Activities of Wogonin Analogs and Other Flavones against Flavobacterium columnare

Article abstract of DOI:10.1002/cbdv.201400181

In our on-going pursuit to discover natural products and natural product-based compounds to control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these compounds, along with flavonoids from natural sources, for their antibacterial activities against two isolates of F. columnare (ALM-00-173 and BioMed) using a rapid bioassay. The flavonoids chrysin (1a), 5,7-dihydroxy-4′-methoxyflavone (11), isorhamnetin (26), luteolin (27), and biochanin A (29), and chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM-00-173 based on minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4′-dihydroxy-7-methoxyflavone), 26, and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon MIC results. The 24-h 50% inhibition concentration (IC₅₀) results revealed that 27 and 29 were the most active compounds against F. columnare ALM-00-173 (IC₅₀ of 7.5 and 8.5 mg/l, resp.), while 26 and 29 were the most toxic compound against F. columnare BioMed (IC₅₀ of 9.2 and 3.5 mg/l, resp.). These IC₅₀ results were lower than those obtained for wogonin against F. columnare ALM-00-173 and F. columnare BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification of the wogonin structure to enhance antibacterial is of interest.

Full text of DOI:10.1002/cbdv.201400181

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
259
Activities of Wogonin Analogs and Other Flavones against Flavobacterium
columnare
by Cheng-Xia Tana)b), Kevin K. Schraderb), Ikhlas A. Khanc), and Agnes M. Rimando*b)
a) Zhejiang University of Technology, College of Chemical Engineering and Materials Science,
Hangzhou 310014, P. R. China
b) U.S. Department of Agriculture, Agricultural Research Service, Natural Products Utilization
Research Unit, P.O. Box 1848, University, Mississippi 38677-1848, USA
(phone: þ1-6629151037; fax: þ1-6629151035; e-mail: agnes.rimando@ars.usda.gov)
c) Thad Cochran Research Center, The University of Mississippi, University, Mississippi 38677, USA
In our on-going pursuit to discover natural products and natural product-based compounds to
control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel
catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these
compounds, along with flavonoids from natural sources, for their antibacterial activities against two
isolates of F. columnare (ALM-00-173 and BioMed) using a rapid bioassay. The flavonoids chrysin (1a),
5,7-dihydroxy-4’-methoxyflavone (11), isorhamnetin (26), luteolin (27), and biochanin A (29), and
chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM-00-173 based on
minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4’-dihydroxy-7-methoxy-
flavone), 26, and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon
MIC results. The 24-h 50% inhibition concentration (IC50) results revealed that 27 and 29 were the most
active compounds against F. columnare ALM-00-173 (IC50 of 7.5 and 8.5 mg/l, resp.), while 26 and 29
were the most toxic compound against F. columnare BioMed (IC50 of 9.2 and 3.5 mg/l, resp.). These IC50
results were lower than those obtained for wogonin against F. columnare ALM-00-173 and F. columnare
BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in
this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification
of the wogonin structure to enhance antibacterial is of interest.
Introduction. – Columnaris disease is one of the most common worldwide bacterial
fish diseases, causing heavy economic losses to various aquaculture industries [1]. In the
Southeastern United States of America, epidemics of columnaris disease can occur in
populations of pond-raised channel catfish (Ictalurus punctatus), resulting in high
mortalities and subsequent large economic losses to producers in the channel catfish
aquaculture industry. The bacterial species that causes columnaris disease is Flavo-
bacterium columnare, a Gram-negative motile rod (2–10 mm in length). Most F.
columnare isolates from diseased channel catfish can be designated as genomovars I (F.
columnare BioMed) or II (F. columnare ALM-00-173), with genomovar II isolates
appearing to be more pathogenic for channel catfish [2].
Catfish producers currently have several available management approaches to
control columnaris disease including the application of medicated feeds (e.g.,
Aquaflorꢀ), attenuated vaccines [3], and non-antibiotic therapeutants such as 35%
Perox-Aidꢀ for external columnaris. One negative aspect of the use of medicated feeds
ꢁ 2015 Verlag Helvetica Chimica Acta AG, Zꢂrich

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in aquaculture is public concern about the potential development of antibiotic-resistant
strains of bacteria in the environment [4]. Perox-Aidꢀ is not recommended for use in
earthen ponds, unless water exchange can be implemented which is usually not
practical in commercial-size (e.g., 4 ha) catfish ponds. Although additional therapeutic
agents such as copper sulfate pentahydrate (CuSO4 ·5 H2O) and potassium permanga-
nate (KMnO4) have been mentioned as potential treatments of columnaris [5], the
efficacy of these therapeutants can be adversely impacted by certain water quality
variables (e.g., copper toxicity to fish is inversely correlated with dissolved organic
matter, pH, and total alkalinity). Therefore, these compounds must be applied
carefully due to their broad-spectrum toxicity towards non-target organisms (e.g.,
channel catfish) [6].
Natural products have been explored as possible therapeutic agents for columnaris
disease. For this purpose, a rapid 96-well microplate bioassay has been developed [7]
and utilized to evaluate numerous natural and natural product-based compounds for
antibacterial activity towards F. columnare. Some promising compounds have been
identified using this assay such as tannic acid [8], and flavone and wogonin [9].
Flavone and wogonin (1 and 2, resp.; Fig.) were found to possess strong
antibacterial activities towards two genomovars of F. columnare, ALM-00-173
(genomovar II) and BioMed (genomovar I). Both of these compounds belong to a
class of natural products called flavones. Flavones possess a wide range of biological
features such as anticancer [10–14], antiviral [15][16], anti-inflammatory [17],
antioxidant [18], and antitumor activities [19][20]. In particular, flavone compounds
in the diet may act as chemopreventive agents against the development of cancers of
the alimentary tract [21]. These interesting properties render flavones a valuable tool
for studying a number of important physiological processes.
To improve the antibacterial activity of wogonin, we synthesized wogonin analogs
3–5 and 14–21, and other flavone analogs 6–10, 12, and 13. The antibacterial activities
of these flavonoids, three synthesized chalcone derivatives 7b–9b, and 13 naturally-
occurring flavonoids, 1a, 2, and 22–32, were evaluated by a 96-well microplate bioassay.
With the exception of wogonin (2), these natural and synthetic flavonoid analogs have
not been previously tested for antibacterial activity against F. columnare. Herein,
results, as well as synthesis of the flavonoids, are presented.
Results and Discussion. – Synthesis of the wogonin analogs was inspired by results
from a previous study [9] that showed its strong antibacterial activity towards two
isolates of Flavobacterium columnare (ALM-00-173 and BioMed). Acetylation,
methylation, and benzoylation were performed envisioning an improvement of the
effects of wogonin (2), as encouraged by studies that revealed that acetylation or
methylation of the parent polyphenolic compound increased inhibitory activity against
prostate cancer cells [22], and that benzoylation resulted in an analog with significant in
vitro inhibitory activity against the malaria parasite comparable with the activity of the
control, chloroquine diphosphate [23]. Acetylated, methylated, and benzoylated
analogs, i.e., 3 and 4, 5, and, 14–21, respectively, were obtained, but these structure
modifications resulted in weaker activities.
Other flavone analogs, 6–10, 12, and 13, were also synthesized likewise triggered by
the previous finding of a strong antibacterial activity of flavone [9]. Introduction of

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
261
Figure. Chemical structures of wogonin and flavone analogs, chalcones, and other flavonoids evaluated
for their activities against Flavobacterium columnare ALM-00 and Flavobacterium columnare BioMed
MeO substituents in ring A of flavone was performed as inspired by reports that
showed P-glycoprotein inhibitory potency of methoxyflavones increased with increas-
ing number of MeO groups and reversed multi-drug resistance in in vitro assays (e.g.,
[24]). The synthetic flavones, along with synthetic chalcones, 7b–9b, natural flavones,

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1a and 22–25, and other natural flavonoids, 29–32, were evaluated for their activities
against the two F. columnare isolates.
