Substituted 2-Imino-5-arylidenethiazolidin-4-one Inhibitors of Bacterial Type III Secretion

Article abstract of DOI:10.1021/jm8004515

Diverse species of pathogenic Gram-negative bacteria use secretion systems to export a variety of protein toxins and virulence factors that help establish and maintain infection. Disruption of such secretion systems is a potentially effective therapeutic strategy. We developed a high-throughput screen and identified a trisaryl substituted 2-imino-5-arylidenethiazolidin-4-one, compound 1, as an inhibitor of the type III secretion system. Expansion of this chemotype enabled us to define the essential pharmacophore for type III secretion inhibition by this structural class. A synthetic diversity set helped us identify N-3 as the most permissive locus and led to the design of a panel of novel N-3-dipeptide-modified congeners with improved activity and physiochemical properties. We now report on the synthesis of these compounds, including a novel solid phase approach to the rapid generation of the dipeptide-thiazolidinone hybrids, and their in vitro characterization as inhibitors of type III secretion in Salmonella enterica serovar Typhimurium.

Full text of DOI:10.1021/jm8004515

J. Med. Chem. 2008, 51, 7065–7074
7065
Articles
Substituted 2-Imino-5-arylidenethiazolidin-4-one Inhibitors of Bacterial Type III Secretion
Toni Kline,*,† Heather B. Felise,*,† Kathleen C. Barry,† Stona R. Jackson,† Hai V. Nguyen,† and Samuel I. Miller†,‡,§
Departments of Genome Sciences, Microbiology, and Medicine, UniVersity of Washington, Seattle, Washington 98195
ReceiVed April 21, 2008
Diverse species of pathogenic Gram-negative bacteria use secretion systems to export a variety of protein
toxins and virulence factors that help establish and maintain infection. Disruption of such secretion systems
is a potentially effective therapeutic strategy. We developed a high-throughput screen and identified a tris-
aryl substituted 2-imino-5-arylidenethiazolidin-4-one, compound 1, as an inhibitor of the type III secretion
system. Expansion of this chemotype enabled us to define the essential pharmacophore for type III secretion
inhibition by this structural class. A synthetic diversity set helped us identify N-3 as the most permissive
locus and led to the design of a panel of novel N-3-dipeptide-modified congeners with improved activity
and physiochemical properties. We now report on the synthesis of these compounds, including a novel
solid phase approach to the rapid generation of the dipeptide-thiazolidinone hybrids, and their in vitro
characterization as inhibitors of type III secretion in Salmonella enterica serovar Typhimurium.
Introduction
Diverse species of pathogenic Gram-negative bacteria use
type II (T2SSa) and type III (T3SS) secretion systems to deliver
virulence factors to host cells to establish and maintain bacterial
infections. Some of the component proteins of these secretory
apparati, such as the outer membrane secretin YscC/InvG, the
inner membrane ring protein YscJ/PrgK, and the ATPase YscN/
InvC/SpaI, demonstrate primary amino acid sequence conserva-
tion.1 Others demonstrate structural conservation.2 A potential
advantage of targeting secretion is that it may result in less
selection for resistant mutants because secretion systems are
not required for bacterial growth.3 Small molecules that inhibit
secretion systems might be indicated for the prevention and/or
treatment of infection from a wide variety of Gram-negative
bacterial species and be applicable to diverse plant and animal
diseases.4,5 The concept of secretion inhibition as a potentially
effective broad-range therapeutic strategy is supported by the
literature reports of in vitro activity against Yersina, Chlamydia,6,7
and Salmonella8 by the salicylhydrazide class of T3SS inhibitors.
The potential to disable the virulence processes of diverse Gram-
negative pathogens9 prompted us to embark on a discovery
project for novel T3SS inhibitors.
Figure 1. Lead structure emerging from our high-throughput screen
for T3SS inhibitors.
A high-throughput screen (HTS) was developed in our
laboratory and used to evaluate 92 000 small molecules for
inhibition of the Salmonella enterica serovar Typhimurium
T3SS. This screen produced 89 initial hits, from which 25
were selected for confirmatory and secondary assays. In
selecting compounds to advance to the next screens, we
placed a priority on compounds likely to be functioning via
our target mechanism of action. We rejected frank cytotoxic
or cytostatic compounds, or transcriptional inhibitors, and
favored those compounds that exhibited synthetic tractability
and suitability for chemotype expansion. The results from
our secondary assays led us to focus on the tris-aryl
substituted 2-imino-5-arylidenethiazolidin-4-one 1 (Figure 1)
as our candidate hit. We assayed for secretion of SipA, a
T3SS-secreted protein, and using both commercially available
and our own synthetic compounds, generated a panel of
analogues with which we were able to define the essential
pharmacophore of this class of secretion inhibitors.10
* To whom correspondence should be addressed. For T.K.: telephone,
(206) 543-4720; fax, (206) 685-7344; e-mail: klinet@u.washington.edu. For
H.F.: telephone, (206) 616-5108; fax, (206) 616-5109; e-mail, hrb@
u.washington.edu.
†
Department of Genome Sciences.
Department of Microbiology.
Department of Medicine.
‡
§
Six distinct structural classes of T3SS inhibitors have been
described previously. Two classes, the glycolipid caminosides
reported by Linington et al.11,12 and the pseudotripeptide
guadinomines recently reported by Iwatsuki et al.,13 are natural
products. Synthetic inhibitor classes include the N-substituted
(2-acylamino-4-aryl)-2-pyridones,14 2,5-disubstituted oxazole-
3-carboxamides,15 substituted (2-alkylamino)-1,3,5-triazines,16
and N-acylhydrazones of salicylaldehydes.17,18 Mechanisms of
action and molecular targets have not been determined for these
compound classes, although currently the acylhydrazones are
a Abbreviations: Ala, alanine; Arg, arginine; ATPase, adenosine triph-
osphate hydrolase; ATCC, American Type Culture Collection; Boc, tert-
butoxycarbonyl; Cbz, benzyloxycarbonyl; Dht, dihydrotryptophan; DIC,
diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMSO, dimethyl
sulfoxide; Fmoc, fluorenylmethyloxycarbonyl; HOAt, hydroxyazabenzot-
riazole; HTS, high-throughput screen; LB, Luria-Bertoni broth, OD, optical
density; Orn, ornithine; Pbf, pentamethylsulfonylbenzofuran; RP-HPLC,
reverse phase high performance liquid chromatography; SDS-PAGE,
sodium dodecyl sulfate polyacrylamide gel electrophoresis; T2SS, type II
secretion system; T3SS, type III secretion system; TCA, trichloroacetic acid;
TFA, trifluoroacetic acid; TIS, triisopropylsilane; TLC, thin layer chroma-
tography; Tyr, tyrosine; Trp, tryptophan; Val, valine; Z, benzyloxycarbonyl.
10.1021/jm8004515 CCC: $40.75
2008 American Chemical Society
Published on Web 10/24/2008

7066 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Kline et al.
Scheme 1
Scheme 2
believed to act by inhibiting transcription of one or more T3SS
proteins.6,7 In contrast, our initial studies with the 2-iminothia-
zolidinones suggest that this class may act directly to disrupt a
protein-protein interaction such as the interactions of the
secretin protein involved in the assembly and/or stability of the
secretion apparatus.10
The thiazolidinone nucleus appears in drug discovery pro-
grams in diverse therapeutic areas. Substituted thiazolidinones
are under investigation as anticancer antagonists of the Rvꢀ3
integrins,19 anti-inflammatory agents,20,21 ꢀ3 adrenergic ago-
nists,22,23 antischistosomal agents,24 general antifungal/antimi-
crobial agents,25-27 and inhibitors of the YycG histidine kinase
of S. epidermidis.28 Many of these thiazolidinones are highly
potent and target-specific, suggesting that the substituents impart
the high degree of selectivity to the common core from which
they are presented. This phenomenon in medicinal chemistry29
may be considered the small-molecule correlate of the observa-
tion that diverse proteins often share a similar fold without any
primary sequence similarity.
only in high micromolar concentrations. In order to enhance
solubility and activity and to gain insight into the ligand-protein
interface, we embarked on a synthetic program to first define,
and then exploit, the permissive or conserved functional groups
on the 2-iminothiazolidinone core. We synthesized a variety of
compounds, with particular emphasis on ω-polar groups (Scheme
1), and subsequently, the dipeptides (Schemes 2 and 3) fused
to the thiazolidinone at N-3. We now present the preparation
of these more hydrophilic 3-substituted 2-imino-5-arylidenethi-
azolidinones and their T3SS inhibitory activity.
Chemistry
Synthetic methods for thiazolidinones are well described in
the literature. Our synthesis of the aminomethylphenyl and
aminohexyl analogues, shown in Scheme 1, is an adaptation of
the method of Klika et al.30
The regiochemical outcome of the cyclization to 2-iminothia-
zolidinone can be controlled by the choice of reagents and
reaction conditions, but unless sufficient differences exist
between the nucleophilic strengths of the two nitrogens, the more
thermodynamically favored 5-arylidene product will be obtained
after the Knovenagel reaction.20,21 Under the conditions in
Our initial panel of thiazolidinones was useful for character-
izing the target and for supporting the hypothesis that very
specific molecular interactions were disrupting a protein-protein
interaction, but these compounds were poorly soluble and active

Thiazolidinone Inhibitors of T3SS
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7067
Scheme 3
Scheme 1 we obtained a single compound from the cyclization
of the alkyl/aryl thioureas but a mixture of regiomers for all
aryl/aryl thioureas. We separated 5 and 7 by preparative reverse
phase HPLC at the protected penultimate stage (optimum for
this procedure) before cleaving the Boc group to, respectively,
the ValTyr (42), AlaTrp (44), and OrnDht (49) analogues. The
D-alanine dipeptides 20b and 22b were obtained from D-alanine,
and the D-biphenylalanine dipeptide 55b was obtained from
D-biphenylalanine.
