Structure-based design, synthesis, and study of potent inhibitors of β-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents

Article abstract of DOI:10.1021/jm049141s

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, β-ketoacyl-ACP synthase III, is a particularly attractiv

Full text of DOI:10.1021/jm049141s

1596
J. Med. Chem. 2005, 48, 1596-1609
Structure-Based Design, Synthesis, and Study of Potent Inhibitors of
â-Ketoacyl-acyl Carrier Protein Synthase III as Potential Antimicrobial Agents
Zhe Nie,*,† Carin Perretta,† Jia Lu,‡ Ying Su,§ Stephen Margosiak,‡ Ketan S. Gajiwala,§ Joseph Cortez,‡
Victor Nikulin,† Kraig M. Yager,† Krzysztof Appelt, and Shaosong Chu*,†
Departments of Medicinal Chemistry, Protein Biochemistry and Structural Biology, Quorex Pharmaceuticals, Inc.,
1890 Rutherford Road, Suite 200, Carlsbad, California 92008
Received October 26, 2004
Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic
pathway have been identified as attractive targets for the development of new antibacterial
agents. FabH, â-ketoacyl-ACP synthase III, is a particularly attractive target, since it is central
to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and
-negative bacteria. Small molecules that inhibit FabH enzymatic activity have the potential to
be candidates within a novel class of selective, nontoxic, broad-spectrum antibacterials. Using
crystallographic structural information on these highly conserved active sites and structure
based drug design principles, a benzoylaminobenzoic acid series of compounds was developed
as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity
against Gram-positive and selected Gram-negative organisms.
Introduction
Fatty acid biosynthesis (FAB) is an essential meta-
bolic process for prokaryotic organisms and is required
for cell viability and growth.1 Targeting this pathway
consequently represents a reasonable approach for
developing new antibacterial agents. Since many of
today’s nosochomial bacterial infections are resistant to
Figure 1. FabH-catalyzed initiation reaction of fatty acid
biosynthesis.
several of the available antibiotics, compounds targeting
1).5 The other bacterial condensing enzymes FabB and
the FAB pathway could fill a serious medical need.
FabF, functioning later in the cycle, differ significantly
Large multifunction proteins termed type I fatty acid
from FabH in that they use acyl-ACP rather than acetyl-
synthases catalyze these essential reactions in eukary-
CoA as the primer for subsequent condensations and
otes.2 In contrast, bacteria use multiple enzymes to
are hence nonredundant. As a result, no other known
accomplish the same goal and are referred to as type
enzyme in the pathway appears to be able to accomplish
II, or dissociated, fatty acid synthases.3 The bacterial
this essential reaction, and thus, FabH appears to play
system and proteins bear little homology to the human
a key role in the bacterial FAB cycle. FabH proteins
system and therefore represent a set of attractive target
from both Gram-positive and -negative bacteria are
proteins for novel antibacterial development. Indeed,
highly conserved at the sequence and structural level
this pathway has been targeted previously by both
while there are no significantly homologous proteins in
natural products (e.g., cerulenin and thiolactomycin)
humans. Importantly, the residues that comprise the
and the synthetic derivatives (e.g., triclosan, isoniazid,
active site are essentially invariant among Gram-
and the diazoborines) that inhibit the condensation
positive and -negative organisms. These attributes
enzymes and the enoyl-ACP reductase activity of the
suggest that small molecule inhibitors of FabH enzy-
FabI gene product, respectively. Unfortunately, these
matic activity could be potential development candidates
agents and/or associated targets have various shortcom-
leading to selective, nontoxic, and broad-spectrum an-
ings and are of limited usefulness in the development
tibacterials.
of broad-spectrum antimicrobials.4
Herein we describe the structure-based drug design
A key enzyme responsible for initiation of bacterial
approach that facilitated generation of potent FabH
fatty acid biosynthesis has so far escaped serious
inhibitors with strong antibacterial activity against
attention by the drug discovery industry. FabH, a
Gram-positive and selected Gram-negative organisms.
This process was initiated using proprietary computa-
â-keto-acyl-ACP synthase, is the bacterial condensing
enzyme in Gram-positive and -negative bacteria that
tional methods to pare down the more than 3 million
initiates the FAB cycle by catalyzing the first condensa-
commercial chemicals available from reputable com-
tion step between acetyl-CoA and malonyl-ACP (Figure
mercial sources to generate a FabH-directed compound
library. These sets of compounds were screened in
* Corresponding author. Current address: Phoenix Pharma-
enzymatic assays to generate leads that were cocrys-
tallized with various pathogenic FabH proteins and
subsequently optimized using structure-guided drug
design methods.
cologics, Inc. Phone: 858-452-6688 ext 109. E-mail: zhenie@
phoenixpharmaco.com.
† Medicinal Chemistry.
‡ Protein Biochemistry.
§ Structural Biology.
10.1021/jm049141s CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/12/2005

Inhibitors of FabH as Antimicrobial Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1597
Scheme 1a
a Reagents: (a) ClSO3H, 65-140 °C; (b) HNEt2, EtOAc; (c) (i) SOCl2, DMF (cat.), reflux; (ii) methyl anthranilate, pyr; (iii) NaOH,
THF/MeOH; (d) PhB(OH)2, Pd(PPh3)4, aq Na2CO3, DME, reflux; (e) amine or alcohol, K2CO3 or NaH, DMF, rt or heat.
In Silico Selection of a FabH-Directed
Compound Library
Chemistry
Syntheses of diethylsulfonamide derivatives (13a-h)
began from commercially available substituted benzoic
acids or benzoyl chlorides (1-4) using procedures
described in the literature (Scheme 1).16 As expected,
p-bromo derivatives (1 and 2) and p-fluoro derivative 4
required much higher temperatures (140 °C vs 65 °C)
to achieve chlorosulfonylation as compared to p-methoxy
derivative 3. Subsequent reaction with diethylamine
provided diethylsulfonamide derivatives (9-12). The
desired 3-diethylsulfamoylbenzoylaminobenzoic acids
(13a, 13c, 13d, and 13e) were then obtained by treating
carboxylic acids 9-12 with thionyl chloride and catalytic
DMF at reflux, followed by reaction with methyl an-
thranilate in pyridine and subsequent ester hydrolysis.
Other activation-coupling strategies with these inter-
mediates were unsuccessful and this efficient, albeit
three-step, sequence became the standard procedure for
making the amide bond of the benzoylaminobenzoic acid
series. The phenylsulfonamide 13b was obtained by
Suzuki17-type palladium-mediated coupling between
bromosulfonamide 13a and phenylboronic acid. Using
fluorosulfonamide 13e as a common intermediate, al-
cohols and amines were used to readily displace the
fluoro group via nucleophilic aromatic substitutions to
provide compounds 13f-h.
Target-based approaches were used for selecting the
initial compound library destined for screening. To apply
target-based selection criteria, all available information
concerning FabH was collected. This knowledge was
then translated into various search queries in order to
mine chemical databases for compounds with an in-
creased potential to bind the FabH active site. Specif-
ically, known inhibitors,6-8 substrates, and the active
site topology from available FabH crystal structures9-12
were used to build FabH-specific queries. Compound
selection proceeded via searches of substructure, 2D
similarity, phamacophore with size and shape con-
straints, and docking with FlexX (Tripos, Inc.). The
pharmacophore models describe specific areas for de-
sired protein-ligand interactions and limit the molec-
ular volume to be appropriate for the FabH active site.
The commercial compound database was prefiltered to
remove members that did not satisfy Lipinski’s guide-
lines13 and that contained undesirable groups that could
potentially generate false positives due to reactivity and
aggregation.14,15 After applying these criteria and meth-
ods, ∼2500 compounds from the prefiltered commercial
database were selected for initial FabH inhibition
screening.
Using our standard procedure, 2-(3-phenoxybenzoyl-
amino)benzoic acid (15) was synthesized from com-
mercially available 3-phenoxybenzoic acid (Scheme 2).
Originally, the synthesis of 2-(3-phenoxybenzoylamino)-
benzoic acids with B ring para substitutions was ac-
complished by assembling the A and B rings, followed
by reaction of the phenolate (generated in situ with
NaH) with bromo benzene to install the C ring. Al-
though operable, the yields were usually poor and the
synthetic sequences were lengthy. Fortunately, develop-
ments in arylboronic acid coupling reactions allow the
formation of diaryl ethers in high yields at room
temperature through copper-catalyzed coupling of aryl-
boronic acids and phenols.18 Accordingly (Scheme 2),
phenol 16 was brominated to provide the desired
intermediate 18 as the major product after purification
by flash column chromatography. Copper-catalyzed
O-arylation of compounds 17 and 18 with phenylboronic
acid proceeded smoothly with 1 equiv of Cu(OAc)2 and
excess base in CH2Cl2. Powdered 4 Å molecular sieves
were added to the reaction mixture to quench any water
generated during the reaction. After alkaline hydrolysis,
compounds 19 and 20 underwent standard coupling
Biological Methods
The potency of FabH inhibition (IC50) was determined
using [3H]- or [14C]-radiolabeled substrates. This was
accomplished at fixed concentrations of acetyl-CoA in a
coupled FabD/FabH assay system that generates the
substrate malonyl-ACP in situ. Subsequently, the FabH
acetylation reaction was performed to exclude the
possible contribution of undesired FabD inhibitory
activity. Compounds with an IC50 < 10 µM and good
aqueous solubility were included in cocrystallization
trials with Entercoccus faecalis and Haemophilus in-
fluenzae FabH proteins and the derived protein-inhibi-
tor cocrystal structures were used to further optimize
the inhibitors.
Biochemical screening of the 2500 select compound
library resulted in 27 hits with IC50 < 10 µM. Of the
four structurally diverse series identified, the benzoyl-
aminobenzoic acid series of compounds was chosen for
optimization. The MIC (minimum inhibitory concentra-
tion) of sufficiently potent compounds was assessed
using both Gram-positive and -negative pathogenic
bacteria.