Chrysin (1a), 5,7-dihydroxy-4’-methoxyflavone (11), isorhamnetin (26), luteolin
(27), and biochanin A (29) exhibited the strongest activities against F. columnare
isolate ALM-00-173 based upon minimum inhibition concentration (MIC) results
(2.5ꢀ0, 1.6ꢀ1.3, 3.2ꢀ0, 2.9ꢀ0, and 2.8ꢀ0 mg/l, resp.; Table 1). Chalcone 8b, which
was an intermediate in the synthesis of compound 8, also showed a strong activity
against this isolate (MIC 3.4ꢀ0 mg/l). Chalcones 7b and 9b showed only moderate
inhibition activities, suggesting that a halogen substitution is not favorable. The MIC
relative-to-drug control (RDC) values for the flavonoids 1a, 11, 26, 27, and 29 were also
strong, with values ranging from 5.5 to 10.8 with respect to the two positive drug
controls, florfenicol (F) and oxytetracycline (O). Compounds 27 and 29 had the lowest
24-h 50% inhibition concentration (IC50) values of 7.5ꢀ0.9 and 8.5ꢀ2.8 mg/l,
respectively, and these values indicated stronger activities than that of wogonin (2;
24-h IC50 28.4ꢀ0 mg/l). Based upon MIC results, none of the synthetic or natural
flavonoids evaluated in this study were as active as 2 (MIC 0.3ꢀ0 mg/l) towards F.
columnare ALM-00-173. Among the active flavonoids, no common structural feature
could be recognized as requirement for activity against this bacterial isolate. Notably,
however, ortho-dihydroxy groups in the B-ring appears to confer selective activity
towards F. columnare ALM-00-173; thereby, 27 was inhibitory to this isolate but not to
F. columnare BioMed.
Evaluation of the flavone analogs for toxic activity towards the F. columnare isolate
BioMed disclosed that 1a, 8, 11, 13 (5,4’-dihydroxy-7-methoxyflavone), 26, and 29 had
strong antibacterial activities against F. columnare BioMed based on MIC results (MIC
2.5ꢀ0, 3.4ꢀ0, 3.0ꢀ0, 2.8ꢀ0, 3.2ꢀ0, and 1.6ꢀ1.3 mg/l, resp.; Table 2). The MIC
RDCF, and RDCO values for these analogs were in the range of 1.0–10.8. Compound
29 had the lowest 24-h IC50 value of 3.5ꢀ2.8 mg/l which was also lower than that of
wogonin (2; 24-h IC50 5.4ꢀ0 mg/l), thereby indicating slightly higher activity. Based
upon MIC results, none of the flavonoids evaluated in this study were as active as 2
(MIC 0.3ꢀ0 mg/l) towards F. columnare BioMed. Of compounds 6–10, only 8 showed
strong inhibition, indicating that a MeO group at C(4’) is favorable over halogenation,
similar to that observed with chalcones 7b and 9b. Of compounds 11–13, 12 was only
moderately inhibitory, suggesting that, for these congeners, an additional MeO group is
an unfavorable substituent. No structural feature stands out as salient for activity
towards this isolate. Curiously, 8 and 13, both bearing a MeO at C(5), are inhibitory
only to this isolate. It may be that this structural feature is important for selective
activity towards F. columnare BioMed.
Four flavonoids, 1a, 11, 26, and 29, showed strong antibacterial activities towards
both isolates. Other than the OH groups in meta-position to each other (at C(5) and
C(7)) and the absence of a functional group at C(6), no other structural features could
be identified as being essential for the activities of these four compounds. A meta-OH
group is also present in 28, but this analog had little or no antibacterial activity at the
concentrations tested. Compound 28 only differs from 26 by lacking a MeO group at
C(3’). Therefore, it appears that the MeO group conferred activity. Indeed, by
examining the structure of wogonin (2), it is observable that the difference between 2
and these four active flavonoids is the presence of a MeO group at C(8) (see atom

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Table 1. Antibacterial Activities of Flavonoids towards Flavobacterium columnare ALM-00-173
a
24-h IC50
)
MICb)
24-h IC50
MIC
[mg/l]
[mg/l]
RDCFc)
RDCOd)
RDCFc)
RDCOd)
10.8 (0)
1a
2
3
4
5
6
7
8
9
>25.4
2.5 (0)
>51.8
51.8 (0)
>51.8
>51.8
>51.8
>51.8
>51.8
15.3 (0.3)
28.0 (2.6)
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
>51.8
20.7 (0)
>51.8
>51.8
13.5 (1.6)
>51.8
15.5 (5.2)
>51.8
>51.8
>51.8
>49.3
49.3 (0)
>49.3
>49.3
>49.3
>49.3
>49.3
14.6 (0.3)
26.6 (2.5)
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
>49.3
19.7 (0)
>49.3
>49.3
12.8 (1.5)
>49.3
14.8 (4.9)
>49.3
>49.3
>49.3
10.0 (0)
28.4 (0)
>36.9
>32.6
>29.8
>31.2
>33.0
10.1 (0.2)
18.8 (1.8)
>28.4
>33.2
>34.4
>34.9
>29.8
>28.4
>28.4
>42.3
>42.3
>42.3
>40.2
>40.2
>40.2
>41.6
>41.6
>57.9
>57.9
0.3 (0)
36.9 (0)
32.6 (0)
29.8
31.2 (0)
>33.0
1.0 (0)
100.0 (0)
100.0 (0)
100.0 (0)
100.0 (0)
1.1 (0)
107.5 (0)
107.5 (0)
107.5 (0)
107.5 (0)
>100.0
>107.5
34.2
100.0 (0)
100.0 (0)
100.0 (0)
55.0 (45.0)
10.0 (0)
>100.0
5.5 (4.5)
100.0 (0)
107.5 (0)
107.5 (0)
107.5 (0)
59.2 (48.4)
10.8 (0)
>107.5
5.9 (4.8)
107.5 (0)
34.7 (0)
28.4 (0)
18.3 (15.0)
3.4 (0)
>34.9
1.6 (1.3)
28.4 (0)
10
7b
8b
9b
11
12
13
14
15
16
17
18
19
20
21
22e)
23
24
25
26
27
28
29
30
31
32
>28.4
>100.0
>107.5
>42.3
>42.3
>42.3
>40.2
>40.2
>40.2
>41.6
>41.6
>57.9
>57.9
15.8 (12.9)
>67.7
3.2 (0)
2.9 (0)
33.8 (0)
2.8 (0)
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
55.0 (45.0)
>100.0
10.0 (0)
10.0 (0)
100.0 (0)
10.0 (0)
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
59.2 (48.4)
>107.5
10.8 (0)
10.8 (0)
107.5 (0)
10.8 (0)
11.4 (0)
>67.7
>31.6
7.5 (0.9)
>33.8
8.5 (2.8)
>41.6
>61.1
>29.0
>41.6
>61.1
>29.0
>100.0
>100.0
>100.0
>107.5
>107.5
>107.5
a) 24-h IC50, 50% Inhibition concentration. b) MIC, Minimum inhibition concentration. c) Relative-to-
drug control florfenicol; values closer to 1.0 indicate higher antibacterial activity compared to florfenicol.
d) Relative-to-drug control oxytetracycline; values closer to 1.0 indicate higher antibacterial activity
compared to oxytetracycline. e) Apigenin 7-O-b-d-(6-O-p-coumaroyl)glucoside. Values in parentheses
are standard errors of the mean.
numbering in Fig. 1) in 2. Interestingly, our observations are similar to those reported
by other groups; i.e., wogonin analogs without MeO group at C(8) are unable to inhibit
prostaglandin E2 production [25], and alkylation at C(5) and C(7) of wogonin (2)
results in analogs with reduced inhibitory activity [26].

