Synthesis and antiviral activity of substituted quercetins

Article abstract of DOI:10.1016/j.bmcl.2011.10.119

Influenza viruses are important pathogens that cause respiratory infections in humans and animals. In addition to vaccination, antiviral drugs against influenza virus play a significant role in controlling viral infections by reducing disease progression and virus transmission. Plant derived polyphenols are associated with antioxidant activity, anti-carcinogenic, and cardio- and neuro-protective actions. Some polyphenols, such as resveratrol and epigallocatechin gallate (EGCG), showed significant anti-influenza activity in vitro and/or in vivo. Recently we showed that quercetin and isoquercetin (quercetin-3-β-d-glucoside), a glucoside form of quercetin, significantly reduced the replication of influenza viruses in vitro and in vivo (isoquercetin). The antiviral effects of isoquercetin were greater than that of quercetin with lower IC₅₀ values and higher in vitro therapeutic index. Thus, we investigated the synthesis and antiviral activities of various quercetin derivatives with substitution of C3, C3′, and C5 hydroxyl functions with various phenolic ester, alkoxy, and aminoalkoxy moieties. Among newly synthesized compounds, quercetin-3-gallate which is structurally related to EGCG showed comparable antiviral activity against influenza virus (porcine H1N1 strain) to that of EGCG with improved in vitro therapeutic index.

Full text of DOI:10.1016/j.bmcl.2011.10.119

Bioorganic & Medicinal Chemistry Letters 22 (2012) 353–356
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiviral activity of substituted quercetins
Mahendra Thapa a, Yunjeong Kim b, John Desper a, Kyeong-Ok Chang b, Duy H. Hua a,
⇑
a Department of Chemistry, Kansas State University, Manhattan, KS 66506, United States
b Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Influenza viruses are important pathogens that cause respiratory infections in humans and animals. In
addition to vaccination, antiviral drugs against influenza virus play a significant role in controlling viral
infections by reducing disease progression and virus transmission. Plant derived polyphenols are associ-
ated with antioxidant activity, anti-carcinogenic, and cardio- and neuro-protective actions. Some poly-
phenols, such as resveratrol and epigallocatechin gallate (EGCG), showed significant anti-influenza
Received 8 October 2011
Revised 27 October 2011
Accepted 31 October 2011
Available online 6 November 2011
activity in vitro and/or in vivo. Recently we showed that quercetin and isoquercetin (quercetin-3-b-D-glu-
Keywords:
coside), a glucoside form of quercetin, significantly reduced the replication of influenza viruses in vitro
and in vivo (isoquercetin). The antiviral effects of isoquercetin were greater than that of quercetin with
lower IC50 values and higher in vitro therapeutic index. Thus, we investigated the synthesis and antiviral
activities of various quercetin derivatives with substitution of C3, C30, and C5 hydroxyl functions with
various phenolic ester, alkoxy, and aminoalkoxy moieties. Among newly synthesized compounds, quer-
cetin-3-gallate which is structurally related to EGCG showed comparable antiviral activity against influ-
enza virus (porcine H1N1 strain) to that of EGCG with improved in vitro therapeutic index.
Ó 2011 Elsevier Ltd. All rights reserved.
Antiviral activity
Influenza virus
Porcine H1N1 strain
Quercetin-3-gallate
Substituted quercetins
Influenza infections are responsible for over 3 million cases of
illness and up to a half of million deaths per year.1 Although antivi-
ral drugs against influenza infections are available, the emergence
of viral resistance to existing antiviral drugs emphasizes the de-
mand for development of new antiviral drugs against influenza
infections. In the past few years, polyphenols including quercetin
(1) and its analogs,2–4 quercetin-3-b-galactoside,5 quercetin 3-
Among the derivatives, quercetin-3-gallate (4) which is structurally
related to EGCG showed comparable antiviral activity against influ-
enza virus porcine H1N1 as that of EGCG with improved in vitro
therapeutic index.
