Discovering potent inhibitors against the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) of helicobacter pylori: Structure-based design, synthesis, bioassay, and crystal structure determination

Article abstract of DOI:10.1021/jm8015602

The discovery of HpFabZ inhibitors is now of special interest in the treatment of various gastric diseases. In this work, three series of derivatives (compounds 3, 4, and 5) were designed, synthesized, and their biological activities were investigated as potential HpFabZ inhibitors in a two phased manner. First, we designed and synthesized two series of derivatives (3a-r and 4a-u) and evaluated the enzyme-based assay against HpFabZ. Five compounds (3i-k, 3m, and 3q) showed potential inhibitory activity, with IC₅₀ values less than 2 μM. Second, a focused combinatorial library containing 280 molecules was designed employing the LD1.0 program. Twelve compounds (5a-l) were selected and synthesized. The activity of the most potent compound 5h (IC ₅₀) 0.86 μM) was 46 times higher than that of the hit 1. The high hit rate and the potency of the new HpFabZ inhibitors demonstrated the efficiency of the strategy for the focused library design and virtual screening.

Full text of DOI:10.1021/jm8015602

J. Med. Chem. 2009, 52, 2465–2481
2465
Discovering Potent Inhibitors Against the ꢀ-Hydroxyacyl-Acyl Carrier Protein Dehydratase
(FabZ) of Helicobacter pylori: Structure-Based Design, Synthesis, Bioassay, and Crystal
Structure Determination†
Lingyan He,‡,§ Liang Zhang,‡,§ Xiaofeng Liu,‡,§ Xianghua Li,§ Mingyue Zheng,§ Honglin Li,§ Kunqian Yu,§ Kaixian Chen,§
Xu Shen,*,§,| Hualiang Jiang,*,§,| and Hong Liu*,§
Center for Drug DiscoVery and Design, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, China, School of Pharmacy, East China UniVersity of Science and Technology,
Shanghai 200237, China
ReceiVed December 10, 2008
The discovery of HpFabZ inhibitors is now of special interest in the treatment of various gastric diseases.
In this work, three series of derivatives (compounds 3, 4, and 5) were designed, synthesized, and their
biological activities were investigated as potential HpFabZ inhibitors in a two phased manner. First, we
designed and synthesized two series of derivatives (3a-r and 4a-u) and evaluated the enzyme-based assay
against HpFabZ. Five compounds (3i-k, 3m, and 3q) showed potential inhibitory activity, with IC50 values
less than 2 µM. Second, a focused combinatorial library containing 280 molecules was designed employing
the LD1.0 program. Twelve compounds (5a-l) were selected and synthesized. The activity of the most
potent compound 5h (IC50 ) 0.86 µM) was 46 times higher than that of the hit 1. The high hit rate and the
potency of the new HpFabZ inhibitors demonstrated the efficiency of the strategy for the focused library
design and virtual screening.
1. Introduction
Helicobacter pylori (H. pyloria) has recently been the subject
of intense interest because it is a major causative factor for
gastrointestinal illnesses such as chronic gastritis and peptic
ulcers.1-5 Additionally, infection of H. pylori is also associated
with adenocarcinomas and stomach lymphomas, increasing the
risk of gastric cancer.6,7 Nevertheless, there is no effective
therapy in eradicating H. pylori infection. Combination therapies
employing one proton pump inhibitor (e.g., omeprazole) and
two or three antibiotics (e.g., amoxicillin, clarithromycin, or
tetracycline) have been used as preferred treatments.8-10
However, the multiple therapy regimens have not been very
effective in the clinical setting because the bacterium is likely
to develop resistance.11-14 Moreover, this treatment may disrupt
the natural population of commensal microorganisms in the
gastrointestinal tract, leading to undesired side effects such as
diarrhea.15,16 Hence, a new level of treatment is needed to
inactivate the key enzymes associated with the bacterial growth.
Recently, the pathway of the type-II fatty acid biosynthesis
system (FAS-II) has been particularly appreciated as an interest-
Figure 1. The structures of compounds 1 and 2.
ing target for antibacterial agent discovery due to the major
differences of the structural architectures and biological func-
tions that exist between the plastid-associated enzymes of FAS-
II found in most microorganisms and the enzymes involved in
FAS-I for mammals and yeast.17-20 FAS-II consists of six key
enzymes: FabD, FabH, FabF (or FabB), FabI, FabZ (or FabA),
and FabG, all of which work in concert during the synthesis of
fatty acid in vivo including the initiation and elongation phases.
During the elongation cycle of FAS II, four consecutive
reactions complete the extension of two carbons.21,22 In the third
step of the elongation cycle, the dehydration of ꢀ-hydroxy-ACP
to trans-2-acyl-ACP is catalyzed by FabZ or FabA. FabA is a
bifunctional enzyme and is only found in Gram-negative bacteria
with its partner, FabB, to participate in the formation of
unsaturated fatty acids.23 However, FabZ has a ubiquitous
distribution in the FAS II pathway and is a primary dehydratase
that participates in the elongation cycles of both saturated and
unsaturated fatty acid biosyntheses. Accordingly, FabZ presents
itself as a suitable, yet unexplored target for the discovery of
compounds against pathogenic microbes.22,23
†
PDB ID codes: 2GLL, 3DOY, 3DOZ, 3DP0, 3DP1, 3DP2, and 3DP3.
* To whom correspondence should be addressed (X.S.) Phone: +86-
21-50806918. Fax: +86-21-50807088. E-mail: xshen@mail.shcnc.ac.cn.
(H.J.) Phone: +86-21-50805873. Fax: +86-21-50807088. E-mail: hljiang@
mail.shcnc.ac.cn. (H.L.) Phone: +86-21-50807042. Fax: +86-21-50807088.
E-mail: hliu@mail.shcnc.ac.cn.
‡
Authors equally contributed to this work.
Center for Drug Discovery and Design, State Key Laboratory of Drug
§
Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences.
|
School of Pharmacy, East China University of Science and Technology.
a Abbreviations: FabZ, ꢀ-hydroxyacyl-acyl carrier protein dehydratase;
Hp, Helicobacter pylori or H. pylori; FAS-I, type I fatty acid biosynthesis;
FabD, malonyl-CoA:ACP transacylase; FabH, ꢀ-ketoacyl-acyl carrier protein
synthase III; FabF, ꢀ-ketoacyl-acyl carrier protein synthase II; FabB,
ꢀ-ketoacyl-acyl carrier protein synthase I; FabI, enoyl-acyl carrier protein
reductase; FabA, ꢀ-hydroxyacyl-acyl carrier protein dehydratase; FabG,
ꢀ-ketoacyl-ACP reductase I; ACP, the acyl carrier protein; CoA, acetyl
coenzyme A; PfFabZ, Plasmodium falciparum FabZ.
So far, the only known small molecule inhibitors of FabZ
are flavonoids discovered by random screening against Plas-
modium falciparum FabZ (PfFabZ).24 Recently, we discovered
two synthetic compounds (1 and 2, Figure 1) as inhibitors of
H. pylori FabZ (HpFabZ) with IC50 values of 39.8 ( 0.35 µM
10.1021/jm8015602 CCC: $40.75
2009 American Chemical Society
Published on Web 03/23/2009

2466 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
and 47.6 ( 0.29 µM, respectively.25 In addition, we also
determined the X-ray crystal structures of HpFabZ in complex
with these inhibitors, which demonstrated the inhibitory mech-
anism of FabZ for the first time.25 In this study, on the basis of
the structures and activities of compounds 1 and 2, and their
interactive models with HpFabZ as well, we identified several
small compounds that inhibit HpFabZ activity with IC50 values
of 0.86-42.7 µM by employing a comprehensive approach
integrating structure-based design, focused-library design, virtual
screening, chemical synthesis, and bioassay.
We approached anti-H. pylori therapeutic development
employing structure-based design to identify small organic
compounds as lead candidates. In the first phase of this study,
we designed and synthesized two series of derivatives of
compounds 1 and 2 according to the structural models of these
inhibitors binding to HpFabZ. Five compounds showed high
inhibition activities with IC50 values less than 2 µM, nearly 28
times more potent than the initial starting hits 1 and 2.
Afterward, the crystal structures of the complexes of six
compounds with the HpFabZ were determined. In the second
phase, we adopted a strategy integrating focused combinatorial
library design, virtual screening, chemical synthesis, and bio-
assay to identify more potent inhibitors based on the result of
the first phase. Using the fragments taken from the HpFabZ
inhibitors identified in the first phase, a focused combinatorial
library containing 280 molecules was designed by employing
the LD1.0 program26 on the basis of the crystal structures of
the inhibitor-HpFabZ complexes. By using docking-based
virtual screening targeting the binding site of HpFabZ, 12
compounds (5a-l) were selected for synthesis and bioassay.
Eight compounds showed HpFabZ enzymatic inhibition activi-
ties with IC50 values of 0.86-42.7 µM. The inhibitory activity
of the most potent compound increased 46 times more than that
of the original compounds. All the inhibitors discovered in this
study showed weak antibacterial activity, overcoming the
shortcomings of the original inhibitors (compounds 1 and 2)
due to improving the druglikeness of the designed compounds.
The high hit rate and the high potency of the new HpFabZ
inhibitors demonstrated the efficiency of the discovery strategy
used in this study. In addition, the chemical entities reported in
this study could be leading factors contributing to the discovery
of new therapies against the FAS-II pathway.
Figure 2. Discovery strategy and experimental flowchart in this study.
back into the binding pockets of HpFabZ and were scored by
virtual screening functions to select candidates for further
synthesis and bioassay. Several new inhibitors were discovered
in this phase of the design. Finally, the bacterial growth
inhibitory activities of all the obtained HpFabZ inhibitors were
assessed. The detailed experimental procedures of each step are
described in the following sections.
2.1.1. Phase I Design. On the basis of the structural feature
and their interaction models with HpFabZ of compounds 1 and
2, 39 new analogues (3a-r and 4a-u, Tables 1 and 2) were
designed and synthesized in the first phase. Keeping the pyridine
ring of compound 1, we obtained compounds 3a-d by the
removal of the hydroxyl group or bromide or by introducing
methoxyl groups to the phenyl ring. Keeping the 2,4-dihydroxy-
3,5-dibromo phenyl ring and replacing the pyridine ring with
the phenyl ring, substituted phenyl ring, methyl, naphthalene
ring, or various heterocycles, we obtained compounds 3e-r.
On the basis of the structural features of compound 2, we
designed compounds 4a-u (Table 2). By changing the sub-
stituent groups of the substitutional phenyl ring R3 of compound
2, we got compounds 4a-c. Compounds 4d-r were obtained
by introducing various electronic, hydrophobic groups to the
ortho-, meta-, or para- position of the unsubstituted phenyl ring
R4 of compound 2 or displacing the R4 group with the naphthalin
ring. Compounds 4s-u were obtained by displacing the
methoxy group with three different amino groups.
2. Materials and Methods
2.1. Discovery Strategy. On the basis of our previously
discovered HpFabZ inhibitors (compounds 1 and 2) and the
crystal structures of the inhibitor-HpFabZ complexes, we
started to design the new generation HpFabZ inhibitors to
increase the enzymatic and antibacterial activities. The flowchart
of the discovery strategy and procedure is outlined in Figure 2.
In general, the inhibitor-designing process was conducted in a
two-phase manner. During the phase I design, a series of
derivatives of compounds 1 and 2 were designed and synthesized
by a structure-based drug design approach according to the
binding models of compounds 1 and 2 with HpFabZ. Several
potent inhibitors were selected via an enzymatic activity assay,
and the crystal structures of the complexes of the most potent
inhibitors with HpFabZ were determined, which was the starting
point for the phase II design. During the phase II design, a
focused library containing 280 compounds was designed by
using the fragments isolated from the inhibitors obtained in the
2.1.2. Phase II Design. Before phase II design, the X-ray
crystal structures of HpFabZ in complex with the inhibitors
produced from phase I design were determined, which provide
inhibitor-enzyme interaction models for further inhibitor design.
A focused library was designed using the structural fragments
isolated by the above discovered inhibitors according to the
inhibitor-enzyme interaction models. The LD1.0 program26,27
was used during the focused library design. The inhibitor-enzyme
interaction models indicate that the HpFabZ inhibitors can be
divided into three parts (Figure 3A): part A is located in the
large pocket (site A), part B interacts with the small pocket of
HpFabZ (site B), and part L is a linker between A and B,
interacting with the “saddle” pocket between sites A and B,
with a length of 5-6 Å (Figure 3A, middle image). From
fragments of HpFabZ inhibitors discovered above and previ-
ously,25,28 the LD1.0 program optimized 14 fragments for part
A, 5 fragments for part B, and 4 linkers for part L. The chemical
structures of the fragments are shown in Figure 4. Then LD1.0
connected these fragments together according to the structure
phase
I design as building blocks according to the
inhibitor-HpFabZ binding models derived from the crystal
structures. The structures in the focused library were docked