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 2. Antibacterial Activities of Flavonoids towards Flavobacterium columnare BioMed
a
24-h IC50
)
MICb)
24-h IC50
MIC
[mg/l]
[mg/l]
RDCFc)
RDCOd)
RDCFc)
RDCOd)
10.8 (0)
1a
2
3
4
5
6
7
8
9
>25.4
2.5 (0)
>42.4
8.1 (0)
>42.4
>42.4
>42.4
>42.4
>42.4
12.3 (0)
33.5 (3.8)
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
>42.4
27.6 (2.2)
>42.4
>42.4
13.6 (3.4)
>42.4
5.1 (4.2)
>42.4
>42.4
>42.4
>58.8
11.2 (0)
>58.8
>58.8
>58.8
>58.8
>58.8
17.1 (0)
46.5 (5.3)
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
>58.8
38.3 (3.0)
>58.8
>58.8
18.8 (4.7)
>58.8
7.1 (5.8)
>58.8
>58.8
>58.8
10.0 (0)
5.4 (0)
>36.9
>32.6
>29.8
>31.2
>33.0
9.9 (0)
27.4 (3.1)
>28.4
>33.2
>34.4
>34.9
>29.8
>28.4
>28.4
>42.2
>42.2
>42.2
>40.2
>40.2
>40.2
>41.6
>41.6
>57.9
>57.9
0.3 (0)
36.9 (0)
32.6 (0)
29.8 (0)
31.2 (0)
33.0 (0)
3.4 (0)
19.1 (15.6)
28.4 (0)
18.3 (15.0)
18.9 (15.5)
19.2 (15.7)
3.0 (0)
1.0 (0)
100.0 (0)
100.0 (0)
100.0 (0)
100.0 (0)
100.0 (0)
10.0 (0)
55.0 (45.0)
100.0 (0)
55.0 (45.0)
55.0 (45.0)
55.0 (45.0)
10.0 (0)
1.1 (0)
107.5 (0)
107.5 (0)
107.5 (0)
107.5 (0)
107.5 (0)
10.8 (0)
59.2 (48.4)
107.5 (0)
59.2 (48.4)
59.2 (48.4)
59.2 (48.4)
10.8 (0)
10
7b
8b
9b
11
12
13
14
15
16
17
18
19
20
21
22e)
23
24
25
26
27
28
29
30
31
32
28.4 (0)
2.8 (0)
42.3 (0)
100.0 (0)
10.0 (0)
>100.0
107.5 (0)
10.8 (0)
>107.5
>42.2
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
55.0 (45.0)
>100.0
10.0 (0)
55.0 (45.0)
100.0 (0)
5.5 (4.5)
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
>107.5
59.2 (48.4)
>107.5
10.8 (0)
59.2 (48.4)
107.5 (0)
5.9 (4.8)
>42.2
>40.2
>40.2
>40.2
>41.6
>41.6
>57.9
>57.9
15.8 (12.9)
>67.7
3.2 (0)
15.8 (12.9)
33.8 (0)
18.6 (1.4)
>67.7
>31.6
9.2 (2.3)
>33.8
3.5 (2.8)
>41.6
>61.1
>29.0
1.6 (1.3)
>41.6
>61.1
>29.0
>100.0
>100.0
>100.0
>107.5
>107.5
>107.5
a) 24-h IC50, 50% Inhibition concentration. b) MIC, Minimum inhibition concentration. c) Relative-to-
drug control florfenicol; values closer to 1.0 indicate higher antibacterial activity compared to florfenicol.
d) Relative-to-drug control oxytetracycline; values closer to 1.0 indicate higher antibacterial activity
compared to oxytetracycline. e) Apigenin 7-O-b-d-(6-O-p-coumaroyl)glucoside. Values in parentheses
are standard error of the mean.
Conclusions. – We have synthesized analogs of wogonin (2) and flavone (1) and,
have evaluated their antibacterial activities towards two F. columnare isolates (ALM-
00-173 and BioMed). While no analog was found to be more active than 2 based
collectively on both 24-h IC50 and MIC results, 27 and 29 were more active than 2

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
265
against F. columnare ALM-00-173, and 29 was more active than F. columnare BioMed
based on 24-h IC50 results. Additionally, we have obtained valuable data for these
synthetic and natural flavonoids, as well as the synthetic chalcones, regarding their
activities against isolates of F. columnare for the first time, and our study showed that
the presence of OH groups at C(5) and C(7) with a MeO group at C(8) appears to be
essential and must be kept intact for the antibacterial activity to be maintained. Our
study provides guidance in the synthesis of further analogs of 1 and 2.
We thank Marcuslene Harries and Phaedra Page for technical assistance. We are also grateful for the
financial support to C.-X. T. from the Chinese Scholarship Council.
Experimental Part
General. Chemicals and solvents were purchased from commercial sources and used without
purification. Reactions were performed in a reaction flask with protection by N2 and were monitored by
TLC. Anal. and prep. TLC: silica gel GF plates (Analtech uniplate; 10ꢁ20 cm, 250 mm, and 20ꢁ20 cm,
250 mm, resp.); visualization by exposure to UV light. 1H- and 13C-NMR spectra: 400 and 100 MHz, resp.,
on an AVANCEIII Bruker spectrometer. HR-ESI-MS: Waters Micromass Q-TOF Micro and JEOL
AccuTOF (JMS-T100LC) spectrometers.
Bactericide Bioassay. Two isolates of Flavobacterium columnare (BioMed (genomovar I) and ALM-
00-173 (genomovar II)) were obtained from Dr. Covadonga Arias (Department of Fisheries and Allied
Aquacultures, Auburn University, Alabama). To assure purity, cultures of F. columnare isolates were
maintained separately on modified Shieh agar plates (pH 7.2–7.4) [27]. Prior to conducting the bioassay,
single colonies of the test cultures were used to prepare the assay culture materials by culturing each
genomovar isolate separately in 75 ml of modified Shieh broth (18 h for BioMed and 24 h for ALM-00-
173) at 29ꢀ18 at 150 rpm on a rotary shaker (model C24KC; New Brunswick Scientific, Edison, New
Jersey).
Compounds were evaluated for antibacterial activity using a rapid 96-well microplate bioassay as
described in [7]. Florfenicol and oxytetracycline HCl, antibiotics that are utilized in medicated feed, were
included as positive drug controls for each bioassay. In addition, control wells (no test compound added)
and wells containing wogonin (2) were included in each assay. Technical-grade MeOH was used to
dissolve the test compounds. Final concentrations of test compounds and drug control in the microplate
wells were 0.01, 0.1, 1.0, 10.0, and 100.0 mm. Three replications were used for each dilution of each test
compound and controls. Final results were converted to units of mg/l in order to allow comparison with
previous studies.
For determination of the 24-h 50% inhibition concentration (IC50) and minimum inhibition
concentration (MIC), sterile 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachu-
setts) with flat-bottom wells were used. Dissolved test compounds were added to microplate wells (10 ml/
well), and solvent was allowed to completely evaporate, before 0.5 MacFarland bacterial culture (see [7])
was added to the microplate wells (200 ml/well). Microplates were incubated at 298 (VWR model 2005
incubator; Sheldon Manufacturing, Inc., Cornelius, Oregon). A Packard model SpectraCount microplate
photometer (Packard Instrument Company, Meriden, Connecticut) was used to measure the absorbance
(630 nm) of the microplate wells at time 0 and 24 h.
