Structure-based redesign of an edema toxin inhibitor

Article abstract of DOI:10.1016/j.bmc.2011.10.091

Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3′,5′-cyclic adenosine mo

Full text of DOI:10.1016/j.bmc.2011.10.091

Bioorganic & Medicinal Chemistry 20 (2012) 368–376
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based redesign of an edema toxin inhibitor
Deliang Chen a, Lili Ma b, John J. Kanalas c, Jian Gao c, Jennifer Pawlik d,e, Maria Estrella Jimenez b,
Mary A. Walter c, Johnny W. Peterson d,e, Scott R. Gilbertson b, Catherine H. Schein a,d,e,f,
⇑
a Sealy Center for Structural Biology and Molecular Biophysics, Department of Biochemistry and Molecular Biology, UTMB, Galveston, TX 77555-0857, USA
b Department of Chemistry, University of Houston, Houston, TX 77004, USA
c Mission Pharmacal Company, San Antonio, TX, USA
d Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infections, UTMB, Galveston, TX 77555, USA
e Department of Microbiology and Immunology, UTMB, Galveston, TX 77555-1070, USA
f Institute for Translational Studies, UTMB, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID cat-
egory B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the
conversion of ATP to 30,50-cyclic adenosine monophosphate (cAMP). We previously identified compound
1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mamma-
lian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of
Received 2 September 2011
Revised 17 October 2011
Accepted 25 October 2011
Available online 16 November 2011
15 lg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of
Keywords:
new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP pro-
duction in murine monocyte-macrophage cells (RAW 264.7). Structure–activity relationship (SAR) anal-
ysis of the bioassay results for 22 compounds indicated positions important for activity. Several
derivatives demonstrated superior pharmacological properties compared to our initial lead compound,
and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-produc-
ing bacteria.
Adenylyl cyclase toxin inhibitor
Non-nucleotide inhibitors
Toxicity profiling
Computer aided design
Cell-based assay
Anthrax
ETEC (enterotoxigenic E. coli)
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
compound selection, molecular docking, and experimental
screening with a cell-based bioassay.5,6 Compound 1 consistently
The toxins produced by many pathogenic bacteria cause mor-
bidity and mortality in infected individuals. For example, the tox-
ins of Bacillus anthracis, a naturally occurring pathogen that has
been used as a bioweapon,1 are important virulence factors. The
toxin complex consists of two classic AB toxins, where enzymatic
moiety A is either a Zn2+ metalloprotease, lethal factor (LF), or an
adenylyl cyclase, edema factor (EF). Both enzymes require a ‘‘B’’
protein, protective antigen (PA), for receptor binding and cell en-
try2; combining PA and EF gives edema toxin (ET). Other groups
have focused on inhibiting the interaction between EF and activa-
tors to control its activity.3,4 We have focused on identifying non-
nucleotide inhibitors that should bind to the active site of EF. These
could be used together with LF inhibitors to treat late stage anthrax
infections, where antibiotics might be unable to prevent death.
These EF inhibitors could also be useful against pathogens which
produce similar toxins, such as Bordetella pertussis, the causative
agent of whooping cough.
inhibited cAMP production induced by edema toxin (IC50 2–9 lM
in cells).7 Compound 1 also reduced diarrhea caused by enterotox-
igenic Escherichia coli (ETEC), at an oral gavage dose of 15
l
g/mouse.8 As we have previously shown that compound 1 inhibits
cholera toxin,9 which is 80% identical to lethal toxin of ETEC,10 and
human ETEC strains can also produce adenylyl cyclase toxins,11 we
attribute this activity to direct inhibition of the E. coli toxins. The
ETEC study also suggested that the compound was working as an
inhibitor of quorum sensing, which in many bacteria is controlled
by cAMP levels12,13, as the levels of bacteria in the colons of the
infected and treated mice were much lower than those who did
not receive the compound.
Here we describe the search for derivatives of Compound 1 that
would have similar or better activity, with improved predicted
aqueous solubility and reduced predicted toxicity. Compound 1
treatment gave no obvious toxic effect in the cell cultures or mouse
studies, and the Ames II™ Mutagenicity Assay determined the
Of three initial ‘hits’, we identified a novel inhibitor of EF, com-
compound
However, there was a positive response for genotoxicity in the
GreenScreen HC [GADD45 (growth arrest and DNA damage
1 to be non-mutagenic in all conditions tested.
pound 130 (Fig. 1), by using a combination of pharmacophore-based
a
gene)-Green Fluorescent Protein (GFP)] assay test on mammalian
cells (Supplementary Fig. 1 and Table 1) at 10–20ꢀ the active dose
⇑
Corresponding author.
E-mail address: chschein@utmb.edu (C.H. Schein).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.091

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
369
Figure 1. Structures for the lead compound 1 and derivatives 2–22 analyzed in this work.
Table 1
Values for the activity of the derivatives of compound 1 (Fig. 1) in inhibiting EF induced secretion of cAMP by cultured cells, compared to their cLogP, binding (BE) and docking
energies (DE) calculated with Autodock.
