Flavone-based analogues inspired by the natural product simocyclinone D8 as DNA gyrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.08.094

The increasing occurrence of drug-resistant bacterial infections in the clinic has created a need for new antibacterial agents. Natural products have historically been a rich source of both antibiotics and lead compounds for new antibacterial agents. The natural product simocyclinone D8 (SD8) has been reported to inhibit DNA gyrase, a validated antibacterial drug target, by a unique catalytic inhibition mechanism of action. In this work, we have prepared simplified flavone-based analogues inspired by the complex natural product and evaluated their inhibitory activity and mechanism of action. While two of these compounds do inhibit DNA gyrase, they do so by a different mechanism of action than SD8, namely DNA intercalation.

Full text of DOI:10.1016/j.bmcl.2013.08.094

Accepted Manuscript
Flavone-based analogues inspired by the natural product simocyclinone D8 as
DNA gyrase inhibitors
Jenson Verghese, Thuy Nguyen, Lisa M. Oppegard, Lauren M. Seivert, Hiroshi
Hiasa, Keith C. Ellis
PII:
S0960-894X(13)01043-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.094
BMCL 20832
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
13 June 2013
22 August 2013
26 August 2013
Please cite this article as: Verghese, J., Nguyen, T., Oppegard, L.M., Seivert, L.M., Hiasa, H., Ellis, K.C., Flavone-
based analogues inspired by the natural product simocyclinone D8 as DNA gyrase inhibitors, Bioorganic &
Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.08.094
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Flavone-based analogues inspired by the
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gyrase inhibitors
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Jenson Verghesea, Thuy Nguyena, Lisa M. Oppegardb, Lauren M. Seivertb, Hiroshi Hiasab, and Keith C. Ellisa

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Flavone-based analogues inspired by the natural product simocyclinone D8 as
DNA gyrase inhibitors.
Jenson Verghesea, Thuy Nguyena, Lisa M. Oppegardb, Lauren M. Seivertb, Hiroshi Hiasab, and Keith C.
Ellisa
aDepartment of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Biotech 1, 800 East Leigh Street, Richmond, VA 23298, USA
bDepartment of Pharmacology, University of Minnesota Medical School, Nils Hasselmo Hall, 312 Church Street, Minneapolis, MN 55455, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The increasing occurrence of drug-resistant bacterial infections in the clinic has created a need
for new antibacterial agents. Natural products have historically been a rich source of both
antibiotics and lead compounds for new antibacterial agents. The natural product simocyclinone
D8 (SD8) has been reported to inhibit DNA gyrase, a validated antibacterial drug target, by a
unique catalytic inhibition mechanism of action. In this work, we have prepared simplified
flavone-based analogues inspired by the complex natural product and evaluated their inhibitory
activity and mechanism of action. While two of these compounds do inhibit DNA gyrase, they
do so by a different mechanism of action than SD8, namely DNA intercalation.
Keywords:
Simocyclinone D8
DNA gyrase
Antibiotics
2009 Elsevier Ltd. All rights reserved.
Natural product
Flavone
Quercetin
The emergence and proliferate spread of drug resistant
bacterial infections in the clinic has created a critical need for
The natural product simocyclinone D8 (SD8, Figure 1)
inhibits DNA gyrase by a unique mechanism of action. While the
fluoroquinolones trap DNA in a covalent complex with the
enzyme, SD8 is a competitive inhibitor of DNA binding to the
enzyme.12 A natural product−protein co-crystal structure shows
that SD8 binds to sites on DNA gyrase that are distinct from the
binding site of the fluoroquinolones.13 SD8 inhibits both wild-
type DNA gyrase and a ciprofloxacin-resistant mutant in a
supercoiling assay, showing that the competitive inhibition
2
new antibacterial agents.1, Drug resistance occurs by several
mechanisms including mutations of the molecular targets of
current antibacterial agents, production of additional bacterial
enzymes that can inactivate the drug or modify the molecular
target, and acquisition or increased expression of drug efflux
pumps.3, 4
DNA gyrase is a validated molecular target for antibacterial
drug development.5 The fluoroquinolone class of DNA gyrase
inhibitors has been widely used in the clinic due to its broad
spectrum of activity.6 However, mutations in DNA gyrase that
confer resistance to the fluoroquinolones have emerged and
diminished their utility. Fluoroquinolone resistance has been
reported in pneumonia,7 influenza,8 MDR/XDR tuberculosis,9
and respiratory tract infections.10 To treat these fluoroquinolone-
resistant infections, there is an urgent need for compounds that
inhibit drug-resistant DNA gyrase mutants. One potential source
of inhibitors of fluoroquinolone-resistant mutants is compounds
that inhibit DNA gyrase by mechanisms of action different from
that of the fluoroquinolones.
mechanism can overcome
a
fluoroquinolone resistance
mutation.12 We have also shown that SD8 can inhibit DNA
gyrase from both E. coli and S. aureus.14
The SD8 molecule consists of three subunits: a coumarin, a
tetraene linker, and an angucyclinone aglycone that is comprised
of an olivose sugar bound to
benz[a]anthracene system by a C-glycoside bond. The SD8-
liganded crystal structure reveals distinct binding pockets for the
coumarin and the benz[a]anthracene system, both of which are
required for maximum inhibition.13
a
highly substituted
Natural products have long been a source of antibiotic drugs
and it is reasonable to expect new natural products to serve as
promising leads for antibiotic drug discovery. An overwhelming
majority (69%) of antibacterial drugs that the FDA approved
between 1981 and 2006 were either natural products or
developed from natural products.11
Figure 1. Structure of the natural product simocyclinone D8 (SD8)