1598 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Nie et al.
Scheme 2a
a Reagents: (a) (i) SOCl2, DMF (cat.), reflux; (ii) methyl anthranilate, pyr; (iii) NaOH, THF/MeOH; (b) Br2, HOAc; (c) (i) PhB(OH)2,
Cu(OAc)2, TEA, 4 Å sieves, CH2Cl2; (ii) NaOH, THF/MeOH; (d) (i) SOCl2, DMF (cat.), reflux; (ii) methyl anthranilate, pyr; (e) NaOH,
THF/MeOH; (f) (i) amines, DMSO, 120 °C; (ii) NaOH, THF/MeOH; (g) (i) ArB(OR3)2, Pd(PPh3)4, aq Na2CO3, DME, reflux; (ii) NaOH,
THF/MeOH.
procedures to yield 21 and 22. Using the fluoro- or
bromobenzoylaminobenzoic acid derivative, the B ring
4-position could be substituted with secondary amines
and aromatic rings, respectively. Due to the electron-
donating o-phenoxy group, nucleophilic replacement of
the fluoro group with amines was sluggish and required
harsh conditions. Nevertheless, compounds 23c-i were
obtained from 21 by heating with an excess of amine in
DMSO (120-140 °C). Aromatic substitutions were ac-
complished by palladium-mediated coupling of 22 with
commercially available arylboronic acids or esters to
yield compounds 24a-p.
Derivatives having various A ring systems (com-
pounds 28a-f) were obtained via the standard proce-
dure and the appropriate aromatic amines (Scheme 3).
For C ring modification, the anion of methyl 3-hydroxy-
benzoate (16), generated with NaH (1 equiv) was heated
with 4-chloropyridine in dry DMF, which after the
saponification of the ester gave acid 25. Coupling as
described previously afforded 2-[3-(pyridin-4-yloxy)ben-
zoylamino]benzoic acid (28g). Compounds 28h-j were
obtained by condensing the appropriate substituted
sodium phenolate with aromatic bromide 27 in the
presence of catalytic CuCl.
hydrolysis with LiOH provided 30. Cleavage of the
methyl ether group by boron tribromide gave compound
31. Compound 33 was obtained by Suzuki coupling of
30 with phenyl boronic acid followed by demethylation.
Results and Discussion
Cocrystallization studies of the benzoylaminobenzoic
acid series of compounds resulted in a 2.6 Å resolution
cocrystal structure of 13a bound to full length E. faecalis
FabH. Key features observed for ligand binding include
an ionic interaction between the negatively charged
carboxylate oxygen of 13a and the γ-NH of the proto-
nated imidazole ring of His250 (Figure 2). Additionally,
both carboxylate oxygens of 13a are involved in hydro-
gen bonding to the side chain NH2 of Asn280. Together,
these interactions define the orientation of the inhibitor
within the active site, thus serving as the inhibitor
“anchor”. The remainder of the molecule aligns along
the active site in an extended conformation, making
hydrophobic interactions with protein residues nearby.
In particular, Phe312 makes van der Waals (VDW)
interactions with the inhibitor A ring (distance 3.3 Å).
The p-bromide occupies a hydrophobic pocket created
by residues Trp38, Ile39, Ile45, Val160, and Leu161
(Figure 2C), while the diethyl-sulfonamide portion of the
inhibitor is largely solvent exposed and surrounded by
several arginine and lysine residues (Figure 2D).
On the basis of information derived from the cocrystal
structure of 13a, our initial focus was to explore the
pocket opening accessed by the B ring para position.
Compounds 31 and 33 were synthesized according to
Scheme 4. Using the general procedure for amide
coupling, 20 was treated with SOCl2 followed by react-
ing with 2-amino-6-methoxybenzoic acid in pyridine.
However, instead of giving the desired amide product
directly, the benzooxazinone 29 was formed, which upon

Inhibitors of FabH as Antimicrobial Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1599
Scheme 3a
a Reagents: (a) NaH, CuCl, pyr/DMF, chloropyridine hydrochloride, 120 °C; (b) (i) SOCl2, DMF (cat.), reflux; (ii) aromatic amines, pyr;
(iii) hydrolysis: NaOH, THF/MeOH; or demethylation: BBr3, CH2Cl2; (c) (i) SOCl2, DMF (cat.), reflux; (ii) methyl anthranilate, pyr; (d)
(i) phenols, NaH, CuCl, pyr, 120 °C; (ii) NaOH, THF/MeOH.
Scheme 4a
a Reagents: (a) (i) SOCl2, DMF (cat.), reflux; (ii) 2-amino-6-methoxybenzoic acid, pyr; (b) LiOH, THF; (c) BBr3, CH2Cl2; (d) PhB(OH)2,
Pd(PPh3)4, aq Na2CO3, DME, reflux.
Modeling studies predicted improved binding affinity by
occupying the hydrophobic space and/or by forming π
stacking interactions with surface Trp38. To explore this
possibility, a set of para-substituted derivatives related
to 13a was synthesized. However, none of these deriva-
tives showed better activity than the parent compound
13a (Table 1).
A more in-depth conformational analysis suggests
that the orientation of the sulfonamide group is influ-
enced by these ortho substituents and as such disfavors
the optimal conformation observed in the cocrystal
structure. Clearly, combinations of the rather large and
rigid sulfonamide group with ortho substituents would
be problematic during optimization. These complica-
tions, along with the instability of the sulfonamide group
to liver microsomes (data not shown), prompted the
search for a suitable replacement. Modeling studies
suggested that replacement of the sulfonamide with a
phenoxy group might provide interaction with Phe224.
To this end, compound 15 was synthesized and dem-
onstrated similar activity (IC50 ) 2.7 µM) against E.
faecalis FabH, proved the principle of this design
strategy.
Using compound 15 as our new lead, optimization of
substituents on the B ring was pursued. The cocrystal
structure of 13a clearly indicated that the B ring and
its amide bond needs to be coplanar, which obviates
substitutions ortho to the amide. The meta carbon on
the B ring is close to the side chain of Leu161 (4.2 Å);
here even a small methyl group at this position resulted
in a 100-fold loss in inhibition activity (13c, Table 1).
The position para to the amide bond, however, offered
a good vector toward an amphiphilic pocket formed by
Trp38, Ile39, Ile45, Val160, and Leu161 at one side and
Arg42 at the other side (Figure 2C). In a recently solved
acetyl-CoA-FabH cocrystal structure (data not shown,
but similar to the published Escherichia coli structure
with CoA),9 we observed that the size of this pocket had

1600 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Nie et al.
Figure 2. Compound 13a (IC50 ) 1.6 µM) bound in the active site of E. faecalis FabH. (A) Green molecular surface shows the
active site cleft in which compound 13a binds. (B) Details of protein-13a interactions. Key side chains lining the binding pocket
are shown. The carboxylate of the compound 13a makes hydrogen-bonding interactions with His250 and Asn280. (C) The bromine
atom points toward a pocket lined with hydrophobic residues: Ile39, Ile45, Val160, and Leu161. (D) The sulfonamide group is
located on the exposed face of the protein that is lined with basic residues.
Table 1. Diethylsulfonamide Derivatives as Inhibitors of FabH
modeling to exclude substituents that might insult
highly conserved protein residues, a set of compounds
(23c-i, 24a-c, Table 2) with diverse rings at the
4-position was prepared. Replacement of the proton of
15 with a simple phenyl group (24c, Table 2) resulted
in dramatic ca. 50-fold improvement in potency (IC50
)
2.7 µM vs 0.056 µM). Charged hydrophilic groups at the
para position were not tolerated and resulted in signifi-
cant decreases in potency (23c, 23d, 23e, and 24a) (IC50
> 20 µM). Consistent with these results, the contour of
an electrostatic map (ZAP, Tripos, Inc.) suggested that
positively charged or partially positively charged groups
in this pocket would be disfavored. Compounds with
weakly basic groups at this position, such as 23h and
23i, showed 10-fold improved binding (IC50 ) 0.29 and
0.27 µM, respectively). A complexed structure of 23i
with FabH (Figure 3) ultimately revealed the nature of
the pocket and its interactions with para substituents.
The dimethylpiperidine ring establishes hydrophobic
interactions with the adjacent protein side chains and
accounts for the observed 10-fold improvement in po-
tency. The 50-fold improvement observed for compound
24c is likely due to a combination of factors: (1) the
phenyl group occupies this hydrophobic space; (2) it also
forms stacking interactions with the indole ring of
Trp38; (3) intramolecular stacking interactions between
the two adjacent aromatic rings on the B ring confor-
mationally preorganized the two rings, leading to favor-
able interactions with the protein active site.
increased due to side chain movements of Trp38 and
Arg42 likely induced by interaction of the CoA’s adenine
ring with the protein. This protein side chain mobility
hampered our efforts to design a specific para functional
group to fit into the binding pocket. Therefore, a more
traditional approach was used to establish structure-
activity relationships in this area. Using molecular
Encouraged by the 24c result, a series of compounds
with different substitutions on the distal arene (Table
3) was synthesized. Most of these compounds show sub-
micromolar IC50 values, the best one being 24i (IC50
)
28 nM). One possible explanation for the rather flat SAR
in this region is that the stronger stacking interaction

Inhibitors of FabH as Antimicrobial Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1601
Table 2. [3-Phenoxybenzoylamino]benzoic Acid Derivatives as
Inhibitors of FabH: Para-Substitutions on the B Ring
Figure 3. Compound 23i (IC50 ) 0.27 µM) bound in the active
site of E. faecalis FabH. (A) Green molecular surface shows
the active site cleft in which compound 23i binds. (B) Details
of protein-23i interactions. Key side chains lining the binding
pocket are shown. The Cys117 side chain is found to be
acetylated in this crystal structure.
Table 3. Aromatic Substitutions on the Para Position of
[3-Phenoxybenzoylamino]benzoic Acid Derivatives
E. faecalis
E. faecalis
FabH
IC50 (µM)
FabH
compd
R
IC50 (µM) compd
R
24c
24d
24e
24f
24g
24h
24i
H
0.056
0.096
0.16
2.1
0.41
0.22
0.028
24j
24k
24l
24m
24n
24o
24p
3-iPr
0.79
0.47
0.24
0.57
0.33
0.25
0.16
4-CF3
3-OCF3
4-Me
4-CO2H
4-OH
4-OEt
4-SO2Me
3-Me-4-F
3-Cl-4-F
3,4-di-F
3-Me-4-Cl
2,4-di-F
resulted in organization of the flexible region around
Trp38; thus, the entropic energy cost may cancel out
any stacking energy gain. This rationale is supported
by the crystal structures of 13a and 23i (Figure 4),
which when overlayed reveals the changes in side chain
conformation by Arg42 and Trp38, although the main
chain is essentially invariant. FabH cocrystal structures
from this series (24a-p) are awaited to confirm the
hypothesis.
The A ring is located deep in the active site pocket and
is oriented to provide optimal ionic interactions between
the ligand COO- and the protonated imidazole side
chain of His250. Intramolecular neutralization of the
carboxylate group by changing the benzene ring to a
pyridine ring (28c and 28d) diminishes the inhibition
activity. Additionally, a thiophene ring at this position
did not improve the inhibitor potency (28a and 28b)
either. In addition to the interaction between the
aromatic COOH and the protein residues, the aromatic
Concurrent with exploration of the hydrophobic pocket
occupied by the para-position substituent of the B ring
was the optimization of the A and C rings (Table 4).

1602 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Nie et al.
Figure 5. Model of compound 28f bound to E. faecalis FabH.
The interaction between the hydroxyl group and the carbonyl
oxygen of Phe312 is likely responsible for the 7-fold improve-
ment in the binding affinity.
Figure 4. Flexibility of the side chains on the protein surface.
Trp38 and Arg42 side chains adopt different conformations
in the 23i bound structure (shown in atom colors) as compared
with 13a bound structure (shown in monochrome green).
Table 5. Potent Inhibitors of FabH: Effect of Adding OH
Ortho to the Carboxylic Acid
Table 4. [3-Phenoxybenzoylamino]benzoic Acid Derivatives as
Inhibitors of FabH: Modifications of Ring A and C
E. faecalis
FabH
E. faecalis
FabH
IC50 (µM)
compd R1 R2 IC50 (µM) compd R1
R2
15
23b
24c
H
Br
Ph
H
H
H
2.7
28f
31
33
H
OH
0.41
0.062
0.004
1.1
Br OH
Ph OH
0.056
remained an open question. Models based on the coc-
rystal structure of 23i suggested that an o-OH group
would be positioned to form a 3.1 Å hydrogen-bonding
interaction with the backbone carbonyl of Phe312
(Figure 5). To test this theory, compound 28f (IC50
)
0.41 µM) was made and indeed showed improved
binding (∼7-fold) as compared to nonhydroxylated com-
pound 15 (IC50 ) 2.7 µM). The same trend was observed
upon comparison of 23b with 31 (IC50 ) 1.1 and 0.062
µM) and 24c with 33 (IC50 ) 0.056 and 0.004 µM), as
shown in Table 5. Adding an o-OH does provide ad-
ditional potency, which makes compound 33 the most
potent compound in the series so far.
Ring C of the inhibitors is located on the surface of
the active site. Besides a few flexible arginine residues,
there is a phenylalanine residue (Phe224). Compounds
28g-j were synthesized with the aim to accomplish one
or both of the following objectives: (1) to strengthen π-π
stacking interaction and (2) to form ionic interactions
with positively charged surface arginine residues. How-
ever, none of these compounds showed improved bind-
ing. Analysis from the cocrystal structure of compound
23i and the enzyme (Figure 2) suggested that the
relative positioning of the benzene C ring to arene of
Phe224 does not provide an optimal π-π stacking
interaction. Interactions with solvent-exposed arginines
do not provide further improvement in binding affinity.
To generate data concerning the broad spectrum
potential of these compounds, the IC50 values of selected
compounds against FabH enzymes from different patho-
gens were determined (Table 6). Although this series is
highly potent against E. faecalis and Streptococcus
pyogenes FabH (IC50 < 100 nM), they only show moder-
ring is also surrounded by a tight array of hydrophobic
residues including Leu194, Ile223, Phe226, Ala227,
Ala252, and Phe312. Whether any substitution ortho
to the COOH on the benzene ring would be tolerated