Compound
R1=
R2=
cLogP
IC50
(
lM)
AutoDock scores
BE DE
Assay 1a
Assay 2a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
–COOH
H
4.29
4.72
2.59
4.77
4.75
3.57
3.57
3.99
3.99
3.57
4.19
4.61
4.91
4.45
5.06
4.64
5.27
5.60
4.77
5.45
1.64–12.25
19.0 ( 2.27)
>100
l
M (many assays)
ꢁ13.28
ꢁ11.94
ꢁ11.60
ꢁ11.72
ꢁ12.80
ꢁ14.45
ꢁ14.28
ꢁ14.11
ꢁ14.53
ꢁ14.34
ꢁ14
ꢁ14.75
ꢁ11.86
ꢁ11.53
ꢁ11.87
ꢁ13.19
ꢁ14.97
ꢁ15.6
See Figure 1
See Figure 1
H
–COOMe
See Figure 1
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
–COOMe
–COOMe
–COOMe
–SMe
–OCF3
–NMe2
–Cl
H
H
28.6 ( 2.97)
11.0 ( 3.05)
80.00 ( 3.05)
6.08 ( 3.09)
5.64 ( 4.84)
6.21 ( 2.86)
1.30 ( 0.23)
5.81 ( 1.60)
11.36 ( 8.55)
9.21 ( 1.14)
>100 ( 2.11)
12.98 ( 2.65)
7.18 ( 0.93)
26.03 ( 1.49)
6.80 ( 0.56)
1.51 ( 2.11)
3.80 ( 2.81)
12.92 ( 4.94)
2.71 ( 0.83)
6-NH2
2-OH
6-OH
5-NH2
4-OMe
2-Me
2-Cl
6-OH
2-Me
4-OMe
H
5.64 ( 5.99)
6.21 ( 2.86)
ꢁ15.47
ꢁ15.7
ꢁ15.53
ꢁ15.3
11.36 ( 0.45)
ꢁ13.73
ꢁ13.59
ꢁ11.39
ꢁ11.46
ꢁ10.95
ꢁ11.08
ꢁ10.69
ꢁ10.66
ꢁ10.99
ꢁ13.15
ꢁ12.21
ꢁ15.46
ꢁ15.18
ꢁ12.91
ꢁ13.12
ꢁ12.65
ꢁ11.88
ꢁ11.42
ꢁ11.97
ꢁ11.78
ꢁ13.10
ꢁ13.34
248.8 ( 2.68)
12.92 ( 2.65)
H
H
6-F
H
6.86 ( 1.33)
1.51 ( 0.46)
–C(O)NHOH
–C(@NH)NH2
H
3.15
2.71 ( 5.29)
The most active compounds are given in bold type.
a
Assays were repeated for compounds that had IC50 <20 lM in the initial assay, or where the results were not consistent with theory. The Descriptive Statics function in
SigmaPlot was used to calculate STD Error for the data points closes to the IC50 concentration.
(62.5
l
g/ml vs active dose in mice of about 3
l
g/ml, assuming 5 ml
designed compounds were then redocked with AutoDock 3.0.14,15
Our previous studies5 indicated that the docking of 3d0ATP to the
crystal structure of EF (PDB structure:1K9016) had low RMSD
(root-mean-square deviations) between the predicted structure
and the crystal structure, and thus, AutoDock 3.0 was reliable
enough to predict the binding mode of the ligands to EF. The syn-
thesized derivatives were dissolved in DMSO and then diluted at
least 100-fold into cell culture medium. Their ability to reduce to-
tal extracellular cAMP production in mammalian cells treated with
protective antigen (PA) and EF (which together17 are called edema
toxin, ET) was determined. This assay gives more variability than
using isolated enzymes, but it is biologically relevant, as the con-
centrations needed to inhibit diarrhea and intestinal damage in
serum/mouse). Further, the cLogP of compound 1 was high (4.29),
it had to be dissolved in DMSO and then diluted into buffer 1:100
fold before adding to cell assays or for gavage. As the results here
show, we were able to identify several derivatives with better
predicted pharmaceutical properties, and equivalent or better
activity in the bioassay for cAMP production induced by treatment
of mammalian cells with ET.
2. Results
Derivatives of compound 1 were designed to add substituents at
positions that had contact with residues important in the active
site, according to docked conformations (Fig. 1 and Table 1). The
the ETEC murine model (7.5–15 lg/mouse) were similar to those

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D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
Table 2
Predicted toxicities (based on results from the on-line program OSIRIS Property
Explorer)
Compound
Expected toxicity
Mutagenic
Tumorigenic
Irritant
Reproduct
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
High
Mid
High
Low
Low
Low
High
Low
Low
High
Mid
Low
Low
Low
Low
Low
Low
Low
High
Low
Mid
Low
Low
Low
Low
Low
Low
Low
High
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Mid
Low
Mid
Low
Low
High
Mid
Low
Low
Mid
Low
Low
Low
Low
Low
Low
Low
Low
High
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Figure 2. Overlay of the docked conformation of 1 A: with the position of 3d0-ATP
in the crystal structure (in 1K90.pdb) and B: with the fragments of the original
pharmacophore (dotted spheres) used to screen the ZINC database.6
indicated by the cell culture IC50 values for compound 1.8 The IC50
values for compound 1 in the assays shown here (Table 1) ranged
from 1.64–12.6 lM, in part due to dilutions being done in 10-fold
steps when large groups of inhibitors were assayed together. As-
says were done in triplicate and samples with IC50 values
<20 lM were assayed at least twice on different days, and re-as-
sayed in two-fold dilution steps to reduce errors at lower doses.
Below we summarize the key points of our structure–activity rela-
tionship analysis of these results.
The most active compounds in Table 1 are given in bold type. None of the com-
pounds were obviously toxic in the cell culture assay when added alone, at the
highest dose tested.
2.1. The fluorenone ring and benzoic acid were essential for the
activity of 1
2.2. The position of the carboxyl group
Moving the carboxyl group to any other position reduced activ-
ity, as shown, for example, by a much higher IC50 for 6 than for 7.
This was also consistent with docking results, where the binding
affinity of 6 was lower than 7. The docked structures suggested
one explanation for this: the angle of the carboxyl of 6 to the metal
ion deviated greatly from ideal geometry (88° versus the optimal
hydrogen bond angle of P120°20,21 for the sp2 oxygen atom; the
angles for 1 and 7 are 121.5° and 117°, respectively).
Compounds 2–5 (Fig 1) were synthesized for the following rea-
sons: 2: reduce the fluorenone ring to a dibenzene (remove the car-
bonyl group) and change the carboxyl group to methyl ester; 3:
reduce the fluorenone ring to a benzene; 4: remove the carboxyl
group of the benzoic acid; and 5 (and others): change this carboxyl
group to a methyl ester. The IC50 for compounds 2–5 were all high-
er than those for 1 (Table 1). Assay results for compounds 2 and 3
(as well as for other compounds that are not shown) indicated the
fluorenone ring (which overlays the position of the purine ring of
the ATP analogue in our dockings) was important for activity. Thus
the bulk of our derivative study concentrated on alterations to the
benzoic acid moiety. We also do not anticipate toxicity from this
moiety, as the fluorenone itself in the interferon inducer tilorone18
and related compounds19 showed no toxic effects in cells.
2.3. Additions to the benzoic acid
Depending on their position, addition of hydrophilic groups
such as –OH (8 and 9), –NH2(7 and 10), and –OMe (11) decreased
the cLogP, and addition of more hydrophobic ones such as –Me
(12) and –Cl (13) increased the cLogP. However, adding –NH2
groups, which had the most beneficial effect on cLogP, also
increased markers of toxicity (Table 2) and, except for addition at
the 5 position (Compound 10), reduced the EF inhibitory activity.
