Design, synthesis, and biological evaluation of a novel series of quercetin diacylglucosides as potent anti-MRSA and anti-VRE agents

Article abstract of DOI:10.1016/j.bmcl.2010.02.060

A series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition of different multi-drug resistant bacterial strains and Escherichia coli DNA gyrase B was investigated. In the series, compound 10h was up to 128-fold more potent against vancomycin-resistant enterococci and more effective than A, which represents a promising new candidate as a potent anti-MRSA and anti-VRE agent.

Full text of DOI:10.1016/j.bmcl.2010.02.060

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5349–5352
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of a novel series of quercetin
diacylglucosides as potent anti-MRSA and anti-VRE agents
b
a
b
b
Abugafar M. L. Hossion a, , Nao Otsuka , Rafiya K. Kandahary , Tomofusa Tsuchiya , Wakano Ogawa ,
*
Akimasa Iwado a, Yoshito Zamami a, Kenji Sasaki a,
a Department of Molecular Design for Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-Naka, Kita-Ku,
*
Okayama 700-8530, Japan
b Department of Molecular Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-Naka, Kita-Ku, Okayama
700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification
on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition of different multi-
drug resistant bacterial strains and Escherichia coli DNA gyrase B was investigated. In the series, com-
pound 10h was up to 128-fold more potent against vancomycin-resistant enterococci and more effective
than A, which represents a promising new candidate as a potent anti-MRSA and anti-VRE agent.
Ó 2010 Published by Elsevier Ltd.
Received 5 January 2010
Revised 10 February 2010
Accepted 13 February 2010
Available online 19 February 2010
Keywords:
Antibacterials
Multi-drug resistance
Quercetin-3-glucoside
The increasing emergence of multi-drug resistant Gram-posi-
tive bacterial pathogens, including methicillin-resistant Staphylo-
coccus aureus (MRSA), vancomycin-resistant enterococci (VRE)
and recently found vancomycin-intermediate resistant S. aureus
(VISA) become a serious global clinical problem for the treatment
of various nosocomial and community-acquired infections.1 In
order to overcome these bacterial resistance problems, numerous
efforts have focused on discovering novel anti-MRSA, anti-VRE,
and anti-VISA agents in recent decades.2 Thus, newly disclosed
antibacterial agents such as tetracycline, linezolid, quinupristin–
dalforpristin, tigecycline, trimethoprim–sulfamethoxazole, and
daptomycin, have all been used alone or in combination with other
agents for the treatment of some MRSA, VRE or VISA infections.
Notwithstanding, the limitations of these drugs have been well
documented in the literature3 and there remains a concern for
the critically ill, high-risk patient population. In view of increasing
resistance of Gram-positive bacteria to currently available antibac-
terial agents, new and potent anti-MRSA, VRE, and VISA agents are
highly desired.
recently been isolated and reported as a potent anti-MRSA agent.8,9
To assist the development of novel anti-MRSA agents, as well as to
investigate structure–activity relationship for the related series of
compound A, a new series of 2-(30,40-dihydroxyphenyl)-5,7-dihy-
droxy-3-b-D
-glucosyl-4H-chromen-4-one 200,300-diesters 10a–h
commonly known as quercetin diacylglucosides was designed
and synthesized for the first time. Meanwhile, the replacements
of kaempferol by quercetin and 2,4-di-O-(4-hydroxycinnamoyl)-
OH
4'
3'
OH
OH
1
HO
O
1a
4a
HO
O
7
3
O
O
O
5
OH O
1"
OH O
6"
O
O
O
O
O
3"
HO
1"'
OH
HOO
O
3"'
O
1"'
1""
O
3"'
Flavonoids are bioactive polyphenolic compounds. They have
significant inhibitions towards the growth of both Gram-positive
and Gram-negative bacterial strains4 and are prospective drug can-
didiates.5 A novel kaempferol diacylrhamnoside A6–9 (Fig. 1) has
1""
OH
4""
F
4""
F
OH
A (MIC: 4 or 8 µg/mL,
10h (MIC: 1 µg/mL,
against VRE strains).
against VRE strains).
* Corresponding authors. Tel.: +81 (0) 86 251 7971; fax: +81 (0) 86 251 7926.
E-mail addresses: chem.lokman@gmail.com (A.M.L. Hossion), ksasaki@phea-
sant.pharm.okayama-u.ac.jp (K. Sasaki).
Figure 1. Kaempferol diacylrhamnoside A and quercetin diacylglucoside 10h.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.02.060

