Novel daidzein analogs and their in vitro anti-influenza activities

Article abstract of DOI:10.1002/cbdv.201400337

A series of novel isoflavonoids were synthesized based on structural modifications of daidzein, an active ingredient of traditional Chinese medicine (TCM) and evaluated for their anti-influenza activity, in vitro, against H1N1 Tamiflu-resistant (H1N1 TR) virus in the MDCK cell line. Among them, 4-oxo-4H-1-benzopyran-8-carbaldehydes 11a-11g were most promising, and they demonstrated better activities and selectivities comparable to those the reference ribarivin, a nucleoside antiviral agent. 3-(4-Bromophenyl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11c) displayed the best inhibitory activity (EC₅₀, 29.0 μM) and selectivity index (SI>10.3). Analysis of the structure￡activity relationships (SAR) indicated that both the non-naturally-occurring Br-substituted B-ring and appropriate CHO and OH groups on the A-ring might be critical for the activity and selectivity against H1N1 TR influenza viruses.

Full text of DOI:10.1002/cbdv.201400337

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Novel Daidzein Analogs and Their in Vitro Anti-Influenza Activities
by Shu-Ting Chunga), Yi-Ting Huangb), Hsin-Yi Hsiungb), Wen-Hsin Huanga)b), Chen-Wen Yao c ), and
An-Rong Lee*a)b)
a) Institute of Medical Sciences, National Defense Medical Center, 161 Minchuan East Rd. Sec. 6,
Taipei, 11490, Taiwan
b) School of Pharmacy, National Defense Medical Center, 161 Minchuan East Rd. Sec. 6, Taipei, 11490,
Taiwan
c) Deparment of Pathology and Parasitology, Tri-Service General Hospital, National Defense Medical
Center, 161 Minchuan East Rd. Sec. 6, Taipei, 11490, Taiwan
(phone: þ886-2-87923100 ext. 18873; fax: þ886-2-87923169; e-mail: lar@mail.ndmctsgh.edu.tw)
A series of novel isoflavonoids were synthesized based on structural modifications of daidzein, an
active ingredient of traditional Chinese medicine (TCM) and evaluated for their anti-influenza activity,
in vitro, against H1N1 Tamiflu-resistant (H1N1 TR) virus in the MDCK cell line. Among them, 4-oxo-
4H-1-benzopyran-8-carbaldehydes 11a–11g were most promising, and they demonstrated better
activities and selectivities comparable to those the reference ribarivin, a nucleoside antiviral agent. 3-
(4-Bromophenyl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11c) displayed the best inhib-
itory activity (EC50, 29.0 mm) and selectivity index (SI>10.3). Analysis of the structureÀactivity
relationships (SAR) indicated that both the non-naturally-occurring Br-substituted B-ring and
appropriate CHO and OH groups on the A-ring might be critical for the activity and selectivity against
H1N1 TR influenza viruses.
Introduction. – Prompted by the success of oseltamivir (ꢀTamifluꢁ) and zanamivir
(ꢀRelenzaꢁ), two major neuraminidase (NA) inhibitors, drug designs for novel anti-
H1N1 viral agents in recent decades have been focused on the transition-state analogs
of substrate molecules. Since the structures of these compounds resemble the transition
state of the sialoside substrate molecule in NA-catalyzed hydrolysis, they would bind
more tightly to the enzyme than the actual substrate, and thus may be potent
competitive inhibitors of neuraminidase [1][2]. Unfortunately, such potent NA
inhibitors are always associated with inevitable adverse effects [3]. Recent studies
also indicate that, owing to the widespread and indiscriminate use of oseltamivir, strains
of pathogenic oseltamivir-resistant pandemic H1N1 viral isolates have gradually
emerged, mainly resulting from the mutation of a single amino acid (His274Tyr in N1)
in NA [4], which may lead to increasing fatality in human beings [5][6].
Recently, there have been several interesting reports concerning the anti-influenza
activity of flavonoids, which possess three aromatic rings designated as ring A, B, and C
based on the sequence of their biogenesis (Fig.) [7–12]. According to their
structureÀactivity relationships (SAR), substituents capable of forming a H-bonding
at C(8) on the A ring reportedly benefits NA inhibitory activity (e.g., 1, IC50, 40.91 mm)
[13]. While flavonoids have a 2-phenyl-4H-1-benzopyran-4-one backbone, isoflavo-
noids possess a 3-phenyl-4H-1-benzopyran-4-one backbone, resulting from the
ꢂ 2015 Verlag Helvetica Chimica Acta AG, Zürich

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Figure. The structures of flavonoids, isoflavonoids, 1, daidzein (2), formononentin (3), and 4–6
flavonoid biogenesis pathway. One of the important traditional Chinese medicine
(TCM) components is daidzein (2), an isoflavone isolated from Kudzu (Pueraria lobata
(Wild.) Ohwi) [14] and abundant in soybeans (Glycine max (L.) Merrill), which
are a major dietary supplement in many Asian countries. This isoflavone has been
reported to display significant antiviral and antidiabetic features [15–18]. A recent
study indicated that two structurally similar isoflavones, daidzein (2; 4’-OH in ring B)
and formononetin (3; 4’-MeO in ring B), isolated from beans had anti-influenza virus
activity through inhibiting NA [19]. Although the anti-influenza virus activity of 2
(IC50 >787 mm) is reportedly more potent than that of 3 (IC50 not determined), neither
of their potencies is strong enough to be considered as a candidate for further
development, and therefore they need optimization.
We have already reported that flavonoids with a B ring bearing properly positioned
Cl-atoms, e.g., compound 4, exhibited significantly improved antiviral activity and
selectivity [20]. We later showed that, after further modification of baicalein, a flavone
ingredient found in the TCM Scutellaria baicalensis Georgi, some synthetic products
showed extremely potent in vitro activities against H1N1 Tamiflu-resistant (TR) and
seasonal H3N2-infected influenza viruses with preserved favorable selectivity [21]. In
this study, non-naturally-occurring Br-substituted B-rings and the OH group on the A-
rings (e.g., 5 and 6) might both have critical effects on the activity and selectivity against
H1N1-Tamiflu-resistant influenza viruses. To the best of our knowledge, however, no
studies have been systematically conducted on the SAR on the isoflavonoid framework
of daidzein (2) analogs. Therefore, in this study we applied the above mentioned
approaches to structural modification on daidzein (2), especially on the A and B rings,
to ameliorate the capability of the compound to inhibit the influenza virus. Again, some
of our synthetic products displayed more potent in vitro activities and higher
selectivities against the H1N1 TR virus than the reference ribavirin. The essential
functional groups and moieties are discussed herein.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Results and Discussion. – Design. Reports on the weakly active daidzein (2), the
active ingredient of the popular TCM Puerariae Radix, prompted this study of
structural optimization on isoflavonoids. By employing similar strategies as in our
previous reports, we envisaged that it would be possible to improve the antiviral activity
and selectivity of the isoflavonoids via subjecting their A and B ring to bear
appropriately positioned electron-donating or -withdrawing groups [20][21]. Herein,
we designed the synthetic routes for preparation of three isoflavone derivatives, 2-[4-
oxo-4H-1-benzopyran-7-yl)oxy]acetates 9, [4-oxo-4H-1-benzopyran-7-yl)oxy]acetoni-
triles 10, and 4-oxo-4H-1-benzopyran-8-carbaldehydes 11. The aim was to use this new
compound library to establish the SAR in anti-influenza virus activity.