Adding functional groups such as –OH (8, 9 and 14), –Me (12
and 15), and –Cl (13) at ortho (2 or 6) positions to the carboxyl
group (or ester) reduced the expected toxicities, with acceptable
binding energies. However, they also affected the IC50 values,
depending on the groups and their positions. Electron donating
groups on carbons 2, 4 and 6 (e.g., –OH) should increase the
electron density on the oxygen atoms of the carboxyl group or
the ester, which can enhance interactions between the ligands
and receptor. However, these additions may also reduce interac-
tions with active site residues by increasing desolvation energies.
Positional effects may be explained by how the additional
groups affect the ability of the carboxyl group (or a carboxy methyl
ester) to interact with metal ions in the active site. For example,
adding –OH at position 6 of 5 (compound 14) renders it completely
inactive, while additions at the 2 and 4 positions (compounds 15
and 16) did not decrease activity significantly. This may be because
interaction between the sp2 oxygen atom of the ester of 14 and the
Comparing compounds 4 and 5 it is clear that removing the car-
boxyl group of 1 had a more drastic effect on activity than changing
it to a methyl ester. The overall decrease in activity was consistent
with docking results that indicated this carboxyl overlaid the posi-
tion of the a
-phosphate of ATP from the crystal structure16 and the
negatively charged oxygen atom interacted with the metal ion to
form an ion–ion interaction (Fig 2A). Our previous dockings indi-
cated that the interaction with metal is the most important hydro-
gen bond or ion–ion interaction (Fig 2B).5,6
With these initial results in mind, further redesign concentrated
on modifying the benzoic acid to increase the hydrogen bond inter-
actions and/or the ion–ion interactions. Compounds 6–22 added
substituents to the benzoic acid ring or replaced the carboxyl
group of the benzoic acid with other groups. All the derivatives
synthesized, compared to compound 1, had lower calculated LogP
(cLogP) values (Table 1), and most had lower expected toxicities,
according to the on-line program OSIRIS Property Explorer
(http://www.organic-chemistry.org/prog) (Table 2). With a few
exceptions, any changes to 1 reduced potency, for reasons that
could be explained by comparing docked conformations.

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
371
metal ion was decreased by an intramolecular hydrogen bond be-
tween the –OH and the sp2 oxygen atom. This effect would not be
anticipated in compound 8, as both oxygen atoms of the carboxyl
group are strong hydrogen bond acceptors, and an intramolecular
hydrogen bond between the –OH and one oxygen atom should
have little effect on the interaction of the other oxygen atom with
the metal ion. Electron withdrawing groups (e.g., Cl) would be
expected to decrease the electron density of the oxygen atoms of
the carboxyl group or the ester, thus reducing interactions be-
tween the ligands and receptors. Adding a methyl group, a hydro-
phobic and weak electron donating group, to the ortho positions to
the carboxyl group (or ester) (e.g., compare 12 with 1 and 15 with
5) did not improve activity, and raised the cLogP considerably.
has no toxicity markers, it was selected to start further testing as
the next generation of EF inhibitor.
3. Discussion
Although early treatment with antibiotics can greatly reduce
the effects of bacterial infections, inhibitors of the toxins could play
a therapeutic role in later stage infections and in preventing diar-
rhea or death, based on the type of infection. Our previously iden-
tified lead compound 1 was active in bioassays in the low
range, and the effective dose to reduce diarrhea in mice (from
ETEC) was also low, on the order of 7.5–15 g when given intraper-
lM
l
itoneally or by gavage.8 Before beginning additional animal stud-
ies, it was necessary to eliminate as many markers of toxicity as
possible, and improve solubility. Our goals in this study were to
optimize activity, by designing compounds to retain biological
activity while improving pharmaceutically important properties,
such as solubility, and to remove known markers of toxicity or
mutagenicity (properties summarized in Tables 1 and 2). These
compounds would allow us to proceed to more advanced animal
trials for inhibiting anthrax in the rabbit model.22 Also, in light of
recent outbreaks of E. coli, for example in Germany,23 a variety of
compounds with similar activities to our Compound 1 should be
available for testing against other toxin producing E. coli strains.
Our initial results indicated that the fluorenone ring contributed
to the inhibitory activity, and thus, we focused this study on the
benzoic acid side chain, which had some markers of toxicity
(Table 2).
2.4. Effect of changing the carboxyl group to other functional
groups
Changing the carboxyl group of 1 and its derivatives to methyl
esters (14–16) reduced the expected toxicities but increased cLogP
values. The reduction in potencies could be due to reduced interac-
tions of the less Lewis basic oxygen atoms of the esters with the
metal ions in the active site. In 14, as in 9, adding an additional
6-OH group rendered the compound inactive.
While a group is necessary at the 1 position (compound 4 is rel-
atively inactive), substitutions of the carboxyl group of 1 with
groups having weaker hydrogen bond forming abilities yielded
the most promising derivatives in this study. For example, adding
–SMe (17) reduced activity, but addition of –OCF3 (18), –NMe2 (19)
and –Cl (20) all gave inhibitory activities similar to those of Com-
pound 1. The latter groups are much weaker hydrogen bond
donors than –SMe. The overall ability of compounds 18–20 to inhi-
bit may thus be due to their more favorable desolvation energies.
The relatively low activity of 21 might be attributable to the hydro-
philic group C(O)NHOH being a weaker hydrogen bond donor than
the carboxyl group.
Substituting the carboxyl with the hydrogen bond donor group
3-carbamimidoyl, –C(@NH)NH2, (22) yielded a compound with a
reduced cLogP, and better than or equivalent activity to 1 in the
bioassay. Docking of both the neutral (–C(@NH)NH2) and the posi-
tively charged (–C@NH2NH2+) forms of 22 to 1K90 (Fig. 3) sug-
gested that this group could make strong hydrogen bonds with
the backbone oxygen atoms from the active site residues of
Lys346, Gly347, Val350 and the carboxyl group of Asp491. As 22
3.1. The importance of the fluorenone for activity
Compound, 1 was chosen as our lead compound as it consis-
tently scored very high in all our library dockings, and proved
the most soluble of all three lead compounds we obtained from
our preliminary screenings.6 In these dockings, the fluorenone ring
overlaid the position of the adenine ring of 3d0-ATP in the crystal
structure of EF, much as other large planar inhibitors fit the ATP
binding pockets.24 The fluorenone could not be replaced with
smaller rings (Compounds 2 and 3), suggesting its size and planar-
ity were important. The carbonyl group on the fluorenone ring
might be more essential for solubility than activity. Removal (com-
pound 2) decreased the potency, when compared to compound 5.