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2467
Table 1. Chemical Structures, HpFabZ Inhibitory Activities, and Antibacterial Activity of Compounds 1 and 3a-r
a Values are means of three determinations and deviation from the mean is <10% of the mean value. b Due to poor solubility without antibacterial activity
bioassay.
of HpFabZ binding site, producing a library containing 280 (14
× 4 × 5) molecules. All molecules in the designed library were
ranked by the scoring functions of DOCK4.0, AutoDock4.0,
and the druglikeness filter of LD1.0 (Figure S1 and Table S1
of the Supporting Information) and analyzed for relatively lower
binding energy, favorable shape complementarity, and potential
in forming hydrogen bonds with HpFabZ. In this way, 12
compounds (5a-1) were finally selected for synthesis and
bioassay.
2.2. Chemical Synthesis. Compounds 3-5 were synthesized
through the routes outlined in Schemes 1 and 2. Compounds 6
and 9 were brominated by Br2 in 95% EtOH at room temper-
ature, affording 7 and 10, respectively.29 Compound 7 was
methylated by (CH3)2SO4 in DMF in the presence of K2CO3 at
100 °C.30 Commercially available acid 11 was successfully
converted to acid hydrazide 12 by a sequential esterification
and hydrazinolysis. Then acid hydrazide 12 was condensed with
6, 7, 8, 9, and 10 in anhydrous EtOH at room temperature,
giving the target compounds 1, 3a-r, and 5a-j, respectively.31
Scheme 2 depicts the sequence of reactions that led to the
preparation of compounds 2, 4a-u, and 5k-l. Treatment of
isothiocyanate 13 with amine 14 in toluene at room temperature
quickly afforded thiourea 15a-q and 5k to produce good
yields.32 Thiourea 15a-q were condensed with chloroacetic acid
and sodium acetate anhydrous in anhydrous EtOH to produce
16a-q. Commercially available substituted amines 17a-b were
esterified to afford esters 18a-b. Compounds 18a-b were
dissolved in hydrochloric acid, and diazotized by the addition
of a sodium nitrite solution at 0 °C and then treated with
2-furfuraldehyde and cupric chloride to give 19a-b as prod-
ucts.33 In the same manner as the preparation of 19a-b, 19c
was prepared from 4-chloro aniline and 2-furfuraldehyde. 19a-c
was condensed with 16a-q in EtOH in the presence of
piperidine and then hydrolyzed in THF in the presence of LiOH,
affording the target compounds 2 and 4a-u. Treatment of
commercially available acid 20 with anilines 21 in the presence
of pyridine produced amides 5l. The details of the synthetic
procedures and structural characterizations are described in the
Experimental Section. The combustion analyses and the purities
of all these compounds are listed in Tables S2 and S3 in the
Supporting Information, respectively.
2.3. Compound Screening. 2.3.1. Virtual Screening. The
binding potential between HpFabZ and molecules enumerated
from the focused library were evaluated by the scoring module
of LD1.0. Docking algorithm and scoring function of
DOCK4.034,35 were adopted in the virtual screening. The scoring
function of AutoDock4.036 was also used to rank the molecules
to avoid the inaccurateness of one scoring function. The X-ray
crystal structure of HpFabZ was used as a target for the virtual
screening. Residues of HpFabZ around the binding site at a
radius of 6.5 Å were isolated for constructing the grids of the
docking screening. The resulting substructure included all
residues of the binding pocket. During the docking simulation,
Kollman-all-atom charges37 were assigned to the protein atoms

2468 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
Table 2. Chemical Structures of Compounds 2 and 4a-u and Their HpFabZ Inhibitory Activities
and Gasteiger-Marsili charges38 were assigned to the ligand
atoms in the library. Conformational flexibility of the molecules
from the library was considered in the docking search.
ꢀ-hydroxybutyryl-CoA. The compounds dissolved in 1% DMSO
were incubated with the enzyme for 1 h before the assays were
started. The 50% inhibitory concentration (IC50) of each inhibitor
was estimated by fitting the inhibition data to a dose-dependent
curve using a logistic derivative equation.41
The druglikeness of molecules from the combinatorial library
was also evaluated by the druglikeness filter of LD1.0.26 The
druglikeness descriptor set encoded in LD1.0 is composed of
the “Rule of Five” and the other three descriptors of the
structural ratio.39,40 For obtaining more information and under-
standing the synthetical alternation, we broadened the constraint
of the molecular weight to 600. The range of evaluation on
druglikeness is between 0 and 1, which stands for nondrug-
likeness and druglikeness, respectively.40 All the parameters
used in the LD1.0 were described in our previous paper.26
2.3.2. Enzymatic Activity Assay. The enzymatic inhibition
assay of HpFabZ was monitored by the spectrophotomeric
method as described previously.21,25 In brief, the activity of
HpFabZ was measured by the detection of the decrease in
absorbance at 260 nm for the conversion of crotonoyl-CoA to
2.3.3. Antibacterial Assay. The bacterial growth inhibition
activity for the compounds was evaluated by using the paper
discus method. Dimethyl sulfoxide and ampicillin paper were
used as negative and positive controls respectively. The
minimum inhibitory concentration (MIC) values were deter-
mined by the standard agar dilution method42 using Columbia
agar supplemented with 10% sheep blood containing 2-fold
serial dilutions of agents. A bacterial suspension of H. pylori
was made by swabbing and suspending the cells in sterile saline
with a turbidity equivalent to a 2.0 McFarland standard.
Compound-free Columbia agar media were used as controls.
Inoculated plates were incubated at 37 °C under microaerobic
conditions (85% N2, 10% CO2, and 5% O2) and examined after

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2469
Figure 3. Crystal structures of HpFabZ in complex with compounds 3i, 3j, 3k, 3m, 3n, and 3q. (A) Binding model A of compounds 3i-k, 3n,
and 3q in pocket A. The electrostatic surface of the HpFabZ active tunnel is generated by Pymol.51 The binding sites of compounds (binding site
A, saddle and site B) and the critical residues that interact with the compounds are shown. Oxygen, nitrogen, and sulfur atoms are colored red, blue,
and orange, respectively. (B) Binding model B of compounds 3i-k, 3n, and 3q in pocket B. (C) Binding model of compound 3m in pocket A. The
schematic picture is prepared by Pymol.51
3 days. The MIC was generally defined as the lowest concentra-
tion of antimicrobial agents that completely inhibits visible
bacterial growth.
phase I design, we modify the two aromatic ends of compounds
1 and 2 to enhance the hydrophobic interactions with the
enzyme, producing 39 compounds (designated as series 3 and
4).
3. Results and Discussion
The chemical structures of the 39 designed compounds (3a-r
and 4a-u) are shown in Tables 1 and 2. These compounds were
synthesized through the routes outlined in Schemes 1 and 2,
and the details of the synthetic procedures and structural
characterizations are described in the Experimental Section. The
primary inhibitory activities of these compounds at 50 µM
against HpFabZ were determined, and the results are also listed
in Tables 1 and 2. Bioassay detail is described in the
Experimental Section.
3.1. Identification of Inhibitors by Phase I Design. In our
previous study,25 we discovered two small molecular inhibitors
of HpFabZ (compounds 1 and 2) and determined the X-ray
crystal structures of the inhibitor-enzyme complexes. This
provided a solid foundation for designing more potent HpFabZ
inhibitors. Compound 1 is composed of a pyridine ring and a
2,4-dihydroxy-3,5-dibromo phenyl ring linked by a formohy-
drazide moiety. The crystal structure revealed that two molecules
of compound 1 bind to one hexamer of HpFabZ with two
distinct interactive models: one molecule fits into the groove
around the entrance (model A, Figure 5) and the other molecule
lies at the active site of the tunnel (model B, Figure 5).
Compound 2 is mainly composed of four rings: a phenyl ring
at one end, a 2-chloro-benzoic acid moiety at the other end,
and two heterocycles (a furan ring and a thiazolidinone
substituted by a 2-methoxy-ethyl chain) in the middle. The
crystal structure illustrated that compound 2 binds to monomer
B of HpFabZ with a binding model similar to model A of
compound 1. In model A, the two aromatic ends of either
compound 1 or 2 are sandwiched between the two “lips” of the
HpFabZ entrance via hydrophobic interactions. Hence, during
The primary data indicate that seven compounds (3h-k, 3m,
3n, and 3q) are more active than the leading hit (compound 1)
(Table 1). Encouraged by this result, we determined the
quantitative inhibition activities (IC50 values) for seven com-
pounds. The results are also listed in Table 1. As IC50 values
indicated, the inhibitory activities of these seven inhibitors
increased about 4-28 times in comparison with that of
compound 1. Structure-activity relationship results indicated
that removal of the bromine substituents of the phenyl ring
(3a-3c) or replacement of the hydroxyl group with methoxyl
(3d) decreases the inhibitory activities of compounds. The
inhibitory activities of compounds (3e, 3f, 3p, and 3r) obtained
by replacing the pyridine ring of 1, respectively, with phenyl

2470 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
potent inhibitors (3h-k, 3m, 3n, and 3q) using X-ray crystal-
lography. Except inhibitor 3h, all other six compounds soaked
into the binding sites of HpFabZ. The structural models are
shown in Figure 3 and Figure S2 in the Supporting Information.
The coordinates and structural factors of these crystal structures
have been deposited in the Protein Data Bank (PDB codes are
3DOY, 3DOZ, 3DP0, 3DP1, 3DP2, and 3DP3, respectively).
The binding models of these compounds with HpFabZ are
shown in Figure 3. The crystal structures were determined by
molecular replacement (MR) to resolutions of 2.3-2.5 Å. The
final structures of inhibitor-HpFabZ are well ordered, and the
electron densities for all residues with the exception of the
N-terminus (residues 1-10) are clearly interpretable. The
statistics of the diffraction data and structure refinement are listed
in Tables S4 and S5 in the Supporting Information. The overall
structure of HpFabZ in the complexes is similar to that of the
native HpFabZ (PDB code is 2GLL),25 displaying a classic
“trimer of dimers” organization. Each HpFabZ dimer forms two
substrate-binding tunnels with catalytic sites on the interface,
which are ∼20 Å away from each other (Figure S2 in the
Supporting Information).
Except compound 3m, five other inhibitors bind to HpFabZ
in a similar way to compound 1.25 For 3i-k, 3n, and 3q, two
molecules of each compound bind to one hexamer of HpFabZ
with two distinct interaction models: one molecule fits into the
groove around the entrance with an extended conformation
(Figure 3A) (designated as model A hereinafter); the other
molecule lies at the active site of the tunnel with a folded
conformation (Figure 3B) (called model B below). For model
A, Tyr100 and Phe83 adopt open and closed conformations,
respectively, thus the R1 substituent (see Table 1) of each
compound is located between the phenol ring of Tyr100 and
the pyrrolidine ring of Pro112′, forming a sandwich structure,
and the R2 substituent at the other end of the inhibitor binds
into a pocket composed of Arg158, Glu159, Phe59′, and Lys62′
through hydrophobic interactions, particularly, the aromatic ring
of R2 substituent form a π· · ·π stacking interaction with the
side chain of Phe69′, and cation · · ·π interactions with the
positively charged side chains of Lys62′ and Arg158 (Figure
3A). For model B, Tyr100 adopted a closed conformation and
Phe83 adopted an open conformation and the whole molecules
of the inhibitors entered into the middle of the tunnel, located
near the active site of HpFabZ (His58 and Glu72′). However,
the orientation of the designed inhibitors inside the tunnel of
enzyme is opposite to that of compound 1: the R1 substituent is
sandwiched between Ile98 and Phe59′, while the R2 substituent
moves deeply to the catalytic site of the tunnel, contacting with
Ile20, Leu21, Pro22, Phe83, Ala94, Lys97, and Val99 via
hydrophobic interactions (Figure 3B).
Figure 4. Building blocks for focused library generation. Fragments
for parts A, B, and L were isolated for our previously discovered
HpFabz inhibitors. The symbols * and + represent the sites where
fragments connect each other to form a complete structure.
ring, furan ring, methyl, and 4-hydroxy benzyl decreased. While
compounds produced by replacing the pyridine ring of 1 with
more hydrophobic groups such as halogen (F, Cl, and Br)
substituted phenyl rings (3g-k), a naphthalene ring (3m) and
a methoxy substituted phenyl ring (3n) show more potent
inhibitory activities than that of compound 1. Slight improve-
ment in inhibitory activities were observed when the pyridine
ring of 1 was displaced by a thiophene ring (3l) and a tert-
butyl substituted phenyl ring (3q). When the position of the
N-atom of the pyridine ring is changed (3o), tremendous
decrease of inhibitory activities was observed.
Table 2 lists the chemical structures and inhibitory data of
compound 2 and its derivatives 4a-u. Unfortunately, no potent
active compounds have been discovered by the modification of
compound 2, most of derivatives were proven to have com-
pletely lost the inhibitory activities. Accordingly, in the fol-
lowing X-ray crystallography study and new inhibitor design,
we only focused on the analogues of compound 1.
Different from the other five inhibitors, compound 3m was
observed to bind to HpFabZ only with model A. The reason is
possibly that the steric hindrance caused by the bulky naph-
thalene prevents the inhibitor from entering further into the
tunnel (Figure 3C). Also, compound 3m fits well into the
entrance of the tunnel with an extended conformation; however,
the 2,4-dihydroxyl-3,5-dibromo phenyl ring binds more tightly
with the hydrophobic pocket lined by Arg158, Glu159, Phe59′,
and Lys62′ so that two hydroxyl groups of 3m form two strong
hydrogen bonds (H-bonds), respectively, with the backbone
carbonyl of Glu159 and the guanidinium group of Arg158.
These additional H-bonds led 3m to be equal or more active
than other compounds although this compound can only bind
to the enzyme with one model.
3.2. Binding Models of the Inhibitors to HpFabZ De-
tected by X-ray Crystallography. To obtain more information
for further inhibitor design and to investigate the potential
inhibition mechanism, we determined the three-dimensional (3D)
structures for the complexes of HpFabZ with the seven most