1
6 and 8. The 1D and 2D H NMR spectra of 6 and 8 clearly
Results and Discussion
indicated that the two were regiomers, and the mass spectra
gave the same parent ions but a different fragmentation pattern
for each compound. Absolute assignment of structure was
obtained by crystallography of 8 (data not shown). The general
method of Scheme 1 was used to generate the compounds in
Table 1.
Compounds were evaluated as T3SS inhibitors by measuring
secretion of the Salmonella enterica serovar Typhimurium
effector protein SipA (Tables 1 and 2 and Figure 2).10
Replacement of the syringyl ring (28-38, 40), with the
exception of p-thiomethylphenyl (27, 39) or 3,4-dimethoxy (38)
was generally a poorly tolerated modification. The 2-imino-
substituted analogue 6 was also somewhat weaker in inhibitory
activity than 1. Amido/imino regiomeric pairs that had proven
difficult to separate (36-40) were each tested as a mixture with
the intention of resolving them only if the inhibitory activity
warranted additional effort. Within this data set, none of these
compounds qualified. In contrast, multiple analogues with
different N-3 groups were active (Table 1), particularly the N-3
ω-carboxy (23) and carbalkoxy (24) analogues that retained a
significant degree of SipA inhibition at concentrations of 1 µM
or less. We used N-3 as the position of choice for a series of
dipeptides with the intention of identifying additional beneficial
functional groups.
Short peptides are an expeditious way to survey functional
groups, stereochemistry, and molecular shape. Significantly for
this work, by appending dipeptides to the nonpeptidic thiazo-
lidinone scaffold, the hydrophilicities of the ensuing hybrid
molecules can be varied across several orders of magnitude. In
order to investigate the SAR of the dipeptide component, we
prepared a diverse set of analogues presenting the dipeptide from
R3 and holding the vinylidene (R1) and 2-imino (R2) groups
constant. Our selection of amino acids emphasized arginine,
tryptophan, and tyrosine in accordance with residues that are
statistically overrepresented in protein-protein interactions.31
Other residues were selected for their neutrality (alanine), charge
(ornithine, glutamic acid), steric demand (valine) or to probe
hydrogen-bonding (thiazolidinylalanine, pyridylalanine) or hy-
drophobic contacts (biphenylalanine, tetrahydroisoquinoline
carboxylate). Suitably protected dipeptides were assembled and
incorporated into the original synthetic route. With arginine in
the terminal position, a protected ornithine was used to provide
a common precursor to both the primary amine 20a and, upon
guanylation,32 22a. The solution synthesis of dipeptidyl thia-
zolidinones is illustrated by the preparation of 22a (Scheme 2).
The linear directionality of the Scheme 2 route, from peptide
C-terminus to complete N3-peptidyl thiazolidinone, prompted
the investigation of a solid phase route for the rapid generation
of dipeptide libraries. A Tentagel Rink resin was loaded with
the C-terminal residue, and the Scheme 1 and Scheme 2 methods
were adapted to solid phase by increasing the stoichiometry of
the reagents (Scheme 3). In the solid phase route, arginine could
be added directly as the pentamethylsulfonylbenzofuran (Pbf)
protected derivative. Yields and purities were comparable to
those obtained in solution chemistry. The chiral integrity was
generally preserved for the dipeptides, with the exception of
The inhibitory potencies of the dipeptides are shown in Table
2. Many of the dipeptides, while having improved solubility,33
retained but did not exceed a level of potency comparable to
that of 1. We selected 1, 7, 8, 52, and 55a as representative
compounds in the 5-substituted-2-aryliminothiazolidinone class
to test for growth inhibitory activity against S. typhimurium,
and no effect was observed at concentrations up to 1 mM (data
not shown). These data support the conclusion that effects of
these compounds are mediated by direct inhibition of the T3SS
secretory process and not a result of inhibition of bacterial
growth.
The more inhibitory dipeptides, 55a and 55b and to a lesser
extent 20a and 20b, have a common motif of a neutral residue
(alanine or biphenylalanine) in the proximal position coupled
to a cationic residue (ornithine or arginine) in the terminal
position. It is interesting that other dipeptides that ostensibly
share this motif are much weaker: Arginine is in the terminal
position of analogues 22a, 22b, 50, and 51, and ornithine is in
the terminal position of 56, but the inhibitory activity of all of
these remains comparable to that of 1. Appending a peptide

7068 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Kline et al.
c
Table 1. Synthetic Diversity Set Generated to Evaluate Modifications of R1, R2, and R3
a R2 and R3 were regiomeric mixtures of p-methoxyphenyl and phenyl, with the major regiomer indicated in the column. b Significant (>50%) inhibition
only at 1 mM. c The IC50 values are calculated from the percent inhibition of SipA secretion, as determined by Western blot, using a minimum of three
concentrations of inhibitor. Compounds 36-40 are regiomeric pairs in which the major isomer has R2 as p-methoxyphenyl and R3 as phenyl, and the minor
isomer has R3 as p-methoxyphenyl and R2 as phenyl.
does not necessarily improve the inhibitory activity of the
3-substituent; glutamyl dipeptide 53, which might be regarded
as a congruent hybrid of two modestly active analogues, acid
25 (66 µM) and biphenyl peptide 52 (13 µM), showed no
significant improvement over acid 25 and was weaker than the
corresponding tert-butyl ester 24. Taken together, these data
suggest that an interactive relationship between the two residues
is likely to be more important than the independent contributions
of the individual side chain functional groups. The two residues
of the epimeric active dipeptides might be acting in concert to
fold the ligand into the correct pose for the binding epitope,
and there may be more than a single solution to the problem of
peptide-induced, or peptide-stabilized, ligand folding. The higher
activities of 52 vs 56 and of 55a/b vs 54 indicate that the
dipeptide’s effect, however subtle and complex, is the result of
specific contacts rather than some general phenomenon such
as increased access to the target.
Protein-protein interactions are extended, often over 1000-
3000 Å2, but are neither random nor unstructured. The interfaces
are frequently coiled coils of helices or other highly defined
secondary structures.34 The residues making the most significant
contributions to this interface may be buried inside the shape
they create, limiting access to external ligands.35 This model
describes quaternary structures in the T3SS, including the 3500
Å2 surface of the EscJ trimeric interface in which 35% of that
interface is buried.36 Strategies to design ligands for protein-
protein interfaces have been described. These strategies em-

Thiazolidinone Inhibitors of T3SS
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7069
Table 2. Dipeptides Presented from N-3a
a The IC50 values are calculated from the percent inhibition of SipA secretion, as determined by Western blot, using a minimum of three concentrations
of inhibitor. The R carbons are all S (retention of the L-amino acid stereochemistry) unless otherwise designated. For the discrete epimers 44a/44b and
49a/49b the absolute configurations were not determined. Dihydrotryptophan analogues 49a/49b and 50, derived from commercially supplied racemic material,
are correspondingly racemic at the γ carbon.
phasize the predominance of hydrophobic interactions and
aromatic residues in the “hotspots” that drive the particular
protein-protein interaction.34,37 In this work we have identified
an epimeric pair of compounds, 55a and 55b, that increase the
T3SS inhibition of our initial HTS hit more than 10-fold. These
dipeptides are characterized by a combination of a cationic group
and a functional group having the potential for hydrophobic
interactions. Our current effort to determine the optimum
consensus pharmacophore takes into account the accumulated
data from the Table 1 and Table 2 compounds. From the
perspective of future drug evolution, it is encouraging to observe
that the more active compounds such as 55a are an order of
magnitude more hydrophilic than 1, suggesting that potent
inhibitors of T3SS can be developed that will have acceptable
pharmacokinetic properties.
Conclusion
Virulence targets represent an emerging concept in antibacte-
rial therapy3,38,39 for which there are examples of compounds
that inhibit virulence functions and have demonstrated efficacy
in mouse40-42 and other8 in vivo models. The challenge for
the further development of antivirulence therapeutics will require
the development of compounds with adequate pharmacokinetic
and activity profiles to promote incentive for further develop-
ment. A key issue when considering a given virulence target is
whether drugs successfully directed against it will have suf-

7070 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Kline et al.
Figure 2. Inhibition of the secretion of the virulence protein SipA into the culture medium by S. typhimurium. Shown are representative Western
blots of SipA secreted into culture medium from S. typhimurium grown in the presence of compound 1 (above) and 52 (below) at the concentrations
(in µM) indicated above each blot. The results for 55a were comparable to those shown for 52.10 Secretion in the absence of compound but the
presence of 5% DMSO is shown (“0”) at the far left.
2H), 3.65 (dd, J ) 6.3, 6.3 Hz, 2H), 7.21-7.57 (m, 5H). MS m/z
374 [M + Na]+, 274 [M - Boc + Na]+.
ficiently broad spectrum efficacy to be clinically useful. This
work suggests that dipeptide derivatives of the thiazolidinone
scaffold may provide a critical step toward the validation of
this strategy and the development of novel therapeutics.