The means and standard deviations of absorbance measurements were calculated, graphed, and
compared to controls to determine the 24-h IC50 and MIC value for each test compound (see [7]). The 24-
h IC50 and MIC results for each compound tested were divided by the respective 24-h IC50 and MIC
results obtained for the positive controls florfenicol and oxytetracycline to determine the relative-to-
drug-control florfenicol (RDCF) and relative-to-drug-control oxytetracycline (RDCO) values.
Preparation and Synthesis of 3–5. Compounds 3 and 4 were prepared by acetylation of wogonin (2;
obtained from AmplaChem Company; Carmel, IN) [28], while 5 was prepared by methylation of 2 as
described in [29].

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8-Methoxy-4-oxo-2-phenyl-4H-chromene-5,7-diyl Diacetate (3) and 7-Hydroxy-8-methoxy-4-oxo-2-
phenyl-4H-chromen-5-yl Acetate (4). The mixture of 2 (20 mg, 0.07 mmol), pyridine (3 ml), and Ac2O
(0.04 ml) in a sealed flask was stirred at r.t. for 30 h, then the reaction was quenched with cold H2O, and
the mixture was extracted with CHCl3 (3ꢁ10 ml). The combined layers were washed with brine and
dried (MgSO4). After removal of the volatiles, the crude product was purified by open column
chromatography (CC; SiO2; AcOEt/hexane 40 :60) to give 3 (3.0 mg, 13%) and 4 (16.8 mg, 65%) as
white needles.
Data of 3. 1H-NMR (CDCl3): 7.89 (d, J¼7.9, HꢂC(2’,6’)); 7.51–7.56 (m, HꢂC(3’,4’,5’)); 6.81 (s,
HꢂC(6)); 6.68 (s, HꢂC(3)); 4.04 (s, MeO); 2.43 (s, C(7)AcO); 2.39 (s, C(5)AcO). 13C-NMR (CDCl3):
176.5 (C(4)); 169.7, 167.9 (CO(5)); CO(7)); 162.2 (C(2)); 151.5 (C(8a)); 146.8 (C(7)); 144.3 (C(5)); 139.1
(C(8)); 131.9 (C(1’)); 131.1 (C4’)); 129.2 (C(3’,5’)); 126.2 (C(2’,6’)); 116.0 (C(4a)); 114.3 (C(6)); 108.5
(C(3)); 61.8 (MeO); 21.0, 20.6 (C(5)AcO, C(7)AcO). HR-ESI-MS: 369.0974 ([MþH]þ , C20H17O7þ ;
calc. 369.0959).
Data of 4. 1H-NMR (CDCl3): 12.4 (br. s, OH); 7.89 (d, J¼8.0, HꢂC(2’,6’)); 7.53–7.58 (m,
HꢂC(3’,4’,5’)); 6.70 (s, HꢂC(6)); 6.62 (s, HꢂC(3)); 4.11 (s, MeO); 2.43 (s, AcO). 13C-NMR (CDCl3):
178.5 (C(4)); 171.8 (AcO); 162.6 (C(2)); 152.5 (C(8a)); 151.3 (C(5)); 150.8 (C(7)); 136.1 (C(8)); 130.9
(C(1’)); 129.7 (C(4’)); 128.8 (C(3’,5’)); 127.2 (C(2’,6’)); 114.0 (C(4a)); 111.3 (C(6)); 105.5 (C(3)); 60.8
(MeO); 21.0 (AcO). HR-ESI-MS: 327.0854 ([MþH]þ , C18H15O6þ ; calc. 327.0868).
5-Hydroxy-7,8-dimethoxy-2-phenyl-4H-chromen-4-one (5). Compound 2 (30 mg, 0.11 mmol), MeI
(7.5 ml, 0.12 mmol), and K2CO3 (30 mg, 0.22 mmol) were dissolved in DMSO (3 ml) (Scheme 1). The
mixture was stirred overnight at r.t. (208). The next day, 30 ml of double-dist. H2O was added, and the
white precipitate was collected by filtration and dried (Na2SO4) in CHCl3. After evaporation, the residue
was subjected to CC (SiO2; AcOEt/hexane 20 :80) to give 5 (27 mg, 82%). Pale-yellow solid. 1H-NMR
(CDCl3): 12.6 (br. s, OH); 7.95 (d, J¼6.5, HꢂC(2’,6’)); 7.5–7.58 (m, HꢂC(3’,4’,5’)); 6.68 (s, HꢂC(3)); 6.44
(s, HꢂC(6)); 3.95 (s, MeO). 13C-NMR (CDCl3): 182.7 (C(4)); 162.7 (C(2)); 160.2 (C(7)); 154.9 (C(5));
141.9 (C(8a)); 135.3 (C(8)); 131.6 (C(1’)); 130.3 (C(3’,5’)); 130.2 (C(2’,6’)); 126.3 (C(4’)); 116.2 (C(4a));
116.0 (C(3)); 96.6 (C(6)); 61.9 (C(8)MeO); 56.1 (C(7)MeO). HR-ESI-MS: 299.0919 ([MþH]þ
,
C17H15Oþ5 ; calc. 299.0919).
5,6,7-Trimethoxy-2-phenyl-4H-chromen-4-one (6). 3,4,5-Trimethoxyphenol (1 g, 5.4 mmol), cinna-
moyl chloride (0.9 g, 5.4 mmol), and BF3 ·Et2O (50 ml) were dissolved in anh. toluene (10 ml)
(Scheme 2). The mixture was stirred and heated to reflux for 2 h, then the reaction was quenched with
excess of H2O, and the mixture was extracted with toluene (3ꢁ10 ml). After removal of the volatiles, the
crude product was purified by CC (SiO2; AcOEt/hexane 30 :70) to give (2E)-1-(6-hydroxy-2,3,4-
trimethoxyphenyl)-3-phenylprop-2-en-1-one (6b; 1 g, 86%). Yellow solid. 1H-NMR (CDCl3): 13.6 (br. s,
OH); 7.95 (d, J¼15.6, C¼CHꢂPh); 7.83 (d, J¼15.6, CH¼CꢂPh); 7.65 (dd, J¼2.3, 2.3, HꢂC(2’,6’)); 7.39–
7.44 (HꢂC(3’,4’,5’)); 6.31 (s, HꢂC(5)); 3.95 (s, MeOꢂC(2)); 3.91 (s, MeOꢂC(4)); 3.85 (s, MeOꢂC(3)).
13C-NMR (CDCl3): 192.9 (C(1)); 162.7, C(4)); 160.2, C(6)); 155.0 (C(2)); 143.1, (CH¼CHꢂPh);
135.5 (C(3)); 135.4 (C(1’)); 130.2 (CH¼CHꢂPh); 128.9 (C(3’,5’)); 128.4 (C(2’,6’)); 126.7 (C(4’)); 108.8
(C(1)); 96.6 (C(5)); 61.8 (MeOꢂC(2)); 61.2 (MeOꢂC(3)); 56.0 (MeOꢂC(4)). HR-ESI-MS: 315.1262
([MþH]þ , C18H19Oþ5 ; calc. 315.1232).
Scheme 1a)
a) DMSO, K2CO3; wogonin/MeI 1:1.1.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
267
Scheme 2
a) BF3 ·Et2O. b) I2, DMSO, 2 h. c) 47% HBr/AcOH.
A mixture of 6b (19.8 mg, 0.06 mmol) and I2 (4.0 mg) in DMSO (4 ml) was stirred and heated at 1008
for 2 h, and then poured into crushed ice/H2O (20 ml). The precipitate was filtered and washed with 20%
aq. Na2SO3 soln. The crude product was purified by CC (SiO2; AcOEt/hexane 30 :70) to give 6 (14 mg,
71%). Yellow solid. 1H-NMR (MeOD): 7.98 (dd, J¼2.3, 2.3, HꢂC(2’,6’)); 7.52–7.57 (m, HꢂC(3’,4’,5’));
7.11 (s, HꢂC(3)); 6.72 (s, HꢂC(8)); 4.01 (s, MeOꢂC(5)); 3.93 (s, MeOꢂC(6)); 3.87 (s, MeOꢂC(7)).