For the synthesis of C3-O-ester analogs of quercetin, a reported
selective protection of the three most reactive hydroxyl functions
at C7, C30, and C40 was adapted.12 Hence, 30,40,7-tri-O-benzylqu-
ercetin (11), derived from tribenzylation of rutin with potassium
carbonate and benzyl bromide in DMF followed by removal of
the C3-rutinose with hydrochloric acid and ethanol, was con-
densed with 3,4,5-tribenzylgallic acid (12),13 N-ethyl-N0-(3-
dimethylaminopropyl)carbodiimide (EDC), and a catalytic amount
of 4-dimethylaminopyridine (DMAP) in dichloromethane to give
ester 16 in 64% yield (Scheme 1). Removal of the benzyl ether pro-
tecting groups of 16 with hydrogen (15 psi) and 5% palladium over
carbon afforded quercetin gallate 4 (68% yield). It should be noted
that C5-hydroxyl function is less reactive than C3-hydroxyl due to
intramolecular hydrogen bonding and resonance conjugation with
C4-keto function.
Similarly, condensation of compound 11 with 2,5-dibenzyloxy-
benzoic acid (13),14,15 4-benzyloxy-3-(N,N-dibenzylamino)benzoic
acid (14), and 3-benzyloxy-4-(N,N-dibenzylamino)benzoic acid
(15) separately gave benzyl esters 17, 18, and 19 (83%, 75%, and
71% yield), respectively, which upon hydrogenation furnished
respectively quercetin analogs 5, 6, and 7 (61%, 58%, and 70% yield)
(Scheme 1). Compounds 12 and 13 are known and were prepared
from a modified procedure of benzylation of gallic acid (20) and
2,5-dihydroxybenzoic acid (21), respectively, with sodium hydride
rhamnoside,6 quercetin 3-b- -glucoside or isoquercetin (2)7
D
(Fig. 1) were reported effective against influenza infections. Among
them, isoquercetin (2) demonstrated a lower IC50 value against
influenza viruses and higher therapeutic index compared to that
of quercetin.7 (ꢀ)-Epigallocatechin-3-gallate (EGCG; 3) is the major
polyphenol in green tea and reported to have anti-influenza virus
activity8,9 and organic anion-transporting enhancing property.10
Quercetin-3-gallate (4) has been reported to be effective for the
treatment of inflammatory bowel disease by inhibiting Na+-K+-
ATPase and/or Na+/H+ exchange activities.11 However, the synthesis
and anti-influenza virus activities of 4 and its hydroxyl aryl analogs
have not been reported. A hybrid of quercetin and gallate or its ana-
logs may show improved antiviral activities. Therefore antiviral
activities of derivatives of quercetin with various substitutions at
the hydroxyl functions were investigated. Herein, we report the
synthesis and anti-influenza virus activities of compounds 4–10
containing C3-dihydroxybenzoate, C3-aminohydroxybenzoates,
C30-gallate, C30-aminopropyloxy, and C5-propyloxy functions.
⇑
Corresponding author. Tel.: +1 785 532 6699; fax: +1 785 532 6666.
E-mail address: duy@ksu.edu (D.H. Hua).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.119

354
M. Thapa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 353–356
OH
OH
OH
OH
OH
OH
HO
O
OH
O
HO
O
O
HO
O
O
O
HO
HO
O
OH
OH
OH
OH
OH
O
OH
OH
OH
Quercetin (1)
Isoquercetin (2)
OH
EGCG (3)
OH
OH
OH
OH
OH
OH
3'
4'
HO
O
HO
O
HO
O
O
2
3
7
5
O
O
O
O
OH
O
OH
OH
OH
O
OH
O
3"
O
R2
4"
OH
R1
HO
OH
6 : R1 = NH2, R2 = OH
7 : R1 = OH, R2 = NH2
5
Quercetin-3-gallate (4)
OH
OH
O
NH2
OH
O
OH
HO
O
OH
OH
HO
O
O
O
HO
O
O
OH
OH
O
OH
OH
OH
O
OH
8
9
10
Figure 1. Quercetin, Isoquercetin, EGCG, quercetin-3-gallate and synthesized quercetin analogs.