Inhibitors of FabH as Antimicrobial Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1603
Table 6. Cross-Genus Inhibitor Activity
IC50 (µM)
broad-spectrum antibacterial activity lies in a detailed
understanding of the FabH active sites. Toward this
end, new structural data is guiding further modifica-
tions of the current series with the hopes of improving
both enzymatic inhibition and physical properties.
compd E. faecalis S. pyogenes S. aureus H. influenzae
24c
24i
31
0.056
0.028
0.062
0.004
0.05
10
> 100
> 100
20
0.003
0.034
0.004
24
12.3
3.8
Experimental Section
33
2.4
General Chemistry Methods. Proton NMR spectra were
collected at 300 MHz on a Bruker Avance 300 spectrometer,
and chemical shifts are reported in parts per million (δ)
downfield from tetramethylsilane as internal standard. At-
mospheric pressure ionization electrospray mass spectra and
LCMS were recorded on an Agilent 1100 Series LC/MSD-SL
1946D spectrometer equipped with an Agilent 100 series
HPLC system. Silica gel 60 (230-400 mesh) from EM Science
was used for column chromatography, and analytical or
preparative thin-layer chromatography was conducted using
EM Science Kieselgel 60 F254 plates. An Agilent 1100 Series
HPLC with an Agilent Zorbax Eclipse XDB-C8 (4.6 × 150 mm)
reversed phase column was used for analytical HPLC analyses.
Preparative RP-HPLC was performed on a Gilson instrument
with a MetaSil AQ 10 µm C18 column. The elution buffer was
an A/B gradient, where A ) H2O-0.1% TFA and B ) ACN-
0.1% TFA. For reactions performed under anhydrous condi-
tions, glassware was either oven- or flame-dried and the
reaction was run under a positive pressure of nitrogen.
Anhydrous solvents were used as purchased from commercial
sources. Except where noted, reagents were purchased from
commercial sources and used without further purification. The
reported yields are the actual isolated yields of purified
material and are not optimized.
4-Bromo-3-diethylsulfamoylbenzoic Acid (9). Repre-
sentative Procedure for 9-12. A mixture of 4-bromobenzoyl
chloride (1, 1 g, 4.56 mmol) in chlorosulfonic acid (3 mL) was
heated to 140 °C for 4 h, after which time HPLC indicated
complete conversion of the starting material to the intermedi-
ate, 4-bromo-3-chlorosulfonylbenzoic acid (5). The reaction
mixture was slowly dripped over ice and filtered. The solid
was dried in vacuo to yield the intermediate (5, 954 mg, 70%),
which was carried forward without further purification. 4-Bro-
mo-3-chlorosulfonylbenzoic acid (5, 954 mg, 3.18 mmol) was
dissolved in EtOAc (20 mL), and diethylamine (0.99 mL, 9.54
mmol) was then added. After stirring at room temperature for
24 h, the reaction mixture was concentrated. The resulting
residue was dissolved in H2O and extracted with EtOAc and
3 N HCl. The combined organic layers were dried over
anhydrous MgSO4 and concentrated in vacuo to yield the
4-bromodiethylsulfamoylbenzoic acid intermediate (9, 413 mg,
39%).
Table 7. Minimum Inhibitory Concentrations (MIC) for Select
Benzoylaminobenzoic Acid FabH Inhibitorsa
MIC (µg/mL)
compd
Sa
Spn
Spy
Ef
Nm
Ec
23i
24c
24d
24i
24j
24l
24o
24p
31
5.6
11.3
5.6
45
2.8
1.4
0.7
45
2.8
ND
2.8
22
2.8
2.8
1.4
2.8
11.3
5.6
11.3
22
2.8
1.4
1.4
5.6
1.4
0.7
0.7
0.36
2.8
1.4
5.6
1.4
0.7
5.6
2.8
0.7
0.7
0.7
2.8
1.4
5.6
>45
5.6
5.6
5.6
5.6
45
1.4
0.7
2.8
2.8
45
5.6
2.8
5.6
11.3
>45
>45
33
22
22
a Sa ) S. aureus MRSA 703; Spn ) S. pneumoniae ATCC 6301;
Spy ) S. pyogenes NZ131; Ef ) E. faecalis VRE 583; Nm )
Neisseria meningitides MC58 (+PMB); Ec ) E. coli TolC-
(+PMB).
ate activity (IC50 ) 4-24 µM) against Staphylococcus
aureus and essentially lack significant activity against
H. influenzae FabH (IC50 ) 2.5f100 µM). Clearly there
is a structural difference in the active sites of these
latter proteins that amino acid sequence conservation
and alignments are not revealing. To provide this
important data, crystal structures were solved for S.
aureus and H. influenzae proteins both with and without
bound inhibitor.19 These structures provided significant
insight into the structural differences, and structure-
based optimization of the compounds in the series with
broad-spectrum FabH inhibition is in progress.
Antibacterial Activity. Determination of minimum
inhibitory concentrations (MIC) for several Gram-posi-
tive and -negative pathogenic bacteria showed that most
of the potent enzyme inhibitors also possessed modest
to strong antibacterial activity against both Gram-
positive and -negative bacterium (Table 7). However,
poor activity was observed for several compounds that
are potent inhibitors of the enzyme in vitro (24i, 31, and
33). These results suggest that these compounds either
fail to penetrate the bacterial cell membranes or are
actively pumped out of the bacterial cells by efflux
pumps. These hypotheses are supported by observed
activity, albeit modest, against the E. coli TolC- mutant
in the presence of the membrane permeablizing agent
polymyxin B.
4-Bromo-3-diethylsulfamoyl-5-methylbenzoic
acid
(10): 1H NMR (DMSO-d6) δ 13.62 (br s, 1H), 8.36 (d, J ) 2.0
Hz, 1H), 8.16 (d, J ) 2.0 Hz, 1H), 4.07 (q, 4H), 2.53 (s, 3H),
1.11 (t, 6H); LCMS (API-ES) m/z 349.9, 351.9 [M + H+]; 347.9,
349.9 [M + H-].
3-Diethylsulfamoyl-4-methoxybenzoic acid (11): 1H
NMR (DMSO-d6) δ 13.14 (br s, 1H), 8.36 (d, J ) 2.2 Hz, 1H),
8.18 (dd, J ) 2.2 and 8.7 Hz, 1H), 7.37 (d, J ) 8.7 Hz, 1H),
4.01 (s, 3H), 3.27 (q, 4H), 1.04 (t, 6H); LCMS (API-ES) m/z
288.0 [M + H+].
3-Diethylsulfamoyl-4-fluorobenzoic acid (12): 1H NMR
(DMSO-d6) δ 13.55 (br s, 1H), 8.30 (m, 1H), 7.76 (m, 1H), 7.64
(m, 1H), 3.35 (q, 4H), 1.10 (t, 6H); LCMS (API-ES) m/z 276.0
[M + H+]; 274.0 [M + H-].
Conclusion
FabH is an emerging target for the development of
novel antibacterial chemotherapeutics. Structure-based
design of a series of benzoylaminobenzoic acid deriva-
tives led to the discovery of potent inhibitors of type II
fatty acid synthase III (FabH). These FabH inhibitors
demonstrated potent antibacterial activity (MIC) and
as such have the potential to be novel and potent
antibacterial agents. Given the unforeseen structural
differences within the active site of some pathogenic
FabH enzymes, the key to discovering inhibitors with
General Procedure for Amide Bond Formation of
Substituted Benzoylaminobenzoic Acid Derivatives. The
acid (1.5 equiv) was converted to the acid chloride by refluxing
in thionyl chloride with a catalytic amount of DMF. After 2-4
h, the reaction mixture was concentrated and suspended in
pyridine. The mixture was cooled to 0 °C under N2, and methyl
anthranilate (1 equiv) was added. After stirring for 24 h at
room temperature, the reaction mixture was concentrated,
neutralized with 1 N HCl, and extracted with EtOAc (3×). The
organics were washed with brine, dried over anhydrous

