Synthesis and anti-influenza activities of novel baicalein analogs

Article abstract of DOI:10.1248/cpb.c13-00897

A series of novel flavones derivatives were synthesized based on modification of the active ingredients of a traditional Chinese medicine Scutellaria baicalensis GEORGI and screened for anti-influenza activity. The synthetic baicalein (flavone) analogs, especially with the B-rings substituted with bromine atoms, were much more potent than oseltamivir or ribavirin against H1N1 Tamiflu-resistant (H1N1 TR) virus and usually with more favorable selectivity. The most promising were 5b, 5c, 6b and 6c, all displaying an 50% effective concentration (EC₅₀) at around 4.0-4.5 μM, and a selective index (SI=50% cytotoxic concentration (CC₅₀)/EC ₅₀)>70. For seasonal H3N2-infected influenza virus, both 5a and 5b with SI >17.3 indicated superior to ribavirin. The flavonoids having both not-naturally-occurring bromo-substituted B-rings and appropriate hydroxyls positioning on the A-rings might be critical in determining the activity and selectivity against H1N1-Tamiflu-resistant infected influenza viruses.

Full text of DOI:10.1248/cpb.c13-00897

May 2014
Regular Article
Chem. Pharm. Bull. 62(5) 415–421 (2014)
415
Synthesis and Anti-influenza Activities of Novel Baicalein Analogs
,a,b
Shu-Ting Chung,a Pei-Yu Chien,b Wen-Hsin Huang,a,b Chen-Wen Yao,c and An-Rong Lee*
a Institute of Medical Sciences, National Defense Medical Center; b School of Pharmacy, National Defense Medical
c
Center; and Deparment of Pathology and Parasitology, Tri-Service General Hospital, National Defense Medical
Center; 161 Minchuan East Rd. Sec. 6, Taipei 11490, Taiwan.
Received November 15, 2013; accepted February 17, 2014; advance publication released online February 25, 2014
A series of novel flavones derivatives were synthesized based on modification of the active ingredients of
a traditional Chinese medicine Scutellaria baicalensis GEORGI and screened for anti-influenza activity. The
synthetic baicalein (flavone) analogs, especially with the B-rings substituted with bromine atoms, were much
more potent than oseltamivir or ribavirin against H1N1 Tamiflu-resistant (H1N1 TR) virus and usually
with more favorable selectivity. The most promising were 5b, 5c, 6b and 6c, all displaying an 50% effective
concentration (EC50) at around 4.0–4.5µM, and a selective index (SI=50% cytotoxic concentration (CC50)/
EC50)>70. For seasonal H3N2-infected influenza virus, both 5a and 5b with SI >17.3 indicated superior to
ribavirin. The flavonoids having both not-naturally-occurring bromo-substituted B-rings and appropriate
hydroxyls positioning on the A-rings might be critical in determining the activity and selectivity against
H1N1-Tamiflu-resistant infected influenza viruses.
Key wordsꢀ flavonoid;ꢀanti-influenzaꢀvirus;ꢀH1N1ꢀTamiflu-resistantꢀvirus;ꢀH3N2ꢀvirus
Influenzaꢀ virusesꢀ areꢀ theꢀ mostꢀ significantꢀ sourceꢀ ofꢀ viralꢀ vegetables, fruits, tea and wine,9,10) and are well known to
respiratory infections in humans worldwide, causing recur- exhibit various favorable pharmacological activities, includ-
rent epidemics and global pandemics that bring about severe ingꢀanti-inflammation,ꢀanti-osteoporosis,ꢀanticancer,ꢀantivirus,ꢀ
morbidity and mortality involving millions of people annu- anti-bacteria and anti-oxidation.11–16) In general, the structures
ally.1)ꢀInfluenzaꢀvirusꢀisꢀanꢀRNAꢀvirusꢀofꢀtheꢀOrthomyxoviri- ofꢀflavonoidsꢀconsistꢀofꢀthreeꢀaromaticꢀringsꢀdesignatedꢀasꢀringꢀ
daeꢀ familyꢀ andꢀ canꢀ beꢀ classifiedꢀ intoꢀ threeꢀ types:ꢀ A,ꢀ B,ꢀ andꢀ A,ꢀ Bꢀ andꢀ Cꢀ basedꢀ onꢀ theꢀ sequenceꢀ ofꢀ theirꢀ biogenesis.ꢀ Theꢀ
C.2)ꢀ Unlikeꢀ influenzaꢀ Bꢀ andꢀ Cꢀ viruses,ꢀ whichꢀ mainlyꢀ infectꢀ plantsꢀ synthesizeꢀ aꢀ two-ringꢀ intermediateꢀ chalconeꢀ firstꢀ andꢀ
humans,ꢀ influenzaꢀ Aꢀ virusesꢀ infectꢀ aꢀ wideꢀ rangeꢀ ofꢀ hosts,ꢀ then form a bicyclic fused A/C ring system appended with
including humans, swine, birds, horses and whales.3) H1N1 aꢀ B-ring.ꢀ Oneꢀ majorꢀ classꢀ ofꢀ flavonoidsꢀ includesꢀ baicaleinꢀ
andꢀH3N2ꢀinfluenzaꢀAꢀvirusesꢀhaveꢀco-circulatedꢀsinceꢀ1977.ꢀ (5,6,7-trihydroxyflavone)ꢀ (Fig.ꢀ 1),ꢀ togetherꢀ withꢀ itsꢀ glycosideꢀ
Inꢀtheꢀspringꢀofꢀ2009,ꢀaꢀnewꢀvirulentꢀpandemicꢀstrainꢀwithꢀtheꢀ baicalin, which are extracted from the root of Scutellaria
H1N1 antigenic subtype appeared and spread globally.4)
baicalensis GEORGI, a traditional Chinese medicine (TCM)
To date, there have been several antiviral agents approved having been used for hundreds of years.14) It is particularly
toꢀtreatꢀandꢀpreventꢀinfluenzaꢀvirusꢀinfection.ꢀAmantadineꢀandꢀ noteworthy that, in recent years, baicalein and baicalin have
rimantadine remain important in this regard. However, the beenꢀ demonstratedꢀ anti-influenzaꢀ virusꢀ activityꢀ withꢀ lowꢀ
rapid development of resistance, the serious toxic effects in toxicity.16–19) Recently, in our investigations on the anti-viral
the autonomic nervous system in addition to their limited an- screening from traditional Chinese medicines, we had isolated
tiviralꢀefficacyꢀotherꢀthanꢀvirusꢀAꢀhaveꢀmadeꢀtheseꢀtwoꢀagentsꢀ twoꢀ veryꢀ potentꢀ flavones,ꢀ kaemferolꢀ fromꢀ Thesium chinense
impracticalꢀinꢀclinicalꢀuse.ꢀTwoꢀmajorꢀspecificꢀneuraminidaseꢀ TURCZꢀ andꢀ licoflavonolꢀ fromꢀ Sophora flavescens AIT (Fig. 1),
(NA)ꢀ inhibitors,ꢀ oseltamivirꢀ (Tamiflu)ꢀ andꢀ zanamivirꢀ (Relen- andꢀ foundꢀ thatꢀ bothꢀ possessedꢀ anti-influenzaꢀ (H1N1ꢀ TR)ꢀ ac-
za),ꢀ haveꢀ beenꢀ successfullyꢀ preparedꢀ byꢀ computer-aidedꢀ drugꢀ tivity with EC50ꢀatꢀasꢀlowꢀasꢀ10.9ꢀµM and 8.8µM, respectively.
design and extensively used.1,5) The structures of NA inhibi- Preliminary data of the molecular modeling study showed that
tors,ꢀinꢀmanyꢀcases,ꢀconsistꢀofꢀaꢀtransition-stateꢀconfiguration.ꢀ bothꢀ kaemferolꢀ andꢀ licoflavonolꢀ bindꢀ toꢀ theꢀ sameꢀ activeꢀ siteꢀ
Therefore, they present their antiviral activity by targeting of neuraminidase as oseltamivir but somehow in a different
neuraminidase and thus effectively abolish the proliferation
andꢀspreadingꢀofꢀviruses.ꢀTheꢀgenesisꢀofꢀoseltamivirꢀandꢀzana-
mivir represent a new generation of antiviral agents associated
withꢀ excellentꢀ efficacyꢀ andꢀ specificity.ꢀ Unfortunately,ꢀ theseꢀ
two potent NA inhibitors are by no means free from adverse
effects.6) Moreover, recent studies have indicated that pandem-
icꢀH1N1ꢀTamiflu-resistantꢀ(H1N1ꢀTR)ꢀviralꢀisolatesꢀhaveꢀbeenꢀ
progressively rising, which may pose a risk for increasing
fatality in human beings.7,8) This means that development of
new therapeutic agents are urgently needed and prompted us
toꢀsynthesizeꢀsomeꢀnovelꢀflavonoidsꢀandꢀexploreꢀtheꢀpossibilityꢀ
asꢀpotentialꢀanti-influenzaꢀagents.
