Synthesis and biological evaluation of 5,7-dihydroxyflavanone derivatives as antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2016.05.003

A series of 5,7-dihydroxyflavanone derivatives were efficiently synthesized. Their antimicrobial efficacy on Gram-negative, Gram-positive bacteria and yeast were evaluated. Among these compounds, most of the halogenated derivatives exhibited the best antimicrobial activity against Gram-positive bacteria, the yeast Saccharomyces cerevisiae, and the Gram-negative bacterium Vibrio cholerae. The cytotoxicities of these compounds were low as evaluated on HepG2 cells using a cell viability assay. This study suggests that halogenated flavanones might represent promising pharmacological candidates for further drug development.

Full text of DOI:10.1016/j.bmcl.2016.05.003

Accepted Manuscript
Synthesis and biological evaluation of 5,7-dihydroxyflavanone derivatives as
antimicrobial agents
Xing Zhang, Omar Khalidi, So Young Kim, Ruitong Wang, Victor Schultz,
Brady F. Cress, Richard A. Gross, Mattheos A.G. Koffas, Robert J. Linhardt
PII:
S0960-894X(16)30483-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.003
BMCL 23857
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2016
2 May 2016
3 May 2016
Please cite this article as: Zhang, X., Khalidi, O., Kim, S.Y., Wang, R., Schultz, V., Cress, B.F., Gross, R.A., Koffas,
M.A.G., Linhardt, R.J., Synthesis and biological evaluation of 5,7-dihydroxyflavanone derivatives as antimicrobial
agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.003
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Synthesis and biological evaluation of 5,7-dihydroxyflavanone
derivatives as antimicrobial agents
Xing Zhanga,#, Omar Khalidib,#, So Young Kimc, Ruitong Wanga, Victor Schultza, Brady F. Cressb,
Richard A. Grossa,d,e, Mattheos A.G. Koffasb,d, *, Robert J. Linhardta,b,c,d,*
a Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York
12180, USA
b Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary
Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA
c
Biochemistry and Biophysics Graduate Program, Center for Biotechnology and Interdisciplinary
Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA
d
Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, New York 12180, USA
e
Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute, Troy, New York 12180, USA
Prepared for Bioorganic & Medicinal Chemistry Letters, March 2016
*Corresponding author Tel.: +1 518 276 2220
E-mail address: koffam@rpi.edu (M.A.G. Koffas)
*Corresponding author Tel.: +1 518 276 3405
E-mail address: linhar@rpi.edu (R.J. Linhardt)
#Equal contributors.

ABSTRACT
A series of 5,7-dihydroxyflavanone derivatives were efficiently synthesized. Their
antimicrobial efficacy on Gram-negative, Gram-positive bacteria and yeast were evaluated.
Among these compounds, most of the halogenated derivatives exhibited the best
antimicrobial activity against Gram-positive bacteria, the yeast Saccharomyces cerevisiae,
and the Gram-negative bacterium Vibrio cholerae. The cytotoxicities of these compounds
were low as evaluated on HepG2 cells using a cell viability assay. This study suggests that
halogenated flavanones might represent promising pharmacological candidates for further
drug development.
Keywords: Synthesis; Halogenated flavanones; Antimicrobial activity; Cytotoxicity

Flavanones are an important class of flavonoids containing a 2-phenyl-benzopyran-4-one
skeleton, and are commonly found in fruits and vegetables.1-3 These natural products
possess a variety of biological activities such as antitumor and anti-inflammatory
properties that are closely related to the skeleton and substitution patterns.2-7 The ability to
manipulate flavonoid activity through structural variation motivates research on the
synthesis of flavanone derivatives and evaluation of their bioactivity.8-16
Pinocembrin (5,7-dihydroxyflavanone) 1, is one of the primary flavanones which is
abundant in propolis and can be extracted from plants, fruits, vegetables, seeds, flowers
and teas (Figure 1).17 A vast range of biological/pharmacological activities for
pinocembrin have been reported, including antimicrobial, anti-inflammatory, anticancer
and antioxidant, as well as neuroprotective potential.17, 18 Pinocembrin is a small molecular
weight natural compound and is a biologically active constituent of honey. Its presence in
food suggests its safety for long-term administration, making it an excellent chemical
template for the design and synthesis of new compounds for pharmaceutical research.
Recently, we reported the antimicrobial efficacy of synthetic flavanone derivatives.19
Derivatives were identified that impede growth of a series of organisms. For instance,
4-chloro-flavanone, when combined with the inhibitor Phe-Arg-β-naphthylamide, exhibits
MIC’s of 30, 30 and 70 μg mL-1 for Saccharomyces cerevisiae, Cryptococcus neoformans
and Escherichia coli, respectively.19 These results led us to hypothesize that halide
substitution of flavanones might show enhanced antimicrobial activity. In addition, there is
increased interest in bioactive halogenated compounds,20-23 Here, we use an improved
synthetic approach to obtain a series of mostly halogenated 5,7-dihydroxyflavanone
derivatives that were assessed for antimicrobial efficacy and mammalian cytotoxicity.