Synthesis. As outlined in Scheme 1, the isoflavonoid daidzein (2) analogs were
synthesized by a modified HoubenÀHoesch approach [22][23]. The yields of the
intermediates 7a–7g obtained by reaction of resorcinol (¼ benzene-1,3-diol) and
various substituted phenylacetonitriles were consistently within the range of 30–50%,
i.e., apparently much lower than the yields of (i.e., >80%) obtained when using
phloroglucinol (¼ benzene-1,3,5-triol) and ClCH2CN as starting materials [22]. The
low yields are likely attributable to the electron-donating effects of the starting
phenolic compounds on reactivity, i.e., the higher the electron density on the benzene
ring of phenols, the higher the reactivity in this electrophilic acylation. In contrast to
phloroglucinol, resorcinol contains two OH groups on its benzene ring, which may
explain its lower reactivity compared to phloroglucinol which possesses three OH
groups. However, other subtle effects on yields such as the moisture of HCl and its flow
rate could not be excluded, since we tended to observe variations of the yields in the
repetition of the same reactions. During the synthesis of intermediate 7g, an
unexpected side product with a lower polarity was repeatedly detected. This side
product, examined under scrutiny, turned out to be ethyl 2-(4-nitrophenyl)acetate, the
structure of which was established by 1H-NMR spectroscopy ((D6)DMSO): 1.18 (t, J¼
7.2, 3 H), 3.86 (s, 2 H), 4.09 (q, J¼7.2, 2 H), 7.55 (d, J¼8.7, 2 H), 8.18 (d, J¼8.7, 2 H))
and mass spectrometry (m/z 209 (Mþ )). We speculate that, before the HoubenÀHoesch
reaction, some starting material 2-(4-nitrophenyl)acetonitrile, highly activated by the
NO2 group, might be first attacked by Et2O, catalyzed by the acids, on the C-atom of the
Scheme 1
i) ZnCl2, HCl, Et2O, 12 h. ii) BF3 ·Et2O, DMF, 508. iii) MeSO2Cl, DMF, 1008, 1 h.

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
CN group, and undergoes subsequent hydrolysis to form the final ester product. The
key intermediates 7a–7g were converted smoothly to isoflavones 8a–8g in tolerably
good yields. Esterification on HOÀC(7) of 8a–8g with ClCH2COOEt afforded 9a–9g
with fairly high yields (65–95%; Scheme 2). Although simple filtration and washing
generally provided sufficient purity, their preparations were time-consuming (ca. 12 h)
and required dry acetone as the reaction solvent. Reaction of 8a–8g with BrCH2CN
under basic conditions produced 10a–10g with yields ranging from 68 to 98%. While
the syntheses of 10a–10g proceeded smoothly with satisfactory purities and yields,
preparation of 11a–11g by formylation at C(8) of 8a–8g was consistently difficult.
Attempts to synthesize 11a–11g by reacting 8a–8g with various reagents, such as
VilsmeierÀHaack reagent (N,N-dimethylaniline/POCl3), paraformaldehyde/MgCl2, or
methenamine/CF3COOH, consistently failed, even under reflux conditions. The
desired products could only be obtained by Duff reaction [24] with methenamine (¼
hexamethylenetetramine¼1,3,5,7-tetrazatricyclo[3.31.13,7]decane; 1.1 equiv.) in glacial
AcOH at 1008. The reaction required 6 h to furnish 11a–11g (Scheme 2). Compounds
9a–9g could be further hydrolyzed [25] smoothly under acidic conditions. The best
results were obtained by hydrolysis with a solution of H2SO4 in glacial AcOH (1:3) for
12 h followed by neutralization by 1n NaOH to give the end products 12a–12g with the
desired carboxylic acid functional groups (Scheme 3). The yields of the 12a–12g were
significantly influenced by pH values; the optimal pH value turned out to be 2 or below.
Residual 9a–9g that were carried over to the end products was effortlessly removed by
simply partitioning the mixture with AcOEt and H2O. After acidifying the H2O layer to
pH 2 or below with 1n HCl, the precipitated products were collected. Higher pH values
usually led to co-precipitation with unidentifiable impurities.
In most cases, crystallization was performed for purification. The structures of
synthetic intermediates and end products were elucidated by various spectroscopic
studies and by high-resolution mass spectra.
Scheme 2
i) K2CO3, acetone, r.t., 1 h. ii) ClCH2COOEt, acetone, 758, 12 h. iii) BrCH2CN, K2CO3, acetone, reflux,
5–6 h. iv) Methenamine, glacial AcOH, 1008, 6 h.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
689
Scheme 3
i) H2SO4, glacial AcOH, 12 h. ii) 1n NaOH, 2 h.
In Vitro Anti-Influenza Virus Assay. Activities of the daidzein (isoflavone) analogs
in this study were expressed as 50% effective concentration (EC50) against H1N1
Tamiflu-resistant virus in the MDCK cell line. The CC50 means the 50% cell cytotoxic
concentration after treatment with the testing compounds, wherein selectivity index
(SI) was expressed as CC50/EC50 and employed in the SAR analysis (Table). In terms of
potency of anti-H1N1 Tamiflu-resistant virus activity, the synthetic isoflavonoids were
ranked as 11>9>12>10. All our synthetic products showed moderate-to-significant
antiviral activities except series 10, which were inactive with both EC50 and CC50 higher
than 300 mm. The hydrolyzed series 12 generally exhibited much lower cytotoxicities
compared to their parents 9, but revealed neither superior inhibitory effects nor more
selective indexes. Compounds 9a, 9e, and 9g displayed inhibitory activities in the range
EC50 75.6–135.7 mm; the most potent of these was 9f with an inhibitory activity of
36.5 mm, but the cellular survival rate decreased to a concentration of 292.1 mm.
Conjugation of bulkier groups, e.g., EtOCOCH2O-, NCCH2O, and HOCOCH2O, with
a OH group at C(7) on the A-ring of isoflavonoids was scarcely advantageous to the
anti-H1N1 TR virus activity.