General method B for generation of 2-iminothiazolidinones
is illustrated for the preparation of (Z)-tert-butyl-4-(4-oxo-2-
phenylthizolidin-3-ylideneamino)benzylcarbamate 3 and (Z)-
tert-butyl4-(4-oxo-3-phenylthizolidin-2-ylideneamino)benzylcar-
bamate 4. To a stirred solution of 2 (435 mg, 1.22 mmol) in 40
mL of CH2Cl2 were added successively DIEA (424 µL, 2.44 mmol)
and methyl bromoacetate (115 µL, 1.22 mmol). The reaction
mixture was stirred overnight at ambient temperature and concen-
trated in vacuo, and the residue was purified via silica gel
chromatography using a gradient from 2% to 20% MeOH in CH2Cl2
to give 3 and 4 (484 mg, 1.2 mmol) as a mixture of regiomers. 1H
NMR (300 MHz, CDCl3, δ): 1.48 (s, 9H), 3.99 (s, 2H), 4.29 (br d,
J ) 5.1 Hz, 1H), 4.37 (br d, J ) 5.7 Hz, 1H), 4.90 (br, 1H),
6.89-7.57 (m, 9H). MS m/z 398 [M + H]+, 420 [M + Na]+, 342
[M - t-Bu + H]+, 364 [M - t-Bu + Na]+.
Experimental Section
Chemistry. General. All reactions were run under an atmosphere
of dry nitrogen. Reagents and solvents were obtained in the highest
available purity and used without further purification unless
1
indicated. H NMR spectra were obtained on a 300 MHz (Bruker
AV300 or AV301) or 500 MHz (Bruker AV500 or Varian)
instrument. 13C NMR spectra were obtained on a 500 MHz Bruker
AV500. Identity of the compounds was confirmed by mass
spectrometry. The compound solution was infused into the elec-
trospray ionization source operating in positive ion mode. Low
resolution spectra were obtained on the Esquire LC ion trap mass
spectrometer (Bruker Daltonics, Billerica, MA). Accurate mass
measurements were performed on the APEX Qe 47 Fourier
transform ion cyclotron resonance mass spectrometer (Bruker
Daltonics, Billerica, MA). LC-MS measurements to determine
log P values were obtained on a Waters Quattro Micro mass
spectrometer interfaced with a Waters Alliance 2795 liquid chro-
matography instrument. Normal phase silica gel purifications were
done using a Biotage SP4 instrument using the cartridges supplied
by Biotage. RP-HPLC was done on a Varian instrument equipped
with a diode array ultraviolet detector. For preparative reverse phase
chromatography a 10 mm × 250 mm C18 5 µm column at a flow
rate of 4.6 mL/min was used. For analytical reverse phase
chromatography a 4.6 mm × 250 mm C18 5 µm column at a flow
rate of 1 mL/min was used. Ultraviolet detection was at 215 and
either 254 or 360 nm. Unless otherwise specified, buffer A was
0.05% TFA in H2O, buffer B was 0.05% TFA in acetonitrile. Thin
layer chromatography was done using 0.2 mm polygram SIL G/UV
plates (Alltech, Deerfield, IL) or Si250F (J. T. Baker, Phillipsburg,
NJ) plates, developed using mobile phases of varying compositions
of ethyl acetate/hexane, MeOH/CH2Cl2, or MeOH/CHCl3 and
visualized by UV light supplemented by vanillin, ninhydrin, and
other solution stains where appropriate.
(Z)-tert-Butyl 6-(4-oxo-2-phenylimino)thiazolidin-3-yl)hexylcar-
bamate 10 was prepared by general method B on a 1.14 mmol scale
1
from 9 to give 471 mg, 1.1 mmol, as a colorless oil. H NMR (300
MHz, CDCl3, δ): 1.51 (s, 9H), 1.38-1.77 (m, 8H), 3.11 (dd, J ) 6.3,
6.3 Hz, 2H), 3.81 (s, 2H), 3.86 (dd, J ) 7.2, 7.2 Hz, 2H), 4.53 (br,
1H), 6.95-7.39 (m, 5H). 13C NMR (500 MHz, CDCl3, δ): 26.4, 26.5,
27.1, 285, 29.9, 32.7, 40.5, 43.07, 79.0, 121.0, 124.6, 133.3, 148.26,
154.4, 155.9, 171.8. MS m/z 392 [M + H]+, 414 [M + Na]+, 336
[M - t-Bu + H]+, 292 [M - Boc + H]+.
General method C to introduce the 5-arylidene or 5-vinylidene
substituent is illustrated by the synthesis of tert-butyl4-((Z)-((Z)-
5-(4-hydroxy-3,5-dimethoxybenzylidine)-4-oxo-3-phenylthiazolidin-
2-ylidene)amino)benzylcarbamate 5 and tert-butyl 4-((Z)-((Z)-5-
(4-hydroxy-3,5-dimethoxybenzylidne)-4-oxo-2-phenylthiazolidin-
3-ylidene)amino)benzylcarbamate 7. A solution of the mixture of 3
and 4 (484 mg, 1.22 mmol), syringaldehyde (222 mg, 1.22 mmol),
and piperidine (163 µL, 1.64 mmol) in 15 mL of EtOH was refluxed
for 19 h. The reaction mixture was cooled to ambient temperature
and treated with hexane to precipitate the product, which was collected,
washed with additional hexane, and dried to give 565 mg of a mixture
of 5 and 7.
Preparative reverse phase HPLC using a gradient of 65-85% B
in A over 15 min enabled the separation of the two pure compounds
as well as an overlap band that was held in reserve for further
purification. 5 (48.5 mg, 0.09 mmol) 1H NMR (300 MHz, DMSO-
d6, δ): 1.41 (s, 9H), 3.77 (s, 6H), 4.06-4.14 (m, 2H), 6.90 (s, 2H),
6.95-7.59 (m, 9H), 7.75 (s, 1H), 9.32 (br, 1H). MS m/z 562 [M +
H]+, 584 [M + Na]+. 7 (54.2 mg, 0.10 mmol) 1H NMR (300 MHz,
DMSO-d6, δ): 1.43 (s, 9H), 3.72 (s, 6H), 4.15-4.23 m, 2H), 6.89
(s, 2H), 7.00-7.64 (m, 9H), 7.76 (s, 1H), 9.31 (br, 1H). MS m/z
562 [M + H]+, 584 [M + Na]+.
tert-Butyl-6-((2Z,5Z)-6-(4-hydroxy-3,5-dimethoxybenzlidene)-
4-oxo-2-(phenylimino)thiazolidin-3-yl)hexylcarbamate 11 was
prepared by general method C to give 548 mg, 0.95 mmol, as an
orange solid. 1H NMR (300 MHz, CDCl3, δ): 1.42 (s, 9H),
1.46-1.54 (m, 6H), 1.71-1.81 (m, 2H), 3.03 (dd, J ) 6.5, 6.6 Hz,
2H), 3.86 (s, 6H), 3.96 (dd, J ) 4.2, 4.2 Hz, 2H), 5.28 (br, 1H),
General method
A for generation of thioureas is
illustrated for the preparation of tert-butyl 4-(3-phenyl-
thioureido)benzylcarbamate 2. To a stirred solution of phenyl
isothiocyanate (244 µL, 2 mmol) in 50 mL of CH2Cl2 was added
(Boc) 4-aminomethylaniline (244 mg, 2 mmol), and the solution
was stirred for 5 days until TLC (95:5 CH2Cl2/MeOH) showed the
reaction to be complete. The solvent was removed in vacuo and
the white solid collected and washed with hexane/diethyl ether to
1
give 2. Yield: 6.14 mg, 1.72 mmol. H NMR (300 MHz, CDCl3,
δ): 1.47 (s, 9H), 4.32 (d, J ) 5.9 Hz, 2H), 4.91 (br, 1H), 7.28-7.86
(m, 9H). MS m/z 358 [M + H]+, 380 [M + Na]+, 324 [M - t-Bu
+ H]+, 346 [M - t-Bu + Na]+.
tert-Butyl 6-(3-phenylthioureido)hexylcarbamate 9 was pre-
pared by general method A to give 9 (593 mg, 1.70 mmol) that
was used without further purification. 1H NMR (300 MHz, CDCl3,
δ): 1.50 (s, 9H), 1.34-1.58 (m, 8H), 3.10 (dd, J ) 6.3, 6.3 Hz,

Thiazolidinone Inhibitors of T3SS
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7071
6.81 (s, 2H), 7.07 (d, J ) 7.5 Hz, 2H), 7.23 (t, J ) 7.4 Hz, 1H),
7.43 (t, J ) 7.9 Hz, 2H), 7.66 (s, 1H). 13C NMR (500 MHz, CDCl3,
δ): 23.6, 24.4, 26.4, 26.5, 27.4, 28.5, 29.5, 42.9, 45.3, 55.7, 56.0,
81.4, 108.0, 121.0, 121.3, 124.5, 129.2, 129.2, 132.6, 148.5, 149.4,
151.0, 167.3. MS m/z 556 [M + H]+, 578 [M + Na]+, 500 [M -
t-Bu + H]+.
1H NMR (300 MHz, CD3OD, δ): 1.45 (s, 9H), 1.51-1.65 (m, 3H),
1.65-1.84 (m, 1H), 3.07 (t, J ) 6.2 Hz, 2H), 3.09-3.24 (m, 1H).