As part of a program to investigate simplified analogues of
SD8 as potential new antibacterial compounds, we have
investigated a set of flavone-based analogues as DNA gyrase
inhibitors (Figure 2). In particular, we sought to identify flavone-
based analogues that act by SD8’s catalytic inhibition
mechanism. We envisioned the bicyclic ring system of the
flavone as an isosteric replacement for the coumarin of
simocyclinone D8 and that the 2-phenyl ring of the flavone could
occupy a hydrophobic cleft formed by Leu98, Gly170, and
Tyr266 that lies adjacent to the coumarin binding site (Figure
3),13 thereby creating additional binding interactions. Flavones
have been previously reported as potential isosteric replacements
for coumarins in Hsp90 inhibitors.15 Docking of flavone scaffolds
into the DNA gyrase crystal structure (2Y3P) with DOCK
demonstrated that this scaffold can be accommodated sterically
by the enzyme, with the flavones scoring as well or better than
the coumarin scaffold.
Figure 3. Binding pocket for the SD8 coumarin on DNA gyrase (PDBID:
2Y3P).13 The angucyclinone aglycone has been omitted for clarity.
Figure 2. General structure of flavone-based analogues.
Scheme 1. Reagents and conditions: (i) BnBr, K2CO3, DMF, overnight, room temp., 50%; (ii) tBuO2C(CH2)5CO2H, EDCI,
EtN(iPr)2, DMAP, THF, overnight, room temp., quantitative; (iii) TFA, Et3SiH, DCM, overnight, room temp., 76%; (iv) a. NEt3,
THF, reflux, 4h, b. N2H4ꢀH2O, EtOH, reflux, 2h, 60-80% over two steps; (v) EDCI, HOBt, NMM, DMF, overnight, room temp.,
77-95%; (vi) for 9a: Pd/C, H2, EtOH/Dioxane (3:1), overnight, room temp., 43%; for 9b: TBAF, THF, overnight, room temp.,
65% then Pd/C, H2, EtOH/Dioxane (3:1), overnight, room temp., 34%; for 9c: TBAF, THF, overnight, room temp., 69% then
Pd/C, H2, EtOH/Dioxane (3:1), overnight, room temp., 34%; for 9d: Pd/C, H2, EtOH/Dioxane (3:1), overnight, room temp., 52%
then TFA, Et3SiH, DCM, overnight, room temp., quantitative; for 9e: Pd/C, H2, EtOH/Dioxane (3:1), overnight, room temp.,
57% then HCl, THF, 90 min, room temp., 36%.

