Defining a minimum pharmacophore for simocyclinone D8 disruption of DNA gyrase binding to DNA

Article abstract of DOI:10.1007/s00044-014-0942-z

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 used a deconstruction-reconstruction approach to prepare analogs of the coumarin subunit of SD8 and evaluated their ability to disrupt binding of the DNA gyrase enzyme to DNA in a surface plasmon resonance assay. This has led to a minimum pharmacophore required for disruption of binding. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0942-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0942-z
ORIGINAL RESEARCH
Defining a minimum pharmacophore for simocyclinone
D8 disruption of DNA gyrase binding to DNA
•
Lauren M. Gaskell Thuy Nguyen
•
Keith C. Ellis
Received: 7 September 2013 / Accepted: 7 February 2014
Ó Springer Science+Business Media New York 2014
Abstract 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 simo-
cyclinone 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 used a deconstruction-reconstruction approach to
prepare analogs of the coumarin subunit of SD8 and
evaluated their ability to disrupt binding of the DNA gyrase
enzyme to DNA in a surface plasmon resonance assay.
This has led to a minimum pharmacophore required for
disruption of binding.
molecular target, and acquisition or increased expression of
´
drug efflux pumps (Martınez, 2012; Wright, 2012).
DNA gyrase is a validated molecular target for anti-
bacterial drug development (Bax et al., 2010). The fluo-
roquinolone class of DNA gyrase inhibitors has been
widely used in the clinic due to its broad spectrum of
activity (Maxwell, 1997). However, mutations in DNA
gyrase that confer resistance to the fluoroquinolones have
emerged and diminished their utility. Fluoroquinolone
resistance has been reported in pneumonia (Brown et al.,
´
´
2004), influenza (Perez-Vazquez et al., 2007), MDR/XDR
tuberculosis (Devasia et al., 2009), and respiratory tract
infections (Reinhardt et al., 2007). To treat these fluor-
oquinolone-resistant infections, there is an urgent need for
compounds that inhibit drug-resistant DNA gyrase
mutants. One potential source of inhibitors of fluor-
oquinolone-resistant mutants is compounds that inhibit
DNA gyrase by mechanisms of action different than that of
the fluoroquinolones.
Keywords Simocyclinone D8 ꢀ DNA gyrase ꢀ
Antibiotics ꢀ Natural product ꢀ Catalytic inhibition
Introduction
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
approved by the Food and Drug Administration (FDA)
between 1981 and 2006 was either natural products or
developed from natural products (Newman and Cragg,
2007).
The emergence and proliferate spread of drug-resistant
bacterial infections in the clinic has created a critical need
for new antibacterial agents (Jones, 2010; Boucher et al.,
2009). 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
The natural product simocyclinone D8 (SD8, Fig. 1)
inhibits DNA gyrase by a unique mechanism of action. In
contrast to the fluoroquinolones, which trap DNA in a
covalent complex with the enzyme, SD8 is a competitive
inhibitor of DNA binding to the enzyme (Flatman et al.,
2005). 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
L. M. Gaskell ꢀ T. Nguyen ꢀ K. C. Ellis (&)
Department of Medicinal Chemistry, School of Pharmacy,
Virginia Commonwealth University, Biotech 1, 800 East Leigh
Street, Richmond, VA 23298, USA
e-mail: kcellis@vcu.edu
123

Med Chem Res
Fig. 1 Structure of the natural
product simocyclinone D8
(SD8)
O
HO
Me
O
Cl
HO
O
O
Me
O
O
OH
AcO
O
O
N
H
OH OH OH
OH
O
Simocyclinone D8 (SD8)
(Edwards et al., 2009). SD8 inhibits both wild-type DNA
gyrase and a ciprofloxacin-resistant mutant in a supercoil-
ing assay, showing that the competitive inhibition mecha-
nism can overcome a fluoroquinolone resistance mutation
(Flatman et al., 2005). We have also shown that SD8 can
inhibit DNA gyrase from both Gram-(-) (E. coli) and
Gram-(?) (S. aureus) bacteria, offering the potential for
broad-spectrum activity (Oppegard et al., 2009).
The SD8 molecule consists of three subunits: a cou-
marin, a tetraene linker, and an angucyclinone aglycone
that consists of an olivose sugar bound to a highly substi-
tuted benz[a]anthracene system by a C-glycoside bond.
The SD8 crystal structure reveals distinct binding pockets
for the coumarin and the benz[a]anthracene system, both of
which are required for maximum inhibition (Edwards
et al., 2009).
Scheme 1 Reagents and conditions: (i) RCOCl, pyridine, 65 % for
2a, 26 % for 2b, 52 % for 2c, 52 % for 2d, 45 % for 2e, 21 % for 2f
The ability of SD8 to inhibit DNA gyrase by a mecha-
nism of action distinct from the fluoroquinolones, its
activity against a fluoroquinolone-resistant DNA gyrase
mutant, and its broad spectrum of activity encouraged us to
investigate the natural product as a potential lead molecule
for a new class of DNA gyrase inhibitors. SD8’s complex
structure and synthetic inaccessibility, however, hindered a
traditional approach of total synthesis and synthesis of
analogs to explore the structure–activity relationships. For
this reason, we have chosen a deconstruction-reconstruc-
tion approach, starting with simple fragments of one sub-
unit and adding structural elements to determine how they
contribute to biological activity. In this manner, we sought
to identify a simplified molecule that acted by SD8’s cat-
alytic inhibition mechanism.
linker terminated with different functional groups (Scheme 1,
2a–2d) by coupling 1 with the appropriate acid chloride in
pyridine. We also prepared an analog where the linker was
replaced with a larger aryl ring (2e) and a dimeric compound
with coumarins on both ends of the fatty acid linker (2f) using
this method. 3-Amino-4-hydroxycoumarin 4 was prepared by
reduction of commercially available 4-hydroxy-3-nitro-
coumarin 3. Analogs of coumarin 4 with substitutions at the
3-amino positionwereagain prepared(5, 6a and 6b)aswellas
the di-coumarin 7 (Scheme 2).
To continue adding substitutions found in the coumarin
subunit of SD8, we prepared 3-amino-7-benzyloxycouma-
rin 11 in six steps (Scheme 3) (Yu et al., 2005). Coumarin
11 was then coupled to acid chlorides to prepare analogs
13a–c, which were deprotected by hydrogenation to afford
analogs 14a–c (Scheme 4). Coumarin 11 was also depro-
tected for testing in biological assays. Finally, 4,7-dihydr-
oxycoumarin 18 was prepared in three steps for use in
biological testing (Scheme 5).
Results and discussion
We began our work exploring analogs of the coumarin
subunit, due to the commercial availability of this scaffold
and the ability to prepare them by synthetic routes. Using
several coumarins with different substitution patterns, we
prepared analogs at the 3-amino position. These substitu-
ents were designed to probe the steric and electronic effects
of the SD8 coumarin and tetraene linker subunits.
We evaluated the coumarin analogs that we prepared for
their ability to disrupt the binding of the DNA gyrase
enzyme to DNA in a surface plasmon resonance assay
(SPR) (Flatman et al., 2005). In this assay, a 147 bp
fragment of 50-biotinylated double-stranded DNA was
immobilized on a streptavidin-coated SPR chip. The DNA
Starting from commercially available 3-aminocoumarin
(1), we prepared several analogs with a saturated fatty acid
123

Med Chem Res
Scheme 2 Reagents and
conditions: (i) 5 % Pd/C, H2,
1 % HCl in MeOH, 85 %. (ii)
Sebacic diacid chloride,
pyridine, 86 % for 5, 17 % for
7. (iii) oxalyl chloride, DMF,
DCM, then ArNH2, NEt3, THF,
80 % for 6a, 39 % for 6b
Scheme 3 Reagents and
conditions: (i) N-acetyl glycine,
NaOAc, Ac2O, 24 %. (ii)
K2CO3, MeOH. (iii) BnBr,
MeCN, 99 % for 2 steps. (iv)
Boc2O, DMAP, THF. (v)
hydrazine, MeOH. (vi) TFA,
DCM, 69 % for 3 steps
Scheme 4 Reagents and
conditions: (i) 5 % Pd/C, H2,
1 % HCl in MeOH, 92 % for
12, 28 % for 14a, 70 % for 14b,
83 % for 14c. (ii) RCOCl,
pyridine, 37 % for 13a, 66 %
for 13b. (iii) Me3SnOH, 1,2-
dichloroethane, 80 °C,
overnight, 60 %
gyrase–DNA interaction was detected as a change in signal
when a solution of DNA gyrase enzyme was flowed over
the chip. To test for the ability of our compounds to disrupt
this interaction, DNA gyrase (100 nM) was treated with
each coumarin analog (100 lM) prior to flowing the
enzyme over the immobilized DNA on the SPR chip. SD8
was used as a control, as it completely disrupted DNA
gyrase binding to DNA in this experiment.