13C-NMR (MeOD): 177.8 (C(4)); 162.3 (C(2)); 158.2 (C(7)); 153.9 (C(5)); 151.4 (C(8a)); 138.3 (C(6));
131.4 (C(1’)); 129.3 (C(3’,5’)); 128.2 (C(2’,6’)); 125.9(C(4’)); 111.9 (C(4a)); 107.0 (C(3)); 96.5 (C(8)); 61.2
(MeOꢂC(5)); 60.4 (MeOꢂC(6)); 55.7 (MeOꢂC(7)). HR-ESI-MS: 313.1056 ([MþH]þ , C18H17NO5þ ;
calc. 313.1076).
General Procedure for the Synthesis of Compounds 7–9, 12, and 13. These compounds were
synthesized as described in [30]. To a soln. of substituted phenol (10 mmol) in dry toluene (10 ml) were
added Ac2O (0.5 ml) and BF3 ·Et2O (5 ml) (Scheme 3). The mixture was stirred at refluxing temp. for 2 h,
Scheme 3
a) Toluene, BF3 ·Et2O. b) Arene-carb aldehydes, KOH, DMSO. c) I2, DMSO, 2 h.

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
and then carefully poured into ice/H2O (30 ml). The precipitate was filtered, washed with brine, and
purified by flash CC (SiO2; AcOEt/hexane 60 :40) to afford 7a–9a.
1-(6-Hydroxy-2,3,4-trimethoxyphenyl)ethanone (7a). Yield: 2.0 g (89%). Yellow solid. 1H-NMR
(CDCl3): 13.4 (s, OH); 6.22 (s, HꢂC(5)); 3.98 (s, MeOꢂC(2)); 3.87 (s, MeOꢂC(3)); 3.77 (s, MeOꢂC(4));
2.64 (s, AcO).
1-(2,6-Dihydroxy-4-methoxyphenyl)ethanone (8a). Yield: 1.8 g (81%). Yellow solid. 1H-NMR
(CDCl3): 13.2 (s, OH); 6.31 (s, HꢂC(5)); 6.29 (s, HꢂC(3)); 3.73 (s, MeO); 2.60 (s, AcO).
Compounds 7b–9b, 12b and 13b were synthesized by a base-catalyzed ClaisenꢂSchmidt condensa-
tion reaction of substituted phenol and substituted aldehydes. To a DMSO soln. of 7a–9a (1.0 equiv) and
substituted aldehydes (1.0 equiv) was added 50% KOH (3.0 equiv). The mixture was stirred overnight at
r.t., and then the pH was adjusted to 3–4 with 2m HCl. The precipitate was collected by filtration and
purified by recrystallization in EtOH.
3-(4-Fluorophenyl)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-en-1-one (7b). Yield: 32 mg
(9.6 mmol, 72%). Pale-yellow solid. 1H-NMR (CDCl3): 13.6 (br. s, OH); 7.86 (d, J¼15.6,
COꢂCH¼CHꢂPh); 7.83 (d, J¼15.6, COꢂCH¼CH); 7.65 (d, J¼7.8, HꢂC(2’,6’)); 7.08–7.13 (m,
HꢂC(3’,5’)); 6.28 (s, HꢂC(5)); 3.92 (s, MeOꢂC(2)); 3.89 (s, MeOꢂC(3)); 3.82 (s, MeOꢂC(4)).
13C-NMR (CDCl3): 192.7 (CO); 165.1 (C(4)); 162.7 (C(4’)); 160.2 (C(6)); 154.9 (C(2)); 141.9
(COꢂC¼CH); 135.3 (C(3)); 131.6 (C(1’)); 130.3 (C(2’)); 130.2 (C(6’)); 126.3 (COꢂC¼CH); 116.2
(C(3’)); 116.0 (C(5’)); 108.7 (C(1)); 96.6 (C(5)); 61.9 (MeOꢂC(2)); 61.3 (MeOꢂC(3)); 56.1
(MeOꢂC(4)). HR-ESI-MS: 333.1111 ([MþH]þ , C18H18FO5þ ; calc. 333.0038).
1-(6-Hydroxy-2,3,4-trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (8b). Yield: 24 mg
(7.0 mmol, 69%). Pale-yellow solid. 1H-NMR (CDCl3): 13.8 (br. s, OH); 7.84 (dd, J¼15.6, 15.6,
CH¼CH); 7.60 (d, J¼8.7, HꢂC(2’,6’)); 6.94 (d, J¼8.7, HꢂC(3’,5’)); 6.29 (s, HꢂC(5)); 3.93 (s, MeOꢂC(2));
3.90 (s, MeOꢂC(3)); 3.86 (s, MeOꢂC(4)); 3.83 (s, MeOꢂC(4’)). 13C-NMR (CDCl3): 192.8 (CO); 162.6
(C(4)); 161.6 (C(6)); 159.9 (C(4’)); 155.0 (C(2)); 143.4 (COꢂC¼CH); 135.3 (C(3)); 130.2 (C(2’,6’)); 128.1
(C(1’)); 124.0 (COꢂC¼CH); 114.4 (C(3’,5’)); 108.8 (C(1)); 96.6 (C(5)); 61.9 (MeOꢂC(2)); 61.3
(MeOꢂC(3)); 56.1 (MeOꢂC(4)); 55.4 (MeOꢂC(4’)). HR-ESI-MS: 345.1319 ([MþH]þ , C19H21O6þ ; calc.
345.1338).
3-(4-Chlorophenyl)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-en-1-one (9b). Yield: 26 mg,
(7.5 mmol, 72%). Pale-yellow solid. 1H-NMR (CDCl3): 13.6 (br. s, OH); 7.90 (d, J¼15.6, COꢂCH¼CH);
7.74 (d, J¼15.6, COꢂCH¼CH); 7.65 (d, J¼8.0, HꢂC(2’,6’)); 7.37 (d, J¼8.0, HꢂC(3’,5’)); 6.29 (s,
HꢂC(5)); 3.92 (s, MeOꢂC(2)); 3.90 (s, MeOꢂC(3)); 3.83 (s, MeOꢂC(4)). 13C-NMR (CDCl3): 192.6
(CO); 162.7 (C(4)); 160.3 (C(6)); 154.9 (C(2)); 141.6 (COꢂC¼CH); 136.1 (C(3)); 135.3 (C(4’)); 133.9
(C(1’)); 129.5 (C(2’,6’)); 129.2 (C(3’,5’)); 127.0 (COꢂC¼CH); 108.7 (C(1)); 96.6 (C(5)); 61.9
(MeOꢂC(2)); 61.3 (MeOꢂC(3)); 56.1 (MeOꢂC(4)). HR-ESI-MS: 349.0826 ([MþH]þ , C18H17ClO5þ ;
calc. 349.0842).
1-(2-Hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (12b). Yield: 22.5 mg
(7.4 mmol, 72%). Pale-yellow solid. 1H-NMR (CDCl3): 13.5 (br. s, OH); 7.86 (dd, J¼15.6, 15.6,
CH¼CH); 7.63 (d, J¼8.6, HꢂC(2’,6’)); 6.92 (d, J¼8.6, HꢂC(3’,5’)); 6.33 (s, HꢂC(5)); 6.30 (s, HꢂC(3));
3.82 (s, MeOꢂC(4)); 3.79 (s, MeOꢂC(4’)). 13C-NMR (CDCl3): 192.4 (CO); 166.2 (C(4)); 162.4 (C(6));
161.8 (C(2)); 159.7 (C(4’)); 143.2 (COꢂC¼CH); 130.4 (C(2’,6’)); 128.2 (C(1’)); 124.5 (COꢂC¼CH); 114.6
(C(3’,5’)); 108.5 (C(1)); 96.6 (C(5)); 96.4 (C(3)); 56.3 (MeOꢂC(4)); 55.2 (MeOꢂC(4’)). HR-ESI-MS:
301.1025 ([MþH]þ , C17H17Oþ5 ; calc. 301.1076).