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2471
Scheme 1a
a Reagents and conditions: (a) Br2, 95% EtOH, 25 °C; (b) (CH3)2SO4, K2CO3, DMF, 100 °C; (c) Br2, 95% EtOH, 25 °C; (d) EtOH, H2SO4, reflux 4 h;
(e) hydrazine hydrate (85%), EtOH, reflux 4-8 h; (f) EtOH, 25 °C.
Scheme 2a
together, a 14 × 4 × 5 focused library was obtained by using
LD1.0.26 Targeting the crystal structure of HpFabZ, the focused
library was screened by using the scoring function of
DOCK4.034,35 that has been encoded in LD1.0. The interaction
energies of these 280 molecules to HpFabZ calculated by the
DOCK4.0 function range from -47.68 to -22.57 kcal/mol
(Figure S1 and Table S1 in the Supporting Information).
However, docking programs and scoring functions have a
tendency to generate a significant number of false positives.43
To reduce the number of false positives in compound selection,
the binding poses of these 280 molecules with HpFabZ were
redocked using AutoDock4.0,36 which has a better ability to
optimize binding poses as well as predict binding energy.44
AutoDock4.0 calculation indicates that the binding free energies
of the compounds to HpFabZ range from -10.16 to -5.96 kcal/
mol (Table S1 and Figure S1 in the Supporting Information).
Considering these two scoring results, structural diversity,
druglikeness, and synthetic feasibility, 12 compounds (5a-l)
a Reagents and conditions: (a) toluene, rt, 1-2 h; (b) ClCH2COOH,
CH3COONa, EtOH, reflux, 12 h; (c) MeOH, H2SO4, reflux; (d) HCl, NaNO2
0 °C; (e) CuCl2, 2-furaldehyde, rt, 4 h; (f) piperidine, ethanol, reflux, 12 h;
(g) LiOH, MeOH, rt, 4 h; (h) pyridine, rt, 5 h.
were selected and synthesized for actual biological testing and
their chemical structures are shown in Table 3.
The primary inhibition activities of compounds 5a-l against
HpFabZ at 10 µM were measured. The results are also listed in
Table 3. Of the 12 compounds, eight showed enhanced inhibition
activities against HpFabZ at 10 µM. Thus, the IC50 values of
these eight compounds were also determined (Table 3) and the
IC50 values of five compounds (5c, 5f, 5g, 5h, and 5j) were
discovered less than 4 µM. The IC50 value of compound 5h
(0.86 ( 0.01 µM) was 46 times higher than that of starting
compound 1 (IC50) 39.8 ( 0.35 µM). The focused library
broadened the diversity of subtituents R1 and R2 and the linkers
between them. The result demonstrated that hydroxyl substituted
naphthalene, 2,4-dihydroxy-3,5-dibromo phenyl ring and me-
thyleneformohydrazide seem to be the optimized fragments for
R1, R2, and linker.
3.4. Antibacterial Activity. Fifteen compounds (3h-k,
3m-n, 3q, 5b-d, and 5f-j) were shown to be inhibitors of
HpFabZ through two cycles design. Their antibacterial activities
were tested against the H. pylori strains SS1 and ATCC4 using
the minimal inhibitory concentrations (MIC) approach. The
results are listed in Tables 1 and 3. Twelve compounds showed
weak antibacterial properties, with MIC values of 50-100 µg/
mL. Because of poor solubility, we did not obtained the MIC
values of compounds 3h and 3n. Compounds 3i and 5b are
inactive in inhibiting the growth of H. pylori strain even at a
concentration of >100 µg/mL. Among the compounds, 3m, 3q,
3.3. Identification of Inhibitors by the Phase II Design.
The two binding models of the phase I designed compounds
with HpFabZ indicate that compounds with bulky substituents
like compound 3m still show high activities despite that only
one binding model (model A) exists. This may be structurally
attributed to that the 2,4-dihydroxyl-3,5-dibromo phenyl ring
binds more tightly with the hydrophobic pocket lined by Arg158,
Glu159, Phe59′, and Lys62′ so that two hydroxyl groups of 3m
form two strong hydrogen bonds (H-bonds), respectively, with
the backbone carbonyl of Glu159 and the guanidinium group
of Arg158. Thus, in the phase II compound design, a focused
library was designed only according to model A (Figure 3A).
Model A clearly illustrates that the inhibitors discovered through
the phase I design could be divided into three parts (Figure 3A):
part A interacts with the large pocket of HpFabZ composed of
Tyr100, Met102, Phe109′, Ile111′, and Pro112′ (site A); part B
resides in the small pocket lined by Lys62′, Arg158, Glu159,
Phe59′, and Pro22 (site B); and part L is a linker between A
and B, interacting with the “saddle” pocket between sites A
and B with a length of 5-6 Å. According to the structural
diversity of the previously discovered inhibitors, we selected
14 fragments for part A, five fragments for part B, and four
linkers for part L (Figure 4). Combining these fragments

2472 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
Figure 5. Schematic picture of the superposition of HpFabZ-compounds complex. Two molecules of each compound (except compound 3m) bind
to one hexamer of HpFabZ with two distinct interaction models. HpFabZ dimer A/B, C/D, compound 1, compounds 3i, 3j, 3k, 3m, 3n, and 3q are
colored yellow/magenta, blue/orange, cyan, and green, respectively. The active-site cavities are produced with the program MOLE52 and shown in
dark-gray surface representations.
Table 3. Chemical Structures, HpFabZ Inhibitory Activities and Antibacterial Activity of The 12 Compounds Selected From The Focused Library
Using Virtual Screening
a Values are the means of three determinations and deviation from the mean is <10% of the mean value.
5c, 5f,and 5j displays encouraging antibacterial activity against
one strain (SS1 or ATCC4) compared with compounds 1 and 2
without any antibacterial activity. Although the antibacterial
activities of the compounds that were determined are not so
high in comparison with existing antibiotics, they provide
leading factors contributing to the discovery of new therapies
against the FAS-II pathway. Although the most potent inhibitor
(compound 5h) discovered in this study is structurally similar

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2473
to the patented antibacterial45 or antimetazoan parasite com-
pounds,46 it is purely incidental. Nevertheless, this further
illustrates that our discovery strategy is reasonable because the
design procedure converged some of the HpFabZ inhibitors into
similar structures of existing antibacterial compounds. In
addition, the molecular targets of the patented antibacterial
compounds are unknown, our study accidentally found that FabZ
may be one of the targets of these compounds. Of note, this
study only implied that HpFabZ is a target of the antibacterial
compounds similar to 5h. The target specificity of these
compounds or whether their antibacterial activity is associated
with other target needs to be further explored.
3.5. Structure-Activity Relationship (SAR). Bioassay
indicates that, of the 12 binders, eight compounds (5b-d and
5f-j) showed strong HpFabZ inhibitory activity with IC50 values
of 0.86-42.7 µM in Table 3 and the activity of the most potent
one 5h (0.86 µM) increased 46 times. Structurally, compounds
5a-j have similar chemical scaffolds with the B1 fragment
(Figure 4), indicating that B1 is the most optimized fragment
for part B. Fragments for part A are structurally more diverse
than fragments for part B; fragments A2-5, A7-10, and A14
are the building blocks of part A for the HpFabZ inhibitors.
Observing the crystal structures, the linker is with a length of
5-6 Å, which can involve 3-4 atoms and is relatively rigid in
a way that reduces the entropy loss when fitting into the steric
restraint binding groove. Considering the length and rigidity of
saddle and synthetic feasibility, the diversity of candidate linkers
is constrained. According to bioisostere replacement, four
representive linkers (L1-L4) were finally selected with similar
length and rigidity in the focused library design. The results
illustrated that compounds with moderate or potent HpFabZ
activity contain the L4 regiment as the linker and indicated that
L4 is the most favorable fragment for the linker between parts
A and B. Accordingly, as the HpFabZ inhibitors, the building
blocks for parts B and L are fixed at fragments B1 and L4 and
the fragment for part A is a little bit flexible.
dihydroxyl-3,5-dibromo phenyl ring binds more tightly with the
hydrophobic pocket lined by Arg158, Glu159, Phe59′, and
Lys62′ so that two hydroxyl groups of 3m form two strong
hydrogen bonds (H-bonds), respectively, with the backbone
carbonyl of Glu159 and the guanidinium group of Arg158. The
binding models of compound 3i-k, 3m, 3n, and 3q with
HpFabZ clearly illustrated that structural modifications that
increase the hydrophobicities of R1 and R2 may increase the
inhibition activities due to enhancement of the binding affinity
(Tables 2 and 3). Compounds with a large hydrophobic group
(naphthalene ring or 9H-fluorene ring) possess the potent
inhibitory activity due to forming two strong hydrogen bonds
(H-bonds), respectively, with the backbone carbonyl of Glu159
and the guanidinium group of Arg158. The additional hydrogen
bond play vital roles for these compounds binding to the
HpFabZ, and this may be an important reason that these
compounds could potentially inhibit HpFabZ. However, no
further activity improvements were observed with even bulkier
fragments (5b, 5e, 5k, and 5l) than the substituent naphthalene;
this probably contributed to the size limit of the pocket (site A
in Figure 3).
4. Conclusions
The discovery of HpFabZ inhibitors is now of special interest
in the treatment of gastric diseases. In the present study, a series
of hydrazine derivatives and a series of novel thiazolidone
derivatives were designed, synthesized based on the screening
hits 1 and 2. All the compounds were evaluated by the cell-
based assay for their inhibitory activities against HpFabZ. Six
compounds (3i, 3j, 3k, 3m, and 3q) for concentration-response
studies showed prominent inhibitory activities, five compounds
with IC50 values less than 2 µM, which is nearly 25 times more
potent than the initial compound 1. To discover novel entries
of HpFabZ inhibitors with more potent activities, on the basis
of the chemical structures of the HpFabZ inhibitors we
discovered previously and the modes of binding to HpFabZ in
the crystal of the complexes of small molecules with the
HpFabZ, a focused library has been designed by using the
combinatorial library design program LD1.0.26 Through virtual
screening and druglike analysis, 12 compounds were selected
from the focused library for chemical synthesis and bioassay.
Remarkably, eight compounds among the 12 molecules show
HpFabZ inhibitor activities, with IC50 values ranging from 0.86
to 42.7 µM (Table 3). The hit rate for inhibitors is 66.7%. In
comparison with the results in the phase I design, inhibitory
activities of the active compounds designed in the phase II are
moderately improved (Table 3). The crystallography study
demonstrated that six compounds (3i-k, 3m-n, and 3q) bind
to the binding pocket of HpFabZ in a similar way: part A fits
into site A, part B is located in site B, and part L interacts with
the saddle site of HpFabZ. The bioassay results indicate that
the fragment combinations for potent HpFabZ inhibitors are
A2-5, A7-10, and L4 in conjunction with B1. In addition,
most of the active compounds discovered in this study are
leading factors contributing to the discovery of new therapies
against the FAS-II pathway, so the encouraging results in vitro
activities may possess potential for the treatment of H. pylori
infections based on new mechanisms.
The reason underlying the structure-activity relationship of
compounds 3 and 5 can be addressed from the X-ray crystal
structures of HpFabZ in complex with 3i-k, 3m, 3n, and 3q
(Figure 3). In model A, inhibitors bind to the entrance of
HpFabZ tunnel: the substituented phenyl ring of compound 3
(except 3m) is located between the phenol ring of Tyr100 and
the pyrrolidine ring of Pro112′, forming a sandwich structure.
The hydrophobic interactions between the phenyl ring of
inhibitors and residues Phe109′, Ile111′, and Met102 stabilized
the inhibitors in the right place, and the 2,4-dihydroxy-3,5-
dibromo phenyl ring at the other end of compound 1 binds with
a pocket formed by Arg158, Glu159, Phe59′, and Lys62′ through
hydrophobic interactions. Extraordinarily, the aromatic ring of
R2 substituent is involved in a π· · ·π stacking interaction with
the side chain of Phe59′ in addition to cation-π interactions
with the positively charged side chains of Lys62′ and Arg158.
In model B, compound 3 (except 3m) entered into the middle
of the tunnel, located near the active site of HpFabZ (His58
and Glu72′): the 2,4-dihydroxy-3,5-dibromo phenyl ring almost
accessed the exit of the tunnel, contacting with Ile20, Leu21,
Pro22, Phe83, Ala94, Lys97, and Val99 via hydrophobic
interactions while the substituented phenyl is sandwiched
between Ile98 and Phe59′. Different from the other five
inhibitors, compound 3m was observed to bind to HpFabZ only
with model A. It exclusively adopted an extended linear
conformation with good shape complementarity with the binding
tunnel a little differently. Because of steric hindrance imposed
by the bulky naphthalene ring of the inhibitor, the 2,4-
5. Experimental Section
Chemistry. The reagents (chemicals) were purchased from
Lancaster, Acros, and Shanghai Chemical Reagent Company and
used without further purification. Analytical thin-layer chromatog-
raphy (TLC) was HSGF 254 (150-200 µm thickness, Yantai