The results of the SPR experiment for the coumarin
analogs that we prepared are shown in Table 1, and a
sample of the data is shown in Fig. 2. The majority of the
compounds did not inhibit DNA gyrase binding to DNA.
There are a few notable exceptions, however. The cou-
marin analogs that contained an 8-carbon spacer and a
carboxylic acid functional group (2a, 5, 14c; Fig. 2c, d) did
disrupt the enzyme–DNA interaction, and did so equally as
123

Med Chem Res
well as SD8. Interestingly, the length of the carbon linker
˚
seems to be a crucial parameter (*15 A), as a similar
compound with a 5-carbon spacer did not disrupt the
enzyme–DNA interaction (2a vs. 2b; Fig. 2c vs. 2e). The
carboxylic acid also seems to be a key functional group for
disrupting the enzyme–DNA interaction. Modification of
this functional group to the ester (2c, 14a) or removal of it
(2d, 14b; Fig. 2f) abolished the ability of these compounds
to disrupt binding. Lastly, the substitutions on the coumarin
itself did not seem to influence the ability of 2a, 5, and 14c
to inhibit binding. The conclusions drawn from these data
are summarized in the pharmacophore model in Fig. 3.
Scheme 5 Reagents and conditions: (i) K2CO3, BnBr, MeCN, reflux,
overnight, 69 %. (ii) NaH, diethyl carbonate, toluene, reflux, over-
night, 63 %. (iii) 5 % Pd/C, H2, 1 % HCl in MeOH, 98 %
Conclusions
In brief, we have explored coumarin analogs based on the
natural product SD8 and defined a minimum pharmaco-
phore for disrupting binding of DNA gyrase to DNA. Work
to evaluate these compounds as functional inhibitors DNA
gyrase is currently in progress.
Table 1 Evaluation of the ability of coumarin analogs to disrupt
DNA gyrase binding to DNA in an SPR assay
Compound
Disrupts DNA gyrase binding
to DNA in SPR assaya
1
No
Yesb
No
No
No
No
2a
2b
2c
2d
2e
2f
Experimental
Chemical reagents and solvents were purchased from
Sigma-Aldrich (MO, USA), Alfa-Aesar (MA, USA), or
Fisher Scientific (PA, USA). Analytical Thin Layer Chro-
matography (TLC) was performed using silica gel GHLF
plates (Analtech Inc., DE, USA). Flash chromatography was
performed on TELEDYNE ISCO CombiFlashÒ Rf instru-
ment using RediSep Rf Normal-phase Flash Columns (4, 12,
24, or 40 g). 1H NMR and 13C NMR spectra were recorded
on a Bruker TopSpin 400 MHz using deuterated chloroform
(CDCl3) or methanol (CD3OD). All chemical shifts are
reported as d in units of parts per million (ppm) relative to
chloroform and methanol residual peaks at 7.26 and 3.31,
respectively (1H NMR spectra); 77.16 and 49.00, respec-
tively (13C NMR spectra). The data is reported as follows:
chemical shifts (ppm), multiplicity (s = singlet, d = dou-
blet, dd = doublet of doublets, t = triplet, q = quartet, and
m = multiplet), coupling constant(s) (Hz), and integral
values. Electrospray ionization mass spectra were obtained
from Perkin Elmer Flexar UPLC/AxION2 TOF Mass
Spectrometer.
c
-
4
No
Yesb
5
6a
6b
7
No
No
c
-
12
14a
14b
14c
18
a
No
No
No
Yesb
No
Each compound (100 lM) was incubated with DNA gyrase (100
nM) for 1 h followed by injection in the SPR chip, the surface of
which was pre-coated with a 147 bp fragment of dsDNA. DNA
gyrase treated with buffer alone was used as positive control. SD8
was used as the negative control
b
Compound that disrupted DNA gyrase binding to DNA produced
sensorgrams similar to that of SD8
c
These compounds increased the binding signal between DNA
gyrase and DNA. Another interesting result is that the di-coumarin
analogs 2f and 7 appeared to promote the binding of DNA gyrase to
DNA. These two compounds gave signal higher than that of control
samples where DNA gyrase interacted with DNA without a coumarin
analog present. It is possible that these compounds are aiding the
dimerization of the GyrA subunits, thereby helping to pre-order the
enzyme for DNA binding or that they are binding to the enzyme
coumarin binding site and intercalating DNA, thereby promoting
binding
10-Oxo-10-((2-oxo-2H-chromen-3-yl)amino)decanoic
acid (2a)
Commercially available sebacid diacid chloride (3.4 mmol,
0.915 mL) was dissolved in 15 mL of pyridine. Commer-
cially available 3-aminocoumarin (1) (3.1 mmol, 0.5 g) in
16 mL of pyridine was added dropwise to the acid chloride
123

Med Chem Res
Fig. 2 Sample sensorgrams for
the SPR assay. a Interaction of
DNA gyrase alone (control)
with DNA results in an increase
in signal. b Pre-incubation of
DNA gyrase with SD8 abolishes
the interaction of DNA gyrase
with immobilized DNA. c,
d Pre-incubation of DNA gyrase
with 2a or 14c abolishes the
interaction of DNA gyrase with
immobilized DNA in a similar
manner to SD8. e, f Pre-
incubation of DNA gyrase with
2b or 2d does not inhibit DNA
gryase binding to DNA and
results in an increase in signal,
similar to control in a. Spikes at
the beginning and end of the
injection are due to the slight
difference in the DMSO
concentration between the
sample and the running buffer
TLC. mp 153 °C dec.; IR m (neat)/cm-1 3329, 3076, 2923,
2853; H NMR (400 MHz, CDCl3): d 1.24–1.37 (m, 12H,
Coumarin Scaffold
is required
1
O
O
~15 Å Linker
required
–(CH2)–), 1.63–1.74 (m, 2H, –(CH2)–), 2.43 (t, 2H,
J = 7.6, 7.6, –(CH2)–), 7.23–7.41 (m, 2H, Ar–H),
7.42–7.46 (m, 1H, Ar–H), 7.50 (dd, 1H, J = 1.2, 7.6, Ar–
H), 8.05 (s, 1H, Ar–H), 8.70 (s, 1H, N–H); 13C NMR
(100 MHz, CDCl3): d 25.25 (–(CH2)–), 29.04 (–(CH2)–),
29.70 (–(CH2)–), 37.69 (–(CH2)–), 116.34 (Ar–C), 119.93
(Ar–C), 123.20 (Ar–C), 125.17 (Ar–C), 127.78 (Ar–C),
129.57 (Ar–C), 149.88 (Ar–C); HRMS C19H23NO5 m/z
[M-H]-Expected: 344.1498, Found: 344.1500.
O
R1
OH
N
H
R2
O
Coumarin Substitutions
are tolerated
COOH
required
Fig. 3 Minimum coumarin pharmacophore for disrupting DNA
gyrase binding to DNA based on the data in Table 1
solution. The reaction was refluxed overnight and then
quenched by the addition of water (30 mL) and 6 M
hydrochloric acid (3 mL). The product was extracted into
an equal volume of ethyl acetate. The organic layer was
then separated, washed with 10 % copper sulfate solution,
and water until the water washes were no longer blue. The
organic layer was washed with brine, dried over sodium
sulfate, and then concentrated under vacuum. The reaction
afforded a white solid (0.69 g, 65 %) that was pure by
7-Oxo-7-((2-oxo-2H-chromen-3-yl)amino)heptanoic
acid (2b)
Pimelic acid (0.0994 g, 0.62 mmol) was dissolved in
0.62 mL dichloromethane. One drop of dimethylformam-
ide was added to the solution before oxalyl chloride
(0.337 mL, 3.1 mmol) was added dropwise. The reaction
was stirred for one hour at room temperature before the
123

Med Chem Res
solvent and excess oxalyl chloride was removed by vacuum.
