phytotoxin too.[3] Whereas the
former originated from plants, the later was synthesized. They
belong to polyphenols, so many benzoflavones such as
methoxylated derivatives can be prepared. Because some
benzoflavones were synthesized before the NMR instruments
were developed, they were identified without NMR data. In this
study, six 7,8-benzoflvones and eight 5,6-benzoflavones were
synthesized (Fig. 1). Among them, four compounds were novel.
The 1H NMR data for four of these compounds, the 13C NMR data
for two of them, and both the 1H NMR data and 13C NMR data for
one of them have been previously reported. Therefore, we
assigned completely the NMR data of the 14 benzoflavones
and compared the literature data with the current data. The
complete NMR data of the benzoflavones can be useful for the
identification of newly isolated or synthesized derivatives.
with water and cold ethanol. The crude solid was purified by
recrystallization using ethanol to give pure 7,8-benzoflavones 1–6.
The same procedures were applied to obtain 5,6-benzoflavones
7–14 from hydroxynaphthochacones (II).
NMR spectra
The synthetic benzoflavone derivatives 1, 2, 4, 6, 7, and 8 were
dissolved in CDCl3; derivatives 3, 5, 9, and 12, in DMSO-d6; and
derivatives 10, 11, 13, and 14, in DMF-d7. The 1H chemical shifts
of the deuterated solvents referenced to TMS were 7.27, 2.50, and
2.75/2.92 ppm, and 13C chemical shifts were 77.2, 39.5, and 29.8/
34.9 ppm, respectively. Their concentrations were adjusted to
approximately 50 mM, and they were transferred to a 2.5-mm
NMR tube. All NMR data were collected on a Bruker Avance 400
spectrometer system (9.4 T; Bruker, Karlsruhe, Germany) at a
1
temperature of 298 K. In the H NMR experiment, the relaxation
delay, spectral width, and 90ꢀ pulse were 1 s, 4873 Hz, and
11.8 ms, respectively. For the 13C NMR experiments, the relaxation
delay, spectral width, and 90ꢀ pulse were 2 s, 21097 Hz, and
*
Correspondence to: Yoongho Lim, Division of Bioscience and Biotechnology,
Konkuk University, Hwayang-Dong 1, Kwangjin-Ku, Seoul, Korea 143–701.
E-mail: yoongho@konkuk.ac.kr
Experimental
Dongsoo Koh, Department of Applied Chemistry, Dongduk Women’s Univer-
sity, Hawolgok-Dong, Seongbuk-Ku, Seoul, Korea 136–714. E-mail: dskoh@
dongduk.ac.kr
Synthesis
All of the benzoflavone derivatives (1–14) were synthesized
as shown in Scheme 1. 1′-Hydroxy-2′-naphthylchalcones (I)
and 2′-hydroxy-1′-naphthylchalcones (II) were prepared to
form 1′-hydroxy-2′-aceto-naphthone and 2′-hydroxy-1′-aceto-
naphthone, respectively, with methoxy-substituted benzaldehydes
as described in a previous report.[4] Hydroxynaphthochalcones (I)
were refluxed in dimethyl sulfoxide (DMSO) in the presence of
a Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul
143-701, Korea
b Swine Science Division, National Institute of Animal Science, RDA, Cheonan
330-801, Korea
c
Department of Applied Chemistry, Dongduk Women’s University, Seoul
136-714, Korea
Magn. Reson. Chem. 2012, 50, 62–67
Copyright © 2012 John Wiley & Sons, Ltd.

Synthesis of methoxybenzoflavones and assignments of their NMR data
R4
8b
R3
7b
7a
8a
B
1'
O
2
9
R2
2'
A
C
R1
3
5
O
Derivative
Nomenclature
R1
H
R2
R3
H
R4
H
7,8-Benzoflavone
H
H
1
2
3
4
5
6
4’-Methoxy-7,8-benzoflavone
2’-Methoxy-7,8-benzoflavone
3’-Methoxy-7,8-benzoflavone
2’,3’-Dimethoxy-7,8-benzoflavone
3’,5’-Dimethoxy-7,8-benzoflavone
H
OCH3
H
H
OCH3
H
H
H
OCH3
OCH3
OCH3
H
H
OCH3
H
H
H
H
OCH3
R4
R3
B
8
1'
O
2
9
R2
2'
A
C
6
R1
3
6a
6b
5
O
5a
5b
Derivative
Nomenclature
R1
R2
H
R3
R4
4’-Methoxy-5,6-benzoflavone
2’-Methoxy-5,6-benzoflavone
H
OCH3
H
H
H
7
OCH3
H
H
8
3’-Methoxy-5,6-benzoflavone
OCH3
OCH3
H
H
H
9
2’,3’-Dimethoxy-5,6-benzoflavone
2’,4’-Dimethoxy-5,6-benzoflavone
2’,3’,4’-Trimethoxy-5,6-benzoflavone
2’,4’,5’-Trimethoxy-5,6-benzoflavone
3’,5’-Dimethoxy-5,6-benzoflavone
OCH3
OCH3
OCH3
OCH3
H
H
H
10
11
12
13
14
OCH3
OCH3
OCH3
H
H
OCH3
H
H
OCH3
OCH3
OCH3
Figure 1. Structures and nomenclature of benzoflavones 1–14.