Series 11 exhibited potent anti-H1N1 TR virus activity in MDCK cells (SI, 8.5–
10.3) and were generally more active than the reference ribavirin. Both 7-OH and 8-
CHO groups were essential for H1N1 TR virus inhibitory activity. Interestingly, the
structure of the most active compound 11c (SI>10.3) bears a Br-atom on the B ring
and a free OH group on the A ring, a reminiscence of the A- and B-ring substitutions in
extremely potent anti-H1N1 TR flavonoids [21]. This indicates that this lipophilic
substituent, 4’-Br on the B ring, might contribute tremendously to anti-H1N1 TR virus
activity.
Conclusion. – We have synthesized a novel series of daidzein analogs and
demonstrated that certain products have inhibiting effects on influenza virus. Notably,
synthetic isoflavonoids with a Br-substituent on the B ring and a CHO and a free OH
group on the A ring consistently revealed potent activities and highly selective indexes.
This report, which presents the first systematic investigation of the use of the effects of
daidzein analogs against H1N1 TR influenza virus in the MDCK cell line, led to the

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Table. In Vitro Anti-Influenza Activities of Isoflavonoids 9, 11, and 12 in MDCK Cells, Determined by
CPE Assaya)b)
Compound
R1
R2
R3
H1N1 Tamiflu-resistant virus
EC50 [mm]
70.63Æ0.01
60.32Æ0.02
>300
>300
135.71Æ0.04
CC50 [mm]
SI
>4.0
2.0
<0.4
<0.5
>2.2
8.0
>2.2
>8.9
>9.0
>10.3
>9.0
>9.3
>8.5
>9.3
>1.0
>1.2
NDc)
ND
9a
9b
9c
9d
9e
EtOCOCH2O
EtOCOCH2O
EtOCOCH2O
EtOCOCH2O
EtOCOCH2O
EtOCOCH2O
EtOCOCH2O
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
CHO
CHO
CHO
CHO
CHO
CHO
CHO
H
H
H
H
H
4’-MeO
3’,4’,5’-(MeO)3
4’-Br
4’-Cl
3’-OCH2O-4’
4’-F
>300
120.74Æ0.03
124.02Æ0.05
139.41Æ0.08
>300
9f
9g
36.54Æ0.01
135.41Æ0.09
33.82Æ0.04
33.34Æ0.06
29.01Æ0.03
33.32Æ0.01
32.21Æ0.07
35.24Æ0.02
32.13Æ0.04
306.51Æ0.06
258.82Æ0.03
292.13Æ0.03
4’-NO2
>300
11a
11b
11c
11d
11e
11f
11g
12a
12b
12c
12d
12e
12f
12g
Ribavirin
4’-MeO
3’,4’,5’-(MeO)3
4’-Br
4’-Cl
3’-OCH2O-4’
4’-F
>300
>300
>300
>300
>300
>300
>300
>300
OH
OH
4’-NO2
HOCOCH2O
HOCOCH2O
HOCOCH2O
HOCOCH2O
HOCOCH2O
HOCOCH2O
HOCOCH2O
4’-MeO
3’,4’,5’-(MeO)3
4’-Br
4’-Cl
3’-OCH2O-4’
4’-F
>300
>300
>300
>300
>300
293.93Æ0.05
>300
300.02Æ0.04
40.94Æ0.02
73.52Æ0.03
0.3
H
H
300
>1.0
>1.0
>7.3
4’-NO2
>300
>300
a) EC50, Mean value the 50% effective concentration; CC50, mean value the 50% cell cytotoxic
concentration; SI, selective index, CC50/EC50. b) In vitro anti-H1N1 influenza virus activity of oseltamivir
(ꢀTamifluꢁ): EC50 ¼32.0 mm, CC50 ¼320.1 mm, SI¼10; oseltamivir is void of activity against H1N1
Tamiflu-resistant virus. c) ND, Not determined. d) All results are expressed as mean ÆSD (n¼6).
discovery of the 4’-Br-substituted product 11c as the most promising. The Br-atoms
might also facilitate the suppression of viral resistance. Considering their facile
preparation, series 11 might be superior to commercially available drugs such as
oseltamivir (ꢀTamifluꢁ) for clinical treatment (prophylaxis and/or therapy) of H1N1-
TR influenza. These results may provide a basis for further design and development of
new anti-H1N1-TR agents. Detailed pharmacological studies are underway and will be
reported subsequently.
We gratefully acknowledge the research grants, NSC 99-2320-B-016-005-MY3 and 100-EC-17-A-20-
S1-028, from the National Science Council and the Ministry of Economic Affairs of the Republic of China.
Prof. Ted Knoy is appreciated for his editorial assistance.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
691
Experimental Part
General. All the chemicals were purchased from AldrichÀSigma (St. Louis, MO, USA), and
AlfaÀAesar (Heysham, LA32XY, England), and used without further purification. TLC: F254 silica-gel
plates (Merck). Column chromatography (CC): silica gel (70–230 mesh; Merck). M.p.: Büchi-530
melting-point apparatus, uncorrected. UV/VIS Spectra: Shimadzu UV-160A spectrophotometer; lmax
(log e) in nm. IR Spectra: Perkin-Elmer FTIR 1610 series infrared spectrophotometer; n˜ in cmÀ1. 1H- and
13C-NMR spectra: Varian Gemini-300 NMR spectrometer; (D6)DMSO; d in ppm rel. to Me4Si as internal
standard, J in Hz. HR-EI-MS: Finnigan MAT-95XL spectrometer; in m/z.
General Procedure for the Synthesis of Isoflavones 8a–8g. Resorcinol (¼ benzene-1,3-diol; 11.0 g,
100 mmol) and ZnCl2 (5.0 g) in Et2O (100 ml) were placed in a two-neck round bottle fitted with an inlet
Teflon tube, through which dry HCl was passed into the stirring mixture. After two liquid layers had
formed in the mixture, a soln. of a substituted phenylacetonitriles (30–80 mmol) was added dropwise to
the mixture. Stirring was continued during the introduction of HCl at least for 12 h. The mixture was
further stirred overnight. The Et2O layer was decanted from the yellow solids formed, and H2O was then
added to half-volume of the flask (ca. 150 ml). The yellow imine hydrochloride was hydrolyzed by
heating to reflux for 1 h. After cooling the mixture overnight, the precipitates were filtered and collected
to obtain the corresponding 1-(2,4-dihydroxyphenyl)-2-(substituted phenyl)ethanones 7a–7g (24–68%).