5350
A. M. L. Hossion et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5349–5352
OH
OH
Ph
Ph
Ph
Ph
O
O
O
O
HO
O
BnO
O
O
HO
O
a, b
c
d
OG
G=
OG
OH
OH O
BnO
OH O
AcO
O
AcO
3
2
1
OAc
AcO
(for 8)
h
Ph
Ph
OH
OH
O
O
O
BnO
O
O
O
O
HO
O
OR1
OH
f
OR1
e
g
h
O
O
OR2
OH
BnO
OH O
O OR1
O
O OH
O
HO OR1
OH
OR2
OR3
8, 9, 10a-h
OH
OH
R3O
OH
5
6a-h
7a-h
4
6,7 a: R1 = COC6H4(4-OEt)
b: R1 = COC6H4(4-Pr)
c: R1 = COC6H4(4-F)
8: R2 = R3 = COCH3
9: R2 = R3 = H
10a: R2 = COC6H4(4-OEt); R3 = H
b: R2 = COC6H4(4-Pr); R3 = H
h (for 9)
d: R1 = COCH2C6H4(4-F)
4
e: R1 = COCH=CHC6H4(4-OCH2Ph)
f: R1 = COCH=CHC6H4(4-Me)
g: R1 = COCH=CHC6H4(4-OEt)
h: R1 = COCH=CHC6H4(4-F)
c: R2 = COC6H4(4-F); R3 = H
d: R2 = COCH2C6H4(4-F); R3 = H
e: R2 = COC2H4C6H4(4-OH); R3 = H
f: R2 = COC2H4C6H4(4-Me); R3 = H
g: R2 = COC2H4C6H4(4-OEt); R3 = H
h: R2 = COC2H4C6H4(4-F); R3 = H
Scheme 1. Reagents and conditions: (a) Ph2CCl2, 170 °C, 8–10 min; (b) 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide, anhydrous K2CO3, anhydrous acetone, 40 °C, 10 h;
(c) BnBr, anhydrous K2CO3, anhydrous DMF, rt, 4 h; (d) (i) MeONa, MeOH–THF, rt, 1.5 h, (ii) Dowex 50 (H+) resin; (e) anhydrous acetone, concd H2SO4, anhydrous CuSO4, rt,
24 h; (f) aliphatic or aromatic carboxylic acids, DMAP, DCC, anhydrous CH2Cl2, 0 °C–rt, 6–8 h; (g) 0.5 M HCl, MeOH–THF, 50–60 °C, 2–3 h; (h) Pd/C (10%), H2, MeOH–EtOAc, rt,
8–10 h.
a
-
L
-rhamnose by 2,3-disubstituted b-
D
-glucopyranose have con-
Synthesized compounds 8, 9, 10a–h, and quercetin were
screened for their potential antibacterial activity in vitro against
eight selected multi-drug resistant Gram-positive and two Gram-
negative bacterial strains, including MRSA, MSSA, VRE, VISA,
Pseodomonas aeruginosa and E. coli strains (Table 1). Norfloxacin,
vancomycin, and novobiocin were used as reference strains stan-
dards. Encouraged by promising inhibition by quercetin of E. coli
DNA gyrase B,13 compounds 9 and 10a–h bearing quercetin moiety
also were investigated for in vitro inhibitions towards same en-
zyme DNA gyrase B from E. coli (Table 1). Furthermore, AutoDock
modeling14 was performed (Table 1) to explore the probable bind-
ing conformation inside the ATP binding site of 24 kDa fragment of
the DNA gyrase B subunit from E. coli (PDB accession code 1AJ6).
Most of the tested compounds showed pronounced inhibitions of
E. coli DNA gyrase B. Among those, compound 10h showed the
siderably enhanced the inhibition towards the growth of bacterial
strains.
Scheme 1 depicts a convenient route for the synthesis of com-
pounds 8, 9, and 10a–h. Quercetin 1 was reacted with
a,a-dic-
hlorodiphenylmethane to protect the 30- and 40-hydroxyl groups.
Then, the 30,40-protected compound was regioselectively glucosy-