OBn
OBn
OBn
OBn
R4
OH
3'
4'
EDC
BnO
O
O
R3
R2
DMAP
BnO
O
O
O
7
+
3
CH2Cl2
O
5
R4
OH
OH
R3
R2
R1
OH
O
12 : R1 = R2 = R3 = OBn, R4 = H
13 : R1 = R4 = OBn, R2 = R3 = H
11 : Bn = CH2Ph
R1
14 : R1 = NBn2, R2 = OBn, R3 = R4 = H
15 : R1 = OBn, R2 = NBn2, R3 = R4 = H
16 : R1 = R2 = R3 = OBn, R4 = H
17 : R1 = R4 = OBn, R2 = R3 = H
18 : R1 = NBn2, R2 = OBn, R3 = R4 = H
19 : R1 = OBn, R2 = NBn2, R3 = R4 = H
H2, Pd/C
4 ~ 7
R4
OH
R5
R4
NaH
PhCH2Br
R3
NaOH
R3
R2
MeOH, H2O
O
O
12 ~ 15
R2
dioxane
DMF
80oC
R1
R1
20 : R1 = R2 = R3 = OH, R4 = H
21 : R1 = R4 = OH, R2 = R3 = H
22 : R1 = NH2, R2 = OH, R3 = R4 = H
23 : R1 = OH, R2 = NH2, R3 = R4 = H
24 : R1 = R2 = R3 = R5 = OBn, R4 = H
25 : R1 = R4 = R5 = OBn, R2 = R3 = H
26 : R1 = NBn2, R2 = R5 = OBn, R3 = R4 = H
27 : R1 = R5 = OBn, R2 = NBn2, R3 = R4 = H
Scheme 1. Synthesis of quercetin 3-O-analogs 4–7.

M. Thapa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 353–356
355
OR
OBn
Na2CO3
DMF
3'
4'
BnO
O
1
7
3
OBn
O
5
PhCH2Cl
80 C
º
OH
28 : R = H (63% yield)
29 : R = Bn (18% yield)
OBn
EDC
O
DMAP
H2, Pd/C
BnO
BnO
O
O
OBn
OBn
28
+
12
8
O
CH2Cl2
(35% yield)
(56% yield)
OBn
OBn
OH
OBn
30
OBn
O
NaH, DMF
1) H2NNH2
(85% yield)
º
80 C (40% yield)
O
O
28
9
O
N
2)H2, Pd/C
(58% yield)
O
O
I
N
OH
32
OBn
(31)
O
OBn
H2, Pd/C
(90% yield)
NaH, DMF
allyl bromide
29
BnO
O
O
10
(88% yield)
OBn
O
33
Scheme 2. Syntheses of quercetin C30 and C5 analogs.
(or sodium carbonate) and benzyl bromide in DMF13,15 followed by
basic hydrolysis with sodium hydroxide (Scheme 1). Carboxylic
acids 22 and 23 were similarly tetrabenzylated and hydrolyzed
to give benzylated arylcarboxylic acids 14 and 15 in 76% and 87%
overall yield, respectively.
To access quercetin analogs containing C30-O- and C5-O-sub-
stituents, we prepared hydroxyl-protected quercetins, compounds
28 and 29, as described previously16 from 1 and sodium carbonate
and 3 equiv of benzyl chloride in DMF (Scheme 2). A mixture of
tribenzyl 28 (63% yield) and tetrabenzyl 29 (18% yield) were iso-
lated after column chromatographic separation. As mentioned
above, due to intramolecular hydrogen bonding, C5-hydroxyl func-
tion is less reactive toward an electrophile than C30-hydroxyl,
hence selective esterification of 28 with tribenzyl gallic acid 12,
EDC, and DMAP afforded C30-gallic ester 30. Deprotection of the
benzyl ether functions of 30 with hydrogen and palladium gave
quercetin C30-gallate 8 (Scheme 2).
Figure 2. An ORTEP drawing of X-ray crystallographically determined structure of
compound 32.
Selective C30-alkylation can similarly be carried out. Hence,
alkylation of 28 with sodium hydride and N-(3-iodopropyl)phthal-
imide (31)17,18 gave compound 32 (40% yield), which was sub-
jected to reduction with hydrazine followed by hydrogen and
palladium afforded 3-aminopropyl quercetin 9 (49% overall yield).
The structure of compound 32 was unequivocally determined by a
single-crystal x-ray analysis (Fig. 2).19 The crystal is triclinic, space
group of P-1, and the R factor of 0.051. A mole of water was
revealed in the crystal structure of 32.
either tribenzyl protected gallic acid 12 or tribenzyl protected gal-
loyl chloride and pyridine or sodium hydride at elevated tempera-
ture. Likely, the bulky 3,4,5-tribenzyloxybenzoyl group could not
fit into the C5-hydroxyl function. However, smaller electrophiles
such as allyl bromide can be attached to C5-hydroxyl group of
29. Alkylation of quercetin 29 with sodium hydride and allyl bro-
mide gave C5-O-allyl quercetin 33 in 88% yield, which upon reduc-
tion with hydrogen and palladium furnished C5-O-propyl
derivative 10 (90% yield). Allyl bromide was chosen as the alkylat-
ing agent instead of propyl iodide because a higher yield was
produced.