The diacid chloride was then redissolved in the necessary
amount of dichloromethane. This solution was added drop-
wise to a mixture of 3-aminocoumarin (1) (0.1 g, 0.62 mmol)
and triethyl amine (0.26 mL, 1.9 mmol) dissolved in tetra-
hydrofuran (2.5 mL, 30.6 mmol). This reaction was refluxed
overnight before being quenched by the addition of water
(2 mL) and 6 M hydrochloric acid (0.5 mL). The product was
extracted into dichlormethane, dried over sodium sulfate, and
concentrated under vacuum. Purification by MPLC (Combi-
Flash, 0–100 % ethyl acetate in hexanes) gave a white solid
(0.049 g, 26 %). mp 187–190 °C; IR m (neat)/cm-1 2470,
2861, 2925 (b), 3324; 1H NMR (400 MHz, CDCl3): d
1.48–1.54 (m, 6H, –(CH2)–), 1.81 (m, 2H, –(CH2)–), 2.47 (t,
2H, J = 7.2, 7.6, –(CH2)–), 7.28–7.33 (m, 2H, Ar–H),
7.42–7.51 (m, 2H, Ar–H), 8.06 (s, 1H, Ar–H), 8.70 (s, 1H, N–
H); 13C NMR (100 MHz, CDCl3): d 24.82 (–(CH2)–), 28.58
(–(CH2)–), 37.32 (–(CH2)–), 116.34 (Ar–C), 119.89 (Ar–C),
123.29 (Ar–C), 123.94 (Ar–C), 125.17 (Ar–C), 127.80 (Ar–
C), 129.61 (Ar–C), 149.89 (Ar–C), 158.82 (C=O), 172.16
(C = O); HRMS C19H17NO5 m/z [M-H]- Expected:
302.1028, Found: 302.1037.
(–(CH2)–), 34.05 (–(CH2)–), 37.72 (–(CH2)–), 51.37 (Me),
116.33 (Ar–C), 123.13 (Ar–C), 124.01 (Ar–C), 125.14
(Ar–C), 127.75 (Ar–C), 129.54 (Ar–C), 172.47 (C=O),
174.19 (C=O); HRMS C20H25NO5 m/z [M-H]-Expected:
358.1654, Found: 358.1664.
N-(2-oxo-2H-chromen-3-yl)decanamide (2d)
Commercially available decanoyl chloride (0.12 mL,
0.68 mmol) was dissolved in pyridine (3.1 mL). To this
solution, 3-aminocoumarin (1) (0.110 g, 0.68 mmol) dis-
solved in pyridine (3.1 mL) was added dropwise. The
reaction was refluxed overnight before being quenched by
the addition of water (5 mL) and 6 M hydrochloric acid
(0.5 mL). The product was extracted into dichloromethane.
The organic layer was then washed with 10 % copper
sulfate solution, followed by water washes until the water
was no longer blue tinted. The organic layer was dried over
sodium sulfate and concentrated under vacuum. Purifica-
tion by MPLC (CombiFlash, 0–100 % ethyl acetate in
hexanes gradient) afforded an off-white solid (0.1158 g,
52 %). mp 106–107 °C; IR m (neat)/cm-1 2850, 2921,
1
3079, 3337; H NMR (400 MHz, CDCl3) d 0.88 (t, 3H,
Methyl 10-oxo-10-((2-oxo-2H-chromen-3-
yl)amino)decanoate (2c)
J = 6.8, 6.8, Me), 1.27–1.38 (m, 12H, –(CH2)–), 1.73
(m, 2H, –(CH2)–), 2.43 (t, 2H, J = 7.6, 7.6, –(CH2)–),
7.28–7.33 (m, 2H, Ar–H), 7.44 (m, 1H, Ar–H), 7.5 (dd, 1H,
J = 1.2, 7.6, Ar–H), 8.04 (s, 1H, Ar–H), 8.70 (s, 1H, N–H);
13C NMR (100 MHz, CDCl3) d 14.08 (–(CH2)–), 22.65
(–(CH2)–), 25.36 (–(CH2)–), 29.17 (–(CH2)–), 29.24
(–(CH2)–), 29.32 (–(CH2)–), 29.40 (–(CH2)–), 31.84
(–(CH2)–), 37.80 (Me), 116.35 (Ar–C), 123.16 (Ar–C),
125.17 (Ar–C), 127.77 (Ar–C), 129.56 (Ar–C); HRMS
C19H25NO3 m/z [M-H]- Expected: 314.1756, Found:
314.1760.
Commercially available sebacic acid methyl ester (0.134 g,
0.631 mmol) was dissolved in dichloromethane (0.621 mL).
To this solution, one drop of dimethylformamide was
added before oxalyl chloride was added dropwise
(0.337 mL, 3.1 mmol). The reaction was allowed to stir
one hour at room temperature before the solvent and excess
oxalyl chloride was removed by vacuum. The acid chloride
was then dissolved in the necessary amount of dichloro-
methane before being added dropwise to a solution of
3-aminocoumarin (1) (0.1 g, 0.62 mmol) and triethylamine
(0.26 mL, 1.8 mmol) in tetrahydrofuran (3.1 mL). This
reaction was refluxed overnight before being quenched by
the addition of water (3 mL) and a few drops of 6 M
hydrochloric acid. The product was extracted into dichlo-
romethane. The organic layer was washed with brine, then
dried over sodium sulfate, and concentrated under vacuum.
Flash silica gel chromatography (20–50 % gradient ethyl
acetate in hexanes) afforded a white solid (0.054 g
25 %). mp 116–117 °C; IR m (neat)/cm-1 2849, 2911,
2925, 3353; 1H NMR (400 MHz, CDCl3) d 1.26–1.40 (m,
8H, –(CH2)–), 1.62 (m, 2H, –(CH2)–), 1.73 (m, 2H,
–(CH2)–), 2.30 (t, 2H, J = 7.2, 8.0, –(CH2)–), 2.43 (t, 2H,
J = 7.2, 7.6, –(CH2)–), 3.67 (s, 3H, Me), 7.30 (m, 2H,
Ar–H), 7.42–7.46 (m, 1H, Ar–H), 7.50 (dd, 1H, J = 1.2,
7.6, Ar–H), 8.03 (s, 1H, Ar–H), 8.70 (s, 1H, N–H); 13C
NMR (100 MHz, CDCl3) d 24.88 (–(CH2)–), 25.27
(–(CH2)–), 29.03 (–(CH2)–), 29.06 (–(CH2)–), 29.08
4-Methoxy-N-(2-oxo-2H-chromen-3-yl)benzamide (2e)
3-Aminocoumarin (1) (0.1 g, 0.62 mmol) was dissolved in
tetrahydrofuran (2.5 mL). To the solution, triethyl amine
(0.26 mL, 1.86 mmol) was added, and the mixture was
brought to reflux. Neat p-methoxy benzoyl chloride
(0.11 g, 0.62 mmol) was added dropwise, and the reaction
was allowed to reflux overnight. The reaction was quen-
ched by the addition of water and a few drops of 6 M
hydrochloric acid. Product was extracted into dichloro-
methane. The organic layer was separated, dried over
sodium sulfate, and concentrated under vacuum, affording
an off-white solid (0.082 g, 45 %). mp 112–115 °C; IR m
1
(neat)/cm-1 2833, 2932, 3007, 3362; H NMR (400 MHz,
CDCl3) d 3.82 (s, 3H, Me), 4.31 (s, 1H, N–H), 6.33 (s, 1H,
Ar–H), 6.90 (d, 2H, J = 8.8, Ar–H), 7.17–7.23 (m, 2H,
Ar–H), 7.26–7.31 (m, 4H, Ar–H); 13C NMR (100 MHz,
123

Med Chem Res
CDCl3) d 55.32 (Me), 105.52 (Ar–C), 114.25 (Ar–C),
116.06 (Ar–C), 121.65 (Ar–C), 124.59 (Ar–C), 125.10
(Ar–C), 125.78 (Ar–C), 126.92 (Ar–C), 128.68 (Ar–C),
129.20 (Ar–C), 132.86 (Ar–C), 147.99 (Ar–C), 159.21
(C=O), 159.58 (C=O). HRMS C17H13NO4 m/z [M-H]-
Expected: 294.0766, Found: 294.0772.
