Coumarin-based inhibitors of bacillus anthracis and staphylococcus aureus replicative DNA helicase: Chemical optimization, biological evaluation, and antibacterial activities

Article abstract of DOI:10.1021/jm300922h

The increasing prevalence of drug-resistant bacterial infections demands the development of new antibacterials that are not subject to existing mechanisms of resistance. Previously, we described coumarin-based inhibitors of an underexploited bacterial tar

Full text of DOI:10.1021/jm300922h

Article
pubs.acs.org/jmc
Coumarin-Based Inhibitors of Bacillus anthracis and Staphylococcus
aureus Replicative DNA Helicase: Chemical Optimization, Biological
Evaluation, and Antibacterial Activities
Bing Li,*,† Ramdas Pai,† Ming Di,† Daniel Aiello,† Marjorie H. Barnes,† Michelle M. Butler,†
Tommy F. Tashjian,† Norton P. Peet,† Terry L. Bowlin,† and Donald T. Moir*,†
†Microbiotix Inc., One Innovation Drive, Worcester, Massachusetts 01605, United States
S
* Supporting Information
ABSTRACT: The increasing prevalence of drug-resistant bacterial infections
demands the development of new antibacterials that are not subject to existing
mechanisms of resistance. Previously, we described coumarin-based inhibitors of an
underexploited bacterial target, namely the replicative helicase. Here we report the
synthesis and evaluation of optimized coumarin-based inhibitors with 9−18-fold
increased potency against Staphylococcus aureus (Sa) and Bacillus anthracis (Ba)
helicases. Compounds 20 and 22 provided the best potency, with IC50 values of 3
and 1 μM, respectively, against the DNA duplex strand-unwinding activities of both
B. anthracis and S. aureus helicases without affecting the single strand DNA-
stimulated ATPase activity. Selectivity index (SI = CC50/MIC) values against S.
aureus and B. anthracis for compound 20 were 33 and 66 and for compound 22 were
20 and 40, respectively. In addition, compounds 20 and 22 demonstrated potent
antibacterial activity against multiple ciprofloxacin-resistant MRSA strains, with MIC
values ranging between 0.5 and 4.2 μg/mL.
structures of the B. anthracis and S. aureus replicative helicases
differ significantly from those of their eukaryotic counter-
parts,14,15 indicating that bacterial-specific inhibitors of helicase
may be identified. The human replicative helicase was described
recently as a complex of 11 proteins, namely Cdc45/MCM2−
7/GINS (“CMG”), none of which have significant homology to
the DnaB family of bacterial hexameric replicative helicases.16,17
Accordingly, inhibitors of B. anthracis helicase are unlikely to
demonstrate target-based toxicity vs mammalian hosts.
For all of the reasons described above, DnaB helicase from
Escherichia coli, S. aureus, and Pseudomonas aeruginosa have been
targeted previously in anti-infective screens. Screening assay
readouts have included electrochemiluminescence,18 fluores-
cence or FRET,19−21 time-resolved FRET,22 scintillation
proximity (SPA),23,24 and radiometric detection of ATPase
inhibition,25 but few hits have been described and none have
progressed further in drug development. A triaminotriazine
structure was recently shown to inhibit P. aeruginosa DnaB, but
it displays significant cytotoxicity and is not selective in MMS
studies.20 A large antibacterial screening effort undertaken by
GSK resulted in no hits for S. aureus replicative helicase.26
While hits were obtained for another essential helicase (PcrA)
in S. aureus, which is involved in DNA repair and plasmid
replication, no lead compounds could be developed from these
hits.26 Inhibitors of the E. coli orthologue of PcrA, namely
INTRODUCTION
■
Bacillus anthracis (Ba), the causative agent of anthrax, is
considered an agent of biological warfare or terrorism because
of its virulence, its stability in aerosol form, and its previous use
in acts of terrorism.1,2 While ciprofloxacin and doxycycline are
effective antidotes if administered immediately after suspected
contact with B. anthracis, there is little doubt that capable
terrorists will develop forms of the bacterium resistant to these
common antibiotics. Moreover, antibiotic resistant pathogens
are not only a problem for biodefense but are also found
increasingly in the clinic, e.g., community-acquired methicillin-
resistant Staphylococcus aureus (CA-MRSA). New antibiotics
based on underexploited targets are critical components for
treating drug-resistant pathogens both in the clinic and for
biodefense because there will be no pre-existing target-based
resistance mechanisms for such new agents. A key example of
such an underexploited target is the bacterial replicative
helicase, which catalyzes an essential rate-limiting step in
DNA replication. Several features of the B. anthracis and S.
aureus replicative DNA helicase make them particularly
attractive as targets for the discovery of new antibacterial
therapeutics for biodefense. First, they are members of a drug-
validated pathway. While gyrase, topoisomerase IV, and DNA
polymerase III have been targeted successfully, helicase remains
an untapped vulnerability in the mechanism of bacterial DNA
replication. Second, they are multifunctional proteins, providing
multiple opportunities for antibacterial intervention.3−9 Third,
helicase activity is essential to bacteria.10−14 Fourth, the primary
Received: July 10, 2012
© XXXX American Chemical Society
A
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helicase IV, have also been described, but no information on
cytotoxicity was provided and they do not appear to have
progressed further.25 Two investigators have described
inhibition of E. coli helicases (DnaB and RepA) by flavones
such as myricetin;27,28 however, myricetin is quite promiscuous
and cytotoxic. Similarly, intercalators and minor groove binders,
which interact with DNA, are potent helicase inhibitors but
they lack bacterial selectivity as well.29
Recently, we reported the discovery and validation of five
different chemotypes of B. anthracis and S. aureus helicase
inhibitors in a high-throughput screening effort. The most
potent inhibitors discovered in this campaign shared a
coumarin scaffold as a common motif (Figure 1),30 but they
were prepared using Suzuki coupling reactions, followed by
ester hydrolysis.
Structure−Activity Relationship (SAR) Studies. Synthe-
sized coumarin analogues were evaluated in a fluorescence
resonance energy transfer (FRET)-based assay to measure
concentration-dependent inhibition of ATP-dependent DNA
strand unwinding catalyzed by the B. anthracis and S. aureus
DNA replicative helicases. To further optimize the potency of
the coumarin-based inhibitors, we extended the preliminary
SAR studies reported previously.30 We maintained the essential
carboxylic acid group attached to the 3-position through an
alkyl linker and systematically investigated substitutents at the
7-position, which had been shown to influence antihelicase
potency significantly. Specifically, we used alkenyl, alkynyl,
aromatic, or heteroaromatic groups and explored substituent
effects on the aromatic or heteroaromatic rings. As shown in
Table 1, compounds 5 and 6 with an alkenyl or an alkynyl
group at the R3 position were inactive vs both helicases, while
compound 7 with a phenyl group at the R3 position exhibited
weak inhibitory activity vs both helicases. However, with
compound 8, bearing a pyridyl group at the same position,
inhibitory activity was undetectable. Compounds with
quinolinyl- or isoquinolinyl-substitution at the R3 position
(10−13) exhibited weak or no inhibitory activity vs DNA
helicases. Interestingly, 1-naphthyl, 2-naphthyl, or anthracenyl
substitution at the R3 position (compounds 1, 9, and 14,
respectively) provided moderate potency, with IC50 values
ranging from 6 to 10 μM. Compounds with biphenyl
substitution at the R3 position were potent DNA helicase
inhibitors, with 1,4-biphenyl substitution providing the best
potency against both B. anthracis and S. aureus helicases
(compound 20 (1,4-biphenyl) vs compounds 15 (1,2-biphenyl)
and 18 (1,3-biphenyl) and compound 19 (1,4-biphenyl) vs
compounds 16 (1,2-biphenyl) and 17 (1,3-biphenyl)). We next
examined the effect of substituents on the biphenyl group.
Substitutions with F, Cl, CF3, and CN on the distal phenyl ring
were all tolerated within a 2-fold range of potency compared to
unsubstituted compound 20. The most potent biphenyl
compound was chloro compound 22, which exhibited an IC50
value of 1.0 μM against S. aureus helicase and a comparable
value against B. anthracis helicase (IC50 = 1.5 μM).
Figure 1. Two coumarin-based helicase HTS hits.
did not inhibit gyrase or the binding of ATP to helicase.
Preliminary SAR studies of the coumarin-based inhibitors
indicated that the substituent at the 7-position dramatically
affects the potency against B. anthracis and S. aureus helicases
and that an ester functionality at the 3-position resulted in
compounds that were inactive against both of the DNA
helicases. Herein we report the chemical optimization,
biological evaluation, and antibacterial activities of this
coumarin-based series of Bacillus anthracis and Staphylococcus
aureus DNA replicative helicase inhibitors.
RESULTS AND DISCUSSION
■
Chemistry. The general synthesis of coumarin helicase
inhibitors is illustrated in Scheme 1. The classic Pechmann
condensation31 of 2-ethylresorcinol (3a), 2-methylresorcinol
(3b), or resorcinol (3c) with various β-keto esters provided 7-
hydroxycoumarin intermediates 4a−e, which were further
derivatized with alkylating agents. Hydrolysis of coumarin
esters provided the corresponding coumarin carboxylic acids.
Amides were also prepared from selected coumarin carboxylic
acids. Synthesis of biphenyl coumarin helicase inhibitors 24−27
is shown in Scheme 2. The 7-[(4-bromo)benzyloxy]coumarin
compound 23 was produced by alkylation of the 7-
hydroxycoumarin precursor 4a. Biphenyl compounds 24−27
The effect of methyl substituents at the 4- and 8-positions
(R2 and R1, Table 1) of the coumarin core on potency was
examined by comparison with unsubstituted or ethyl-
substituted counterparts. We found that removal of the methyl
group from the 4-position of the coumarin core dramatically
decreased the potency (33 vs 20), while unsubstituted 8-
position compounds exhibited a decreased potency of about 2−
3-fold (15 vs 16, 18 vs 17, and 20 vs 19). Ethyl substitution was
well-tolerated at the 8-position, as evidenced by the low
a
Scheme 1. General Synthesis of Coumarin Helicase Inhibitors
a
Reagents and conditions: (a) CH3COCH(CO2Et)(CH2)nCO2Et (n = 1−3), H2SO4, 0 °C, or HCOCH(CO2Et)(CH2)2CO2Et, H2SO4, RT; (b)
R3CH2X, Na2CO3, DMF, RT; (c) 2N NaOH, RT; (d) (COCl)2, DMF, THF; (e) RNH2.