13C NMR (500 MHz, CD3OD, δ): 29.8, 31.4, 36.3, 43.6, 58.0, 82.5,
158.6, 180.3. MS m/z 232 [M + H]+, 254 [M + Na]+. Tetrahy-
drofuran (5 mL) was added to a dry mixture of Cbz L-alanine (406
mg, 1.82 mmol) and carbonyldiimidazole (353 mg, 2.18 mmol),
and the reaction mixture was allowed to stir for 1.5 h. To this was
added amine 14, along with DIEA (350 µL, 2.00 mmol), and the
reaction mixture was stirred for 3 days until determined complete
by TLC (95:5 CH2Cl2/MeOH). The volatiles were removed in
vacuo, and a 4% aqueous solution of NaHCO3 (10 mL) was added
to precipitate the product. The crude solid was collected and purified
on silica gel using a gradient from 0% to 10% MeOH in CH2Cl2
to give R-Cbz-δ-Boc dipeptide 15 (226 mg, 0.52 mmol). 1H NMR
(300 MHz, CD3OD, δ): 1.36 (d, J ) 7.2 Hz, 3H), 1.44 (s, 9H),
1.48-1.74 (m, 3H), 1.76-1.94 (m, 1H), 2.97-3.12 (m, 2H), 4.15
(q, J ) 7.1 Hz, 1H), 4.27-4.41 (m, 1H), 5.13 (s, 2H), 7.26-7.43
(m, 5H). MS m/z 437 [M + H]+, 459 [M + Na]+. Hydrogenolysis
of the Cbz group over 10% palladium on carbon (225 mg) in 38
mL of 14:1 EtOH/DMF for 2 h gave R-amino δ-Boc dipeptide
General method D for deprotection of acid-labile protecting
groups is illustrated by the preparation of (2Z,5Z)-2-(4-(ami-
nomethyl)phenylimino)-5-(4-hydroxy-3,5-dimethoxybenzyidene)-
3-phenylthiazolidin-4-one 6. Ice cold TFA (0.5 mL) was added
to neat 5 (4 mg, 0.007 mmol) cooled in an ice bath. The reaction
was complete by HPLC in 15 min, and the reaction mixture was
concentrated in vacuo to a sticky brown oil. Trituration with diethyl
ether gave a solid that was purified on reverse phase HPLC
(65-85% B in A over 12 min) to give 6 (3.0 mg, 0.004 mmol) as
1
the TFA salt. H NMR (500 MHz, CD3CN, δ): 3.83 (s, 6H), 4.25
(s, 2H), 6.85 (s, 2H), 7.02 (d, J ) 8.5 Hz, 2H), 7.22 (t, J ) 8.2 Hz,
1H), 7.42 (t, J ) 8.5 Hz, 2H), 7.60 (d, J ) 8.4 Hz, 2H), 7.67 (d,
J ) 8.4 Hz, 2H), 7.74 (s, 1H). MS m/z 462 [M + H]+. HRMS
(m/z): [M + H]+ calcd for C25H23N3O4S, 462.1488; found 462.1491.
(2Z,5Z)-3-(4-(Aminomethyl)phenylimino)-5-(4-hydroxy-3,5-
dimethoxybenzyidene)-2-phenylthiazolidin-4-one 8 was obtained
from 7 (6.9 mg, 0.01 mmol) by general method D and purified on
reverse phase HPLC (65-85% B in A over 12 min) to give 8 (5.5
1
amide 16 (147.8 mg, 0.49 mmol). H NMR (300 MHz, CD3OD,
δ): 1.30 (d, J ) 6.9 Hz, 3H), 1.45 (s, 9H), 1.50-1.75 (m, 3H),
1.77-1.89 (m, 1H), 3.08 (td, J ) 6.6, 1.7 Hz, 2H), 3.48 (q, J )
6.9 Hz, 1H), 4.30-4.49 (m, 1H). MS m/z 303 [M + H]+.
General method G for the formation of N-phenyl-N′-amido-
dipeptidyl thioureas is illustrated by the preparation of tert-
butyl (S)-5-amino-5-oxo-4-((S)-2-(3-phenylthioureido)propan-
amido)pentylcarbamate 17. To a solution of 16 (144 mg, 0.48
mmol) in 1.5 mL of MeOH was added phenyl isothiocyanate (180
µL, 0.95 mmol), and the reaction mixture was stirred overnight,
during which time the product precipitated. The volatiles were
removed in vacuo, hexane was added, and the suspension was
cooled at 4 °C for 1 h. The crude thiourea was collected and washed
with diethyl ether to give 17 (204 mg, 0.47 mmol). 1H NMR (500
MHz, CD3OD, δ): 1.45 (s, 9H), 1.46 (d, J ) 7.4 Hz, 3H), 1.51-1.65
(m, 2H), 1.65-1.76 (m, 1H), 1.85-1.94 (m, 1H), 2.98-3.17 (m,
2H), 4.32-4.44 (m, 1H), 4.97 (q, J ) 7.0 Hz, 1H), 7.22 (t, J ) 7.3
Hz, 1H), 7.39 (t, J ) 7.9 Hz, 2H), 7.45 (d, J ) 7.9 Hz, 2H). MS
m/z 438 [M + H]+, 460 [M + Na]+.
tert-Butyl (S)-5-amino-5-oxo-4-((S)-2-((Z)-4-oxo-2-(phenylimi-
no)thiazolidin-3-yl)propanamido)pentylcarbamate 18 was pre-
pared by general method B in THF on a 1.77 mmol scale to give,
after silica gel chromatography using a gradient from 0% to 10%
MeOH in CH2Cl2, 603.8 mg (1.27 mmol) of product. 1H NMR
(300 MHz, CD3OD, δ): 1.44 (s, 9H), 1.49-1.78 (m, 3H), 1.66 (d,
J ) 7.0 Hz, 3H), 1.82-2.01 (m, 1H), 2.50-3.10 (m, 2H), 3.97 (s,
2H), 4.33-4.49 (m, 1H), 5.25 (q, J ) 7.0 Hz, 1H), 6.98 (d, J )
7.9 Hz, 2H), 7.14 (t, J ) 7.4 Hz, 1H), 7.35 (t, J ) 7.8 Hz, 2H).
13C NMR (500 MHz, CDCl3, δ): 17.7, 31.0, 32.5, 32.8, 36.8, 43.3,
56.5, 56.9, 83.5, 125.1, 129.0, 133.4, 151.3, 157.5, 160.8, 173.0,
175.6, 178.2. MS m/z 478 [M + H]+, 500 [M + Na]+.
tert-Butyl (S)-5-amino-4-((S)-2-((2Z,5Z)-5-(4-hydroxy-3,5-
dimethoxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)-
propanamido)-5-oxopentylcarbamate 19 was prepared by general
method C from the corresponding thiazolidinone on a 0.44 mmol
scale. The crude solid was purified via silica gel chromatography
using a gradient from 0% to 10% MeOH in CH2Cl2 to give 224
mg (0.35 mmol) of product. 1H NMR (300 MHz, CD3OD, δ): 1.41
(s, 9H), 1.47-1.68 (m, 3H), 1.72 (d, J ) 7.0 Hz, 3H), 1.81-2.02
(m, 1H), 3.03 (t, J ) 6.5 Hz, 2H), 3.83 (s, 6H), 4.35-4.45 (m,
1H), 5.43 (q, J ) 7.0 Hz, 1H), 6.81 (s, 2H), 7.07 (d, J ) 7.4 Hz,
2H), 7.20 (t, J ) 7.4 Hz, 1H), 7.41 (t, J ) 7.8 Hz, 2H), 7.71 (s,
1H). 13C NMR (500 MHz, CDCl3, δ): 18.0, 30.82, 32.4, 32.9, 43.3,
56.5, 57.0, 60.4, 83.4, 111.4, 122.3, 125.2, 125.3, 129.1, 129.2,
133.4, 136.3, 141.2, 151.39, 153.3, 160.7, 170.4, 173.2, 178.2. MS
m/z 642 [M + H]+, 664 [M + Na]+.
1
mg, 0.009 mmol) as the TFA salt. H NMR (500 MHz, CD3CN,
δ): 3.84 (s, 6H), 4.14 (s, 2H), 6.86 (s, 2H), 7.05 (d, J ) 8.3 Hz,
2H), 7.46-4.62 (m, 7H) 7.75 (s, 1H). MS m/z 462 [M + H]+.
HRMS (m/z): [M + H]+ calcd for C25H23N3O4S, 462.1488; found
462.1486.
(2Z,5Z)-3-(6-Aminohexyl)-5-(4-hydroxy-3,5-dimethoxyben-
zylidene)-2-(phenylimino)thiazolidin4-one 12 was obtained from
11 on a 0.95 mmol scale to give, after two silica gel chromatog-
raphies, the first using a gradient from 0% to 10% MeOH in CHCl3
and the second from 1% to 50% MeOH in CHCl3, 456 mg (0.80
mmol). 1H NMR (300 MHz, CDCl3, δ): 1.42-1.45 (m, 4H),
1.64-1.66 (m, 2H), 1.77-1.79 (m, 2H), 2.65 (br, 2H), 2.89 (t, J
) 7.5 Hz, 2H), 3.84 (s, 6H), 3.97 (t, J ) 7.5 Hz, 2H), 6.71 (s, 2H),
6.99 (d, J ) 8.4 Hz, 2H), 7.14-7.19 (m, 1H), 7.28-7.38 (m, 2H),
7.63 (s, 1H). 13C NMR (500 MHz, DMF-d7, δ): 30.2, 30.5, 31.3,
43.6, 47.0, 53.0, 60.3, 112.5, 120.6, 122.1, 123.0, 125.5, 129.0,
133,5, 133.6, 135.5, 152.6, 152.8, 154.4, 162.9, 163.1, 163.4, 166.1,
166.4, 166.6, 170.5. MS m/z 456 [M + H]+. HRMS (m/z): [M +
H]+ calcd for C24H30N3O4S, 456.1957; found 456.1946.