Flavonoids are important polyphenolic constituents of
Theꢀauthorsꢀdeclareꢀnoꢀconflictꢀofꢀinterest.
Fig.ꢀ 1.ꢀ TheꢀStructuresꢀofꢀBaicalein,ꢀKaemferol,ꢀandꢀLicoflavonol
© 2014 The Pharmaceutical Society of Japan
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀlar@ndmctsgh.edu.tw

416
Vol. 62, No. 5
Reagentsꢀandꢀconditions:ꢀ(a)ꢀDMF,ꢀDCM,ꢀrt;ꢀ(b)ꢀBF3–Et2O,ꢀreflux;ꢀ(c)ꢀI2,ꢀDMSO,ꢀreflux
Chart 1. Synthesis of Compounds 2a–m
Chart 2. Synthesis of Compounds 3a–j
manner (unpublished result). These might explain the reason eties are discussed herein.
whyꢀ kaemferolꢀ andꢀ licoflavonolꢀ areꢀ stillꢀ activeꢀ againstꢀ theꢀ
H1N1ꢀTamiflu-resistantꢀstrain.ꢀWeꢀhadꢀreportedꢀthatꢀkaemferolꢀ
appendedꢀwithꢀaꢀB-ringꢀbearingꢀproperlyꢀpositioningꢀchlorineꢀ
Results and Discussion
Chemistry The synthetic approaches and molecular struc-
atomsꢀ couldꢀ significantlyꢀ improveꢀ theirꢀ anti-viralꢀ activityꢀ tures are shown in Charts 1–3. In our laboratories various
and selectivity.20) In this study, based on the structures of the derivedꢀ flavonoidsꢀ relatedꢀ toꢀ baicaleinꢀ wereꢀ readilyꢀ obtainedꢀ
activeꢀflavonoidsꢀmentionedꢀ above,ꢀ weꢀ wishꢀ toꢀ reportꢀ ourꢀ re- accordingꢀ toꢀ ourꢀ publishedꢀ proceduresꢀ withꢀ slightꢀ modifica-
sults of synthesis of novel baicalein analogs, especially with tions.14,20,21) In this study, we determined the data of spectra
B-ringsꢀ substitutedꢀ withꢀ bromineꢀ atoms,ꢀ andꢀ theirꢀ abilityꢀ ofꢀ and elemental analyses and anti-viral activities of reported 2l,
inhibitingꢀinfluenzaꢀvirus.ꢀAgain,ꢀsomeꢀofꢀtheꢀsyntheticꢀprod- 2m, 5b and 6c, which have been known to possess activities
ucts showed still superior activity and excellent selectivity, in againstꢀ inducibleꢀ nitricꢀ oxideꢀ synthaseꢀ (iNOS)ꢀ andꢀ cyclooxy-
vitro,ꢀagainstꢀH1N1ꢀTamiflu-resistantꢀvirusꢀandꢀseasonalꢀH3N2ꢀ genase-2ꢀ (COX-2).22,23) However, they were published in the
virus, respectively. The necessary functional groups and moi- form of a letter and none of their physico-chemical properties

May 2014
417
Reagentsꢀandꢀcoditions:ꢀ(a)ꢀBF3–Et2O,ꢀ45ꢀmin;ꢀ(b)ꢀ47%ꢀHBr,ꢀAcOH,ꢀ2.5ꢀh;ꢀ(c)ꢀ47%ꢀHBr,ꢀAcOH,ꢀ48–54ꢀh
Chart 3. Synthesis of Compounds 4a–6c
haveꢀbeenꢀcharacterized.
Ofꢀ theꢀ syntheticꢀ routesꢀ towardꢀ flavonoids,ꢀ theꢀ mostꢀ ef-
years.16–18)
In our previous report,20)ꢀweꢀhaveꢀdemonstratedꢀthatꢀflavo-
ficientꢀ wasꢀ toꢀ directꢀ Friesꢀ acylation,ꢀ catalyzedꢀ byꢀ BF3–Et2Oꢀ noidsꢀwithꢀhalide-substituentsꢀsuchꢀasꢀClꢀonꢀtheꢀB-ringꢀconsis-
forꢀ 10ꢀminꢀ underꢀ reflux,ꢀ ofꢀ trimethoxyphenolꢀ withꢀ substi- tentlyꢀpossessedꢀstrongerꢀanti-influenzaꢀactivityꢀthanꢀbaicalein,ꢀ
tuted cinnamoyl chlorides, obtainable by in-situ treatment especially at the 4′-Cl but not at the 2′-Cl. This prompted us
of the corresponding cinnamic acids with oxalyl chloride in to systematically investigate the baicalein analogs with bromo-
dichloromethane at room temperature for 2h, to produce the substitutedꢀ B-rings,ꢀ togetherꢀ withꢀ theꢀ hydroxylꢀ functionalityꢀ
chalcone intermediates 1a–mꢀ inꢀ excellentꢀ yields.ꢀ Sequentialꢀ onꢀtheꢀA-ring,ꢀtoꢀtreatꢀtheꢀinfectionꢀofꢀspecificꢀinfluenzaꢀvirus-
oxidativeꢀcyclizationꢀofꢀabove-mentionedꢀchalconeꢀwithꢀiodineꢀ esꢀofꢀH1N1ꢀTamiflu-resistantꢀandꢀseasonalꢀH3N2ꢀstrains.ꢀMostꢀ
inꢀdimethylꢀsulfoxideꢀ(DMSO)ꢀforꢀ3ꢀhꢀunderꢀrefluxꢀledꢀtoꢀtheꢀ compounds displayed salient anti-viral activities (Tables 1, 2).
isolationꢀ ofꢀ theꢀ desiredꢀ substitutedꢀ 5,6,7-trimethoxyflavonidsꢀ
Table 1 showed that the most potent compounds were 2l,
2a–m in good overall yields which is illustrated in Chart 1. 4b, 4c, 5b, 5c, 6b, and 6c that revealed an effective anti-
Ofꢀ theꢀ productsꢀ obtained,ꢀ compoundsꢀ 2a–j were subject to H1N1ꢀ Tamiflu-resistantꢀ virusꢀ activityꢀ withꢀ EC50 at around
exhaustiveꢀ demethylation.ꢀ Thus,ꢀ treatmentꢀ withꢀ 47%ꢀ HBrꢀ 4.0–4.5µM and selectivity index >70 except for 2l and 4b
inꢀ aceticꢀ acidꢀ (1ꢀ:ꢀ2,ꢀ v/v)ꢀ underꢀ refluxꢀ forꢀ 48ꢀh,ꢀ cooling,ꢀ andꢀ (SI >8 and 16, respectively). Compounds 2k, 4a, 5a, and 6a,
additionꢀ ofꢀ iceꢀ waterꢀ andꢀ filtrationꢀ gaveꢀ precipitatedꢀ crudeꢀ however, exhibited a slightly less inhibitory activity in the
products 3a–jꢀwhichꢀwereꢀre-crystallizedꢀfromꢀethanolꢀtoꢀyieldꢀ range of 8.6–16.0µMꢀ againstꢀ H1N1ꢀ Tamiflu-resistantꢀ virus.ꢀ
very pure compounds (Chart 2). The bromo-substituted 2k, 2l, Amongꢀ them,ꢀ extensiveꢀ placementꢀ ofꢀ OCH3 groups onto the
and 2m were subject to hydrolytic demethylation, dependent A-ringꢀ ofꢀ bromoflavonesꢀ undesirablyꢀ ledꢀ toꢀ augmentationꢀ
upon the meticulously controlled conditions, to yield various in cytotoxicity and reduction in inhibition (2k, 2l, and 2m).
functionalizedꢀ targetꢀ flavonoidsꢀ 4a–c, 5a–c, and 6a–c, in ex- Interestingly,ꢀ flavonoidsꢀ withꢀ B-ringsꢀ substitutedꢀ withꢀ eitherꢀ
cellentꢀyieldsꢀafterꢀpurificationꢀbyꢀcolumnꢀchromatographyꢀonꢀ 2′-Brꢀ orꢀ 3′-Brꢀ wereꢀ notꢀ affected.ꢀ Introductionꢀ ofꢀ OHꢀ groupsꢀ
silica gel (Chart 3).
ontoꢀ theꢀ A-ringꢀ ofꢀ bromoflavonesꢀ dramaticallyꢀ facilitatedꢀ theꢀ
Inꢀ mostꢀ cases,ꢀ crystallizationꢀ orꢀ columnꢀ chromatographyꢀ inhibitory activity and diminished the cytotoxicity, no matter
wasꢀ necessaryꢀ forꢀ purification.ꢀ Theꢀ structuresꢀ ofꢀ syntheticꢀ whatꢀpositionsꢀofꢀtheꢀbromineꢀatomꢀareꢀonꢀtheꢀB-ringsꢀ(5b, 5c,
intermediates and end products were established by various 6b and 6c), and thus seemed to be advantageous in general.