1-(2-Hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (13b). Yield: 21.6 mg
(7.5 mmol, 67%). Pale-yellow solid. 1H-NMR (CDCl3): 13.7 (br. s, OH); 7.85 (dd, J¼15.4, 15.4,
CH¼CH); 7.58 (d, J¼8.6, HꢂC(2’,6’)); 6.74 (d, J¼8.6, HꢂC(3’,5’)); 6.30 (s, HꢂC(5)); 6.28 (s, HꢂC(3));
3.82 (s, MeOꢂC(4)). 13C-NMR (CDCl3): 192.3 (CO); 166.1 (C(4)); 162.3 (C(6)); 161.7 (C(2)); 156.7
(C(4’)); 143.3 (COꢂC¼CH), 130.8 (C(2’,6’)); 128.5 (C(1’)); 124.4 (COꢂC¼CH); 115.3 (C(3’,5’)); 108.4
(C(1)); 96.3 (C(5)); 96.0 (C(3)); 56.2 (MeOꢂC(4)). HR-ESI-MS: 287.0943 ([MþH]þ , C16H15O5þ ; calc.
287.0919).
Compounds 7–9, 12, and 13 were prepared from 7b–9b, 12b, and 13b, resp. A mixture of 7b–9b, 12b,
or 13b (0.06 mmol) and I2 (4.0 mg) in DMSO (4 ml) was stirred and heated at 1008 for 2 h, and then
poured into crushed ice H2O (20 ml). The precipitate was filtered and washed with 20% aq. Na2SO3 soln.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
269
The crude product was purified by CC (SiO2; AcOEt/hexane 30 :70) to give corresponding compounds
7–9, 12, or 13 as a pale-yellow solid.
2-(4-Fluorophenyl)-5,6,7-trimethoxy-4H-chromen-4-one (7). Yield: 14.5 mg (4.8 mmol, 80%). Pale-
1
yellow solid. H-NMR (CDCl3): 7.87 (dd, J¼5.3, 5.3, HꢂC(2’,6’)); 7.17–7.22 (m, HꢂC(3’,5’)); 6.80 (s,
HꢂC(3)); 6.61 (s, HꢂC(8)); 3.98 (s, MeOꢂC(5), MeOꢂC(6)); 3.92 (s, MeOꢂC(7)). 13C-NMR (CDCl3):
177.1 (C(4)); 163.2 (C(2)); 162.3 (C(4’)); 159.8 (C(7)); 154.5 (C(8a)); 153.9 (C(5)); 138.1 (C(6)); 128.2
(C(2’,6’)); 127.8 (C(1’)); 116.3 (C(3’,5’); 111.9 (C(4a)); 105.0 (C(3)); 96.5 (C(8)); 62.2 (MeOꢂC(5)); 61.5
(MeOꢂC(6)); 56.3 (MeOꢂC(7)). HR-ESI-MS: 331.0945 ([MþH]þ , C18H16FO5þ ; calc. 331.0981).
5,6,7-Trimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (8). Yield: 15.7 mg (4.6 mmol, 74%).
1
Pale-yellow solid. H-NMR (CDCl3): 7.81 (d, J¼8.8, HꢂC(2’,6’)); 7.00 (d, J¼8.8, HꢂC(3’,5’)); 6.79 (s,
HꢂC(3)); 6.59 (s, HꢂC(8)); 3.98 (s, MeOꢂC(5), MeOꢂC(6)); 3.91 (s, (MeOꢂC(7)); 3.87 (s, MeOꢂC(4’)).
13C-NMR (CDCl3): 177.2 (C(4)); 162.1 (C(2)); 161.2 (C(4’)); 157.6 (C(7)); 154.5 (C(8a)); 152.5 (C(5));
140.3 (C(6)); 127.62 (C(2’,6’)); 123.8 (C(1’)); 114.4 (C(3’,5’)); 112.9 (C(4a)); 107.0 (C(3)); 96.2 (C(8));
62.2 (MeOꢂC(5)); 61.5 (MeOꢂC(6)); 56.3 (MeOꢂC(7)); 55.5 (MeOꢂC(4’)). HR-ESI-MS: 343.1155
([MþH]þ , C19H19Oþ6 ; calc. 343.1181).
2-(4-Chlorophenyl)-5,6,7-trimethoxy-4H-chromen-4-one (9). Yield: 16 mg (4.7 mmol, 74%). Pale-
yellow solid. 1H-NMR (CDCl3): 7.81 (d, J¼8.6, HꢂC(2’,6’)); 7.48 (d, J¼8.6, HꢂC(3’,5’); 6.81 (s, HꢂC(3));
6.65 (s, HꢂC(8)); 3.99 (s, MeOꢂC(5), MeOꢂC(6)); 3.92 (s, MeOꢂC(7)). 13C-NMR (CDCl3): 177.0
(C(4)); 160.0 (C(2)); 157.9 (C(4’)); 154.5 (C(7)); 152.6 (C(8a)); 140.5 (C(5)); 137.5 (C(6)); 130.0 (C(1’));
129.3 (C(2’,6’)); 127.2 (C(3’,5’)); 111.9 (C(4a)); 108.5 (C(3)); 96.2 (C(8)); 62.2 (MeOꢂC(5)); 61.5
(MeOꢂC(6)); 56.3 (MeOꢂC(7)). HR-ESI-MS: 347.0661 ([MþH]þ , C18H15ClO5þ ; calc. 347.0686).
5,7-Dihydroxy-6-methoxy-2-phenyl-4H-chromen-4-one (10). A soln. of 6 (20 mg, 0.06 mmol) in 47%
HBr (2 ml) and glacial AcOH (3 ml) was refluxed for 3 h, and then added to ice-water (30 ml). The pale-
yellow precipitate was filtered and collected. The crude product was purified by CC (SiO2; AcOEt/
hexane 30 :70) to give 10 (14 mg, 77%). Pale-yellow solid. 1H-NMR ((D6)DMSO): 12.5 (br. s,
HOꢂC(5)); 7.98 (d, J¼6.3, HꢂC(2’,6’)); 7.53–7.57 (m, HꢂC(3’,4’,5’)); 6.70 (s, HꢂC(3)); 6.41 (s, HꢂC(8));
3.85 (s, MeO). 13C-NMR ((D6)DMSO): 182.4 (C(4)); 163.3 (C(2)); 156.3 (C(7)); 153.6 (C(5)); 151.9
(C(8a)); 131.5 (C(6)); 130.6 (C(1’)); 128.3 (C(3’,5’)); 127.2 (C(2’,6’)); 127.0(C(4’)); 106.2 (C(4a)); 105.0
(C(3)); 96.1 (C(8)); 60.3 (MeO). HR-ESI-MS: 285.0765 ([MþH]þ , C16H13O5þ ; calc. 285.0763).
5,7-Dihydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (¼ 5,7-Dihydroxy-4’-methoxyflavone; 11).
