Design, synthesis, and structure - Activity relationships of benzophenone-based tetraamides as novel antibacterial agents

Article abstract of DOI:10.1021/jm900519b

The increase in the incidence of both hospital- and community-acquired antibiotic-resistant infections is a major concern to the healthcare community. There have been only two new classes of antibiotics approved by the FDA over the past 40 years, and clea

Full text of DOI:10.1021/jm900519b

5020 J. Med. Chem. 2009, 52, 5020–5031
DOI: 10.1021/jm900519b
Design, Synthesis, and Structure-Activity Relationships of Benzophenone-Based Tetraamides as Novel
Antibacterial Agents
Sunil K. Vooturi,† Chrissy M. Cheung,‡,§ Michael J. Rybak,‡ and Steven M. Firestine*,†
†Department of Pharmaceutical Sciences and ‡Anti-Infective Research Laboratory, Eugene Applebaum College of Pharmacy, Wayne State
University, Detroit, Michigan 48201. §Current Address: The Asian Liver Center, Division of General Surgery, Stanford University School of
Medicine, Stanford, California 94305.
Received April 23, 2009
The increase in the incidence of both hospital- and community-acquired antibiotic-resistant infections is
a major concern to the healthcare community. There have been only two new classes of antibiotics
approved by the FDA over the past 40 years, and clearly there is a growing need for additional
antimicrobial agents. In this paper, we present our work on the discovery of a class of benzophenone
containing compounds that possess good activity against MRSA, VISA, VRSA, and VRE and
moderate activity against E. coli. These compounds display MIC values in the 0.5-2.0 mg/L range
and are not cytotoxic against mammalian cells. Extensive structure-activity relationship studies
revealed that the benzophenone was absolutely essential for antibacterial activity as was the presence
of a cationic group. Although these agents display DNA binding activity, we observed that these
compounds do not inhibit any macromolecular synthesis reliant upon DNA nor do they inhibit lipid or
cell wall biosynthesis. Instead, we found that these agents cause membrane depolarization, indicating
that the bacterial membrane was the primary site of action for these agents. Our studies suggest that
caution should be taken in assigning the mechanism of action for DNA binding antibiotics.
Introduction
agents contained at least one cationic group usually at the
C-terminus, which interacted with the negatively charged
phosphate groups in DNA. In some cases, additional groups
at the N-terminus of the molecule were frequently observed to
enhance binding affinity.11-13,19
The rapid emergence of bacteria resistant to treatment with
standard antibiotics constitutes a serious public threat.1-6
Recent studies have shown an increase in antibiotic resistance
of Gram-positive bacterial strains, and resistance has been
detected to antibiotics ranging from the β-lactams, macro-
lides, and quinolones to the glycopeptides and oxazolidi-
nones.7 While the incidence of antibiotic-resistant infections
continues to increase, the pharmaceutical industry has decrea-
sed its research on new antibacterials.5,8 Indeed, in past 40 years,
only two antibiotics (linezolid and daptomycin) with novel
mechanisms of action have made it to the market.9 Clearly,
there is an increasing need for novel antibacterial agents that
arelesspronetobacterial resistance anddirected towardnovel
biological targets.
Beginning in 2002, a series of reports were published on a
new set of molecules that bound to DNA and display potent
antimicrobial activity.10-19 These DNA binding antibiotics
(DBAa) were based upon the natural products distamycin A
and netropsin and consisted of a series of N-methylpyrrole
amino acids connected via an amide bond. All of the active
Given the fact that these agents possessed DNA binding,
most researchers postulated that the antimicrobial activity of
these agents was due to disruption of critical biochemical
processes involving DNA (i.e., replication, transcription, gene
regulation). Surprisingly, however, few studies have been
directed at validating this mechanism of action.20,21 In this
paper, we describe the results of our studies on a unique set of
benzophenone containing tetraamides with DNA binding
affinity and potent antibacterial activity. These compounds
are active against antibiotic-resistant Gram-positive microbes
with an MIC value of ∼0.5 mg/L, have moderate activity
against Gram-negative microbes, and are not cytotoxic to
mammalian cells. Mechanistic studies on these compounds
reveal that they do not inhibit bacterial growth by binding
to DNA. Instead, these agents result in depolarization of
the bacteria leading to cell death. Our results indicate that
care should be taken in assigning the mechanism of action
for DBA.
*To whom correspondence should be addressed. Phone: 1-313-577-
0455. Fax: 1-313-577-2033. E-mail: sfirestine@wayne.edu.
a Abbreviations: DBA, DNA binding antibiotic; MIC, minimal
inhibitory concentration; VRSA, vancomycin sensitive Staphylococcus
aureus; VISA, vancomycin intermediate Staphylococcus aureus; GISA,
glycopeptide intermediate Staphylococcus aureus; VRE, vancomycin
resistant Enterococci; MRSA, vancomycin resistant Staphylococcus
aureus; MOA, mechanism of action; MHB, Mueller-Hinton broth;
MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium; diSC3(5), 3,30-dipropylthiadicarbocyanine
iodide.
Results
Discovery of a New Antibacterial Agent. Previous work in
our laboratory focused on the role that the three-dimen-
sional structure of DNA played on gene expression.22 One
portion of these studies focused on the development of minor
groove DNA binding agents with the potential to bend
DNA. The design hypothesis of these agents consisted of
r
pubs.acs.org/jmc
Published on Web 08/04/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5021
Table 1. DNA Binding Affinity and in Vitro Antimicrobial Activity
of 1-10
Figure 1. Conceptual breakdown of potent compound 1. The arrow
indicates the axis of symmetry for the molecule.
connecting the sequence recognition properties of N-methyl-
pyrroles to sterically bulky, hydrophobic groups that could
force open the minor groove of DNA, resulting in a bend. As
part of this project, approximately a dozen compounds were
prepared, and on the basis of previous reports of antimicro-
bial activity of DNA binding agents, we examined these
compounds for antibacterial activity against both Gram-
positive and Gram-negative bacteria. Most of the agents
displayed no antibacterial activity up to a concentration of
32 mg/L. However, one compound, 1, gave MIC values in
the 8-16 mg/L range against VRSA, MRSA, and GISA. No
activity was observed against Gram-negative bacteria. This
result is similar to published examples of DBA.10-19
Although 1 did display antibacterial activity, the MIC values
were too high to be useful. Thus, we decided to prepare a
series of analogues of the lead agent in an effort to identify
more potent molecules and to elucidate some of the key
structural features of this molecule that were necessary for
activity.
a Association constant for binding to AT hairpin (50-CGAAAAA-
CAAAAAGTTTTTCG-30) as determined by the ethidium bromide
displacement assay.
Table 2. DNA Binding Affinity and in Vitro Antimicrobial Activity
for 11-16
Design of Compounds. The lead agent, 1, can conceptually
be divided into distinct regions (Figure 1). The first, called
the tail region, is the portion of the molecule that contain
cationic groups. At the time, we reasoned that the positive
charge of the tail interacted with the negative charge on the
phosphate backbone of DNA. We also were interested in the
hydrophobic character of this region, since the tail region
also interacts with the hydrophobic portion of the sugar
residue located in the minor groove of DNA. Previous
studies have shown that enhancing the hydrophobicity of
the cationic tail could increase the binding affinity of the
molecule to DNA and aid in drug transport.14,16,18 In addi-
tion, the work of others has shown tremendous differences
in antibiotic activity depending upon the chemical nature
of the tail substituents.10-14,19 On the basis of previous
investigations, we chose to change the tail region to a methyl-
piperazine, dimethylaniline, morpholine, piperidine, or pyr-
rolidine (Table 1).
The second region is the heterocycle region. Previous
researchers have shown that the nature of the heterocycle
can change the antibacterial activity of the molecule.11,13,16,23
We decided to investigate inclusion of N-methylimidazole,
since previous researchers have shown that the inclusion of
imidazole into DNA binding agents would result in im-
proved antibacterial activity (Table 1). We also examined
the role that the heterocycle played in activity by removing
this portion of the molecule.
The final region is the linker region, which is unique to our
molecule. To investigate this region, we reduce the ketone
and replaced the linker with an aliphatic linker of approxi-
mately the same length as the benzophenone linker (Table 2).
a Association constant for binding to AT hairpin (50-CGAAAAA-
CAAAAAGTTTTTCG-30) as determined by the ethidium bromide
displacement assay.
Finally, 1 is symmetrical about the ketone (Figure 1). We
wondered whether this symmetry was necessary for activity,
and therefore, we prepared a series of molecules that essen-
tially mimicked one-half of the molecule. These compounds
should allow us to determine whether both parts of the
molecule are needed for antibacterial activity (Table 3).
Chemistry. We prepared 21 new compounds clustered into
three categories based upon the regions discussed above.

5022 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Vooturi et al.
Synthesis of these molecules was done essentially according
to literature protocols and as outlined in Schemes 1-5. The
key heterocycle building blocks, N-methylpyrrole (22a) and
N-methylimidazole (22b), were synthesized according to
literature methods.24,25 The synthesis of compounds 1-10
were done according to Scheme 1. Reaction of 22a,b with
commercially available amines 23a-f resulted in the desired
amides (24a-j) via the haloform reaction.26 The nitro group
was reduced by hydrogenation, and the resulting amine was
used without further purification.26 Coupling was accom-
plished by activating the appropriate diacid followed by
coupling with amine to generate the desired final products.