spectroscopyꢀ andꢀ theꢀ specificꢀ dataꢀ ofꢀ highꢀ resolutionꢀ massꢀ The presence and appropriately positioning of these hydroxyl
spectra.
residues at C-5, C-7 and/or C-6 of the A-ring appeared to be
In-Vitro Anti-influenza Virus Activity In-vitro anti- critical determinants of anti-viral potency as was proved to
influenzaꢀvirusꢀactivityꢀ wasꢀ expressedꢀ asꢀ selectiveꢀ indexꢀ(SI)ꢀ beꢀ trueꢀ inꢀ bromoflavonesꢀ 4a–c, 5a–c, and 6a–c. Substitution
which is the value of 50% cytotoxic concentration (CC50) on of a bromine atom at the meta or paraꢀpositionꢀofꢀtheꢀB-ringꢀ
Madin-Darbyꢀ canineꢀ kidneyꢀ (MDCK)ꢀ cellsꢀ dividedꢀ byꢀ theꢀ ofꢀ 5,7-dihydroxy-6-methoxyflavoneꢀ orꢀ 5,6,7-trihydroxyflavoneꢀ
50% effective concentration (EC50) on H1N1virus (SI=CC50/ (5a, 5b, 5c, 6a, 6b, and 6c) resulted in highest in vitro anti-
EC50). The higher the selective index is, the more potent or influenzaꢀ virusꢀ activityꢀ againstꢀ theꢀ H1N1ꢀ Tamiflu-resistantꢀ
less toxic the test compound is.
Evenꢀ thoughꢀ flavonoidsꢀ areꢀ aꢀ kindꢀ ofꢀ well-knownꢀ C6-C3-
strain and selective indexes, even superior to ribavirin.
In the screening test for anti-H3N2 virus activity, all the
C6ꢀ phenylbenzopyroneꢀ scaffold,ꢀ veryꢀ fewꢀ reportsꢀ haveꢀ beenꢀ syntheticꢀflavonoidsꢀillustratedꢀlessꢀpotent.ꢀHowever,ꢀintroduc-
focusedꢀ onꢀ theꢀ anti-influenzaꢀ virusꢀ activitiesꢀ ofꢀ severalꢀ fla- tionꢀ ofꢀ aꢀ Brꢀ substituentꢀ situatedꢀ atꢀ meta- or para-position of
vonoids derived from plants or Chinese medicines in the past theꢀB-ringꢀofꢀ5,7-dihydroxy-6-methoxyflavoneꢀ(5a, 5b) result-

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Table 1. In VitroꢀAnti-influenzaꢀActivitiesꢀofꢀFlavonoidsꢀwithꢀBromo-SubstitutedꢀBꢀRingsꢀinꢀMDCKꢀCellsꢀUsingꢀCPEꢀAssaya,c)
H1N1ꢀTamiflu-resistant
H3N2
Compd
R1
R2
R3
EC50
(μM)
CC50
(μM)
EC50
(μM)
CC50
(μM)
SI
SI
2k
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
16.0
4.0
128.2
32.1
8.0
>8.0
0.7
64.1
128.2
64.1
2.0
0.5
2l
2m
128.2
b)
192.3
16.1
4.2
128.2
133.0
—
>300
199.5
—
4a
8.3
133.0
133.0
—
1.5
4b
OH
66.5
>15.8
>71.4
>34.9
>69.8
>70.0
>33.3
>66.7
>66.7
>6.5
66.5
0.5
4c
OH
4.2
>300
>300
—
5a
OH
8.6
>300
>300
>300
>300
>300
>300
>300
>300
>300
17.3
17.3
34.5
71.8
35.9
35.9
92.3
103.9
25.6
>300
>300
>300
>300
>300
>300
>300
>300
>300
>17.3
>17.3
>8.7
>4.2
>8.4
>8.4
>3.3
>2.9
>11.7
5b
OH
OH
4.3
5c
OH
OH
4.3
6a
OH
OH
9.0
6b
OH
OH
OH
4.5
6c
OH
OH
OH
4.5
Baicalein
Baicalin
Ribavirin
46.2
69.3
25.6
>4.3
>11.7
a) EC50:ꢀ50%ꢀeffectiveꢀconcentration;ꢀCC50:ꢀ50%ꢀcytotoxicꢀconcentration;ꢀSIꢀ(selectiveꢀindex)=CC50/EC50. b)ꢀ—:ꢀcellꢀviabilityꢀbelowꢀ50–75%.ꢀc) In vitroꢀanti-H1N1ꢀinfluenzaꢀ
virusꢀactivityꢀofꢀoseltamivirꢀ(Tamiflu):ꢀEC50=32.0µM, CC50=320.1µM, SI=10;ꢀoseltamivirꢀisꢀvoidꢀofꢀactivityꢀagainstꢀH1N1ꢀTamiflu-resistantꢀvirus.
Table 2. In Vitroꢀ Anti-influenzaꢀ Activitiesꢀ ofꢀ Flavonoidsꢀ 3 and 6 in
At this stage, we turned to focus our effort on the sub-
stitutionꢀ effectꢀ onꢀ theꢀ B-ringꢀ ofꢀ 5,6,7-trihydroxyflavoneꢀ
(Table 2). Various functionalities with different electronic
effects were thus introduced and examined their anti-H1N1
Tamiflu-resistantꢀvirusꢀactivity.ꢀOnceꢀagain,ꢀallꢀtheꢀflavonoids-
displayedꢀ highꢀ anti-viralꢀ potencyꢀ withoutꢀ significantꢀ cellularꢀ
toxicity. However, their effective concentrations somehow ap-
peared to distribute in a wide range. Particularly noteworthy
is that, of the compounds tested, 6a–c with a bromine atom
attachedꢀ onꢀ anyꢀ positionꢀ ofꢀ theꢀ B-ringꢀ wouldꢀ exhibitꢀ aꢀ sig-
nificantlyꢀhigherꢀinhibitoryꢀactivity,ꢀinꢀtheꢀrangeꢀofꢀ4.5–9.0ꢀµM,
than the others (3a–j). It is known that the bromine atom,
owing to its intrinsic electronegativity, displays negative
inductive effect, and among other things, would manifest a
positive resonance effect after conjugation with aromatic rings
by increasing the electronic density in the conjugated systems.
Ourꢀfavorableꢀresultꢀdistinctlyꢀrevealedꢀtheꢀsignificanceꢀofꢀtheꢀ
impactꢀ ofꢀ bromo-substitutionꢀ inꢀ theꢀ B-ring,ꢀ whichꢀ wasꢀ neverꢀ
foundꢀ inꢀ theꢀ structuresꢀ ofꢀ naturally-occurringꢀ flavonoids,ꢀ onꢀ
theꢀ anti-H1N1ꢀ Tamiflu-resistantꢀ virus.ꢀ Interestingly,ꢀ parallel-
ingꢀtheꢀbromo-substitutedꢀflavonoidsꢀ(6a–c), two of the mono-
chloro substituted products (3b and 3d) still demonstrated
highly promising in terms of their prominent selective index-
es.ꢀAsꢀmentionedꢀabove,ꢀintroductionꢀofꢀaꢀBr-substituentꢀontoꢀ
theꢀ B-ringꢀ ofꢀ 5,6,7-trihydroxyflavoneꢀ (6a–c) exhibited much
betterꢀinhibitionꢀofꢀH1N1ꢀTamiflu-resistantꢀvirusꢀthanꢀtheꢀcor-
responding Cl-substituent (3b–d), especially on the 3′-position
ofꢀ theꢀ B-ring.ꢀ Surprisingly,ꢀ inꢀ theꢀ casesꢀ ofꢀ theꢀ flavonoidsꢀ
substitutedꢀ withꢀ twoꢀ halogensꢀ onꢀ theꢀ B-ringsꢀ (3e–g), all of
them unexpectedly displayed rather poor anti-viral potency,
MDCKꢀCellsꢀUsingꢀCPEꢀAssaya,c)
H1N1ꢀTamiflu-resistantꢀvirus
Compd
R
EC50 (μM)
CC50 (μM)
SI
3a
H
92.3
10.2
50.0
10.2
73.7
>300
>300
>300
>300
>300
—
>3.3
>29.4
>6.0
>29.4
>4.1
—
3b
2′-Cl
3′-Cl
4′-Cl
3c
3d
3e
2,′4′-(Cl)2
3,′4′-(Cl)2
3′-Br,ꢀ4′-F
4′-COOH
4′-Ph
4′-NO2
2′-Br
3′-Br
b)
3f
—
3g
204.4
39.7
72.0
18.1
9.0
>300
>300
>300
>300
>300
>300
>300
>300
>300
>1.5
>7.6
>4.2
>16.6
>33.3
>66.7
>66.7
>3.3
>11.7
3h
3i
3j
6a
6b
4.5
6c
4′-Br
H
4.5
Baicalein
Ribavirin
92.3
25.6
a) EC50:ꢀ 50%ꢀ effectiveꢀ concentration;ꢀ CC50:ꢀ 50%ꢀ cellꢀ cytotoxicꢀ concentra-
tion; SI (selective index)=CC50/EC50. b)ꢀ—:ꢀ cellꢀ viabilityꢀ belowꢀ 50–75%.ꢀ c) In
vitroꢀ anti-H1N1ꢀ influenzaꢀ virusꢀ activityꢀ ofꢀ oseltamivirꢀ (Tamiflu):ꢀ EC50=32.0µM,
CC50=320.1µM, SI=10;ꢀ oseltamivirꢀ isꢀ voidꢀ ofꢀ activityꢀ againstꢀ H1N1ꢀ Tamiflu-
resistant virus.
ed in better inhibition (EC50=17.3 µM, SI >17.3) than ribavirin. even one of the substituted functionality was deliberately in-
All these compounds presented higher in vitro anti-H1N1 troduced as a bromine atom. Enhancement of the anti-H1N1
Tamiflu-resistantꢀvirusꢀactivityꢀthanꢀanti-H3N2ꢀvirusꢀactivity.