1604 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Nie et al.
MgSO4, and concentrated in vacuo. The methyl ester was then
hydrolyzed with 1 N NaOH in THF/MeOH (2:1). The final
compound was purified using RP-HPLC.
compound 13g (15 mg, 46%): 1H NMR (300 MHz, DMSO-d6)
δ 13.95 (br s, 1H) 12.29 (s, 1 H), 8.69 (d, J ) 7.7 Hz, 1H), 8.55
(d, J ) 2.3 Hz, 1H), 8.17 (dd, J ) 2.3 and 8.7 Hz, 1H), 8.09
(dd, J ) 1.5 and 7.9 Hz, 1H), 7.72 (t, J ) 8.1 Hz, 1H), 7.56 (m,
2H), 7.10-7.38 (m, 5H), 3.35 (q, J ) 7.1 Hz, 4H), 1.12 (t, J )
7.1 Hz, 3H); LCMS (API-ES) m/z 469.0 [M + H+]; 467.0 [M +
H-].
2-[(2-Diethylsulfamoyl-biphenyl-4-carbonyl)amino]ben-
zoic Acid (13b). A solution of 2-(4-bromo-3-diethylsulfamoyl-
benzoylamino)benzoic acid methyl ester (13a, 40 mg, 0.098
mmol), phenylboronic acid (13.1 mg, 0.108 mmol), Pd(PPh3)4
(6 mg, 0.005 mmol), and 2 M Na2CO3 (0.147 mL, 0.294 mmol)
in DME (3 mL) was refluxed under N2 for 24 h. Upon cooling
the reaction to room temperature, it was diluted with EtOAc
and washed with brine and water, dried over anhydrous
MgSO4, and concentrated in vacuo. The methyl ester was then
hydrolyzed, and the crude material was acidified with 1 N HCl
and purified using RP-HPLC to afford the final compound 13b
(12 mg, 27%): 1H NMR (300 MHz, DMSO-d6) δ 13.95 (br s,
1H), 12.40 (s, 1H), 8.74 (d, J ) 8.4 Hz, 1H), 8.60 (s, 1H), 8.25
(d, J ) 7.9 Hz, 1H), 8.12 (d, J ) 7.9 Hz, 1H), 7.75 (t, J ) 7.7
Hz, 1H), 7.62 (d, J ) 6.4 Hz, 1H), 7.45 (m, 5H), 7.30 (t, J )
7.3 Hz, 1H), 2.84 (q, J ) 6.9 Hz, 4H), 0.94 (t, J ) 7.1 Hz, 6H);
LCMS (API-ES) m/z 453.0 [M + H+]; 451.0 [M + H-].
2-(4-Bromo-3-diethylsulfamoyl-5-methylbenzoylamino)-
benzoic acid (13c) was prepared according to the general
procedure for amide bond formation: 1H NMR (300 MHz,
DMSO-d6) δ 13.95 (br s, 1H), 12.35 (s, 1H), 8.67 (d, J ) 8.1
Hz, 1H), 8.42 (s, 1H), 8.20 (s, 1H), 8.10 (d, J ) 8.0 Hz, 1H),
7.71 (m, 1H), 7.27 (m, 1H), 3.51 (q, J ) 6.9 Hz, 4H), 2.59 (s,
3H), 1.12 (t, J ) 7.0 Hz, 6H); LCMS (API-ES) m/z 470.8 [M +
H+]; 467.9 [M + H-].
2-(3-Diethylsulfamoyl-4-methoxybenzoylamino)ben-
zoic acid (13d) was prepared according to the general
procedure for amide bond formation: 1H NMR (300 MHz,
DMSO-d6) δ 14.05 (br s, 1H), 12.31 (s, 1H), 8.72 (d, J ) 8.0
Hz, 1H), 8.45 (d, J ) 2.3 Hz, 1H), 8.22 (dd, J ) 2.4 and 8.7
Hz, 1H), 8.09 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.71 (t, J ) 7.1 Hz,
1H), 7.49 (d, J ) 8.8 Hz, 1H), 7.26 (t, J ) 8.1 Hz, 1H), 4.04 (s,
3H), 3.31 (q, J ) 7.0 Hz, 4H), 1.07 (t, J ) 7.1 Hz, 6H); LCMS
(API-ES) m/z 407.0 [M + H+]; 405.0 [M + H-].
2-(3-Diethylsulfamoyl-4-fluorobenzoylamino)benzo-
ic acid (13e) was prepared according to the general procedure
for amide bond formation: 1H NMR (300 MHz, DMSO-d6) δ
13.95 (br s, 1H), 12.29 (s, 1H), 8.65 (d, J ) 7.7 Hz, 1H), 8.43
(dd, J ) 2.4 and 6.7 Hz, 1H), 8.31 (m, 1H), 8.09 (dd, J ) 1.5
and 7.9 Hz, 1H), 7.76 (m, 2H), 7.31 (t, J ) 7.9 Hz, 1H), 3.33
(q, J ) 7.0 Hz, H), 1.12 (t, J ) 7.1 Hz, 6H); LCMS (API-ES)
m/z 395.0 [M + H+]; 393.0 [M + H-].
2-(3-Diethylsulfamoyl-4-piperidin-1-ylbenzoylamino)-
benzoic Acid (13f). To a stirred solution of 2-(3-diethylsul-
famoyl-4-fluorobenzoylamino)benzoic acid (13e, 30 mg, 0.07
mmol) and piperidine (0.015 mL, 0.15 mmol) in DMF was
added potassium carbonate (28 mg, 0.2 mmol). The reaction
mixture was allowed to heat at 130 °C for 24 h. The reaction
mixture was cooled; diluted with EtOAc, H2O, and 1 N HCl;
and extracted with EtOAc (2×). Combined organic layers were
washed with brine and H2O, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude material was purified using
RP-HPLC to afford the final compound 13f (25 mg, 78%): 1H
NMR (300 MHz, DMSO-d6) δ 13.95 (br s, 1H) 12.29 (s, 1 H),
8.72 (d, J ) 7.8 Hz, 1H), 8.50 (d, J ) 2.2 Hz, 1H), 8.16 (dd, J
) 2.2 and 8.4 Hz, 1H), 8.09 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.71
(t, 1H), 7.64 (d, J ) 8.4 Hz, 1H), 7.26 (t, J ) 7.9 Hz, 1H), 3.35
(q, J ) 7.1 Hz, 4H), 3.00 (m, 4H), 1.60 (m, 6H), 1.00 (t, J ) 7.1
Hz, 3H); LCMS (API-ES) m/z 460.0 [M + H+]; 458.0 [M + H-].
2-(4-Cyclopentylmethoxy-3-diethylsulfamoylbenzoy-
lamino)benzoic Acid (13h). To a stirred solution of 2-(3-
diethylsulfamoyl-4-fluorobenzoylamino)benzoic acid (13e, 30
mg, 0.076 mmol) and cyclopentane-methanol (1 mL, 0.924
mmol) was added potassium carbonate (32 mg, 0.228 mmol).
The reaction mixture was allowed to heat at 135 °C for 24 h.
After confirming the product by LCMS, the reaction mixture
was cooled; diluted with EtOAc, H2O, and 1 N HCl; and
extracted with EtOAc (2×). Combined organic layers were
washed with brine and H2O, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude material was purified using
RP-HPLC to afford the final compound 13h (12.1 mg, 34%):
1H NMR (300 MHz, DMSO-d6) δ 12.32 (s, 1H), 8.72 (d, J )
8.3 Hz, 1H), 8.46 (d, J ) 2.3 Hz, 1H), 8.19 (dd, J ) 2.3, 8.7 Hz,
1H), 8.09 (dd, J ) 1.4 and 7.9 Hz, 1H), 7.70 (dt, J ) 1.5 and
7.8 Hz, 1H), 7.48 (d, J ) 8.8 Hz, 1H), 7.25 (t, J ) 7.9 Hz, 1H),
4.13 (d, J ) 7.2 Hz, 2H), 2.54 (overlap with DMSO, 4H), 1.86
(m, 1H), 1.63 (m, 4H), 1.43 (m, 1H), 1.28 (m, 1H), 1.07 (t, J )
7.1 Hz, 6H), 0.90 (m, 1H); LCMS (API-ES) m/z 475.0 [M +
H+]; 473.0 [M + H-].
2-(3-Phenoxybenzoylamino)benzoic acid (15) was pre-
pared according to the general procedure for amide bond
formation: 1H NMR (300 MHz, DMSO-d6) δ 12.28 (s, 1H), 8.69
(d, J ) 8.3 Hz, 1H), 8.08 (d, J ) 7.9 Hz, 1H), 7.74 (t, J ) 7.4
Hz, 1H), 7.68 (d, J ) 4.7 Hz, 1H), 7.64 (d, J ) 7.7 Hz, 1H),
7.57 (s, 1H), 7.48 (t, J ) 7.8 Hz, 2H), 7.32 (dd, J ) 2.4, 7.8 Hz,
1H), 7.25 (t, J ) 7.4 Hz, 2H), 7.14 (d, J ) 8.5 Hz, 2H); LCMS
(API-ES) m/z 334.0 [M + H+]; 332.0 [M + H-].
Methyl 4-Bromo-3-hydroxybenzoate (18). To methyl
3-hydroxybenzoate (16, 10 g, 66 mmol) in acetic acid (40 mL)
was added bromine (3.4 mL, 66 mmol) dropwise. After stirring
at room temperature for 4 h, the reaction mixture was diluted
with water and extracted three times with EtOAc. Organics
were dried over anhydrous MgSO4. Purification by flash
column chromatography (10% EtOAc/hexane) provided 18 as
a white solid (6.8 g, 44%): 1H NMR (300 MHz, DMSO-d6) δ
10.78 (s, 1H), 7.67 (d, J ) 8.2 Hz, 1H), 7.56 (d, J ) 2.0 Hz,
1H), 7.33 (dd, J ) 2.0 and 8.2 Hz, 1H), 3.86 (s, 3H); LCMS
(API-ES) m/z 228.9, 230.9 [M + H-].
4-Fluoro-3-phenoxybenzoic Acid (19). A mixture of
4-fluoro-3-hydroxybenzoic acid (17, 641 mg, 4.0 mmol), phe-
nylboronic acid (975 mg, 8 mmol), copper acetate (726.6 mg, 4
mmol), TEA (2.78 mL, 20 mmol), and 4 Å molecular sieves in
DCM (40 mL) was stirred at room temperature for 2 days.
Once the reaction was complete, the reaction mixture was
filtered and the filtrate concentrated in vacuo. The resulting
residue was diluted with EtOAc and acidified with 1 N HCl.
The aqueous layer was extracted two times with EtOAc, and
the organics were washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. Purification by flash
column chromatography (20% hexane/EtOAc with 0.1% MeOH)
provided the acid (19, 300 mg, 32%): 1H NMR (300 MHz,
DMSO-d6) δ 7.77 (m, 1H), 7.61 (d, J ) 8.4 Hz, 1H), 7.39 (m,
3H), 7.20 (t, J ) 6.6 Hz, 1H), 7.04 (d, J ) 8.0 Hz, 2H); LCMS
(API-ES) m/z 231.1 [M + H-].
2-(3-Diethylsulfamoyl-4-phenoxybenzoylamino)ben-
zoic Acid (13g). To a stirred solution of phenol (14 mg, 0.15
mmol) in anhydrous DMF was added NaH (60%, 8 mg, 0.2
mmol). The reaction mixture was stirred at room temperature
for 0.5 h and then 2-(3-diethylsulfamoyl-4-fluorobenzoylami-
no)benzoic acid methyl ester (30 mg, 0.07 mmol) was added.
The reaction mixture was allowed to heat at 120 °C for 24 h.
The reaction mixture was cooled; diluted with EtOAc, H2O,
and 1 N HCl; and extracted with EtOAc (2×). Combined
organic layers were washed with brine and H2O, dried over
anhydrous MgSO4, and concentrated in vacuo. The crude
material was purified using RP-HPLC to afford the final
4-Bromo-3-phenoxybenzoic Acid (20). A mixture of
4-bromo-3-hydroxybenzoic acid methyl ester (18, 2.1 g, 9.1
mmol), phenyl boronic acid (2.2 g, 18 mmol), copper acetate
(1.65 g, 9.1 mmol), TEA (6.26 mL, 45 mmol), and 4 Å molecular
sieves in DCM (100 mL) was stirred at room temperature for
20 h. Once the reaction was complete, the reaction mixture
was filtered and the filtrate concentrated in vacuo. The
resulting residue was diluted with EtOAc and saturated
NaHCO3. The aqueous layer was extracted twice with EtOAc,
and the organics were washed with brine, dried over anhy-
drous MgSO4, and concentrated in vacuo. Purification by flash
column chromatography (12.5% EtOAc/hexane) gave the in-