Among five hydroxyl moieties, C5-hydroxyl function is the least
reactive hydroxyl group of quercetin. We were unable to esterify
C5-hydroxyl function of tetrabenzyl protected quercetin 29 with

356
M. Thapa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 353–356
cetin 11 and various arylcarboxylic acids, and C30- and C5-
substituted quercetins were synthesized from C3,C40,C7-O-triben-
zyl- and C3,C30,C40,C7-O-tetrabenzyl-protected quercetin, respec-
tively. Subsequent global removal of benzyl protecting groups
afforded substituted quercetins. C5-Esterification with gallic acid
12 failed, but alkylation was possible. The synthetic sequence is
short and amenable to large scale synthesis, and the synthesized
C3-analogs have comparable antiviral activity as that of EGCG
implying further modification at C3 is possible in improving
efficacy.
Table 1
Values of effective dose required to reduce the replication of virus by 50% (ED50) and
toxic dose for 50% cell death (TD50) of various natural and synthetic compounds
Compound
ED50 value
in
TD50 value
in
Therapeutic
index (TI)
lM
lM
Quercetin (1)
Isoquercetin (2)
EGCG (3)
Quercetin-3-gallate (4)
5
6
7
8
48.2
1.2
8.3
83.4
45.1
45.5
90.2
45.2
60.1
54.8
NDa
ND
1.7
37.6
5.5
9.9
2.3
2.7
2.3
—
9.1
19.4
22.6
24.1
>50
>50
>50
>50
Acknowledgments
9
10
29
—
—
—
ND
ND
This work has been supported by the National Institutes of
Health, National Institute of Allergy and Infectious Diseases (U01
AI081891).
a
ND: not determined due to high ED50 values. Note: Quercetin, isoquercetin and
EGCG were purchased from Sigma–Aldrich (St. Louis, MO).
Supplementary data
The inhibition of influenza
A virus (A/swine/OH/511445/
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.119.
2007[H1N1], Oh7) by compounds 1–10 and 29 was evaluated fol-
lowing a procedure reported previously.7 In brief, Madin-Darby ca-
nine kidney (MDCK) cells were infected with influenza virus Oh7
and incubated with each compound at various concentrations from
References and notes
0.1–150 lM in the presence of trypsin (10 lg/mL) for up to 4 days.
1. Russell, C. A.; Jones, T. C.; Barr, I. G.; Cox, N. J.; Garten, R. J.; Gregory, V.; Gust, I.
D.; Hampson, A. W.; Hay, A. J.; Hurt, A. C.; de Jong, J. C.; Kielso, A.; Klimov, A. I.;
Kageyama, T.; Komadina, N.; Lapedes, A. S.; Lin, Y. P.; Mosterin, A.; Obuchi, M.;
Odagiri, T.; Osterhaus, A. D.; Rimmelzwaan, G. F.; Shaw, M. W.; Skepner, E.;
Stohr, K.; Tashiro, M.; Fouchier, R. A.; Smith, D. J. Vaccine 2008, 26, D31.
2. Liu, A. –L.; Wang, H. –D.; Lee, S. M.; Wang, Y. –T.; Du, G. –H. Bioorg. Med. Chem.
2008, 16, 7141.
3. Lin, C. –W.; Tsai, F. –J.; Tsai, C. –H.; Lai, C. –C.; Wan, L.; Ho, T. –Y.; Hsieh, C. –C.;
Chao, P. –D. L. Antiviral Res. 2005, 68, 36.
4. Ryu, Y. B.; Kim, J. H.; Park, S. –J.; Chang, J. S.; Rho, M. –C.; Bae, K. –H.; Park, K. H.;
Lee, W. S. Bioorg. Med. Chem. Lett. 2010, 20, 971.
Cytopathic effects (CPE) of the cells were observed for each com-
pound. The effective doses for 50% virus reduction (ED50) and toxic
doses at 50% cell death (TD50) of the above compounds are summa-
rized in Table 1. Viral replication was also confirmed by immuno-
fluorescence assay and Western blot analysis with antibodies to
the viral nucleoprotein or whole influenza virus, respectively.