However, as this introduced one more rotatable bond than
Figure 3. Effect of protonation state of the benzene side chain side chain –C(@NH)NH2 on potential interactions with EF residues. Docking poses are shown for 22 when
neutral (A) or positively charged (B). In A, this group interacts with the metal ion, the backbone oxygen atom of Lys346, Gly347, Val50 and the carboxyl group of Asp491,
while in B it interacts with the backbone oxygen atoms of Lys346, Gly347, Val50 and the carboxyl group of Asp491.

372
D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
compound 5, this would result in a predicted free energy cost for
rotor restrictions of about 3.5–5.0 kJ/mol per rotor.25 This addi-
tional rotation should increase the IC50 value considerably, and this
alone should increase the IC50 4.1–7.5 times.
formed by the apolar hydrogen and carbon atoms from the side
chain of Leu348, Asn583, Gly547, etc, where the purine of 3d0ATP
is located in the 1K90 structure, and the fluorenone in the docked
structures of all derivatives is located. The third, negatively-
charged region (red circle) includes the backbone oxygen atoms
from the residues of Lys346, Gly347, Val350 and the carboxyl
group of Asp491. The –C(@NH)NH2 of 22 could form hydrogen
bonds with EF in this last region, thus accounting for its relatively
good activity. This structure–activity analysis, following traditional
approaches,26–28 suggests that activity could be further improved
by adding both hydrogen bond acceptors and hydrogen bond do-
nors to the benzene ring so that the ligands could interact with
positively-charged residues and negatively-charged backbone oxy-
gen atoms. Adding both hydrogen bond acceptors and hydrogen
bond donors to the benzene ring could increase the activity, as
illustrated by 10, which was among the most active compounds.
Docking suggested that the negatively charged carboxyl group
can interact with positively charged residues, while the amino
group –NH2, in its protonated form, interacts with the negatively
charged region. The activity of 1 would also be improved by chang-
ing its carboxyl group to even larger hydrogen bond acceptors and/
or negatively-charged groups, to fully occupy the positively-
charged region (blue circle in Fig 4). A single carboxyl group could
not occupy this region as effectively as the tri-phosphate group of
ATP (thick lines).
Both hydrogen bond acceptors and hydrogen bond donors on
the benzene ring improved the predicted cLogP, without changing
activity. Substitutions at the benzoic acid affected the optimal
docking conformation of closely related compounds on EF, suggest-
ing that there are many ways that these compounds could bind
within the active site and still serve as inhibitors. We initially as-
sumed that the carboxyl group was essential, as the docked confor-
mation indicated it interacted with the metal ion and/or positively
charged residues such as Arg329, Lys346, Lys353, and Lys372 in
the active site, similar to the phosphate oxygens of 3d0-ATP in
the crystal structure (which it overlaid). Methoxy analogues in
general did not have as high an inhibitory activity as their car-
boxyl-counterparts, which seemed to confirm the need for a hydro-
philic, metal ion binding group. Thus it was surprising that the
carboxyl group of the benzoic acid could be exchanged with one
that had a much lower affinity for metal ions, 3-carbamimidoyl-
phenyl, without losing activity (compound 22 in Table 1). Dockings
indicated this inhibitor could target other areas of the active site
and might simply better fill the substrate-binding area than the
smaller carboxyl group. The dockings also suggested the hydrogen
atoms of the group interacted with the backbone oxygen atoms
from the residues of Lys346, Gly347, Val350 and carboxyl group
of Asp491(Fig. 3).
3.3. Docking scores do not directly correlate with EF-inhibitory
activity, especially when the ligands can adopt multiple proton-
ated states
3.2. Further analysis of the docked conformation suggested
other changes that could lead to a more active molecule
The results in Table 1 show that there is no direct relationship
between docking scores and activities, although compound 10,
which had the best activity of the series, also had the lowest
docking energy. However, compound 6 had a similarly low docking
energy, but was inactive (as discussed in Section 2.2). An additional
factor in docking is the need to account for variable protonation
states of ligands, as we previously discussed.5 For example, the
active compounds 10, and 19 have basic groups that can be proton-
ated, allowing their interaction with negatively charged residues in
the active site (red circle in Fig. 4). Other reasons are that the scor-
ing functions of AutoDock does not fully consider the desolvation
energies of ligands binding to the active site, and that flexible side
chain interactions were not considered in our dockings. A final
complicating factor in our bioassay is possible interactions with
media or cellular components.
The docked conformations of the compounds we tested showed
strong binding to three regions of the active site, which are circled
in Figure 4. The first is the positively charged region (blue circle)
which includes the metal ion and four conserved positively
charged residues (Arg329, Lys346, Lys353, and Lys372) that inter-
act with the triphosphate group of 3d0-ATP in the 1K90 crystal
structure. The second is the hydrophobic region (green circle)
3.4. Optimization must take into account potential toxicity and
balance interactions with the solvent and within the active site
As noted at the outset, we modified compound 1 as it proved to
have some mutagenic potential in the Genescreen assay. As this
toxicity was consistent with predictions from the Osiris property
predictor (Table 2), we also used these predictions to eliminate
some active derivatives. Compounds 10 and 19, while among the
more active compounds in inhibiting EF, were rejected based on
their high mutagenic and irritant potential, although neither
caused obvious toxicity to the cultured cells.