lated with one equiv. of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl
bromide in the presence of 1 equiv of K2CO3 in dry DMF to give
the compound 210 as the major product (61%). Since the higher
reactivity at position 3 of starting compound allows the selective
glucosylation, trace amounts of 3,7-di-glucosylated quercetin was
detected and ignored. The free hydroxyl groups of compound 2
were then protected with excess benzyl bromide in the presence
of anhydrous K2CO3 in dry DMF to give compound 3. The removal
of acetyl group of 3 with MeONa in MeOH followed by treatment
with Dowex 50 (H+) ion exchange resin gave compound 4. Reaction
of 4 with acetone in the presence of anhydrous copper sulfate and a
catalytic amount of sulfuric acid gave a key intermediate 5. The
200,300-dihydroxy groups of compound 5 were esterified by Steglich
esterification11 with different aliphatic or aromatic carboxylic
acids in the presence of DMAP and DCC to obtain compounds
6a–h. The isopropylidene protecting group of 6a–h was removed
by hydrolysis with 0.5 M hydrochloric acid in a mixture of MeOH
and THF to give compounds 7a–h. Finally, the benzyl and diph-
enylbenzo protecting groups of 7a–h, 3, and 4 were removed in
one step by catalytic hydrogenation with 10% Pd/C in MeOH–EtOAc
under hydrogen atmosphere to give the compounds 10a–h, 8, and
9. During this treatment, the carbon–carbon double bond adjacent
to carbonyl group in the R1 of compounds 7e–h were selectively
hydrogenated and were converted into compounds 10e–h. The
structure of all compounds were confirmed by 1H NMR, 13C NMR,
FAB-MS, and/ or elemental analysis.
most pronounced inhibition (IC50: 0.19 lM) that was increased
concentration-dependently as shown in Figure 2. Docking simula-
tion also supports the inhibition and the superposition on native li-
gand (novobiocin)15 as shown in Figure 3 where the carbonyl
group of 3-(4-fluorophenyl)propanoyloxy at 300-position and the
hydroxyl group at 400-position of compound 10h could form H-
bonds with ASN46 and SER121 of ATP binding site in the 24 kDa
of E. coli DNA gyrase B, respectively. On the contrary, screened
compounds did not show reasonable inhibitions towards the
growth of Gram-negative bacterial strains, which may be caused
by difficulty in outer-membrane permeability of Gram-negative
organisms. Compounds bearing 3-(4-substitutedphenyl)propa-
noyloxy groups (e.g., 10e–h) and (4-fluorophenyl)acetyloxy groups
(e.g., 10d) at 200,300-positions tend to show comparatively enhanced
activity against the growth of Gram-positive bacterial strains.
Among these compounds, 3-(4-fluorophenyl) propanoyloxy deriv-
ative 10h has the most pronounced inhibition (0.25–1
lg/mL) to-
wards the growth of VRE, VISA, MRSA or MSSA strains and was