B
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a
Scheme 2. Synthesis of Biphenyl Coumarin Helicase Inhibitors 24−27
a
Reagents and conditions: (a) 4-bromobenzyl bromide, Na2CO3, DMF, RT; (b) Ar−B(OH)2, Pd(PPh3)4, Na2CO3, DME, 85 °C; (c) 2N NaOH,
RT.
micromolar IC50 values for compounds 34 and 35. We also
substitution is tolerated, while 2′,4′-difluoro-substitution is
detrimental to the antibacterial activities (see compound 27).
Compound 34, bearing an ethyl group at the 8-position of
the coumarin ring, displayed antibacterial activities comparable
to its 8-methyl analogue 20, while compound 33, without a
substituent at the 4-position, displayed a higher MIC value vs S.
aureus. Helicase inhibitors with different linkers at the 3-
position of the coumarin core generally exhibited Bacillus
growth inhibitory activity. However, compounds with a
propylene linker exhibited much higher MIC values for S.
aureus than did those with ethylene or methylene linkers (32
and 35 vs 20 and 31).
varied the length of the aliphatic linker between the coumarin
core and the carboxylic acid functionality at the 3-position.
Compared to compound 20, which bears an ethylene
(−CH2CH2−) linker between the coumarin core and the
carboxylic acid functionality, compound 31, with a methylene
(−CH2−) linker, demonstrated 3−4-fold less potency against
both DNA helicases. Interestingly, compounds 32 and 35,
bearing a propylene (−CH2CH2CH2−) linker between the
coumarin core and the carboxylic acid functionality, exhibited
potency comparable to that of compound 20.
In general, the carboxylic acid functionality plays an
important role in helicase inhibition for the coumarin
compounds, suggesting that the carboxylic acid makes favorable
interactions with the bacterial DNA helicase target. Unlike the
acid compounds, neutral amide compounds 28 and 29 were
less active vs B. anthracis helicase (see Table 1: for 28, IC50
(Ba) >100 μM; for 29, IC50 (Ba) >100 μM) and exhibited a
substantial reduction in potency against S. aureus helicase (see
Table 1: for 28, IC50 (Sa) = 38 μM; for 29, IC50 (Sa) >100
μM). Surprisingly, amide compound 30, with an additional
flexible linker tethered to a tertiary amine moiety, exhibited
even better helicase inhibitory activity than observed for
compound 20. This is an exception to the general SARs. In the
absence of cocrystal structural information, it is not possible to
provide a definitive explanation for the antihelicase potency of
this compound. However, we propose that the positively
charged amine moiety with a flexible long linkage may be
extending into a solvent accessible region or a hydrophilic
surface to make additional interactions with the enzyme, thus
increasing affinity.
Antibacterial Activity of Helicase Inhibitors. Com-
pounds which showed DNA helicase inhibitory activities were
further evaluated in bacterial growth inhibition assays against B.
anthracis and S. aureus (Table 2). Biphenyl compounds
exhibited potent antibacterial growth activities, which corre-
lated well with their helicase inhibitory activities. 1,4-Biphenyl
compounds showed better antibacterial activities, especially for
S. aureus growth inhibition, and less mammalian cytotoxicity
than did 1,2- and 1,3-biphenyl isomeric compounds. For
example, compound 20 exhibited MIC values of 1.25 and 2.5
μM versus B. anthracis and S. aureus, respectively, and the
selectivity index (SI) values were 66 and 33, respectively.
The substituents on the biphenyl group dramatically affected
the antibacterial activity of members of this series. Fluoro-,
chloro-, or cyano-monosubstitution was generally tolerated (see
compounds 21, 22, and 23); however, compound 25, bearing a
trifluoromethyl group at the 4′-position, showed a higher MIC
value vs S. aureus than did compound 20. A 3′,4′-dichloro-
Several compounds, such as 9, 14, 25, 32, 33, and 35,
exhibited much higher MIC values for S. aureus than they did
for B. anthracis, even though they exhibited potent S. aureus
helicase inhibitory activity. To explore whether the poor S.
aureus growth inhibition was due to efflux, we added the efflux
pump inhibitor reserpine to the growth inhibition assay (20 μg/
mL).32 The addition of reserpine did not affect the MIC values
for compounds 9, 14, 32, 33, or 35, suggesting that the higher
MIC values vs S. aureus are due to influx deficiencies rather
than to efflux. However, addition of reserpine did improve the
MIC value for compound 25 by about 8-fold, indicating that
efflux is a factor in the sensitivity of S. aureus cells to this
compound.
Consistent with its lack of antihelicase activity, amide 28
exhibited no bacterial growth inhibitory activity. However,
amide 30 exhibited potent MIC values comparable to that of
20. Surprisingly, it also demonstrated significant HeLa cell
cytotoxicity, suggesting that the carboxylic acid functionality
may be necessary for maximizing the antibacterial activity and
minimizing the cytotoxicity in the coumarin-type helicase
inhibitors. In this regard, it is important to note that in general,
antihelicase potency and cytotoxicity are not correlated. In fact,
a scatter plot of the helicase strand unwinding inhibition
potency (IC50) vs the HeLa cytotoxicity in serum-free medium
(CC50) for the 21 coumarin analogues in Table 2 revealed no
significant correlation (r2 = 0.008) (Figure S1, Supporting
Information). These results suggest that antihelicase potency
can be optimized without concomitant increases in cytotoxicity.
The structure−activity relationships (SARs) of the coumarin-
based bacterial DNA helicase inhibitors for their antihelicase
activities and antibacterial activities are summarized in Figure 2.
Mode of Helicase Inhibition by Coumarins. The effect
of different concentrations of each of the two helicase
substrates ATP and DNA on inhibitor IC50 values was used
to assess the mode of inhibition of the most selective coumarin
inhibitor 20 in the DNA duplex strand-unwinding assay. The
IC50 values for compound 20 were only modestly affected by
C
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Table 1. Bacillus anthracis and Staphyloccocus aureus Helicase Inhibition of Coumarin Compounds
D
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Table 1. continued
Table 2. Antibacterial Activity of Coumarin-Based Helicase Inhibitors
a
compd ID
MIC B. anthracis (μM)
MIC S. aureus (μM)
HeLa CC50 in SFM (μM)
SI (CC50/MIC) Ba
SI (CC50/MIC) Sa
1
65
5
>100
>100 (>100)
20 (50)
10
6
0.09
7.6
<0.06
<1
9
38
14
15
16
17
18
19
20
21
22
24
25
26
27
28
30
31
32
33
34
35
2.5
8.3
3.3
0.4
0.4
0.6
1.0
0.3
6.8
33
2.5
4.4
1.8
5
12.5
7.8
1.6
2.5
10
10.1
5.6
4.0
0.625
2.5
20
9.0
3.125
21.2
82
8.5
1.25
1.25
0.625
<0.8
<0.8
3
2.5
66
2.5
19.5
25
15.6
40
7.8
20
1.25
3
19.2
20.3
19.2
>24
>25.4
6.4
6.4
a
50 (6.3)
3 (6.3)
>100
3.2
6.4
>100
>100
2.5
>100
90.7
1.1
0.91
0.44
46
0.91
0.22
14
5
2.5
8.2 (12.5)
>100 (>100)
>100 (>100)
5.0
115
31
1.25
2.5
25
<0.8
2.5
>100
42
>40
34
1.25
2.5
8.4
>100 (>100)
15.4
6.2
3.1
a
MIC value (μM) with efflux pump inhibitor reserpine.
27-fold variations in the oligonucleotide concentrations (IC50
=
We also examined the effect of compounds 20 and 22 on the
single-strand DNA stimulated ATPase activity of S. aureus
helicase. Neither compound inhibited the ATPase activity
significantly at a concentration that is 25−50-fold greater than
its IC50 in the strand-unwinding assay (6% and 8% inhibition by
compounds 20 and 22 at 50 μM, respectively; see Table 3).
Taken together, these results indicate that the coumarin-type
helicase inhibitors bind at sites distinct from both the DNA and
ATP substrates, and the mechanism of strand-unwinding
inhibition does not involve reduction of the energy supply
(ATP hydrolysis) for translocation and strand unwinding. This
mechanism is distinct from that of the known DNA helicase
1.8−2.2 μM) (Figure 3A) or by 16-fold variations in ATP
concentration (IC50 = 1.5−4.1 μM) (Figure 3B). As described
by Wei et al.,33 insensitivity of IC50 values to variations in the
substrates is indicative of a noncompetitive mode of inhibition
for compound 20 vs both substrates. Consistent with these
results, a fit of the initial velocities to standard, nonlinear four-
parameter curves demonstrated that the data were best
represented by a noncompetitive mode of inhibition, with Ki
values of 4.0 and 2.4 μM for compound 20 vs oligonucleotide
and ATP, respectively.
E
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Figure 2. Summary of SARs of the coumarin-based bacterial DNA helicase inhibitors.
information is available for other inhibitors of bacterial DnaB
type replicative helicases.
Effect of Coumarin Helicase Inhibitor 20 on Bacterial
Viability. S. aureus cells were incubated with various
concentrations of compound 20 or the control bactericidal
antibiotic ciprofloxacin, and aliquots of the culture were diluted
and plated on rich medium without inhibitors to determine the
number of viable cells capable of forming colonies. The
resulting killing curve (Figure 4) confirmed the cidality of
Figure 3. Dependence of IC50 values for compound 20 on
concentrations of substrates. The concentration dependence of
compound 20 inhibition of S. aureus helicase was determined in the
FRET-based DNA duplex strand-unwinding assay in the presence of
various concentrations of the two substrates, DNA (A) and ATP (B)
as noted. RFU/min values were normalized to the slope of the
uninhibited control. Lines were drawn based on four-parameter
nonlinear curve fitting (GraphPad Prizm 5.0).
Figure 4. Effect of compound 20 and ciprofloxacin on the viability of
S. aureus cells. S. aureus (ATCC 25923) cells were incubated for the
indicated time with no drug (open circles), 1× MIC (triangles), or 4×
MIC (diamonds) of compound 20 (solid lines) or ciprofloxacin
(dashed lines) and spread on LB agar plates at various times indicated
on the abscissa to determine the number of viable colony-forming
units (CFU). For ciprofloxacin, 1× MIC was 0.5 μg/mL, and 4× MIC
was 2.0 μg/mL; for compound 20, 1× MIC was 2.5 μM and 4× MIC
was 10 μM (Table 2).