To the respective ethanones benzylketones 7a–7g (1.0 equiv.) in dry DMF (20 ml) was added dropwise
BF3 ·Et2O (5 equiv.), and then the mixture was warmed to 508. To the mixture was slowly added a soln. of
MsCl (3 equiv.) in dry DMF (5 ml). The resulting mixture was heated to 1008 for 1 h. After cooling, it
was poured into H2O (100 ml) and left overnight to precipitate. After filtration, the precipitates were
collected to give the corresponding isoflavones 8a–8g: 7-hydroxy-3-(4-methoxyphenyl)-4H-1-benzopyr-
an-4-one (8a; 70%), 7-hydroxy-3-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-4-one (8b; 62%), 3-(4-
bromophenyl)-7-hydroxy-4H-1-benzopyran-4-one (8c; 54%), 3-(4-chlorophenyl)-7-hydroxy-4H-1-ben-
zopyran-4-one (8d; 59%), 3-(benzo[d][1,3]dioxol-5-yl)-7-hydroxy-4H-1-benzopyran-4-one (8e; 56%),
3-(4-fluorophenyl)-7-hydroxy-4H-1-benzopyran-4-one (8f; 83%), 7-hydroxy-3-(4-nitrophenyl)-4H-1-
benzopyran-4-one (8g; 87%).
General Procedure for the Synthesis of Isoflavones 9a–9g. Various isoflavones derivatives 8a–8g
(1.0 equiv.) and anh. K2CO3 (3 equiv.) were placed in dry acetone (100 ml), and then the mixture was
stirred for 1 h. ClCH2COOEt (1.5 equiv.) was added dropwise to the mixture, which was heated to 758
and maintained at this temp. for 12 h. Then, the mixture was evaporated under vacuum to give solid
residues, to which H2O (100 ml) was poured. The precipitates were filtered off and washed with cool H2O
to get the corresponding esters. Recrystallization from EtOH afforded pure 9a–9g (65–95%).
Ethyl 2-{[3-(4-Methoxyphenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetate (9a). Yield: 81%. M.p.
140–1428. Rf (hexane/AcOEt 1:1) 0.38. UV (MeOH): 263 (4.77). IR (KBr): 2996, 1746, 1622, 1252, 1180,
832. 1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 3.78 (s, 3 H); 4.19 (q, J¼7.2, 2 H); 4.98 (s,
2 H); 6.99 (dd, J¼6.9, 2.1, 2 H); 7.12 (dd, J¼9.0, 2.7, 1 H); 7.18 (d, J¼2.7, 1 H); 7.52 (dd, J¼6.9, 2.1, 2 H);
8.04 (d, J¼9.0, 1 H); 8.42 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 14.2; 55.5; 61.6; 66.2; 102.4; 114.5;
115.4; 119.8; 125.3; 125.4; 128.3; 131.0; 153.6; 158.6; 160.7; 163.4; 168.7; 175.7. HR-EI-MS: 354.1100 (Mþ,
C20H18Oþ6 ; calc. 354.1103).
Ethyl 2-{[4-Oxo-3-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-7-yl]oxy}acetate (9b). Yield: 67%.
M.p. 127–1298. Rf (hexane/AcOEt 1:1) 0.18. UV (MeOH): 220 (4.46). IR (KBr): 2986, 1764, 1634, 1198,
1124, 835. 1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 3.68 (s, 3 H); 3.80 (s, 6 H); 4.19 (q, J¼
7.2, 2 H); 4.99 (s, 2 H); 6.91 (s, 2 H); 7.13 (dd, J¼8.7, 2.4, 1 H); 7.20 (d, J¼2.4, 1 H); 8.02 (d, J¼8.7, 1 H);
8.50 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 13.9; 56.2; 60.1; 61.3; 65.8; 102.0; 107.6; 115.0; 119.4;
125.0; 127.9; 128.2; 139.3; 153.8; 153.9; 158.1; 163.0; 168.3; 175.1. HR-EI-MS: 414.1308 (Mþ, C22H22O8þ ;
calc. 414.1315).
Ethyl 2-{[3-(4-Bromophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetate (9c). Yield: 65%. M.p. 163–
1648. Rf (hexane/AcOEt 1:1) 0.50. UV (MeOH): 254 (4.55). IR (KBr): 3069, 1760, 1637, 1196, 1062, 824.
1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 4.19 (q, J¼7.2, 2 H); 4.99 (s, 2 H); 7.13 (dd, J¼
9.0, 2.4, 1 H); 7.21 (d, J¼2.4, 1 H); 7.55 (dd, J¼6.6, 2.1, 2 H); 7.63 (dd, J¼6.6, 2.1, 2 H); 8.05 (d, J¼9.0,
1 H); 8.52 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 14.3; 61.7; 66.2; 102.5; 115.6; 119.7; 122.4; 124.5;

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
128.3; 131.8; 132.1; 132.6; 154.5; 158.7; 163.7; 168.7; 175.2. HR-EI-MS: 402.0101 (Mþ, C19H1579BrO5þ ; calc.
402.0103), 404.0101 (Mþ, C19H1581BrO5þ ; calc. 404.0082).
Ethyl 2-{[3-(4-Chlorophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetate (9d). Yield: 89%. M.p. 135–
1378. Rf (hexane/AcOEt 1:1) 0.50. UV (MeOH): 206 (4.56). IR (KBr): 2991, 1755, 1628, 1198, 1090, 850.
1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 4.19 (q, J¼7.2, 2 H); 4.99 (s, 2 H); 7.13 (dd, J¼
9.0, 2.4, 1 H); 7.21 (d, J¼2.4, 1 H); 7.50 (d, J¼8.7, 2 H); 7.62 (d, J¼8.7, 2 H); 8.05 (d, J¼9.0, 1 H); 8.52 (s,
1 H). 13C-NMR (75 MHz, (D6)DMSO): 14.3; 61.7; 66.2; 102.5; 115.6; 119.8; 124.5; 128.3; 129.1; 131.5;
132.2; 134.3; 154.6; 158.7; 163.7; 168.7; 175.3. HR-EI-MS: 358.0607 (Mþ, C19H15ClO5þ ; calc. 358.0608).
Ethyl 2-{[3-(1,3-Benzodioxol-5-yl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetate (9e). Yield: 73%. M.p.