2474 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
Huiyou Company, China). Yields were not optimized. Melting
points were measured in capillary tube on a SGW X-4 melting point
apparatus without correction. Nuclear magnetic resonance (NMR)
spectra were performed on a Bruker AMX-400 and AMX-300 NMR
(IS as TMS). Chemical shifts were reported in parts per million
(ppm, δ) downfield from tetramethylsilane. Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). Low- and high-resolution mass spectra
(LRMS and HRMS) were given with electric, electrospray, and
matrix-assisted laser desorption ionization (EI, ESI, and MALDI)
produced by Finnigan MAT-95, LCQ-DECA spectrometer, and
IonSpec 4.7 T, respectively. An Agilent 1100 Series HPLC with
an Agilent Zorbax Eclipse XDB-C18 (4.6 mm × 50 mm, 5 µm)
reversed phase column was used for analytical HPLC analyses.
Preparative RP-HPLC was performed on an Agilent 1200 Series
HPLC with an Agilent Zorbax Eclipse XDB-C18 (9.4 mm × 250
mm, 5 µm) column. The elution buffer was an A/B gradient, where
A ) H2O-0.1% TFA and B ) CH3OH-0.1% TFA. Elemental
analyses were performed on an Elementar Vario EL I analyzer.
3,5-Dibromo-2,4-dihydroxy-benzaldehyde (7). Representa-
tive Procedure for 7 and 10. To a stirred solution of 6 (2.76 g, 10
mmol) in EtOH (50 mL, 95%), Br2 (2.5 mL, 50 mmol) was added
dropwise at room temperature. Some pale-yellow solid appeared
as Br2 was added. Stirring was continued for 30 min. Thereafter,
the suspension was poured into H2O (100 mL), filtered under
suction, and the precipitate was washed with H2O for three times
and recrystallized from EtOH/H2O (1:1 v/v) to give 7 as brown
(d, J ) 8.7 Hz, 1H), 7.53-7.66 (m, 2H), 7.85 (t, J ) 15.6 Hz,
2H), 8.26 (dt, J ) 1.9 Hz and J ) 1.6 Hz, 1H).
Biphenyl-4-carboxylic Acid Hydrazide (12b). 1H NMR (DMSO-
d6): δ 7.38-7.43 (m, 1H), 7.49 (t, J ) 15.0 Hz, 2H), 7.71-7.77
(m, 4H), 7.95 (d, J ) 8.4 Hz, 2H).
6-Bromo-2-naphthoic Acid Hydrazide (12c). 1H NMR (DMSO-
d6): δ 7.71 (dd, J ) 1.5 Hz and J ) 1.5 Hz, 1H), 7.80-7.94 (m,
3H), 8.28 (d, J ) 2.1 Hz, 1H), 8.45 (s, 1H).
4-Phenoxy-benzoic Acid Hydrazide (12d). 1H NMR (DMSO-
d6): δ 7.00-7.11 (m, 4H), 7.19-7.24 (m, 1H), 7.41-7.48 (m, 2H),
7.84-89 (m, 2H).
6-Hydroxy-2-naphthoic Acid Hydrazide (12e). 1H NMR (DM-
SO-d6): δ 7.05-7.17 (m, 2H), 7.58-7.74 (m, 1H), 7.80-7.87 (m,
2H), 8.31 (s, 1H).
9H-Fluorene-2-carboxylic Acid Hydrazide (12f). 1H NMR
(DMSO-d6): δ 7.35-7.45 (m, 2H), 7.63 (d, J ) 7.2 Hz, 1H), 7.88
(d, J ) 8.4 Hz, 1H), 7.97 (d, J ) 7.5 Hz, 2H), 8.06 (s, 1H). EI-MS
m/z 224 (M+) 193 (100%).
4′-Hydroxy-biphenyl-4-carboxylic Acid Hydrazide (12g). 1H
NMR (DMSO-d6): δ 6.83-6.90 (m, 2H), 7.56 (d, J ) 8.1 Hz, 2H),
7.67 (d, J ) 8.4 Hz, 2H), 7.87-7.94 (m, 2H).
Nicotinic Acid (3,5-Dibromo-2,4-dihydroxy-benzylidene)-hy-
drazide (1). General Procedure for 1, 3a-r, and 5a-j. The
mixture of nicotinic acid hydrazide (137 mg, 1 mmol) and 7 (296
mg, 1 mmol) was dissolved in EtOH, and after a while, plenty of
yellow precipitation appeared. The mixture was stirred at room
temperature for 3 h. The precipitation was collected by filtration
and washed with EtOH and purified using RP-HPLC to afford the
final compound 1 (391 mg, 94.9%) as a yellow solid: mp 255-256
1
needles (3.49 g, 59.0%): mp 210-213 °C. H NMR (300 MHz,
CD3OD): δ 5.52 (s, 0.45H, OH), 7.39 (s, 0.45H, OH), 7.86 (s, 1H),
9.69 (s, 1H, -CHdO). EI-MS m/z 294 (M+) 294 (100%). HRMS
(EI) m/z calcd for C7H4Br2O3 (M+) 293.8527, found 293.8527.
3,5-Dibromo-4-hydroxy-benzaldehyde (10). Melting point
181-183 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.08 (s, 2H), 9.80
(s, 1H, -CHdO). EI-MS m/z 278 (M+) 278 (100%). HRMS (EI)
m/z calcd for C7H4Br2O2 (M+) 277.8578, found 277.8577.
3,5-Dibromo-2,4-dimethoxy-benzaldehyde (8). Compound 7
(148 mg, 0.5 mmol) was dissolved in DMF, and then K2CO3 (700
mg, 5.0 mmol) and dimethyl sulfate (3.2 mL, 40.0 mmol) were
added in sequence. The suspension was heated to 100 °C and stirred
for 3 h until TLC indicated the completion of the reaction. After
K2CO3 was filtered off, the filtrate was poured into water and
extracted with EtOAc for three times, and the combined organic
layer were washed, dried over MgSO4, filtered, and concentrated
to yield 8 (136 mg, 84.5%): mp 98-100 °C. 1H NMR (300 MHz,
CDCl3): δ 3.96 (s, 3H, -OCH3), 3.99 (s, 3H, -OCH3), 8.03 (s,
1H), 10.23 (s, 1H, -CHdO). EI-MS m/z 321 (M+) 306 (100%).
HRMS (EI) m/z calcd for C9H8Br2O3 (M+) 321.8840, found
321.8840.
1
°C. H NMR (300 MHz, DMSO-d6): δ 7.59 (dd, J ) 5.1 and 7.8
Hz, 1H), 7.79 (s, 1H), 8.28 (dt, J ) 7.8 and 1.8 Hz, 1H), 8.46 (s,
1H), 8.78 (dd, J ) 5.1 and 1.2 Hz, 1H), 9.07 (d, J ) 1.8 Hz, 1H).
EI-MS m/z 413 (M+) 106 (100%). HRMS (EI) m/z calcd for M+,
412.9011, found 412.8972. Anal. (C13H9Br2N3O3) calcd, C 37.62,
H 2.19, N 10.12; found, C 37.99, H 2.20, N 10.03.
Nicotinic Acid (2,4-Dihydroxy-benzylidene)-hydrazide (3a).
In the same manner as described in the preparation of 1, 3a was
prepared from nicotinic acid hydrazide and 2,3-dihydroxy-benzal-
1
dehyde (6). Yield: 53.8%: mp 277-278 °C. H NMR (300 MHz,
DMSO-d6): δ 6.35 (m, 2H), 7.33 (d, J ) 8.4 Hz, 1H), 7.57 (dd, J
) 4.5 and 8.1 Hz, 1H), 8.24 (dt, J ) 8.1 and 1.8 Hz, 1H), 8.49 (s,
1H), δ 8.75 (dd, J ) 4.5 and 1.8 Hz, 1H), 9.04 (d, J ) 1.5 Hz,
1H). EI-MS m/z 257 (M+) 106 (100%). HRMS (EI) m/z calcd for
M+ 257.0080, found 257.0790. Anal. (C13H11N3O3) calcd C 60.70,
H 4.31, N 16.33; found C 60.31, H 4.22, N 16.33.
Nicotinic Acid (3,5-Dibromo-4-hydroxy-benzylidene)-hy-
drazide (3b). In the same manner as described in the preparation
of 1, 3b was prepared from nicotinic acid hydrazide and 3,5-
dibromo-4-hydroxy-benzaldehyde (10). Yield: 59.6%; mp 275-276
1-Hydroxy-2-naphthoic Acid Hydrazide (12a). Representa-
tive Procedure for 12a-g. 1-Hydroxy-2-naphthoic acid (190 mg,
1mmol) was dissolved in EtOH, then H2SO4 (catalysis) was added.
The solution was refluxed for 8 h, and then the solvent was
evaporated in vacuo. Water was added and the aqueous layer was
back-extracted with ethyl acetate. The organic layer was washed
with saturated sodium bicarbonate and brine. The combined organic
extracts were dried over MgSO4, filtered through cotton, and
concentrated in vacuo and then purified by chromatography with
EtOAc/petroleum ether (1:20, v/v) to give ethyl 1-hydroxy-2-
1
°C. H NMR (300 MHz, DMSO-d6): δ 7.56 (dd, J ) 5.1 and 7.8
Hz, 1H), 7.94 (s, 2H), 8.24 (dt, J ) 7.8 and 2.1 Hz, 1H), 8.28 (s,
1H), δ 8.75 (dd, J ) 5.1 and 1.5 Hz,1H), 9.03 (d, J ) 1.8 Hz, 1H).
EI-MS m/z 397 (M+) 106 (100%). HRMS (EI) m/z calcd for M+
396.9061, found 396.9058. Anal. (C13H9Br2N3O2) calcd, C 39.13,
H 2.27, N 10.53; found C 39.06, H 2.43, N 10.46.
Nicotinic Acid (4-Hydroxy-benzylidene)-hydrazide (3c). In the
same manner as described in the preparation of 1, 3c was prepared
from nicotinic acid hydrazide and 4-hydroxy-benzaldehyde (9).
1
1
naphthoate as yellow-white oil. Yield 90.0%. H NMR (CDCl3):
Yield: 64.2%; mp 247-249 °C. H NMR (300 MHz, DMSO-d6):
δ 1.47(t, J ) 14.4 Hz, 3H), 3.71 (q, 2H), 7.29 (dd, J ) 2.1 Hz and
J ) 2.1 Hz, 1H), 7.54 (t, J ) 15.2 Hz, 1H), 7.59 (t, J ) 16.6 Hz,
1H), 7.79 (d, J ) 15.0 Hz, 2H), 8.44 (d, J ) 9.3 Hz, 1H), 12.10 (s,
1H).
Ethyl 1-hydroxy-2-naphthoate was dissolved in hydrazine hydrate
and heated to reflux for 8 h, and then the solvent was evaporated.
Water was added, and the aqueous layer was back-extracted with
ethyl acetate. The organic layer was washed with saturated sodium
bicarbonate and brine. The combined organic extracts were dried
over MgSO4, filtered through cotton, and concentrated in vacuo
and then purified by chromatography with EtOAc/petroleum ether
(1:15, v/v) to give 12a. Yield 72.5%. 1H NMR (DMSO-d6): δ 7.36
δ 6.84 (d, J ) 8.4 Hz, 2H), 7.57 (m, 3H), 8.23 (dt, J ) 7.8 and 2.1
Hz, 1H), 8.33 (s, 1H), 8.74 (dd, J ) 4.8 and 1.5 Hz, 1H), 9.03 (d,
J ) 1.8 Hz, 1H). EI-MS m/z 241 (M+) 106 (100%). HRMS (EI)
m/z calcd for M+ 241.0851, found 241.0860. Anal. (C13H11N3O2)
calcd C 64.72, H 4.60, N 17.42; found C 64.49, H 4.48, N 17.39.
Nicotinic Acid (3,5-Dibromo-2,4-dimethoxy-benzylidene)-hy-
drazide (3d). In the same manner as described in the preparation
of 1, 3d was prepared from nicotinic acid hydrazide and intermedi-
1
ate 8, yielding, 61.8%; mp 186-189 °C. H NMR (300 MHz,
DMSO-d6): δ 3.85 (s, 6H), 7.59 (dd, J ) 4.2 and 8.4 Hz, 1H),
8.13 (s, 1H), 8.26 (d, J ) 8.4 Hz, 1H), 8.61 (s, 1H), 8.77 (d, J )
4.2 Hz, 1H), 9.08 (s, 1H). EI-MS m/z 441 (M+) 321 (100%). HRMS