(C=O); HRMS C9H7NO2 m/z [M-H]- Expected: 176.0348,
Found: 176.0353.
10-((4-Hydroxy-2-oxo-2H-chromen-3-yl)amino)-10-
oxodecanoic acid (5)
4-Hydroxy-3-aminocoumarin (4) (0.20 g, 0.97 mmol) was
dissolved in 10 mL pyridine and heated to reflux. Sebacic
diacid chloride (0.26 mL, 0.97 mmol) was added dropwise,
and the reaction was allowed to reflux overnight. The
reaction was quenched by the addition of water and 1 mL
6 M hydrochloric acid. Product was extracted into
dichloromethane and washed with 10 % copper sulfate
solution. The organic layer was then washed repeatedly
with water until they no longer were blue tinted, next was
dried over sodium sulfate, and concentrated under vacuum
to afford an off-white solid (0.315 g, 86 %). mp
N1,N10-Bis(2-oxo-2H-chromen-3-yl)decanediamide
(2f)
3-Aminocoumarin (1) (0.1 g, 0.62 mmol) was dissolved in
pyridine (3.1 mL) and heated to reflux. Sebacic acid
chloride (0.083 mL, 0.31 mmol) was added dropwise to
the solution. The reaction was allowed to reflux overnight
before being quenched by the addition of water (3 mL) and
a few drops of 6 M hydrochloric acid. Dichloromethane
was added to extract the product from the aqueous layer.
The organic layer was washed with 10 % copper sulfate,
followed by water washes until the wash was no longer
blue. The organic was dried over sodium sulfate and con-
centrated under vacuum. Purification by MPLC (Combi-
Flash, 0–100 % ethyl acetate in hexanes gradient) afforded
a white solid (0.032 g, 21 %). mp 213–217 °C; IR m (neat)/
cm-1 2853, 2927, 3077, 3329; 1H NMR (400 MHz,
CDCl3) d 1.35–1.40 (m, 8H, –(CH2)–), 1.68–1.75 (m, 4H,
–(CH2)–), 2.43 (t, 4H, J = 7.6, 7.2, –(CH2)–), 7.23–7.33
(m, 4H, Ar–H), 7.42–7.46 (m, 2H, Ar–H), 7.50 (dd, 2H,
J = 1.2, 7.6, Ar–H), 8.05 (s, 2H, Ar–H), 8.70 (s, 2H, N–H);
13C NMR (100 MHz, CDCl3) d 25.24 (–(CH2)–), 29.02
(–(CH2)–), 29.05 (–(CH2)–), 37.68 (–(CH2)–), 116.32
(Ar–C), 119.94 (Ar–C), 123.16 (Ar–C), 124.01 (Ar–C),
125.14 (Ar–C), 127.75 (Ar–C), 129.54 (Ar–C), 149.90
(Ar–C), 158.83 (C=O), 172.46 (C=O); HRMS C28H28N2O6
m/z [M-H]- Expected: 487.1869, Found: 487.1875.
1
85.5–87 °C; IR m (neat)/cm-1 2851, 2918 (b); H NMR
(400 MHz, DMSO) d 1.00–1.49 (m, 12H, –(CH2)–), 1.81
(m, 2H, –(CH2)–), 2.99 (t, 2H, J = 7.2, 7.6, –(CH2)–), 7.49
(t, 1H, J = 7.2, Ar–H), 7.62 (d, 1H, J = 8.4, Ar–H),
6.67–7.71 (m, 1H, Ar–H), 7.94 (d, 1H, J = 1.6, Ar–H),
11.95 (s, 1H, COOH); 13C NMR (100 MHz, DMSO) d
26.57 (–(CH2)–), 28.27 (–(CH2)–), 28.81 (–(CH2)–), 28.87
(–(CH2)–), 111.67 (Ar–C), 117.64 (Ar–C), 121.31 (Ar–C),
124.75 (Ar–C), 131.39 (Ar–C), 152.88 (Ar–C), 156.08
(C=O); HRMS C19H23NO6 m/z [M-H]-?OH- Expected:
377.1475, Found: 377.1810.
N1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)–N10-
phenyldecanediamide (6a)
Compound 5 (0.05 g, 0.138 mmol) was dissolved in
0.13 mL dichlormethane. To this solution, a drop of
dimethylformamide was added, followed by oxalyl chlo-
ride (0.075 mL, 0.69 mmol). The reaction was allowed to
stir an hour before the solvent was removed under vacuum.
The acid chloride was dissolved in the necessary amount of
dichloromethane, and added dropwise to a refluxing solu-
tion of aniline (0.014 mL, 0.152 mmol) and triethyl amine
(0.058 mL, 0.415 mmol) dissolved in 1 mL tetrahydrofu-
ran. The coupling reaction was allowed to reflux overnight
before being quenched by the addition of 1 mL water and
few drops of 6 M hydrochloric acid. Flash silica gel
chromatography (20–100 % ethyl acetate in hexanes) gave
an off-white solid (0.048 g, 80 %). mp 123.7–124.8 °C; IR
m (neat)/cm-1 2850, 2917, 3060 (w), 3323; 1H NMR
(400 MHz, DMSO) d 1.25–1.4 (m, 8H, –(CH2)–), 1.58 (m,
2H, –(CH2)–), 1.81 (m, 2H, –(CH2)–), 2.28 (t, 2H, J = 7.2,
–(CH2)–), 2.98 (t, 2H, J = 7.2, –(CH2)–), 7.0 (dd, 1H,
J = 7.2, 7.6, Ar–H), 7.26 (dd, 2H, J = 7.6, 8.4, Ar–H),
7.48 (ddd, 1H, J = 0.8, 7.2, 8.0, Ar–H), 7.58 (dd, 3H,
J = 7.2, 7.6, Ar–H), 7.68 (ddd, 1H, J = 1.6, 7.2, 1.2, 7.6,
3-Amino-4-hydroxy-2H-chromen-2-one (4)
4-Hydroxy-3-nitrocoumarin (3) (1 g, 4.8 mmol) was dis-
solved in 75 mL 1 % HCl in methanol. This solution was
cooled on ice and purged with nitrogen prior to the addition
of 5 % palladium on carbon (0.16 g). The reaction vessel
was sealed, placed in a Parr shaker hydrogenator, and
pressurized with hydrogen to 60 psi. The reaction was
monitored by TLC until it was complete, about 24 h. The
catalyst was filtered off and washed with methanol. The
filtrate was concentrated under vacuum to give a light
yellow solid (0.78 g, 92 %). mp 192 °C; IR m (neat)/cm-1
1
2602 (w), 2856, 2927, 3077, 3328; H NMR (400 MHz,
DMSO) d 7.32 (dd, 2H, J = 7.2, 7.6, Ar–H), 7.56 (ddd,
1H, J = 1.2, 7.4, 8.0, Ar–H), 7.96 (d, 1H, J = 8.0, Ar–H);
13C NMR (100 MHz, DMSO) d 117.47 (Ar–C), 122.46
(Ar–C), 125.52 (Ar–C), 131.42 (Ar–C), 133.32 (Ar–C),
135.17 (Ar–C), 153.24 (Ar–C), 159.54 (Ar–C), 180.64
123

Med Chem Res
Ar–H), 7.92 (dd, 1H, J = 1.6, 8.0, Ar–H), 9.82 (s, 1H, N–
H); 13C NMR (100 MHz, DMSO) d 25.04 (–(CH2)–),
25.96 (–(CH2)–), 27.42 (–(CH2)–), 28.25 (–(CH2)–), 28.45
(–(CH2)–), 28.54 (–(CH2)–), 36.35 (–(CH2)–), 111.14 (Ar–
C), 117.16 (Ar–C), 121.46 (Ar–C), 122.85 (Ar–C), 124.00
(Ar–C), 125.09 (Ar–C), 128.57 (Ar–C), 131.71 (Ar–C),
139.31 (Ar–C), 152.22 (Ar–C), 155.02 (Ar–C), 155.40
(Ar–C), 166.86 (C = O), 171.20 (C = O); HRMS
C25H28N2O5 m/z [M-H]- Expected: 435.1920, Found:
435.1924.