Magn. Reson. Chem. 2012, 50, 62–67
Copyright © 2012 John Wiley & Sons, Ltd.
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D. Hwang et al.
1
1
the H peak at 6.92 ppm should be H-3, and the six H peaks
were assigned H-5, H-6, H-7a, H-7b, H-8a, and H-8b. Among
these six peaks, the peak at 8.16 ppm was long-range coupled
to the 13C peak at 178.3 ppm, C-4, so it was assigned H-5. The
five remaining proton peaks, H-6, H-7a, H-7b, H-8a, and H-8b,
were determined based on the interpretation of the COSY
and HMBC spectra. Likewise, C-7, C-8, C-9, and C-10 were de-
termined based on the long-ranged 1H–13C couplings of
HMBC. As a result, the 13C peak at 133.8 ppm should be
C-1′. The complete assignments of the 1H and 13C chemical
shifts for derivative 6, which was determined to be 3′,5′-
dimethoxy-7,8-benzoflavone, are listed in Tables 1 and 2,
respectively.
X
X
8
OH
O
O
I2
7
in DMSO
O
I
1 = H
2 = 4'-OCH3
3 = 2'-OCH3
4 = 3'-OCH3
5 = 2', 3'-di-OCH3
6 = 3', 5'-di-OCH3
X
OH
X
O
I2
6
in DMSO
The 13C NMR spectrum of derivative 13, which was
5
O
O
determined
to
be
2′,4′,5′-trimethoxy-5,6-benzoflavone,
II
7 = 4'-OCH3
contained 22 peaks (Suppl. Fig. 5). The most deshielded peak
at 179.9 ppm was C-4. Both the 1H and 13C peaks of three
methoxy groups were easily determined, but their positions
were still unknown. One of these peaks, the 1H peak at
4.07 ppm, showed NOE cross-peaks in the NOESY spectrum
8 = 2'-OCH3
9 = 3'-OCH3
10 = 2', 3'-di-OCH3
11 = 2', 4'-di-OCH3
12 = 2', 3', 4'-tri-OCH3
13 = 2', 4', 5'-tri-OCH3
14 = 3', 5'-di-OCH3
1
with two H peaks at 7.01 and 7.23 ppm, which were attached
Scheme 1. Synthetic methods used to prepare 5,6-benzoflavones and
7,8-benzoflavones.
directly to the 13C peaks at 98.6 and 113.8 ppm in HMQC,
respectively (Suppl. Figs 6 and 7). Likewise, the 1H peaks at
3.96 and 4.01 ppm, which belonged to the methoxy groups,
showed NOE peaks with the 1H peaks at 7.72 and 7.01 ppm,
respectively. Because the 1H peak at 7.01 ppm showed NOE
peaks with two methoxy groups, 4.01 and 4.07 ppm, it was
determined to be H-3′. One methoxy group, 2′-OCH3, showed
15.0 ms, respectively. Two-dimensional experiments, including
COSY, NOESY, HMQC, and HMBC, were performed by acquiring
2048 data points for t2 and 256 data points for t1. The long-range
coupling time for HMBC was 70 ms, and the relaxation time for
the NOESY experiment was 1 s. Prior to Fourier transformation,
zero-filling of 2 K and a sine-squared bell window function were
applied using XWin-NMR (Bruker).[6] Analysis of the NMR data
was carried out using Sparky.[7]
1
NOE peaks with two protons, H-3′ and H-3; thus, the H peak
at 4.07 ppm was assigned to 2′-OCH3. The 1H peak at
7.23 ppm was long-range coupled with two singlet carbons
at 110.7 and 116.6 ppm in the HMBC spectrum (Suppl. Fig. 8);
therefore, this proton was determined to be H-3. Among the
three protons mentioned above, two protons at 7.01
and 7.23 ppm corresponded to H-3′ and H-3, respectively.