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2475
(EI) m/z calcd for M+ 440.9324, found 440.9342. Anal.
(C15H13Br2N3O3) calcd C 40.66, H 2.96, N 9.48; found C 40.41 H
2.79, N 9.11.
acid hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7).
Yield: 54.8%; mp 244-246 °C. H NMR (300 MHz, DMSO-d6):
δ 7.25 (t, J ) 4.5 Hz, 1H), 7.76 (s, 1H), 7.91 (d, J ) 4.8 Hz, 2H),
8.43 (s, 1H). EI-MS m/z 418 (M+) 111 (100%). HRMS (EI) m/z
calcd for M+ 417.8622, found 417.8607. Anal. (C14H9Br3N2O3)
calcd C 34.31, H 1.92, N 6.67; found C 34.61, H 2.01, N 6.70.
1
Benzoic Acid (3,5-Dibromo-2,4-dihydroxy-benzylidene)-hy-
drazide (3e). In the same manner as described in the preparation
of 1, 3e was prepared from benzoic acid hydrazide and 3,5-dibromo-
2,4-dihydroxy-benzaldehyde (7). Yield: 21.4%; mp 194-196 °C.
1H NMR (300 MHz, DMSO-d6): δ 7.62 (m, 3H), 7.76 (s, 1H),
7.93 (d, J ) 6.9 Hz, 2H), 8.48 (s, 1H). EI-MS m/z 412 (M+) 105
(100%). HRMS (EI) m/z calcd for M+ 411.9058, found 411.9067.
Anal. (C14H10Br2N2O3) calcd C 40.61, H 2.43, N 6.77; found C
40.84, 2.50, 6.70.
Naphthalene-2-carboxylic Acid (3,5-Dibromo-2,4-dihydroxy-
benzylidene)-hydrazide (3m). In the same manner as described
in the preparation of 1, 3m was prepared from naphthalene-2-
carboxylic acid hydrazide and 3,5-dibromo-2,4-dihydroxy-benzal-
1
dehyde (7). Yield: 44.9%; mp 243-244 °C. H NMR (300 MHz,
DMSO-d6): δ 7.66 (m, 2H), 7.78 (s, 1H), 8.04 (m, 4H), 8.52 (s,
1H), 8.57 (s, 1H). EI-MS m/z 462 (M+) 155 (100%). HRMS (EI)
m/z calcd for M+ 461.9215, found 461.9207. Anal. (C18H12Br2N2O3)
calcd C 46.58, H 2.61, N 6.04; found C 46.69, H 2.56, N 6.09.
Furan-2-carboxylic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3f). In the same manner as described in the
preparation of 1, 3f was prepared from furan-2-carboxylic acid
hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7). Yield:
4-Methoxy-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3n). In the same manner as described in the
preparation of 1, 3n was prepared from 4-methoxy-benzoic acid
hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7). Yield:
1
52.3%; mp 247-249 °C. H NMR (300 MHz, DMSO-d6): δ 6.74
(dd, J ) 2.1 and 3.0 Hz, 1H), 7.34 (d, J ) 3.0 Hz, 1H), 7.74 (s,
1H), 7.98 (d, J ) 2.1 Hz, 1H), 8.47 (s, 1H). EI-MS m/z 402 (M+)
95 (100%). HRMS (EI) m/z calcd for M+ 401.8851, found
401.8848. Anal. (C12H8Br2N2O4) calcd, C 35.67, H 2.00, N 6.93;
found C 35.92, H 2.10, N 6.82.
1
64.5%; mp 240-242 °C. H NMR (300 MHz, DMSO-d6): δ 3.82
(s, 3H), 7.05 (d, J ) 9.0 Hz, 2H), 7.71 (s, 1H), 7.89 (d, J ) 9.0
Hz, 2H), 8.42 (s, 1H). EI-MS m/z 442 (M+) 135 (100%). HRMS
(EI) m/z calcd for M+ 441.9164, found 441.9135. Anal.
(C15H12Br2N2O4) calcd C 40.57, H 2.72, N 6.31; found C 40.88, H
2.69, N 5.82.
3-Fluoro-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3g). In the same manner as described in the
preparation of 1, 3g was prepared from 3-fluoro-benzoic acid
hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7). Yield:
1
Isonicotinic Acid (3,5-Dibromo-2,4-dihydroxy-benzylidene)-
hydrazide (3o). In the same manner as described in the preparation
of 1, 3o was prepared from isonicotinic acid hydrazide and 3,5-
dibromo-2,4-dihydroxy-benzaldehyde (7). Yield: 34.9%. HPLC:
100%, tR ) 2.86 min, mp 242-244 °C. 1H NMR (300 MHz,
DMSO-d6): δ 7.81 (s, 1H), 7.85 (d, J ) 6.0 Hz, 2H), 8.49 (s, 1H),
8.81 (d, J ) 6.0 Hz, 2H). EI-MS m/z 413 (M+) 106 (100%). HRMS
(EI) m/z calcd for C13H9Br2N3O3 (M+) 412.9011, found 412.8996.
45.4%; mp 247-249 °C. H NMR (300 MHz, DMSO-d6): δ 7.62
(m, 5H), 8.46 (s, 1H). EI-MS m/z 430 (M+) 123 (100%). HRMS
(EI) m/z calcd for M+ 429.8964, found 429.8961. Anal.
(C14H9Br2FN2O3) calcd 38.92, H 2.10, N 6.48; found C 39.08, H
2.06, N 6.51.
4-Fluoro-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3h). In the same manner as described in the
preparation of 3c, 3h was prepared from 4-fluoro-benzoic acid
hydrazide and and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7).
Acetic Acid (3,5-Dibromo-2,4-dihydroxy-benzylidene)-hy-
drazide (3p). In the same manner as described in the preparation
of 1, 3p was prepared from acetic acid hydrazide and 3,5-dibromo-
2,4-dihydroxy-benzaldehyde (7). Yield: 86.3%; mp 246-248 °C.
1H NMR (300 MHz, DMSO-d6): δ 1.98 (s, 3H), 7.72 (s, 1H), 8.18
(s, 1H). EI-MS m/z 350 (M+) 293 (100%). HRMS (EI) m/z calcd
for M+ 349.8902, found 349.8902. Anal. (C9H8Br2N2O3) calcd C
3.071, H 2.29, N 7.96; found C 30.76, H 2.28, N 7.52.
1
Yield: 48.6%; mp 232-234 °C. H NMR (300 MHz, DMSO-d6):
δ 7.26 (t, J ) 8.7 Hz, 2H), 7.52 (s, 1H), 7.96 (m, 2H), 8.33 (s,
1H). EI-MS m/z 430 (M+) 123 (100%). HRMS (EI) m/z calcd for
M+ 429.8964, found 429.8955. Anal. (C14H9Br2FN2O3) calcd C
38.92, H 2.10, N 6.48; found C 39.26, H 2.40, N 6.55.
4-Chloro-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3i). In the same manner as described in the
preparation of 1, 3i was prepared from 4-chloro-benzoic acid
hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7). Yield:
4-tert-Butyl-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3q). In the same manner as described in the
preparation of 1, 3q was prepared from 4-tert-butyl-benzoic acid
hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7). Yield:
1
16.2%; mp 208-211 °C. H NMR (300 MHz, DMSO-d6): δ 7.64
(d, J ) 8.4 Hz, 2H), 7.78 (s, 1H), 7.96 (d, J ) 8.4 Hz, 2H), 8.46
(s, 1H). EI-MS m/z 446 (M+) 139 (100%). HRMS (EI) m/z calcd
for M+ 445.8668, found 445.8653. Anal. (C14H9Br2ClN2O3) calcd
C 37.49, H 2.02, N 6.25; found C 37.40, H 2.18, N 6.02.
1
50.7%; mp 240-242 °C. H NMR (300 MHz, DMSO-d6): δ 1.32
(s, 9H), 7.56 (d, J ) 8.4 Hz, 2H), 7.75 (s, 1H), 7.87 (d, J ) 8.4
Hz, 2H), 8.46 (s, 1H). EI-MS m/z 468 (M+) 161 (100%). HRMS
(EI) m/z calcd for M+ 467.9684, found 467.9682. Anal.
(C18H18Br2N2O3) calcd C 45.98, H 3.86, N 5.96; found C 46.23,
4.14, N 5.65.
4-Bromo-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3j). In the same manner as described in the
preparation of 1, 3j was prepared from 4-bromo-benzoic acid
hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7). Yield:
1
(4-Hydroxy-phenyl)-acetic Acid (3,5-Dibromo-2,4-dihydroxy-
benzylidene)-hydrazide (3r). In the same manner as described in
the preparation of 1, 3r was prepared from (4-hydroxy-phenyl)-
acetic acid hydrazide and 3,5-dibromo-2,4-dihydroxy-benzaldehyde
(7). Yield: 45.7%; HPLC: 99.45%, tR ) 3.53 min, mp 241-242
31.3%; mp 219-222 °C. H NMR (300 MHz, DMSO-d6): δ 7.75
(m, 3H), 7.85 (d, J ) 8.4 Hz, 2H), 8.44 (s, 1H); EI-MS m/z 490
(M+) 183 (100%). HRMS (EI) m/z calcd for M+, 489.8163, found
489.8148. Anal. (C14H9Br3N2O3) calcd C 34.11, H 1.84, N 5.68;
found C 34.31, H 1.88, N 6.55.
1
°C. H NMR (300 MHz, DMSO-d6): δ 3.43 (s, 2H), 6.69 (d, J )
3-Bromo-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (3k). In the same manner as described in the
preparation of 3c, 3k was prepared from 3-bromo-benzoic acid
hydrazide and and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7).
8.4 Hz, 2H), 7.09 (d, J ) 8.4 Hz, 2H), 7.72 (s, 1H), 8.23 (s, 1H).
EI-MS m/z 442 (M+) 107 (100%). HRMS (EI) m/z calcd for
C15H12Br2N2O4 (M+) 441.9164, found 441.9171.
1
Yield: 34.4%; mp 214-216 °C. H NMR (300 MHz, DMSO-d6):
4-Hydroxy-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (5a). In the same manner as described in the
preparation of 1, 5a was prepared from 4-hydroxy-benzoic acid
hydrazide and and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7).
Yield:76.1%; mp 275-276 °C. 1H NMR (300 MHz, DMSO-d6): δ
6.88 (s, 1H), 6.91 (s, 1H), 7.74 (s, 1H), 7.84 (d, J ) 9.0 Hz, 2H),
8.44 (s, 1H). LRMS (EI) m/z 428 (M+) 121 (100%). HRMS (EI)
m/z calcd for M+ 427.9007, found 427.9026. Anal. (C14H10Br2N2O4)
calcd C 39.10, H 2.34, N 6.51; found C 39.27, H 2.48, N 6.38.
δ 7.44 (t, J ) 7.8 Hz,1H), 7.52 (s, 1H), 7.75 (d, J ) 8.4 Hz, 1H),
7.90 (d, J ) 7.5 Hz, 1H), 8.09 (s, 1H), 8.34 (s, 1H). EI-MS m/z
490 (M+) 183 (100%). HRMS (EI) m/z calcd for M+ 489.8163,
found 489.8147. Anal. (C14H9Br3N2O3) calcd C 34.11, H 1.84, N
5.68; found C 34.31, H 1.79, N 5.64.
Thiophene-2-carboxylic Acid (3,5-Dibromo-2,4-dihydroxy-
benzylidene)-hydrazide (3l). In the same manner as described in
the preparation of 1, 3l was prepared from thiophene-2-carboxylic