One other result of our SAR analysis was the need to balance
adding charged groups to increase ligand-receptor hydrogen
bonds, which also decreased the favorable desolvation energies ob-
tained from binding of a more hydrophobic molecule. This fact may
account for the basic ability of 1 (with a cLogP of 4) to inhibit ATP
(which has a cLogP of about ꢁ8) from binding to the active site of
EF: while the tri-phosphate group of ATP probably interacts better
with the metal ion and active site positively charged residues, its
low cLogP and high negative charge (ꢁ4) would indicate an
Figure 4. Schematic drawing, showing the position of 30dATP (thick ball and stick
figure) in the 1K90 crystal structure, overlaid with the docking pose of 22 (green
thin ball and stick figure). The silver point indicates the position of the metal ion,
the blue and red points indicate positive and negative charged groups, respectively,
from EF side chains or backbone oxygens. The blue circle (top left) indicates a
positively charged region, where negatively charged groups, for example, the
triphosphate of ATP, could be added to enhance inhibitory activity. The red circle
(top right) indicates a negatively charged region formed by the backbone oxygen
atoms of Lys346, Gly347 Val350 and the carboxyl group of Asp491, where the –
C(@NH)NH2 side chain of the 3-carbamimidoylphenyl group of 22 lies. The Green
circle (bottom) indicates a hydrophobic region formed by the side chains of Leu348,
and other residues that are not explicitly shown, where the purine of ATP and the
fluorenone of 22 lie.

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
373
unfavorable net desolvation energy. It may also explain why deriv-
atives of 1 with two carboxyl groups on the benzene ring had re-
duced activity (data not shown). Further redesign of this
molecule will need to take such tradeoffs into account.
filtered, washed with H2O, CH2Cl2, and dried to give moderate-
to-good yields (64–95%).
5.1.5. 3-(9-Oxo-9H-fluorene-1-carboxamido)benzoic acid (1)
Synthesized from methyl 3-(9-oxo-9H-fluorene-1-carboxa-
mido)benzoate according to the general procedure described above
for ester cleavage. Yield 98%. 1H NMR (DMSO-d6, 300 MHz): d 13.00
(br, 1H), 10.69 (s, 1H), 8.34 (t, J = 1.8 Hz, 1H), 7.85–7.96 (m, 3H),
7.70 (t, J = 7.7 Hz, 2H), 7.61–7.64 (m, 2H), 7.52 (dd, J = 6.6, 0.8 Hz,
1H), 7.48 (t, J = 7.7 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H). LCMS m/z
[M+1] 343.08 .
4. Conclusions
Modifying the benzoic acid ring of the lead compound 1
produced two compounds (8 and 22) with improved or similar
inhibition of EF affects on cultured cells, increased aqueous solubil-
ity and reduced expected toxicities. The switch from a hydrogen-
bond donor group (–COOH) to a hydrogen-bond acceptor group
(–C(@NH)NH2) produced the most promising compound, 22, when
aqueous solubility (cLogP) and low potential toxicity is weighted
above the other evaluation criteria. To further improve 22 binding
to the three regions in the active site of EF, one could test the
effects of adding additional hydrogen bond acceptors or electron-
donating groups to the benzimidamide ring of 22.
5.1.6. 3-[(Biphenyl-3-carbonyl)-amino]-benzoic acid (2)
To a microwave vessel containing 3-[(biphenyl-3-carbonyl)-
amino]-benzoic acid methyl ester (1.0 equiv, 55 mg, 0.17 mmol)
in 5 ml of THF–H2O (3:1) was added LiOH (6.0 equiv, 43 mg,
1.02 mmol). The mixture was allowed to warm to 60 °C for 1.5 h.
Upon reaction completion, the solvent was evaporated and the
aqueous layers were acidified with 0.5 M HCl. The white precipi-
tate was washed with cold water yielding the product in 43% yield.
1H NMR (DMSO, 300 MHz): d 12.98 (bs, 1H), 10.49 (s, 1H), 8.40 (t,
J = 2.3 Hz, 1H), 8.24 (t, J = 2.3 Hz, 1H), 8.08–8.05 (m, 1H), 7.95 (bd,
J = 7.7 Hz, 1H), 7.88 (bd, J = 7.7 Hz, 1H), 7.77 (d, J = 6.8 Hz, 2H),
7.69–7.59 (m, 2H), 7.53–7.38 (m, 4H). LCMS m/z [M+1] 317.11.
5. Experimental Section
5.1. Synthesis
5.1.1. General procedure for the amide coupling
In
a reaction vessel, the corresponding carboxylic acids
5.1.7. 3-Benzoylamino-benzoic acid (3)
(0.91 mmol) were dissolved in CH3CN (10 mL), then BOP (1 mmol,
442 mg, 1.1 equiv) was added followed by diisopropyl ethyl amine
(DIPEA) (2.73 mmol, 0.47 ml, 3.0 equiv). Upon addition of DIPEA
the mixture became a homogeneous solution. After 5 min stirring
at room temperature the relevant amines (1 mmol, 1.1 equiv) were
added. The reaction was monitored by TLC and LCMS (1 hr to over-
night) and elevated temperature was used when necessary. Upon
completion, the product precipitated out. The precipitates were
filtered and washed with CH2Cl2, H2O, affording the desired prod-
ucts in moderate to good yields (55–92%). All compounds were
purified to >95% as assessed by HPLC.
Synthesized from the corresponding methyl ester according to
the general procedure described above for ester cleavage. Yield
50%. 1H NMR (MeOD, 300 MHz): d 8.36 (t, J = 1.7 Hz, 1H), 7.97
(dd, J = 2.2, 1.1 Hz, 2H), 7.95–7.92 (m, 2H), 7.80 (dt, J = 7.7,
1.4 Hz, 1H), 7.61–7.43 (m, 5H). LCMS m/z [M+1] 242.02.
5.1.8. 9-Oxo-N-phenyl-9H-fluorene-1-carboxamide (4)
Synthesized from 9-fluorenone-1-carboxilic acid and aniline
according to the general procedure describe above for amide cou-
pling. Yield 68%. 1H NMR (DMSO-d6, 300 MHz): d 10.52 (s, 1H), 7.43
(d, J = 7.5 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.60–7.71 (m, 5H), 7.51
(d, J = 7.7 Hz, 1H), 7.32–7.43 (m, 3H), 7.10 (t, J = 7.2 Hz, 1H). 13C
NMR (CDCl3, 75 MHz): d 196.2, 161.5, 145.6, 143.5, 138.7, 136.0,
135.3, 134.9, 132.9, 132.6, 129.9, 129.6, 128.9, 125.2, 124.2,
123.2, 120.5, 120.1. LCMS m/z [M+1] 300.09.