Therefore, the remaining proton at 7.72 ppm must be H-6′.
Because H-3′ showed NOE cross-peaks with the two methoxy
protons at 4.07 and 4.01 ppm, the later was determined to be
4′-OCH3. The remaining methoxy proton, 5′-OCH3 was at
3.96 ppm. The 5′-OCH3 proton showed a long-range coupling
with the 13C peak at 143.7 ppm, so it was assigned C-5′. Like-
wise, the 13C peak showing long-range coupling with 4′-OCH3
at 153.5 ppm was determined to be C-4′, and from the long-
range coupling of the 2′-OCH3 proton C-2′ corresponded to
the peak at 154.4 ppm. Because H-6′ showed long-range
coupling with the 13C peak at 110.7 ppm, it was assigned
C-1′. In the HMBC spectrum, long-range coupling between
H-3 and the 13C peak at 116.6 ppm was observed; thus, this
carbon was assigned C-10. Because H-6′ showed long-range
coupling with the 13C peak at 158.7 ppm, it was determined
to be C-2. Among the undetermined carbons, the most
deshielded peak at 157.7 ppm was assigned C-9. The two
proton peaks at 7.92 and 8.37 ppm were long-range coupled
to C-10 and C-9, respectively. Therefore, they were assigned
H-8 and H-7, respectively. C-7 (135.6 ppm) was determined
based on the HMBC spectrum, which was long-range coupled
to the 1H peak at 8.12 ppm. Thus, this 1H peak was deter-
mined to be H-6a. Based on the interpretation of the COSY
and HMBC spectra, the remaining protons and carbons were
determined (Suppl. Fig. 9). In order to confirm the positions
of three methoxy groups determined based on the HMBC
data, the NOESY spectrum was interpreted, where two NOE
Results and Discussion
The structures and nomenclature of the benzoflavone derivatives
(1–14) are shown in Fig. 1. Among the 14 compounds, 6, 9, 13,
and 14 were novel. The 1H NMR data of four derivatives 2, 8,
10, and 12, the 13C NMR data of two derivatives 7 and 11, and
both the 1H and 13C NMR data of derivative 1 have been
previously reported.[1,8–25] However, NMR data for three derivatives
3, 4, and 5 have not yet been reported.[14,17,26,27]
Here, the assigning procedures used for derivative 6, which
was identified as 7,8-benzoflavone, and derivative 13, which
was identified as 5,6-benzoflavone, are described in detail
because they have not been reported yet. Eighteen peaks
were observed in the 13C NMR spectrum of derivative 6
(Suppl. Fig. 1). Of these peaks, the intensity of three peaks
at 55.6, 104.5, and 161.3 ppm were twice as high as
their neighboring signals. These peaks were assigned
3′-OCH3/5′-OCH3, C-2′/C-6′, and C-3′/C-5′, respectively. From
the interpretation of HMQC, the 1H peak at 7.12 ppm was
determined to be H-2′/H-6′ (Suppl. Fig. 2). This proton was
long-range coupled to the 13C peak at 103.1 ppm in the
HMBC spectrum (Suppl. Fig. 3); therefore, it was assigned
C-4′. Another 13C long-range coupling with H-2′/H-6′ was
observed at 162.4 ppm in HMBC, and it was determined to
be C-2. The most deshielded peak at 178.3 ppm should be
C-4. Six proton peaks, excluding the peak at 6.92 ppm,
showed 1H–1H correlations in COSY (Suppl. Fig. 4). Therefore,
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Synthesis of methoxybenzoflavones and assignments of their NMR data
Magn. Reson. Chem. 2012, 50, 62–67
Copyright © 2012 John Wiley & Sons, Ltd.
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D. Hwang et al.