Table 3. Effect of Helicase Strand Unwinding Inhibitors on
the Helicase ATPase
reaction
average A630 Stdev
% inhibition
complete
−ATP
−PhiX174
−helicase
0.75 0.01
0.09 0.01
0.19 0.06
0.15 0.05
0.70 0.01
0.69 0.02
88
75
80
6
ciprofloxacin at concentrations of both 1× and 4× MIC (>3 log
decrease in viability over 24 h). However, compound 20 was
bacteriostatic at concentrations of 1× and 4× its MIC value,
and some regrowth was observed at 24 h in 1× compound 20,
possibly due to cellular metabolism of the inhibitor or
mutations in the population. While it is somewhat surprising
that a DNA synthesis inhibitor should appear bacteriostatic, the
complete + 50 μM compd 20
complete + 50 μM compd 22
8
inhibitor myricetin, which also inhibits ATPase activity in a
noncompetitive manner.27 No clear mechanism of inhibition
F
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potency of compound 20 may simply be too low, and much
higher concentrations or a more potent helicase inhibitor could
be bactericidal. Nevertheless, many bacteriostatic agents, such
as the tetracycline, oxazolidinone, and macrolide classes of
protein synthesis inhibitors, are very useful antibiotics. So,
further development of coumarin-type helicase inhibitors may
provide novel antibiotics with important clinical applications.
Spectrum of Antibacterial Activity. The antibacterial
spectra of the two most potent helicase inhibitors 20 and 22
were examined. The results shown in Table 4 revealed
respectively, versus both B. anthracis and S. aureus helicases.
Selectivity index (SI = CC50/MIC) values were 20−66.
Moreover, compounds 20 and 22 demonstrated potent
antibacterial activity against multiple ciprofloxacin-resistant
MRSA strains with MIC values ranging between 0.5 and 4.2
μg/mL. Enzyme kinetic studies demonstrated that these
coumarin-based helicase inhibitors act noncompetitively with
both the DNA and ATP substrates to inhibit the DNA duplex
strand-unwinding activity of helicase, but they do not inhibit
the single-strand stimulated ATPase activity of helicase, which
is the energy source for translocation and unwinding. These
results indicate that further optimization of coumarin-based
helicase inhibitors may provide a new class of antibacterial
agents.
Table 4. Average MIC (μg/mL) Values vs Gram(+) Bacterial
Strains for Compounds 20, 22, and Ciprofloxacin
MIC (μg/mL)
bacterial strain
compd 20
compd 22
ciprofloxacin
EXPERIMENTAL SECTION
■
Bacillus subtilis BD54
B. anthracis ΔANR
B. thuringiensis ATCC 10792
B. anthracis Sterne
4.2
0.65
0.65
0.52
0.39
5.2
3.1
0.65
0.52
0.17
0.098
6.3
0.078
0.078
0.078
0.039
0.039
0.625
0.625
0.313
0.469
>20
General Procedures. All commercially obtained solvents and
reagents were used as received. Melting points were determined in
open capillary tubes with an EZ-Melt (Stanford Research Systems)
apparatus and are uncorrected. 1H NMR spectra were determined on a
Bruker 300 MHz instrument. Chemical shifts are given in δ values
referenced to the internal standard tetramethylsilane. LC-MS analyses
were performed using a Shimadzu LC-10 AD VP HPLC, with Waters
micromass Quattro Ultima triple quad MS. Analytical HPLC analyses
were performed with a Gilson LC system. Elemental analyses were
performed by Columbia Analytical Services. All of the compounds
tested in vitro showed >95% purity either by LC-MS or HPLC.
Ethyl 3-(8-Ethyl-7-hydroxy-4-methyl-2-oxo-2H-chromen-3-yl)-
propanoate (4a). Dry HCl gas was passed through a solution of 2-
ethylresorcinol (3a) (2.00 g, 0.0150 mol) and diethyl 2-acetylglutarate
(3.33 g, 0.0150 mol) in absolute ethanol (40 mL) at 0 °C for 1 h. The
reaction mixture was stirred at room temperature for 24 h, the volume
was reduced to about 20 mL and the mixture was poured into water.
The resulting precipitate was collected by filtration, washed with water,
and dried at 50 °C under vacuum overnight to give an off-white solid
(3.92 g, 86% yield) of the desired product, mp = 156−157 °C, Rf =
0.21 (1:99 MeOH:DCM). 1H NMR (300 MHz, CDCl3) δ 7.29 (d, J =
9.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.56 (br, 1H), 4.14 (q, J = 6.0
Hz, 2H), 2.97 (t, J = 9.0 Hz, 2H), 2.85 (q, J = 9.0 Hz, 2H), 2.63 (t, J =
9.0 Hz, 2H), 2.41 (s, 3H), 1.25 (t, J = 6.0 Hz, 3H), 1.18 (t, J = 9.0 Hz,
3H). LC/MS m/z 304.8 (M + H+).
Ethyl 3-(7-Hydroxy-4,8-dimethyl-2-oxo-2H-chromen-3-yl)-
propanoate (4b). 2-Methylresorcinol (3b) was treated with diethyl
2-acetylglutarate as above. Compound 4b was obtained as an off-white
powder, mp = 169−170 °C, Rf = 0.29 (2:98 MeOH:DCM). 1H NMR
(300 MHz, DMSO-d6) δ 10.29 (s, 1H), 7.45 (d, J = 8.7 Hz, 1H), 6.85
(d, J = 8.7 Hz, 1H), 4.05 (q, J = 7.2 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H),
2.47 (t, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.15 (s, 3H), 1.16 (t, J = 7.2 Hz,
3H). LC/MS m/z 290.9 (M + H+).
Ethyl 3-(7-Hydroxy-4-methyl-2-oxo-2H-chromen-3-yl)-
propanoate (4c). 2-Resorcinol (3c) was treated with diethyl 2-
acetylglutarate as above. Compound 4c was obtained as an off-white
powder, mp = 118−119 °C, Rf = 0.71 (5:95 MeOH:DCM). 1H NMR
(300 MHz, CDCl3) δ 10.41 (s, 1H), 7.59 (d, J = 9.0 Hz, 1H), 6.79
(dd, J = 1.8, 8.7 Hz, 1H), 6.68 (d, J = 1.8 Hz, 1H), 4.06 (q, J = 6.9 Hz,
2H), 2.79 (t, J = 7.2 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H), 2.36 (s, 3H),
1.17 (t, J = 6.6 Hz, 3H). LC/MS m/z 276.9 (M + H+).
B. lichenformis ATCC 14580
E. faecalis ATCC 29212
VRE faecalis ATCC 51575
VRE faecalis 51299
>25
18.8
>25
16.7
2.6
25
4.2
VRE faecalis 700802
VRE faecalis F118
16.7
3.6
E. faecium ATCC 19434
VRE faecium B42762
VRE faecium 1644
12.5
9.4
10.0
14.6
2.1
>20
10.4
0.78
0.78
1.56
0.65
0.65
0.52
1.04
2.1
>20
S. aureus ATCC 25923
MRSA 1234522733
MRSA 1234543349
MRSA 1234544081
MRSA 1234547263
MRSA 1234549404
MRSA 1234558336
MRSA 1094
1.56
2.6
0.235
10.0
4.2
>20.0
0.313
>20
1.56
2.1
1.56
3.1
0.469
>20
12.5
15.0
considerable breadth across the Bacillus and Staphylococcus
genera, with detectable but reduced potency versus species of
the enterococcus genus. Importantly, both 20 and 22 showed
potent antibacterial activity against multiple ciprofloxacin-
resistant MRSA strains, with MIC values ranging between 0.5
and 4.2 μg/mL.
CONCLUSIONS
■
The replicative DNA helicase catalyzes a rate-limiting step in
DNA replication and is essential for bacterial growth. However,
the DNA helicase is an underexploited biological target for the
discovery of new antibacterial therapeutics for biodefense and
clinical use, and very few small molecule inhibitors have been
identified that can potently inhibit helicase activity and bacterial
growth. In a previous high-throughput screening campaign, we
discovered several coumarin-based inhibitors that inhibit both
Bacillus anthracis and Staphylococcus aureus DNA helicases.
Chemical optimization of the coumarin helicase inhibitor series
identified several derivatives exhibiting more potency and
selectivity. The structure−activity relationships are responsive
to alterations in the scaffold and exhibit clear trends, such as
preferences for a hydrophobic group at position 7, an acid
group attached to position 3 through an ethylene linker, and for
a methyl group at position 4. Biphenyl coumarins 20 and 22 are
the most potent compounds, with IC50 values of 3 and 1 μM,
Ethyl 2-(7-Hydroxy-4,8-dimethyl-2-oxo-2H-chromen-3-yl)acetate
(4d). 2-Methyl resorcinol (3b) was treated with diethyl 2-
acetylsuccinate as above. Compound 4d was obtained as an off-
white powder, 86% yield, mp = 199−200 °C, Rf = 0.31 (1:9
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 10.38 (s, 1H),
7.51 (d, J = 9.0 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 4.08 (q, J = 7.2 Hz,
2H), 3.64 (s, 2H), 2.34 (s, 3H), 2.16 (s, 3H), 1,18 (t, J = 6.9 Hz, 3H).
LC/MS m/z 276.9 (M + H+).
Ethyl 4-(8-Ethyl-7-hydroxy-4-methyl-2-oxo-2H-chromen-3-yl)-
butanoate (4e). 2-Ethylresorcinol (3a) was treated with diethyl 2-
acetylhexanoate as above. Compound 4e was obtained as an off-white
G
dx.doi.org/10.1021/jm300922h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
powder, 74% yield, mp =130−131 °C, Rf = 0.13 (1:4 EtOAc:hexanes).
1H NMR (300 MHz, DMSO-d6) δ 10.21 (s, 1H), 7.45 (d, J = 8.7 Hz,
2H), 2.43−2.38 (m, 5H), 2.24 (s, 3H). HRMS (ESI-TOF) m/z calcd
for C21H21O5 (M +H)+ 353.1389, found 353.1385.