Among the tested compounds, isoquercetin, EGCG, quercetin-3-
gallate appear to have similar ED50 values against H1N1 virus,
and isoquercetin (2) is the most active compound with a ED50 va-
5. Chen, L.; Li, J.; Luo, C.; Liu, H.; Xu, W.; Chen, G.; Liew, O. W.; Zhu, W.; Puah, C.
M.; Shen, X.; Jiang, H. Bioorg. Med. Chem. 2006, 14, 8295.
lue of 1.2
mined at 8.3 and 9.1
l
M. The ED50 of EGCG and quercetin-3-gallate was deter-
M, respectively, and therapeutic index (TI)
6. Choi, J. J.; Song, J. H.; Park, K. S.; Kwon, D. H. Eur. J. Pharm. Sci. 2009, 37, 329.
7. Kim, Y.; Narayanan, S.; Chang, K. –O. Antiviral Res. 2010, 88, 227.
8. Song, J. –M.; Lee, K. –H.; Seong, B. –L. Antiviral Res. 2005, 68, 66.
9. Nagle, D. G.; Ferreira, D.; Zhou, Y. –D. Phytochemistry 2006, 67, 1849.
10. Roth, M.; Timmermann, B. N.; Hagenbuch, B. Drug Metab. Dispos. 2011, 39, 920.
11. Soares-Da-Silva, P. M. V. A. PCT Int. Appl. WO 2004/084886, 2004, 36 pages.
12. Huang, H.; Jia, Q.; Ma, J.; Qin, G.; Chen, Y.; Xi, Y.; Lin, L.; Zhu, W.; Ding, J.; Jiang,
H.; Liu, H. Eur. J. Med. Chem. 2009, 44, 1982.
13. Belin, F.; Barthelemy, P.; Ruiz, K.; Lacombe, J. M.; Pucci, B. Helv. Chim. Acta 2003,
86, 247.
14. Hayashi, K.; Hamada, Y.; Shioiri, T. Tetrahedron Lett. 1992, 33, 5075.
15. Yamada, K.; Ikezaki, M.; Umino, N.; Ohtsuka, H.; Itoh, N.; Ikezawa, K.;
Kiyomoto, A.; Iwakuma, T. Chem. Pharm. Bull. 1981, 29, 744.
16. Wang, R. E.; Kao, J. L.-F.; Hilliard, C. A.; Pandita, R. K.; Roti Roti, J. L.; Hunt, C. R.;
Taylor, J.-S. J. Med. Chem. 2009, 52, 1912.
17. Kaji, E.; Zen, S. Bull. Chem. Soc. Jpn. 1973, 46, 337.
18. Shi, A.; Nguyen, T. A.; Battina, S. K.; Rana, S.; Takemoto, D. J.; Chiang, P. K.; Hua,
D. H. Bioorg. Med. Chem. Lett. 2008, 18, 3364.
19. The authors have deposited atomic coordinates for the structures, compound
32 with the Cambridge Crystallographic Data Centre as supplementary
publication number CCDC 175022. The coordinates can be obtained on
request, from the Director, Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge, CB2 1EZ, U.K.
l
value of quercetin-3-gallate of 9.9 is slightly better than that of
EGCG of 5.5. C3-Dihydroxyl and hydroxylaminobenzoate analogs,
compounds 5–7, have ED50 values of 20–24 lM and similar TD50
values as that of EGCG indicating modification of the gallate moiety
(of EGCG and quercetin-3-gallate) retains the anti-influenza activ-
ity. Quercetin (1), without gallate function, is less effective with
ED50 and TD50 values of 48.2 and 83.4 lM, respectively. Despite
having a gallate function at C30, compound 8, and C30-3-aminopro-
pyloxy analog 9 and C5-propyloxy compound 10 are not effective
up to 50
effective up to 50
l
M. Benzylated analogs such as compound 29 are not
M. Therefore the TD50 values of compounds
l
8–10 and 29 were not determined. It appears that derivatization
of C30 and C5 led to lower antiviral activity.
In conclusion, various C3, C30, and C5 substituted quercetins
were synthesized and their anti-H1N1 activities were examined.
C3-Substituted quercetins were derived from a carbodiimide-acti-
vated coupling reaction of C30,C40,C7-O-tribenzyl-protected quer-