N-Methylaminopyrroles were coupled to the diacids using
predominantly two methods. The first was the pentafluoro-
trifluoroacetate methodology as described by Dyatkina et al.18
The second method was the use of a carbodiimide coupling
method (EDC, DMAP).27 Coupling of N-methylaminoimi-
dazoles was achieved using HBTU coupling conditions as
described in Scheme 1.16 Finally, the ketone group of benzo-
phenone was reduced to hydroxyl group by using sodium
borohydride and Pd/C chemistry (Scheme 2).28
Structure-Activity Relationships. We first prepared a
series of analogues in which the cationic “tail” region of
the molecule was altered. The substitutions that we chose
were based upon work conducted by previous researchers
which showed that the nature of the cationic tail had a
profound influence on antibacterial activity.10-13 Two com-
pounds lost all antibacterial activity, while two showed
dramatic improvements in activity. Compounds 4 and 5
possessed MIC values in the 0.5-2 mg/L range against VRSA,
MRSA, and GISA and gained activity against VRE (4 mg/L)
and E. coli (16 mg/L). This activity is comparable to vanco-
mycin and linezolid against MRSA but better than these
antibiotics against VRSA and GISA. Daptomycin is roughly
2-4 times as potent as our agents.
Table 3. DNA Binding Affinity and in Vitro Antimicrobial Activity of
Compounds 17-20
Replacement of N-methylpyrrole with N-methylimidazole
did result in active compound, but the antibacterial activity
was roughly 8-fold lower than for the pyrrole containing
agents (7-10, Table 1). This suggested that the heterocycle
portion of the molecule could tolerate modification. Next,
we removed the heterocycle portion and found that activity is
completely lost (21), suggesting that heterocycle portion is
required for the activity.
We also examined the role that the linker played in
antibacterial activity. Reduction of the ketone to an alcohol
resulted in complete loss of activity (11 and 12, Table 2).
Examination of one of the alcohols revealed a substantial
loss in DNA binding activity (compare 12 to 5). Complete
reduction to the methylene (13 and 14) also resulted in loss of
activity (Table 2). Replacement of the benzophenone with a
long, flexible linker of approximately the same length as the
a Association constant for binding to AT hairpin (50-CGAAAAA-
CAAAAAGTTTTTCG-30) as determined by the ethidium bromide
displacement assay.
Scheme 1. Synthesis of Benzophenone-Based Compounds Containing N-Methylpyrrole and N-Methylimidazole Units

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5023
benzophenone (15 and 16) resulted in loss of antibacterial
and DNA binding activity (Scheme 3, Table 2). Thus, we
concluded that the benzophenone linker is important for
both the DNA binding and the antibacterial activity of the
compounds.
were interested in determining whether these agents were
cytotoxic against mammalian cells. To do this, we incubated
4 and 5 against CHO cells at concentrations ranging from
25 to 100 ꢀ MIC levels (using MRSA as the standard MIC
value). After incubation for 24 h, we measured cell viability
using the MTS assay (Figure 2). Incubation of compounds 4
and 5 with mammalian cells results in no change in cell
viability for either compound. These studies indicate that the
antibacterial activity of the compounds is not due to general
cytotoxic but rather due to selective killing of bacterial cells.
Studies of DNA Displacement in Bacteria. An examination
of the data presented in Tables 1-3 reveals that there is no
correlation between DNA binding affinity and antibacterial
activity. Previous researchers studying DBA have also ob-
served this lack of correlation and have posited that this was
due to differences in transport of these molecules into the
bacterium.12,16,27 To examine whether this was the case with
our agents, we examined the ability of our compounds to
bind to DNA in vivo using a Hoescht dye competition assay.
Hoescht dye was incubated with bacteria, resulting in fluor-
escent bacteria. Individual agents were titrated into a solu-
tion containing the fluorescent bacteria, and the change in
fluorescence due to Hoechst displacement was measured as a
decrease in total fluorescence. As shown in Figure 3, all
agents resulted in approximately the same degree of compe-
tition, with the Hoechst dye indicating that these agents are
transported into the bacteria. Importantly, all compounds,
including those lacking antibacterial activity, resulted in a
fluorescence decrease. Furthermore, approximately the
same degree of competition was observed for both Gram-
positive and Gram-negative bacteria even though our agents
are approximately 16 times as potent against Gram-positive
Compound 1 can be thought of as symmetrical molecules
with an axis of symmetry bisecting the ketone group of the
benzophenone linker. We wondered if both halves of the
molecule were necessary for activity. To address this issue,
we prepared several compounds (17-20, Scheme 4) that
consisted of only one-half of the lead agent 1. These agents
bound to DNA with affinities comparable to that of the lead
agent; however, none of the compounds displayed activity
against MRSA and VRSA (Table 3). Thus, we concluded
that both halves of the molecule were necessary for anti-
bacterial activity but not required for DNA binding affinity.
Cytotoxicity Studies. Compounds 4 and 5 display good
activity against a variety of Gram-positive, antibiotic-resis-
tant bacteria. These agents also displayed moderate activity
against some Gram-negative microbes including E. coli. We
Scheme 2. Synthesis of Compounds in Which Ketone Group of
Benzophenone Was Reduced to Hydroxyl Group
Scheme 3. Synthesis of Compounds with Different Linkers
Scheme 4. Synthesis of Compounds Mimicking Only One-Half of Compound 5

5024 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Vooturi et al.
microbes. Taken together, these results gave us misgivings
that DNA was the target responsible for antibacterial activity.
Compound 5 Does Not Inhibit DNA, RNA, Protein, Lipid,
or Cell Wall Biosynthesis. Of the compounds reported here, 5
displayed the most potent antibacterial activity, and thus, we
chose this as our lead agent. To investigate the mechanism of
action (MOA) of 5, we examined the effect of 5 on macro-
molecule synthesis using radiolabel incorporation assays.29
Staphylococcus aureus (ATCC 29213) was grown to early log
phase and treated, in triplicate, with various concentrations
of 5 ((0.5-8) ꢀ MIC) or a DMSO (10%) control. The cells
were incubated for 5 min before radiolabeled precursors
were added ([3H]thymidine (DNA synthesis), [3H]uridine
(RNA synthesis), [3H]leucine (protein synthesis), [3H]N-
acetylglucosamine (cell wall), [3H]glycerol (lipid synthesis).
After 30 or 60 min (for lipid synthesis), cells were treated with
TCA and the amount of radiolabel in the acid precipitate was
determined by a scintillation counter.
the macromolecular syntheses examined (Figure 4). In con-
trast, all positive controls gave the appropriate results. This
indicates that 5 exerts its antibacterial effects on some path-
way other than those commonly employed by antibiotics.
This result is important because it establishes that our
compounds have a MOA distinct from that of commonly
used antibiotics.
Compounds 4 and 5 Depolarize Staphylococcal Cells. Since
our studies indicated that 5 does not affect metabolic path-
ways commonly targeted by antibiotics, we began to examine
other potential targets. One potential target is the bacterial
membrane itself.30 The bacterial membrane is the target for a
number of cationic peptides, and previous researchers have
produced antibacterial peptide mimetics.31,32 Frequently,
these compounds are composed of amphipathic molecules
containing a cationic residue that interacts with negatively
charged membrane components.31,32 Although our com-
pounds have no structural relationship to existing antibac-
terial peptide mimetics, we hypothesized that the cationic
nature of 5 might enable it to bind to the membrane. This
hypothesis was supported by the fact that removal of the
cationic tail from 5 destroyed antibacterial activity.
Treatment of Staphylococcus with 5 up to a concentration
of 8ꢀMIC resulted in no dose-dependent inhibition of any of
Many compounds that target bacterial membranes induce
pore formation resulting in the depolarization of the cell.31,33,34
Thus, we examined whether 4 and 5 depolarized bacterial
membranes.35 To conduct these studies, Staphylococcus
aureus was grown in MHB supplemented with MgSO4 to
early log phase, the cells collected by centrifugation and
resuspended in 5 mM HEPES buffer (pH 7.2) containing
5 mM glucose. The cells were diluted to an OD600 of 0.05, and
Scheme 5. Synthesis of Null Molecule with Respect to the
Heterocycle Unit
Figure 2. MTS assay of compounds 4 and 5 at three different
concentrations (25, 50, and 100 mg/L) in CHO cell line. Untreated
cells (with 5% DMSO) were used as control. Error bars are the
standard deviation of each triplicate experiment.
Figure 3. In vivo competition of Hoescht dye with active and inactive antibacterial compounds. Compounds 4 (O) and 9 (9) are active, while 2
(2) is inactive. All compounds were tested at five different concentrations (25, 50, 100, 150, and 200 mg/L). The background fluorescence of
cells not treated with the agents is also shown (b). Error bars are the standard deviation of two independent experiments: (A) competition of
compounds with Hoescht dye in Gram-positive bacteria (S. aureus); (B) competition of compounds with Hoescht dye in Gram-negative
bacteria (E. coli).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5025
Figure 4. Effect of compound 5 on inhibition of macromolecular synthesis in S. aureus cells. Compound 5 was tested at five different
concentrations ((0.5-8.0) ꢀ MIC), while the respective control inhibitor was used at 8.0 ꢀ MIC.
to be a correlation between antibacterial activity and the
kinetics of depolarization. We are conducting further studies
to examine the depolarization caused by our agents.