3-(4,8-Dimethyl-2-oxo-7-(pyridin-4-ylmethoxy)-2H-chromen-3-
yl)propanoic Acid (8). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
light-brown powder, 64% yield, mp = 154−155 °C, Rf = 0.59 (5:95
1H), 6.84 (d, J = 8.7 Hz, 1H), 4.03 (dd, J = 3.0, 7.2 Hz, 2H), 2.72 (dd,
J = 6.9, 14.4 Hz, 2H), 2.60−2.51 (dd, J = 7.2, 15.3 Hz, 2H), 2.36 (s,
3H), 1,73−1.68 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H), 1.09 (t, J = 7.2 Hz,
3H). LC/MS m/z 318.9 (M + H+).
Methyl 4-(7-Hydroxy-4,8-dimethyl-2-oxo-2H-chromen-3-yl)-
butanoate (4f). 2-Methyl resorcinol (3b) was treated with diethyl
2-acetylhexanoate as above. Compound 4e was obtained as an off-
white powder, 95% yield, mp = 152−153 °C, Rf = 0.23 (1:99
MeOH:CH2Cl2). 1H NMR (300 MHz, CDCl3) δ 7.33 (d, J = 9.0 Hz,
1H), 6.82 (d, J = 8.7 Hz, 1H), 4.14 (q, J = 7.2 Hz, 2H), 2.70 (t, J = 6.6
Hz, 2H), 2.44−2.39 (m, 5H), 2.32 (s, 3H), 1.92−1.82 (m, 2H), 1.26
(t, J = 7.2 Hz, 3H). LC/MS m/z 304.8 (M + H+).
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 8.64 (d, J = 6.0
Hz, 2H), 7.42 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 5.7 Hz, 2H), 6.81 (d, J
= 9.0 Hz, 1H), 5.2 (s, 2H), 4.12 (q, J = 7.2 Hz, 2H), 2.97 (t, J = 7.5
Hz, 2H), 2.61 (t, J = 7.8 Hz, 2H), 2.43 (s, 3H), 2.41 (s, 3H), 1.24 (t, J
= 7.2 Hz, 3H). LC/MS m/z 382.0 (M + H+). Compound 8 was
obtained as a light-brown powder after NaOH hydrolysis of the ester
intermediate, 71% yield, mp = 277−278 °C, Rf = 0.19 (80:18:2
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.19
3-(7-(Allyloxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl)propanoic
Acid (5). A mixture of compound 4b (0.2 g, 0.69 mmol), allyl bromide
(0.125 g, 1 mmol), and potassium carbonate (0.19 g, 1.4 mmol) in
acetone (25 mL) was heated at reflux for 24 h. The volume of acetone
was reduced by 50%, and the solution was poured into water. The
resulting precipitate was collected by filtration and dried at 50 °C
under vacuum overnight to give 0.286 g (89%) of the coumarin ester
(br s, 1H), 8.59 (dd, J = 1.5, 4.5 Hz, 2H), 7.62 (d, J = 9.0 Hz, 1H),
7.46 (d, J = 5.7 Hz, 2H), 7.06 (d, J = 9.0 Hz, 1H), 5.35 (s, 2H), 2.79 (t,
J = 7.2 Hz, 2H), 2.39 (m, 5H), 2.29 (s, 3H). LC/MS m/z 353.9 (M +
H+). Anal. (C20H19NO5·0.2H2O): C, 67.29; H, 5.48; N, 3.92. Found:
C, 67.07; H, 5.30; N, 3.95.
3-(4,8-Dimethyl-7-(naphthalen-2-ylmethoxy)-2-oxo-2H-chro-
men-3-yl)propanoic Acid (9). The synthetic procedure used was the
same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 94% yield, mp = 145−146 °C, Rf =
0.60 (1:99 MeOH:CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ 8.00−
7.92 (m, 4H), 7.60 (dd, J = 2.4, 10.5 Hz, 2H), 7.55−7.52 (m, 2H),
7.16 (d, J = 8.7 Hz, 1H), 5.43 (s, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.81 (t,
J = 7.5 Hz, 2H), 2.47 (t, J = 7.5 Hz, 2H), 2.37 (s, 3H), 2.27 (s, 3H),
1.15 (t, J = 7.2 Hz, 3H). LC/MS m/z 431.1 (M + H+). Compound 9
was obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 70% yield, mp = 205−206 °C, Rf = 0.27 (80:18:2
1
intermediate, mp 112−113 °C, Rf = 0.58 (1:99 MeOH:CH2Cl2). H
NMR (300 MHz, DMSO-d6) δ 7.61 (d, J = 9.0 Hz, 1H), 7.03 (d, J =
9.0 Hz, 1H), 6.15−6.02 (m, 1H), 5.43 (dd, J = 1.8, 17.4 Hz, 1H), 5.29
(dd, J = 1.5, 10.5 Hz, 1H), 4.70 (d, J = 5.1 Hz, 2H), 4.05 (q, J = 7.2
Hz, 2H), 2.82 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H), 2.39 (s,
3H), 2.21 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). LC/MS m/z 330.9 (M +
H+). A mixture of the coumarin ester intermediate (0.15g g, 0.45
mmol) and NaOH (0.020 g, 0.50 mmol) in water (2 mL) and ethanol
(8 mL) was heated at reflux for 1 h, cooled to room temperature,
diluted with water (10 mL), and then neutralized to pH 7 with 1N
HCl. The resulting precipitate was collected by filtration, washed with
water, and dried at 50 °C overnight to give a white powder (0.109 g,
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.20
(s, 1H), 8.00−7.91 (m, 4H), 7.61 (d, J = 9.0 Hz, 2H), 7.55−7.52 (m,
2H), 7.17 (d, J = 9.0 Hz, 1H), 5.44 (s, 2H), 2.79 (t, J = 7.2 Hz, 2H),
2.43−2.39 (m, 5H), 2.28 (s, 3H). LC/MS m/z 403.2 (M + H+). Anal.
(C25H22O5): C, 74.61; H, 5.51. Found: C, 74.47; H, 5.30.
80% yield), mp
= 181−182 °C, Rf = 0.23 (80:18:2
CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6) δ
12.17 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 1H),
6.15−6.02 (m, 1H), 5.43 (dd, J = 1.8, 17.3 Hz, 1H), 5.29 (dd, J = 1.5,
10.7 Hz, 1H), 4.70 (dd, J = 1.8, 3.6 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H),
2.43−2.38 (m, 5H), 2.21 (s, 3H). LC/MS m/z 303.0 (M + H+). Anal.
(C17H18O5): C, 67.54; H, 6.00. Found: C, 67.31; H, 5.94.
3-(7-((7-Chloroquinolin-2-yl)methoxy)-4,8-dimethyl-2-oxo-2H-
chromen-3-yl)propanoic Acid (10). The synthetic procedure used
was the same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 89% yield, mp = 204−205 °C, Rf = 0.47
1
(1:99 MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6 + TFA-d) δ
3-(4,8-Dimethyl-2-oxo-7-(prop-2-ynyloxy)-2H-chromen-3-yl)-
propanoic Acid (6). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white solid, 98% yield, mp = 153−154 °C, Rf = 0.59 (1:99
MeOH:CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ 7.66 (d, J =
9.0 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 4.96 (d, J = 2.1 Hz, 2H), 4.05
(q, J = 7.2 Hz, 2H), 3.61 (t, J = 2.1 Hz, 1H), 2.83 (t, J = 7.5 Hz, 2H),
2.48 (t, J = 7.5 Hz, 2H), 2.41 (s, 3H), 2.19 (s, 3H), 1.16 (t, J = 7.2 Hz,
3H). LC/MS m/z 328.9 (M + H+). Compound 6 was obtained as a
beige powder after NaOH hydrolysis of the ester intermediate, 87%
8.73 (d, J = 8.4 Hz, 1H), 7.96 (s, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.76
(d, J = 8.7 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.32 (d, J = 9.0 Hz, 1H),
6.74 (d, J = 9.0 Hz, 1H), 5.43 (s, 2H), 3.85 (q, J = 7.2 Hz, 2H), 2.71 (t,
J = 6.9 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H), 2.15 (s, 3H), 2.04 (s, 3H),
0.88 (t, J = 7.2 Hz, 3H). LC/MS m/z 466.5 (M + H+). Compound 10
was obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 57% yield, mp = 249−250 °C (decomposed), Rf = 0.31
1
(80:18:2 CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6
+ TFA-d) δ 8.55 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.7 Hz, 2H), 7.79 (d,
J = 8.4 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H),
7.11 (d, J = 8.1 Hz, 1H), 5.55 (s, 2H), 2.82 (t, J = 6.0 Hz, 2H), 2.46−
2.39 (m, 5H), 2.34 (s, 3H). LC/MS m/z 438.0 (M + H+).
y i e l d , m p
= 1 9 0 − 1 9 1 ° C , R f = 0 . 2 3 ( 8 0 : 1 8 : 2
CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6) δ
12.18 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H),
4.96 (d, J = 2.1 Hz, 2H), 3.61 (t, J = 2.1 Hz, 1H), 2.79 (t, J = 7.5 Hz,
2H), 2.43−2.38 (m, 5H), 2.19 (s, 3H). LC/MS m/z 300.9 (M + H+).
Anal. (C17H16O5·0.2H2O): C, 67.19; H, 5.44. Found: C, 67.03; H,
5.28.