General method E for amidation is illustrated by the prep-
aration of (S)-benzyl 1-amino-5-tert-butoxycarbonylamino-1-
oxopentan-2-ylcarbamate 13. To a suspension of Z-Orn(Boc)-
OH (733 mg, 2.0 mmol) in 3.5 mL of THF was added
N-methylmorpholine (0.48 mL, 4.4 mmol). The reaction mixture
was cooled in an ice bath, and isobutyl chloroformate (0.31 mL,
2.4 mmol) was added. After the mixture was stirred for 1 h on ice,
concentrated NH4OH (0.60 mL, 10 mmol) was added, and the
reaction mixture was stirred an additional 30 min. The reaction
mixture was partitioned between 4:1 CHCl3/isopropanol and
saturated aqueous sodium carbonate. The organic phase was
separated, washed with saturated brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified on silica
gel using a gradient from 0% to 25% MeOH in CH2Cl2 to give 13
1
670.2 mg (1.84 mmol). H NMR (500 MHz, CD3OD, δ): 1.46 (s,
9H), 1.51-1.72 (m, 3H), 1.72-1.88 (m, 1H), 2.99-3.16 (m, 2H),
4.07-4.18 (m, 1H), 5.05-5.22 (m, 2H), 7.26-7.46 (m, 5H). 13C
NMR (500 MHz, CD3OD, δ): 30.08, 31.4, 33.3, 43.4, 58.5, 70.3,
82.55, 131.5, 131.6, 132.1, 140.8, 161.1, 180.2. MS m/z 366 [M +
H]+, 388 [M + Na]+.
General method F for hydrogenolysis, coupling, and subse-
quent deprotection to the free r-amino dipeptide amide is
illustrated by the synthesis of tert-butyl (S)-5-amino-4-((S)-2-
aminopropanamido)-5-oxopentylcarbamate 16. N-Protected 13
(664 mg, 1.82 mmol) was hydrogenated over 10% palladium on
carbon (650 mg) in 50 mL of 4:1 EtOH/DMF for 2.5 h. The catalyst
was filtered off and the filtrate concentrated in vacuo to give 14
(420 mg, 1.82 mmol) that was used without further purification.
(S)-5-Amino-2-((S)-2-((2Z,5Z)-5-(4-hydroxy-3,5-dimethoxy-
benzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propanami-
do)pentanamide 20a was obtained from 19 (102 mg, 0.16 mmol)
by general method D and purified on reverse phase HPLC (10-95%
B in A over 25 min) to give 20a (5.1 mg, 0.008 mmol) as the TFA

7072 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Kline et al.
1
salt. H NMR (300 MHz, CD3OD, δ): 1.75 (d, J ) 7.0 Hz, 3H),
(S)-5-Guanidino-2-((R)-2-((2Z,5Z)-5-(4-hydroxy-3,5-dimethoxy-
benzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propanamido)-
pentanamide 22b was prepared by general method H via the free
amine 20b (200 mg, 0.37 mmol) and purified via silica gel chroma-
tography using a gradient from 1% to 20% MeOH in CH2Cl2 to give
the bis-Boc guanidine 21b (226 mg, 0.29 mmol). 1H NMR (300 MHz,
CD3OD, δ): 1.47 (s, 9H), 1.52 (s, 9H), 1.61-1.85 (m, 3H), 1.76 (d, J
) 6.9 Hz, 3H), 1.88-2.07 (m, 1H), 3.22-3.46 (br, solvent envelope
over CH2), 3.82 (s, 6H), 4.45-4.56 (m, 1H), 5.37-5.55 (m, 1H), 6.79
(s, 2H), 7.04 (d, J ) 7.7 Hz, 2H), 7.19 (t, J ) 7.2 Hz, 1H), 7.39 (t, J
) 7.1 Hz, 2H), 7.71 (s, 1H). 13C NMR (500 MHz, CD3OD, δ): 16.9,
29.5, 30.9, 30.9, 31.2, 43.9, 56.6, 57.2, 59.5, 83.0, 87.0, 110.6, 111.7,
121.8, 124.9, 128.5, 128.7, 133.0, 135.9, 151.9, 152.2, 156.6, 156.7,
160.3, 167.2, 170.7, 174.2, 179.3. MS m/z 785 [M + H]+; 807 [M +
Na]+. The free guanidine, purified on reverse phase HPLC (10-75%
B in A over 30 min), gave 22b (2.0 mg, 0.003 mmol) as the TFA salt.
1H NMR (300 MHz, CD3OD, δ): 1.57-1.73 (m, 3H), 1.75 (d, J )
7.0 Hz, 3H), 1.90-2.10 (m, 1H), 3.10 (t, J ) 6.3 Hz, 2H), 3.83 (s,
6H), 4.42-4.58 (m, 1H), 5.45 (q, J ) 7.0 Hz, 1H), 6.80 (s, 2H), 7.06
(d, J ) 7.4 Hz, 2H), 7.21 (t, J ) 7.5 Hz, 1H), 7.41 (t, J ) 7.8 Hz,
2H), 7.72 (s, 1H). 13C NMR (500 MHz, CD3OD, δ): 16.8, 29.0, 32.4,
44.5, 56.6, 56.8, 59.5, 111.7, 119.5, 121.9, 124.9, 128.8, 133.1, 136.0,
151.8, 152.3, 153.9, 161.2, 165.7, 166.0, 174.4, 179.1. MS m/z 584
[M + H]+. HRMS (m/z): [M + H]+ calcd for C27H34N7O6S, 584.2286;
found 584.2279; [M + Na]+ calcd for C27H33N7O6NaS, 606.2105;
found 606.2115.
2-((2Z,5Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)acetic acid 23 (329 mg, 0.70 mmol)
was obtained by general method D and purified by silica gel
chromatography using a gradient from 0% to 10% MeOH in CH2Cl2
to give 23 (235.7 mg, 0.57 mmol). 1H NMR (300 MHz, DMF-d7,
δ): 3.70 (br s, 1H), 4.02 (s, 6H), 4.85 (s, 2H), 7.11 (s, 2H), 7.25
(d, J ) 7.4 Hz, 2H), 7.39 (t, J ) 7.4 Hz, 1H), 7.62 (t, J ) 7.8 Hz,
2H), 7.95 (s, 1H), 9.62 (br s, 1H). 13C NMR (500 MHz, DMF-d7,
δ): 53.1, 60.3, 112.4, 112.6, 122.0, 125.5, 128.3, 129.1, 133.6, 133.7,
126.0, 143.5, 152.2, 152.8, 154.1, 170.2. MS m/z 415 [M + H]+,
437 [M + Na]+. HRMS (m/z): [M + Na]+ calcd for C20H18N2O6-
NaS, 437.0783; found 437.0787.
4-((2Z,5Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-
(phenylimino)thiazolidin-3-yl)butanoic acid 25 (190 mg, 0.38
mmol) was obtained by general method D and purified by silica
gel chromatography using a gradient from 0% to 10% MeOH in
CHCl3 to give 25 (89 mg, 0.20 mmol). 1H NMR (300 MHz, CDCl3,
δ): 1.86-2.14 (m, 4H), 2.62 (br s, 1H), 4.00 (s, 6H), 4.07-4.22
(m, 2H), 6.83 (s, 2H), 7.34 (d, J ) 6.9 Hz, 2H), 7.47-7.64 (m,
3H), 7.84 (s, 1H). 13C NMR (500 MHz, CDCl3, δ): 13.8, 30.3,
60.6, 68.9, 111.6, 119.8, 128.5, 131.8, 133.9, 134.0, 134.8, 136.5,
137.7, 139.2, 142.0, 151.6, 176.5. MS m/z 443 [M + H]+, 465 [M
+ Na]+. HRMS (m/z): [M + Na]+ calcd for C22H22N2O6NaS,
465.1096; found 465.1106.
(S)-5-Amino-N-((S)-1-amino-3-(biphenyl-4-yl)-1-oxopropan-
2-yl)-2-((2Z,5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-
2-(phenylimino)thiazolidin-3-yl)pentanamide 52. Compound 52
was obtained by general method D on a 0.052 mmol scale and
purified on reverse phase HPLC (10-75% B in A over 30 min) to
give 52 (6.0 mg, 0.007 mmol) as the TFA salt. 1H NMR (300 MHz,
CD3OD, δ): 1.60-1.87 (m, 2H), 2.35 (q, J ) 7.6 Hz, 2H),
2.94-3.13 (m, 3H), 3.16-3.28 (m, 1H), 3.77 (s, 6H), 4.56-4.71
(m, 1H), 5.35 (t, J ) 7. Five Hz, 1H), 6.72 (s, 2H), 6.91 (d, J )
7.5 Hz, 2H), 7.17 (t, J ) 7.4 Hz, 1H), 7.21-7.37 (m, 8H), 7.40 (d,
J ) 8.1 Hz, 2H), 7.67 (d, J ) 7.1 Hz, 1H), 7.69 (s, 1H). 13C NMR
(500 MHz, CD3OD, δ): 28.1, 28.2, 40.7, 43.0, 58.6, 59.4, 59.9,
111.6, 120.9, 124.9, 128.2, 128.9, 130.3, 130.8, 130.8, 132.3, 133.0,
133.2, 136.5, 139.9, 142.50, 143.5, 144.4 151.3, 152.2, 153.5, 170.4,
172.9, 178.8. MS m/z 694 [M + H]+. HRMS (m/z): [M + H]+
calcd for C38H40N5O6S, 694.2694; found 694.2687; [M + Na]+
calcd for C38H39N5O6NaS, 716.2523; found 716.2513.