Discussion
In this paper, we have presented our work on the discovery
of a class of benzophenone-containing DNA binding agents
that display potent antibacterial activity against both Gram-
positive and Gram-negative bacteria. We have not been the
first group to identify potent antibacterial DNA binding
antibiotics. Suckling and colleagues as well as several compa-
nies have published extensively on the identification of DNA
binding agents that display potent antimicrobial activity.10-19
These groups have elucidated a number of the key structural
features necessary for activity, and in a few cases, detailed
Figure 5. Depolarization studies in S. aureus, as measured by the
membrane potential-sensitive dye diSC3(5). Compounds 4 (2, 8.0
pharmacokinetic studies of the compounds have also been
reported. One area that has not been adequately addressed is
mg/mL), 5 ([, 4.0 mg/L), 15 (9, 32 mg/L), and nisin (b, 25 mg/L)
the mechanism of action of the compounds. Most researchers
were used. Cells without any drug were used as a negative control.
have postulated that these compounds bind to DNA and
Changes in membrane potential were calculated as a percentage of
the nisin control which was set to 100%.
prevent DNA replication, transcription, or gene expression.20,21
However, a number of groups, including ours, have noted that
there is no correlation between DNA binding affinity and
antibacterial activity.12,16,27 While some researchers have
proposed that transport differences account for this lack of
correlation, we have shown in this report (via the in vivo DNA
displacement assay) that there are no qualitative differences in
transport between antibacterial versus inactive agents. This
suggests that the interaction with DNA is not responsible for
the antibacterial activity of these agents, a result validated
by studies showing that our agents do not disrupt critical
biochemical pathways dependent upon DNA. Instead, we
found that our agents result in membrane depolarization,
suggesting that the membrane was the primary target for these
400 nM membrane potential-sensitive dye diSC3(5) was
added. The cells were placed into a 37 °C fluorescence
cuvette, stirred, and equilibrated for 5 min before addition
of 100 μM KCl to equilibrate the intra- and extracellular
potassium levels. The compounds to be tested were added
(8 ꢀ MIC), and the change in fluorescence was monitored
over time. As seen in Figure 5, 4 and 5 resulted in depolar-
ization whereas a structurally related compound (15) did not.
These compounds induced about 50% of the depolarization
observed for nisin, which was used as a positive control
(Figure 5). In addition, the time to reach a fixed level of
depolarization was slower for our agents and there appeared

5026 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Vooturi et al.
compounds. This indicates that simply being able to bind
DNA does not clearly provide the mechanism for antibacter-
ial activity. Our results suggest caution is warranted regarding
the mechanism of action of antimicrobial agents that bind
DNA.
Table 4. HPLC Conditions Used To Verify Purity of Compounds 1-
21a
isocratic method
compd
solvent A, % solvent B, % purity, %
7, 16-18
70
75
78
80
30
25
22
20
>95
>95
>95
>95
Why are our agents selective for bacterial versus mamma-
lian membranes? Bacterial membranes contain a number of
unique characteristics that make bacteria more disposed to
the action of membrane targeting drugs.32 First, and most
importantly, the lipid composition of prokaryotes is different
from that of eukaryotes.32,36,37 Prokaryotic membranes
contain a large amount of negatively charged phospholipids,
whereas eukaryotic membranes contain zwitterionic phos-
pholipids.34 Furthermore, prokaryotic membranes are fre-
quently decorated with polyanionic groups such as
lipoteichoic acids and lipopolysaccharides, further increasing
the negative charge of the membrane.38 Thus, cationic com-
pounds are selectively targeted to bacterial membranes be-
cause they form favorable electrostatic interactions with the
negatively charged membrane (see ref 32 for a discussion). In
addition, bacteria possess a large transmembrane potential
and this has been shown to help insertion and pore formation
of membrane binding compounds.39 Indeed, it has been
suggested that the electronegativity of the membrane could
actually attract cationic molecules to the membrane.40 This
literature evidence indicates that a positive charge is a require-
ment for selective association to bacterial versus mammalian
membranes.
1, 4, 5, 6, 9, 12- 15, 19, 20
8, 10
2, 11, 21
a Solvent A: 0.1% TFA in water. Solvent B: 0.1% TFA in acetonitrile.
on the bacterial membrane. However, an examination of
pharmacophoric features for both DNA binding agents and
membrane active antibiotics indicates that they share similar
features. Both classes of compounds require a cationic region
for interacting with negatively charged phosphate groups
present in either the backbone of DNA or the lipid bilayer.
Hydrophobic groups are frequently seen in DNA binding
agents to maximize contacts with deoxyribose, and these
groups are obviously necessary for interaction with the lipid
tail. Finally, both classes utilize hydrogen bonding. In the case
of DNA binding agents, hydrogen bonding is used to recog-
nize specific base pairs within DNA, whereas in membrane
targeted antibiotics, hydrogen bonds are frequently used to
form specific secondary structures or to aid in supramolecular
assembly of pore complexes. Other researchers have also
noticed the similarity between agents that bind DNA and
agents that interact with negatively charged membrane com-
ponents.42
The evidence presented within this paper indicates that a
cationic charge is required for activity by our benzophenone-
containing antibiotics but is not the sole determinant of
antibacterial efficacy. For example, compound 3, which is
inactive, is not charged at physiological pH and yet 2, which is
charged, is also inactive. Other factors must be involved. One
of these is likely hydrophobicity, since our data indicate that
these agents interact with the membrane. Like charge, the
activity of our agents is not simply correlated to the hydro-
phobicity of the compound (as measured by cLogP).
For example, compounds 4 and 9 have similar cLogP
values (2.69 vs 2.58) and yet display a 4-fold difference
in MIC values. We consider it probable that the relation-
ship between the antibacterial activity of these agents and
their physicochemical properties will likely be due to a com-
bination of charge, hydrophobicity, and as yet unknown
factors.
The mechanism of action of the compounds outlined in this
report, as well as the difficulty in understanding the struc-
ture-function-activity relationships of these agents, is similar
to that proposed for cationic antimicrobial peptides.32 These
peptides, which are commonly found in nature, have been
shown to depolarize bacterial membranes by pore formation
or by destabilizing or disintegrating the membrane.33,34,41 In
thecaseof our agents, wecurrently donot havedirectevidence
indicating whether our agents result in pore formation. How-
ever, we have observed that treatment of bacteria with our
agents does not lead to the release of intracellular ATP,
indicating that the compounds do not dissolve the bacterial
membrane (S. Firestine, unpublished data). Thus, at the
present time, we propose that the benzophenone-containing
antibiotics described in this report are coordinated into a pore
complex within the membrane and the resulting pore leads to
membrane depolarization.
Conclusion
We have described the synthesis, structure-activity rela-
tionships, and microbiological and mechanistic studies of a
novel class of antibacterial compounds that have potent
activity against both Gram-positive and Gram-negative bac-
teria. These compounds display MIC values in the range of
0.5-1.0 mg/L and are active against a wide range of anti-
biotic-resistant bacterial including MRSA, VRSA and VRE.
The compounds do not display toxicity against mammalian
cells. Mechanistic studies revealed that although these agents
have modest DNA binding affinity, their antibacterial activity
is not related to any biochemical process that involves this
macromolecule. Instead, these agents appear to selectively
interact with the bacterial membrane, resulting in membrane
depolarization. Our findings suggest that others should be
cautious in assigning a mechanism of action for agents that
interact with DNA. We are actively working on understand-
ing the nature of the membrane interaction and hope to report
on these findings in due course.
Experimental Section
1
Chemistry. H NMR and 13C NMR spectra were obtained
with a Varian DRX400 at 400 and 100 MHz, respectively. Mass
measurements were carried out at the central instrumentation
facility at Wayne State University, Detroit, MI, using a electro-
spray mass spectrometry (Waters-Micromass ZQ qudrupole).
The radiolabeled incorporation assays were conducted by Mi-
cromyx (Kalamazoo, MI). Purity of all final compounds was
˚
determined by HPLC using a PRP-1, 100 A column from
Hamilton (4.1 mmꢀ150 mm, 10 μm) with an isocratic gradient
of 0.1% TFA in water and 0.1% TFA in acetonitrile (see Table 4
for details and percent purities). All compounds were more than
95% pure.
General Method for Coupling Amines with 22a,b26. To a
solution of 22a (1.0 mmol) in DMF under argon, 1.2 mmol of
Finally, we note that it was an unexpected finding to
observe that DNA binding agents could have direct effects

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5027
the amine (23a-f) was added dropwise, and the reaction mixture
was stirred at room temperature for 2 h to obtain the desired
products (24a-f). Compound 22b was also reacted in similar
way with amines (23c-f) to obtain products 24g-j.
N-(3-(Dimethylamino)propyl)-1-methyl-4-nitro-1H-pyrrole-2-
carboxamide (24a). This compound was prepared from 22a and
23a in 95% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=0.4).
1H NMR (400 MHz, CDCl3): δ 8.68 (s, 1H, NH), δ 7.51 (d, J=
2.0 Hz, 1H, Ar-H), 6.96 (d, J=1.5 Hz, 1H, Ar-H), 3.99 (s, 3H,
CH3), 3.48 (q, J=5.2 Hz, 2H, CH2), 2.55 (t, J=6.4 Hz, 2H, CH2),
2.35 (s, 6H, 2ꢀCH3), 1.74-1.68 (m, 2H, CH2). 13C NMR (100
MHz, CDCl3): δ 160.7, 135.1, 131.1, 128.1, 117.9, 58.7, 47.0,
36.3, 26.4.