2476 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
4′-Hydroxy-biphenyl-4-carboxylic Acid (3,5-Dibromo-2,4-
dihydroxy-benzylidene)-hydrazide (5b). In the same manner as
described in the preparation of 1, 5b was prepared from 4′-hydroxy-
biphenyl-4-carboxylic acid hydrazide (12g) and 3,5-dibromo-2,4-
dihydroxy-benzaldehyde (7). Yield: 68.1%; HPLC: 99.61%, tR )
6-Hydroxy-naphthalene-2-carboxylic Acid (3,5-Dibromo-2,4-
dihydroxy-benzylidene)-hydrazide (5i). In the same manner as
described in the preparation of 1, 5i was prepared from 6-hydroxy-
2-naphthoic acid hydrazide (12e) and 3,5-dibromo-2,4-dihydroxy-
benzaldehyde (7). Yield: 52%; HPLC: 95.73%, tR ) 2.56 min, mp
113-114 °C. 1H NMR (300 MHz, DMSO-d6): δ 7.20 (t, 2H), 7.76
(d, J ) 5.4 Hz, 1H), 7.83 (d, J ) 8.7 Hz, 1H), 7.93 (t, 2H), 8.46
(s, 1H), 8.51 (s, 1H). LRMS (EI) m/z 478 (M+) 171 (100%). HRMS
(EI) m/z calcd C18H12Br2N2O4 (M+) 477.9164, found 477.9139.
6-Bromo-naphthalene-2-carboxylic Acid (3,5-Dibromo-2,4-
dihydroxy-benzylidene)-hydrazide (5j). In the same manner as
described in the preparation of 1, 5j was prepared from 6-bromo-
2-naphthoic acid hydrazide (12c) and 3,5-dibromo-2,4-dihydroxy-
1
3.15 min, mp 113-114 °C. H NMR (300 MHz, DMSO-d6): δ
6.91 (d, J ) 7.8 Hz, 2H), 7.61 (d, J ) 8.1 Hz, 2H), 7.77 (d, J )
5.4 Hz, 3H), 8.00 (d, J ) 6.9 Hz, 2H), 8.50 (s, 1H). LRMS (EI)
m/z 504 (M+) 197 (100%). HRMS (EI) m/z calcd for C20H14Br2N2O4
(M+) 503.9320, found 503.9295.
3,5-Bis(trifluoromethyl)-benzoic Acid (3,5-Dibromo-2,4-di-
hydroxy-benzylidene)-hydrazide (5c). In the same manner as
described in the preparation of 1, 5c was prepared from 3,5-
bis(trifluoromethyl)-benzoic acid hydrazide and 3,5-dibromo-2,4-
1
benzaldehyde (7). Yield: 59.4%; mp 166-167 °C. H NMR (300
1
dihydroxy-benzaldehyde (7). Yield: 61.5%; mp 240-242 °C. H
MHz, DMSO-d6): δ 7.75 (t, 2H), 8.07 (d, J ) 7.8 Hz, 3H), 8.32
(s, 1H), 8.51 (s, 1H), 8.59 (s, 1H). LRMS (EI) m/z 540 (M+) 235
(100%). HRMS (EI) m/z calcd for M+ 539.8320, found 539.8250.
Anal. (C18H11Br3N2O3) calcd C 39.81, H 2.04, N 5.16; found C
40.06, H 2.28, N 5.01.
NMR (300 MHz, DMSO-d6): δ 7.78 (d, J ) 12.0 Hz, 1H), 8.34 (s,
1H), 8.48 (s, 1H), 8.57 (s, 2H). LRMS (EI) m/z 548 (M+) 241
(100%). HRMS (EI) m/z calcd for M+ 547.8806, found 547.8818.
Anal. (C16H8Br2F6N2O3) calcd C 34.94, H 1.47, N 5.09; found
35.30, H 1.47, N 5.09.
N-(3,5-Bis(trifluoromethyl)phenyl)-3-(2,3,4-trimethoxyphe-
nyl) Acrylamide (5l). 3-(2,3,4-Trimethoxyphenyl)acrylic acid (238
mg, 1.0 mmol) and 3,5-bis(trifluoromethyl)aniline (229 mg, 1.0
mmol) was dissolved in CH2Cl2 and pyridine. After 5 h, plenty of
water was added and the organic layer was extracted with EtOAc
for three times, and the combined organic layers were washed and
then dried over MgSO4. After flash chromatography with ethyl
acetate-petroleum (1:5, v/v) and purified using RP-HPLC to afford
the final compound 5l was obtained as a white solid, yield 67.6%;
HPLC: 99.00%, tR ) 4.00 min. 1H NMR (300 MHz, DMSO-d6): δ
3.78 (s, 3H), 3.85 (s, 6H), 6.75 (d, J ) 15.6 Hz, 1H), 6.93 (d, J )
8.4 Hz, 1H), 7.38 (d, J ) 8.7 Hz, 1H), 7.75 (d, J ) 15.9 Hz, 1H),
8.36 (s, 2H). EI-MS m/z 449 (M+) 221 (100%). HRMS (EI) m/z
calcd for M+ 449.1062, found 449.1035.
4-Phenoxy-benzoic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (5d). In the same manner as described in the
preparation of 1, 5d was prepared from 4-phenoxy-benzoic acid
hydrazide (12d) and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7).
1
Yield: 79.2%; mp 113-114 °C. H NMR (300 MHz, DMSO-d6):
δ 7.09-7.15 (m, 3H), 7.24 (t, 1H), 7.43 -7.50 (m, 3H), 7.76 (s,
1H), 7.99 (d, J ) 9 Hz, 2H), 8.46 (s, 1H). LRMS (EI) m/z 504
(M+) 197 (100%). HRMS (EI) m/z calcd for M+ 503.9320, found
503.9302. Anal. (C20H14Br2N2O4) calcd C 47.46, H 2.79, N 5.53;
found C 47.56, H 2.71, N 5.74.
Biphenyl-4-carboxylic Acid (3,5-Dibromo-2,4-dihydroxy-ben-
zylidene)-hydrazide (5e). In the same manner as described in the
preparation of 1, 5e was prepared from biphenyl-4-carboxylic acid
hydrazide (12b) and 3,5-dibromo-2,4-dihydroxy-benzaldehyde (7).
1-(2-Methoxy-ethyl)-3-phenyl-thiourea (15a). Representa-
tive Procedure for 15a-q and 5k. Isothiocyanato-benzene (1.35
g, 0.01 mol) was dissolved in toluene, and then 2-methoxy-
ethylamine (751 mg, 0.01 mol) was added, and after a while, plenty
of white precipitation appeared. Stirring was continued for 1 h.
The precipitation was collected by filtration, washed with toluene,
and dried to afford 15a (1.997 g, 95.0%) as white powder. 1H NMR
(CDCl3): δ 3.30 (s, 3H), 3.53 (t, J ) 8.4 Hz, 2H), 3.82 (s, br, 2H),
7.19-7.29 (m, 3H), 7.38-7.43 (m, 2H).
1
Yield: 77.2%; mp 178-179 °C. H NMR (300 MHz, DMSO-d6):
δ 7.44 (t, 1H), 7.52 (t, 2H), 7.77 (t, 3H), 7.87 (d, J ) 8.7 Hz, 2H),
8.06 (d, J ) 8.4 Hz, 2H), 8.51 (s, 1H). LRMS (EI) m/z 488 (M+)
181 (100%). HRMS (EI) m/z calcd for M+, 487.9371, found
487.9348. Anal. (C20H14Br2N2O3) calcd, C 49.01, H 2.88, N 5.72;
found C 48.96, H 2.79, N 5.67.
9H-Fluorene-2-carboxylic Acid (3,5-Dibromo-2,4-dihydroxy-
benzylidene)-hydrazide (5f). In the same manner as described in
the preparation of 1, 5f was prepared from 9H-fluorene-2-carboxylic
acid hydrazide (12f) and 3,5-dibromo-2,4-dihydroxy-benzaldehyde
(7). Yield: 65.6%. 1H NMR (300 MHz, DMSO-d6): δ 3.40 (s, 2H),
7.42(s, 2H), 7.64 (s, 1H), 7.76 (s, 1H), 8.03 (d, J ) 6.6 Hz, 2H),
8.18 (s, 1H), 8.49 (s, 1H). EI-MS m/z 500 (M+) 193 (100%). HRMS
(EI) m/z calcd for M+ 499.9371, found 499.9381. Anal.
(C21H14Br2N2O3) calcd C 50.23, H 2.81, N 5.58; found C 50.38, H
2.87, N 5.47.
1-Hydroxy-naphthalene-2-carboxylic Acid (3,5-Dibromo-2,4-
dihydroxy-benzylidene)-hydrazide (5g). In the same manner as
described in the preparation of 1, 5g was prepared from 1-hydroxy-
2-naphthoic acid hydrazide (12a) and 3,5-dibromo-2,4-dihydroxy-
benzaldehyde (7). Yield: 68.3%; HPLC: 96.58%, tR ) 13.43 min,
mp 132-133 °C. 1H NMR (300 MHz, DMSO-d6): δ 7.47 (d, J )
8.7 Hz, 1H), 7.59 (t, 1H), 7.68 (t, 1H), 7.80 (s, 1H), 7.89-7.97
(m, 2H), 8.31 (d, J ) 8.1 Hz, 1H), 8.57 (s, 1H). LRMS (EI) m/z
478 (M+) 171 (100%). HRMS (EI) m/z calcd C18H12Br2N2O4 (M+)
477.9164, found 477.9139.
3-Hydroxy-naphthalene-2-carboxylic Acid (3,5-Dibromo-2,4-
dihydroxy-benzylidene)-hydrazide (5h). In the same manner as
described in the preparation of 1, 5h was prepared from 3-hydroxy-
2-naphthoic acid hydrazide and 3,5-dibromo-2,4-dihydroxy-ben-
zaldehyde (7). Yield: 62.7%; mp 241-242 °C. 1H NMR (300 MHz,
DMSO-d6): δ 7.28-7.75 (m, 2H), 7.76-7.71 (m, 1H), 7.82 (dd, J
) 9.6 Hz and J ) 9 Hz, 2H), 7.98 (dd, J ) 10.8 Hz and J ) 7.8
Hz, 1H), 8.43 (d, J ) 15.3 Hz, 1H), 8.57 (m, 1H). LRMS (EI) m/z
478 (M+) 171 (100%); HRMS (EI) m/z calcd for M+ 477.9164,
found 477.9153. Anal. (C18H12Br2N2O4) calcd C 45.03, H 2.52, N
5.83; found C 44.67, H 2.45, N 5.76.
1-(2-Methoxy-ethyl)-3-(4-methoxy-phenyl)-thiourea (15b). 1H
NMR (CDCl3): δ 3.29 (s, 3H), 3.52 (t, J ) 9.9 Hz, 2H), 3.80-3.82
(m, 5H), 6.93 (d, J ) 9.0 Hz, 2H), 7.14 (d, J ) 8.7 Hz, 2H).
1
1-(4-Bromo-phenyl)-3-(2-methoxy-ethyl)-thiourea (15c). H
NMR (CDCl3): δ 3.34 (s, 3H), 3.55 (t, 2H), 3.81 (t, 2H), 7.11 (d,
J ) 9.3 Hz, 2H), 7.53 (d, J ) 8.4 Hz, 2H).
1-(2-Methoxy-ethyl)-3-naphthalen-1-yl-thiourea (15d). 1H NMR
(CDCl3): δ 3.13 (s, 3H), 3.44 (t, J ) 9.6 Hz, 2H), 3.79 (t, J ) 3.9
Hz, 2H), 7.43-7.59 (m, 4H), 7.87-7.99 (m, 3H).
1-(2-Methoxy-ethyl)-3-(4-trifluoromethyl-phenyl)-thiourea
(15e). 1H NMR (CDCl3): δ 3.37 (s, 3H), 3.59 (t, J ) 9.3 Hz, 2H),
3.81 (t, J ) 7.2 Hz, 2H), 7.40 (d, J ) 8.1 Hz, 2H), 7.65 (d, J ) 8.1
Hz, 2H).
1-(3-Dimethylamino-propyl)-3-phenyl-thiourea (15f). 1H NMR
(CDCl3): δ 1.29 (t, 2H), 1.40 (s, 6H), 1.96 (t, 2H), 3.38 (t, 2H),
6.85-6.94 (m, 2H), 7.02-7.07 (m, 2H), 7.36 (s, 1H).
1
1-(2-Diethylamino-ethyl)-3-phenyl-thiourea (15g). H NMR
(CDCl3): δ 0.78 (t, 6H), 2.39 (q, 4H), 2.52 (t, J ) 15.6 Hz, 2H),
3.60 (t, 2H), 7.12-7.21 (m, 2H), 7.36-7.41 (m, 2H), 7.76 (s, 1H).
1-Phenyl-3-(2-pyrrolidin-1-yl-ethyl)-thiourea (15h). 1H NMR
(CDCl3): δ 1.53 (m, 4H), 2.40 (m, 4H), 2.58 (t, J ) 5.7 Hz, 2H),
3.51 (t, J ) 5.7 Hz 2H), 7.07-7.16 (m, 2H), 7.19-7.24 (m, 2H),
7.77 (s, 1H).
1-(2-Methoxy-ethyl)-3-(2-methoxy-phenyl)-thiourea (15i). 1H
NMR (CDCl3): δ 3.33 (s, 3H), 3.56 (t, J ) 10.2 Hz, 2H), 3.85 (s,
5H), 6.98-6.95 (m, 2H), 7.22(m, 1H), 7.26 (m, 2H).
1
1-(2-Methoxy-ethyl)-3-(3-methoxy-phenyl)-thioure (15j). H
NMR (CDCl3): δ 3.32 (s, 3H), 3.54 (t, J ) 9.6 Hz, 2H), 3.80 (s,
3H), 3.83 (t, 2H), 6.76-6.83 (m, 3H), 7.28-7.33 (m, 1H).