164–1678. Rf (hexane/AcOEt 1:1) 0.40. UV (MeOH): 205 (4.78). IR (KBr): 2990, 1755, 1644, 1194, 1099,
832. 1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 4.19 (q, J¼7.2, 2 H); 4.98 (s, 2 H); 6.04 (s,
2 H); 6.98 (d, J¼8.1, 1 H); 7.06 (dd, J¼8.1, 1.8, 1 H); 7.11 (dd, J¼8.7, 2.4, 1 H); 7.15 (d, J¼1.8, 1 H); 7.18
(d, J¼2.4, 1 H); 8.03 (d, J¼8.7, 1 H); 8.42 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 14.3; 61.7; 66.2;
102.10; 102.5; 108.8; 110.5; 115.5; 119.9; 122.3; 123.3; 125.4; 127.1; 128.4; 148.6; 153.9; 158.7; 163.6; 168.7;
175.6. HR-EI-MS: 368.0894 (Mþ, C20H16O7þ ; calc. 368.0896).
Ethyl 2-{[3-(4-Fluorophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetate (9f). Yield: 90%. M.p. 131–
1338. Rf (hexane/AcOEt 1:1) 0.48. UV (MeOH): 248 (4.50). IR (KBr): 3599, 2988, 1755, 1633, 1223,
1068, 825. 1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 4.19 (q, J¼7.2, 2 H); 4.99 (s, 2 H); 7.13
(dd, J¼9.0, 2.4, 1 H); 7.20 (d, J¼2.4, 1 H); 7.27 (t, J¼8.7, 2 H); 7.63 (dd, J¼8.7, 5.4, 2 H); 8.05 (d, J¼9.0,
1 H); 8.49 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 13.8; 60.7; 65.2; 101.8; 114.8; 114.9; 115.1; 118.1;
122.9; 127.1; 128.2; 128.2; 130.9; 131.0; 154.2; 157.3; 162.2; 168.0; 174.4. HR-EI-MS: 342.0907 (Mþ,
C19H15FOþ5 ; calc. 342.0904).
Ethyl 2-{[3-(4-Nitrophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetate (9g). Yield: 95%. M.p. 192–
1938. Rf (hexane/AcOEt 1:1) 0.38. UV (MeOH): 219 (4.34). IR (KBr): 3002, 1752, 1629, 1351, 1199, 851.
1H-NMR (300 MHz, (D6)DMSO): 1.22 (t, J¼7.2, 3 H); 4.19 (q, J¼7.2, 2 H); 5.00 (s, 2 H); 7.16 (dd, J¼
9.0, 2.4, 1 H); 7.24 (d, J¼2.4, 1 H); 7.91 (dd, J¼7.2, 2.1, 2 H); 8.07 (d, J¼9.0, 1 H); 8.29 (dd, J¼7.2, 2.1,
2 H); 8.68 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 14.3; 61.3; 65.7; 102.4; 115.7; 118.6; 122.5; 123.7;
127.7; 130.5; 139.6; 147.5; 156.3; 157.8; 162.9; 168.6; 174.5. HR-EI-MS: 369.0856 (Mþ, C19H15NO7þ ; calc.
369.0849).
General Procedure for the Synthesis of Nitriles 10a–10g. The respective isoflavone 8a–8g (1.0 equiv.)
with anh. K2CO3 (3 equiv.) was placed in dry acetone (100 ml). BrCH2CN (1.1 equiv.) was added
dropwise to the mixture, which was heated to reflux for 5–6 h and then evaporated under vacuum to give
solid residues, to which H2O (100 ml) was poured the precipitates were filtered off to obtain the
corresponding 10a–10g (68–98%) after recrystallization from EtOH.
2-{[3-(4-Methoxyphenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetonitrile (10a). Yield: 89%. M.p.
203–2048. Rf (hexane/AcOEt 1:1) 0.43. UV (MeOH): 330 (3.01). IR (KBr): 3074, 2963, 2942, 2841,
1638. 1H-NMR (300 MHz, (D6)DMSO): 3.72 (s, 3 H); 5.37 (s, 2 H); 7.00 (d, J¼8.7, 2 H); 7.21 (dd, J¼9,
2.4, 1 H); 7.39 (d, J¼2.4, 1 H); 7.54 (d, J¼9, 2 H); 8.14 (d, J¼9, 1 H); 8.48 (s, 1 H). 13C-NMR (75 MHz,
(D6)DMSO): 54.7; 55.8; 102.9; 114.4; 115.4; 116.7; 119.7; 124.4; 124.7; 128.3; 130.8; 1584.4; 157.8; 159.6;
161.3; 175.4. HR-EI-MS: 307.0842 (Mþ, C18H13NO4þ ; calc. 307.0845).
2-{[4-Oxo-3-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-7-yl]oxy}acetonitrile (10b). Yield: 90%.
M.p. 196–1988. Rf (hexane/AcOEt 1:1) 0.22. UV (MeOH): 330 (3.04). IR (KBr): 3070, 1643, 1621,
1578. 1H-NMR (300 MHz, (D6)DMSO): 3.69 (s, 3 H); 3.81 (s, 6 H); 5.38 (s, 2 H); 6.93 (s, 2 H); 7.22 (dd,
J¼8.7, 2.4, 1 H); 7.41 (d, J¼2.4, 1 H); 8.12 (d, J¼8.7, 1 H); 8.56 (s, 1 H). 13C-NMR (75 MHz,
(D6)DMSO): 54.7; 56.7; 60.7; 102.9; 107.2; 107.5; 115.5; 116.7; 119.7; 124.5; 127.9; 128.3; 138.5; 153.2;
153.4;. 155.2; 157.8; 161.3; 175.2. HR-EI-MS: 367.1056 (Mþ, C20H17NO6þ ; calc. 367.1056).
2-{[3-(4-Bromophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetonitrile (10c). Yield: 69%. M.p. 217–
2198. Rf (hexane/AcOEt 1:1) 0.63. UV (MeOH): 332 (2.18). IR (KBr): 3063, 2952, 1936, 1902, 1847,
1
1642. H-NMR (300 MHz, (D6)DMSO): 5.38 (s, 2 H); 7.23 (dd, J¼8.7, 2.4, 1 H); 7.41 (d, J¼2.4, 1 H);
7.54 (d, J¼8.4, 2 H); 7.65 (d, J¼8.4, 2 H); 8.12 (d, J¼8.7, 1 H); 8.58 (s, 1 H). 13C-NMR (75 MHz,
(D6)DMSO): 54.8; 103.1; 115.6; 116.7; 119.7; 121.9; 123.6; 128.3; 131.7; 131.8; 155.4; 157.9; 161.4; 174.9.
HR-EI-MS: 354.9841 (Mþ, C17H1079BrNO3þ ; calc. 354.9844), 356.9822 (Mþ, C17H1081BrNOþ3 ; calc.
356.9793).