1.84-1.70 (m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ
157.99, 137.69, 129.24, 128.43, 54.94, 54.27, 38.54, 37.27, 23.71.
1-Methyl-4-nitro-N-(2-(piperidin-1-yl)ethyl)-1H-imidazole-2-
carboxamide (24i). This compound was prepared from 22b and
23e in 82% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=0.4).
1H NMR (400 MHz, CDCl3): δ 7.78 (s, 1H, Ar-H), 7.65 (s, 1H,
NH), 4.10 (s, 3H, CH3), 3.45 (q, J=6.0 Hz, 2H, CH2), 2.48 (t, J=
6.4 Hz, 2H, CH2), 2.42-2.32 (m, 4H, 2 ꢀ CH2), 1.59-1.48 (m,
4H, 2 ꢀ CH2), 1.46-1.33 (m, 2H, CH2). 13C NMR (100 MHz,
CDCl3): δ 157.93, 137.71, 124.43, 57.52, 54.67, 37.20, 36.59,
26.05, 24.51.
1-Methyl-4-nitro-N-(2-(morpholin-1-yl)ethyl)-1H-imidazole-
2-carboxamide (24j). This compound was prepared from 22b
and 23f in 83% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=
0.45). 1H NMR (400 MHz, CD3OD): δ 7.78 (s, 1H, Ar-H), 7.60
(s, 1H, NH), 4.14 (s, 3H, CH3), 3.71 (t, J=4.4 Hz, 4H, 2ꢀCH2),
3.50 (q, J=6.0 Hz, 2H, CH2), 2.55 (t, J=6.0 Hz, 2H, CH2),
2.52-2.43 (m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ
157.99, 137.57, 124.37, 67.08, 57.33, 53.70, 37.24, 36.14.
General Method for the Activation of Dicarboxylic Acids (25/
27a,b)18. The dicarboxylic acid (1.0 mmol) was suspended in
4.0 mL of dry DMF followed by the addition of diisoproylethy-
leneamine (DIEA, 2.2 mmol). The solution was stirred for 5 min
before the addition of pentafluorotrifluoroacetate (2.2 mmol).
The mixture was stirred at room temperature for 1 h, and solvent
was evaporated. Flash silica gel chromatography (toluene/ethyl
acetate, 9:1) provided the corresponding pentafluorodiester.
Bis(perfluorophenyl) 4,40-Methylenedibenzoate (27a). This com-
pound was prepared from 26a in quantitative yield. TLC (95:
5 hexane/ethyl acetate, Rf=0.75). 1H NMR (400 MHz, DMSO):
δ 8.16 (d, J=6.8 Hz, 4H, Ar-H), 7.38 (d, J=7.2 Hz, 4H, Ar-H),
3-(4-Methylpiperazin-1-yl)propan-1-amine)-4-nitro-1H-pyr-
role-2-carboxamide (24b). This compound was prepared from
22a and 23b in quantitative yield. TLC (97:2:1 CH2Cl2/MeOH/
1
Et3N, Rf=0.4). H NMR (400 MHz, CD3OD): δ 7.82 (d, J=
2.0 Hz, 1H, Ar-H), 7.26 (d, J=2.0 Hz, 1H, Ar-H), 3.93 (s, 3H,
CH3), 3.39-3.25 (m, 4H, 2ꢀCH2), 2.8-2.41 (m, 6H, 3ꢀCH2),
1.80-1.69 (m, 2H, CH2). 13C NMR (100 MHz, CD3OD): δ
160.7, 135.1, 131.1, 128.1, 117.9, 57.6, 53.7, 46.6, 36.3.
N-(4-(Dimethylamino)phenyl)-1-methyl-4-nitro-1H-pyrrole-
2-carboxamide (24c). This compound was prepared from 22a
and 23c in 70% yield. TLC (1:1 ethyl acetate/hexane, Rf=0.55).
1H NMR (400 MHz, CDCl3): δ 7.58 (d, J=1.6 Hz, 1H, Ar-H),
7.50 (s, 1H, NH), 7.38 (d, J=9.2 Hz, 2H, Ar-H), 7.17 (s, 1H,
NH), 6.72 (d, J=8.8 Hz, 2H, Ar-H), 4.01 (s, 3H, CH3), 2.93
(s, 6H, 2ꢀCH3). 13C NMR (100 MHz, CDCl3): δ 158.64, 148.61,
127.07, 126.93, 126.62, 122.47, 113.07, 107.11, 40.96, 38.125.
1-Methyl-4-nitro-N-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrrole-2-
carboxamide (24d). This compound was prepared from 22a and
23d in 95% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=0.55).
1H NMR (400 MHz, CDCl3): δ 7.44 (d, J=1.6 Hz, 1H, Ar-H),
7.15 (d, J=2.0 Hz, 1H, Ar-H), 3.80 (s, 3H, CH3), 3.30 (t, J=
6.4 Hz, 2H, CH2), 2.52 (t, J=6.0 Hz, 2H, CH2), 2.52-2.41 (m,
4H, 2ꢀCH2), 1.71-1.65 (m, 4H, 2ꢀCH2). 13C NMR (100 MHz,
CDCl3): δ 160.58, 126.81, 126.7, 107.14, 54.51, 54.06, 38.20,
38.05, 23.71.
1-Methyl-4-nitro-N-(2-(piperidin-1-yl)ethyl)-1H-pyrrole-2-
carboxamide (24e). This compound was prepared from 22a and
23e in 90% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=0.6).
1H NMR (400 MHz, CD3OD): δ 7.82 (d, J=2.0 Hz, 1H, Ar-H),
7.29 (d, J=2.0 Hz, 1H, Ar-H), 3.94 (s, 3H, CH3), 3.46 (t, J=
7.2 Hz, 2H, CH2), 2.59-2.45 (m, 6H, 3 ꢀ CH2), 1.68-1.56 (m,
4H, 2 ꢀ CH2), 1.58-1.46 (m, 2H, CH2). 13C NMR (100 MHz,
CD3OD): δ 160.78, 127.20, 107.44, 57.56, 54.34, 36.73, 36.01,
25.34, 23.92.
1-Methyl-4-nitro-N-(2-(morpholino-1-yl)ethyl)-1H-pyrrole-2-
carboxamide (24f). This compound was prepared from 22a and
23f in 90% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=0.6).
1H NMR (400 MHz, CDCl3): δ 7.55 (d, J=2.0 Hz, 1H, Ar-H),
7.04 (d, J=2.0 Hz, 1H, Ar-H), 6.54 (s, NH, 2H), 3.98 (s, 3H,
CH3), 3.73 (t, J=4.4 Hz, 4H, 2ꢀCH2), 3.47 (q, J=5.6 Hz, 2H,
CH2), 2.57 (t, J=6.4 Hz, 2H, CH2), 2.56-2.43 (m, 4H, 2ꢀCH2).
13C NMR (100 MHz, CDCl3): δ 160.52, 126.81, 126.71, 106.98,
67.16, 56.97, 53.56, 38.07, 35.76.
N-(4-(Dimethylamino)phenyl)-1-methyl-4-nitro-1H-imida-
zole-2-carboxamide (24g). This compound was prepared from
22b and 23c in 78% yield. TLC (1:1 ethyl acetate/methylene
chloride, Rf=0.55). 1H NMR (400 MHz, CDCl3): δ 7.99 (s, 1H,
Ar-H), 7.50 (d, J = 2.0 Hz, 2H, CH2), 6.74 (d, J = 2.0 Hz,
2H, Ar-H), 4.14 (s, 3H, CH3), 2.9 (s, 6H, 2ꢀCH3). 13C NMR
(100 MHz, CDCl3): δ 155.50, 148.66, 126.99, 125.14, 121.86,
113.31, 40.86, 37.14.
4.19 (s, 2H, CH2). 19F NMR (400 MHz, CDCl3): -152.78 (m),
13
-157.50 (t), -162.23 (m).
C NMR (100 MHz, CDCl3): δ
165.2, 146.7, 142.4, 142.0, 140.1, 139.3, 130.8, 129.2, 127.7, 41.3.
Bis(perfluorophenyl) Undecanedioate (27b). 27b was prepared
from 26b with quantitative yield. TLC (95:5 hexane/ethyl acet-
1
ate, Rf=0.55). H NMR (400 MHz, CDCl3): δ 2.71-2.58 (m,
4H, 2ꢀCH2), 1.85-1.72 (m, 4H, 2ꢀCH2), 1.40-1.20 (m, 12H,
6ꢀCH2). 13C NMR (100 MHz, CDCl3): δ 169.81, 33.55, 29.61,
29.52, 29.31, 29.04, 24.97.
General Method for the Coupling of the N-Methylpyrrole
Amines with Dicarboxylic Acids18,26. To a stirred solution of
nitropyrrole derivatives (24a-f, 0.5 mmol) in methanol (30 mL),
10% Pd/C (15% weight of the nitro derivative) was added. The
flask was evacuated, flushed three times with hydrogen, and
filled with hydrogen to 35-40 psi. The resultant suspension was
shaken vigorously for 1.5 h. The suspended material was filtered
through Celite, and the filtrate was evaporated to dryness. The
resulting aminopyrrole derivatives were used immediately in
the next reaction. Freshly prepared aminopyrrole derivatives
were dissolved in 3 mL of dry DMF, and a dipentafluorester
(0.2 mmol) generated from 25/26a/26b was added. The mixture
was stirred for 15 h at 55 °C and cooled, and the DMF was
evaporated. The final product was purified by silica gel using
column chromatography.