We chose quercetin (1, Scheme 1) as a flavone building block,
due to its commercial availability. For ease of synthesis we
utilized a flexible linker comprised of pimelic acid and glycine.
In order to potentially create binding interactions in the
benz[a]anthracene binding site, we coupled the flavone-linker
unit to substituted anilines. We envisioned that the aromatic ring
of the anilines could π−stack with His80 of DNA gyrase, ~15 Å
away.
Table 1: Effects of flavone analogues on E. coli DNA gyrase
Compound
Supercoiling Assay
IC50 (µM)a
48.6 0.8
84.7 5.5
>200
DNA Cleavage
(fold-stimulation)b
9a
9b
7.8
5.0
3.0
1.8
1.9
<1
9c
To begin our work, protection of quercetin to afford tribenzyl-
quercetin 2 was followed by coupling to the t-butyl mono ester of
pimelic acid to afford 3. Deprotection of t-butyl ester under
acidic conditions afforded carboxylic acid 4 for use in coupling
to the anilines.
9d
>200
9e
>200
10
>200
SD8
0.41
<114
Suitable protected anilines 6a-e were coupled to N-
phthaloylglycine acid chloride 5, followed by phthalide
deprotection with hydrazine to afford primary amines 7a-e. This
strategy was chosen to avoid difficult and low yield couplings of
the aniline nitrogen directly with the carboxylic acid of the
flavone-linker subunit.
a. The IC50 values were determined based on the results from
two independent experiments. b. Fold-stimulation in the
generation of linear DNA at 200 μM of the compound over the
amount of linear DNA in the absence of any compound. The
level of stimulation was determined based on the results from
two independent experiments.
Coupling of the (BnO)3-quercetin-pimelic acid fragment 4
with glycinyl aniline fragments 7a-e was achieved in 77-95%
yields to afford 8a-e. These compounds were then deprotected
using suitable conditions to afford target analogues 9a-e. As a
control compound, we also synthesized quercetin-sebacic methyl
ester analogue 10 from tribenzyl-quercetin 2 as shown in Scheme
2. This analogue lacks the aromatic ring that we envisioned is
needed for binding in the benz[a]anthracene pocket.
Table 2: Effects of flavone analogues on human Topo II
Compound
Decatenation Assay
IC50 (µM)a
149 3.2
>200
DNA Cleavage
(fold-stimulation)b
9a
9b
9c
9d
9e
10
2.7
2.4
2.1
<1
<1
<1
>200
>200
78.9 1.4
>200
Scheme 2. Reagents and conditions: (i) MeO2C(CH2)8CO2H,
EDCI, EtN(iPr)2, DMAP, THF, overnight, room temp., 58%;
(ii) Pd/C, H2, EtOH/Dioxane (3:1), overnight, room temp.,
27%.
a. The IC50 values were determined based on the results from
two independent experiments. b. Fold-stimulation in the
generation of linear DNA at 200 μM of the compound over the
amount of linear DNA in the absence of any compound. The
level of stimulation was determined based on the results from
two independent experiments.
We first assessed the ability of the flavone analogues 9a-e and
10 to inhibit E. coli DNA gyrase. A supercoiling activity assay14
was performed in the presence of various concentrations of these
compounds and the 50% inhibitory concentration (IC50) was
determined (Table 1). Compounds 9a and 9b exhibited modest
inhibitory effects on the supercoiling activity of gyrase with IC50
values of 48.6 0.8 μM and 84.7 5.5 μM, respectively, but the
remaining compounds were not active (i.e., IC50 value greater
than 200 μM). The activities of these compounds against E. coli
DNA gyrase were significantly lower than those of SD8 (IC50
value of 0.41 μM)14 and quercetin alone (IC50 value of 0.14 μM)16
but approximately equal to the activity of coumain-linker subunit
of SD8 (MGD8N2A, Figure 4, IC50 value of 50 μM).13
Compound 10, the flavone-based analogue most similar to
MGD8N2A, was inactive, suggesting that quercetin is a poor
isosteric replacement for the coumarin of SD8.
To determine whether compounds 9a and 9b inhibit E. coli DNA
gyrase by a catalytic mechanism of action similar to SD8, these
compounds were tested for their ability to disrupt binding of
DNA gyrase to DNA by surface Plasmon resonance (SPR).12
Neither 9a nor 9b were active in this assay and no disruption of
the DNA-enzyme interaction was observed (see Supporting
Information). These results suggested that while compounds 9a
and 9b inhibited the activity of DNA gyrase, they did so by a
mechanism different from that of SD8.
To further investigate the mechanism of action of these
flavone analogues, we tested the effect of the compounds on the
DNA cleavage activity14 of E. coli DNA gyrase (Table 1). All
compounds, except 10, modestly stimulated gyrase-catalyzed
cleavage, with compounds 9a and 9b exhibiting the highest
levels of stimulation (7.8-fold and 5.0-fold stimulation in the
generation of linear DNA at 200 μM, respectively). These results
suggested that compounds 9a and 9b inhibited the activity of
DNA gyrase by poisoning it.
To assess the potential toxicity of these compounds, we
measured the activity of these compounds against human Topo
II. We first examined the effect of the compounds on the
Figure 4. MGD8N2A, a degradation product of SD8, also inhibits DNA
gyrase (IC50 value of 50 µM).13