5.1.2. General procedure for the methyl ester cleavage
To a reaction vessel containing the corresponding methyl esters
(0.5 mmol) in 30 mL of THF–H2O (2:1 ratio) was added LiOHꢂH2O
(1.5 mmol, 3 equiv). The mixture was refluxed overnight. Upon
completion, monitored by TLC, the solvent was evaporated; the
mixture was diluted in CH2Cl2 and acidified with 0.5 M HCl until
pH 2. The precipitated solid was then filtered and washed with
CH2Cl2, H2O affording the acid in good yields (75–99%).
5.1.9. Methyl 3-(9-oxo-9H-fluorene-1-carboxamido)benzoate (5)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 91%. 1H NMR (DMSO-d6,
300 MHz): d 10.72 (s, 1H), 8.39 (d, J = 1.8 Hz, 1H), 7.94 (t, J = 7.3
Hz, 2H), 7.88 (dd, J = 7.3, 0.8 Hz, 1H), 7.50–7.76 (m, 6H), 7.42 (t,
J = 7.3 Hz, 1H), 3.88 (s, 3H). LCMS m/z [M+1] 357.73.
5.1.3. General procedure for the acid chloride formation
To a dried and tared 50 mL round bottom reaction flask
equipped with a stirring bar was added 3-[(9-oxo-9H-fluorene-1-
carbonyl)-amino]-benzoic acid (16 mmol, 3.59 g) and thionyl
chloride (20 ml). The mixture was refluxed 3 h. Upon completion,
monitored by TLC, the thionyl chloride was removed by distilla-
tion. The resulting solid was dried overnight under vacuum to
remove trace amounts of thionyl chloride, affording the crude acid
chloride in good yields (91–98%).
5.1.10. 5-Amino-2-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (6)
Synthesized from 9-oxo-9H-fluorene-1-carbonyl chloride and
the corresponding amine according to the procedure described
above for the amide coupling via acid chloride. A small amount
of product was dissolved in DMSO and purified by reverse-phase
HPLC (Vydac C18, UV 254) with a linear gradient of 0–100% aceto-
nitrile (0.1% TFA) over 30 min at 4 mL/min. Relevant fractions were
combined, and the solvent was removed by freeze-drying to afford
the desired product in 15% yield. 1H NMR (DMSO-d6, 300 MHz): d
11.13 (s, 1H), 8.27 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.86
(d, J = 7.7 Hz, 1H), 7.58–7.73 (m, 3H), 7.51 (d, J = 7.7 Hz, 1H), 7.40
(td, J = 7.3, 0.8 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 7.3 Hz,
1H). LCMS: m/z [M+1] 359.17.
5.1.4. General procedure for the amide bond formation via acid
chloride
To a stirred solution of amine (1.1 mmol, 1.1 equiv) and 0.46 mL
triethylamine (3.3 mmol, dissolved in 2 mL dry CH2Cl2 at 0 °C, was
added dropwise a solution of acid chloride (1 mmol, 1 equiv) in
2 mL dry CH2Cl2. Stirring was continued at 0 °C for 1 h and at room
temperature for 3 h. The reaction mixture was diluted with 2–5 mL
CH2Cl2, and acidified by 2 N HCl until pH 4. The precipitate was

374
D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
5.1.11. 2-Amino-5-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (7)
5.1.17. 2-Chloro-3-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (13)
Synthesized from 9-fluorenone-1-carboxilic chloride and the
corresponding amine according to the procedure for amide cou-
pling via acid chloride described above. A small amount of product
was dissolved in DMSO and purified by reverse-phase HPLC (Vydac
C18, UV 254) with a linear gradient of 0–100% acetonitrile (0.1%
TFA) over 30 min at 4 mL/min. Relevant fractions were combined,
and the solvent was removed by freeze-drying to afford the desired
product in 8% yield. 1H NMR (DMSO-d6, 300 MHz): d 10.28 (s, 1H),
8.27 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.86 (d, J = 7.7 Hz,
1H), 7.58–7.73 (m, 3H), 7.51 (d, J = 7.7 Hz, 1H), 7.40 (td, J = 7.3,
0.8 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 7.3 Hz, 1H). LCMS
m/z [M+1] 359.18.
Synthesized from 9-oxo-9H-fluorene-1-carbonyl chloride and
the corresponding amine according to the procedure described
above for the amide coupling via acid chloride. Yield 80%. 1H
NMR (DMSO-d6, 300 MHz): d 10.50 (s, 1H), 7.96 (t, J = 6.9 Hz, 2H),
7.86 (d, J = 7.4 Hz, 1H), 7.39–7.73 (m, 7H). LCMS, m/z [M+1] 378.08.
5.1.18. Methyl 2-hydroxy-5-(9-oxo-9H-fluorene-1-carboxa
mido)benzoate (14)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 72%. 1H NMR (DMSO-d6,
300 MHz): d 10.48 (s, 1H), 8.24 (d, J = 2.4 Hz, 1H), 7.92 (d,
J = 7.4 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.60–7.74 (m, 4H), 7.48 (d,
J = 7.4 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), 5.73
(s, 1H), 3.91 (s, 3H). LCMS m/z [M+1] 374.12.
5.1.12. 2-Hydroxy-3-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (8)
Synthesized from 9-fluorenone-1-carboxilic chloride and the
corresponding amine according to the general procedure describe
above for amide coupling via acid chloride. Yield 95%. 1H NMR
(DMSO-d6, 300 MHz): d 10.52 (s, 1H), 8.33 (dd, J = 7.7, 1.1 Hz,
1H), 7.50 (dd, J = 7.0, 1.5 Hz, 1H), 7.85 (d, J = 7.3 Hz, 1H), 7.59–
7.70 (m, 5H), 7.40 (t, J = 7.3 Hz, 1H), 6.95 (t, J = 8.1 Hz, 1H). LCMS
m/z [M+1] 360.10.
5.1.19. Methyl 2-methyl-3-(9-oxo-9H-fluorene-1-carboxamido)
benzoate (15)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 80%. 1H NMR (DMSO-d6,
300 MHz): d 10.24 (s, 1H), 7.89 (dd, J = 10.6, 7.7 Hz, 3H), 7.62–
7.75 (m, 4H), 7.51 (d, J = 7.7 Hz, 1H), 7.41 (m, 2H), 3.85 (s, 3H),
2.44 (s, 3H). LCMS m/z [M+1] 371.98.