Inhibitors of FabH as Antimicrobial Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1605
termediate, 4-bromo-3-phenoxybenzoic acid methyl ester (1.26
g), which was then hydrolyzed using 1 N NaOH (10.28 mL,
10.28 mmol) and THF/MeOH (25 mL/12.5 mL) to provide the
acid (20, 1.08 g, 40% in two steps): 1H NMR (300 MHz, DMSO-
d6) δ 13.39 (s, 1H), 7.91 (d, J ) 8.3 Hz, 1H), 7.67 (dd, J ) 1.9
and 8.3 Hz, 1H), 7.47 (m, 2H), 7.42 (d, J ) 1.9 Hz, 1H), 7.26
(m, 1H), 7.08 (m, 2H); LCMS (API-ES) m/z 290.9, 292.9 [M +
H-].
(300 MHz, DMSO-d6) δ 13.83 (br s, 1H), 12.16 (s, 1H), 8.99
(m, 1H), 8.69 (d, J ) 8.4 Hz, 1H), 8.49 (m, 1H), 8.08 (d, J )
7.9 Hz, 1H), 7.81 (d, J ) 8.4 Hz, 1H), 7.68 (t, J ) 7.9 Hz, 1H),
7.56 (s, 1H), 7.44 (t, J ) 7.7 Hz, 2H), 7.36 (d, J ) 8.5 Hz, 1H),
7.20 (m, 2H), 7.03 (d, J ) 8.4 Hz, 2H), 1.24 (d, J ) 6.4 Hz,
6H); LCMS (API-ES) m/z 446.0 [M + H+]; 444.0 [M + H-].
2-(4-Morpholin-4-yl-3-phenoxybenzoylamino)benzo-
ic acid (23f) was prepared according to the general procedure
for replacement of fluoro group: 1H NMR (300 MHz, DMSO-
d6) δ 12.14 (s, 1H), 8.69 (d, J ) 7.8 Hz, 1H), 8.07 (dd, J ) 1.5
and 7.9 Hz, 1H), 7.79 (dd, J ) 2.1 and 8.5 Hz, 1H), 7.67 (m,
1H), 7.53 (d, J ) 2.1 Hz, 1H), 7.42 (m, 2H), 7.14-7.27 (m, 3H),
6.99 (m, 2H), 3.17 (m, 5H); LCMS (API-ES) m/z 419.0 [M +
H+]; 417.0 [M + H-].
2-(3-Phenoxy-4-thiomorpholin-4-ylbenzoylamino)ben-
zoic acid (23g) was prepared according to the general
procedure for replacement of fluoro group:1H NMR (300 MHz,
DMSO-d6) δ 12.14 (s, 1H), 8.69 (d, J ) 8.6 Hz, 1H), 8.07 (d, J
) 7.8 Hz, 1H), 6.99-7.77 (m, 8H), 6.95 (m, 2H); LCMS (API-
ES) m/z 433.0 [M + H-].
2-(4-Fluoro-3-phenoxybenzoylamino)benzoic
Acid
Methyl Ester (21). Using the general procedure for making
amide, the acid (19, 600 mg, 3 mmol) was first converted to
the acid chloride and then reacted with methyl anthranilate
(0.389 mL, 3 mmol). The crude material was purified by flash
column chromatography (5-10% EtOAc/hexane) to yield 21
(420 mg, 38%): 1H NMR (300 MHz, DMSO-d6) δ 11.51 (s, 1H),
8.43 (d, J ) 7.7 Hz, 1H), 8.02 (dd, J ) 1.5 and 7.9 Hz, 1H),
8.01 (m, 1H), 7.66-7.73 (m, 3H), 7.48 (m, 2H), 7.29 (m, 2H),
7.14 (d, J ) 7.8 Hz, 2H), 3.88 (s, 3H); LCMS (API-ES) m/z
366.0 [M + H+].
2-(4-Bromo-3-phenoxybenzoylamino)benzoic
Acid
Methyl Ester (22). Using the general procedure for making
amide, the acid (20, 1.08 g, 3.67 mmol) was first converted to
the acid chloride and then reacted with methyl anthranilate
(0.347 mL, 2.68 mmol). The crude material was purified by
flash column chromatography (6.25-12.5% EtOAc/hexane) to
yield 22 (1.12 g, 98%): 1H NMR (300 MHz, DMSO-d6) δ 11.53
(s, 1H), 8.44 (d, J ) 8.0 Hz, 1H), 7.98-8.04 (m, 2H), 7.66-
7.72 (m, 2H), 7.47-7.54 (m, 3H), 7.24-7.30 (m, 2H), 7.10-
7.13 (m, 2H), 3.85 (s, 3H); LCMS (API-ES) m/z 425.9, 427.9
[M + H+]; 425.9 [M + H-].
2-(4-Fluoro-3-phenoxybenzoylamino)benzoic Acid (23a).
The ester (21) was saponified using 1 N NaOH in THF/MeOH
to provide the acid (23a): 1H NMR (300 MHz, DMSO-d6) δ
12.17 (s, 1H), 8.62 (d, J ) 8.3 Hz, 1H), 8.07 (dd, J ) 1.5 and
7.9 Hz, 1H), 7.83 (m, 1H), 7.66-7.72 (m, 3H), 7.47 (m, 2H),
7.25 (m, 2H), 7.12 (d, 2H); LCMS (API-ES) m/z 352.0 [M +
H+]; 350.0 [M + H-].
2-(4-Bromo-3-phenoxybenzoylamino)benzoic Acid (23b).
The ester (22) was saponified using 1 N NaOH in THF/MeOH
to provide the acid (23b): 1H NMR (300 MHz, DMSO-d6) δ
12.19 (s, 1H), 8.60 (d, J ) 7.6 Hz, 1H), 8.06 (m, 2H), 7.71 (m,
2H), 7.56 (s, 1H), 7.45 (m, 2H), 7.24 (m, 2H), 7.10 (m, 2H);
LCMS (API-ES) m/z 409.9, 411.8 [M + H-].
General Procedure for Replacement of Fluoro Group
with Amines To Make Derivatives 23c-i. A mixture of
2-(4-fluoro-3-phenoxybenzoylamino)benzoic acid methyl ester
(21, 1 equiv) and the amine (5 equiv) was heated at 120 °C in
a scintillation vial with DMSO (1 mL) for a few days. After
that the reaction mixture was diluted with H2O and EtOAc.
Brine was used to help separate the layers. The combined
organics were dried over anhydrous MgSO4 and concentrated
in vacuo. The ester was then saponified using 1 N NaOH in
THF/MeOH and purified using RP-HPLC to afford the final
compound.
2-(3-Phenoxy-4-piperazin-1-ylbenzoylamino)benzoic
acid (23c) was prepared according to the general procedure
for replacement of fluoro group: 1H NMR (300 MHz, DMSO-
d6) δ 8.67 (d, J ) 7.4 Hz, 1H), 8.07 (d, J ) 8.1 Hz, 1H), 7.53
(d, J ) 8.6 Hz, 1H), 7.42 (m, 1H), 7.42 (m, 3H), 7.33 (d, J )
8.3 Hz, 1H), 7.20 (t, J ) 7.3 Hz, 2H), 7.04 (d, J ) 8.4 Hz, 2H),
3.53 (m, 2H), 3.11 (m, 2H), 2.62 (m, 4H); LCMS (API-ES) m/z
418.0 [M + H+].
2-[4-(4-Methylpiperazin-1-yl)-3-phenoxybenzoylamino-
benzoic acid (23d) was prepared according to the general
procedure for replacement of fluoro group: 1H NMR (300 MHz,
DMSO-d6) δ 12.14 (s, 1H), 9.75 (br s, 1H), 8.67 (d, J ) 8.4 Hz,
1H), 8.07 (d, J ) 8.1 Hz, 1H), 7.77 (d, J ) 8.7 Hz, 1H), 7.67 (t,
J ) 8.1 Hz, 1H), 7.46 (m, 3H), 7.36 (d, J ) 8.4 Hz, 1H), 7.22
(t, J ) 7.2 Hz, 2H), 7.07 (d, J ) 8.4 Hz, 2H), 3.83 (m, 2H),
3.12 (m, 2H), 2.86 (s, 4H), 2.11 (s, 3H); LCMS (API-ES) m/z
432.0 [M + H+].
2-(3-Phenoxy-4-piperidin-1-ylbenzoylamino)benzoic
acid (23h) was prepared according to the general procedure
for replacement of fluoro group: 1H NMR (300 MHz, DMSO-
d6) δ 13.97 (br s, 1H), 12.14 (s, 1H), 8.70 (d, J ) 8.5 Hz, 1H),
8.07 (d, J ) 7.9 Hz, 1H), 7.77 (d, J ) 8.5 Hz, 1H), 7.67 (t, J )
7.8 Hz, 1H), 7.53 (d, J ) 1.9 Hz, 1H), 7.39 (m, 2H), 7.11-7.26
(m, 3H), 6.97 (m, 2H), 3.15 (s, 5H), 2.77 (s, 1H), 2.61 (s, 1H);
LCMS (API-ES) m/z 417.0 [M + H+]; 415.0 [M + H-].
2-[4-(3,5-Dimethylpiperidin-1-yl)-3-phenoxybenzoyl-
amino]benzoic acid (23i) was prepared according to the
general procedure for replacement of fluoro group: 1H NMR
(300 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.71 (d, J ) 8.2 Hz, 1H),
8.07 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.78 (dd, J ) 2.2 and 8.4
Hz, 1H), 7.67 (t, J ) 7.8 Hz, 1H), 7.56 (d, J ) 2.2 Hz, 1H),
7.38 (t, J ) 8.0 Hz, 2H), 7.21 (m, 2H), 7.12 (d, J ) 7.3 Hz,
1H), 6.93 (m, 2H), 3.45 (overlap with DMSO water), 2.47 (m,
2H), 1.71 (m, 1H), 1.21 (m, 2H), 0.84 (d, 6H); LCMS (API-ES)
m/z 445.0 [M + H+]; 443.0 [M + H-].
General Procedure for the Preparation of Derivatives
24a-p via Pd-Mediated Cross-Coupling. A mixture of 2-(4-
bromo-3-phenoxybenzoylamino)benzoic acid methyl ester (22,
1 equiv), aryl boronic acid or ester (1.5 equiv), 2 M aqueous
NaCO3 (3 equiv), and Pd(PPh3)4 (3 mol %), in DME (to 0.5 M
the ester, compound 22), was heated at 85 °C under an inert
atmosphere for 5 h or until HPLC or TLC analysis indicates
complete reaction. The mixture was then concentrated in
vacuo, dissolved in EtOAc (20 mL), washed with H2O (2 × 5
mL) and brine (1 × 5 mL), and dried over anhydrous MgSO4.
The methyl ester was then hydrolyzed, and the crude material
was purified using RP-HPLC.
2-[3-Phenoxy-4-(1H-pyrazol-4-yl)benzoylamino]ben-
zoic acid (24a) was prepared according to the general Pd-
mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 12.21 (s, 1H), 8.67 (d, J ) 8.1 Hz, 1H), 8.18 (s,
2H), 8.07 (d, J ) 8.1 Hz, 2H), 7.84 (d, J ) 7.7 Hz, 1H), 7.69
(m, 1H), 7.58 (d, J ) 1.7 Hz, 1H), 7.41 (t, J ) 8.5 Hz, 2H),
7.17 (m, 2H), 7.07 (m, 2H); LCMS (API-ES) m/z 400.0 [M +
H+]; 398.0 [M + H-].
2-(3-Phenoxy-4-pyridin-3-ylbenzoylamino)benzoic acid
(24b) was prepared according to the general Pd-mediated
cross-coupling procedure: 1H NMR (300 MHz, DMSO-d6) δ
12.24 (s, 1H), 8.88 (d, J ) 1.6 Hz, 1H), 8.67 (d, J ) 8.4 Hz,
1H), 8.62 (d, J ) 8.1 Hz, 1H), 8.10 (m, 2H), 7.87 (m, 2H), 7.70
(t, J ) 7.9 Hz, 1H), 7.57 (m, 2H), 7.43 (t, J ) 8.0 Hz, 2H), 7.27
(t, J ) 7.3 Hz, 1H), 7.20 (t, J ) 7.4 Hz, 1H), 7.08 (d, J ) 8.7
Hz, 2H); LCMS (API-ES) m/z 411.0 [M + H+]; 309.0 [M + H-].
2-[(2-Phenoxybiphenyl-4-carbonyl)amino]benzoic acid
(24c) was prepared according to the general Pd-mediated
cross-coupling procedure: 1H NMR (300 MHz, DMSO-d6) δ
12.27 (s, 1H), 8.68 (d, J ) 8.4 Hz, 1H), 8.08 (dd, J ) 1.5 and
7.9 Hz, 1H), 7.88 (dd, J ) 1.7 and 8.1 Hz, 1H), 7.77 (d, J ) 8.0
Hz, 1H), 7.38-7.69 (m, 9H), 7.26 (t, J ) 7.8 Hz, 1H), 7.17 (t,
J ) 7.9 Hz, 1H), 7.05 (m, 2H); LCMS (API-ES) m/z 410.0 [M
+ H+]; 408.0 [M + H-].
2-[4-(3,5-Dimethylpiperazin-1-yl)-3-phenoxybenzoyl-
amino]benzoic acid (23e) was prepared according to the
general procedure for replacement of fluoro group: 1H NMR