This compound was generously supplied by Dr. Hankui Wu, School of Pharmacy, The University of
Mississippi, Oxford, MS. 1H-NMR ((D6)DMSO): 13.0 (br. s, HOꢂC(5)); 7.95 (d, J¼8.9, HꢂC(2’,6’)); 6.91
(d, J¼8.8, HꢂC(3’,5’)); 6.84 (s, HꢂC(3)); 6.77 (s, HꢂC(8)); 6.37 (s, HꢂC(6)); 3.86 (s, MeO). 13C-NMR
((D6)DMSO): 182.0 (C(4)); 162.3 (C(7)); 161.2 (C(2)); 160.9 (C(5)); 158.3(C(4’)); 141.9 (C(8a)); 128.2
(C(2’,6’)); 121.6 (C(1’)); 116.3 (C(3’,5’)); 106.2 (C(4a)); 106.0 (C(3)); 98.6 (C(6)); 98.6 (C(8)); 56.1
(MeO). ESI-HR-MS: 285.0767 ([MþH]þ , C16H13O5þ ; calc. 285.0763).
5-Hydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (¼ 5-Hydroxy-4’,7-dimethoxy-fla-
1
vone; 12). Yield: 14 mg (4.6 mmol, 78%). Pale-yellow solid. H-NMR ((D6)DMSO): 12.9 (br. s, OH);
8.07 (d, J¼9.0, HꢂC(2’,6’)); 7.12 (d, J¼8.6, HꢂC(3’,5’)); 6.95 (s, HꢂC(3)); 6.81 (s, HꢂC(8)); 6.39 (s,
HꢂC(6)); 3.87 (s, MeOꢂC(7)); 3.86 (s, MeOꢂC(4’)). 13C-NMR ((D6)DMSO): 182.2 (C(4)); 163.5 (C(7));
162.7 (C(2)); 160.3 (C(5)); 159.83(C(4’)); 141.9 (C(8a)); 128.2 (C(2’,6’)); 122.5 (C(1’)); 114.3 (C(3’,5’));
104.8 (C(4a)); 104.3 (C(3)); 98.5 (C(6)); 94.8 (C(8)); 56.8 (MeOꢂC(7)); 56.3 (MeOꢂC(4’)). HR-ESI-
MS: 299.0919 ([MþH]þ , C17H15O5þ ; calc. 299.0919).
5-Hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one (¼ 5,4’-Dihydroxy-7-methoxyfla-
vone, 13). Yield: 12.8 mg (4.5 mmol, 75%). Pale-yellow solid. 1H-NMR ((D6)DMSO): 12.9 (br. s,
HOꢂC(5)); 8.03 (d, J¼8.8, HꢂC(2’,6’)); 7.10 (d, J¼8.9, HꢂC(3’,5’)); 6.87 (s, HꢂC(3)); 6.50 (s, HꢂC(8));
6.19 (s, HꢂC(6)); 3.85 (s, MeO). 13C-NMR ((D6)DMSO): 182.3 (C(4)); 163.3 (C(7)); 162.2 (C(2)); 160.5
(C(5)); 159.3(C(4’)); 141.7 (C(8a)); 128.0 (C(2’,6’)); 122.6 (C(1’)); 115.5 (C(3’,5’)); 106.2 (C(4a)); 106.0
(C(3)); 98.3 (C(6)); 94.6 (C(8)); 56.8 (MeO). HR-ESI-MS: 285.0762 ([MþH]þ , C16H13O5þ ; calc.
285.07630).
General Procedure for the Synthesis of Compounds 14–21. Substituted benzoyl chloride and DMAP
(1.05 mg, 0.01 mmol) were added to a soln. of 2 (15 mg, 0.053 mmol) in dry pyridine (2 ml; Scheme 4).
The mixture was stirred at r.t. for 24 h and then carefully poured into ice H2O (30 ml). The precipitate

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
was filtered and washed with brine. Purification was performed by flash CC (SiO2; AcOEt/hexane
30 :70).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 2-Chlorobenzoate (14). Yield: 21.2 mg
(4.9 mmol, 92%). Pale-yellow solid. 1H-NMR (CDCl3): 12.4 (br. s, OH); 8.12 (d, J¼7.8, HꢂC(6’’));
7.96 (d, J¼7.4, HꢂC(2’,6’)); 7.52–7.58 (m, HꢂC(3’,5’,3’’,4’’,5’’)); 7.40–7.44 (m, HꢂC(4’)); 6.79 (s, HꢂC(3));
6.71 (s, HꢂC(6)); 4.00 (s, MeO). 13C-NMR (CDCl3): 183.0 (C(4)); 164.4 (CO); 161.9 (C(2)); 156.6
(C(5)); 150.8 (C(8a)); 149.1 (C(7); 134.8 (C(8)); 133.7 (C(4’’)); 132.7 (C(2’’)); 132.3 (C(1’’)); 132.1
(C(6’’)); 131.5 (C(1’)); 130.9 (C(3’’)); 129.3 (C(3’,5’)); 128.3 (C(4’)); 126.9 (C(5’’)); 126.4 (C(2’,6’)); 109.3
(C(4a)); 106.1 (C(6); 105.9 (C(3)); 62.0 (MeO). HR-ESI-MS: 423.06625 ([MþH]þ , C23H16ClO6;
423.0635).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 3-Chlorobenzoate (15). Yield: 19.4 mg
(4.5 mmol, 87%). Pale-yellow solid. 1H-NMR (CDCl3): 12.4 (br. s, OH), 8.22 (s, HꢂC(2’’)); 8.12 (d,
J¼7.8, HꢂC(6’’)); 7.96 (d, J¼6.6, HꢂC(2’,6’)); 7.66 (d, J¼7.1, HꢂC(4’’)); 7.51–7.56 (m,
HꢂC(3’,4’,5’,3’’,5’’)); 6.79 (s, HꢂC(3)); 6.68 (s, HꢂC(6)); 3.96 (s, MeO). 13C-NMR (CDCl3): 183.0
(C(4)); 164.4 (CO), 162.9 (C(2)); 156.6 (C(5)); 149.9 (C(8a)); 149.3 (C(7)); 135.0 (C(8)); 134.1 (C(3’’));
132.6 (C(4’’)); 132.3 (C(1’’)); 130.9 (C(1’)); 130.4 (C(2’’)); 130.3 (C(6’’)); 129.3 (C(3’,5’)); 128.5 (C(4’));
126.4 (C(2’,6’)); 109.7 (C(4a)); 106.1 (C(6)); 105.9 (C(3)); 62.0 (MeO). HR-ESI-MS: 423.0627 ([Mþ
H]þ , C23H16ClOþ6 ; calc. 423.06354).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 4-Chlorobenzoate (16). Yield: 20.3 mg
1
(4.7 mmol, 89%). Pale-yellow solid. H-NMR (CDCl3): 12.4 (br. s, OH); 8.17 (d, J¼8.6, HꢂC(2’’,6’’));
7.95 (d, J¼8.3, HꢂC(3’’,5’’)); 7.51–7.56 (m, HꢂC(2’,3’,4’,5’,6’)); 6.99 (s, HꢂC(3)); 6.88 (s, HꢂC(6)); 3.95 (s,
MeO). 13C-NMR (CDCl3): 183.0 (C(4)); 164.4 (CO); 163.3 (C(2)); 156.6 (C(5)); 149.9 (C(8a)); 149.4
(C(7)); 140.7 (C(4’’)); 132.6 (C(8)); 132.3 (C(1’’)); 131.8 (C(2’’,6’’)); 130.9 (C(1’)); 129.3 (C(3’’,5’’)); 129.2
(C(3’,5’)); 127.0 (C(4’)); 126.4 (C(2’,6’)); 109.7 (C(4a)); 106.1 (C(6)); 105.9 (C(3)); 62.0 (MeO). HR-ESI-
MS: 423.0650 ([MþH]þ , C23H16ClOþ6 ; calc. 423.06354).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 2-Methylbenzoate (17). Yield: 18.0 mg