5.1.13. 2-Hydroxy-5-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (9)
5.1.20. Methyl 4-methoxy-3-(9-oxo-9H-fluorene-1-carboxamido)
benzoate (16)
Synthesized from the corresponding methyl ester according to
the general procedure described above for ester cleavage. Yield
99%. 1H NMR (DMSO-d6, 300 MHz): d 10.47 (s, 1H), 8.22 (d,
J = 2.6 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H),
7.60–7.77 (m, 4H), 7.50 (d, J = 6.6 Hz, 1H), 7.42 (t, J = 7.3 Hz, 1H),
6.96 (d, J = 8.2 Hz, 1H), 5.74 (s, 1H). LCMS m/z [M+1] 360.09.
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 90%. 1H NMR (DMSO-d6,
300 MHz): d 10.50 (s, 1H), 8.81 (d, J = 1.8 Hz, 1H), 7.95 (dd, J = 7.2,
1.1 Hz, 1H), 7.86 (d, J = 7.5 Hz, 1H), 7.80 (dd, J = 8.5, 2.2 Hz, 1H),
7.62–7.73 (m, 4H), 7.40 (td, J = 7.5, 1.0 Hz, 1H), 7.21 (d, J = 8.6 Hz,
1H), 3.92 (s, 3H), 3.86 (s, 3H). LCMS m/z [M+1] 388.15.
5.1.14. 3-Amino-5-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (10)
Synthesized from 9-oxo-9H-fluorene-1-carbonyl chloride and
the corresponding amine according to the procedure described
above for the amide coupling via acid chloride. Yield 85%. 1H
NMR (DMSO-d6, 300 MHz): d 10.76 (s, 1H), 8.37 (s, 1H), 8.12 (d,
J = 2.2 Hz, 1H), 7.94 (d, J = 7.3 Hz, 1H), 7.87 (d, J = 7.0 Hz, 1H),
7.62–7.73 (m, 4H), 7.53 (d, J = 7.7 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H).
LC–MS (ESI): mass calcd for (C21H14N2O4), m/z 358.10; measured
5.1.21. N-(3-(methylthio)phenyl)-9-oxo-9H-fluorene-1-
carboxamide(17)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described above
for amide coupling. Yield 92%. 1H NMR (DMSO-d6, 300 MHz): d 10.53
(s, 1H), 7.94 (dd, J = 7.4 Hz, 0.8 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 7.06–
7.72 (m, 4H), 7.39–7.51 (m, 3H), 7.30 (t, J = 7.7 Hz, 1H), 7.00 (dt,
J = 7.7, 0.8 Hz, 1H), 2.48 (s, 3H). LCMS m/z [M+1] 346.11.
[M+H]+ m/z 359.18.
,
5.1.22. 9-Oxo-N-(3-(trifluoromethoxy)phenyl)-9H-fluorene-1-
carboxamide (18)
5.1.15. 4-Methoxy-3-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (11)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 77%. 1H NMR (DMSO-d6,
300 MHz): d 10.77 (s, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.86 (d,
J = 7.2 Hz, 2H), 7.56–7.72 (m, 4H), 7.38–7.51 (m, 3H), 7.10 (d,
J = 8.0 Hz, 1H). LCMS m/z [M+1] 384.01.
Synthesized from the corresponding methyl ester according to
the general procedure described above for ester cleavage. Yield
75%. 1H NMR (DMSO-d6, 300 MHz): d 12.72 (br, 1H),10.44 (s, 1H),
8.77 (d, J = 1.8 Hz, 1H), 7.95 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 7.3 Hz,
1H), 7.77 (dd, J = 8.4, 1.9 Hz, 1H), 7.62–7.73 (m, 4H), 7.41 (t,
J = 7.5 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 3.91 (s, 3H). LCMS m/z
[M+1] 374.13.
5.1.23. N-(3-(dimethylamino)phenyl)-9-oxo-9H-fluorene-1-
carboxamide (19)
5.1.16. 2-Methyl-3-(9-oxo-9H-fluorene-1-carboxamido)benzoic
acid (12)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 85%. 1H NMR (DMSO-d6,
300 MHz): d 10.36 (s, 1H), 7.92 (d, J = 6.9 Hz, 1H), 7.86 (d,
J = 7.2 Hz, 1H), 7.60–7.71 (m, 3H), 7.50 (d, J = 7.7 Hz, 1H), 7.40 (t,
J = 7.4 Hz, 1H), 7.02–7.17 (m, 3H), 6.49 (dd, J = 7.8, 2.2 Hz, 1H),
2.90 (s, 6H). LCMS m/z [M+1] 343.17.
Synthesized from the corresponding methyl ester according to
the general procedure described above for ester cleavage. Yield
94%. 1H NMR (DMSO-d6, 300 MHz): d 10.17 (s, 1H), 7.92 (d,
J = 7.3 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.58–7.71 (m, 4H), 7.51 (d,
J = 7.7 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 2.44
(s, 3H). LCMS m/z [M+1] 358.14.

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 368–376
375
5.1.24. N-(3-chloro-4-fluorophenyl)-9-oxo-9H-fluorene-1-
carboxamide (20)
bearing the upstream promoter region and regulatory genes
sequences of the human GADD45 gene operatively linked to a hu-
a
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Yield 91%. 1H NMR (DMSO-d6,
300 MHz): d 10.70 (s, 1H), 8.00 (dd, J = 6.8, 2.5 Hz, 1H), 7.95 (d,
J = 7.4 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 7.23–7.55 (m, 4H), 7.38–
7.51 (m, 3H). LCMS m/z [M+1] 351.98.
man codon optimized EGFP gene. The control cell line (GenM-C01)
is TK6 cells tranfected with an identical plasmid except that 4 bp
have been removed at the start of the EGFP gene, such that a func-
tional GFP protein is not produced. The plasmids are stably main-
tained in TK6 cells by addition of 200 lg/ml hygromycin B to the
cultures. Further assay details are included in Supplementary data.