1606 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Nie et al.
2-[(2-Phenoxy-4′-trifluoromethylbiphenyl-4-carbonyl)-
amino]benzoic acid (24d) was prepared according to the
general Pd-mediated cross-coupling procedure: 1H NMR (300
MHz, DMSO-d6) δ 13.98 (br s, 1H), 12.30 (s, 1H), 8.67 (d, J )
8.3 Hz, 1H), 8.08 (dd, J ) 1.4 and 7.9 Hz, 1H), 7.82-7.91 (m,
6H), 7.67 (t, J ) 7.7 Hz, 1H), 7.57 (d, J ) 1.3 Hz, 1H), 7.43 (t,
J ) 7.9 Hz, 2H), 7.19 (t, J ) 7.4 Hz, 1H), 7.11 (t, J ) 7.0 Hz,
1H), 6.98 (d, J ) 8.5 Hz, 2H); LCMS (API-ES) m/z 478.0 [M +
H+]; 476.0 [M + H-].
2-[(4′-Methyl-2-phenoxybiphenyl-4-carbonyl)amino]-
benzoic acid (24e) was prepared according to the general
Pd-mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 13.85 (br s, 1H), 12.25 (s, 1H), 8.68 (d, J ) 8.4
Hz, 1H), 8.08 (d, J ) 7.9 Hz, 1H), 7.87 (d, J ) 8.1 Hz, 1H),
7.72 (m, 2H), 7.55 (d, J ) 8.3 Hz, 3H), 7.40 (t, J ) 7.8 Hz,
2H), 7.26 (m, 3H), 7.15 (t, J ) 7.3 Hz, 1H), 7.03 (d, J ) 8.5
Hz, 2H), 2.36 (s, 3H); LCMS (API-ES) m/z 424.0 [M + H+];
422.0 [M + H-].
4′-(2-Carboxyphenylcarbamoyl)-2′-phenoxybiphenyl-
4-carboxylic acid (24f) was prepared according to the general
Pd-mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 13.89 (br s, 1H), 13.08 (br s, 1H), 12.27 (s, 1H),
8.67 (d, J ) 7.8 Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H),
8.03 (d, J ) 8.4 Hz, 2H), 7.89 (dd, J ) 1.6 and 8.1 Hz, 1H),
7.80 (t, J ) 9.1 Hz, 3H), 7.70 (dt, J ) 1.6 and 7.9 Hz, 1H),
7.55 (ds, J ) 1.5 Hz, 1H), 7.42 (t, J ) 8.0 Hz, 2H), 7.23 (dt, J
) 1.0 and 7.8 Hz, 1H), 7.17 (t, J ) 7.4 Hz, 1H), 7.07 (dd, J )
1.0 and 8.6 Hz, 2H); LCMS (API-ES) m/z 454.0 [M + H+]; 452.0
[M + H-].
) 7.3 Hz, 1H), 7.18 (t, J ) 7.4 Hz, 1H), 7.07 (d, J ) 7.9 Hz,
2H); LCMS (API-ES) m/z 493.9 [M + H+]; 491.9 [M + H-].
2-[(4′-Fluoro-3′-methyl-2-phenoxybiphenyl-4-carbonyl)-
amino]benzoic acid (24l) was prepared according to the
general Pd-mediated cross-coupling procedure: 1H NMR (300
MHz, DMSO-d6) δ 13.98 (br s, 1H), 12.24 (s, 1H), 8.68 (d, J )
8.2 Hz, 1H), 8.08 (dd, J ) 1.6 and 7.9 Hz, 1H), 7.86 (dd, J )
1.7 and 8.1 Hz, 1H), 7.76 (d, J ) 6.1 Hz, 1H), 7.69 (dt, J ) 1.6
and 7.9 Hz, 1H), 7.57 (dd, J ) 2.0 and 8.3 Hz, 2H), 7.50 (m,
1H), 7.41 (t, J ) 7.6 Hz, 2H), 7.14-7.27 (m, 3H), 7.04 (d, J )
7.7 Hz, 2H), 2.29 (s, 3H); LCMS (API-ES) m/z 442.0 [M + H+];
440.0 [M + H-].
2-[(3′-Chloro-4′-fluoro-2-phenoxybiphenyl-4-carbonyl)-
amino]benzoic acid (24m) was prepared according to the
general Pd-mediated cross-coupling procedure: 1H NMR (300
MHz, DMSO-d6) δ 13.79 (br s, 1H), 12.29 (s, 1H), 8.67 (d, J )
8.5 Hz, 1H), 8.08 (d, J ) 7.9 Hz, 1H), 7.83 (m, 3H), 7.69 (m,
2H), 7.55 (m, 2H), 7.44 (t, J ) 7.0 Hz, 2H), 7.22 (m, 2H), 7.08
(d, J ) 8.0 Hz, 2H); LCMS (API-ES) m/z 461.9, 463.9 [M +
H+]; 460.0, 461.9 [M + H-].
2-[(3′,4′-Difluoro-2-phenoxybiphenyl-4-carbonyl)ami-
no]benzoic acid (24n) was prepared according to the general
Pd-mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 13.98 (br s, 1H), 12.26 (s, 1H), 8.66 (d, J ) 8.3
Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.69-7.85 (m, 4H),
7.52-7.58 (m, 3H), 7.43 (t, J ) 7.9 Hz, 2H), 7.25 (t, J ) 8.2
Hz, 1H), 7.25 (t, J ) 7.6 Hz, 1H), 7.07 (d, J ) 8.6 Hz, 2H);
LCMS (API-ES) m/z 446.0 [M + H+]; 443.9 [M + H-].
2-[(4′-Chloro-3′-methyl-2-phenoxybiphenyl-4-carbonyl)-
amino]benzoic acid (24o) was prepared according to the
general Pd-mediated cross-coupling procedure: 1H NMR (300
MHz, DMSO-d6) δ 13.86 (br s, 1H), 12.25 (s, 1H), 8.67 (d, J )
8.1 Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.86 (dd, J )
1.7 and 8.1 Hz, 1H), 7.77 (d, J ) 8.0 Hz, 1H), 7.69 (t, J ) 8.1
Hz, 1H), 7.64 (s, 1H), 7.56 (d, J ) 1.6 Hz, 1H), 7.50 (d, J ) 1.1
Hz, 2H), 7.40 (m, 2H), 7.26 (t, J ) 7.9 Hz, 1H), 7.18 (t, J ) 7.4
Hz, 1H), 7.04 (d, J ) 7.7 Hz, 2H), 2.39 (s, 3H); LCMS (API-
ES) m/z 458.0, 460.0 [M + H+]; 455.9, 458.0 [M + H-].
2-[(2′,4′-Difluoro-2-phenoxybiphenyl-4-carbonyl)ami-
no]benzoic acid (24p) was prepared according to the general
Pd-mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 13.85 (br s, 1H), 12.25 (s, 1H), 8.66 (d, J ) 7.6
Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.85 (dd, J ) 1.7
and 8.0 Hz, 1H), 7.71 (d, J ) 8.1 Hz, 2H), 7.61 (m, 1H), 7.53
(ds, J ) 1.6 Hz, 1H), 7.43 (m, 3H), 7.23 (m, 3H), 7.04 (d, J )
7.7 Hz, 2H); LCMS (API-ES) m/z 446.0 [M + H+]; 444.0 [M +
H-].
2-[(4′-Hydroxy-2-phenoxybiphenyl-4-carbonyl)amino]-
benzoic acid (24g) was prepared according to the general
Pd-mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 12.23 (s, 1H), 9.69 (s, 1H), 8.69 (d, J ) 8.4 Hz,
1H), 8.07 (d, J ) 7.9 Hz, 1H), 7.84 (d, J ) 8.0 Hz, 1H), 7.69
(m, 2H), 7.56 (s, 1H), 7.49 (d, J ) 7.9 Hz, 2H), 7.40 (t, J ) 7.5
Hz, 2H), 7.25 (t, J ) 7.7 Hz, 1H), 7.14 (t, J ) 7.1 Hz, 1H), 7.01
(d, J ) 8.0 Hz, 2H), 6.84 (d, J ) 8.0 Hz, 2H); LCMS (API-ES)
m/z 425.9 [M + H+]; 424.0 [M + H-].
2-[(4′-Ethoxy-2-phenoxybiphenyl-4-carbonyl)amino]-
benzoic acid (24h) was prepared according to the general
Pd-mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 12.29 (s, 1H), 8.68 (d, J ) 8.1 Hz, 1H), 8.07 (dd,
J ) 1.4 and 7.9 Hz, 1H), 7.85 (dd, J ) 1.6 and 8.1 Hz, 1H),
7.73 (d, J ) 8.0 Hz, 1H), 7.68 (t, J ) 7.9 Hz, 1H), 7.56 (m,
3H), 7.40 (t, J ) 7.9 Hz, 2H), 7.17 (t, J ) 7.8 Hz, 1H), 7.15 (t,
J ) 7.6 Hz, 1H), 7.00 (m, 4H), 4.07 (q, J ) 6.9 Hz, 2H), 1.37
(t, J ) 6.9 Hz, 3H); LCMS (API-ES) m/z 454.0 [M + H+]; 452.0
[M + H-].
Pyridin-4-yloxybenzoic Acid (25). To a solution of methyl
3-hydroxybenzoate (16, 609 mg, 4 mmol) in dry pyridine was
added NaH (60%, 200 mg, 5 mmol). The reaction mixture was
stirred at room temperature for 0.5 h. Then chloropyridine
hydrochloride (600 mg, 4 mmol) was added, followed with a
catalytic amount of CuCl (100 mg). The reaction mixture was
allowed to heat at 120 °C for 24 h. The reaction mixture was
cooled and separated between EtOAc and H2O. Combined
organic layers were washed with brine and H2O, dried over
anhydrous MgSO4, and concentrated in vacuo. The crude
material was then saponified and purified using RP-HPLC:
1H NMR (300 MHz, DMSO-d6) δ 8.58 (dd, J ) 4.8 Hz, 2H),
7.93 (dd, J ) 1.3 and 7.6 Hz, 1H), 7.70 (m, 2H), 7.56 (m, 1H),
7.08 (m, 2H); LCMS (API-ES) m/z 216.1 [M + H+]; 214.1 [M
+ H-].
2-(3-Bromobenzoylamino)benzoic Acid Methyl Ester
(27). Using the general procedure for amide formation, starting
material 3-bromobenzoic acid (26, 2 g, 10 mmol) was first
converted to acetyl chloride, followed by reaction with methyl
anthranilate (1.16 mL, 9 mmol) to give 2.6 g of product (27,
78%): 1H NMR (300 MHz, DMSO-d6) δ 11.50 (s, 1H), 8.45 (d,
J ) 8.3 Hz, 1H), 8.14 (t, J ) 3.5 Hz, 1H), 8.03 (dd, J ) 1.5 and
7.9 Hz, 1H), 7.98 (md, J ) 7.3 Hz, 1H), 7.92 (md, J ) 7.5 Hz,
1H), 7.72 (dt, J ) 8.1 Hz, 1H), 7.62 (t, J ) 7.9 Hz, 1H), 7.34
(dt, J ) 7.9 Hz, 1H), 3.92 (s, 3H); LCMS (API-ES) m/z 333.9,
335.9 [M + H+]; 331.9, 333.9 [M + H-].
2-[(4′-Methanesulfonyl-2-phenoxybiphenyl-4-carbony-
l)amino]benzoic acid (24i) was prepared according to the
general Pd-mediated cross-coupling procedure: 1H NMR (300
MHz, DMSO-d6) δ 13.85 (br s, 1H), 12.25 (s, 1H), 8.66 (d, J )
8.0 Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H), 8.04 (d, J ) 8.4
Hz, 2H), 7.94 (d, J ) 8.4 Hz, 2H), 7.89 (dd, J ) 1.5 and 8.1
Hz, 1H), 7.83 (d, J ) 8.0 Hz, 1H), 7.70 (dt, J ) 1.5 and 7.9 Hz,
1H), 7.55 (ds, J ) 1.4 Hz, 1H), 7.45 (t, J ) 7.7 Hz, 2H), 7.23
(p, J ) 7.5 Hz, 2H), 7.12 (d, J ) 7.8 Hz, 2H), 3.30 (s, 3H);
LCMS (API-ES) m/z 488.0 [M + H+]; 485.9 [M + H-].
2-[(3′-Isopropyl-2-phenoxybiphenyl-4-carbonyl)amino]-
benzoic acid (24j) was prepared according to the general Pd-
mediated cross-coupling procedure: 1H NMR (300 MHz,
DMSO-d6) δ 13.85 (br s, 1H), 12.26 (s, 1H), 8.68 (d, J ) 8.1
Hz, 1H), 8.08 (d, J ) 7.8 Hz, 1H), 7.88 (d, J ) 8.2 Hz, 1H),
7.79 (m, 2H), 7.61 (s, 1H), 7.41 (m, 5H), 7.28 (m, 2H), 7.18 (t,
J ) 7.6 Hz, 1H), 7.03 (d, J ) 8.1 Hz, 2H), 2.80 (m, 1H), 1.21
(d, J ) 3.4 Hz, 6H); LCMS (API-ES) m/z 452.0 [M + H+]; 450.0
[M + H-].
2-[(2-Phenoxy-3′-trifluoromethoxybiphenyl-4-carbonyl)-
amino]benzoic acid (24k) was prepared according to the
general Pd-mediated cross-coupling procedure: 1H NMR (300
MHz, DMSO-d6) δ 13.85 (br s, 1H), 12.26 (s, 1H), 8.67 (d, J )
8.1 Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.89 (dd, J )
1.5 and 8.1 Hz, 1H), 7.83 (d, J ) 8.0 Hz, 1H), 7.70 (m, 2H),
7.64 (m, 2H), 7.58 (m, 1H), 7.42 (t, J ) 7.8 Hz, 3H), 7.26 (t, J