3-(7-((7-Chloroquinolin-2-yl)methoxy)-4-methyl-2-oxo-2H-chro-
men-3-yl)propanoic Acid (11). The synthetic procedure used was the
same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 93% yield, mp = 148−149 °C, Rf =
1
0.19 (1:99 MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 8.48
3-(7-(Benzyloxy)-4, 8-dimethyl-2-oxo-2H-chromen-3-yl)-
propanoic Acid (7). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
off-white powder, 91% yield, mp = 116−117 °C, Rf = 0.62 (1:99
(d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 9.5 Hz, 2H),
7.66 (dd, J = 2.1, 8.7 Hz, 1H), 7.11−7.07 (m, 2H), 5.49 (s, 2H), 4.04
(q, J = 7.2 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H), 2.47 (t, J = 7.5 Hz, 2H),
2.39 (s, 3H), 1.15 (s, 3H). LC/MS m/z 452.2 (M + H+). Compound
11 was obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 25% yield, mp = 216−217 °C, Rf = 0.25 (80:18:2
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 7.61 (d, J = 9.0
Hz, 1H), 7.49−7.31 (m, 5H), 7.22 (d, J = 9.0 Hz, 1H), 5.26 (s, 2H),
4.04 (q, J = 6.9 Hz, 2H), 2.82 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.2 Hz,
2H), 2.39 (s, 3H), 2.23 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). LC/MS m/z
381.2 (M + H+). Compound 7 was obtained as a white powder after
NaOH hydrolysis of the ester intermediate, 85% yield, mp = 232−233
°C, Rf = 0.23 (80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300
MHz, DMSO-d6) δ 12.17 (s, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.49−7.34
(m, 5H), 7.11 (d, J = 9.0 Hz, 1H), 5.26 (s, 2H), 2.79 (t, J = 7.5 Hz,
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.19
(br s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 9.0 Hz, 2H), 7.73 (dd,
J = 2.7, 9.0 Hz, 2H), 7.67 (dd, J = 2.1, 8.7 Hz, 1H), 7.10−7.07 (m,
2H), 5.49 (s, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.39 (m, 5H). LC/MS m/z
424.2 (M + H+). Anal. (C23H18ClNO5): C, 65.18; H, 4.28; N, 3.30.
Found: C, 65.01; H, 4.38; N, 3.26.
H
dx.doi.org/10.1021/jm300922h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3-(7-(Isoquinolin-1-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chromen-
3-yl)propanoic Acid (12). The synthetic procedure used was the same
as described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 93% yield, mp = 130−131 °C, Rf = 0.54 (3:97
z 429.5 (M + H+). Anal. (C27H24O5·0.3H2O): C, 74.74; H, 5.71.
Found: C, 74.44; H, 5.69.
3-(7-(Biphenyl-2-ylmethoxy)-4-methyl-2-oxo-2H-chromen-3-yl)-
propanoic Acid (16). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
gummy material, 81% yield, Rf = 0.70 (1:99 MeOH:CH2Cl2). 1H
NMR (300 MHz, DMSO-d6) δ 7.61−7.26 (m, 10H), 6.82 (dd, J = 2.4,
9.0 Hz, 1H), 6.70 (d, J = 2.7 Hz, 1H), 4.99 (s, 2H), 4.11 (q, J = 6.9 Hz,
2H), 2.94 (t, J = 7.5 Hz, 2H), 2.58 (t, J = 7.8 Hz, 2H), 2.41 (s, 3H),
1.23 (t, J = 7.2 Hz, 3H). LC/MS m/z 443.5 (M + H+). Compound 16
was obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 94% yield, mp = 172−173 °C, Rf = 0.27 (80:18:2
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 8.50 (d, J = 5.7
Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.87 (d, J
= 5.7 Hz, 1H), 7.84−7.71 (m, 2H), 7.62 (d, J = 9.0 Hz, 1H), 7.30 (d, J
= 9 Hz, 1H), 5.84 (s, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.80 (t, J = 7.5 Hz,
2H), 2.47 (t, J = 7.5 Hz, 2H), 2.38 (s, 3H), 2.14 (s, 3H), 1.15 (t, J =
6.9 Hz, 3H). LC/MS m/z 432.1 (M + H+). Compound 12 was
obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 99% yield, mp = 206−207 °C, Rf = 0.32 (80:18:2
1
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.17
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 8.50
(s, 1H), 7.68 (d, J = 9.6 Hz, 1H), 7.62 (dd, J = 7.8, 1.8 Hz, 1H), 7.48−
7.34 (m, 8H), 6.92 (d, J = 2.4 Hz, 1H), 6.89 (s, 1H), 5.04 (s, 2H), 2.77
(t, J = 7.5 Hz, 2H), 2.42−2.37 (m, 5H). LC/MS m/z 415.6 (M + H+).
Anal. (C26H22O5): C, 75.35; H, 5.35. Found: C, 75.28; H; 5.37.
3-(7-(Biphenyl-3-ylmethoxy)-4-methyl-2-oxo-2H-chromen-3-yl)-
propanoic Acid (17). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 87% yield, mp = 81−82 °C, Rf = 0.86 (1:99
(d, J = 5.7 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H),
7.87 (d, J = 5.7 Hz, 1H), 7.84−7.71 (m, 2H), 7.59 (d, J = 9.0 Hz, 1H),
7.28 (d, J = 9.0 Hz, 1H), 5.82 (s, 2H), 2.75 (t, J = 7.5 Hz, 2H), 2.37 (s,
3H), 2.29 (t, J = 8.1 Hz, 2H), 2.13 (s, 3H). LC/MS m/z 404.1 (M +
H+). Anal. (C24H21NO5·0.4H2O): C, 70.20; H, 5.35; N, 3.41. Found:
C, 69.96; H, 5.01; N, 3.40.
3-(7-(Isoquinolin-1-ylmethoxy)-4-methyl-2-oxo-2H-chromen-3-
yl)propanoic Acid (13). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 94% yield, mp = 99−100 °C, Rf = 0.56 (3:97
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 7.63−7.31 (m,
10H), 6.93 (dd, J = 2.7, 8.7 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 5.15 (s,
2H), 4.11 (q, J = 7.2 Hz, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.58 (t, J = 7.2
Hz, 2H), 2.39 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H). LC/MS m/z 443.4 (M
+ H+). Compound 17 was obtained as a white powder after NaOH
hydrolysis of the ester intermediate, 70% yield, mp = 156−157 °C, Rf
= 0.34 (80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz,
DMSO-d6) δ 12.14 (s, 1H), 7.77−7.63 (m, 5H), 7.53−7.46 (m, 4H),
7.41−7.36 (m, 1H), 7.09−7.04 (m, 2H), 5.30 (s, 2H), 2.78 (t, J = 7.5
Hz, 2H), 2.42−2.37 (m, 5H). LC/MS m/z 415.5 (M + H+). Anal.
(C26H22O5): C, 75.35; H, 5.35. Found: C, 75.19; H; 5.23.
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 8.52 (d, J = 5.7
Hz, 1H), 8.33 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.88 (d, J
= 5.7 Hz, 1H), 7.85−7.67 (m, 3H), 7.17 (d, J = 2.4 Hz, 1H), 7.06 (dd,
J = 2.4, 9.0 Hz, 1H), 5.80 (s, 2H), 4.05 (q, J = 7.2 Hz, 2H), 2.81 (t, J =
7.5 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.38 (s, 3H), 1.16 (t, J = 7.2 Hz,
3H). LC/MS m/z 418.4 (M + H+). Compound 13 was obtained as a
white powder after NaOH hydrolysis of the ester intermediate, 74%
y i e l d , m p
= 1 9 5 − 1 9 6 ° C , R f = 0 . 3 2 ( 8 0 : 1 8 : 2
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 8.52
(d, J = 5.7 Hz, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H),
7.88 (d, J = 5.7 Hz, 1H), 7.85−7.69 (m, 3H), 7.18 (d, J = 2.4 Hz, 1H),
7.07 (dd, J = 2.7, 9.0 Hz, 1H), 5.8 (s, 2H), 2.78 (t, J = 7.5 Hz, 2H),
2.43−2.37 (m, 5H). LC/MS m/z 390.1 (M + H+). Anal.
(C23H19NO5·0.2H2O): C, 70.29; H, 4.98; N, 3.56. Found: C, 70.33;
H, 4.94; N, 3.50.
3-(7-(Biphenyl-3-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chromen-3-
yl)propanoic Acid (18). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 90% yield, mp = 126−127 °C, Rf = 0.86 (1:99
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 7.66−7.36 (m,
10H), 6.91 (d, J = 9.0 Hz, 1H), 5.24 (s, 2H), 4.12 (q, J = 7.2 Hz, 2H),
2.97 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 7.8 Hz, 2H), 2.42 (s, 3H), 2.39 (s,
3H), 1.23 (t, J = 7.2 Hz, 3H). LC/MS m/z 457.6 (M + H+).
Compound 18 was obtained as a white powder after NaOH hydrolysis
of the ester intermediate, 94% yield, mp = 200−201 °C, Rf = 0.34
(80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6)
δ 12.17 (s, 1H), 7.77 (s, 1H), 7.69−7.62 (m, 4H), 7.53−7.46 (m, 4H),
7.41−7.36 (m, 1H), 7.16 (d, J = 9.0 Hz, 1H), 5.35 (s, 2H), 2.79 (t, J =
7.2 Hz, 2H), 2.43−2.38 (m, 5H), 2.26 (s, 3H). LC/MS m/z 429.4 (M
+ H+). Anal. (C27H24O5·0.2H2O): C, 75.05; H, 5.69. Found: C, 74.98;
H; 5.44.
3-(7-(Anthracen-9-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chromen-
3-yl)propanoic Acid (14). The synthetic procedure used was the same
as described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 15% yield, mp = 187−188 °C, Rf = 0.60 (1:99
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H),
8.39 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 9.0 Hz,
1H), 7.64−7.54 (m, 5H), 6.19 (s, 2H), 4.05 (q, J = 7.2 Hz, 2H), 2.82
(t, J = 7.5 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 2.41 (s, 3H), 1.97 (s, 3H),
1.16 (t, J = 7.2 Hz, 3H). LC/MS m/z 481.4 (M + H+). Compound 14
was obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 77% yield, mp = 190−191 °C, Rf = 0.27 (80:18
3-(7-(Biphenyl-4-ylmethoxy)-4-methyl-2-oxo-2H-chromen-3-yl)-
propanoic Acid (19). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 85% yield, mp = 102−103 °C, Rf = 0.84 (1:99
MeOH:CH2Cl2). 1H NMR (300 MHz, CDCl3) δ 7.64−7.33 (m,
10H), 6.95 (dd, J = 2.7, 8.9 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 5.16 (s,
2H), 4.12 (q, J = 7.2 Hz, 2H), 2.96 (t, J = 7.5 Hz, 2H), 2.59 (t, J = 8.1
Hz, 2H), 2.43 (s, 3H), 1.23 (t, J = 7.2 Hz, 3H). LC/MS m/z 443.3 (M
+ H+). Compound 19 was obtained as a white powder after NaOH
hydrolysis of the ester intermediate, 81% yield, mp = 219−220 °C, Rf
= 0.34 (80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz,
DMSO-d6) δ 12.19 (s, 1H), 7.74−7.66 (m, 5H), 7.56 (d, J = 8.4 Hz,
2H), 7.47 (t, J = 7.5 Hz, 2H), 7.39−7.35 (m, 1H), 7.08−7.03 (m, 2H),
2.79 (t, J = 7.2 Hz, 2H), 2.43−2.38 (m, 5H). LC/MS m/z 415.3 (M +
H+). Anal. (C26H22O5): C, 75.35; H, 5.35. Found: C, 75.39; H; 5.16.