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2477
1-(2-Methoxy-ethyl)-3-(2-trifluoromethyl-phenyl)-thiourea
(15k). 1H NMR (CDCl3): δ 3.31 (s, 3H), 3.54 (t, J ) 9.9 Hz, 2H),
3.79 (t, 2H), 7.40-7.51 (m, 2H), 7.54-7.84 (m, 2H).
3-(2-Diethylamino-ethyl)-2-phenylimino-thiazolidin-4-one
(16g). H NMR (CDCl3): δ 0.76 (t, J ) 14.1 Hz, 6H), 2.38 (q,
4H), 2.56 (t, J ) 13.5 Hz, 2H), 3.47 (s, 2H), 3.64 (t, J ) 14.1 Hz,
2H), 6.61 (d, J ) 8.1 Hz, 2H), 6.78 (t, J ) 4.7 Hz, 1H), 6.99 (t,
2H).
1
1-(2-Methoxy-ethyl)-3-(3-trifluoromethyl-phenyl)-thiourea
(15l). 1H NMR (CDCl3): δ 3.34 (s, 3H), 3.57 (t, J ) 4.2 Hz, 2H),
3.80 (t, 2H), 7.486 (m, 3H), 7.54 (s, 1H).
2-Phenylimino-3-(2-pyrrolidin-1-yl-ethyl)-thiazolidin-4-one
1
(16h). H NMR (CDCl3): δ 1.92 (m, 4H), 3.01 (m, 4H), 3.14 (t,
1
1-(2-Bromo-phenyl)-3-(2-methoxy-ethyl)-thiourea (15m). H
2H), 3.87 (s, 2H), 4.09 (t, J ) 12.6 Hz, 2H), 6.98 (dd, J ) 1.2 Hz,
and J ) 1.5 Hz, 2H), 7.11 (m, 1H), 7.30-7.34 (m, 2H).
3-(2-Methoxy-ethyl)-2-(2-methoxy-phenylimino)-thiazolidin-
NMR (CDCl3): δ 3.32 (s, 3H), 3.56 (t, J ) 9.9 Hz, 2H), 3.82 (t,
2H), 7.16 (m, 1H), 7.36-7.43 (m, 2H), 7.67 (d, 1H).
1
1-(3-Bromo-phenyl)-3-(2-methoxy-ethyl)-thiourea (15n). H
1
4-one (16i). H NMR (CDCl3): δ 3.40 (s, 3H), 3.75 (t, J ) 11.1
NMR (CDCl3): δ 3.36 (s, 3H), 3.56 (t, J ) 9.9 Hz, 2H), 3.81 (t,
Hz, 2H), 3.82 (m, 5H), 4.12 (t, J ) 11.1 Hz, 2H), 6.89-6.95 (m,
2H), 7.19 (m, 1H), 7.25-7.30 (m, 1H), 7.38-7.40 (m, 2H).
3H), 7.13 (m, 1H).
1
1-(2-Fluoro-phenyl)-3-(2-methoxy-ethyl)-thiourea (15o). H
3-(2-Methoxy-ethyl)-2-(3-methoxy-phenylimino)-thiazolidin-
NMR (CDCl3): δ 3.33 (s, 3H), 3.56 (t, J ) 9.6 Hz, 2H), 3.80 (t,
1
4-one (16j). H NMR (CDCl3): δ 3.39 (s, 3H), 3.71 (t, J ) 11.1
2H), 6.52-6.61 (m, 1H), 7.13-7.36 (m, 2H), 7.56-7.57 (m, 1H).
Hz, 2H), 3.82 (s, 2H), 4.07 (t, J ) 11.4 Hz, 2H), 6.51-6.57 (m,
2H), 6.68-6.71 (m, 1H), 7.20-7.22 (m, 1H).
1
1-(4-Fluoro-phenyl)-3-(2-methoxy-ethyl)-thiourea (15p). H
NMR (CDCl3): δ 3.32 (s, 3H), 3.54 (t, J ) 9.9 Hz, 2H), 3.81 (t,
3-(2-Methoxy-ethyl)-2-(2-trifluoromethyl-phenylimino)-thia-
1
2H), 7.08-7.13 (m, 2H), 7.21-7.27 (m, 2H).
zolidin-4-one (16k). H NMR (CDCl3): δ 3.38 (s, 3H), 3.72 (t, J
1
1-(3-Fluoro-phenyl)-3-(2-methoxy-ethyl)-thiourea (15q). H
) 11.4 Hz, 2H), 3.87 (s, 2H), 4.07 (t, J ) 11.7 Hz, 2H), 7.02 (d,
J ) 8.1 Hz, 1H), 7.22-7.26 (m, 1H), 7.50 (m, 1H), 7.67 (d, J )
7.8 Hz, 1H).
NMR (CDCl3): δ 3.36 (s, 3H), 3.58 (t, J ) 9.9 Hz, 2H), 3.84 (t,
2H), 6.97-7.05 (m, 3H), 7.37-7.39 (m, 1H).
3-(2-Methoxy-ethyl)-2-(3-trifluoromethyl-phenylimino)-thia-
zolidin-4-one (16l). 1H NMR (CDCl3): δ 3.39 (s, 3H), 3.71 (t, J )
11.4 Hz, 2H), 3.86 (s, 2H), 4.10 (t, 2H), 7.16 (d, 1H), 7.24 (d,
1H), 7.41-7.46 (m, 2H).
1-(3,4,5-Trimethoxybenzyl)-3-(3,5-bis(trifluoromethyl)phe-
nyl) Thiourea (5k). In the same manner as described in the
preparation of 15a, 5k was prepared from 1-isothiocyanato-3,5-
bis(trifluoromethyl) benzene and (3,4,5-trimethoxyphenyl) metha-
1
2-(2-Bromo-phenylimino)-3-(2-methoxy-ethyl)-thiazolidin-4-
namine. Yield: 90.5%; HPLC: 100.00%, tR ) 2.39 min. H NMR
1
one (16m). H NMR (CDCl3): δ 3.40 (s, 3H), 3.79 (t, J ) 11.4
(300 MHz, DMSO-d6): δ 3.65 (s, 3H), 3.78 (s, 6H), 4.68 (s, 2H),
6.72 (s, 2H), 7.76 (s, 1H), 8.27 (d, J ) 3.3 Hz, 1H). EI-MS m/z
468 (M+) 181 (100%). HRMS (EI) m/z calcd for C15H12Br2N2O4
(M+) 468.0942, found 468.0936.
Hz, 2H), 3.86 (s, 2H), 4.11 (t, J ) 11.4 Hz, 2H), 6.95-6.97 (m,
2H), 7.27 (t, J ) 7.8 Hz, 1H), 7.61 (d, J ) 8.1 Hz, 1H).
2-(3-Bromo-phenylimino)-3-(2-methoxy-ethyl)-thiazolidin-4-
one (16n). 1H NMR (CDCl3): δ 3.38 (s, 3H), 3.69 (t, J ) 11.1 Hz,
2H), 3.85 (s, 2H), 4.06 (t, J ) 11.1 Hz, 2H), 6.90 (d, 1H), 7.13 (s,
1H), 7.21-7.23 (m, 1H), 7.26 (m, 1H).
3-(2-Methoxy-ethyl)-2-phenylimino-thiazolidin-4-one (16a).
Representative Procedure for 16a-q. 1-(2-Methoxy-ethyl)-3-
phenyl-thiourea (15a) (2.120 g, 16.08 mmol) was dissolved in
EtOH, and then anhydrous sodium acetate (1.643 g, 32.17 mmol)
and chloroacetic acid (1.190 g, 20.10 mmol) were added in
sequence. The suspension was refluxed for 12 h, and then the
solvent was evaporated. Water was added and the aqueous layer
was back-extracted with ethyl acetate. The organic layer was washed
with saturated sodium bicarbonate and brine. The combined organic
extracts were dried over Na2SO4, filtered through cotton, and
concentrated in vacuo, and then purified by chromatography with
EtOAc/petroleum ether (1:15, v/v) to give 2.235 g (89.3%) of 16a
2-(2-Fluoro-phenylimino)-3-(2-methoxy-ethyl)-thiazolidin-4-
one (16o). 1H NMR (CDCl3): δ 3.39 (s, 3H), 3.73 (t, J ) 11.4 Hz,
2H), 3.86 (s, 2H), 4.10 (t, J ) 11.1 Hz, 2H), 6.97-7.00 (m, 1H),
7.10-7.12 (m, 3H).
2-(4-Fluoro-phenylimino)-3-(2-methoxy-ethyl)-thiazolidin-4-
one (16p). 1H NMR (CDCl3): δ 3.39 (s, 3H), 3.70 (t, J ) 11.4 Hz,
2H), 3.83 (s, 2H), 4.06 (t, J ) 11.4 Hz, 2H), 6.89-6.95 (m, 2H),
7.00-7.06 (m, 2 H).
2-(3-Fluoro-phenylimino)-3-(2-methoxy-ethyl)-thiazolidin-4-
one (16q). 1H NMR (CDCl3): δ 3.38 (s, 3H), 3.69 (t, J ) 8.4 Hz,
2H), 3.84 (s, 2H), 4.06 (t, J ) 8.4 Hz, 2H), 6.66-6.70 (m, 1H),
6.73-6.75 (m, 1H), 6.81-6.86 (t, 1H), 7.26-7.32 (m, 1H).
5-Amino-2-chloro-benzoic Acid Methyl Ester (18a). A 100
mL round-bottom flask with a magnetic stir bar was charged with
5-amino-2-chloro-benzoic acid (3.433 g, 0.02 mol), H2SO4 (ca-
talysis), and 50 mL of MeOH, and the solution was refluxed for
5 h. MeOH was evaporated in vacuo. Water was added, and the
aqueous layer was back-extracted with ethyl acetate. The organic
layer was washed with saturated sodium bicarbonate and brine. The
combined organic extracts were dried over Na2SO4, filtered through
cotton, and concentrated in vacuo, yielding 18a (3.19 g, 93.0%) as
1
as yellow-white oil. H NMR (CDCl3): δ 3.39 (s, 3H), 3.71 (t, J
) 11.4 Hz, 2H), 3.83 (s, 2H), 4.09 (t, J ) 11.4 Hz, 2H), 6.95-6.97
(m, 2H), 7.14 (t, 1H), 7.32-7.37 (m, 2H).
3-(2-Methoxy-ethyl)-2-(4-methoxy-phenylimino)-thiazolidin-
1
4-one (16b). H NMR (CDCl3): δ 3.39 (s, 3H), 3.70 (t, J ) 11.1
Hz, 2H), 3.81 (s, 2H), 4.07 (t, J ) 11.1 Hz, 2H), 6.89-6.90 (m,
4H).
2-(4-Bromo-phenylimino)-3-(2-methoxy-ethyl)-thiazolidin-4-
one (16c). 1H NMR (CDCl3): δ 3.38 (s, 3H), 3.69 (t, J ) 11.1 Hz,
2H), 3.84 (s, 2H), 4.06 (t, J ) 10.8 Hz, 2H), 6.84 (d, J ) 8.7 Hz,
2H), 7.45 (d, J ) 8.7 Hz, 2H).
1
yellow oil. H NMR (CDCl3): δ 3.97 (s, 3H), 6.98 (d, 1H), 7.55
(d, 1H), 7.86 (d, 1H).
3-(2-Methoxy-ethyl)-2-(naphthalen-1-ylimino)-thiazolidin-4-
1
3-Amino-4-chloro-benzoic acid methyl ester (18b). In the same
manner as described in the preparation of 18a, 18b was prepared
from 3-amino-4-chloro-benzoic acid. Yield: 87.7%. 1H NMR
(CDCl3): δ 3.90 (s, 3H), 7.15 (d, 2H), 7.53 (d, 1H).
one (16d). H NMR (CDCl3): δ 3.46 (s, 3H), 3.83 (m, 4H), 4.24
(t, J ) 5.7 Hz, 2H), 7.04 (d, J ) 7.5, 1H), 7.40-7.51 (m, 3H),
7.66 (d, J ) 8.4 Hz, 1H), 7.84-7.88 (m, 1H), 7.94 (d, J ) 7.8 Hz,
1H).
2-Chloro-5-(5-formyl-furan-2-yl)-benzoic Acid Methyl Ester
(19a). A mixture of 5-amino-2-chloro-benzoic acid methyl ester
(18a) (8.67 mmol), hydrochloric acid (15%, 5.2 mL), and water (9
mL) was heated to get a clear solution. The solution was cooled to
0 °C and was diazotized by the addition of sodium nitrite solution
(30%, 2.0 mL). The cold clear solution of the diazonium salt was
collected by filtration and was treated with 2-furfuraldehyde (0.72
mL, 8.67 mmol) and water (5 mL). To this, an aqueous solution of
cupric chloride (219 mg in 1 mL of water) was added dropwise
with stirring. Stirring was continued for 4 h at room temperature
3-(2-Methoxy-ethyl)-2-(4-trifluoromethyl-phenylimino)-thia-
zolidin-4-one (16e). H NMR (CDCl3): δ 3.38 (s, 3H), 3.70 (tJ
1
11.1 Hz, 2H) 3.84 (s, 2H), 4.07 (t, J ) 11.1 Hz, 2H), 7.03 (d, J )
8.1 Hz, 2H), 7.59 (d, J ) 8.1 Hz, 2H).
3-(3-Dimethylamino-propyl)-2-phenylimino-thiazolidin-4-
one (16f). 1H NMR (CDCl3): δ 1.80 (t, J ) 15.3 Hz, 2H), 2.23 (s,
6H), 2.49 (t, J ) 15.9 Hz, 2H), 3.54 (s, 2H), 3.63 (t, J ) 14.1 Hz,
2H), 6.67 (d, J ) 8.1 Hz, 2H), 6.87 (t, J ) 7.5 Hz, 1H), 6.99-7.09
(m, 1H).