4,40-Carbonylbis(N-(N1,N1-dimethylpropane-1,3-diamino)-
1H-pyrrole)benzamide (1). 1 was prepared from 25 and 24a with
78% yield. TLC (89:10:1 CH2Cl2/MeOH/Et3N, Rf=0.55). H
1
NMR (400 MHz, CD3OD): δ 8.05 (d, J=8.4 Hz, 4H, Ar-H),
7.90 (d, J=8.4 Hz, 4H, Ar-H), 7.32 (d, J=2.0 Hz, 1H, Ar-H),
6.99 (d, J=1.6 Hz, 1H, Ar-H), 3.99 (s, 6H, 2ꢀCH3), 3.41 (t, J=
6.4 Hz, 4H, 2 ꢀ CH2), 3.13 (t, J=7.2 Hz, 4H, 2 ꢀ CH2), 2.87
(s, 12H, 4 ꢀ CH3), 2.03-1.93 (m, 4H, 2 ꢀ CH2). 13C NMR
(100 MHz, CD3OD and CDCl3): δ 164.04, 162.76, 139.41,
138.35, 130.25, 127.66, 122.83, 122.18, 120.02, 104.82, 62.93,
56.32, 52.90, 43.70, 36.75, 36.39, 25.32. ES-MS [Mþ1] found
683.3, calculated for C37H46N8O5 682.4.
1-Methyl-4-nitro-N-(2-(pyrrolidin-1-yl)ethyl)-1H-imidazole-
2-carboxamide (24h). This compound was prepared from 22b
and 23d in 91% yield. TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=
0.55). 1H NMR (400 MHz, CDCl3): δ 7.78 (s, 1H, Ar-H),
7.61 (s, 1H, NH), 4.16 (s, 3H, CH3), 3.51 (q, J=6.0 Hz, 2H,
CH2), 2.68 (t, J=6.4 Hz, 2H, CH2), 2.59-2.46 (m, 4H, 2ꢀCH2),
4,40-Carbonylbis(N-(2-(4-methylpiperazin-1-yl)propyl)-1H-
pyrrole)benzamide (2). 2 was prepared from 25 and 24b in 70%
yield. TLC (89:10:1 CH2Cl2/MeOH/Et3N, Rf=0.45). 1H NMR

5028 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Vooturi et al.
(400 MHz, CDCl3): δ 9.13 (s, 2H, NH), 7.87 (d, J=8.4 Hz, 4H,
Ar-H), 7.64 (d, J=8.0 Hz, 4H, Ar-H), 7.34 (t, J=8.4 Hz, 2H,
NH), 7.18 (d, J=1.6 Hz, 2H, Ar-H), 6.81 (d, J=1.6 Hz, 2H,
Ar-H), 3.83 (s, 6H, 2 ꢀ CH3), 3.45-3.37 (m, 4H, 2 ꢀ CH2),
2.50-2.39 (m, 16H, 8ꢀCH2), 2.22 (s, 6H, 2ꢀCH3), 1.72-1.66
(m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ 195.34,
164.03, 162.05, 139.20, 138.58, 130.22, 127.45, 124.25, 121.78,
119.07, 104.20, 57.59, 55.10, 53.35, 46.05, 39.46, 36.76, 25.54.
ES-MS [Mþ1] found 793.3, calculated for C43H56N10O5 792.4.
4,40-Carbonylbis(N-(N1,N1-dimethylbenzene-1,4-diamine)-1H-
pyrrole)benzamide (3). 3 was prepared from 25 and 24c in 60%
yield. TLC (94:5:1 CH2Cl2/MeOH/Et3N, Rf=0.35). 1H NMR
(400 MHz, CDCl3): δ 9.04 (s, 1H, NH), 8.05 (d, J=7.6 Hz, 4H,
Ar-H), 7.88 (d, J=8.0 Hz, 4H, Ar-H), 7.48-7.39 (m, 4H, Ar-
H), 7.32 (s, 2H, Ar-H), 6.98 (s, 2H, Ar-H), 6.78 (d, J=8.8 Hz,
4H, Ar-H), 3.91 (s, 6H, 2ꢀCH3), 2.9 (s, 12H, 4ꢀCH3). ES-MS
[Mþ1] found 751.1, calculated for C43H42N8O5 750.3.
2 ꢀ CH2), 2.40-2.28 (m, 8H, 4 ꢀ CH2), 1.58-1.46 (m, 8H, 4 ꢀ
CH2), 1.43-1.36 (m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz,
CDCl3): δ 164.81, 161.89, 144.31, 132.89, 129.40, 127.98,
127.54, 123.92, 121.60, 119.27, 103.39, 57.35, 54.47, 41.76,
36.84, 35.98, 26.14, 24.50. ES-MS [Mþ1] found 721.4, calculated
for C41H52N8O4 720.4.
N1,N1-Bis(1-methyl-5-(2-(pyrrolidin-1-yl)ethylcarbamoyl)-1H-
pyrrol-3-yl)undecanediamide (15). 15 was prepared from 27b and
24d in 69% yield. TLC (89:10:1 CH2Cl2/MeOH/Et3N, Rf =
0.35). 1H NMR (400 MHz, CDCl3): δ 7.32 (s, 1H, CONH), 7.09
(d, J=2.0 Hz, 2H, Ar-H), 6.49 (d, J=1.6 Hz, 2H, Ar-H), 3.87
(s, 6H, 2 ꢀ CH3), 3.45 (q, J=5.6 Hz, 4H, 2 ꢀ CH2), 2.64 (t, J=
6.4 Hz, 4H, 2 ꢀ CH2), 2.61-2.49 (m, 8H, 4 ꢀ CH2), 2.27 (t, J=
7.2 Hz, 4H, 2 ꢀ CH2), 1.71-1.64 (m, 4H, 2 ꢀ CH2), 1.41 -1.20
(m, 12H, 6 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ 170.86,
162.01, 123.76, 121.48, 118.89, 103.38, 54.95, 54.13, 38.10, 37.14,
36.76, 29.38, 29.35, 29.30, 25.89, 23.73. ES-MS [Mþ1] found
681.4, calculated for C37H60N8O4 680.4.
4,40-Carbonylbis(N-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrrole)ben-
zamide (4). 4 was prepared from 25 and 24d in 80% yield. TLC
(92:7:1 CH2Cl2/MeOH/Et3N, Rf=0.45). 1H NMR (400 MHz,
CD3OD): δ 8.03 (d, J=7.6 Hz, 4H, Ar-H), 7.90 (d, J=8.0 Hz,
4H, Ar-H), 7.31 (d, J=1.6 Hz, 2H, Ar-H), 6.91 (d, J=1.6 Hz,
2H, Ar-H), 3.88 (s, 6H, 2 ꢀ CH3), 3.49 (t, J=6.8 Hz, 4H, 2 ꢀ
CH2), 2.88-2.70 (m, 12H, 6 ꢀ CH2), 1.93-1.82 (m, 8H, 4 ꢀ
CH2). 13C NMR (100 MHz, CD3OD): δ 195.63, 13.33, 161.73,
139.40, 138.86, 130.59, 128.31, 123.90, 122.47, 119.02, 104.87,
55.69, 54.31, 38.48, 36.74, 23.80. ES-MS [Mþ1] found 707.3,
calculated for C39H46N8O5 706.4.
N1,N11-Bis(1-methyl-5-(2-(piperidin-1-yl)ethylcarbamoyl)-1H-
pyrrol-3-yl)undecanediamide (16). 16 was prepared from 27b and
24e in 64% yield. TLC (89:10:1 CH2Cl2/MeOH/Et3N, Rf =
1
0.50). H NMR (400 MHz, CDCl3): δ 7.20 (s, 2H, CONH),
7.14 (d, J=6.0 Hz, 2H, Ar-H), 6.62-2.54 (m, 2H, CONH), 6.43
(d, J=1.6 Hz, 2H, Ar-H), 3.88 (s, 6H, 2 ꢀ CH3), 3.43 (q, J=
5.2 Hz, 4H, 2ꢀCH2), 2.49 (t, J=6.4 Hz, 4H, 2ꢀCH2), 2.49-2.34
(m, 8H, 4ꢀCH2), 2.27 (t, J=7.6 Hz, 4H, 2ꢀCH2), 1.74-1.64 (m,
4H, 2 ꢀ CH2), 1.62-1.50 (m, 8H, 4 ꢀ CH2), 1.49-1.39 (m, 4H,
2ꢀCH2), 1.35 - 1.21 (m. 12H, 6ꢀCH2). 13C NMR (100 MHz,
CDCl3): δ 170.76, 161.90, 123.75, 121.45, 119.00, 103.07, 57.4,
54.48, 37.17, 36.78, 35.96, 29.36, 29.31, 26.18, 25.90, 24.54. ES-
MS [Mþ1] found 709.4, calculated for C39H64N8O4 708.5.