Tamiflu-resistantꢀ virusꢀ activityꢀ withꢀ para substitution on the

May 2014ꢀ
419
B-ringsꢀ followedꢀ theꢀ orderꢀ Br>Cl>NO2>COOH>Ph. The the National Tsing-Hua University, Taiwan, using a Finnigan
influenceꢀ ofꢀ theꢀ electron-releasingꢀ orꢀ electron-withdrawingꢀ MAT-95XL.ꢀAllꢀtheꢀsolventsꢀandꢀreagentsꢀwereꢀobtainedꢀfromꢀ
groupsꢀ inꢀ theꢀ B-ringsꢀ ofꢀ theꢀ flavonoidsꢀ isꢀ apparentꢀ andꢀ com- commercialꢀsourcesꢀandꢀpurifiedꢀbeforeꢀuseꢀifꢀnecessary.
plex but nevertheless not very clear.
General Procedure for the Synthesis of Compounds
Byꢀ analyzingꢀ theꢀ EC50 and CC50 values shown in the 2a–m To a solution of cinnamic acid (10mmol) in dichlo-
tables,ꢀ weꢀ noticedꢀ thatꢀ thereꢀ existedꢀ twoꢀ generalizationsꢀ inꢀ romethane (30mL) in an ice bath was added a mixture of
theꢀ followingꢀ structure–activityꢀ relationships:ꢀ (1)ꢀ theꢀ bromo- oxalyl chloride (1.5mL, 17.5mmol) and dimethylformamide (2
flavonoidsꢀ wereꢀ generallyꢀ moreꢀ activeꢀ andꢀ selectiveꢀ thanꢀ theꢀ drops). After the mixture was stirred for 2h at room tempera-
otherꢀflavonoids,ꢀevenꢀtheꢀcorrespondingꢀchloro-flavonoids;ꢀ(2)ꢀ ture, the solvent was removed under reduced pressure to give
substitutionꢀ ofꢀ aꢀ bromineꢀ atomꢀ atꢀ anyꢀ carbonꢀ inꢀ theꢀ B-rings,ꢀ cinnamoyl chloride which was mixed with 3,4,5-trimethoxy-
togetherꢀ withꢀ allꢀ freeꢀ hydroxylsꢀ orꢀ withꢀ C6-OCH3 in the A- phenolꢀ(1.8ꢀg,ꢀ10ꢀmmol)ꢀandꢀthenꢀdissolvedꢀinꢀboronꢀtrifluorideꢀ
rings, consistently led to tremendous improvements in activity etherateꢀ(10ꢀmL)ꢀandꢀheatedꢀtoꢀrefluxꢀforꢀ10ꢀmin.ꢀAfterꢀcooling,ꢀ
andꢀselectivityꢀinꢀtheꢀflavonoids.
the mixture was poured into ice water and then the precipitate
wasꢀfilteredꢀandꢀwashedꢀwithꢀwaterꢀuntilꢀfreeꢀfromꢀacid.ꢀTheꢀ
resulting precipitate was washed with hexane and then with
Conclusion
Weꢀhaveꢀsuccessfullyꢀpreparedꢀaꢀnovelꢀseriesꢀofꢀflavonoids,ꢀ ether to obtain the corresponding chalcones 1a–mꢀ (89–97%)ꢀ
and demonstrated that certain members of the products pos- as orange-yellow powders. A mixture of chalcones 1a–m
sessedꢀsignificantꢀanti-influenzaꢀviralꢀactivity.ꢀItꢀisꢀnoteworthyꢀ (10ꢀmmol)ꢀandꢀiodineꢀ(1.0ꢀeq)ꢀinꢀDMSOꢀ(25ꢀmL)ꢀwasꢀheatedꢀtoꢀ
thatꢀ theꢀ syntheticꢀ flavonoidsꢀ withꢀ theꢀ bromineꢀ substitutedꢀ onꢀ refluxꢀforꢀ3ꢀh.ꢀAfterꢀcooling,ꢀtheꢀmixtureꢀwasꢀpouredꢀintoꢀiceꢀ
theꢀ B-ringꢀ andꢀ hydroxylsꢀ onꢀ theꢀ A-rings,ꢀ consistentlyꢀ dis- water.ꢀTheꢀprecipitateꢀwasꢀfilteredꢀandꢀwashedꢀwithꢀsaturatedꢀ
played extremely potent and very high selective indexes. This sodium thiosulfate solution. The residue was chromatographed
isꢀtheꢀfirstꢀreportꢀonꢀsystematicꢀinvestigationꢀaboutꢀtheꢀbromo- onꢀaꢀsilicaꢀgelꢀcolumnꢀwithꢀhexane/EtOAcꢀ(2ꢀ:ꢀ1)ꢀasꢀtheꢀeluentꢀ
bearingꢀ flavonidsꢀ asꢀ novelꢀ anti-H1N1-TR-infectedꢀ influenzaꢀ to give pure 2a–m as pale yellow solids. The preparation of
virusꢀ inꢀ aꢀ MDCKꢀ cellꢀ line.ꢀ Amongꢀ theꢀ productsꢀ obtained,ꢀ 3a–j (Chart 2) were essentially according to our published
the 3′-Brꢀ substitutedꢀ (5b, 6b) and the 4′-Brꢀ substitutedꢀ (5c, procedure.20) All the physicochemical and spectroscopic char-
6c) products have shown the most promising. We believed acteristics of products 2a–j and 3a–j are in agreement with
that the substituted bromine atoms might also contribute to those reported.20)
the inhibition of resistance in viruses. Taking their facile syn-
2-(2-Bromophenyl)-5,6,7-trimethoxy-4H-chromen-4-one
theses into consideration, they were superior to the licensed (2k)ꢀ ꢀYield:ꢀ 61%.ꢀ mp:ꢀ 122–123°C.ꢀ UVꢀ λmaxꢀ (MeOH)ꢀ nmꢀ
oseltamivirꢀ (Tamiflu)ꢀ inꢀ everyꢀ experimentꢀ andꢀ mightꢀ beꢀ fa- (logε):ꢀ 215ꢀ (4.56).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 2994,ꢀ 2934,ꢀ 2346,ꢀ 1648,ꢀ
vorable alternatives in the management of H1N1-TR-infected 1604. HR-electron impact (EI)-MS m/z:ꢀ 390.0110ꢀ (M+), Calcd
influenzaꢀ inꢀ clinicꢀ practice.ꢀ Whileꢀ theꢀ roleꢀ ofꢀ theꢀ bromineꢀ for C18H15BrO5:ꢀ 390.0103.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ
atomꢀinꢀ theꢀaromaticꢀ B-rings,ꢀ however,ꢀ isꢀ toꢀ beꢀ clarified,ꢀtheꢀ 3.77ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.81ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.91ꢀ (3H,ꢀ s,ꢀ OCH3),
results gleaned from this study have provided a basis to facili- 6.34ꢀ (1H,ꢀ s,ꢀ ArH),ꢀ 7.07ꢀ (1H,ꢀ s,ꢀ CHCO),ꢀ 7.48–7.59ꢀ (2H,ꢀ m,ꢀ
tate the design and further development of new agents for the ArH), 7.71 (1H, dd, J=9.5,ꢀ5.7ꢀHz,ꢀArH),ꢀ7.82ꢀ(1H,ꢀdd,ꢀJ=9.5,ꢀ
treatmentꢀ (prophylaxisꢀ and/orꢀ therapy)ꢀ ofꢀ influenza.ꢀ Weꢀ haveꢀ 5.7ꢀHz,ꢀ ArH).ꢀ 13C-NMRꢀ (75ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 56.4,ꢀ 60.9,ꢀ
chosen these four compounds (5b, 5c, 6b, 6c) for further phar- 61.7,ꢀ97.1,ꢀ112.1,ꢀ112.7,ꢀ121.1,ꢀ128.1,ꢀ131.2,ꢀ132.4,ꢀ133.2,ꢀ133.4,ꢀ
macokinetic investigations and their preliminary results were 140.1,ꢀ151.8,ꢀ154.4,ꢀ157.9,ꢀ161.2,ꢀ175.3.
alsoꢀ satisfactory.ꢀ Detailedꢀ pharmacokinetic,ꢀ pharmacologicalꢀ
2-(3-Bromophenyl)-5,6,7-trimethoxy-4H-chromen-4-one
and toxicological studies are in progress and will be reported (2l)ꢀ ꢀYield:ꢀ 59%.ꢀ mp:ꢀ 190–191°C.ꢀ UVꢀ λmaxꢀ (MeOH)ꢀ nmꢀ
subsequently.