10 % copper sulfate solution, and then water washes until
the water was no longer tinted blue. The organic layer was
then dried over sodium sulfate and concentrated under
vacuum. Flash silica gel chromatography afforded a pale
yellow oil (0.050 g, 17 % yield). IR m (neat)/cm-1 2850,
2917 (s), 3080 (w), 3424 (b); 1H NMR (400 MHz, DMSO)
d 1.22–1.45 (m, 10H, –(CH2)–), 1.82 (m, 3H, –(CH2)–),
2.99 (t, 3H, J = 8.0, –(CH2)–), 7.48 (dd, 2H, J = 7.2, 7.6,
Ar–H), 7.59 (d, 2H, J = 8.4, Ar–H), 7.69 (ddd, 2H,
J = 1.6, 7.6, 8.0, Ar–H), 7.92 (d, 2H, J = 8.0, Ar–H).
HRMS C28H28N2O8 m/z [M-H]- Expected: 519.1767,
Found: 519.1770.
N1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-N10-(4-
hydroxyphenyl)decanediamide (6b)
3-Acetamido-2-oxo-2H-chromen-7-yl acetate (9)
Compound 5 (0.05 g, 0.138 mmol) was dissolved in
0.15 mL dichlormethane. To this solution, a drop of
dimethylformamide was added, followed by oxalyl chlo-
ride (0.075 mL, 0.69 mmol). The reaction was allowed to
stir an hour before the solvent was removed under vacuum.
The acid chloride was dissolved in the necessary amount of
dichloromethane, and added dropwise to a refluxing solu-
tion of 4-aminophenol (0.017 g, 0.15 mmol) and triethyl
amine (0.054 mL, 0.39 mmol) dissolved in 0.6 mL tetra-
hydrofuran. The coupling reaction was allowed to reflux
overnight before being quenched by the addition of 1 mL
water and few drops of 6 M hydrochloric acid. Flash silica
gel chromatography (0–10 % methanol in dichlorometh-
ane) afforded an off-white solid (0.024 g, 39 %). mp
181–184.7 °C; IR m (neat)/cm-1 2850, 2918, 3296 (w),
Compound 9 was synthesized according to the previously
reported procedure (Kudale et al., 2007). 2,4-Dihydroxy-
benzaldehyde (8) (10 g, 72.4 mmol) was dissolved in
362 mL acetic anhydride. Sodium acetate (23.76 g,
290 mmol) and N-acetylgycine were added to the solution
and the reaction was refluxed overnight. The reaction was
quenched by cooling on ice and slowly adding ice-cold
water. Precipitate was filter off and triturated with ice-cold
ethyl acetate to remove impurities and afford a pale pink
1
solid (4.54 g, 24 %). H NMR (400 MHz, CDCl3) d 2.24
(s, 3H, –O–CO–Me), 2.33 (s, 3H, –NH–CO–Me), 7.07 (dd,
1H, J = 2.0, 8.4, Ar–H), 7.13 (d, 1H, J = 2.0, Ar–H), 7.50
(d, 1H, J = 8.4, Ar–H), 8.02 (s, 1H, Ar–H), 8.67 (s, 1H, N–
13
H);
C NMR (100 MHz, CDCl3) d 21.09 (Me), 24.71
1
3448 (w); H NMR (400 MHz, DMSO) d 1.32 (m, 9H,
(Me), 110.04 (Ar–C), 117.63 (Ar–C), 119.12 (Ar–C),
122.74 (Ar–C), 123.57 (Ar–C), 128.36 (Ar–C), 150.15
(Ar–C), 151.39 (Ar–C), 158.49 (C=O), 168.83 (C=O),
169.33 (C=O).
–(CH2)–), 1.56 (m, 1H, –(CH2)–), 1.81 (m, 2H, –(CH2)–),
2.22 (t, 2H, J = 7.6, –(CH2)–), 2.99 (t, 2H, J = 7.6,
–(CH2)–), 6.65 (d, 2H, J = 8.8, Ar–H), 7.34 (d, 2H, J = 8.8,
Ar–H), 7.49 (dd, 1H, J = 8.0, 8.8, Ar–H), 7.93 (dd, 1H,
J = 1.6, 7.6, Ar–H), 9.10 (s, 1H, Ar–H), 9.56 (s, 1H, Ar–
H); 13C NMR (100 MHz, DMSO) d 25.17 (–(CH2)–),
25.97 (–(CH2)–), 27.42 (–(CH2)–), 28.26 (–(CH2)–), 28.59
(–(CH2)–), 111.15 (Ar–C), 114.92 (Ar–C), 117.17 (Ar–C),
120.77 (Ar–C), 121.48 (Ar–C), 125.09 (Ar–C), 131.71
(Ar–C), 152.23 (Ar–C), 155.03 (Ar–C), 166.87 (C=O).
HRMS C25H28N2O6 m/z [M-H]- Expected: 451.1869,
Found: 451.1876.
N-(7-(Benzyloxy)-2-oxo-2H-chromen-3-yl)acetamide
(10)
Compound 10 was synthesized according to the previously
reported procedure (Kudale et al., 2007). Compound 9
(4 g, 15.3 mmol) was dissolved in 30.5 mL methanol.
Potassium carbonate (2.75 g, 20 mmol) was added to the
solution and the reaction was allowed to stir 30 min. The
solvent was then removed by rotary evaporation. The solids
were then suspended in 77 mL acetonitrile before the
addition of benzyl bromide (2 mL, 16.8 mmol). This sec-
ond reaction was refluxed overnight. The reaction was then
diluted with water, and neutralized with 6 M HCl. Ethyl
acetate was then added to precipitate the product. Product
was filtered off and rinsed with water and then dried under
N1,N10-Bis(4-hydroxy-2-oxo-2H-chromen-3-
yl)decanediamide (7)
4-Hydroxy-3-aminocoumarin (4) (0.1 g, 0.565 mmol) was
dissolved in 2.8 mL pyridine and heated to reflux. Sebacic
diacid chloride (0.076 mL, 0.282 mmol) was added drop-
wise and the reaction was allowed to continue overnight.
The reaction was quenched by the addition of 3 mL water
and 0.5 mL 6 M hydrochloric acid. Product was extracted
with dichloromethane. The organic layer was washed with
1
vacuum to give a pale yellow solid (4.69 g, 99 %). H
NMR (400 MHz, CDCl3) d 2.22 (s, 3H, Me), 5.12 (s, 2H,
benzyl CH2), 6.89 (d, 1H, J = 2.0, Ar–H), 6.96 (dd, 1H,
J = 2.0, 8.4, Ar–H), 7.30–7.44 (m, 6H, Ar–H), 7.95 (s, 1H,
123

Med Chem Res
Ar–H), 8.63 (s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d
24.62 (Me), 70.56 (Ph–CH2–), 101.89 (Ar–C), 113.32 (Ar–
C), 113.86 (Ar–C), 121.69 (Ar–C), 123.99 (Ar–C), 127.47
(Ar–C), 128.33 (Ar–C), 128.65 (Ar–C), 128.73 (Ar–C),
135.95 (Ar–C), 151.34 (Ar–C), 159.00 (Ar–C), 160.35
(C = O), 169.11 (C = O).
3-Amino-7-hydroxy-2H-chromen-2-one (12)
Compound 11 (0.15 g, 0.561 mmol) was dissolved in
0.56 mL 1 % HCl methanol. The solution was cooled on ice
and the vessel purged with nitrogen gas before the addition of
5 % palladium on carbon (0.0075 g) catalyst. The reaction
vessel was placed under vacuum, and then backfilled with
1 atm hydrogen gas. The reaction was monitored by TLC.
Once TLC indicated no starting material remained, the cata-
lyst was filtered off and rinsed with methanol. The filtrate was
concentrated under vacuum to give an off-white solid
(0.091 g, 92 %). mp 212 °C dec.; IR m (neat)/cm-1 2598,
2851 (b), 3233 (b); 1H NMR (400 MHz, DMSO) d 6.68 (d,
1H, J = 1.6, Ar–H), 6.71 (dd, 1H, J = 1.6, 8.4, Ar–H), 7.29
(d, 1H, J = 8.4, Ar–H), 8.28 (s, 1H, Ar–H); 13C NMR
(100 MHz, DMSO) d 101.91 (Ar–C), 112.89 (Ar–C), 113.21
(Ar–C), 114.22 (Ar–C), 126.46 (Ar–C), 127.69 (Ar–C),
150.09 (Ar–C), 157.07 (Ar–C), 158.83 (C=O); HRMS
C9H7NO3 m/z[M-H]- Expected:176.0348, Found:176.0356.