General Method for Coupling Amino-N-methylimidazole with
Dicarboxylic Acid16. To a stirred solution of nitroimidazole
derivatives 24g-j (3.0 mmol) in methanol (30 mL), 10% Pd/C
(15% weight of the nitro derivative) was added. The flask was
evacuated, flushed three times with hydrogen, and filled with
hydrogen to 35-40 psi. The resultant suspension was shaken
vigorously for 1.5 h. The suspended material was filtered
through Celite, and the filtrate was evaporated to dryness. The
resulting aminoimidazole derivative was used immediately with-
out purification. To a flame-dried flask, 25 (1.0 mmol), HBTU
(3.0 mmol), and DIEA (5.0 mmol) were added followed by
addition of the aminoimidazole. The mixture was stirred under
argon overnight at room temperature before the solvent was
removed by evaporation. The final product was obtained by
silica gel column chromatography.
4,40-Carbonylbis(N-(2-(piperdin-1-yl)ethyl)-1H-pyrrole)benza-
mide (5). 5 was prepared from 25 and 24e in 82% yield. TLC
(89:10:1 CH2Cl2/MeOH/Et3N, Rf=0.55). 1H NMR (400 MHz,
CD3OD): δ 8.04 (d, J=8.4 Hz, 4H, Ar-H), 7.88 (d, J=8.4 Hz,
4H, Ar-H), 7.30 (d, J=2.0 Hz, 2H, Ar-H), 6.85 (d, J=2.0 Hz,
2H, Ar-H), 3.89 (s, 6H, 2 ꢀ CH3), 3.46 (t, J=6.4 Hz, 4H, 2 ꢀ
CH2), 2.64-2.50 (m, 12H, 6 ꢀ CH2), 1.70-1.59 (m, 8H, 4 ꢀ
CH2), 1.52-1.42 (m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz,
DMSO-d6): δ 163.25, 161.63, 139.31, 138.77, 130.27, 128.23,
123.86, 122.39, 118.91, 104.68, 58.41, 54.66, 46.26, 36.76,
36.44, 26.19, 24.63. ES-MS [Mþ1] found 735.3, calculated for
C41H50N8O5 734.4.
4,40-Carbonylbis(N-(2-(morpholino-1-yl)ethyl)-1H-pyrrole)-
benzamide (6). 6 was prepared from 25 and 24f in 80% yield.
TLC (89:10:1 CH2Cl2/MeOH/Et3N, Rf =0.5). H NMR (400
1
MHz, CDCl3): δ 8.54 (s, 2H, NH), 7.91 (d, J=8.4 Hz, 4H, Ar-
H), 7.74 (d, J=8.4 Hz, 4H, Ar-H), 7.97 (d, J=1.6 Hz, 2H, Ar-
H), 6.70 (d, J=2.0 Hz, 2H, Ar-H), 6.48 (s, J=8.4 Hz, 2H, NH),
3.90 (s, 6H, 2ꢀCH3), 3.70 (t, J=4.4 Hz, 8H, 4ꢀCH2), 3.50 (q,
J=5.6 Hz, 4H, 2ꢀCH2), 2.54 (t, J=6.0 Hz, 4H, 2ꢀCH2), 2.55-
2.39 (m, 8H, 4ꢀCH2). 13C NMR (100 MHz, CDCl3): δ 195.44,
164.05, 161.88, 139.43, 138.51, 130.39, 127.38, 124.09, 121.56,
119.42, 103.69, 103.62, 67.19, 57.19, 53.54, 36.92, 36.85, 35.72.
ES-MS [Mþ1] found 739.2, calculated for C39H46N8O7 738.3.
4,40-Methylenebis(N-(2-(pyrrolidin-1-yl)ethyl)benzamide (13).
13 was prepared from 27a and 24d in 70% yield. TLC (89:10:1
CH2Cl2/MeOH/Et3N, Rf=0.40). 1H NMR (400 MHz, CDCl3):
δ 8.56 (s, 1H, NH), 7.71 (d, J=8.0 Hz, 4H, Ar-H), 7.23 (s, 1H,
Ar-H), 7.11 (d, J=7.6 Hz, 4H, Ar-H), 6.73 (s, 2H, NH), 6.66
(s, 2H, Ar-H), 3.95 (s, 2H, CH2), 3.82 (s, 6H, 2ꢀCH3), 3.43 (q,
J=5.2 Hz, 4H, 2ꢀCH2), 2.64 (t, J=5.6 Hz, 4H, 2ꢀCH2), 2.62-
2.49 (m, 8H, 4ꢀCH2), 1.79-1.66 (m, 8H, 4ꢀCH2). 13C NMR
(100 MHz, CDCl3): δ 164.98, 162.09, 144.26, 129.26, 127.62,
123.68, 121.86, 119.32, 103.90, 55.12, 54.15, 37.94, 36.78, 23.64.
ES-MS [Mþ1] found 693.3, calculated for C39H48N8O4 692.4.
4,40-Methylenebis(N-(2-(piperdin-1-yl)ethyl)benzamide (14).
14 was prepared from 27a and 24e in 65% yield. TLC (92:7:1
CH2Cl2/MeOH/Et3N, Rf=0.45). 1H NMR (400 MHz, CDCl3):
δ 8.01 (s, 2H, NH), 7.70 (d, J=8.0 Hz, 4H, Ar-H), 7.22 (s, 2H,
Ar-H), 7.15 (d, J=8.0 Hz, 4H, Ar-H), 6.59-6.51 (m, 2H, NH),
6.51 (d, J=1.2 Hz, 2H, Ar-H), 3.98 (s, 2H, CH2), 3.82 (s, 6H, 2ꢀ
CH3), 3.68 (q, J=5.6 Hz, 4H, 2ꢀCH2), 2.43 (t, J=6.0 Hz, 4H,
4,40-Carbonylbis(N-(N1,N1-dimethylbenzene-1,4-diamine)-1H-
imidazole)benzamide (7). 7 was prepared from 25 and 24g in 60%
yield. TLC (1:1 ethyl acetate/CHCl3, Rf=0.6). 1H NMR (400 MHz,
CDCl3): δ 8.78 (s, 2H, NH), 8.63 (s, 2H, NH), 8.01 (d, J=8.0 Hz,
4H, Ar-H), 7.88 (d, J=8.0 Hz, 4H, Ar-H), 7.60 (s, 2H, Ar-H),
7.47 (d, J=8.0 Hz, 4H, Ar-H), 6.72 (d, J=8.0 Hz, 4H, Ar-H),
4.09 (s, 6H, 2 ꢀ CH3), 2.92 (s, 12H, 4 ꢀ CH3). 13C NMR (100
MHz, CDCl3): δ 195.07, 163.37, 156.49, 139.96, 137.33, 135.83,
134.80, 130.56, 127.50, 121.61, 114.93, 113.19, 36.14. ES-MS
[Mþ1] found 753.1, calculated for C41H40N10O5 752.3.
4,40-Carbonylbis(N-(2-(pyrrolidin-1-yl)ethyl)-1H-imidazole)-
benzamide (8). 8 was prepared from 25 and 24h in 52% yield TLC
(94:5:1 CH2Cl2/MeOH/Et3N, Rf = 0.45). H NMR (400 MHz,
1
CDCl3): δ 8.77 (s, 2H, NH), 8.04 (d, J=8.8 Hz, 4H, Ar-H), 7.91
(d, J=8.8 Hz, 4H, Ar-H), 7.59 (s, 2H, NH), 7.56 (s, 2H, Ar-H),
4.06 (s, 6H, 2ꢀCH3), 3.53 (q, J=6.0 Hz, 4H, 2ꢀCH2), 2.73 (t,
J=6.4 Hz, 4H, 2ꢀCH2), 2.66-2.55 (m, 8H, 4ꢀCH2), 1.85-1.76
(m, 8H, 4 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ 195.14,
163.44, 159.16, 139.95, 137.48, 135.93, 134.68, 130.55, 127.52,
114.44, 55.13, 54.25, 38.00, 35.94, 23.73. ES-MS [Mþ1] found
709.2, calculated for C37H44N10O5 708.3.
4,40-Carbonylbis(N-(2-(piperdin-1-yl)ethyl-1H-imidazole)-
benzamide (9). 9 was prepared from 25 and 24i in 57% yield.
TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf=0.5 1H NMR (400 MHz,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5029
CD3OD): δ 8.08 (d, J=8.0 Hz, 4H, Ar-H), 7.91 (d, J=8.4 Hz,
4H, Ar-H), 7.55 (s, 2H, Ar-H), 4.02 (s, 6H, 2 ꢀCH3), 3.69 (t,
J=6.0 Hz, 4H, 2 ꢀ CH2), 3.26-3.14 (m, 12H, 6 ꢀ CH2), 1.90-
1.81 (m, 8H, 4ꢀCH2), 1.69-1.63 (m, 4H, 2ꢀCH2). 13C NMR
(100 MHz, CD3OD): δ 164.75, 160.77, 139.79, 137.71, 136.71,
136.73, 133.95, 130.23, 127.74, 115.38, 57.29, 53.94, 35.32, 34.36,
23.57, 21.87. ES-MS [M þ 1] found 737.3, calculated for
C39H48N10O5 736.4.
δ 164.95, 162.06, 134.65, 131.76, 128.88, 127.18, 121.64, 119.46,
103.87, 55.22, 54.22, 37.66, 36.89, 23.66. ES-MS [M þ 1] found
341.2, calculated for C19H24N4O2 340.2.