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2-{[3-(4-Chlorophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetonitrile (10d). Yield: 92%. M.p. 169–
1708. Rf (hexane/AcOEt 1:1) 0.49. UV (MeOH): 332 (3.22). IR (KBr): 3073, 2956, 1773. 1H-NMR
(300 MHz, (D6)DMSO): 5.39 (s, 2 H); 7.23 (dd, J¼9, 2.1, 1 H); 7.42 (d, J¼2.1, 1 H); 7.51 (d, J¼8.4, 2 H);
7.64 (d, J¼8.7, 2 H); 8.12 (d, J¼9, 1 H); 8.58 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 42.7; 52.6; 65.8;
102.5; 115.7; 118.9; 123.4; 127.9; 128.9; 131.4; 131.6; 155.2; 158.0; 162.9; 169.3; 175.0. HR-EI-MS:
311.0348 (Mþ, C17H10ClNOþ3 ; calc. 311.0349).
2-{[3-(1,3-Benzodioxol-5-yl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetonitrile (10e). Yield: 78%. M.p.
236–2388. Rf (hexane/AcOEt 1:1) 0.43. UV (MeOH): 330 (3.16), 206 (2.61). IR (KBr): 3078, 2952, 2892,
1618, 1590, 1500, 1246, 1196. 1H-NMR (300 MHz, (D6)DMSO): 5.37 (s, 2 H); 6.06 (s, 2 H); 6.99 (d, J¼
8.1, 1 H); 7.08 (dd, J¼8.1, 1.5, 1 H); 7.17 (s, J¼1.8, 1 H); 7.21 (dd, J¼9, 2.4, 1 H); 7.38 (d, J¼2.4, 1 H);
8.11 (d, J¼9, 1 H); 8.48 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 54.7; 101.8; 102.9; 108.8; 110.1; 115.5;
116.7; 119.7; 123.2; 124.4; 126.1; 128.3; 147.8; 154.7; 157.8; 161.3; 157.3. HR-EI-MS: 321.0635 (Mþ,
C18H11NOþ5 ; calc. 321.0637).
2-{[3-(4-Fluorophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetonitrile (10f). Yield: 98%. M.p. 175–
1778. Rf (hexane/AcOEt 1:1) 0.63. UV (MeOH): 332 (2.51), 206 (2.54). IR (KBr): 3073, 2956, 2362,
1
1762. H-NMR (300 MHz, (D6)DMSO): 6.88 (d, J¼2.1, 1 H); 6.94 (dd, J¼8.7, 2.1, 1 H); 7.25 (t, J¼9,
2 H); 7.61 (dd, J¼8.7, 5.7, 2 H); 7.97 (d, J¼8.7, 1 H); 8.39 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 54.7;
102.9; 115.5; 115.8; 116.6; 119.6; 123.7; 128.2; 128.8; 128.8; 131.6; 131.7; 155.1; 157.9; 161.1; 161.3; 164.3;
175.1. HR-EI-MS: 295.0643 (Mþ, C17H10FNOþ3 ; calc. 295.0645).
2-{[3-(4-Nitrophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetonitrile (10g). Yield: 68%. M.p. 224–
2258. Rf (hexane/AcOEt 1:1) 0.50. UV (MeOH): 334 (3.78), 206 (2.96). IR (KBr): 3067, 1650, 1625,
1568. 1H-NMR (300 MHz, (D6)DMSO): 5.39 (s, 2 H); 7.26 (dd, J¼9, 2.4, 1 H); 7.46 (d, J¼2.4, 1 H); 7.93
(d, J¼9, 2 H); 8.15 (d, J¼9, 1 H); 8.32 (d, J¼9, 2 H); 8.74 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO):
50.7; 70.4; 103.1; 115.8; 116.6; 119.6; 122.8; 123.9; 128.3; 130.6; 139.6; 147.75; 156.6; 157.8; 161.6; 174.7.
HR-EI-MS: 322.0588 (Mþ, C17H10N2O5þ ; calc. 322.0590).
General Procedure for the Synthesis of Carbaldehydes 11a–11g. Compound 8a–8g (1.0 equiv.) with
methenamine (¼ hexamethylenetetramine¼1,3,5,7-tetraaza[3.3.1.13,7]decane; 1.1 equiv.) was placed in
glacial AcOH (100 ml), and then the mixture was heated to 1008 for 6 h. The resulting hot reddish brown
liquid was treated with HCl (1:1, 100 ml) and again heated to 1008 for 5 min. After dilution with H2O
(150 ml), it was allowed to stand overnight. The yellow precipitates were collected, washed with H2O, and
dried. The dry solids were extracted with hot benzene (2Â100 ml) and the residues were discarded. The
benzene soln. was evaporated under reduced presure to obtain the crude solids. Recrystallization from
EtOH afforded pure 11a–11g (11–65%).
7-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11a). Yield: 33%. M.p.
160–1638. Rf (hexane/AcOEt 1:1) 0.50. UV (MeOH): 334 (4.00). IR (KBr): 2923, 1657. 1H-NMR
(300 MHz, (D6)DMSO): 3.79 (s, 3 H); 7.00 (d, J¼8.7, 2 H); 7.11 (d, J¼8.7, 1 H); 7.53 (d, J¼8.7, 2 H); 8.25
(d, J¼9, 1 H); 8.48 (s, 1 H); 10.49 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 55.8; 110.5; 114.4; 116.6;
117.2; 124.2; 124.9; 130.8; 153.7; 157.6; 160.0; 166.7; 174.7; 190.8. HR-EI-MS: 296.0681 (Mþ, C17H12O5þ,
calc. 296.0685).
7-Hydroxy-4-oxo-3-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-8-carboxaldehyde (11b). Yield:
51%. M.p. 172–1768. Rf (hexane/AcOEt 1:1) 0.73. UV (MeOH): 336 (3.85). IR (KBr): 3434, 2941,
1
1643. H-NMR (300 MHz, (D6)DMSO): 3.69 (s, 3 H); 3.79 (s, 6 H); 6.94 (s, 2 H); 7.13 (dd, J¼9, 1 H);
8.26 (d, J¼9, 1 H); 8.59 (s, 1 H); 10.50 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 54.7; 55.8; 102.9;
114.4; 115.4; 116.7; 119.7; 124.3; 124.6; 128.3; 130.8; 154.5; 157.8; 159.9; 161.2; 175.4. HR-EI-MS:
356.0891 (Mþ, C19H16Oþ7 ; calc. 356.0896).
3-(4-Bromophenyl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11c). Yield: 65%. M.p.
102–1058. Rf (hexane/AcOEt 1:1) 0.62. UV (MeOH): 392 (2.63), 333 (3.34). IR (KBr): 3067, 2248, 1638.
1H-NMR (300 MHz, (D6)DMSO): 7.13 (d, J¼9, 1 H); 7.57 (d, J¼8.4, 2 H); 7.65 (d, J¼8.4, 2 H); 8.26 (d,
J¼9, 1 H); 8.59 (s, 1 H); 10.50 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 22.48; 110.61; 116.89; 117.19;
119.47; 121.51; 112.15; 131.04; 131.45; 131.58; 131.68; 131.88; 132.56; 131.23; 154.66; 166.81; 174.31;
190.72. HR-EI-MS: 343.9682 (Mþ, C16H979BrO4þ ; calc. 343.9684), 345.9663 (Mþ, C16H981BrOþ4 ; calc.
345.9664).