3-(7-(Biphenyl-4-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chromen-3-
yl)propanoic Acid (20). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 91% yield, mp = 139−140 °C, Rf = 0.84 (1:99
MeOH:CH2Cl2). 1H NMR (300 MHz, CDCl3) δ 7.63−7.33 (m,
10H), 6.89 (d, J = 8.7 Hz, 1H), 5.21 (s, 2H), 4.12 (q, J = 7.2 Hz, 2H),
2.97 (t, J = 7.5 Hz, 2H), 2.60 (t, J = 7.8 Hz, 2H), 2.42 (s, 3H), 2.39 (s,
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.25
(s, 1H), 8.72 (s, 1H), 8.39 (d, J = 8.7 Hz, 2H), 8.16 (dd, J = 1.5, 9.3
Hz, 2H), 7.69 (d, J = 9.0 Hz, 1H), 7.64−7.54 (m, 5H), 6.19 (s, 2H),
2.80 (t, J = 7.2 Hz, 2H), 2.45−2.39 (m, 5H), 1.97 (s, 3H). HRMS
(ESI-TOF) m/z calcd for C29H25O5 (M + H)+ 453.1702, found
453.1697.
3-(7-(Biphenyl-2-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chromen-3-
yl)propanoic Acid (15). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 90% yield, mp = 133−134 °C, Rf = 0.44 (1:9
1
MeOH:CH2Cl2). H NMR (300 MHz, DMSO-d6) δ 7.65−7.62 (m,
1H), 7.54 (d, J = 9.0 Hz, 1H), 7.47−7.33 (m, 8H) 6.87 (d, J = 9.0 Hz,
1H), 5.12 (s, 2H), 4.04 (q, J = 6.9 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H),
2.47 (t, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.11 (s, 3H), 1.15 (t, J = 7.2 Hz,
3H). LC/MS m/z 480.5 (M + H+). Compound 15 was obtained as a
white powder after NaOH hydrolysis of the ester intermediate, 90%
yield, mp = 87−88 °C, Rf = 0.27 (80:18:2 CHCl3:CH3OH:CH3NH2).
1H NMR (300 MHz, DMSO-d6) δ 7.65−7.62 (m, 1H), 7.52 (d, J = 9.0
Hz, 1H), 7.47−7.33 (m, 8H), 6.85 (d, J = 9.0 Hz, 1H), 5.11 (s, 2H),
2.77 (t, J = 7.5 Hz, 2H), 2.39−2.34 (m, 5H), 2.11 (s, 3H). LC/MS m/
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3H), 1.23 (t, J = 7.2 Hz, 3H). LC/MS m/z 457.4 (M + H+).
Compound 20 was obtained as a white powder after NaOH hydrolysis
of the ester intermediate, 90% yield, mp = 221−222 °C, Rf = 0.34
(80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6)
δ 12.16 (s, 1H), 7.72−7.55 (m, 7H), 7.47 (t, J = 6.9 Hz, 2H), 7.39−
7.35 (m, 1H), 7.15 (d, J = 9.3 Hz, 1H), 5.32 (s, 2H), 2.79 (t, J = 7.5
Hz, 2H), 2.43−2.38 (m, 5H), 2.26 (s, 3H). LC/MS m/z 429.3 (M +
H+). Anal. (C27H24O5·0.35H2O): C, 74.59; H, 5.73. Found: C, 74.63;
H; 5.74.
δ 12.16 (s, 1H), 7.74−7.55 (m, 7H), 7.29 (t, J = 9.0 Hz, 2H), 7.15 (d, J
= 9.3 Hz, 1H), 5.32 (s, 2H), 2.79 (t, J = 7.2 Hz, 2H), 2.43−2.38 (m,
5H), 2.26 (s, 3H). HRMS (ESI-TOF) m/z calcd for C27H24FO5 (M +
H)+ 447.1608, found 447.1604.
3-(4,8-Dimethyl-2-oxo-7-((4′-trifluoromethylbiphenyl-4-yl)-
methoxy)-2H-chromen-3-yl)propanoic Acid (25). The ester inter-
mediate: white powder, 41% yield, mp = 172−173 °C, Rf = 0.38 (1:3
1
EtOAc:hexanes). H NMR (300 MHz, DMSO-d6) δ 7.89 (d, J = 8.1
Hz, 2H), 7.82−7.76 (m, 4H), 7.61 (d, J = 8.4 Hz, 3H), 7.13 (d, J = 9.0
Hz, 1H), 5.32 (s, 2H), 4.05 (q, J = 7.2 Hz, 2H), 2.81 (t, J = 7.5 Hz,
2H), 2.48 (t, J = 7.5 Hz, 2H), 2.38 (s, 3H), 2.25 (s, 3H), 1.16 (t, J =
7.2 Hz, 3H). LC/MS m/z 525.0 (M + H+). Compound 25 was
obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 89% yield, mp = 240−241 °C, Rf = 0.21 (80:18:2
3-(7-((2′-Cyanobiphenyl-4-yl)methoxy)-4,8-dimethyl-2-oxo-2H-
chromen-3-yl)propanoic Acid (21). The synthetic procedure used
was the same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 81% yield, mp = 158−159 °C, Rf = 0.34
(1:99 MeOH:CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ 7.96 (d, J =
7.5 Hz, 1H), 7.83−7.78 (m, 1H), 7.66−7.57 (m, 7H), 7.17 (d, J = 9.0
Hz, 1H), 5.37 (s, 2H), 4.05 (q, J = 7.2 Hz, 2H), 2.83 (t, J = 7.2 Hz,
2H), 2.49 (t, J = 7.5 Hz, 2H), 2.40 (s, 3H), 2.28 (s, 3H), 1.16 (t, J =
7.2 Hz, 3H). LC/MS m/z 482.2 (M + H+). Compound 21 was
obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 14% yield, mp = 223−224 °C, Rf = 0.23 (80:18:2
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.16
(s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.84−7.77 (m, 4H), 7.63 (t, J = 6.6
Hz, 3H), 7.15 (d, J = 9.0 Hz, 1H), 5.35 (s, 2H), 2.79 (t, J = 7.2 Hz,
2H), 2.43−2.38 (m, 5H), 2.27 (s, 3H). LC/MS m/z 497.1 (M + H+).
3-(7-((3′,4′-Dichlorobiphenyl-4-yl)methoxy)-4,8-dimethyl-2-oxo-
2H-chromen-3-yl)propanoic Acid (26). Coumarin ester intermediate:
light-yellow powder, 30% yield, mp = 241−242 °C, Rf = 0.77 (1:1
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.18
1
EtOAc:hexanes). H NMR (300 MHz, DMSO-d6) δ 8.03 (s, 1H),
(s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.83−7.78 (m, 1H), 7.67−7.57 (m,
7H), 7.17 (d, J = 9.0 Hz, 1H), 5.37 (s, 2H), 2.79 (t, J = 9.0 Hz, 2H),
2,43−2.38 (m, 5H), 2.28 (s, 3H). HRMS (ESI-TOF) m/z calcd for
C28H24NO5 (M + H)+ 454.1654, found 454.1651.
7.84−7.63 (m, 7H), 7.21 (d, J = 8.4 Hz, 1H), 5.40 (s, 2H), 4.10 (q, J =
7.2 Hz, 2H), 2.88 (t, J = 6.6 Hz, 2H), 2.51 (t, J = 6.6, 2H), 2.46 (s,
3H), 2.32 (s, 3H), 1.22 (t, J = 6.6 Hz, 3H). LC/MS m/z 525.1 (M +
H+). Compound 26 was obtained as a white powder after NaOH
hydrolysis of the ester intermediate, 73% yield, mp = 241−242 °C, Rf
= 0.01 (1:5 EtOAc:hexanes). 1H NMR (300 MHz, DMSO-d6) δ 12.16
(s, 1H), 7.98 (s, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.71 (s, 2H), 7.64−7.57
(m, 3H), 7.14 (d, J = 1H), 5.33 (s, 2H), 2.79 (t, J = 7.2 Hz, 2H), 2.40−
2.37 (m, 5H), 2.26 (s, 3H). HRMS (ESI-TOF) m/z calcd for
C27H23Cl2O5 (M + H)+ 497.0923, found 497.0925.
7-[(2′,4′-Difluorobiphenyl-4-yl)methoxy]-4,8-dimethyl-3-(3-car-
boxypropyl)-2-oxo-2H-chromene (27). The ester intermediate: white
powder, 37% yield, mp = 151−152 °C, Rf = 0.39 (1:3 EtOAc:hexanes).
1H NMR (300 MHz, DMSO-d6) δ 7.64−7.57 (m, 6H), 7.36 (dt, J =
2.4, 10.2 Hz, 1H), 7.23−7.13 (m, 2H), 5.32 (s, 2H), 4.05 (q, J = 7.2
Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H), 2.39 (s,
3H), 2.26 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). LC/MS m/z 492.9 (M +
H+). Compound 27 was obtained as a white powder after NaOH
hydrolysis of the ester intermediate, 70% yield, mp = 225−226 °C, Rf
= 0.21 (80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz,
DMSO-d6) δ 12.17 (s, 1H), 7.65−7.54 (m, 6H), 7.37 (dt, J = 2.1, 10.0
Hz, 1H), 7.24−7.14 (m, 2H), 5.34 (s, 2H), 2.79 (t, J = 7.2 Hz, 2H),
2.50−2.38 (m, 5H), 2.27 (s, 3H). HRMS (ESI-TOF) m/z calcd for
C27H23F2O5 (M + H)+ 465.1514, found 465.1509.