4-Benzamido-1-methyl-N-(2-(piperidin-1-yl)ethyl)-1H-pyrrole-
2-carboxamide (20). 20 was prepared using 28b and 24e in a
manner similar to that of 19. The final product was obtained in
1
58% yield. TLC (94:5:1 CH2Cl2/MeOH/Et3N, Rf =0.50). H
NMR (400 MHz, CDCl3): δ 7.88-7.81 (m, 2H, Ar-H), 7.76 (s,
1H, CONH), 7.52-7.44 (m, 3H, Ar-H), 7.29 (d, J=1.6 Hz, 1H,
Ar-H), 6.53 (s, 1H, CONH), 6.52 (d, J=2.0 Hz, 1H, Ar-H),
3.92 (s, 3H, CH3), 3.43 (q, J=6.0 Hz, 2H, CH2), 2.50 (t, J=
6.0 Hz, 2H, CH2), 2.45-2.37 (m, 4H, 2 ꢀ CH2), 1.62-1.55 (m,
4H, 2 ꢀ CH2), 1.47-1.43 (m, 2H, CH2). 13C NMR (100 MHz,
CDCl3): δ 164.94, 161.81, 134.71, 131.83, 128.95, 127.09, 121.44,
119.25, 103.22, 57.58, 54.47, 36.86, 36.03, 26.32, 24.62. ES-MS
[Mþ1] found 355.2, calculated for C20H26N4O2 354.2.
4,40-Carbonylbis(N-(2-(morpholin-1-yl)ethyl-1H-imidazole)ben-
zamide (10). 10 was prepared from 25 and 24j in 65% yield. TLC
(97:2:1 CH2Cl2/MeOH/Et3N, Rf =0.7). H NMR (400 MHz,
1
CDCl3): δ 8.52 (s, 2H, NH), 8.04 (d, J=8.0 Hz, 4H, Ar-H), 7.92
(d, J=8.8 Hz, 4H, Ar-H), 7.58 (s, 2H, NH), 7.57 (s, 2H, Ar-H),
4.07 (s, 6H, 2ꢀCH3), 3.78 (m, 8H, 4ꢀCH2), 3.55 (q, J=5.6 Hz,
4H, 2ꢀCH2), 2.71-2.58 (m, 12H, 6ꢀCH2). 13C NMR (100 MHz,
CDCl3): δ 163.30, 159.15, 140.02, 137.40, 130.59, 127.47, 114.53,
66.82, 57.52, 53.64, 35.98, 35.50. ES-MS [Mþ1] found 741.2,
calculated for C37H44N10O7 740.3.
4,40-(Hydroxymethylene)bis(N-(2-(pyrrolidin-1-yl)ethyl)benza-
mide (11). Compound 4(0.065 mmol, 30 mg) was dissolved in 3 mL
of MeOH. Pd/C (3.0 mg) was added. The solution was cooled to
0 °C, and sodium borohydride (0.162 mmol, 6 mg) was added. The
mixture was stirred for 1 h under argon. The excess hydride was
neutralized with glacial acetic acid and then filtered through Celite.
The filtrate was concentrated in vacuo, to yield the product (58%).
TLC (89:10:1 CH2Cl2/MeOH/Et3N, Rf=0.3). 1HNMR(400MHz,
CD3OD & CDCl3): δ 7.83 (d, J=7.6 Hz, 4H, Ar-H), 7.47 (d, J=
7.6 Hz, 4H, Ar-H), 7.24 (d, J=1.6 Hz, 2H, Ar-H), 6.82 (d, J=
1.6 Hz, 2H, Ar-H), 5.86 (s, 1H, CH), 3.85 (s, 6H, 2ꢀCH3), 3.46 (t,
J=6.8 Hz, 4H, 2ꢀCH2), 2.73 (t, J=6.8 Hz, 4H, 2ꢀCH2), 2.72-
2.69 (m, 6H, 3 ꢀ CH2), 1.87-1.79 (m, 6H, 3 ꢀ CH2). 13C NMR
(100 MHz, CD3OD and CDCl3): δ 165.92, 162.96, 147.94, 133.65,
127.47, 126.69, 123.31, 122.18, 119.71, 104.98, 74.93, 55.23, 54.06,
37.69, 36.06, 23.13. ES-MS [M þ 1] found 709.3, calculated for
C39H48N8O5 708.4.
4-Benzoyl-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (17). To a
dry flask, 28a (113 mg, 0.5 mmol) was activated using penta-
fluorotrifluoroacetate according to methods described above.
The resulting activated ester was coupled with the amine derived
from 24d (141.6 mg, 0.6 mmol) in DMF for 15 h. The solvent was
evaporated and the residue was purified by column chromatog-
raphy to generate the final product in 75% yield. TLC (89:10:1
CH2Cl2/MeOH/Et3N, Rf=0.55). 1H NMR (400 MHz, CDCl3):
δ 8.16 (s, 1H, CONH), 7.97 (d, J=8.0 Hz. 2H, Ar-H), 7.87 (d,
J=8.0 Hz. 2H, Ar-H), 7.85-7.78 (m, 2H, Ar-H), 7.63 (s, 1H,
Ar-H), 7.54-7.49 (m, 2H, Ar-H), 7.35 (d, J=1.6 Hz, 1H, Ar-
H), 6.82-6.76 (m, 1H, CONH), 6.71 (d, J=1.6 Hz, 1H, Ar-H),
3.94 (s, 3H, CH3), 3.52 (q, J=5.6 Hz, 2H, CH2), 2.77 (t, J=5.6
Hz, 2H, CH2), 2.71-2.60 (m, 4H, 2ꢀCH2), 2.59-2.52 (m, 2H,
CH2), 1.85 (m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ
196.47, 164.26, 162.24, 140.60, 138.17, 137.49, 133.43, 130.74,
130.595, 128.97, 127.45, 124.12, 121.64, 119.81, 104.12, 55.48,
54.55 38.01, 37.21, 23.98, 23.94. ES-MS [M þ1] found 445.1,
calculated for C26H28N4O3 444.2.
4-Benzoyl-N-(2-(piperidin-1-yl)ethyl)benzamide (18). 18 was
prepared using 28a and 24e in a manner similar to that of 17. The
final product was obtained in 62% yield. TLC (97:2:1 CH2Cl2/
MeOH/Et3N, Rf=0.6). 1H NMR (400 MHz, CDCl3): δ 8.10 (s,
1H, CONH), 7.95 (d, J=7.6 Hz, 2H, Ar-H), 7.85 (d, J=8.0 Hz,
2H, Ar-H), 7.80 (d, J=8.8 Hz, 2H, Ar-H), 7.64-7.58 (m, 1H,
Ar-H) 7.52-7.47 (m, 2H, Ar-H), 7.33 (d, J=1.2 Hz, 1H, Ar-
H), 6.67-6.61 (m, 1H, CONH), 6.60 (d, J=1.6 Hz, 1H, Ar-H),
3.93 (s, 3H, CH3), 3.44 (q, J=6.0 Hz, 2H, CH2), 2.5 (t, J=6.0 Hz,
2H, CH2), 2.45-2.39 (m, 4H, 2ꢀCH2), 1.16-1.11 (m, 2H, CH2).
13C NMR (100 MHz, CDCl3): δ 196.21, 164.02, 161.83, 140.33,
133.97, 137.21, 133.18, 130.46, 130.22, 128.71, 127.17, 124.17,
121.30, 119.40, 109.99, 103.41, 57.38, 54.5, 46.44, 36.92, 36.02,
26.24, 24.56. ES-MS [M þ 1] found 459.2, calculated for
C27H30N4O3 458.2.
4,40-(Hydroxymethylene)bis(N-(2-(piperdin-1-yl)ethyl)benza-
mide (12). Compound 12 was prepared in a manner similar to the
preparation of 11 but using 5 as the starting material. TLC
(89:10:1 CH2Cl2/MeOH/Et3N, Rf=0.25), 60% yield. 1H NMR
(400 MHz, CD3OD): δ 7.85 (d, J=8.4 Hz, 4H, Ar-H), 7.49 (d,
J=8.4 Hz, 4H, Ar-H), 7.27 (d, J=1.6 Hz, 2H, Ar-H), 6.85 (d,
J=1.6 Hz, 2H, Ar-H), 5.88 (s, 1H, CH), 3.84 (s, 6H, 2ꢀCH3),
3.43 (t, J=6.8 Hz, 4H, 2ꢀCH2), 2.56-2.41 (m, 12H, 6ꢀCH2),
1.66-1.52 (m, 8H, 4 ꢀ CH2), 1.49-1.41 (m, 4H, 2 ꢀ CH2). 13
C
NMR (100 MHz, CD3OD): δ 179.26, 165.87, 162.89, 148.18,
133.61, 127.38, 126.59, 123.46, 122.24, 119.54, 104.90, 74.81,
57.94, 54.35, 55.93, 35.65, 25.38, 23.95, 23.07. ES-MS [Mþ1]
found 737.3, calculated for C41H52N8O5 736.4.
4,40-Carbonylbis(N-(2-(piperidin-1-yl)ethyl)benzamide) (21).
In an argon flushed flask, 25 (81 mg, 0.3 mmol) was dissolved
in 4.0 mL of dry DMF. To this solution, 24e (177 mg, 0.75 mmol)
was added under argon, and the mixture was heated at 55 °C
overnight. The solvent was evaporated and the residue purified by
column chromatography to give the final product in 78% yield.