(logε):ꢀ 265ꢀ (4.24),ꢀ 216ꢀ (4.48).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 3088,ꢀ 2941,ꢀ
2834, 1654, 1604. HR-EI-MS m/z:ꢀ 390.0095ꢀ (M+), Calcd for
C18H15BrO5:ꢀ 390.0103.ꢀ 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ3.77ꢀ
Experimental Section
Chemistry All the chemicals were purchased from (3H,ꢀ s,ꢀ OCH3),ꢀ 3.80ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.95ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 6.89ꢀ
Aldrich-Sigmaꢀ Chemicalꢀ Companyꢀ (St.ꢀ Louis,ꢀ MO,ꢀ U.S.A.),ꢀ (1H,ꢀs,ꢀArH),ꢀ7.30ꢀ(1H,ꢀs,ꢀCHCO),ꢀ7.51ꢀ(1H,ꢀt,ꢀJ=8.3ꢀHz,ꢀArH),ꢀ
andꢀ Alfa-Aesarꢀ Chemicalꢀ Companyꢀ (Heysham,ꢀ LA32XY,ꢀ 7.77 (1H, dd, J=8.3ꢀHz,ꢀ 1.2ꢀHz,ꢀ ArH),ꢀ 8.07ꢀ (1H,ꢀ d,ꢀ J=8.3ꢀHz,ꢀ
England)ꢀ andꢀ usedꢀ withoutꢀ furtherꢀ purification.ꢀ Allꢀ reactionsꢀ ArH), 8.27 (1H, t, J=2.0ꢀHz,ꢀ ArH).ꢀ 13C-NMRꢀ (75ꢀMHz,ꢀ
were routinely monitored by TLC on Merck F254 silica gel DMSO-d6) δ:ꢀ56.4,ꢀ60.8,ꢀ61.6,ꢀ97.4,ꢀ108.3,ꢀ112.1,ꢀ122.4,ꢀ125.0,ꢀ
plates. Merck silica gel (70–230 mesh) was used for chro- 128.4,ꢀ 131.0,ꢀ 133.3,ꢀ 134.0,ꢀ 140.0,ꢀ 151.5,ꢀ 153.93,ꢀ 157.7,ꢀ 158.5,ꢀ
matography.ꢀ Meltingꢀ pointsꢀ wereꢀ measuredꢀ onꢀ aꢀ Büchi-530ꢀ 175.5.
melting point apparatus. UV-VIS spectra were recorded on
2-(4-Bromophenyl)-5,6,7-trimethoxy-4H-chromen-4-one
aꢀ Shimazuꢀ UV-160ꢀAꢀ UV-Visbleꢀ recordingꢀ spectrophotom- (2m)ꢀ ꢀYield:ꢀ 67%.ꢀ mp:ꢀ 165–166°C.ꢀ UVꢀ λmaxꢀ (MeOH)ꢀ nmꢀ
eter. IR spectra were registered on a Perkin-Elmer FTIR 1610 (logε):ꢀ 310ꢀ (4.34),ꢀ 268ꢀ (3.32),ꢀ 216ꢀ (4.50).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ
seriesꢀ infraredꢀ spectrophotometerꢀ inꢀ KBrꢀ discs.ꢀ Theꢀ 1H- and 3084,ꢀ2945,ꢀ2841,ꢀ1656,ꢀ1604.ꢀHR-EI-MSꢀm/z:ꢀ 390.0110ꢀ (M+),
13C-NMR spectra were determined on a Varian Gemini-300 Calcd for C18H15BrO5:ꢀ 390.0103.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-
NMRꢀ instrumentꢀ inꢀ DMSO-d6 unless otherwise noted. d6) δ:ꢀ 3.76ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.79ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.94ꢀ (3H,ꢀ s,ꢀ
Chemical shifts (δ) were reported as parts per million (ppm) OCH3),ꢀ 6.86ꢀ (1H,ꢀ s,ꢀ ArH),ꢀ 7.23ꢀ (1H,ꢀ s,ꢀ CHCO),ꢀ 7.76ꢀ (2H,ꢀ
downfieldꢀ fromꢀ tetramethylsilaneꢀ (TMS)ꢀ asꢀ theꢀ internalꢀ stan- d, J=8.6ꢀHz,ꢀ ArH),ꢀ 8.01ꢀ (2H,ꢀ d,ꢀ J=8.6ꢀHz,ꢀ ArH).ꢀ 13C-NMR
dard (σ 0.00), and coupling constants (J)ꢀwereꢀgivenꢀinꢀhertzꢀ (75ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 56.3,ꢀ 60.8,ꢀ 61.6,ꢀ 97.2,ꢀ 107.8,ꢀ 112.1,ꢀ
(Hz).ꢀHighꢀresolutionꢀmassꢀspectraꢀ(HR-MS)ꢀwereꢀperformedꢀ 125.0,ꢀ 127.9,ꢀ 130.2,ꢀ 132.0,ꢀ 134.0,ꢀ 151.6,ꢀ 153.9,ꢀ 157.7,ꢀ 159.2,ꢀ
in the Instrument Center of the National Science Counsel at 175.6.

420
Vol. 62, No. 5
General Procedure for the Synthesis of Compounds 4a–c 3588, 3061, 2363, 1670, 1615. HR-EI-MS m/z:ꢀ 361.9789ꢀ
1
Flavones 2k–mꢀ (1.0ꢀmmol)ꢀ wereꢀ placedꢀ inꢀ BF3–Et2Oꢀ (3ꢀmL)ꢀ (M+), Calcd for C16H11BrO5:ꢀ 361.9790.ꢀ H-NMRꢀ (300ꢀMHz,ꢀ
andꢀthenꢀheatedꢀtoꢀrefluxꢀforꢀ45ꢀmin,ꢀrespectively.ꢀAfterꢀcool- DMSO-d6) δ:ꢀ 3.92ꢀ (3H,ꢀ s,ꢀ OCH3), 7.02 (1H, s, ArH), 7.07
ing, the mixture was poured into ice water. The precipitate (1H,ꢀ s,ꢀ CHCO),ꢀ 7.52ꢀ (1H,ꢀ t,ꢀ J=8.0ꢀHz,ꢀ ArH),ꢀ 7.79ꢀ (1H,ꢀ dd,ꢀ
wasꢀfilteredꢀandꢀwashedꢀwithꢀwater.ꢀTheꢀresidueꢀwasꢀchroma- J=9.3,ꢀ 6.9ꢀHz,ꢀ ArH),ꢀ 8.10ꢀ (1H,ꢀ d,ꢀ J=8.1ꢀHz,ꢀ ArH),ꢀ 8.29ꢀ (1H,ꢀ
tographedꢀonꢀaꢀsilicaꢀgelꢀcolumnꢀwithꢀhexane/EtOAcꢀ(3ꢀ:ꢀ1)ꢀasꢀ t, J=1.8ꢀHz,ꢀ ArH),ꢀ 8.77ꢀ (1H,ꢀ s,ꢀ OH),ꢀ 12.40ꢀ (1H,ꢀ s,ꢀ OH).ꢀ
the eluent to give pure 4a–c as pale yellow solids.
13C-NMRꢀ (75ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 56.2,ꢀ 91.4,ꢀ 105.4,ꢀ 105.5,ꢀ
2-(2-Bromophenyl)-5-hydroxy-6,7-dimethoxy-4H-chro- 122.4,ꢀ 125.3,ꢀ 128.7,ꢀ 130.2,ꢀ 131.1,ꢀ 133.2,ꢀ 134.4,ꢀ 146.2,ꢀ 149.8,ꢀ
men-4-one (4a)ꢀ ꢀYield:ꢀ 69%.ꢀ mp:ꢀ 221–222°C.ꢀ UVꢀ λmax 154.8, 161.4, 182.2.