Figure 1. Chemical structure of Pinocembrin (1).
Scheme 1. Reagents and conditions: (a) Methoxymethyl (MOM) chloride, N, N-diisopropylethylamine,
DCM, 0 oC, 12 h, 95%; (b) KOH, substituted benzaldehyde, MeOH, 16 h; (c) NaOAc, MeOH, reflux, 24
h; (d) 6 N HCl, MeOH, 60 oC, 42-56% yield over 3-steps.
A modified synthetic approach was developed based on a conventional route to efficiently
obtain a small library of flavanones.24 Synthesis commenced with partial methoxymethyl
(MOM) protection of 2,4,6-trihydroxy acetophenone monohydrate to produce phenol 10
(Scheme 1). Aseries of substituted benzaldehydes were reacted with phenol 10, through a
Claisen-Schmidt condensation, to prepare a series of chalcone intermediates 11 with
different substituents on their B-ring. The corresponding chalcones 11 were cyclized with
sodium acetate to provide the protected flavanones 12. Finally, acidic hydrolysis of the
MOM groups afforded the flavanone derivatives 2-8 (Table 1). Compared to the
conventional synthetic method, the efficiency of this synthesis is demonstrated by only one

column chromatography step being required for the conversion of phenol 10 to the target
flavanones 2-8. The structures of flavanone derivatives 2-8 were confirmed from their
spectral (1H- and 13C-nuclear magnetic resonance (NMR) and high resolution-mass
spectrometry (MS)) properties (see the Supplementary data).
Table 1. Target compounds
Compd.
R1
H
R2
F
R3
H
Yield(%)a
225
3
45
56
52
45
47
42
45
F
F
H
F
F
F
4
F
Cl
H
5
626
7
Cl
OH
H
Cl
H
OH
MeO
OH
H
827
a Isolated yield from compound 10 over 3 steps.
The antimicrobial activities of 5,7-dihydroxyflavanone derivatives 2-8 were evaluated
by measuring their inhibitory effect against a series of Gram-negative and Gram-positive
bacteria and yeast and their minimum inhibitory concentration (MIC) values were
determined.
Natural flavanones as bacteriostatic agents have been investigated in the past, and have
shown no inhibition against E. coli,19 which is a Gram-negative bacterium. However,
natural flavanones did show inhibition against Bacillus subtilis,19 which is a Gram-positive
organism. Normally, compounds show greater potency against Gram-positive than
Gram-negative bacteria, since the Gram-negative bacteria have both an outer and an inner
membrane making the cell permeability of an antimicrobial agent much more difficult.
Results herein are consistent with a previous report on the antimicrobial activity of

natural flavanones.19 Non-natural flavanones 2-8 were ineffective at inhibiting growth of
the Gram-negative bacteria E. coli and Pseudomonas aeruginosa (Table 2).
Table 2. Non-natural flavanone MIC values in μg mL-1(μM)
Organism
Compound
5
2
3
4
6
7
8
Gram-negative Bacteria
Escherichia coli
>140a(500)b >290(1000) >310(1000) >80(250) >40(125) >300(1000) >290(1000)
BW25113
Pseudomonas
>140(500)
30(125)
>290(1000) >310(1000) >80(250) >40(125) >300(1000) >290(1000)
aeruginosa PA01
Vibrio
40(125)
>310(1000)
40(125)
40(125)
>300(1000) >290(1000)
cholerae0395N1
Gram-positive Bacteria
Bacillus subtilis
30(125)
30(125)
40(125)
>310(1000)
40(125)
20(62.5) >300(1000) >290(1000)
1012
Bacillus anthracis
20(62.5)
>310(1000) 20(62.5) 10(31.25) >300(1000) >290(1000)
>310(1000) 30(93.75) 10(31.25) >300(1000) >290(1000)
delta sterne
Bacillus cereus
30(125)
30(125)
40(125)
40(125)
10987
Stappylococcusaureus
>310(1000)
40(125)
20(62.5) >300(1000) >290(1000)
10(31.25) >300(1000) >290(1000)
ATCC 33807
Eukaryote
Saccharomyces
60(250)
>290(1000)
40(125)
80(250)
cerevisiae INVSc1
a concentration unit is μg mL-1; b concentration unit is μM.