TLC (97:2:1 CH2Cl2/MeOH/Et3N, Rf = 0.55). 1HNMR(400MHz,
CDCl3): δ 7.88 (d, J=7.2 Hz, 4H, ArH), 7.81 (d, J=8.0 Hz, 4H,
ArH), 3.52 (q, J=5.6 Hz, 4H, 2ꢀCH2), 2.56 (t, J=4.8 Hz, 4H, 2ꢀ
CH2), 2.48-2.36 (m, 8H, 4 ꢀ CH2), 1.61-1.55 (m, 8H, 4 ꢀ CH2),
1.46-1.41 (m, 4H, 2 ꢀ CH2). 13C NMR (100 MHz, CDCl3): δ
195.51, 166.50, 161.40, 139.52, 138.57, 130.73, 127.30, 57.12, 57.01,
54.45, 36.73, 34.81, 26.22, 26.13, 24.47. ES-MS [Mþ1] found 491.2,
calculated for C29H38N4O3 490.3.
Ethidium Bromide Displacement Assay. DNA hairpin oligo-
nucleotide 50-CGAAAAACAAAAAGTTTTTCG-30 was pur-
chased from Trilink Biotechnologies, San Diego, CA, and
prepared for use according to the literature procedures.43 To
a 1.0 mL quartz cell containing Tris buffer (0.1 M Tris HCl, 25 mM,
NaCl, pH 7.0), ethidium bromide (4.0 μM) and the AT hairpin
oligonucleotides (8.0 μM) were added. The fluorescence was
determined using an excitation wavelength of 545 nm and
emission wavelength of 595 nm. Various concentrations of the
agent were added, the solution was mixed, and the change in the
4-Benzamido-1-methyl-N-(2-(pyrrolidin-1-yl)ethyl)-1H-pyr-
role-2-carboxamide (19). To a dry flask under argon, 28b (128 mg,
0.5 mmol), EDC (143 mg, 0.75 mmol), and DMAP (91.5 mg,
0.75 mmol) were added followed by 4 mL of dry DMF. The mix-
ture was stirred for 30 min, and then the freshly prepared 24d,
dissolved in 2 mL of dry DMF, was added dropwise to the
activated acid. The mixture was allowed to stir at room tempera-
ture for 15 h. The DMF was evaporated, and the residue was
dissolved in methylene chloride (10 mL) and washed with brine
and saturated sodium bicarbonate solution. The organic layer was
concentrated in vacuo. The product was purified using column
chromatography to yield the final product in 60% yield. TLC
1
(94:5:1 CH2Cl2/MeOH/Et3N, Rf = 0.55). H NMR (400 MHz,
CDCl3): δ 8.02 (s, 1H, CONH), 7.82-7.77 (m, 2H, Ar-H), 7.42-
7.37 (m, 3H, Ar-H), 7.19 (d, J=0.8 Hz, 1H, Ar-H), 6.91 (s, 1H,
CONH), 6.61(s, 1H, Ar-H), 3.84 (s, 3H, CH3), 3.45 (q, J=5.6 Hz,
2H, CH2), 2.71 (t, J=5.6 Hz, 2H, CH2), 2.65-2.59 (m, 4H, 2 ꢀ
CH2), 1.82-1.77 (m, 4H, 2ꢀCH2). 13C NMR (100 MHz, CDCl3):

5030 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Vooturi et al.
fluorescence at 595 nm was recorded after a 5 min incubation
period. The data were corrected for differences in volume due to
addition of the drug, and binding constants were determined
using a competitive binding model: K=KEB[E]/[agent], where K
is the binding constant, KEB is the binding constant for ethidium
bromide, [E] is the total concentration of ethidium bromide, and
[agent] is the concentration of the drug that reduces fluorescence
(protein synthesis) was added at 0.5-1.0 μCi per mixture,
depending on the experiment. Reactions were allowed to pro-
ceed at room temperature for 15-30 min (60 min for cell wall
synthesis) and then stopped by adding 12 μL of cold 5%
trichloroacetic acid (TCA) or 5% TCA/2% caesamino acids
(protein synthesis only). Mixtures were incubated on ice for
30 min, and the TCA precipitated material was collected on a
25 mm GF/A filter. After being washed three times with 5 mL of
cold 5% TCA, the filters were rinsed two times with 5 mL of
100% ethanol, allowed to dry, and then counted using a Beck-
man LS 3801 liquid scintillation counter. The following com-
pounds served as positive control agents for the macromolecular
synthesis assays: DNA (ciprofloxacin), RNA (rifampicin), cell
wall (vancomycin), protein (linezolid).
For lipid synthesis, the following method was employed. Cells
(0.1 mL) in early exponential growth phase were added to a 1.5 mL
Eppendorf tube (in triplicate) containing various concentrations
(0.25-4.0 μg/mL) of compound 5, or control antibiotic
(cerulenin). Following a 5 min incubation at room temperature,
[3H]glycerol was added at 0.5 μCi per mixture. Reactions were
allowed to proceed at room temperature for 15 min and then
stopped through the addition of 375 μL of chloroform/methanol
(1:2) followed by vortexing for 20 s after each addition. Chloro-
form (125 μL) was then added to each mixture and vortexed
followed by the addition of 125 μL of distilled H2O and
vortexing. Mixtures were centrifuged at 13 000 rpm for 10
min, and 150 μL of the organic phase was transferred to a
scintillation vial and allowed to dry in a fume hood for at least 1
h. Samples were then counted via liquid scintillation counting.
by 50%. KEB for the AT hairpin is 0.27 ꢀ10-6 M-1
.
MTS Assay. Chinese hamster ovary cell line (ATCC, Mana-
ssas, VA) and growth medium RPMI-1640 (ATCC, Manassas,
VA) were used. Cells were seeded in 96-well plates at a seeding
density of 5000 cell/well per 0.1 mL of medium and allowed to
attach overnight. Cells were then treated with medium contain-
ing the agent in different concentrations (25, 50, and 100 mg/L)
and incubated for 24 h. MTS assay reagent (20 μL) was added to
each well and incubated for 120 min, and the absorbance was
measured at 505 nm using a microplate reader (Molecular
Devices, kinetic microplate reader, Sunnyvale, CA). In this
assay, the absorbance is proportional to the number of viable
cells. Untreated cell (with or without 5% DMSO in medium)
were used as controls. Results were presented as percentage
cytotoxicity compared to controls. Experiments were repeated
at least three times.
Depolarization Studies35. S. aureus bacteria were grown at
37 °C with shaking to mid-logarithmic phase (OD600=0.4-0.5).
Bacteria were collected by centrifugation, washed with buffer
(5 mM HEPES, 5 mM glucose, pH 7.2), and suspended in the
same buffer to an OD600 of 0.05. The cell suspension was
incubated with 0.4 μM diSC3(5) for an hour at 37 °C followed
by the addition of 100 mM KCl to equilibrate the cytoplasmic
and external Kþ concentrations. Cell suspension (1 mL) was
placed in a plastic cuvette with a stir bar and placed into a
thermostated cuvette holder (heated to 37 °C). The desired
concentration of the test agent was added, and the fluorescence
readings were monitored for 60 min using a FluoroMax 3 fluore-
scence spectrophotometer (Horiba Jobin Yvon, Edison, NJ) set
to an excitation wavelength of 622 nm and emission wavelength
of 670 nm. Nisin was used as a positive control and a blank with
only cells, and dye was used to subtract the background.
In Vivo Competition Studies with Hoescht Dye. S. aureus
bacteria were grown at 37 °C with shaking to mid-logarithmic
phase (OD600=0.4-0.5). Cells were collected by centrifugation
and suspended in PBS pH 7.2 buffer. Hoescht dye was added to
a final concentration of 1.0 μg/mL, and the cells were incubated
for 30 min at 37 °C. The bacteria were then placed into a
microtiter plate such that each drug concentration was done in
triplicate. Test compounds (25, 50, 100, 150, 200 mg/L) were
added, and the fluorescence was measured using microplate
reader set to an emission wavelength of 461 nm (Molecular
Devices, kinetic microplate reader, Sunnyvale, CA). Cells in-
cubated with Hoescht but not treated with the test compounds
were used as a control.
Incorporation Studies for the Inhibition of Macromolecular
Synthesis29. S. aureus cells were grown at 35 °C overnight on TS
agar. An small amount of bacteria was taken from the plate
and used to inoculate 10 mL of Mueller-Hinton broth II
(MHB). The culture was grown to early exponential growth
phase (OD600=0.2-0.3) in a shaker incubator set to 35 °C.
For DNA, RNA, cell wall, and protein synthesis the follow-
ing protocol was used. When cells reached early exponential
phase, 100 μL of culture was added to triplicate wells containing
various concentrations of (0.25-4.0 μg/mL) of compound 5,
and control antibiotics (5 μL) at 20ꢀ final concentration in
100% DMSO. A 5% DMSO treated culture served as the
“no drug” control for all experiments. Cells were added in
MHB at 105% to account for the volume of drug added to each
mixture or in M9 minimal medium for protein synthesis reac-
tions. Following a 5 min incubation at room temperature,
[3H]thymidine (DNA synthesis), [3H]uridine (RNA synthesis),
[3H]N-acetylglucosamine (cell wall synthesis), or [3H]leucine
Acknowledgment. We thank Micromyx LLC, Kalamazoo,
MI, for the macromolecular synthesis studies and Dr. Yogesh
Patil for helping us with the MTS assay. We thank Wayne
State University for funding.
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