7-[(4′-Chlorobiphenyl-4-yl)methoxy]-4,8-dimethyl-3-[3-(methyla-
mino)-3-oxopropyl]-2-oxo-2H-chromene (28). 3-(7-((4′-Chlorobi-
phenyl-4-yl)methoxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl)-
propanoic acid (22) (217 mg, 0.47 mmol) was suspended in dry THF
(3 mL) and oxalyl chloride (0.12 mL, 0.14 mmol) was added followed
by 2 drops of DMF. The reaction mixture was stirred at room
temperature for 18 h and evaporated to dryness. The residue was dried
at 50 °C under vacuum for 1 h, dissolved in dry THF (5 mL), and
cooled to 0 °C in an ice bath. Methylamine (29 mg, 0.94 mmol) was
added into the solution and stirred at room temperature for 3 h. The
reaction mixture was then diluted with water, and the resulting
precipitate was collected by filtration, washed with water, and dried at
50 °C under vacuum for 24 h to the crude product, which was purified
by recrystallization from DMSO. The crystals were washed with ethyl
acetate and dried at 50 °C under vacuum for 24 h to give a white
powder 28 (45 mg, 20% yield), mp = 166−167 °C, Rf = 0.25
(CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ 7.71 (d, J = 8.4 Hz, 4H),
7.64−7.50 (m, 5H), 7.14 (d, J = 8.7 Hz, 1H), 5.32 (s, 2H), 3.59 (s,
3H), 2.82 (t, J = 7.5 Hz, 2H), 2.52 (t, J = 7.5 Hz, 2H), 2.39 (s, 3H),
2.25 (s, 3H). HRMS (ESI-TOF) m/z calcd for C28H27ClNO4 (M +
H)+ 476.1629, found 476.1623.
3-(7-((4′-Chlorobiphenyl-4-yl)methoxy)-4,8-dimethyl-2-oxo-2H-
chromen-3-yl)propanoic Acid (22). The synthetic procedure used
was the same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 82% yield, mp = 139−140 °C, Rf = 0.49
(1:99 MeOH:CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ 7.72 (d, J =
1.8 Hz, 4H), 7.69−7.50 (m, 5H), 7.14 (d, J = 9.0 Hz, 1H), 5.32 (s,
2H), 4.04 (q, J = 6.9 Hz, 2H), 2.82 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.5
Hz, 2H), 2.39 (s, 3H), 2.25 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). LC/MS
m/z 491.0 (M + H+). Compound 22 was obtained as a white powder
after NaOH hydrolysis of the ester intermediate, 94% yield, mp =
260−261 °C, Rf = 0.32 (80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR
(300 MHz, DMSO-d6) δ 12.18 (s, 1H), 7.71 (d, J = 8.4 Hz, 4 H),
7.63−7.50 (m, 5H), 7.13 (d, J = 9.0 Hz, 1H), 5.32 (s, 2H), 2.79 (t, J =
7.2 Hz, 2H), 2.42−2.37 (m, 5H), 2.25 (s, 3H). LC/MS m/z 463.0 (M
+ H+). Anal. (C27H23ClO5): C, 70.05; H, 5.01. Found: C, 70.13; H;
4.89.
Ethyl 3-(7-((4-Bromobenzyl)oxy)-4,8-dimethyl-2-oxo-2H-chro-
men-3-yl)propanoate (23). The synthetic procedure used was the
same as described for compound 5. White powder, 100% yield, mp =
1
182−183 °C, Rf = 0.59 (1:99 MeOH:CH2Cl2). H NMR (300 MHz,
DMSO-d6) δ 7.63−7.59 (m, 3H), 7.44 (d, J = 8.4 Hz, 2H), 7.09 (d, J =
9.0 Hz, 1H), 5.25 (s, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.82 (t, J = 7.5 Hz,
2H), 2.48 (t, J = 7.2 Hz, 2H), 2.39 (s, 3H), 2.23 (s, 3H), 1.15 (t, J =
7.2 Hz, 3H). LC/MS m/z 459.0 (M + H+).
General Procedure for Suzuki Coupling Used in Scheme 2
for Synthesis of Compounds 24−27. A mixture of compound 23
(200 mg, 0.44 mmol, 1 equiv), the corresponding boronic acid (1.5
equiv), tetrakis(triphenylphosphine)palladium(0) (0.1 equiv), and
sodium carbonate (3 equiv) in 1:1 dimethoxyethane:water (6 mL)
was heated at 85 °C for 4 h, and the solvents were evaporated to
dryness. The residue was sonicated for 15 min with 50 mL of EtOAc
and filtered. Silica gel (1 g) was added to the filtrate, and the solvents
were evaporated to dryness. The residue was applied to a silica gel
column and eluted using EtOAc−hexane as the mobile phase to give
the biphenyl coumarin ester intermediates.
3-(7-((4′-Fluorobiphenyl-4-yl)methoxy)-4,8-dimethyl-2-oxo-2H-
chromen-3-yl)propanoic Acid (24). The ester intermediate: white
powder, 51% yield, mp = 156−157 °C, Rf = 0.22 (1:4 EtOAc:hexanes).
1H NMR (300 MHz, DMSO-d6) δ 7.95−7.54 (m, 6H), 7.29 (t, J = 8.7
Hz, 1H), 7.21−7.11 (m, 2H), 5.29 (s, 2H), 4.05 (q, J = 6.9 Hz, 2H),
2.82 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.8 Hz, 2H), 2.38 (s, 3H), 2.24 (s,
3H), 1.16 (t, J = 7.2 Hz, 3H). LC/MS m/z 474.9 (M + H+).
Compound 24 was obtained as a white powder after NaOH hydrolysis
of the ester intermediate, 74% yield, mp = 248−249 °C, Rf = 0.23
(80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6)
N-(2-Acetylaminoethyl)-3-(7-(biphenyl-4-ylmethoxy)-4,8-dimeth-
yl-2-oxo-2H-chromen-3-yl)propanamide, TFA Salt (29). The syn-
thetic procedure used was the same as described for compound 27:
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Compound 29 was obtained as a white powder after HPLC
purification, 14% yield, mp = 230−231 °C (decomposed), Rf = 0.94
(5:95 CH3OH:CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ 7.90 (br s,
1H), 7.83 (br s, 1H), 9.69 (t, J = 8.4 Hz, 4H), 7.62 (d, J = 9.0 Hz, 1H),
7.57 (d, J = 8.4 Hz, 2H), 7.47 (t, J = 7.5 Hz, 2H), 7.38 (d, J = 7.2 Hz,
1H), 7.15 (d, J = 9.3 Hz, 1H), 5.32 (s, 2H), 3.03 (s, 4H), 2.78 (t, J =
7.2 Hz, 2H), 2.38 (s, 3H), 2.27 (s. 5H), 1.76 (s, 3H). HRMS (ESI-
TOF) m/z calcd for C31H33N2O5 (M + H)+ 513.2389, found
513.2384.
3-(7-(Biphenyl-4-ylmethoxy)-8-ethyl-4-methyl-2-oxo-2H-chro-
men-3-yl)propanoic Acid (34). The synthetic procedure used was the
same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 27% yield, mp = 133−134 °C, Rf =
0.25 (1:4 EtOAc:hexanes). 1H NMR (300 MHz, CDCl3) δ 7.64−7.36
(m, 10H), 6.91 (d, J = 9.0 Hz, 1H), 5.23 (s, 2H), 4.12 (q, J = 7.2 Hz,
2H), 3.00−2.93 (m, 4H), 2.61 (t, J = 7.8 Hz, 2H), 2.42 (s, 3H), 1.26−
1.19 (m, 6H). LC/MS m/z 470.9 (M + H+). Compound 34 was
obtained as a white powder after NaOH hydrolysis of the ester
intermediate, 95% yield, mp = 224−225 °C, Rf = 0.32 (80:18:2
3-(7-(Biphenyl-4-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chromen-3-
yl)-N-(2-(dimethylamino)ethyl)propanamide, Trifluoroacetic Acid
Salt (30). The synthetic procedure for the used was the same as
described for compound 27: Compound 30 was obtained as a
colorless film after HPLC purification, 5% yield, Rf = 0.71 (80:18:2
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 12.17
(s, 1H), 7.69 (t, J = 8.0 Hz, 4H), 7.63 (d, J = 9.0 Hz, 1H), 7.56 (d, J =
8.0 Hz, 2H), 7.47 (t, J = 7.0 Hz, 2H), 7.37 (t, J = 7.0 Hz, 1H), 7.16 (d,
J = 9.0 Hz, 1H), 5.33 (s, 2H), 2.83−2.77 (m, 4H), 2.44−2.40 (m, 5H),
1.14 (t, J = 9.0 Hz, 3H). LC/MS m/z 442.9 (M + H+). Anal.
(C28H26O5); C, 76.00; H, 5.92. Found: C, 75.82; H, 5.80.
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 9.37
(s, 1H), 8.14 (t, J = 6.0 Hz, 1H), 7.72−7.62 (m, 5H), 7.57 (d, J = 9.0
Hz, 2H), 2.78 (s, 8H), 2.39 (s, 3H), 2.34−2.27 (m, 5H). LC/MS m/z
499.3 (M + H+).
4-(7-([1,1′-Biphenyl]-4-ylmethoxy)-8-ethyl-4-methyl-2-oxo-2H-
chromen-3-yl)butanoic Acid (35). The synthetic procedure used was
the same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: light-yellow powder, 26% yield, mp = 112−113 °C, Rf =
0.20 (1:4 EtOAc:hexanes). 1H NMR (300 MHz, CDCl3) δ 7.61 (t, J =
8.4 Hz, 4H), 7.52−7.36 (m, 6H), 6.90 (d, J = 9.0 Hz, 1H), 5.23 (s,
2H), 4.13 (q, J = 7.2 Hz, 2H), 2.97 (q, J = 7.5 Hz, 2H), 2.70 (dd, J =
7.5, 9.9 Hz, 2H), 2.43−2.39 (m, 5H), 1.92−1.84 (m, 2H), 1.26 (t, J =
7.2 Hz, 3H), 1.22 (q, J = 7.5 Hz, 3H). LC/MS m/z 485.1 (M + H+).
Compound 35 was obtained as a white powder after NaOH hydrolysis
of the ester intermediate, 50% yield, mp = 217−218 °C, Rf = 0.48
(80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6)
δ 12.04 (s, 1H), 7.70 (t, J = 8.4 Hz, 4H), 7.62 (d, J = 8.7 Hz, 1H), 7.56
(d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.38 (m, 1H), 7.16 (d, J =
9.0 Hz, 1H), 5.33 (s, 2H), 3.31 (s, 3H), 2.79 (q, J = 7.2 Hz, 2H), 2.58
(t, J = 7.5 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 1.73−1.66 (m, 2H), 1.14
(t, J = 7.5 Hz, 3H). LC/MS m/z 456.9 (M + H+).