However, halogenated derivatives 2, 3, 5 and 6 showed significant activities (30-40 μg
mL-1) against Vibrio cholera, a Gram-negative organism. To the best of our knowledge,
this is one of the rare examples of the inhibition of Gram-negative bacteria by flavanones.
Next, the activity of flavanones 2-8 was tested against the following Gram-positive
bacteria: Bacillus subtilis 1012, Bacillus anthracis, Bacillus cereus 10987 and
Stappylococcus aureus ATCC 33807. The lowest MIC values observed were obtained for
the flavanones 2, 5 and 6, which contain halide substituents. The dichlorinated flavanone
6 provided the greatest inhibition of the growth of all the Gram-positive strains tested,
exhibiting MIC values ranging from 10 to 20 μg mL-1. Flavanones without halogen
substituents did not inhibit the growth of Gram-positive strains, and not all flavanones
having halogens inhibited microbial growth. For example, the trifluorinated flavanone 4
had no effect on growth for Gram-positive bacteria, while the monofluorinated and
difluorinated flavanone, 2 and 3, afforded low MIC values. Inhibition of microbial growth
was observed for trifluorinated compound 4 when tested against S. cerevisiae, affording a
MIC of 40 μg mL-1. Most importantly, the MIC values for some of the flavanones tested
were better than that of their natural analogs, approaching MIC values observed for
traditional antibiotics. All of the antimicrobial results were confirmed in triplicate using a
96-well plates bioassay.
We next used a MTS 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-
3-(4-sulfophenyl) tetrazolium inner salt cell viability assay to evaluate mammalian
cytotoxicity of 5,7-Dihydroxyflavanone derivatives in HepG2 cells. HepG2 is a
well-established human hepatocellular carcinoma cell line. Approximately 50,000 cells
were seeded and incubated for 24 h before addition of 5,7-dihydroxyflavanone to samples
and to the controls. MTS reagent was added at 6 and 24 hours of incubation and cell
viability was determined by measuring Abs490. The poor water solubility of many
flavanones limited the range of concentrations that could be evaluated.
The
5,7-dihydroxyflavanone derivatives generally had low water solubility, ranging from 50 to

2000 μM in growth medium containing 1% dimethylsulfoxide (DMSO). The maximal
solubility for compounds 6 and 7 was 63 μM, for compounds 4 and 5 was 500 μM, and for
compound 2 and 3, 1000 μM. Methoxy-flavanone 8 had the highest solubility at 2000 μM.
All of the flavanones showed dose-dependent toxicity towards HepG2 cells. Because of
the limited solubility of the flavanones, it was only possible to obtain an IC50 value for
compound 4 of 95 μM after 24 h. At a concentration of 50 μM (14-16 μg mL-1), all the
derivatives were completely soluble and exhibited low to no cytotoxicity towards HepG2
cells (Figure 2). At this concentration, the order of their relative
2
3
4
5
6
7
8
0
25
50
75
100
125
150
175
Cell viability (%)
Figure 2. Cytotoxicity of 5,7-dihydroxyflavanone derivatives on HepG2 cells. Cell viability was
measured after incubating HepG2 cells with 50 M of flavanone derivative by MTS assay. Cell viability
was normalized based on the negative control (100% growth medium) and positive control (100%
DMSO). Flavonone 3 shows least toxicity out of the compounds that were tested. Open bars correspond
to 24 h and closed bars correspond to 6 h. A single experiment was performed with three replicate
determinations. The error bars included in the figure are too small to be seen.
cytotoxicity to HepG2 cells is as follows: compound 4 > compound 3 > compound 2 >
compound 5 > compound 8 > compound 7 > compound 6. Toxicity increased with greater
number of fluorine substituents, while OMe, OH, and Cl substituents promoted the
proliferation of HepG2 cells. The dichlorinated flavanone 6 which has the antibacterial

activity against all of the microorganisms tested, showed the lowest level of cytotoxicity
and promoted proliferation of HepG2 cells by 17% at 6 h, and 57% at 24 h over that
obtained in cell culture growth medium.
In summary, a series of 5,7-dihydroxyflavanone derivatives 2-8 were designed and
efficiently synthesized. Their antimicrobial activity on Gram-negative, Gram-positive
bacteria and yeast were evaluated as well as their cytotoxicity against HepG2 cells. In this
study, most of the halogenated compounds showed greater antimicrobial activity (lower
MIC values) than their natural analogs. Further discovery through target design and
synthesis, as well as mechanistic studies are underway.
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Graphical abstract