(MeOH)ꢀ nmꢀ (logꢀε):ꢀ 211ꢀ (4.50).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 3092,ꢀ
2-(4-Bromophenyl)-5,7-dihydroxy-6-methoxy-4H-chro-
3023,ꢀ 2945,ꢀ 1645.ꢀ HR-EI-MSꢀ m/z:ꢀ 375.9946ꢀ (M+), Calcd for men-4-one (5c)ꢀ ꢀYield:ꢀ 85%.ꢀ mp:ꢀ 275–276°C.ꢀ UVꢀ λmax
C17H13BrO5:ꢀ375.9946.ꢀ1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ3.74ꢀ (MeOH)ꢀ nmꢀ (logꢀε):ꢀ 282ꢀ (4.16),ꢀ 218ꢀ (4.08).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ
(3H,ꢀ s,ꢀ OCH3),ꢀ 3.90ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 6.59ꢀ (1H,ꢀ s,ꢀ ArH),ꢀ 6.85ꢀ 3370,ꢀ3084,ꢀ2364,ꢀ2321,ꢀ1669,ꢀ1609.ꢀHR-EI-MSꢀm/z:ꢀ 361.9792ꢀ
(1H,ꢀs,ꢀCHCO),ꢀ7.50–7.60ꢀ(2H,ꢀm,ꢀArH),ꢀ7.73ꢀ(1H,ꢀdd,ꢀJ=9.2,ꢀ (M+), Calcd for C16H11BrO5:ꢀ 361.9790.ꢀ H-NMRꢀ (300ꢀMHz,ꢀ
1
5.9ꢀHz,ꢀArH),ꢀ7.84ꢀ(1H,ꢀdd,ꢀJ=9.2,ꢀ5.9ꢀHz,ꢀArH),ꢀ12.61ꢀ(1H,ꢀs,ꢀ DMSO-d6) δ:ꢀ3.91ꢀ(3H,ꢀs,ꢀOCH3),ꢀ6.96ꢀ(1H,ꢀs,ꢀArH),ꢀ7.04ꢀ(1H,ꢀ
OH).ꢀ13C-NMRꢀ(75ꢀMHz,ꢀDMSO-d6) δ:ꢀ56.4,ꢀ59.9,ꢀ91.6,ꢀ105.3,ꢀ s,ꢀCHCO),ꢀ7.79ꢀ(2H,ꢀd,ꢀJ=8.9ꢀHz,ꢀArH),ꢀ8.04ꢀ(2H,ꢀd,ꢀJ=8.9ꢀHz,ꢀ
110.2,ꢀ 120.9,ꢀ 128.1,ꢀ 131.4,ꢀ 132.4,ꢀ 132.7,ꢀ 133.0,ꢀ 133.5,ꢀ 152.1,ꢀ ArH),ꢀ8.79ꢀ(1H,ꢀs,ꢀOH),ꢀ12.70ꢀ(1H,ꢀs,ꢀOH).ꢀ13C-NMRꢀ(75ꢀMHz,ꢀ
153.2,ꢀ159.2,ꢀ164.4,ꢀ182.0.
DMSO-d6) δ:ꢀ 56.2,ꢀ 91.3,ꢀ 105.0,ꢀ 105.3,ꢀ 125.6,ꢀ 128.2,ꢀ 130.2,ꢀ
2-(3-Bromophenyl)-5-hydroxy-6,7-dimethoxy-4H-chro- 130.2,ꢀ132.1,ꢀ146.2,ꢀ149.8,ꢀ154.8,ꢀ162.1,ꢀ182.3.
men-4-one (4b)ꢀ ꢀYield:ꢀ 64%.ꢀ mp:ꢀ 188–189°C.ꢀ UVꢀ λmax
General Procedure for the Synthesis of Compounds 6a–c
(MeOH)ꢀnmꢀ(logꢀε):ꢀ272ꢀ(4.26),ꢀ215ꢀ(4.41).ꢀIRꢀ(KBr)ꢀν cm−1:ꢀ3071,ꢀ Toꢀ aꢀ solutionꢀ ofꢀ flavonesꢀ 2k–m (1.0mmol) in glacial acetic
2931,ꢀ2836,ꢀ1654,ꢀ1622.ꢀHR-EI-MSꢀm/z:ꢀ375.9933ꢀ(M+), Calcd acid (20mL) in the ice bath was added 47% hydrobromic acid
for C17H13BrO5:ꢀ 375.9946.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ (10ꢀmL).ꢀ Theꢀ mixtureꢀ wasꢀ heatedꢀ toꢀ refluxꢀ forꢀ 48–54ꢀh.ꢀ Afterꢀ
3.73ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.93ꢀ (3H,ꢀ s,ꢀ OCH3), 7.03 (1H, s, ArH), cooling, the mixture was poured into ice water. The resulting
7.11ꢀ (1H,ꢀ s,ꢀ CHCO),ꢀ 7.52ꢀ (1H,ꢀ t,ꢀ J=8.1ꢀHz,ꢀ ArH),ꢀ 7.80ꢀ (1H,ꢀ precipitateꢀwasꢀfilteredꢀandꢀwashedꢀwithꢀwaterꢀuntilꢀfreeꢀfromꢀ
dd, J=8.1ꢀHz,ꢀ1.5ꢀHz,ꢀArH),ꢀ8.10ꢀ(1H,ꢀd,ꢀJ=8.1ꢀHz,ꢀArH),ꢀ8.29ꢀ acidꢀ asꢀ monitoredꢀ byꢀ theꢀ pHꢀ paper.ꢀ Re-crystallizationꢀ fromꢀ
(1H, t, J=1.8ꢀHz,ꢀArH),ꢀ12.66ꢀ(1H,ꢀs,ꢀOH).ꢀ13C-NMRꢀ(75ꢀMHz,ꢀ ethanol afforded pure 6a–c as yellow-brown solids.
DMSO-d6) δ:ꢀ56.4,ꢀ59.9,ꢀ91.8,ꢀ105.4,ꢀ105.8,ꢀ122.4,ꢀ125.4,ꢀ128.8,ꢀ
2-(2-Bromophenyl)-5,6,7-trihydroxy-4H-chromen-4-one
(6a)ꢀ ꢀYield:ꢀ 75%.ꢀ mp:ꢀ 249–251°C.ꢀ UVꢀ λmaxꢀ (MeOH)ꢀ nmꢀ
131.1,ꢀ132.2,ꢀ133.0,ꢀ134.6,ꢀ152.0,ꢀ152.7,ꢀ159.0,ꢀ161.7,ꢀ182.3.
2-(4-Bromophenyl)-5-hydroxy-6,7-dimethoxy-4H-chro- (logε):ꢀ 213ꢀ (4.55).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 3416,ꢀ 3378,ꢀ 3053,ꢀ 1665,ꢀ
men-4-one (4c)ꢀ ꢀYield:ꢀ 72%.ꢀ mp:ꢀ 206–208°C.ꢀ UVꢀ λmax 1618. HR-EI-MS m/z:ꢀ 347.9637ꢀ (M+), Calcd for C15H9BrO5:ꢀ
(MeOH)ꢀ nmꢀ (logꢀε):ꢀ 278ꢀ (4.19),ꢀ 215ꢀ (4.30).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 347.9633.ꢀ H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 6.46ꢀ (1H,ꢀ s,ꢀ
1
2919,ꢀ2852,ꢀ1670,ꢀ1624.ꢀHR-EI-MSꢀm/z:ꢀ375.9929ꢀ(M+), Calcd ArH),ꢀ6.50ꢀ(1H,ꢀs,ꢀCHCO),ꢀ7.48–7.59ꢀ(2H,ꢀm,ꢀArH),ꢀ7.71ꢀ(1H,ꢀ
for C17H13BrO5:ꢀ 375.9946.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ dd, J=9.0,ꢀ 5.7ꢀHz,ꢀ ArH),ꢀ 7.82ꢀ (1H,ꢀ sd,ꢀ J=8.3ꢀHz,ꢀ ArH),ꢀ 8.82ꢀ
3.73ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 3.93ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 6.99ꢀ (1H,ꢀ s,ꢀ ArH),ꢀ (1H,ꢀs,ꢀOH),ꢀ10.59ꢀ(1H,ꢀs,ꢀOH),ꢀ12.48ꢀ(1H,ꢀs,ꢀOH).ꢀ13C-NMR
7.08ꢀ (1H,ꢀ s,ꢀ CHCO),ꢀ 7.79ꢀ (2H,ꢀ d,ꢀ J=8.6ꢀHz,ꢀ ArH),ꢀ 8.05ꢀ (2H,ꢀ (75ꢀMHz,ꢀDMSO-d6) δ:ꢀ93.9,ꢀ104.2,ꢀ109.6,ꢀ121.0,ꢀ128.1,ꢀ129.5,ꢀ
d, J=8.6ꢀHz,ꢀ ArH),ꢀ 12.70ꢀ (1H,ꢀ s,ꢀ OH).ꢀ 13C-NMRꢀ (75ꢀMHz,ꢀ 131.3,ꢀ132.6,ꢀ133.3,ꢀ133.5,ꢀ147.1,ꢀ150.2,ꢀ153.9,ꢀ163.8,ꢀ181.8.
DMSO-d6) δ:ꢀ56.4,ꢀ59.9,ꢀ91.7,ꢀ105.3,ꢀ105.4,ꢀ125.8,ꢀ128.3,ꢀ129.6,ꢀ
129.9,ꢀ132.1,ꢀ152.0,ꢀ152.8,ꢀ159.0,ꢀ162.5,ꢀ182.3.