1.79-1.92 (m, 3H), 1.96-2.18 (m, 1H), 2.85-3.05 (m, 2H), 3.83
(s, 6H), 4.44-4.57 (m, 1H), 5.46 (q, J ) 7.0 Hz, 1H), 6.80 (s,
2H), 7.07 (d, J ) 7.3 Hz, 2H), 7.22 (t, J ) 7.4 Hz, 1H), 7.42 (t, J
) 7.8 Hz, 2H), 7.71 (s, 1H). 13C NMR (500 MHz, CD3OD, δ):
16.7, 27.7, 31.9, 42.7, 56.4, 56.6, 59.5, 111.7, 121.5, 125.0, 128.4,
128.9, 133.1, 136.0, 142.5, 151.8, 152.2, 154.0, 170.6, 174.4, 178.7.
MS m/z 542 [M + H]+, 564 [M + Na]+. HRMS (m/z): [M + H]+
calcd for C26H32N5O6S, 542.2073; found 542.2070; [M + Na]+
calcd for C26H31N5O6NaS, 564.1893; found 564.1890.
(S)-5-Amino-2-((R)-2-((2Z,5Z)-5-(4-hydroxy-3,5-dimethoxy-
benzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propanami-
do)pentanamide 20b. Compound 20b was prepared by general
methods C and D on a 0.58 mmol scale and purified via silica gel
chromatography using a gradient from 1% to 10% MeOH in
CH2Cl2. Fractions containing protected penultimate intermediate
(1H NMR (300 MHz, CDCl3, δ) 1.35 (s, 9H), 1.50-1.74 (m, 2H),
1.76 (d, J ) 7.0 Hz, 3H), 1.97-2.15 (m, 1H), 3.00-3.39 (m, 2H),
3.85 (s, 3H), 3.98 (s, 3H), 4.70-5.10 (m, 1H), 5.34 (br s, 1H),
5.44 (q, J ) 6.9 Hz, 1H), 6.64 (s, 2H), 6.93 (br s, 1H), 7.00 (d, J
) 7.5 Hz, 2H), 7.19 (t, J ) 7.0 Hz, 2H), 7.37 (t, J ) 7.7 Hz, 2H),
7.65 (s, 1H); MS m/z 642 [M + H]+, 664 [M + Na]+) were treated
with neat TFA for 45 min to give, after silica gel chromatography
using a gradient from 1% to 10% MeOH in CH2Cl2, 20b (224 mg,
0.34 mmol) as the TFA salt. 1H NMR (300 MHz, CD3OD, δ):
1.74 (d, J ) 7.0 Hz, 3H), 1.77-1.91 (m, 3H), 1.93-2.13 (m, 1H),
2.93 (t, J ) 6.9 Hz, 2H), 3.79 (s, 6H), 4.44-4.62 (m, 1H), 5.45 (q,
J ) 7.0 Hz, 1H), 6.73 (s, 2H), 7.06 (d, J ) 7.4 Hz, 2H), 7.20 (t,
J ) 7.4 Hz, 1H), 7.40 (t, J ) 7.8 Hz, 2H), 7.68 (s, 1H). 13C NMR
(500 MHz, CD3OD, δ): 16.8, 27.7, 32.1, 42.8, 56.4, 56.6, 59.4,
111.7, 121.5, 124.9, 128.4, 128.8, 133.1, 136.1, 142.3, 151.8, 152.1,
153.9, 170.8, 174.3, 178.0. MS m/z 542 [M + H]+. HRMS (m/z):
[M + H]+ calcd for C26H32N5O6S, 542.2068; found 542.2064; [M
+ Na]+ calcd for C26H31N5O6NaS, 564.1887; found 564.1882.
(S)-5-Guanidino-2-((S)-2-((2Z,5Z)-5-(4-hydroxy-3,5-dimethox-
ybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)propana-
mido)pentanamide (21a). General method H for the formation of
bis-Boc-guanidine and subsequent deprotection to the free guanidine
is illustrated by the synthesis of 22a. 1,3-Bis-(Boc)-2-methyl-2-
thiopseudourea (80 mg, 0.28 mmol), triethylamine (114 µL, 0.82
mmol), and HgCl2 (90 mg, 0.33 mmol) were added to a solution
of 20a (150 mg, 0.23 mmol) in 2.8 mL of DMF at 0 °C. The
reaction mixture was stirred for 1 h at 0 °C and then at room
temperature overnight. The reaction mixture was diluted with ethyl
acetate and filtered over Celite, and the filtrate was concentrated
in vacuo. The crude solid was purified via silica gel chromatography
using a gradient from 0% to 50% MeOH in CH2Cl2 to give 21a
1
(80.7 mg, 0.10 mmol). H NMR (300 MHz, (CD3)2CO, δ): 1.45
(s, 9H), 1.50 (s, 9H), 1.60-1.72 (m, 3H), 1.75 (d, J ) 7.1 Hz,
3H), 1.83-1.96 (m, 1H), 3.24-3.51 (m, 2H), 3.83 (s, 6H),
4.48-4.62 (m, 1H), 5.40 (q, J ) 7.0 Hz, 1H), 6.47 (br s, 1H), 6.86
(s, 2H), 7.01 (br s, 1H), 7.08 (d, J ) 7.3 Hz, 2H), 7.19 (t, J ) 7.4
Hz, 1H), 7.41 (t, J ) 7.8 Hz, 2H), 7.67 (s, 1H), 7.69-7.77 (m,
1H), 8.32 (br s, 1H). 13C NMR (500 MHz, (CD3)2CO, δ): 17.3
29.7, 31.3, 31.6, 31.7, 44.1, 56.8, 59.9, 60.0, 82.2, 86.8, 112.1,
122.4, 125.3, 128.6, 128.8, 133.3, 135.4, 142.7, 152.0, 152.2, 153.3,
160.2, 167.7, 169.6, 169.9, 172.3, 177.4. MS m/z 784 [M + H]+,
806 [M + Na]+. 21a was taken up in 50:50 CH2Cl2/TFA (3 mL)
and stirred at room temperature for 1 h, then concentrated in vacuo.
The crude solid was purified by preparative reverse phase HPLC
using a gradient from 10% to 50% B in A over 30 min to give 22a
(2 mg, 0.003 mmol) as the TFA salt. 1H NMR (300 MHz, CD3OD,
δ): 1.57-1.73 (m, 3H), 1.74 (d, J ) 7.0 Hz, 3H), 1.93-2.12 (m,
1H), 3.07-3.28 (m, 2H), 3.82 (s, 6H), 4.43-4.55 (m, 1H), 5.45
(q, J ) 6.9 Hz, 1H), 6.78 (s, 2H), 7.06 (d, J ) 7.6 Hz, 2H), 7.21
(t, J ) 7.4 Hz, 1H), 7.41 (t, J ) 7.7 Hz, 2H), 7.68 (s, 1H). 13C
NMR (500 MHz, CD3OD, δ): 16.8, 29.0, 32.4, 44.5, 56.7, 57.0,
59.4, 111.6, 121.5, 125.0, 128.4, 128.9, 133.1, 136.0, 142.2, 151.7,
152.1, 153.7, 161.2, 170.5, 174.5, 179.2. MS m/z 584 [M + H]+.
HRMS (m/z): [M + H]+ calcd for C27H34N7O6S, 584.2286; found
584.2306.
General method I for the solid phase synthesis is illustrated by
the preparation of 41. TentaGel S Ram (Advanced ChemTech, 500
mg, 0.25 mmol/g, 0.125 mmol) was swollen in 15 mL of CH2Cl2
for 30 min, then deprotected by two 10 min cycles of 20%

Thiazolidinone Inhibitors of T3SS
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7073
piperidine/DMF (10 mL) with two DMF washes in between. The
resin was washed successively with 10 mL each of CH2Cl2 (3×),
DMF (3×), MeOH (2×), and CH2Cl2 (3×). A solution of Fmoc
L-alanine (117 mg, 0.375 mmol) in 5 mL of CH2Cl2 was treated
successively with hydroxyazabenzotriazole (HOAt, 51 mg, 0.375
mmol), diisopropylcarbodiimide (DIC, 58 mL, 0.375 mmol), and
diisopropylethylamine (109 mL, 0.525 mmol), and the reaction
mixture was stirred for 5 min, then added to the deprotected, washed
resin, rinsing with an additional 10 mL of CH2Cl2. The resin was
shaken overnight, drained, and subjected to the above standard
washing protocol. A sample of the beads gave a negative Kaiser
test. After deprotection as above, the sample of beads gave a positive
Kaiser test, and the second Fmoc L-alanine was added by the
identical procedure. Overnight shaking gave a resin showing, after
washing, a negative Kaiser test. The resin was deprotected, washed,
checked, and treated with 30 µL (0.25 mmol) of phenyl isothio-
cyanate in 10 mL of CH2Cl2. Kaiser test showed the reaction to be
incomplete after overnight shaking, and two additional 150 µL (1.25
mmol) of phenyl isothiocyanate in 10 mL were added over 2 days
to complete the reaction. After washing, a sample of beads were
removed and cleaved with 95:5 TFA/CH2Cl2 for 90 min, and the
filtrate was analyzed by mass spectrometry to show predominant
peaks for the N-amidoAlaAla-N′-phenylthiourea at m/z 317 [M +
Na]+, 295 [M + H]+. The resin was treated with methyl
bromoacetate (115 µL, 1.25 mmol) and DIEA (436 µL, 2.5 mmol)
in 10 mL of CH2Cl2, washed, and an aliquot was cleaved and
examined by mass spectrometry as above to show a predominant
[M + H]+ molecular ion of 335. To the resin was added a solution
of syringaldehyde (229 mg, 1.25 mmol) and piperidine (173 µL,
1.75 mmol) in 10 mL of EtOH, and the suspension was refluxed
for 9 h. After the mixture was drained and washed, a cleaved aliquot
showed the presence of unreacted starting material, and the charge
and reflux were repeated. The drained, washed resin was then
cleaved by a 90 min treatment with 95:5:5 TFA/H2O/triisopropyl-
silane (10 mL). After the resin was filtered and after two washings
with 10 mL of MeOH each, the filtrate was concentrated in vacuo
to give 39 mg of a crude product that was purified on RP-HPLC
(20-95% B in A over 18 min) to provide 9.84 mg (0.02 mmol) of
41 that was identical to the 41 obtained by solution chemistry under
Hz, 2H), 7.19-7.62 (m, 16H). MS m/z 694 [M + H]+. HRMS
(m/z): [M + H]+ calcd for C38H40N5O6S, 694.2694; found 694.2681.