Inhibitors of FabH as Antimicrobial Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1607
3-(3-Phenoxybenzoylamino)thiophene-2-carboxylic
acid (28a) was prepared according to the general procedure
for amide bond formation: 1H NMR (300 MHz, DMSO-d6) δ
13.70 (br s, 1H), 11.23 (s, 1H), 8.09 (d, J ) 5.4 Hz, 1H), 7.96
(d, J ) 5.0 Hz, 1H), 7.68 (m, 2H), 7.52 (m, 1H), 7.48 (d, J )
7.6 Hz, 2H), 7.34 (m, 1H), 7.26 (dt, J ) 1.0 and 7.4 Hz, 1H),
7.15 (d, J ) 7.7 Hz, 2H); LCMS (API-ES) m/z 340.0 [M + H+];
338.0 [M + H-].
this time, 2-(3-bromobenzoylamino)benzoic acid methyl ester
(27, 334 mg, 1.00 mmol) and CuCl (99 mg, 1.00 mmol) were
added, and the reaction mixture was stirred at 120 °C for 2
days. The reaction mixture was removed from the heat,
neutralized with 1 N HCl, and extracted with EtOAc (3×). The
organics were washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. LCMS confirmed the
formation of the hydrolyzed material (in the case of 28i and
28j, final compounds were obtained after sponification). This
material was then purified using RP-HPLC to afford the final
compound 28h (54 mg, 14%): 1H NMR (300 MHz, DMSO-d6)
δ 13.85 (br s, 1H), 13.25 (br s, 1H), 12.21 (s, 1H), 8.69 (dd, J
) 0.7 and 7.7 Hz, 1H), 8.09 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.80
(m, 2H), 7.70 (m, 2H), 7.63 (m, 1H), 7.60 (d, J ) 7.9 Hz, 1H),
7.54 (m, 1H), 7.40 (m, 2H), 7.26 (t, J ) 7.3 Hz, 1H); LCMS
(API-ES) m/z 377.9 [M + H+]; 376.0 [M + H-].
2-[3-(4-Carboxyphenoxy)benzoylamino]benzoic acid
(28i): 1H NMR (300 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.69 (d,
J ) 8.3 Hz, 1H), 8.00 (m, 3H), 7.73 (d, J ) 7.5 Hz, 1H), 7.66
(m, 3H), 7.45 (dd, J ) 2.2 and 8.0 Hz, 1H), 7.26 (t, J ) 7.6 Hz,
1H), 7.16 (m, 2H); LCMS (API-ES) m/z 377.9 [M + H+]; 376.0
[M + H-].
2-[3-(4-Fluorophenoxy)benzoylamino]benzoic acid
(28j): 1H NMR (300 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.69 (dd,
J ) 0.8 and 8.4 Hz, 1H), 8.08 (dd, J ) 1.5 and 7.9 Hz, 1H),
7.61-7.75 (m, 3H), 7.54 (t, J ) 2.0 Hz, 1H), 7.18-7.35 (m, 6H);
LCMS (API-ES) m/z 352.0 [M + H+]; 350.0 [M + H-].
2-(4-Bromo-3-phenoxybenzoylamino)-6-methoxyben-
zoic acid (30). The acid (20, 668 mg, 2.28 mmol) was
converted to the acid chloride by refluxing in thionyl chloride
with 3 drops of DMF. After 2 h, the reaction mixture was
concentrated and suspended in pyridine. The mixture was
cooled to 0 °C under N2, and 2-amino-6-methoxybenzoic acid
(334 mg, 2 mmol) was added. After stirring for 24 h at room
temperature, the reaction mixture was concentrated, neutral-
ized with 1 N HCl, and extracted with EtOAc (3×). The
organics were washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The cyclized intermediate
(29) was then hydrolyzed with 1 N LiOH (7 mL, 6.84 mmol)
in THF overnight. The reaction mixture was concentrated,
acidified by 1 N HCl (10 mL), and extracted by EtOAc. The
final compound was purified using RP-HPLC to provide 150
mg of product (30, 15% in three steps): 1H NMR (300 MHz,
DMSO-d6) δ 13.05 (br s, 1H), 10.76 (s, 1H), 8.00 (d, J ) 8.2
Hz, 1H), 7.70 (dd, J ) 2.0 and 8.3 Hz, 1H), 7.56 (d, J ) 2.0
Hz, 1H), 7.40-7.49 (m, 3H), 7.18-7.28 (m, 2H), 6.92-7.07 (m,
3H), 3.90 (s, 3H); LCMS (API-ES) m/z 441.4, 443.5 [M + H+];
439.5, 441.5 [M + H-].
2-(4-Bromo-3-phenoxybenzoylamino)-6-hydroxyben-
zoic Acid (31). To compound 30 (50 mg, 0.11 mmol) in DCM
(3 mL) at 0 °C was added boron tribromide (1 M in DCM, 0.17
mL, 0.17 mmol) dropwise. After stirring at room temperature
for 20 h, MeOH was added to quench the reaction. Reaction
mixture was trituated three times with MeOH and the crude
material was purified using RP-HPLC to give 20 mg of product
(42%): 1H NMR (300 MHz, DMSO-d6) δ 13.37 (s, 1H), 7.99 (d,
J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.2 Hz, 1H), 7.74 (d, J ) 8.3 Hz,
1H), 7.56 (d, J ) 1.7 Hz, 1H), 7.47 (t, J ) 7.9 Hz, 2H), 7.24-
7.32 (m, 2H), 7.07 (d, J ) 8.4 Hz, 2H), 6.60 (d, J ) 8.2 Hz,
1H); LCMS (API-ES) m/z 427.4, 429.5 [M + H+]; 425.5, 427.4
[M + H-].
2-(3-Phenoxybenzoylamino)thiophene-3-carboxylic
acid (28b) was prepared according to the general procedure
for amide bond formation: 1H NMR (300 MHz, DMSO-d6) δ
13.45 (br s, 1H), 12.08 (s, 1H), 7.69 (m, 2H), 7.50 (dt, J ) 1.2
and 7.1 Hz, 3H), 7.37 (m, 1H), 7.27 (m, 2H), 7.14 (q, J ) 8.4
Hz, 3H); LCMS (API-ES) m/z 340.0 [M + H+]; 338.0 [M + H-].
3-(3-Phenoxybenzoylamino)pyridine-2-carboxylic acid
(28c) was prepared according to the general procedure for
amide bond formation: 1H NMR (300 MHz, DMSO-d6) δ 12.62
(s, 1H), 9.12 (d, J ) 8.3 Hz, 1H), 8.47 (d, J ) 3.9 Hz, 1H), 7.83
(q, J ) 4.7 Hz, 1H), 7.77 (d, J ) 7.8 Hz, 1H), 7.67 (t, J ) 8.0
Hz, 1H), 7.58 (s, 1H), 7.49 (t, J ) 7.9 Hz, 2H), 7.35 (dd, J )
2.1, 7.8 Hz, 1H), 7.25 (t, J ) 7.4 Hz, 1H), 7.14 (d, J ) 7.9 Hz,
2H); LCMS (API-ES) m/z 335.0 [M + H+]; 333.0 [M + H-].
2-(3-Phenoxybenzoylamino)nicotinic acid (28d) was
prepared according to the general procedure for amide bond
formation: 1H NMR (300 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.61
(d, J ) 8.3 Hz, 1H), 8.26 (dd, J ) 1.8 and 7.7 Hz, 1H), 7.80 (d,
J ) 7.8 Hz, 1H), 7.59 (m, 2H), 7.47 (m, 2H), 7.24 (m, 2H), 7.14
(t, J ) 7.6 Hz, 1H), 7.05 (m, 2H); LCMS (API-ES) m/z 335.0
[M + H+]; 333.0 [M + H-].
2-Fluoro-6-(3-phenoxybenzoylamino)benzoic acid (28e)
was prepared according to the general procedure for amide
bond formation: 1H NMR (300 MHz, DMSO-d6) δ 13.75 (br s,
1H), 11.16 (s, 1H), 7.92 (d, J ) 8.2 Hz, 1H), 7.74 (d, J ) 7.8
Hz, 1H), 7.62 (m, 2H), 7.57 (m, 1H), 7.49 (t, J ) 8.1 Hz, 2H),
7.30 (dd, J ) 2.5 and 8.1 Hz, 1H), 7.24 (t, J ) 7.6 Hz, 1H),
7.13 (m, 3H); LCMS (API-ES) m/z 352.0 [M + H+]; 350.0 [M
+ H-].
2-Hydroxy-6-(3-phenoxybenzoylamino)benzoic Acid
(28f). To a suspension of 3-phenoxybenzoyl chloride (550 mg,
2.37 mmol) in dry pyridine (3 mL) was added 2-amino-6-
methoxybenzoic acid methyl ester (264 mg, 1.58 mmol) at 0
°C. After stirring for 24 h at room temperature, the reaction
mixture was concentrated, taken up in EtOAc, and washed
with H2O and 1 N HCl. The organics were washed with brine,
dried over anhydrous MgSO4, and concentrated in vacuo. The
residue was then dissolved in THF (4 mL) and treated with 1
N LiOH (3.46 mL, 3.46 mmol). After stirring overnight at room
temperature, the reaction mixture was concentrated and
separated between EtOAc and H2O. The organic layers were
dried and concentrated to give 382 mg of the intermediate,
2-methoxy-6-(3-phenoxybenzoylamino)benzoic acid (67%). To
this intermediate (161 mg, 0.44 mmol) in DCM (5 mL) at 0 °C
was added boron tribromide (1 M in DCM, 0.66 mL, 0.66 mmol)
dropwise. After stirring at room temperature for 2 h, MeOH
was added to quench the reaction. The reaction mixture was
trituated (3×) with MeOH and the crude material was purified
using RP-HPLC to give 20 mg of product (13%): 1H NMR (300
MHz, DMSO-d6) δ 13.23 (s, 1H), 7.98 (d, J ) 8.2 Hz, 1H), 7.76
(d, J ) 8.2 Hz, 1H), 7.57-7.63 (m, 2H), 7.45-7.51 (m, 2H),
7.21-7.33 (m, 3H), 7.13 (m, 2H), 6.59 (d, J ) 8.1 Hz, 1H);
LCMS (API-ES) m/z 349.6 [M + H+]; 347.6 [M + H-].
2-[3-(Pyridin-4-yloxy)benzoylamino]benzoic acid (28g)
was prepared according to the general procedure for amide
2-Hydroxy-6-[(2-phenoxybiphenyl-4-carbonyl)amino]-
benzoic Acid (33). Using the general Pd-mediated cross-
coupling procedure, 100 mg of compound 30 (0.22 mmol) was
coupled with phenylboronic acid (50 mg, 0.4 mmol) to give the
intermediate (32, 37 mg). This was then treated with boron
tribromide (1 M in DCM, 0.16 mL, 0.16 mmol) in DCM.
Purification by RP-HPLC provided 10 mg of final product (33,
11%): 1H NMR (300 MHz, DMSO-d6) δ 13.79 (s, 1H), 7.97 (d,
J ) 7.8 Hz, 1H), 7.89 (dd, J ) 1.4 and 8.1 Hz, 1H), 7.72 (d, J
) 8.0 Hz, 1H), 7.66 (m, 2H), 7.57 (d, J ) 1.5 Hz, 1H), 7.40-
7.49(m, 5H), 7.26 (t, J ) 8.2 Hz, 1H), 7.14 (t, J ) 8.2 Hz, 1H),
7.04 (d, J ) 8.0 Hz, 2H), 6.56 (dd, J ) 0.8 and 8.2 Hz, 1H);
LCMS (API-ES) m/z 425.5 [M + H+]; 423.6 [M + H-].
1
bond formation using 25 as starting material: H NMR (300
MHz, DMSO-d6) δ 12.24 (s, 1H), 8.75 (d, J ) 6.4 Hz, 2H), 8.69
(d, J ) 8.0 Hz, 1H), 8.09 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.98 (d,
J ) 8.0 Hz, 1H), 7.84 (m, 2H), 7.72 (dt, 1H), 7.64 (dd, 1H),
7.40 (m, 2H), 7.28 (dt, 1H); LCMS (API-ES) m/z 335.0 [M +
H+]; 333.0 [M + H-].
2-[3-(3-Carboxyphenoxy)benzoylamino]benzoic Acid
(28h). Representative Procedure for 28h-j. To a stirred
solution of methyl 3-hydroxybenzoate (152 mg, 1.00 mmol) in
pyridine (5 mL) was added NaH (60 mg, 1.5 mmol), and the
reaction mixture was stirred for 30 min. At the conclusion of