2-(3-Bromophenyl)-5,6,7-trihydroxy-4H-chromen-4-one
(6b)ꢀ ꢀYield:ꢀ 72%.ꢀ mp:ꢀ 255–256°C.ꢀ UVꢀ λmaxꢀ (MeOH)ꢀ nmꢀ
General Procedure for the Synthesis of Compounds 5a–c (logε):ꢀ 276ꢀ (4.23),ꢀ 211ꢀ (4.41).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 3358,ꢀ 3100,ꢀ
Toꢀ aꢀ solutionꢀ ofꢀ flavonesꢀ 2k–m (1.0mmol) in glacial acetic 2893.0,ꢀ 1654,ꢀ 1618.ꢀ HR-EI-MSꢀ m/z:ꢀ 347.9631ꢀ (M+), Calcd for
acid (20mL) in the ice bath was added 47% hydrobromic acid C15H9BrO5:ꢀ 347.9633.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 6.65ꢀ
(10ꢀmL).ꢀ Theꢀ mixtureꢀ wasꢀ heatedꢀ toꢀ refluxꢀ forꢀ 2.5ꢀh.ꢀ Afterꢀ (1H,ꢀs,ꢀArH),ꢀ7.00ꢀ(1H,ꢀs,ꢀCHCO),ꢀ7.51ꢀ(1H,ꢀt,ꢀJ=8.1ꢀHz,ꢀArH),ꢀ
cooling, the mixture was poured into ice water. The resulting 7.78 (1H, dd, J=8.1,ꢀ1.5ꢀHz,ꢀArH),ꢀ8.06ꢀ(1H,ꢀdd,ꢀJ=8.1,ꢀ1.5ꢀHz,ꢀ
precipitateꢀwasꢀfilteredꢀandꢀwashedꢀwithꢀwaterꢀuntilꢀfreeꢀfromꢀ ArH), 8.24 (1H, t, J=1.5ꢀHz,ꢀ ArH),ꢀ 8.83ꢀ (1H,ꢀ s,ꢀ OH),ꢀ 10.59ꢀ
acidꢀ asꢀ monitoredꢀ byꢀ theꢀ pHꢀ paper.ꢀ Re-crystallizationꢀ fromꢀ (1H,ꢀ s,ꢀ OH),ꢀ 12.56ꢀ (1H,ꢀ s,ꢀ OH).ꢀ 13C-NMRꢀ (75ꢀMHz,ꢀ DMSO-
ethanol afforded pure 5a–c as yellow solids.
d6) δ:ꢀ 94.1,ꢀ 104.3,ꢀ 105.4,ꢀ 122.4,ꢀ 125.3,ꢀ 128.7,ꢀ 129.4,ꢀ 131.1,ꢀ
2-(2-Bromophenyl)-5,7-dihydroxy-6-methoxy-4H-chro- 133.4,ꢀ134.3,ꢀ147.0,ꢀ149.9,ꢀ153.8,ꢀ161.2,ꢀ182.0.
men-4-one (5a)ꢀ ꢀYield:ꢀ 77%.ꢀ mp:ꢀ 161–162°C.ꢀ UVꢀ λmax
2-(4-Bromophenyl)-5,6,7-trihydroxy-4H-chromen-4-one
(MeOH)ꢀnmꢀ(logꢀε):ꢀ211ꢀ(4.54).ꢀIRꢀ(KBr)ꢀν cm−1:ꢀ3092,ꢀ3066,ꢀ (6c)ꢀ ꢀYield:ꢀ 80%.ꢀ mp:ꢀ 301–303°C.ꢀ UVꢀ λmaxꢀ (MeOH)ꢀ nmꢀ
3005, 1680, 1622. HR-EI-MS m/z:ꢀ 361.9791ꢀ (M+), Calcd for (logε):ꢀ 265ꢀ (4.19),ꢀ 216ꢀ (4.27).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ 3412,ꢀ 3102,ꢀ
C16H11BrO5:ꢀ 361.9790.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 1654, 1618. HR-EI-MS m/z:ꢀ 347.9642ꢀ (M+), Calcd for
3.89ꢀ (3H,ꢀ s,ꢀ OCH3),ꢀ 6.53ꢀ (1H,ꢀ s,ꢀ ArH),ꢀ 6.83ꢀ (1H,ꢀ s,ꢀ CHCO),ꢀ C15H9BrO5:ꢀ 347.9633.ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 6.61ꢀ
7.51–7.58 (2H, m, ArH), 7.73 (1H, dd, J=6.3,ꢀ 1.8ꢀHz,ꢀ ArH),ꢀ (1H,ꢀ s,ꢀ ArH),ꢀ 6.96ꢀ (1H,ꢀ s,ꢀ CHCO),ꢀ 7.76ꢀ (2H,ꢀ dd,ꢀ J=7.8ꢀHz,ꢀ
7.83 (1H, dd, J=5.4ꢀHz,ꢀ1.5ꢀHz,ꢀArH),ꢀ8.83ꢀ(1H,ꢀs,ꢀOH),ꢀ12.34ꢀ 1.8ꢀHz,ꢀ ArH),ꢀ 8.01ꢀ (2H,ꢀ dd,ꢀ J=6.9,ꢀ 2.1ꢀHz,ꢀ ArH),ꢀ 8.80ꢀ (1H,ꢀ
(1H,ꢀ s,ꢀ OH).ꢀ 13C-NMRꢀ (75ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 56.3,ꢀ 91.2,ꢀ s,ꢀ OH),ꢀ 10.57ꢀ (1H,ꢀ s,ꢀ OH),ꢀ 12.58ꢀ (1H,ꢀ s,ꢀ OH).ꢀ 13C-NMR
105.2,ꢀ 109.9,ꢀ 121.0,ꢀ 128.1,ꢀ 130.4,ꢀ 131.3,ꢀ 132.6,ꢀ 133.2,ꢀ 133.5,ꢀ (75ꢀMHz,ꢀDMSO-d6) δ:ꢀ94.0,ꢀ104.4,ꢀ104.8,ꢀ125.5,ꢀ128.2,ꢀ129.0,ꢀ
146.3,ꢀ150.2,ꢀ154.9,ꢀ164.1,ꢀ182.0.
129.4,ꢀ130.3,ꢀ131.4,ꢀ132.1,ꢀ147.1,ꢀ149.9,ꢀ153.8,ꢀ161.9,ꢀ182.1.
2-(3-Bromophenyl)-5,7-dihydroxy-6-methoxy-4H-chro-
Assay of Cytopathic Effect (CPE) The anti-viral activ-
men-4-one (5b)ꢀ ꢀYield:ꢀ 72%.ꢀ mp:ꢀ 249–250°C.ꢀ UVꢀ λmax ityꢀ ofꢀ flavonoidsꢀ wasꢀ measuredꢀ byꢀ theꢀ CPEꢀ assayꢀ andꢀ usingꢀ
(MeOH)ꢀ nmꢀ (logꢀε):ꢀ 276ꢀ (4.33),ꢀ 216ꢀ (4.46).ꢀ IRꢀ (KBr)ꢀ ν cm−1:ꢀ ribavirin as a positive control. The CPE inhibition assays used

May 2014
421
in this study were performed as described previously.24) Two
influenzaꢀ virusꢀ strains,ꢀ namelyꢀ Tamiflu-resistantꢀ 2009ꢀ pan-
demicꢀ influenzaꢀ Aꢀ (H1N1)ꢀ virus,ꢀ whichꢀ detectedꢀ theꢀ H275Yꢀ
mutationꢀ (N1ꢀ numbering)ꢀ inꢀ neuraminidase,ꢀ andꢀ influenzaꢀ
A/Newꢀ York/469/2004-likeꢀ fluꢀ (H3N2)ꢀ virus,ꢀ wereꢀ providedꢀ
byꢀ Centersꢀ forꢀ Diseaseꢀ Controlꢀ (CDC),ꢀ Taiwan,ꢀ andꢀ adaptedꢀ
for evaluating the in vitro anti-viral activities of the synthetic
flavonoids.
(2011).
ꢀ 4)ꢀ MonsalvoꢀA.ꢀC.,ꢀBatalleꢀJ.ꢀP.,ꢀLopezꢀM.ꢀF.,ꢀKrauseꢀJ.ꢀC.,ꢀKlemencꢀ
J.,ꢀ Hernandezꢀ J.ꢀ Z.,ꢀ Maskinꢀ B.,ꢀ Bugnaꢀ J.,ꢀ Rubinsteinꢀ C.,ꢀ Aguilarꢀ
L.,ꢀ Dalurzoꢀ L.,ꢀ Libsterꢀ R.,ꢀ Savyꢀ V.,ꢀ Baumeisterꢀ E.,ꢀ Aguilarꢀ L.,ꢀ
Cabralꢀ G.,ꢀ Fontꢀ J.,ꢀ Solariꢀ L.,ꢀ Wellerꢀ K.ꢀ P.,ꢀ Johnsonꢀ J.,ꢀ Echavarriaꢀ
M.,ꢀEdwardsꢀK.ꢀM.,ꢀChappellꢀJ.ꢀD.,ꢀCroweꢀJ.ꢀE.ꢀJr.,ꢀWilliamsꢀJ.ꢀV.,ꢀ
Melendi G. A., Polack F. P., Nat. Med., 17,ꢀ195–199ꢀ(2011).