(4.5 mmol, 85%). Pale-yellow solid. 1H-NMR (CDCl3): 12.4 (br. s, OH), 8.20 (d, J¼7.4, HꢂC(6’’));
7.95 (d, J¼6.6, HꢂC(2’,6’)); 7.55–7.59 (m, HꢂC(3’,5’,4’’,5’’)); 7.34–7.38 (m, HꢂC(4’,3’’)); 6.78 (s, HꢂC(3));
6.68 (s, HꢂC(6)); 3.98 (s, MeO), 2.70 (MeꢂC(2’’)). 13C-NMR (CDCl3): 183.0 (C(4)); 164.5 (CO); 164.3
(C(2)); 156.5 (C(5)); 149.9 (C(8a)); 149.7 (C(7)); 141.7 (C(2’’)); 133.2 (C(8)); 132.8 (C(4’’)); 132.3
(C(1’’)); 132.1 (C(3’’)); 131.4 (C(1’)); 131.0 (C(6’’)); 129.3 (C(3’,5’)); 127.7 (C(4’)); 126.4 (C(2’,6’)); 126.1
(C(5’’)); 109.5 (C(4a)); 106.2 (C(6)); 106.0 (C(3)); 61.9 (MeO); 21.9 (MeꢂC(2’’)). HR-ESI-MS: 403.1204
([MþH]þ , C24H19Oþ6 ; calc. 403.1181).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 3-Methylbenzoate (18). Yield: 17.3 mg
1
(4.3 mmol, 81%). Pale-yellow solid. H-NMR (CDCl3): 12.4 (br. s, OH), 8.02–8.06 (m, HꢂC(2’’,6’’));
7.95 (d, J¼6.6, HꢂC(2’,6’)); 7.55–7.59 (m, HꢂC(3’,5’,4’’)); 7.48–7.51 (m, HꢂC(5’’)); 7.41–7.47 (m,
HꢂC(4’)); 6.78 (s, HꢂC(3)); 6.68 (s, HꢂC(6)); 3.96 (s, MeO); 2.63 (Me). 13C-NMR (CDCl3): 183.0
(C(4)); 164.3 (CO); 164.3 (C(2)); 156.5 (C(5)); 149.9 (C(8a)); 149.7 (C(7)); 138.7 (C(3’’)); 134.9 (C(8));
132.8 (C(4’’)); 132.3 (C(2’’)); 131.0 (C(1’)); 130.9 (C(1’’)); 129.3 (C(3’,5’)); 128.6 (C(5’’)); 128.5 (C(4’));
Scheme 4a)
a) 4-(Dimethylamino)pyridine (DMAP), pyridine; wogonin/substituted benzoyl chloride 1:1.1.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
271
127.6 (C(6’’)); 126.4 (C(2’,6’)); 109.5 (C(4a)); 106.08 (C(6)); 106.05 (C(3)); 62.0 (MeO), 21.3 (Me). HR-
ESI-MS: 403.1190 ([MþH]þ , C24H19O6þ ; calc. 403.1181).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 4-Methylbenzoate (19). Yield: 18.5 mg
1
(4.6 mmol, 87%). Pale-yellow solid. H-NMR (CDCl3): 12.4 (br. s, OH); 8.14 (d, J¼7.8, HꢂC(2’,6’));
7.96 (d, J¼7.9, HꢂC(3’’,5’’)); 7.56–7.61 (m, HꢂC(3’,4’,5’)); 7.36 (d, J¼7.8, HꢂC(2’’,6’’)); 6.79 (s, HꢂC(3));
6.70 (s, HꢂC(6)); 3.96 (s, MeO), 2.48 (Me). 13C-NMR (CDCl3): 183.0 (C(4); 164.3 (CO); 164.1 (C(2));
156.5 (C(5)); 149.85 (C(8a)); 149.79 (C(7)); 145.0 (C(4’’)); 132.8 (C(8)); 132.3 (C(1’)); 131.0 (C(1’’));
130.5 (C(2’’,6’’)); 129.5 (C(3’’,5’’)); 129.3 (C(3’,5’)); 126.4 (C(2’,6’)); 125.8 (C(4’)); 109.5 (C(4a)); 106.1
(C(6)); 106.0 (C(3)); 61.9 (MeO); 21.8 (Me). HR-ESI-MS: 403.1212 ([MþH]þ , C24H19O6þ ; calc.
403.1181).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 2,3-Dimethylbenzoate (20). Yield: 16.6 mg
(4.0 mmol, 75%). Pale-yellow solid. 1H-NMR (CDCl3): 12.4 (br. s, OH); 7.96 (d, J¼7.2, HꢂC(2’,6’,6’’));
7.38–7.40 (m, HꢂC(3’,5’,4’’)); 7.41–7.43 (m, HꢂC(4’)); 7.21–7.26 (m, HꢂC(5’’)); 6.78 (s, HꢂC(3)); 6.68 (s,
HꢂC(6)); 3.99 (s, MeO); 2.58 (MeꢂC(3’’)); 2.39 (MeꢂC(2’’)). 13C-NMR (CDCl3): 183.0 (C(4)); 165.5
(CO); 164.3 (C(2)); 156.5 (C(5)); 149.9 (C(8a)); 149.7 (C(7)); 139.2 (C(2’’)); 138.4 (C(3’’)); 134.4 (C(8));
132.8 (C(4’’)); 132.3 (C(1’’)); 131.0 (C(1’)); 129.3 (C(3’,5’)); 128.9 (C(5’’)); 128.6 (C(4’)); 126.4 (C(2’,6’));
125.5 (C(6’’)); 109.5 (C(4a)); 106.2 (C(6)); 106.0 (C(3)); 61.9 (MeO); 20.6 (MeꢂC(3’’)); 16.7
(MeꢂC(2’’)). HR-ESI-MS: 417.1361 ([MþH]þ , C25H21O6þ ; calc. 417.13381).
5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H-chromen-7-yl 2,4-Dimethylbenzoate (21). Yield: 17.1 mg
(4.1 mmol, 77%). Pale-yellow solid. 1H-NMR (CDCl3): 12.4 (br. s, OH); 8.11 (d, J¼8.4, HꢂC(6’’)); 7.96
(d, J¼7.2, HꢂC(2’,6’)); 7.54–7.60 (m, HꢂC(3’,4’,5’)); 7.17 (m, HꢂC(3’’,5’’)); 6.78 (s, HꢂC(3)); 6.68 (s,
HꢂC(6)); 3.96 (s, MeO); 2.67 (MeꢂC(2’’)); 2.42 (MeꢂC(4’’)). 13C-NMR (CDCl3): 183.0 (C(4)); 164.5
(CO); 164.3 (C(2)); 156.5 (C(5)); 149.9 (C(8a)); 149.8 (C(7)); 144.0 (C(2’’)); 142.0 (C(4’’)); 132.9 (C(8));
132.8 (C(3’’)); 132.2 (C(1’)); 131.6 (C(6’’)); 131.0 (C(1’’)); 129.3 (C(3’,5’)); 126.8 (C(4’)); 126.4 (C(2’,6’));
124.7 (C(5’’)); 109.5 (C(4a)); 106.3 (C(6)); 106.0 (C(3)); 61.9 (MeO), 21.9 (MeꢂC(2’’)); 21.5
(MeꢂC(4’’)). HR-ESI-MS: 417.1334 ([MþH]þ , C25H21O6þ ; calc. 417.1338).
The naturally-occurring flavonoids apigenin 7-O-b-d-(6-O-p-coumaroyl)glucoside (22), echinaticin
(23), scutellarein (24), icariin (25), isorhamnetin (26), luteolin (27), quercetin (28), biochanin A (29),
daidzin (30), hesperedin (31), and epicatechin (32) were isolated from plants at the National Center for
Natural Products Research, University of Mississippi, and have been chemically characterized. Their
chemical structures and spectroscopic data were in agreement with those reported in the literature.
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Received May 12, 2014