Sip A Secretion Assay. Salmonella typhimurium Secretion
Assays. Bacteria were grown for 18 h in LB broth, and bacterial
cells were pelleted by centrifugation. Culture supernates were
filtered through a 0.2 µm polyethersulfone syringe filter. Then 10%
TCA was added to culture supernates to precipitate proteins and
incubated on ice for 30 min and spun at 16K rpm for 20 min. Pellets
were washed with acetone and resuspended in SDS-PAGE sample
buffer. Proteins were separated by SDS-PAGE and Western blotted
with polyclonal anti-SipA antibodies.43
MIC Determinations. To 30 µL aliquots of a 2 mL overnight
culture of S. typhimurium (ATCC 14028) were added 0.1, 1.0, 2.0,
3.0, 6.0, and 15.0 µL of each test compound in DMSO. The treated
cultures were incubated overnight, and growth was determined by
reading the OD600. Each sample was run in triplicate. DMSO alone
was used as a control and gave partial growth inhibition at 20%
v/v in LB (comparable to 2 mM compound) and complete growth
inhibition at 50% v/v (comparable to 5 mM compound).
Acknowledgment. The authors thank Ross Lawrence (De-
partment of Medicinal Chemistry) for the assistance with the
high resolution mass spectroscopy data, and Martin Sadilek
(Department of Chemistry) for mass spectroscopy, particularly
in the determinations of the log P values. The crystal structure
of 8 was obtained by Werner Kaminsky (Department of
Chemistry). V. Koronakis (Department of Pathology, University
of Cambridge) generously provided the SipA antibodies. We
are grateful to Wei Deng (Department of Biochemistry) for
helpful discussions regarding the SAR data. The support of
NIAID (Grant U54 A105714) is acknowledged. The investiga-
tors are members of the Northwest Regional Center of Excel-
lence for Biodefense and Emerging Infectious Diseases Research.
Supporting Information Available: Experimental data for the
intermediates and final compounds not given in the main body of
the text; table of HPLC retention times and conditions. This material
is available free of charge via the Internet at http://pubs.acs.org.
1
Scheme 2. H NMR (500 MHz, CD3CN, δ): 1.35 (d, J ) 5.8 Hz,
3H), 1.67 (d, J ) 7.1 Hz, 1H), 3.83 (s, 6H), 4.35 (dq, J ) 7.2, 7.2
Hz, 1H), 5.32 (q, J ) 7.2 Hz, 1H), 5.7 (br, 1H), 7.01 (s, 2H),
7.05-7.46 (m, 5H), 7.70 (s, 1H). MS m/z 499 [M + H]+, 521 [M
+ Na]+ 411 [M Ala + H]+. HRMS (m/z): [M + H]+ calcd for
C24H26N4NaO6S, 521.1465; found 521.1467.
An earlier-eluting minor peak was identified as the D-Ala-L-
AlaNH2 diastereomer m/z 499 [M + H]+, 521 [M + Na]+, 411
[M - Ala + H]+, 428 [M - C(CH3)CONH2 + H]+.
(S)-2-((S)-3-(Biphenyl-4-yl)-2-((2Z,5Z)-5-(4-hydroxy-3,5-
dimethoxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-yl)-
propanamido)-5-guanidinopentanamide 55a. 1H NMR (500
MHz, CD3CN, δ): 1.65-1.70 (m, 2H), 3.12-3.20 (m, 2H), 3.61
(s, 2H), 3.80 (s, 6H), 4.29-4.33 (m, 1H), 5.52 (dd, J ) 6.4, 6.3
Hz, 1H), 5.86 (br, 1H), 6.75 (s, 2H), 6.85 (d, J ) 7.8 Hz, 2H),
7.19-7.62 (m, 13H), 7.87 (br, 1H). MS m/z 736 [M + H]+. HRMS
(m/z): [M + H]+ calcd for C39H42N7O6S, 736.2912; found 736.2888.
(S)-N-((S)-1-Amino-3-(biphenyl-4-yl)-1-oxopropan-2-yl)-5-
guanidino-2-((2Z,5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-
4-oxo-2-(phenylimino)thiazolidin-3-yl)pentanamide 54. 1H NMR
(500 MHz, CD3CN, δ): 1.46-1.50 (m, 2H), 2.91-3.10 (m, 2H),
3.12-3.20 (m, 2H), 3.65 (s, 2H), 4.30-4.45 (m, 1H), 5.10 (t, J )
7.6 Hz, 1H), 5.82 (br, 1H), 6.62 (s, 2H), 6.85 (d, J ) 7.8 Hz, 2H),
6.81-7.56 (m, 13H). MS m/z 736 [M + H]+. HRMS (m/z): [M +
H]+ calcd for C39H42N7O6S, 736.2912; found 736.2884.
References
(1) Cornelis, G. R. The type III secretion injectisome. Nat. ReV. Microbiol.
2006, 4, 811–825.
(2) Rosqvist, R.; Hakansson, S.; Forsberg, A.; Wolf-Watz, H. Functional
conservation of the secretion and translocation machinery for virulence
proteins of yersiniae, salmonellae and shigellae. EMBO J. 1995, 14,
4187–4195.
(3) Smith, P. A.; Romesberg, F. E. Combating bacteria and drug resistance
by inhibiting mechanisms of persistence and adaptation. Nat. Chem.
Biol. 2007, 3, 549–556.
(4) Baron, C.; Coombes, B. Targeting bacterial secretion systems: benefits
of disarmament in the microcosm. Infect. Disord. Drug Targets 2007,
7, 19–27.
(5) Preston, G. M. Metropolitan microbes: type III secretion in multihost
symbionts. Cell Host Microbe 2007, 2, 291–294.
(6) Bailey, L.; Gylfe, A.; Sundin, C.; Muschiol, S.; Elofsson, M.;
Nordstrom, P.; Henriques-Normark, B.; Lugert, R.; Waldenstrom, A.;
Wolf-Watz, H.; Bergstrom, S. Small molecule inhibitors of type III
secretion in Yersinia block the Chlamydia pneumoniae infection cycle.
FEBS Lett. 2007, 581, 587–595.
(7) Negrea, A.; Bjur, E.; Ygberg, S. E.; Elofsson, M.; Wolf-Watz, H.;
Rhen, M. Salicylidene acylhydrazides that affect type III protein
secretion in Salmonella enterica serovar typhimurium. Antimicrob.
Agents Chemother. 2007, 51, 2867–2876.
(8) Hudson, D. L.; Layton, A. N.; Field, T. R.; Bowen, A. J.; Wolf-Watz,
H.; Elofsson, M.; Stevens, M. P.; Galyov, E. E. Inhibition of type III
secretion in Salmonella enterica serovar typhimurium by small-
molecule inhibitors. Antimicrob. Agents Chemother. 2007, 51, 2631–
2635.
(9) Troisfontaines, P.; Cornelis, G. R. Type III secretion: more systems
than you think. Physiology (Bethesda) 2005, 20, 326–339.
(10) Felise, H. B.; Nguyen, H. V.; Pfuetzner, R. A.; Barry, K. C.; Jackson,
S. R.; Blanc, M. P.; Brotnstein, P. A.; Kline, T.; Miller, S. I. An
inhibitor of bacterial virulence protein secretion. Cell Host Microbe,
in press.
(S)-5-Amino-2-((S)-3-(biphenyl-4-yl)-2-((2Z,5Z)-5-(4-hydroxy-
3,5-dimethoxybenzylidene)-4-oxo-2-(phenylimino)thiazolidin-3-
yl)propanamido)pentanamide 56. 1H NMR (500 MHz, CD3CN,
δ): 1.77-1.80 (m, 2H), 1.97-1.99 (m, 2H), 3.06-3.10 (m, 2H),
3.60-3.64 (m, 2H), 3.78 (s, 6H), 4.42-4.55 (m, 1H), 5.64 (dd, J
) 10.3, 5.4 Hz, 1H), 6.08 (br, 1H), 6.74 (s, 2H), 6.82 (d, J ) 7.6

7074 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Kline et al.
(11) Linington, R. G.; Robertson, M.; Gauthier, A.; Finlay, B. B.; van Soest,
R.; Andersen, R. J. Caminoside A, an antimicrobial glycolipid isolated
from the marine sponge Caminus sphaeroconia. Org. Lett. 2002, 4,
4089–4092.
(12) Linington, R. G.; Robertson, M.; Gauthier, A.; Finlay, B. B.;
MacMillan, J. B.; Molinski, T. F.; van Soest, R.; Andersen, R. J.
Caminosides B-D, antimicrobial glycolipids isolated from the marine
sponge Caminus sphaeroconia. J. Nat. Prod. 2006, 69, 173–177.
(13) Iwatsuki, M.; Uchida, R.; Yoshijima, H.; Ui, H.; Shiomi, K.; Kim,
Y. P.; Hirose, T.; Abe, A.; Tomoda, H.; Omura, S. Guadinomines,
type III secretion system inhibitors, produced by Streptomyces, sp.
K01-0509. J. Antibiot. 2008, 61, 230–236.