decatenation activity of human Topo II17 and found that
compounds 9a and 9e exhibited modest activities (IC50 values of
149 3.2μM and 78.9 1.4 μM, respectively). It may be worth
noting that while compound 9a exhibited inhibitory effects on
both E. coli gyrase and human Topo II, compound 9e inhibited
only human Topo II and compound 9b inhibited only E. coli
DNA gyrase.
intercalation was observed with compound 9b, but the level of
intercalation with the other compounds was limited (Fig. 5A).
Compounds 9a and 9b exhibited the highest levels of poisoning
activity among these compounds against both E. coli gyrase
andhuman Topo II, indicating that compounds 9a and 9b might
poison these topoisomerases through their abilities to intercalate
into DNA.
We also examined the effect of the compounds on the DNA
cleavage activity of human Topo II (Table 2).18 Similar to the
results observed with E. coli DNA gyrase (Table 1), compounds
9a and 9b exhibited the highest levels of poisoning activity
against human Topo II, although the levels of stimulation of
DNA cleavage for human Topo II were lower than those for E.
coli DNA gyrase. Thus, compounds 9a and 9b appeared to affect
the activity of the topoisomerases by poisoning, as does
quercetin.16 However, compound 9e acted as a modest inhibitor
of human Topo II without poisoning it, suggesting a different
mode of action for compound 9e.
In conclusion, we have prepared flavone-based analogues
inspired by the complex natural product simocyclinone D8.
While two of these compounds do inhibit DNA gyrase, they do
not act as catalytic inhibitors as SD8 does. The flavone-based
analogues 9a and 9b were determined to be topoisomerase
poisons and DNA intercalators.
Acknowledgments
Funding for this work was provided by the VCU/MCV A.D.
Williams Trust Fund, the Thomas F. and Kate Miller Jeffress
Memorial Trust Fund, and the VCU School of Pharmacy (to
K.C.E.). The author thanks Professor Richard A. Glennon and
Professor John C. Hackett for helpful discussions and Professor
Glen Kellogg for assistance in preparing this manuscript.
Since quercetin can intercalate into DNA19 and some of the
compounds appeared to affect topoisomerase activity through
their quercetin moiety, we decided to examine if these
compounds can intercalate into DNA. We employed a DNA
unwinding assay, where the extent of intercalation of
a
compound can be measured as the level of negative supercoils
introduced into the covalently closed, relaxed circular substrate
DNA.20 We found that some of these compounds could
intercalate into DNA and their levels of intercalation varied
widely (Fig. 5A). Compound 9a exhibited the highest level of
intercalation and the level of intercalation by compound 9a was
similar to that of quercetin (Fig. 5B). A modest level of
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Figure 5. The flavone compounds 9a-e and 10 are able to intercalate
into DNA to varying degrees. DNA unwinding assays contain relaxed
plasmid DNA as the substrate, calf thymus topoisomerase I (Topo I), and the
indicated concentrations of the compounds as previously described. (A)
Intercalation of compounds 9a-e and 10. (B) Intercalation of compound 9a as
compared to intercalation by quercetin. Reactions were performed in
duplicate and essentially identical results were obtained.