Table 2. The 13C NMR chemical shifts of benzoflavones 1–14
Position
1a
2a
3b
4a
5b
6a
2
162.7
108.8
178.3
120.8
125.4
136.0
128.3
129.3
124.1
122.4
127.2
153.5
120.3
132.0
126.3
129.2
131.6
129.2
126.3
—
162.8
107.5
178.3
120.9
125.2
136.0
128.3
129.2
124.2
122.4
127.1
153.5
120.2
124.3
128.0
114.7
162.4
114.7
128.0
—
160.0
112.7
176.9
120.0
125.3
135.4
128.3
129.6
123.5
122.2
127.7
153.1
119.3
119.8
157.7
112.6
132.9
121.0
129.3
56.0
162.5
109.0
178.3
120.7
125.4
136.0
128.3
129.3
124.1
122.3
127.2
153.5
120.2
133.2
112.0
160.1
116.9
130.3
118.7
—
160.5
112.6
176.8
119.9
125.4
135.4
128.3
129.6
123.4
122.1
127.7
153.1
119.4
125.6
147.3
153.0
115.9
124.7
120.6
60.6
162.4
109.2
178.3
120.7
125.4
136.0
128.3
129.3
124.1
122.4
127.2
153.5
120.3
133.8
104.5
161.3
103.1
161.3
104.5
—
3
4
5
6
7
7a
7b
8
8a
8b
9
10
1′
2′
3′
4′
5′
6′
2′- OCH3
3′- OCH3
—
—
—
55.5
56.0
55.6
4′- OCH3
—
55.6
—
—
—
—
5′- OCH3
—
7a
—
8a
—
9b
—
10c
—
11c
55.6
12b
Position
13c
14c
2
160.9
109.1
180.3
130.6
127.2
129.1
130.5
128.1
126.5
135.2
117.6
157.2
117.1
123.6
127.7
114.5
162.2
114.5
127.7
—
158.0
115.6
180.8
130.6
127.2
129.1
130.5
128.1
126.4
135.2
117.7
157.6
117.0
120.4
158.2
111.7
132.3
120.8
129.1
55.7
159.9
110.0
179.2
129.6
126.0
129.0
130.3
128.5
126.5
135.7
118.2
156.9
116.2
132.1
111.3
159.7
117.5
130.3
118.4
—
159.7
114.8
179.7
130.6
126.7
129.3
131.2
129.0
127.0
136.1
118.6
158.0
116.81
126.1
148.2
153.9
116.1
124.9
121.0
60.7
158.8
113.6
179.9
130.6
126.8
129.1
131.1
128.9
126.7
135.7
118.6
157.8
116.6
112.6
160.2
99.3
158.7
112.9
179.1
129.7
126.0
129.0
130.3
128.6
126.5
135.6
118.1
157.1
115.9
117.5
152.2
142.3
156.1
108.4
124.2
61.1
158.7
113.8
179.9
130.6
126.8
129.1
131.1
128.9
126.7
135.6
118.7
157.7
116.6
110.7
154.4
98.6
161.0
111.0
180.0
130.7
127.2
129.5
131.4
129.2
126.9
136.3
118.9
158.0
117.2
133.8
104.8
162.0
104.0
162.0
104.8
—
3
4
5
5a
5b
6
6a
6b
7
8
9
10
1′
2′
3′
4′
163.9
106.5
130.5
56.1
153.5
143.7
112.5
56.61
—
5′
6′
2′- OCH3
3′- OCH3
4′- OCH3
5′- OCH3
—
—
55.4
56.2
—
60.5
56.0
55.5
—
—
—
55.8
56.1
56.1
—
—
—
—
—
—
—
56.62
56.0
Note: d in ppm.
aDerivatives 1, 2, 4, 6, 7, and 8 were dissolved in CDCl3.
bDerivatives 3, 5, 9, and 12 in DMSO-d6.
cDerivatives 10, 11, 13, and 14 in DMF-d7.
cross-peaks between H-3 and 2′-OCH3, and H-6′ and 5′-OCH3
were observed. The complete assignments of the 1H and
13C chemical shifts for derivative 13, which was identified as
2′,4′,5′-trimethoxy-5,6-benzoflavone, are listed in Tables 1 and 2,
benzoflavones were acquired and are listed in Tables 1 and 2,
respectively.
Because benzoflavones can have polysubstituted positions, the
NMR data reported here can help identify the structures of new
benzoflavones.
1
respectively. Similarly, the H and 13C NMR data of the other 12
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Copyright © 2012 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2012, 50, 62–67

Synthesis of methoxybenzoflavones and assignments of their NMR data
[10] M. F. Springsteel, L. J. Galietta, T. Ma, K. By, G. O. Berger, H. Yang, C.
Acknowledgements
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This work was supported by the Priority Research Centers Pro-
gram (NRF 2009–0093824), the Agenda Program (RDA 8-21-52),
the Bio & Medical Technology Development Program (NRF
2011–0027754), the next-generation Biogreen21 Program
(PJ007982), and the NRF funded by MEST (2010–0020966).
Doseok Hwang and Geunhyeong Jo contributed equally to this
work.
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