(14) Li, X. Pyridone Compounds as Inhibitors of Bacterial Type II Protein
Secretion Systems. US 7,987,622 B2, 2006.
(15) Li, X. Inhibitors of Bacterial Type III Secretion Systems. U.S. Patent
0272784 A1, 2005.
(16) Li, X.; Kang, F.-A.; Macielag, M. J. Triazine Compounds as Inhibitors
of Bacterial Type III Protein Secretion Systems. PCT/US2005/015808,
WO2005/111017, 2005.
(17) Nordfelth, R.; Kauppi, A. M.; Norberg, H. A.; Wolf-Watz, H.;
Elofsson, M. Small-molecule inhibitors specifically targeting type III
secretion. Infect. Immun. 2005, 73, 3104–3114.
(18) Kauppi, A. M.; Andersson, C. D.; Norberg, H. A.; Sundin, C.;
Linusson, A.; Elofsson, M. Inhibitors of type III secretion in Yersinia:
design, synthesis and multivariate QSAR of 2-arylsulfonylamino-
benzanilides. Bioorg. Med. Chem. 2007, 15, 6994–7011.
(19) Dayam, R.; Aiello, F.; Deng, J.; Wu, Y.; Garofalo, A.; Chen, X.;
Neamati, N. Discovery of small molecule integrin alphavbeta3
antagonists as novel anticancer agents. J. Med. Chem. 2006, 49, 4526–
4534.
(20) Ottana, R.; Maccari, R.; Barreca, M. L.; Bruno, G.; Rotondo, A.; Rossi,
A.; Chiricosta, G.; Di Paola, R.; Sautebin, L.; Cuzzocrea, S.; Vigorita,
M. G. 5-Arylidene-2-imino-4-thiazolidinones: design and synthesis of
novel anti-inflammatory agents. Bioorg. Med. Chem. 2005, 13, 4243–
4252.
(21) Ottana, R.; Maccari, R.; Ciurleo, R.; Vigorite, M. G.; Panico, A. M.;
Cardile, V.; Garufi, F.; Ronsivalle, S. Synthesis and in vitro evalutation
of 5-arylidene-3-hydroxyalkyl-2phenylimino-4-thiazolidinones with
antigenerative activity on human chondrocyte cultures. Bioorg. Med.
Chem. 2007, 15, 7618–7625.
(22) Hu, B.; Malamas, M.; Ellingboe, J.; Largis, E.; Han, S.; Mulvey, R.;
Tillett, J. New oxadiazolidinedione derivatives as potent and selective
human beta3 agonists. Bioorg. Med. Chem. Lett. 2001, 11, 981–984.
(23) Hu, B.; Malamas, M.; Ellingboe, J. Synthesis of heterocycle substituted
1-aryl-4-piperidones. Heterocycles 2002, 57, 857–865.
(24) Manhi, F. M.; Manhoud, M. R. Studies on the reactivity of fused
thiazole toward nucleophilic reagents: synthesis of new thiazolo-
derivatives of potential antischistomsomal activity. Heteroat. Chem.
2007, 16, 121–131.
(25) Khare, R. K.; Srivastava, M. K.; Singh, H. Synthesis and fungicidal
activity of some 5-methylene-2-[5′-aryl-1′,3′,4′-oxa(thia)-diazol-[2′-
yl]amino-4-thiazolones. Indian J. Chem. 1995, 34B, 828–831.
(26) Rajanarendar, E. A.; Karunakar, D. Synthesis of isoxazolylpryra-
zolo[3,4-d]thiazoles and isoxazolylthiazoles and their antibacterial and
antifungal activity. Indian J. Chem. 2004, 43B, 168–173.
(27) Merja, B. C.; Joshi, A. M.; Parikh, K. A.; Parikh, A. R. Synthesis and
biological evaluation of pyrido-[1,2-1]pyrimidie an disoxazoline
derivatives. Indian J. Chem. 2004, 43B, 909–912.
(30) Klika, K. D.; Valtamo, P.; Janovec, L.; Suchar, G.; Kristian, P.; Imrich,
J.; Kivela, H.; Alfoldi, J.; Pihlaja, K. Regioselective syntheses,
structural characterization, and electron ionization mass spectrometric
behavior of regioisomeric 2,3-disubstituted 2-imino-1,3-thiazolidin-
4-ones. Rapid Commun. Mass Spectrom. 2004, 87–95.
(31) Yin, H.; Hamilton, A. D. Strategies for targeting protein-protein
interactions with synthetic agents. Angew. Chem., Int. Ed. 2005, 44,
4130–4163.
(32) Tian, X.; Field, T. B.; Switzer, A. G.; Mazur, A. W.; Ebetino, F. H.;
Wos, J. A.; Berberich, S. M.; Jayasinghe, L. R.; Obringer, C. M.;
Dowty, M. E.; Pinney, B. B.; Farmer, J. A.; Crossdoersen, D.; Sheldon,
R. J. Design, synthesis, and evaluation of proline and pyrrolidine based
melanocortin receptor agonists. A conformationally restricted dipeptide
mimic approach. J. Med. Chem. 2006, 49, 4745–4761.
(33) Calculated log P values for the dipeptides, using the algorithm provided
by ChemDraw, version 9.01, gave a range of values from 0.98 (24,
48) to 4.47 (54, 55a), compared to a calculated log P value of 4.87
for 1. Experimentally derived values were somewhat lower, although
the ranking did not change. These values were determined for 55a
(log P ) 2.63) and 1 (log P ) 3.34) by monitoring the respective
molecular ions on LC-MS and using calibration curves ranging from
0.1 to 10 µM (MeOH) to obtain the concentration of each compound
partitioned into octanol and water from the respective dry samples.
The LC gradient was 25-95% Bin A over 15 min, where A was 94:
5:1 water/acetonitrile/acetic acid and B was 99:1 acetonitrile/acetic
acid. The column was a Zorba SB-C18 (2.1 mm × 100 mm)
maintained at 30 °C. All samples were analyzed in positive ionization
mode using the respective specific precursor/product ions 433.1/195.2
(dwell 0.1 min, cone 45, coll energy 35 V) for 1 and 736.3/563.2
(dwell 0.1 min, cone 25, coll energy 33 V) for 55a.
(34) Xu, Y.; Lu, H.; Kennedy, J. P.; Yan, X.; McAllister, L. A.; Yamamoto,
N.; Moss, J. A.; Boldt, G. E.; Jiang, S.; Janda, K. D. Evaluation of
“credit card” libraries for inhibition of HIV-1 gp41 fusogenic core
formation. J. Comb. Chem. 2006, 8, 531–539.
(35) Jager, M.; Michalet, X.; Weiss, S. Protein-protein interactions as a
tool for site-specific labeling of proteins. Protein Sci. 2005, 14, 2059–
2068.
(36) Yip, C. K.; Kimbrough, T. G.; Felise, H. B.; Vuckovic, M.; Thomas,
N. A.; Pfuetzner, R. A.; Frey, E. A.; Finlay, B. B.; Miller, S. I.;
Strynadka, N. C. Structural characterization of the molecular platform
for type III secretion system assembly. Nature 2005, 435, 702–707.
(37) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling,
L. L. Influencing receptor-ligand binding mechanisms with multi-
valent ligand architecture. J. Am. Chem. Soc. 2002, 124, 14922–14933.
(38) Alksne, L. E.; Projan, S. J. Bacterial virulence as a target for
antimicrobial chemotherapy. Curr. Opin. Biotechnol. 2000, 11, 625–
636.
(39) Marra, A. Targeting virulence for antibacterial chemotherapy: identify-
ing and characterising virulence factors for lead discovery. Drugs R&D
2006, 7, 1–16.
(40) Bowser, T. E.; Bartlett, V. J.; Grier, M. C.; Verma, A. K.; Warchol,
T.; Levy, S. B.; Alekshun, M. N. Novel anti-infection agents: small-
molecule inhibitors of bacterial transcription factors. Bioorg. Med.
Chem. Lett. 2007, 17, 5652–5655.
(41) Liu, C. I.; Liu, G. Y.; Song, Y.; Yin, F.; Hensler, M. E.; Jeng, W. Y.;
Nizet, V.; Wang, A. H.; Oldfield, E. A cholesterol biosynthesis
inhibitor blocks Staphylococcus aureus virulence. Science 2008, 319,
1391–1394.
(42) Hilleringmann, M.; Pansegrau, W.; Doyle, M.; Kaufman, S.; MacK-
ichan, M. L.; Gianfaldoni, C.; Ruggiero, P.; Covacci, A. Inhibitors of
Helicobacter pylori ATPase Cagalpha block CagA transport and cag
virulence. Microbiology 2006, 152, 2919–2930.
(28) Qin, Z.; Zhang, J.; Xu, B.; Chen, L.; Wu, Y.; Yang, X.; Shen, X.;
Molin, S.; Danchin, A.; Jiang, H.; Qu, D. Structure-based discovery
of inhibitors of the YycG histidine kinase: new chemical leads to
combat Staphylococcus epidermidis infections. BMC Microbiol. 2006,
6, 96.
(29) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger,
R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; erson, P. S.;
Chang, R. S.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.;
Kunkel, K. A.; Springer, J. P.; Hirshfield, J. Methods for drug
discovery: development of potent, selective, orally effective chole-
cystokinin antagonists. J. Med. Chem. 1988, 31, 2235–2246.
(43) Pegues, D. A.; Hantman, M. J.; Behlau, I.; Miller, S. I. PhoP/PhoQ
transcriptional repression of Salmonella typhimurium invasion genes:
evidence for a role in protein secretion. Mol. Microbiol. 1995, 17,
169–181.
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