3-Amino-7-(benzyloxy)-2H-chromen-2-one (11)
Compound 11 was synthesized according to the previously
reported procedure (Kudale et al., 2007). Compound 10
(4.5 g, 14.5 mmol) was suspended in 73 mL tetrahydro-
furan. To the solution, dimethylamino pyridine (8.89 g,
72.7 mmol) was added followed by di-tert-butyl dicar-
bonate (3.18 g, 14.5 mmol). The reaction was allowed to
stir until the yellow suspension became a clear, brown-
colored, solution, about 30 min. To the solution, hydrazine
(3.64 mL, 72.7 mmol) was added, followed by 60 mL
methanol. This second reaction was allowed to stir until the
reaction became bright orange, about 20 min. The solvents
were then removed by rotary evaporation, and the solids
dissolved in the necessary amount of dichloromethane. The
organic solution was washed with 1 M HCl, 10 % copper
sulfate, saturated sodium bicarbonate, and followed by a
brine wash. The organic layer was then dried over sodium
sulfate and concentrated under vacuum. 1H NMR
(400 MHz, CDCl3) d 1.53 (s, 9H, tBu), 5.11 (s, 2H, benzyl
CH2), 6.89 (d, 1H, J = 2.0, Ar–H), 6.92 (dd, 1H, J = 2.0,
8.4, Ar–H), 7.38 (m, 7H, Ar–H), 8.23 (s, 1H, N–H); 13C
NMR (100 MHz, CDCl3) d 20.87 (tBu), 70.52 (Ph-CH2-),
81.48 (tBu), 101.81 (Ar–C), 113.57 (Ar–C), 113.73 (Ar–
C), 121.17 (Ar–C), 122.28 (Ar–C), 127.50 (Ar–C), 128.12
(Ar–C), 128.30 (Ar–C), 128.73 (Ar–C), 136.04 (Ar–C),
150.88 (Ar–C), 152.59 (Ar–C), 158.86 (C=O), 159.83
(C=O). The Boc protected intermediate (5 g, 13.6 mmol)
was then dissolved in 68 mL dichloromethane. To this
solution, trifluoroacetic acid (20.8 mL, 272 mmol) was
added dropwise. The reaction was allowed to stir over night
at room temperature. Once TLC confirmed reaction had
gone to completion, the mixture was neutralized with sat-
urated sodium bicarbonate. The organic layer was sepa-
rated from the aqueous, and dried over sodium sulfate. The
organic layer was then dried over sodium sulfate and
concentrated under vacuum. Flash silica gel chromatogra-
phy (0–100 % ethyl acetate in hexanes gradient) gave
product (2.67 g, 69 %). 1H NMR (400 MHz, CDCl3) d
4.05 (broad s, 2H, NH2), 5.09 (s, 2H, benzyl CH2), 6.69 (s,
1H, Ar–H), 6.89 (m, 2H, Ar–H), 7.19 (d, 1H, J = 9.2, Ar–
H), 7.31–7.41 (m, 5H, Ar–H); 13C NMR (100 MHz,
CDCl3) d 70.47 (Ph-CH2-), 101.94 (Ar–C), 112.04 (Ar–C),
113.30 (Ar–C), 114.65 (Ar–C), 123.40 (Ar–C), 125.82
(Ar–C), 127.48 (Ar–C), 128.19 (Ar–C), 128.69 (Ar–C),
136.33 (Ar–C), 150.27 (Ar–C), 158.14 (Ar–C).
Methyl 10-((7-(benzyloxy)-2-oxo-2H-chromen-3-
yl)amino)-10-oxodecanoate (13a)
Sebacic acid methyl ester (0.0808 g, 0.374 mmol) was dis-
solved in 0.5 mL dichloromethane. A drop of dimethyl-
formamide was added before the addition of oxalyl chloride
(0.0016 mL, 1.87 mmol). The reaction was stirred for one
hour before the solvent was removed by rotary evaporation.
Once the acid chloride was concentrated to an oil, it was
dissolved in the necessary amount of dichloromethane
before being added dropwise to a solution of 11 (0.1 g,
0.374 mmol) and triethylamine (0.156 mL, 1.12 mmol) in
1.5 mL tetrahydrofuran. The coupling reaction was refluxed
overnight before being quenched by the addition of 2 mL
water and a few drops of 6 M HCl. Dichloromethane was
used to extract the product. The organic was dried over
sodium sulfate and concentrated under vacuum. Flash silica
gel chromatography (Rf = 0.68, 1:1 ethyl acetate to hex-
anes) gave of a white solid (0.064 g, 66 %). mp 123–126 °C;
IR m (neat)/cm-1 2851, 2921, 3033 (w), 3064 (w), 3318; 1H
NMR (400 MHz, CDCl3) d 1.25–1.40 (m, 8H, –(CH2)–),
1.62 (m, 2H, –(CH2)–), 1.72 (m, 2H, –(CH2)–), 2.30 (t, 2H,
J = 7.2, 7.6, –(CH2)–), 2.40 (t, 2H, J = 7.6, 7.6, –(CH2)–),
3.66 (s, 3H, Me), 5.12 (s, 2H, benzyl CH2), 6.89 (d, 1H,
J = 2.4, Ar–H), 6.96 (dd, 1H, J = 2.4, 8.4, Ar–H),
7.35–7.45 (m, 6H, Ar–H), 7.92 (s, 1H, Ar–H), 8.65 (s, 1H, N–
H).
N-(7-(Benzyloxy)-2-oxo-2H-chromen-3-yl)decanamide
(13b)
Commercially available decanoyl chloride (0.70 mL,
0.4 mmol) was dissolved in 1.75 mL pyridine and heated
123

Med Chem Res
to reflux. Compound 11 was dissolved in 1.75 mL pyridine
and added dropwise to the acid chloride solution. The
reaction was refluxed overnight before being quenched by
the addition of an equal volume of water and 0.5 mL 6 M
hydrochloric acid. The product was extracted into dichlo-
romethane and washed with 10 % copper sulfate solution,
and then water until the water was no longer blue-tinted.
The organic was then dried over sodium sulfate and con-
centrated under vacuum. Flash silica gel chromatography
(Rf = 0.90 in 1:1 ethyl acetate in hexanes) afforded an oil
(0.115 g, 66 %). IR m (neat)/cm-1 2853, 2923, 2952 (w),
3038 (w), 3316; 1H NMR (400 MHz, CDCl3) d 0.88 (t, 3H,
J = 6.8, 6.8, Me), 1.26 (m, 12H, –(CH2)–), 1.61 (m, 2H,
–(CH2)–), 2.30 (t, 2H, J = 7.2, 7.6, –(CH2)–), 5.12 (s, 2H,
benzyl CH2), 6.89 (d, 1H, J = 2.4, Ar–H), 6.95 (dd, 1H,
J = 2.4, 8.4, Ar–H), 7.40 (m, 6H, Ar–H), 7.93 (s, 1H,
Ar–H), 8.65 (s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d
14.03 (Me), 22.26 (–(CH2)–), 25.39 (–(CH2)–), 29.13
(–(CH2)–), 29.21 (–(CH2)–), 29.30 (–(CH2)–), 29.66
(–(CH2)–), 31.83 (–(CH2)–), 37.75 (–(CH2)–), 70.56
(Ph-CH2-), 101.88 (Ar–C), 113.41 (Ar–C), 113.83 (Ar–C),
121.73 (Ar–C), 123.87 (Ar–C), 127.47 (Ar–C), 128.31
(Ar–C), 128.59 (Ar–C), 128.72 (Ar–C), 174.27 (C=O),
206.68 (C=O).
was filtered off and rinsed with methanol. The filtrate was
concentrated under vacuum. Flash silica gel chromatogra-
phy (0–100 % ethyl acetate in hexanes gradient) gave a
white solid (0.013 g, 27 %). mp 152.0–154.0 °C; IR m
(neat)/cm-1 2849, 2918, 3251 (b), 3316; 1H NMR
(400 MHz, CDCl3) d 1.22–1.41 (m, 8H, –(CH2)–), 1.62 (m,
4H, –(CH2)–), 1.70 (m, 2H, –(CH2)–), 2.30 (t, 2H, J = 7.6,
–(CH2)–), 3.66 (s, 3H, Me), 6.81 (m, 2H, Ar–H), 7.37 (d,
1H, J = 8.8, Ar–H), 7.93 (s, 1H, Ar–H) 8.65 (s, 1H, N–H);
13C NMR (100 MHz, DMSO) d 24.33 (–(CH2)–), 24.96
(–(CH2)–), 28.31 (–(CH2)–), 28.39 (–(CH2)–), 28.42
(–(CH2)–), 28.47 (–(CH2)–), 33.24 (–(CH2)–), 35.81
(–(CH2)–), 41.30 (–(CH2)–), 51.11 (Me), 101.91 (Ar–C),
111.37 (Ar–C), 120.81 (Ar–C), 126.21 (Ar–C), 128.92
(Ar–C), 151.49 (Ar–C), 157.93 (Ar–C), 159.45 (C=O),
172.84 (C=O), 173.48 (C=O); HRMS C20H25NO6 m/z [M-
H]- Expected: 374.1604, Found: 374.1625.