ꢀ 5)ꢀ NeumannꢀG.,ꢀNodaꢀT.,ꢀKawaokaꢀY.,ꢀNature (London), 459,ꢀ931–939ꢀ
(2009).
Inꢀbrief,ꢀvirusꢀatꢀ100ꢀtissueꢀcultureꢀinfectiousꢀdoseꢀ(TCID50)
wereꢀ inoculatedꢀ ontoꢀ nearꢀ confluentꢀ MDCKꢀ cellꢀ monolay-
ers (1×105ꢀ cells/well)ꢀ forꢀ 1ꢀh.ꢀ Afterꢀ beingꢀ incubatedꢀ atꢀ 37°Cꢀ
for 2h, the virus solution was removed, and 100µLꢀ sequen-
tialꢀ 2-foldꢀ serialꢀ dilutionsꢀ ofꢀ theꢀ respectiveꢀ flavonoidsꢀ andꢀ
reference compound ribavirin were added to each well of
theꢀ 96-wellꢀ cultureꢀ plates,ꢀ usingꢀ theꢀ maximalꢀ non-cytotoxicꢀ
concentration (MNCC, i.e.,ꢀ 90%ꢀ viableꢀ cells)ꢀ asꢀ theꢀ highestꢀ
concentration.ꢀ Anꢀ infectionꢀ controlꢀ withoutꢀ flavonoidsꢀ wasꢀ
alsoꢀ included.ꢀ Theꢀ platesꢀ wereꢀ incubatedꢀ atꢀ 37°Cꢀ inꢀ aꢀ 75%ꢀ
humidityꢀ ofꢀ 5%ꢀ CO2 atmosphere for 24h, and then the CPE
6) Fuyuno I., Nature (London), 446,ꢀ358–359ꢀ(2007).
7) Moscona A., N. Engl. J. Med., 360,ꢀ953–956ꢀ(2009).
ꢀ 8)ꢀ GooskensꢀJ.,ꢀJongesꢀM.,ꢀClaasꢀE.ꢀC.,ꢀMeijerꢀA.,ꢀvanꢀdenꢀBroekꢀP.ꢀJ.,ꢀ
KroesꢀA.ꢀM.,ꢀJAMA, 301,ꢀ1042–1046ꢀ(2009).
ꢀ 9)ꢀ ChimentiꢀF.,ꢀFioravantiꢀR.,ꢀBolascoꢀA.,ꢀChimentiꢀP.,ꢀSecciꢀD.,ꢀRossiꢀ
F.,ꢀYanezꢀM.,ꢀOralloꢀF.,ꢀOrtusoꢀF.,ꢀAlcaroꢀS.,ꢀCirilliꢀR.,ꢀFerrettiꢀR.,ꢀ
Sanna M. L., Bioorg. Med. Chem., 18,ꢀ1273–1279ꢀ(2010).
10)ꢀ ChoiꢀH.ꢀJ.,ꢀSongꢀJ.ꢀH.,ꢀParkꢀK.ꢀS.,ꢀKwonꢀD.ꢀH.,ꢀEur. J. Pharm. Sci.,
37,ꢀ329–333ꢀ(2009).
11)ꢀ WooꢀK.ꢀJ.,ꢀLimꢀJ.ꢀH.,ꢀSuhꢀS.ꢀI.,ꢀKwonꢀY.ꢀK.,ꢀShinꢀS.ꢀW.,ꢀKimꢀS.ꢀC.,ꢀ
ChoiꢀY.ꢀH.,ꢀParkꢀJ.ꢀW.,ꢀKwonꢀT.ꢀK.,ꢀImmunobiology, 211,ꢀ359–368ꢀ
(2006).
wasꢀobserved.ꢀTheꢀvirus-inducedꢀCPEꢀwasꢀscoredꢀasꢀfollows:ꢀ 12)ꢀ OrhanꢀD.ꢀD.,ꢀOzcelikꢀB.,ꢀOzgenꢀS.,ꢀErgunꢀF.,ꢀMicrobiol. Res., 165,
496–504ꢀ(2010).
13) Friedman M., Mol. Nutr. Food Res., 51, 116–134 (2007).
14) Huang W. H., Lee A. R., Yang C. H., Biosci. Biotechnol. Biochem.,
70, 2371–2380 (2006).
15)ꢀ NevesꢀM.ꢀP.,ꢀCidadeꢀH.,ꢀPintoꢀM.,ꢀSilvaꢀA.ꢀM.ꢀS.,ꢀGalesꢀL.,ꢀDamasꢀ
A. M., Lima R. T., Vasconcelos M. H., Nascimento M. S. J., Eur. J.
Med. Chem., 46, 2562–2574 (2011).
16)ꢀ NagaiꢀT.,ꢀMiyaichiꢀY.,ꢀTomimoriꢀT.,ꢀSuzukiꢀY.,ꢀYamadaꢀH.,ꢀAntivi-
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 mul-
tiplication was calculated as a percentage of the virus control
(% virus control=CPEexp/CPEvirus control×100). The IC50 of
the CPE with respect to virus control was estimated using the
Reed–Muench method and was expressed in μM. The selectiv-
ity index (SI) was calculated from the ratio CC50/IC50.
ral Res., 19,ꢀ207–217ꢀ(1992).
Statistical Analysis
17)ꢀ NagaiꢀT.,ꢀMoriguchiꢀR.,ꢀSuzukiꢀY.,ꢀTomimoriꢀT.,ꢀYamadaꢀH.,ꢀAnti-
viral Res., 26,ꢀ11–25ꢀ(1995).
18)ꢀ XuꢀG.,ꢀDouꢀJ.,ꢀZhangꢀL.,ꢀGuoꢀQ.,ꢀZhouꢀC.,ꢀBiol. Pharm. Bull., 33,
238–243 (2010).
19)ꢀ ChenꢀL.,ꢀDouꢀJ.,ꢀSuꢀZ.,ꢀZhouꢀH.,ꢀWangꢀH.,ꢀZhouꢀW.,ꢀGuoꢀQ.,ꢀZhouꢀ
C., Antiviral Res., 91, 314–320 (2011).
20) Lee A. R., Liao C. W., Chang W. L., Yao C. W., Huang W. H.,
JCMRD, 1, 28–36 (2012).
Statistical calculations were carried out with Microsoft
Excel 2007 version. Results are expressed as the means ꢀS .D .ꢀ
of six independent experiments.
Acknowledgments We gratefully acknowledge the re-
searchꢀ Grants,ꢀ NSCꢀ 99–2320-B-016–005-MY3ꢀ andꢀ 100-EC-
17-A-20-S1-028, supported from the National Science Coun-
cil and the Ministry of Economic Affairs of the Republic of
China.
21) Huang W. H., Chien P. Y., Yang C. H., Lee A. R., Chem. Pharm.
Bull., 51,ꢀ339–340ꢀ(2003).
22)ꢀ KimꢀD.ꢀH.,ꢀYunꢀC.ꢀH.,ꢀKimꢀM.ꢀH.,ꢀNaveenꢀKumarꢀC.,ꢀYunꢀB.ꢀH.,ꢀ
ShinꢀJ.ꢀS.,ꢀAnꢀH.ꢀJ.,ꢀLeeꢀY.ꢀH.,ꢀYunꢀY.ꢀD.,ꢀRimꢀH.ꢀK.,ꢀYooꢀM.ꢀS.,ꢀ
LeeꢀK.ꢀT.,ꢀLeeꢀY.ꢀS.,ꢀBioorg. Med. Chem. Lett., 22, 700–705 (2012).
23)ꢀ Phamꢀ T.ꢀ A.,ꢀ Cheꢀ H.,ꢀ Phanꢀ P.ꢀ T.,ꢀ Leeꢀ J.ꢀ W.,ꢀ Kimꢀ S.ꢀ S.,ꢀ Parkꢀ H.,ꢀ
Bioorg. Med. Chem. Lett., 22, 2534–2535 (2012).
References
ꢀ 1)ꢀ DeꢀClercqꢀE.,ꢀNat. Rev. Drug Discov., 5, 1015–1025 (2006).
2) Lambert L. C., Fauci A. S., N. Engl. J. Med., 363, 2036–2044
(2010).
24) Mosmann T., J. Immunol. Methods, 65,ꢀ55–63ꢀ(1983).
3) Medina R. A., Garcia-Sastre A., Nat. Rev. Microbiol., 9,ꢀ 590–603ꢀ