A. M. L. Hossion et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5349–5352
5351
Table 1
Antibacterial activity (MICs) against multi-drug resistant Gram-positive and Gram-negative bacterial strains and DNA gyrase inhibition (IC50) against DNA gyrase supercoiling kit
from E. coli and the best docking results on the inhibitions constants (Ki)
MICa
(lg/mL, Gram-positive)
IC50
(l
M, E. coli DNA
Kik
constants)
(
l
M, inhibitions
MICa
Gram-negative)
(lg/mL,
j
Compound
gyrase B inhibitions)
VRE
VISA
MRSA
OM584f
MSSA
209Pi
PA01l
K-12m
FN-1b
NCTC 12201c
Mu50d
OM481e
N315g
COLh
8
9
10a
10b
10c
10d
10e
10f
64
>128
16
8
8
8
8
8
2
1
>128
nt
>128
nt
8
64
>128
16
8
8
8
8
8
4
1
>128
nt
>128
nt
4
128
>128
16
16
2
2
4
4
1
128
>128
32
32
16
8
4
8
2
2
>128
64
0.25
0.25
1
128
>128
32
32
16
8
4
4
2
2
>128
128
0.25
0.25
2
128
>128
8
8
2
2
4
2
1
0.25
>128
2
0.25
0.125
1
>128
>128
>128
16
16
8
8
8
4
1
>128
1
0.25
0.25
1
128
>128
8
8
2
2
4
2
1
0.25
>128
0.5
0.25
0.125
0.5
nt
nc
>128
64
>128
32
0.10
1.10
0.34
0.21
0.28
1.21
1.23
0.46
0.19
0.14
0.09
nt
0.05 ꢀ 10ꢁ3
6.61
2.87
1.48
2.64
40.08
46.96
2.77
>128
>128
>128
>128
>128
>128
>128
>128
>128
nt
>128
>128
>128
>128
>128
>128
>128
>128
64
10g
10h
1
1.93
Quercetin
Norfloxacin
Vancomycin
Novobiocin
A
>128
nt
8
nt
nt
0.18 ꢀ 10ꢁ3
0.09 ꢀ 10ꢁ3
nc
0.25
nt
8
>128
16
>128
0.05
nt
nc
nc
nt
nt, not tested; nc, not calculated.
a
Microdilution method,12 MIC determined after 24 h.
Vancomycin-resistant enterococci FN-1.
Vancomycin-resistant enterococci NCTC 12201.
Vancomycin intermediate-resistant Staphylococcus aureus Mu50.
Methicillin-resistant S. aureus OM481.
Methicillin-resistant S. aureus OM584.
Methicillin-resistant S. aureus N315.
Methicillin-resistant S. aureus COL.
Methicillin sensitive S. aureus 209P.
IC50 the concentration of the drugs that inhibits 50% of supercoiling activity.
Ki values were calculated by computer aided AutoDock 4.0 soft.
Pseodomonas aeruginosa PA01.
b
c
d
e
f
g
h
i
j
k
l
m
Escherichia coli K-12.
up to 128 times more potent than vancomycin as well as eight
times more potent than A. On the other hand, compounds bearing
comparatively shorter 4-substituted benzoyloxy groups (e.g., 10a–
c) on the same positions have reduced activity against the same
bacterial strains. Moreover, most recently Tsuchiya and co-workers
reported, the site of action of compound A8,9 might be at DNA topo-
isomerase IV and/or DNA gyrase, and the primary target would be
DNA topoisomerase IV. Correspondingly, the site of action of com-
pounds 10a–h would be similar to compound A in S. aureus (e.g.,
Gram-positive). Therefore, it seems that compounds 10a–h have
divergent modes of action in Gram-negative and Gram-positive
organisms.
In conclusion, we have discovered a novel series of quercetin
diacylglucosides that have remarkable and acute antibacterial
properties. A range of diverse substituents at 200,300-positions of
quercetin-3-b-D-glucoside modulate antibacterial activity, and
makes it worthy for consideration to be developed as a drug candi-
date for nosocomial infections caused by multi-drug resistant
Gram-positive pathogens. Compound 10h appears to be an
attractive candidate for further investigations to provide a new
antibacterial agent for use against nosocomial multi-drug resistant
For an antibacterial agent to be effective, it must penetrate the
bacterial cell to reach its target. The biological evaluation revealed
that the size and lipophilicity of the substituent on quercetin-3-b-
D
-glucoside should be considered the key factors in determining its
antibacterial activity. Physical properties including relative hydro-
phobicity and molecular mass are important for penetration into
the bacterial cell and have a different role in Gram-negative and
Gram-positive bacteria. Thus, increasing molecular mass and bulk-
iness of substituent at 200,300-positions hinder penetration of quer-
cetin diacylglucosides into Gram-negative organisms through the
porin channels.16 The accumulation of compound in Gram-positive
bacteria (e.g., S. aureus) is thought to take place by simple diffusion
across the cytoplasmic membrane.17 Accordingly, screened com-
pounds 10a–h having high molecular mass and bulky side chains
at 200,300-positions were accumulated in Gram-positive organisms
more favorably than Gram-negative organisms. Furthermore, halo-
gen such as fluorine is very useful to modulate the electronic ef-
fects on phenyl rings of compounds 10c, 10d, 10h and may also
influence the steric characteristics and the hydrophilic–hydropho-
bic balance of the molecules.
Figure 2. DNA supercoiling assay by gyrase from E. coli strains JMtacA and JMtacB
(Hallett et al., 1990) of compound 10h. o.c., open-circular DNA; lin., linear DNA; s.c.,
supercoiled DNA.

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A. M. L. Hossion et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5349–5352
Figure 3. (A) Superposition of native ligand (yellow color) and 10h (Blue color) in the complex with 24 kDa fragment of the DNA gyrase B by using the AutoDock 4.0 software
tool. (B) AutoDock-modeled binding of 10h inside ATP binding site of DNA gyrase B. H-Bonds are displayed as sphere.
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We thank Professor Adrian L. Schwan of University of Guelph
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Supplementary data
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