Bacterial Strains. The bacterial strains and plasmids used in this
study and their sources are as described earlier.30 Standard strains used
for profiling analogues and their sources are as follows: B. anthracis
Sterne 34F2 (Colorado Serum Co.), S. aureus ATCC 25923 (ATCC).
Fluorescent Resonance Energy Transfer (FRET) Assays of
Helicase DNA Duplex Strand-Unwinding Activity. The FRET-
based helicase activity assay was performed essentially as previously
described20,21 using labeled annealed oligodeoxynucleotides Hel-
5′FAM:Hel-3′BHQ.30 The assay is based on the helicase-mediated
dissociation of two annealed oligonucleotides, one with a fluorescent
label, the other bearing a quencher moiety. The replicative helicases of
B. anthracis and S. aureus were prepared as previously described.30
Mode of inhibition studies and Ki value determinations were done in
triplicate, and data were analyzed using GraphPad Prizm 5.0 for four-
parameter nonlinear curve fitting.
Malachite Green Assays of S. aureus Helicase ATPase
Activity. The single-strand DNA stimulated ATPase activity of S.
aureus helicase was measured with a colorimetric assay for the release
of inorganic phosphate, essentially as developed by Baykov et al.
(1988).34 Samples were incubated in clear 96-well microplates in 50
μL comprised of 30 mM Tris-HCl(pH 8.0), 25 mM NaCl, 2 mM
MgCl2, 0.01% Triton X-100, 2.5 mM ATP, S. aureus helicase, and 22
nM phiX174 single-stranded DNA. Samples were incubated for 30 min
at 37 °C. Then, 150 μL of a 0.45 μm filtered 3:1 mixture of 0.045%
(w/w) Malachite Green (carbinol hydrochloride salt):4.2% (w/w)
ammonium molybdate plus 0.2% Brij 35 (w/w) was added. After 10
min incubation at room temperature, the absorbency at 630 nm was
measured for each sample in a Molecular Devices SpectraMax plate
reader. ATPase activity was dependent on the presence of ATP, S.
aureus helicase, and phiX174 DNA. About 20% of the ATP was
hydrolyzed under these conditions, yielding a signal/background ratio
of 4−5.
2-(7-([1,1′-Biphenyl]-4-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chro-
men-3-yl)acetic Acid (31). The synthetic procedure used was the
same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 79% yield, mp = 155−156 °C, Rf =
0.69 (1:99 CH3OH:CH2Cl2). 1H NMR (300 MHz, DMSO-d6) δ
7.69−7.63 (m, 5H), 7.55 (d, J = 7.8 Hz, 2H), 7.47−7.42 (m, 2H),
7.37−7.32 (m, 1H), 7.15 (d, J = 9.3 Hz, 1H), 5.31 (s, 2H), 4.07 (q, J =
6.6 Hz, 2H), 3.64 (s, 2H), 2.35 (s, 3H), 2.24 (s, 3H), 1.16 (t, J = 7.2
Hz, 3H). LC/MS m/z 443.4 (M + H+). Compound 31 was obtained
as a white powder after NaOH hydrolysis of the ester intermediate,
91% yield, mp
= 215−216 °C, Rf = 0.29 (80:18:2
CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz, DMSO-d6) δ
7.70−7.63 (m, 5H), 7.56 (d, J = 8.4 Hz, 2H), 7.48−7.43 (m, 2H),
7.38−7.33 (m, 1H), 7.16 (d, J = 9.0 Hz, 1H), 5.32 (s, 2H), 3.57 (s,
2H), 2.35 (s, 3H), 2.25 (s, 3H). LC/MS m/z 415.3 (M + H+). Anal.
(C26H22O5·0.2H2O): C, 74.70; H, 5.40. Found: C, 74.44; H; 5.15.
4-(7-([1,1′-Biphenyl]-4-ylmethoxy)-4,8-dimethyl-2-oxo-2H-chro-
men-3-yl)butanoic Acid (32). The synthetic procedure used was the
same as described for compound 5. 7-Hydroxycoumarin ester
intermediate: white powder, 89% yield, mp = 127−128 °C, Rf =
0.22 (1:4 EtOAc:hexanes). 1H NMR (300 MHz, DMSO-d6) δ 7.69 (t,
J = 7.8 Hz, 4H), 7.58 (t, J = 7.5 Hz, 3H), 7.47 (t, J = 7.8 Hz, 2H), 7.38
(d, J = 6.9 Hz, 1H), 7.13 (d, J = 9.0 Hz, 1H), 5.31 (s, 2H), 4.02 (q, J =
6.9 Hz, 2H), 2.58 (t, J = 6.9 Hz, 2H), 2.37−2.33 (m, 5H), 2.25 (s,
3H), 1.76−1.67 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H). LC/MS m/z 470.8
(M + H+). Compound 32 was obtained as a white powder after NaOH
hydrolysis of the ester intermediate, 91% yield, mp = 238−239 °C, Rf
= 0.47 (80:18:2 CHCl3:CH3OH:CH3NH2). 1H NMR (300 MHz,
DMSO-d6) δ 12.04 (s, 1H), 7.69 (t, J = 8.1 Hz, 4H), 7.61 (d, J = 9.0
Hz, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.38 (d, J =
7.2 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 5.32 (s, 2H), 2.58 (t, J = 7.2 Hz,
2H), 2.39 (s, 3H), 2.30−2.26 (m 5H), 1.73−1.64 (m, 2H). LC/MS
m/z 442.9 (M + H+). Anal. (C28H26O5): C, 76.00; H, 5.92. Found: C,
76.02; H, 5.92.
3-(7-(Biphenyl-4-ylmethoxy)-8-methyl-2-oxo-2H-chromen-3-yl)-
propanoic Acid (33). The synthetic procedure used was the same as
described for compound 5. 7-Hydroxycoumarin ester intermediate:
white powder, 81% yield, mp = 209−210 °C, Rf = 0.21 (1:4
EtOAc:hexanes). 1H NMR (300 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.69
(t, J = 8.1 Hz, 4H), 7.57 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 8.1 Hz, 3H),
7.38 (d, J = 6.9 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 5.30 (s, 2H), 4.05
(q, J = 6.9 Hz, 2H), 2.73−2.59 (m, 4H), 2.25 (s, 3H), 1.15 (t, J = 6.9
Hz, 3H). LC/MS m/z 443.0 (M + H+). Compound 33 was obtained
as a white powder after NaOH hydrolysis of the ester intermediate,
96% yield, mp
= 224−225 °C, Rf = 0.20 (80:18:2
1
CHCl3:CH3OH:CH3NH2). H NMR (300 MHz, DMSO-d6) δ 7.79
(s, 1H), 7.69 (t, J = 8.4 Hz, 4H), 7.57 (d, J = 8.1 Hz, 2H), 7.47 (t, J =
8.1 Hz, 3H), 7.38 (d, J = 7.2 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 5.30 (s,
2H), 2.67 (t, J = 6.9 Hz, 2H), 2.55 (t, J = 6.3 Hz, 2H), 2.26 (s, 3H).
LC/MS m/z 415.0 (M + H+). Anal. (C26H22O5·0.2H2O): C, 74.70; H,
5.40. Found: C, 74.66; H; 5.28.
Minimum Inhibitory Concentration (MIC) and Bactericidal
Assays. MIC values were determined by the broth microdilution
method described in the CLSI (formerly NCCLS) guidelines.35 MIC
values were expressed in μM to facilitate comparisons with IC50 and
CC50 values (Table 2) or in μg/mL for comparison to ciprofloxacin
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(Table 4) and were determined in duplicate using a 10-point curve
consisting of 2-fold dilutions of inhibitory compound from 100 to 0.2
μM. For bactericidality tests, inhibitors were examined in a standard
method of LB broth culture of S. aureus ATCC 25923 cells followed by
plating on LB agar media and counting colony-forming units.36
Determination of Mammalian Cytotoxicity. The cytotoxic
concentration (CC50) of compounds versus mammalian cells (HeLa)
cultured in serum-free medium was determined as the concentration of
compound that inhibits 50% of the conversion of MTS to
formazan.37,38 Values were determined in duplicate using a 10-point
curve consisting of 2-fold dilutions of inhibitory compound from 100
to 0.2 μM. The “selectivity index” (SI) of a given agent is defined as
the ratio of its mammalian cell cytotoxicity to its MIC value against B.
anthracis or S. aureus (e.g., CC50/MIC).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Scatter plot of potency vs cytotoxicity for 21 coumarin-type
helicase inhibitors. Scatter plot of anti-helicase potency vs
cytotoxicity for 21 coumarin-type inhibitor analogues. Data are
from Tables 1 (IC50) and 2 (CC50). Compound numbers are
shown to the right of each data point. No significant correlation
is observed between these two variables. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For B. Li: phone, +1 508 757 2800; fax, +1 508 757 1999; E-
mail, bli@microbiotix.com. For D. T. Moir: E-mail, dmoir@
microbiotix.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Shawna M. Rotoli and Dr. Subhasis B. Biswas
(Department of Molecular Biology, University of Medicine and
Dentistry of New Jersey (UMDNJ)) and Dr. Esther E. Biswas-
Fliss (UMDNJ and Department of Bioscience Technologies,
Jefferson School of Health Professions, Thomas Jefferson
University) for providing B. anthracis helicase, for confirmation
of the B. anthracis helicase inhibition potency of compound 20,
and for helpful discussions. We thank Steven C. Cardinale at
Microbiotix for assistance with the Malachite Green-based
ATPase assay. This work was supported by the National
Institutes of Health/National Institute of Allergy and Infectious
Diseases (AI064974). The content of this publication does not
necessarily reflect the views or policies of the Department of
Health and Human Services.
ABBREVIATIONS USED
■
Sa, Staphylococcus aureus; Ba, Bacillus anthracis; CA-MRSA,
community-acquired methicillin-resistant S. aureus; FRET,
fluorescence resonance energy transfer; SPA, scintillation
proximity; SAR, structure−activity relationship; MIC, minimal
inhibitory concentration; SI, selectivity index
(17) Takahashi, T. S.; Wigley, D. B.; Walter, J. C. Pumps, paradoxes
and ploughshares: mechanism of the MCM2−7 DNA helicase. Trends
Biochem. Sci. 2005, 30, 437−444.
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