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3-(4-Chlorophenyl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11d). Yield: 14%. M.p.
158–1638. Rf (hexane/AcOEt 1:1) 0.62. UV (MeOH): 336 (3.74). IR (KBr): 3074, 1638. 1H-NMR
(300 MHz, (D6)DMSO): 7.13 (d, J¼9, 1 H); 7.51 (d, J¼8.7, 2 H); 7.63 (d, J¼8.4, 2 H); 8.25 (d, J¼8.7,
1 H); 8.58 (s, 1 H); 10.49 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 110.6; 116.8; 117.1; 124.1; 128.9;
131.0; 133.6; 134.2; 154.7; 57.6; 166.8; 174.3; 190.6. HR-EI-MS: 300.0185 (Mþ, C16H9ClO4þ ; calc.
300.0189).
3-(1,3-Benzodioxol-5-yl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11e). Yield: 49%.
M.p. 242–2438. Rf (hexane/AcOEt 1:1) 0.46. UV (MeOH): 334 (3.53). IR (KBr): 3069, 1643. 1H-NMR
(300 MHz, (D6)DMSO): 6.06 (s, 1 H); 6.99 (d, J¼8.1, 1 H); 7.08 (dd, J¼8.1, 1.5, 1 H); 7.12 (d, J¼9, 1 H);
7.16 (d, J¼1.5, 1 H); 8.25 (d, J¼9, 1 H); 8.50 (s, 1 H); 10.49 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO):
101.8; 108.8; 110.0; 116.7; 117.2; 123.2; 124.9; 125.7; 130.4; 134.3; 147.5; 147.9; 154.0; 166.7; 190.7. HR-EI-
MS: 310.0477 (Mþ, C17H10Oþ6 ; calc. 310.0477).
3-(4-Fluorophenyl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11f). Yield: 11%. M.p.
163–1658. Rf (hexane/AcOEt 1:1) 0.57. UV (MeOH): 336 (3.70). IR (KBr): 3076, 1642. 1H-NMR
(300 MHz, (D6)DMSO): 7.13 (d, J¼9, 1 H); 7.29 (t, J¼9, 2 H); 7.64 (dd, J¼8.7, 5.4, 2 H); 8.25 (d, J¼8.7,
1 H); 8.56 (s, 1 H); 10.49 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 110.6; 115.6; 115.9; 116.8; 117.2;
124.4; 128.5; 131.7; 131.8; 134.3; 154.4; 157.7; 166.8; 174.6; 190.9. HR-EI-MS: 284.0485 (Mþ, C16H9FO4þ ;
calc. 284.0485).
7-Hydroxy-3-(4-nitrophenyl)-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11g). Yield: 59%. M.p.
234–2368. Rf (hexane/AcOEt 1:1) 0.43. UV (MeOH): 332 (4.21). IR (KBr): 3077, 1652. 1H-NMR
(300 MHz, (D6)DMSO): 7.15 (d, J¼8.7, 1 H); 7.91 (d, J¼9, 5.4, 2 H); 8.25–8.31 (m, 3 H); 8.74 (s, 1 H);
10.49 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 110.7; 117.0; 123.4; 123.9; 130.7; 134.1; 139.2; 147.7;
155.9; 157.5; 166.9; 174.0; 190.4. HR-EI-MS: 311.0427 (Mþ, C16H9NO6þ ; calc. 311.0430).
General Procedure for the Synthesis of Acids 12a–12g. The respective ester 9a–9g was added in a
mixture of conc. H2SO4 (4 ml) and glacial AcOH (12 ml), and stirred for 12 h. A soln. of 1n NaOH
(20 ml) was then added dropwise to this mixture, which was for 2 h, and the precipitate was filtered off to
obtain the corresponding acid. Recrystallization from EtOH afforded pure 12a–12g (48–95%).
2-{[3-(4-Methoxyphenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetic Acid (12a). Yield: 55%. M.p.
224–2268. Rf (MeOH/CH2Cl2 1:5) 0.48. UV (MeOH): 262 (4.84). IR (KBr): 2903, 1738, 1623, 1249,
1182, 827. 1H-NMR (300 MHz, (D6)DMSO): 3.83 (s, 3 H); 4.90 (s, 2 H); 6.99 (d, J¼9.0, 2 H); 7.05 (d, J¼
2.4, 1 H); 7.15 (dd, J¼9.0, 2.4, 1 H); 7.51 (d, J¼8.7, 2 H); 8.16 (s, 1 H); 8.27 (d, J¼9.0, 1 H). 13C-NMR
(75 MHz, (D6)DMSO): 55.5; 65.8; 102.4; 114.5; 115.4; 119.8; 125.3; 125.5; 128.3; 131.0; 153.6; 158.7;
160.7; 163.5; 169.4; 175.7. HR-EI-MS: 326.0811 (Mþ, C18H14O6þ ; calc. 326.0790).
2-{[4-Oxo-3-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-7-yl]oxy}acetic Acid (12b). Yield: 95%.
M.p. 152–1558. Rf (MeOH/CH2Cl2 1:5) 0.54. UV (MeOH): 219 (4.46). IR (KBr): 3448, 2944, 1741,
1625, 1197, 1130, 833. 1H-NMR (300 MHz, (D6)DMSO): 3.68 (s, 3 H); 3.79 (s, 6 H); 4.28 (s, 2 H); 6.88 (d,
J¼2.4, 1 H); 6.91 (s, 2 H); 7.00 (dd, J¼9.0, 2.4, 1 H); 7.98 (d, J¼8.7, 1 H); 8.46 (s, 1 H). 13C-NMR
(75 MHz, (D6)DMSO): 56.7; 60.7; 69.3; 70.4; 101.8; 107.6; 116.0; 117.7; 124.1; 127.1; 128.3; 138.5; 153.4;
154.6; 158.0; 164.8; 170.0; 175.2. HR-EI-MS: 386.1008 (Mþ, C20H18O8þ ; calc. 386.1002).