N-(7-Hydroxy-2-oxo-2H-chromen-3-yl)decanamide
(14b)
Compound 13b (0.1 g, 0.230 mmol) was dissolved in 0.3
mL1 % HCl methanol. The solution was cooled on ice and
the vessel purged with nitrogen gas before the addition of
5 % palladium on carbon (0.0005 g) catalyst. The reaction
vessel was placed under vacuum, and then backfilled with
1 atm hydrogen gas. The reaction was monitored by TLC
(Rf = 0.42, 1:1 ethyl acetate in hexanes). Once TLC
indicated no starting material remained, the catalyst was
filtered off and rinsed with methanol. The filtrate was
concentrated under vacuum. Flash silica gel chromatogra-
phy (0–100 % ethyl acetate in hexanes gradient) gave an
off-white solid (0.028 g, 70 %). mp 138.4–142.6 °C; IR m
(neat)/cm-1 2852, 2920, 3146 (b), 3310; 1H NMR
(400 MHz, CDCl3) d 0.88 (t, 3H, J = 6.8, 6.8, Me), 1.27
(m, 12H, –(CH2)–), 1.71 (t, 2H, J = 7.6, 7.6, –(CH2)–),
2.41 (t, 2H, J = 7.6, 7.6, –(CH2)–), 6.81 (m, 2H, Ar–H),
7.37 (d, 1H, J = 9.2, Ar–H), 7.93 (s, 1H, Ar–H), 8.65 (s,
1H, N–H); 13C NMR (100 MHz, CDCl3) d 14.04 (Me),
22.62 (–(CH2)–), 25.41 (–(CH2)–), 29.16 (–(CH2)–), 29.21
(–(CH2)–), 29.29 (–(CH2)–), 29.38 (–(CH2)–), 31.82
(–(CH2)–), 37.77 (–(CH2)–), 103.06 (Ar–C), 113.72 (Ar–
C), 124.03 (Ar–C), 128.95 (Ar–C), 157.25 (Ar–C), 159.05
(C = O); HRMS C19H25NO4 m/z [M-H]- Expected:
330.1705, Found: 330.1724.
10-((7-(benzyloxy)-2-oxo-2H-chromen-3-yl)amino)-
10-oxodecanoic acid (13c)
To a solution of 13a (0.125 g, 0.277 mmol) in 1,2-
dichloroethane (1 mL) was added to trimethyltin hydroxide
(0.151 g, 0.832 mmol) and heated at 80 °C for 16 h. The
reaction mixture was then concentrated in vacuo and the
residue was purified by column chromatography (silica gel,
methanol:dichloromethane 1:9) to give compound 13c as
cream-colored solid (0.060 g, yield 60 %). 1H NMR
(400 MHz, CDCl3) d 1.34 (m, 8H, –(CH2)–), 1.62–1.72
(m, 4H, –(CH2)–), 2.35 (t, J = 7.2 Hz, 2H, –(CH2)–), 2.41
(t, J = 7.4 Hz, 2H, –(CH2)–), 5.12 (s, 2H, benzyl CH2),
6.89 (d, 1H, J = 2.3 Hz, Ar–H), 6.95 (dd, 1H, J = 2.3 Hz,
8.7 Hz, Ar–H), 7.35–7.44 (m, 6H, Ar–H), 7.99 (s, 1H, Ar–
H), 8.66 (s, 1H, N–H).
Methyl 10-((7-hydroxy-2-oxo-2H-chromen-3-
yl)amino)-10-oxodecanoate (14a)
Compound 13a (0.060 g, 0.129 mmol) was dissolved in
0.3 mL 1 % HCl in methanol. The solution was cooled on
ice and purged with nitrogen gas prior to the addition of
5 % palladium on carbon catalyst (0.010 g). The reaction
vessel was placed under vacuum, and then backfilled with
1 atm hydrogen gas. The reaction was monitored by TLC
(Rf = 0.64, 1:1 ethyl acetate in hexanes). Once TLC
indicated that no starting material remained, the catalyst
10-((7-hydroxy-2-oxo-2H-chromen-3-yl)amino)-10-
oxodecanoic acid (14c)
To a solution of 13c (0.018 g, 0.04 mmol) in ethyl acetate
(4 ml) underwent hydrogenolysis under a hydrogen balloon
in the presence of 10 % w/w Pd/C (0.002 g) at room
temperature for 16 h. The mixture was filtered through a
123

Med Chem Res
Celite pad. The mother liquor was concentrated in vacuo
and the residue was purified by column chromatography
(silica gel, methanol:dichloromethane 1:9) to give the
desired product 14c as cream-colored solid (0.012 g,
combined organic washes were washed with brine and
dried over sodium sulfate before being concentrated under
vacuum. Flash silica gel chromatography (0–10 % metha-
nol in dichloromethane gradient) afforded an off-white
solid with a 63 % yield, which was directly carried into the
next step.
1
83 %). H NMR (400 MHz, DMSO-d6) d 1.25 (m, 8H,
–(CH2)–), 1.50 (m, 4H, –(CH2)–), 2.18 (t, J = 7.3 Hz, 2H,
–(CH2)–), 2.42 (t, J = 7.3 Hz, 2H, –(CH2)–), 6.72 (d, 1H,
J = 2.1 Hz, Ar–H), 6.78 (dd, 1H, J = 2.2, 8.5 Hz, Ar–H),
7.49 (d, 1H, J = 8.3 Hz, Ar–H), 8.50 (s, 1H, Ar–H), 9.44
(s, 1H, N–H), 10.34 (s, 1H, OH), 11.94 (br, 1H, COOH).
13C NMR (100 MHz, DMSO-d6) d 24.4 (–(CH2)–), 25.0
(–(CH2)–), 28.4 (–(CH2)–), 28.5 (–(CH2)–), 28.5 (–(CH2)–),
28.6 (–(CH2)–), 33.6 (–(CH2)–), 35.8 (–(CH2)–), 101.9
(Ar–C), 111.4 (Ar–C), 113.5 (Ar–C), 120.9 (Ar–C), 127.2
(Ar–C), 128.9 (Ar–C), 151.4 (Ar–C), 157.9 (Ar–C), 159.4
(C=O), 172.7 (C=O), 174.4 (C=O); HRMS C19H22NO6 m/z
[M-H]- found 360.1460, expected 360.1447.
4,7-Dihydroxy-2H-chromen-2-one (18)
Compound 17 (0.2 g, 0.745 mmol) was dissolved in 3 mL
methanol. The solution was cooled on ice and the reaction
vessel purged with nitrogen gas before the addition of 5 %
palladium on carbon (0.01 g). The reaction vessel was
placed under vacuum, and then backfilled with 1 atm
hydrogen gas. The reaction was monitored by TLC. Once
TLC indicated reaction was complete, the catalyst was
filtered off and rinsed with methanol until the filtrate was
no longer colored. The filtrate was concentrated under
vacuum. Flash silica gel chromatography (0–10 % metha-
nol in dichloromethane gradient) gave a white solid
(0.130 g, 98 %). mp 216 °C dec.; IR m (neat)/cm-1 2600
1-(4-(Benzyloxy)-2-hydroxyphenyl)ethanone (16)
Mono protection of the 2,4-dihydroxyacetophenone (15)
was carried out according to the previously reported pro-
cedure (Jung et al., 2003). 2,4-Dihydroxyacetophenone
(15) (10 g, 65.7 mmol) was dissolved in 131 mL acetoni-
trile. Potassium carbonate (9.99 g, 72.3 mmol) was added
to the solution, and the mixture was refluxed for one hour.