Compound 1 was determined to be non-mutagenic in all condi-
tions tested (Supplementary data Table 1b). All criteria for a valid
test were met. Compound 1 was determined to be cytotoxic at
5.1.25. N-(3-(hydroxycarbamoyl)phenyl)-9-oxo-9H-fluorene-1-
carboxamide (21)
To a 50 ml round bottom flask was added methyl 3-(9-oxo-9H-
fluorene-1-carboxamido)benzoate (0.05 mmol, 17.87 mg, 1 equiv),
hydroxylamine hydrochloride (0.4 mmol, 28 mg, 8 equiv) and
20 mL MeOH. After the dropwise addition of KOH/MeOH (1 M,
0.5 mL), the mixture was refluxed overnight. Upon reaction com-
pletion monitored by TLC, the methanol was evaporated. The mix-
ture was diluted in CHCl3 and yellow hydrolyzed products
precipitated. After filtration, the filtrate was collected and the crude
product was purified by chromatography (1% MeOH/CHCl3) to give
the desired product as white solid in 78% yields. 1H NMR (DMSO-d6,
300 MHz): d 12.59 (s, 1H), 10.70 (s, 1H), 8.48 (t, J = 1.6 Hz, 1H), 8.35
(d, J = 7.3 Hz, 1H), 7.89–7.98 (m, 3H), 7.67 (dt, J = 7.7, 1.4 Hz, 1H),
7.38–7.54 (m, 5H). LCMS m/z [M+15] 373.17.
62.5
at 62.5
l
g/ml and was determined to have some genotoxic activity
g/ml (Supplementary data Fig. 1 and Table 1a). The
l
positive and negative controls were within expected ranges. Com-
pound 1 had a doubling in the number of revertants in one dose
with tester strain TAMix with metabolic activation. There was no
dose response trend and the doubling was within historical nega-
tive control data range.
5.4. Docking
The lead compound (1) and its derivatives (2–22) were docked
to a crystal structure of edema factor 1K90.pdb (resolution 2.75 Å,
r-value 0.225) with AutoDock3.0.5. The ‘Lamarckian’ genetic algo-
rithm (LGA) was used for all docking. The number of iterations was
set to 200 and population size to 100. The ligand and solvent mol-
ecules were removed from the crystal structure to obtain the dock-
ing grid and the active site was defined using AutoGrid. The grid
size was set to 90 ꢀ 90 ꢀ 90 points with grid spacing of 0.375 Å.
The grid box was centered on the center of the 3d0ATP from the
corresponding crystal structure complexes. The Yb3+ ion in the ac-
tive site coordinates carboxyl groups of residues Asp491, Asp493
5.1.26. N-(3-carbamimidoylphenyl)-9-oxo-9H-fluorene-1-
carboxamide (22)
Synthesized from 9-fluorenone-1-carboxilic acid and the corre-
sponding amine according to the general procedure described
above for amide coupling. Upon reaction completion, the reaction
solvent was evaporated and CH2Cl2 was added. The mixture was
acidified by 2 N HCl. Then the water layer was separated and sat
at room temperature for a certain time during which yellow pre-
cipitates formed. The solid was filtered and washed with H2O,
CH2Cl2 to give the desired product in 60% yield. 1H NMR (DMSO-
d6, 300 MHz): d 10.58 (s, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.84 (d,
J = 7.4 Hz, 1H), 7.57–7.70 (m, 3H), 7.19–7.40 (m, 4H), 6.94 (d,
J = 6.6 Hz, 1H). LCMS m/z [M+1] 342.16.
and His577 (Yb-N:2.78 Å) and an oxygen atom of the a-phosphate
group of the 30dATP ligand. For all the dockings using the 1K90
crystal structure, Yb3+ was replaced with the more physiological
Mg2+ at the same position, which allowed better comparison of
the energy data.
Acknowledgments
5.2. General pAssay for inhibition of EF
Support for this project was from NIAID grant U01AI5385802,
and from Mission Pharmacal, San Antonio TX., and in part by grant
1UL1RR029876-01 from the National Center for Research Re-
sources, NIH to the Institute for Translational Studies of the UTMB,
(to CHS for pilot #809). The computational facilities of the Sealy
Center for Molecular Biophysics and Structural Biology were used
for this project.
Inhibition of EF was done as previously described6, by treating
murine monocyte/macrophage cells (RAW 264.7) with PA
(2.5 lg/ml) and EF (0.625 lg/ml) in the presence of DMEM med-
ium or compounds initially dissolved in DMSO and diluted in
DMEM (w/o phenol red) containing isobutylmethylxanthine
(IBMX). Experiments included controls for DMSO and IBMX. Plates
were then incubated for 4 h at 37 °C in 5% CO2. Following incuba-
tion, the culture supernatants were removed and assayed for cAMP
with a cAMP-specific ELISA from Assay Designs, Inc. (Ann Arbor,
Michigan) per manufacturer directions.
Supplementary data
Supplementary data (Table 1a: Greenscreen assay description
and results; Table 1b: AmesII mutagenicity assay results; Table
2s: Table 1 reordered according to activity of the compound, illus-
trating the lack of correlation between absolute docking scores and
activity.) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2011.10.091.
5.3. Assays for toxicity and mutagenicity of Compound 1
(GADD45a-GFP, GreenScreen HC and the AMES IIT Mutagenic-
ity) were performed under Good Laboratory Practice (GLP) by BioR-
eliance (Rockville, MD).29 The genotoxicity potential of 1 was
assessed using the Green Screen HC [GADD45
a-GFP [Human Cells
References and notes
(HC) GADD45 (growth arrest and DNA damage gene)-Green Fluo-
a
1. Migone, T. S.; Subramanian, G. M.; Zhong, J.; Healey, L. M.; Corey, A.;
Devalaraja, M.; Lo, L.; Ullrich, S.; Zimmerman, J.; Chen, A.; Lewis, M.; Meister,
G.; Gillum, K.; Sanford, D.; Mott, J.; Bolmer, S. D. N. Engl. J. Med. 2009, 361, 135.
2. Sweeney, D. A.; Cui, X.; Solomon, S. B.; Vitberg, D. A.; Migone, T. S.; Scher, D.;
Danner, R. L.; Natanson, C.; Subramanian, G. M.; Eichacker, P. Q. J. Infect. Dis.
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Compositions to Inhibit Edema Factor and Adenylyl Cyclase, 2011) as FIV-50; it
was also called DC-5 in our earlier publications. The names for other
compounds are: 4: FIV-61; 5: FIV-64; 6: FIV-72; 7: FIV-73; 8: FIV-39; 9: FIV-
35; 10: FIV-75; 11: FIV-59; 12: FIV-55; 13: FIV-71; 14: FIV-53; 15: FIV-54; 16:
FIV-58; 17: FIV-66; 18: FIV-67; 19: FIV-65; 20: FIV-60; 21: FIV-46; 22: FIV-68.