2-{[3-(4-Bromophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetic Acid (12c). Yield: 92%. M.p. 241–
2428. Rf (MeOH/CH2Cl2 1:5) 0.63. UV (MeOH): 252 (4.55). IR (KBr): 3065, 1711, 1624, 1248, 1197, 828.
1H-NMR (300 MHz, (D6)DMSO): 4.95 (s, 2 H); 7.10 (d, J¼2.1, 1 H); 7.14 (dd, J¼8.7, 2.4, 1 H); 7.60 (s,
4 H); 8.13 (d, J¼8.7, 1 H); 8.35 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 65.5; 102.2; 115.5; 116.0;
122.2; 124.3; 128.1; 131.6; 131.9; 132.4; 154.4; 158.5; 163.5; 169.2; 175.1. HR-EI-MS: 373.9789 (Mþ,
C17H1179BrOþ5 ; calc. 373.9790), 375.9767 (Mþ, C17H1181BrO5þ ; calc. 375.9769).
2-{[3-(4-Chlorophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetic Acid (12d). Yield: 48%. M.p. 233–
2348. Rf (MeOH/CH2Cl2 1:5) 0.53. UV (MeOH): 206 (4.50). IR (KBr): 2907, 1709, 1624, 1249, 1197, 1090,
828. 1H-NMR (300 MHz, (D6)DMSO): 4.96 (s, 2 H); 7.11 (d, J¼2.1, 1 H); 7.15 (dd, J¼8.7, 2.4, 1 H); 7.46
(d, J¼8.7, 2 H); 7.67 (d, J¼8.7, 2 H); 8.14 (d, J¼8.4, 1 H); 8.35 (s, 1 H). 13C-NMR (75 MHz,
(D6)DMSO): 65.5; 102.2; 115.3; 119.4; 124.2; 128.0; 128.7; 131.2; 131.9; 133.9; 154.2; 158.4; 163.4; 169.1;
175.0. HR-EI-MS: 330.0298 (Mþ, C17H11ClOþ5 ; calc. 330.0295).
2-{[3-(1,3-Benzodioxol-5-yl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetic Acid (12e). Yield: 90%. M.p.
241–2438. Rf (MeOH/CH2Cl2 1:5) 0.45. UV (MeOH): 206 (4.40). IR (KBr): 3504, 2906, 1639, 1249,

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1050, 818. 1H-NMR (300 MHz, (D6)DMSO): 4.90 (s, 2 H); 5.99 (s, 2 H); 6.87 (d, J¼8.1, 1 H); 7.02 (dd,
J¼8.1, 1.8, 1 H); 7.04 (d, J¼2.4, 1 H); 7.09 (d, J¼1.8, 1 H); 7.15 (dd, J¼9.0, 2.4, 1 H); 8.15 (s, 1 H); 8.26
(d, J¼9.0, 1 H). 13C-NMR (75 MHz, (D6)DMSO): 65.5; 101.8; 102.1; 108.5; 110.2; 115.2; 119.4; 123.0;
125.0; 126.8; 128.0; 148.2; 153.7; 158.4; 163.3; 169.3; 175.4. HR-EI-MS: 340.0578 (Mþ, C18H12O7þ ; calc.
340.0583).
2-{[3-(4-Fluorophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetic Acid (12f). Yield: 92%. M.p. 209–
2118. Rf (MeOH/CH2Cl2 1:5) 0.38. UV (MeOH): 238 (4.43). IR (KBr): 3523, 3078, 1644, 1248, 1191, 817.
1H-NMR (300 MHz, (D6)DMSO): 4.89 (s, 2 H); 7.05 (d, J¼2.4, 1 H); 7.15 (dd, J¼9.0, 2.4, 1 H); 7.16 (t,
J¼8.7, 2 H); 7.59 (dd, J¼8.7, 5.4, 2 H); 8.19 (s, 1 H); 8.27 (d, J¼9.0, 1 H). 13C-NMR (75 MHz,
(D6)DMSO): 65.5; 102.2; 115.3; 115.3; 115.6; 119.5; 124.4; 128.0; 129.3; 131.6; 131.7; 154.0; 158.4; 163.4;
169.1; 175.2. HR-EI-MS: 314.0592 (Mþ, C17H11FO5þ ; calc. 314.0591).
2-{[3-(4-Nitrophenyl)-4-oxo-4H-1-benzopyran-7-yl]oxy}acetic Acid (12g). Yield: 95%. M.p. 273–
2748. Rf (MeOH/CH2Cl2 1:5) 0.35. UV (MeOH): 220 (4.49). IR (KBr): 3588, 3076, 1746, 1624, 1246, 851.
1H-NMR (300 MHz, (D6)DMSO): 4.90 (s, 2 H); 7.15 (dd, J¼9.0, 2.4, 1 H); 7.22 (d, J¼2.7, 1 H); 7.91 (d,
J¼8.7, 2 H); 8.07 (d, J¼9.0, 1 H); 8.29 (d, J¼9.0, 2 H); 8.68 (s, 1 H). 13C-NMR (75 MHz, (D6)DMSO):
65.8; 102.5; 115.9; 118.6; 122.6; 123.9; 127.8; 130.6; 139.8; 147.6; 156.4; 158.0; 163.3; 170.1; 174.7. HR-EI-
MS: 341.0540 (Mþ, C17H11NOþ7 ; calc. 341.0536).
Cytopathic Effect (CPE) Assay. The antiviral activities of flavonoids were by the CPE assay and
using ribavirin as a positive control. The CPE inhibition assays used in this study were performed as
described [26]. The influenza virus strains, namely Tamiflu-resistant 2009 pandemic influenza A (H1N1)
virus, which contained the H275Y mutation (N1 numbering) in neuraminidase, was provided by Centers
for Disease Control (CDC), Taiwan, and used for evaluating the in vitro antiviral activities of the
synthetic flavonoids.
In brief, virus at 100 TCID50 (tissue culture infectious dose) were inoculated onto near confluent
MDCK cell monolayers (1Â105 cells/well) for 1 h. After incubation at 378 for 2 h, the virus soln. was
removed, and 100 ml of sequential twofold serial dilutions of the respective flavonoids and reference
compound ribavirin were added to each well of the 96-well culture plates, using the maximal non-
cytotoxic concentration (MNCC, i.e., 90% viable cells) as the highest concentration. An infection control
without flavonoids was also included. The plates were incubated at 378 in a 75% humidity of 5% CO2
atmosphere for 24 h, and then the CPE was observed. The virus-induced CPE was scored as follows:
scores: 0, 0% CPE; 1, 0–25% CPE; 2, 25–50% CPE; 3, 50–75% CPE; and 4, 75–100% CPE. The
reduction in virus multiplication was calculated as a percentage of the virus control (% virus control¼
CPE(exp)/CPE(virus control)Â100). The IC50 value of the CPE with respect to virus control was
estimated using the ReedÀMuench method and was expressed in mm. The selectivity index (SI) was
calculated from the ratio CC50/IC50
.
Statistical Analysis. Statistical calculations were carried out with Microsoft Excel 2007 version.
Results are expressed as the mean ÆSD of six independent experiments.
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Received September 15, 2014