After one hour, benzyl bromide (7.66 mL, 64.4 mmol)
dissolved in 13.4 mL acetonitrile was added dropwise to
the mixture. The protection reaction was then refluxed an
additional 3 h before being quenched by the removal of the
potassium carbonate. Ethyl acetate was added to the filtrate
and was washed with dilute HCl. Silica gel chromatogra-
phy (20 % ethyl acetate in hexanes) afforded 16 (15.74 g,
1
(b), 2852, 2921, 2953 (w); H NMR (400 MHz, MeOD) d
5.44 (s, 1H, a-CH), 6.69 (d, 1H, J = 2.0, Ar–H), 7.79 (dd,
1H, J = 2.0, 8.4, Ar–H), 7.73 (d, 1H, J = 8.4, Ar–H); 13C
NMR (100 MHz, MeOD) d 30.73 (a-C–H), 103.17 (C–
OH), 109.49 (Ar–C), 114.11 (Ar–C), 125.93 (Ar–C),
157.20 (Ar–C), 163.67 (Ar–C), 167.04 (Ar–C), 169.22
(C=O); HRMS C9H6O4 m/z [M-H]- Expected: 177.0188,
Found: 177.0193.
SPR Experimental Procedure
Linear, 50 biotinylated DNA was dissolved in SPR buffer
(10 mM HEPES pH 7.3, 3 mM EDTA, 0.005 % surfactant
P20) to give a concentration of 600 nM. The DNA was
immobilized on the surface of flow cell two of a strepta-
vidin-coated sensor chip by flowing 50 lL of the solution
through at a rate of 10 lL per minute. Flow cells one and
two were then capped with 100 lL of a 1 mg/mL solution
of biotin in SPR buffer. The BioCore T200 instrument was
then primed with SPR buffer containing 1 % (v/v) DMSO,
and the flow rate was increased to 50 lL/min. To test the
compound’s ability to inhibit the DNA–DNA gyrase
binding interaction, a premixed solution of DNA gyrase
(100 nM) with or without compound (100 lM) was
injected over flow cells one and two for 7 min. After
allowing four minutes for dissociation, the surface of the
flow cell was regenerated using 1 M NaCl in 50 mM
NaOH for 30 s.
1
99 %). H NMR (400 MHz, CDCl3) d 2.56 (s, 3H, Me),
5.10 (s, 2H, benzyl CH2), 6.50 (s, 1H, Ar–H), 6.53 (d, 1H,
J = 2.48, Ar–H), 7.30–7.47 (m, 5H, Ar–H), 7.64 (d, 1H,
J = 9.12, Ar–H), 12.73 (s, 1H, OH).
7-(Benzyloxy)-4-hydroxy-2H-chromen-2-one (17)
Closing of the coumarin ring of the monoprotected aceto-
phenone 16 was carried out according to the previously
reported procedure (Jung et al., 2003). Sodium hydride
(60 % in mineral oil, 0.70 g, 17.5 mmol) was suspended in
30 mL of toluene. Added to the suspension was 4-benzy-
loxy-2-hydroxyacetophenone 16 (1.5 g, 5.9 mmol) dis-
solved in 15 mL toluene. Once the basic acetophenone
mixture stopped bubbling, diethyl carbonate (1.06 mL,
8.8 mmol) in 7.5 mL toluene was added dropwise. The
reaction was refluxed overnight before being cooled to
0 °C and quenched with water and the necessary 6 M HCl
to bring the pH to 1. The aqueous solution was washed with
ethyl acetate multiple times to extract the product. The
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 Phar-
macy (to K.C.E.). The author thanks Professor Richard A. Glennon,
123

Med Chem Res
Professor B. Frank Gupton, and Professor John C. Hackett for helpful
discussions and Professor Glen Kellogg for assistance in preparing
this manuscript.
Jones D (2010) The antibacterial lead discovery challenge. Nat Rev
Drug Discov 9:751–752
Jung S-H, Cho S-H, Hung Dang T, Lee J-H, Ju J-H, Kim M-K, Lee
S-H, Ryu J-C, Kim
Y (2003) Structural requirement of
isoflavonones for the inhibitory activity of interleukin-5. Eur J
Med Chem 38:537–545
Kudale AA, Kendall J, Warford CC, Wilkins ND, Bodwell GJ (2007)
Hydrolysis-free synthesis of 3-aminocoumarins. Tetrahedron
Lett 48:5077–5080
References
Bax BD, Chan PF, Eggleston DS, Fosberry A, Gentry DR, Gorrec F,
Giordano I, Hann MM, Hennessy A, Hibbs M, Jianzhong H, Jones
E, Jones J, Brown KK, Lewis CJ, May EW, Saunders MR, Singh O,
Spitzfaden CE, Shen C (2010) Type IIA topoisomerase inhibition
by a new class of antibacterial agents. Nature 466:935–940
´
Martınez JL (2012) The antibiotic resistome: challenge and oppor-
tunity for therapeutic intervention. Future Med Chem 4:347–359
Maxwell A (1997) DNA gyrase as a drug target. Trends Microbiol
5:102–109
Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice
LB, Scheld M, Spellberg B, Bartlett J (2009) Bad bugs, no drugs:
no ESKAPE! an update from the infectious diseases society of
America. Clin Infect Dis 48:1–12
Newman DJ, Cragg GM (2007) Natural products as sources of new
drugs over the last 25 years. J Nat Prod 70:461–477
Oppegard LM, Hamann BL, Streck KR, Ellis KC, Fiedler H-P,
Khodursky AB, Hiasa H (2009) In vivo and in vitro patterns of
the activity of simocyclinone D8, an angucyclinone antibiotic
from streptomyces antibioticus. Antimicrob Agents Chemother
53:2110–2119
Brown SD, Farrell DJ, Morrissey I (2004) Prevalence and molecular
analysis of macrolide and fluoroquinolone resistance among
isolates of streptococcus pneumoniae collected during the
2000–2001 PROTEKT US Study. J Clin Microbiol 42:4980–4987
Devasia RA, Blackman A, Gebretsadik T, Griffin M, Shintani A, May
C, Smith T, Hooper N, Maruri F, Warkentin J, Mitchel E,
Sterling TR (2009) Fluoroquinolone resistance in mycobacte-
rium tuberculosis: the effect of duration and timing of fluoro-
quinolone exposure. Am J Respir Crit Care Med 180:365–370
Edwards MJ, Flatman RH, Mitchenall LA, Stevenson CEM, Le TBK,
Clarke TA, McKay AR, Fiedler H-P, Buttner MJ, Lawson DM,
Maxwell A (2009) A crystal structure of the bifunctional antibiotic
simocyclinone D8, bound to DNA gyrase. Science 326:1415–1418
Flatman RH, Howells AJ, Heide L, Fiedler H-P, Maxwell A (2005)
Simocyclinone D8, an inhibitor of DNA gyrase with a novel
mode of action. Antimicrob Agents Chemother 49:1093–1100
´
´
´
´
Perez-Vazquez M, Roman F, Garcıa-Cobos S, Campos J (2007)
Fluoroquinolone resistance in haemophilus influenzae is asso-
ciated with hypermutability. Antimicrob Agents Chemother
51:1566–1569
Reinhardt A, Ko¨hler T, Wood P, Rohner P, Dumas J-L, Ricou B, van
Delden C (2007) Development and persistence of antimicrobial
resistance in pseudomonas aeruginosa: a longitudinal observa-
tion in mechanically ventilated patients. Antimicrob Agents
Chemother 51:1341–1350
Wright GD (2012) Antibiotics: a new hope. Chem Biol 19:3–10
Yu XM, Shen G, Neckers L, Blake H, Holzbeierlein J, Cronk B,
Blagg BSJ (2005) Hsp90 inhibitors identified from a library of
novobiocin analogues. J Am Chem Soc 127:12778–12779
123