1608 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Nie et al.
Computational Chemistry Methods. Protein and small
molecule modeling visualization was accomplished with Sybyl
v. 6.7 (Tripos, Inc. St. Louis, MO.) and Macromodel v. 4.1
(Schro¨dinger, Inc. Portland, OR.). Handling of the small
molecule virtual library, calculations of Lipinski properties,
conversion of the library from 2D to 3D structures, similarity/
substructure search, and pharmacophore modeling were ac-
complished by the Unity 4.2, CONCORD, and HiVol modules
in the Tripos suite (Tripos, Inc. St. Louis, MO.). Docking was
done using FlexX in Sybyl v. 6.7. Conformational analysis and
clustering were performed in Macromodel v. 4.1 (Schro¨dinger,
Inc. Portland, OR.).
Expression and Purification of ACP, ACPS, FabD, and
FabH. Full-length E. coli acyl carrier protein (ACP), acyl
carrier protein synthase (ACPS), malonyl-CoA:ACP transacy-
lase (FabD), and 3-ketoacyl-ACP synthase III (FabH) were
individually cloned into pET expression vectors (Novagen) with
an N-terminal His-tag (ACP, ACPS in pET19; FabD, FabH in
pET28). E. coli FabH was also cloned in a tagless version in
pET30 vector. E. faecalis, S. pyogenes, S. aureus., H. influenzae
FabHs were also cloned and expressed in pET vector in both
His-tagged (pET28) and tagless (pET30) versions.
ACPs can be detected by native polyacrylamide gel electro-
phoresis as described.21
FabD/FabH Coupled Assay. All FabH proteins purified,
tagged or tagless, can use E.coli holo-ACP as their substrate.
There is no detectable difference between tagged and tagless
FabHs in terms of their enzyme activity. We coupled the FabD
and FabH reactions because malonyl-ACP, the product of FabD
reaction, is unstable. In a final 100-µL reaction, 100 mM
Na2HPO4/NaH2PO4, pH 7.0, 2 mM DTT, 1 mM MgCl2, and 12.5
µM holo-ACP were mixed with 10 nM FabD and 1-20 nM
FabH (0.5-1 nM for E. coli FabH, 3-5 nM for E. faecalis and
H. influenzae FabHs, 10 nM for S. pyogenes FabH, and 20 nM
for Staph. aureus FabH), and H2O was added to 80 µL. FabD
reaction was initialized by adding 10 µL of 1 mΜ malonyl-
CoA. After 1 min incubation, a 10 µL mixture of 125 µM acetyl-
CoA, 0.75 µCi [3H]acetyl-CoA (Amersham), 2.5 mM NADH, and
2.5 mM NADPH was added for FabH reaction for 19 min. The
reaction was stopped by adding 100 µL of ice-cold 50% TCA,
incubating for 5 min on ice, and centrifuging to pellet the
protein. The pellet was washed with 10% ice-cold TCA and
resuspended with 20 µL of 2 M NaOH. The incorporation of
3
the H signal in the final product was read by liquid scintil-
lation. When determining the inhibition constant (IC50),
inhibitors were added from a concentrated DMSO stock such
that the final concentration of DMSO did not exceed 2%.
All proteins were expressed in E. coli strain BL21(DE3)
(Invitrogen). Harvested cells containing His-tagged ACP,
ACPS, FabD, and FabHs were lysed by sonication in 20 mM
Tris, pH 7.6, 5 mM imidazole, 0.5 M NaCl and centrifuged at
20 000 rpm for 30 min. The supernatant was applied to a Ni-
NTA agarose column (Qiagen), washed, and eluted using a
5-500 mM imidazole gradient over 20 column volumes. Eluted
protein was dialyzed against 20 mM Tris, pH 7.6, 1 mM DTT,
and 100 mM NaCl. Purified FabD and FabHs were concen-
trated up to 2 mg/mL and stored at -80°C in 20 mM Tris, pH
7.6, 100 mM NaCl, 1 mM DTT, and 20% glycerol for enzymatic
assays.
Harvested cells expressing tagless FabHs were sonicated
in 25 mM Tris, pH 8.0, 20 mM NaCl, 2 mM DTT, 1 mM EDTA,
50 mM â-ME, and 0.5 mL (per 100 mL lysate) protease
inhibitor cocktail (Roche). The crude lysate was centrifuged
at 20 000 rpm for 30 min, and the soluble fraction was
separated by anion exchange chromatography (HiPrep 16/10
Q XL, Amersham Pharmacia Biotech, (APB)) using a 0-1 M
NaCl gradient over 10 column volumes. Protein fractions were
checked by SDS-PAGE, and appropriate fractions were
dialyzed against 25 mM Tris, pH 8.0, 2 mM DTT, and 50 mM
â-ME. Dialyzed protein was loaded onto a 10-mL Source Q-15
column (APB) and further separated by anion-exchange chro-
matography using a 0-500 mM NaCl gradient over 20 column
volumes. Protein fractions were checked by SDS-PAGE, and
FabH-containing fractions were pooled and loaded onto an
8-mL hydroxyapaptite column (Bio-Rad) and eluted with a
0-250 mM gradient of a pH 8.0 potassium phosphate buffer
over 15 column volumes. FabH-containing fractions were
concentrated up to 2 mg/mL and stored at -80 °C in 20 mM
Tris, pH 7.6, 100 mM NaCl, 1 mM DTT, and 20% glycerol for
enzymatic assays or concentrated up to 20 mg/mL for crystal-
lization trials.
Conversion of Apo-ACP to Holo-ACP. Purified ACP
contains approximately 50% of the apo-form that needs to be
converted into the holo-form. The conversion reaction is
catalyzed by ACP synthase (ACPS) and transfers a panto-
thenoate prosthetic group to apo-ACP to form holo-ACP. This
method was described previously20 and modified as follows:
In the final volume of 50 mL, 50 mg ACP, 50 mM Tris, pH
9.0, 2 mM DTT, 10 mM MgCl2, 600 µM CoA, and 0.2 µM ACPS
was incubated for 1 h at 37 °C. The pH of the reaction was
then adjusted to approximately 7.0 using 1 M potassium
phosphate, pH 6. Holo-ACP was purified by fractionation of
the reaction mixture by Source Q-15 (Amersham Pharmacia)
ion exchange chromatography using a 0-500 mM NaCl
gradient over 25 column volumes. This step not only removes
ACPS, it also eliminates minor contamination of FabD and
FabH-like activity that causes the elevation of background in
subsequent FabD or FabH assays. Separated apo- and holo-
Acetylation of FabH by Acetyl-CoA. To ensure that
FabD was not inhibited during this coupled assay, inhibition
of FabH acetylation was demonstrated. The first step of the
FabH catalytic reaction is the acetylation of the catalytic
cysteine residue at the active site. In the absence of holo-ACP,
the reaction can be arrested at this intermediate stage. The
acetylation reaction can be detected by mixing 1 µM FabH
(freshly desalted to remove DTT by a PD10 desalting column
(Pierce)), 100 mM, pH 7.0 sodium phosphate buffer, 1 mM
MgCl2 in 10-100 µL volume and initiated by adding 1-5 µM
[3H]acetyl-CoA (Amersham). The reaction proceeded from 1
to 10 min before being stopped by ice-cold 50% TCA, and the
incorporation of radiolabel was determined by liquid scintil-
lation as described in the previous section. A dose-dependent
decrease in incorporated radiolabel indicated specific FabH
inhibition.
Crystallization of FabH. All crystals were obtained by
vapor diffusion using 20 mg/mL protein solution in the hanging
drop setups at 15° C. The cocrystal structure of E. faecalis
FabH with bound compound 13a was obtained using the
N-terminally His-tagged protein. The crystallization conditions
consisted of 0.2 M lithium sulfate, 0.1 M Bis-Tris, pH 6.5, 25%
PEG3350. The structure of E. faecalis FabH with bound
compound 23i was determined using the tagless protein with
two specific mutations (D36A, E37A) crystallized with 25%
PEG 3350, 0.2 M ammonium sulfate and 0.1 M Bis-Tris, pH
5.5.
Crystallization, Data Collection and Structure Deter-
mination. All data were collected at 110 K using MAR image
plate and rotating anode X-ray generator. Crystals were frozen
in synthetic mother liquor with 25% glycerol. Data were
integrated, reduced, and scaled using the HKL program
suite.22 CNX was used for molecular replacement and refine-
ment of structures. The structure of the E. coli FabH was first
determined by molecular replacement using the 1.8 Å pub-
lished structure of the same protein10 as the starting model.
All the subsequent structures were determined by molecular
replacement, using the E. coli FabH structure as the starting
model.
Acknowledgment. The authors thank Dr. Paul
Brett for invaluable contributions to the project and Dr.
James Levin and Cindy Atwell of our Preclinical Mi-
crobiology Department for MIC determinations and
fruitful discussions. The authors also want to thank
Miss Lucy Aguirre for her assistance in crystallization
of FabH.

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