2478 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
and the precipitated solid was collected by filtration. The crude
compound 19a was recrystallized from a mixture of ethanol and
dioxane. Yield: 67.7%. 1H NMR (CDCl3): δ 3.98 (s, 3H), 6.91 (d,
J ) 3.6 Hz, 1H), 7.345 (d, J ) 3.9 Hz, 1H), 7.54 (d, J ) 8.4 Hz,
1H), 7.85 (d, J ) 2.1 Hz, 1H), 8.26 (d, J ) 2.4 Hz, 1H), 9.68 (s,
1H).
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-2-(4-methoxy-phenylimino)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid (4e).
In the same manner as described in the preparation of 2, 4e was
prepared from 2-chloro-5-{5-[3-(2-methoxy-ethyl)-2-(4-methoxy-
phenylimino)-4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-ben-
zoic acid methyl ester (4d). Yield: 89.9%. HPLC: 97.53%, tR
)
1
4.30 min, mp 202-205 °C. H NMR (DMSO-d6): δ 3.29 (s, 3H),
3.67 (t, J ) 11.4 Hz, 3H), 3.81 (s, 3H), 4.05 (t, 2H), 7.04 (s, 4H),
7.11 (d, J ) 3.6 Hz, 1H), 7.30 (d, J ) 3.6 Hz, 1H), 7.49 (d, J )
8.4 Hz, 1H), 7.56 (s, 1H), 7.63 (d, J ) 2.4 Hz, 1H), 7.98 (d, J )
2.1 Hz, 1H). EI-MS m/z 512 (M+) 278 (100%). HRMS (EI) m/z
calcd for C25H21ClN2O6S (M+) 512.0813, found 512.0809.
5-{5-[2-(4-Bromo-phenylimino)-3-(2-methoxy-ethyl)-4-oxo-
thiazolidin-5-ylidenemethyl]-furan-2-yl}-2-chloro-benzoic Acid
Methyl Ester (4f). In the same manner as described in the
preparation of 4c, 4f was prepared from 2-(4-bromo-phenylimino)-
3-(2-methoxy-ethyl)-thiazolidin-4-one (16c), 2-chloro-5-(5-formyl-
furan-2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine.
Yield: 69.8%. HPLC:100.00%, tR ) 17.13 min, mp 151-153 °C.
1H NMR (DMSO-d6): δ 3.29 (s, 3H), 3.68 (t, J ) 11.1 Hz, 2H),
3.85 (s, 2H), 4.06 (t, 2H), 7.05 (s, 1H), 7.08 (s, 1H), 7.19 (d, J )
3.3 Hz, 1H), 7.38 (d, J ) 3.6 Hz, 1H), 7.61-7.65 (m, 4H), 7.82
(d, J ) 2.1 Hz, 1H), 8.04 (d, J ) 2.7 Hz, 1H). EI-MS m/z 574
(M+) 292 (100%); HRMS (EI) m/z calcd for C25H20BrClN2O5S
(M+) 573.9956, found 573.9965.
4-Chloro-3-(5-formyl-furan-2-yl)-benzoic Acid Methyl Ester
(19b). In the same manner as described in the preparation of 19a,
19b was prepared from 3-amino-4-chloro-benzoic acid methyl ester
(18b) and 2-furfuraldehyde, and then the precipitated solid was
collected by filtration and then purified by chromatography with
EtOAc-petroleum ether (1:15, v/v). Yield: 65.3%. 1H NMR
(CDCl3): δ 3.97 (s, 3H), 7.34-7.38 (m, 2H), 7.58 (d, J ) 8.1 Hz,
1H), 7.99 (dd, J ) 2.1 Hz and 1.8 Hz, 1H), 8.64 (d, J ) 2.4 Hz,
1H), 9.76 (s, 1H).
5-(4-Chloro-phenyl)-furan-2-carbaldehyde (19c).47 In the same
manner as described in the preparation of 19a, 19c was prepared
from 4-chloro aniline and 2-furfuraldehyde, and then the precipitated
solid was collected by filtration and then purified by chromatog-
1
raphy with EtOAc/petroleum ether (1:15, v/v). Yield: 60.1%. H
NMR (CDCl3): δ 6.83 (d, J ) 3.9 Hz, 1H), 7.32(d, J ) 3.9 Hz,
1H), 7.42 (d, J ) 8.7 Hz, 2H), 7.76 (d, J ) 8.7 Hz, 2H), 9.67 (s,
1H).
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-4-oxo-2-phenylimino-thia-
zolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Methyl Ester
(4c). A mixture of 3-(2-methoxy-ethyl)-2-phenylimino-thiazolidin-
4-one (16a) (1.254 g, 5 mmol), 2-chloro-5-(5-formyl-furan-2-yl)-
benzoic acid methyl ester (19a) (1.327 g, 5 mmol), hexahydropy-
ridine (0.58 mL, 5.75 mmol), and 35 mL of EtOH were refluxed
for 11-12 h. The reaction mixture was cooled to room temperature
and the precipitated solid was collected by filtration, washed with
EtOH, and purified using RP-HPLC to afford the final compound
5-{5-[2-(2-Bromo-phenylimino)-3-(2-methoxy-ethyl)-4-oxo-
thiazolidin-5-ylidenemethyl]-furan-2-yl}-2-chloro-benzoic Acid
Methyl Ester (4g). In the same manner as described in the
preparation of 4c, 4g was prepared from 2-(2-bromo-phenylimino)-
3-(2-methoxy-ethyl)-thiazolidin-4-one (16m), 2-chloro-5-(5-formyl-
furan-2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine.
Yield: 62.5%. HPLC: 96.12%, tR ) 11.49 min, mp 145-146 °C.
1H NMR (DMSO-d6): δ 3.30 (s, 3H), 3.78 (t, J ) 11.1 Hz, 2H),
3.82 (s, 3H), 4.10 (t, J ) 11.1 Hz, 2H), 7.18-7.23 (m, 3H), 7.37
(d, J ) 9.6 Hz, 1H), 7.47 (m, 1H), 7.58 (d, 1H), 7.65 (s, 1H),
7.75-7.786 (m, 2H), 7.94 (d, J ) 2.7 Hz, 1H). EI-MS m/z 574
(M+) 292 (100%); HRMS (EI) m/z calcd for C25H20BrClN2O5S
(M+) 573.9939, found 573.9965.
5-{5-[2-(3-Bromo-phenylimino)-3-(2-methoxy-ethyl)-4-oxo-
thiazolidin-5-ylidenemethyl]-furan-2-yl}-2-chloro-benzoic Acid
Methyl Ester (4h). In the same manner as described in the
preparation of 4c, 4h was prepared from 2-(3-bromo-phenylimino)-
3-(2-methoxy-ethyl)-thiazolidin-4-one (16n), 2-chloro-5-(5-formyl-
furan-2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine.
Yield: 65.0%. HPLC: 100.00%, tR ) 16.39 min, mp 120-122 °C.
1H NMR (DMSO-d6): δ 3.29 (s, 3H), 3.67 (t, J ) 12.0 Hz, 2H),
3.82 (s, 3H), 4.06 (t, J ) 11.4 Hz, 2H), 7.10 (d, J ) 7.5 Hz, 1H),
7.18 (d, J ) 3.9 Hz, 1H), 7.31-7.37 (m, 2H), 7.41-7.44 (m, 2H),
7.58-7.63 (m, 2H), 7.77 (dd, J ) 2.1 Hz and J ) 2.1 Hz, 1H),
8.01 (d, J ) 2.1 Hz, 1H). EI-MS m/z 574 (M+) 292 (100%). HRMS
(EI) m/z calcd for C25H20BrClN2O5S (M+) 573.9971, found
573.9965.
1
4c as yellow powder. Yield: 60.1%; mp 150-151 °C. H NMR
(DMSO-d6): δ 3.30 (s, 3H), 3.68 (t, J ) 11.7 Hz, 2H), 3.81 (s,
3H), 4.06 (t, J ) 5.4 Hz, 2H), 7.05-7.07 (m, 2H), 7.12 (d, J ) 3.3
Hz, 1H), 7.24 (m, 1H), 7.32 (d, J ) 1.8 Hz, 1H), 7.45 (t, 2H), 7.56
(d, J ) 7.2 Hz, 2H), 7.72 (m, 1H), 7.94 (d, J ) 2.4 Hz, 1H). EI-
MS m/z 496 (M+) 292 (100%). HRMS (EI) m/z calcd for M+
496.0860, found 496.0860. Anal. (C25H21ClN2O5S) calcd C 60.42,
H 4.26, N 5.64; found C 60.26, H 4.05, N 5.57.
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-4-oxo-2-phenylimino-thia-
zolidin-5-ylidenemethyl]-furan-2-yl}-benzoic acid (2). 2-Chloro-
5-{5-[3-(2-methoxy-ethyl)-4-oxo-2-phenylimino-thiazolidin-5-yliden-
emethyl]-furan-2-yl}-benzoic acid methyl ester (4c) (200 mg, 0.40
mmol) in a solvent of THF (15 mL) was added aqueous LiOH
(84.4 mg, 2.01 mmol) solution, and the mixture was stirred at room
temperature for 6 h. After the removal of THF, water was added
to the mixture and then 10% HCl was added until PH ) 6 and
yellow solid was precipitated. The precipitation was collected by
filtration and washed with MeOH and purified using RP-HPLC to
afford the final compound 2 (182 mg, 91.2%) as yellow powder:
mp 210-212 °C. HPLC: 97.28%, tR ) 4.22 min. 1H NMR (DMSO-
d6): δ 3.30 (s, 3H), 3.68 (t, J ) 11.7 Hz, 2H), 4.06 (t, J ) 5.7 Hz,
2H), 7.07 (d, J ) 7.5 Hz, 2H), 7.13 (d, J ) 3.6 Hz, 1H), 7.25 (m,
1H), 7.34 (d, J ) 3.0 Hz, 1H), 7.46 (t, 2H), 7.56 (t, 2H), 7.69 (m,
1H), 7.96 (d, J ) 2.1 Hz, 1H). EI-MS m/z 482 (M+) 278 (100%).
HRMS (EI) m/z calcd for C24H19ClN2O5S (M+) 482.0687, found
482.0703.
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-2-(naphthalen-1-ylimino)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4i). In the same manner as described in the preparation
of 4c, 4i was prepared from 3-(2-methoxy-ethyl)-2-(naphthalen-1-
ylimino)-thiazolidin-4-one (16d), 2-chloro-5-(5-formyl-furan-2-yl)-
benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
1
62.3%. HPLC: 100.00%, tR ) 16.27 min, mp 126-128 °C. H
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-2-(4-methoxy-phenylimino)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4d). In the same manner as described in the preparation
of 4c, 4d was prepared from 3-(2-methoxy-ethyl)-2-(4-methoxy-
phenylimino)-thiazolidin-4-one (16b), 2-chloro-5-(5-formyl-furan-
2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine (0.58
mL, 5.75 mmol). Yield: 64.6%; mp 86-88 °C. HPLC: 95.99%, tR
NMR (DMSO-d6): δ 3.36 (s, 3H), 3.73 (s, 2H), 3.80(t, J ) 11.1
Hz, 2H), 4.22 (t, 2H), 7.09 (d, J ) 3.9 Hz, 1H), 7.19 (d, J ) 6.9
Hz, 1H), 7.27 (d, J ) 3.6 Hz, 1H), 7.50-7.59 (m, 5H), 7.65 (d, J
) 2.1 Hz, 1H), 7.80-7.87 (m, 3H), 7.97 (t, J ) 6.9 Hz,2H). EI-
MS m/z 546 (M+) 292 (100%). HRMS (EI) m/z calcd for
C29H23ClN2O5S (M+) 546.1019, found 546.1016.
1
) 8.76 min. H NMR (DMSO-d6): δ 3.29 (s, 3H), 3.67 (t, J )
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-2-(naphthalen-1-ylimino)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid (4j).
In the same manner as described in the preparation of 2, 4j was
prepared from 2-chloro-5-{5-[3-(2-methoxy-ethyl)-2-(naphthalen-
1-ylimino)-4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzo-
ic acid methyl ester (4i). Yield: 90.1%. HPLC: 97.98%, tR ) 7.41
11.1 Hz, 2H), 3.79 (s, 6H), 4.05 (t, J ) 11.4 Hz, 2H), 7.01 (s, 4H),
7.12 (d, J ) 4.2 Hz, 1H), 7.32 (d, J ) 3.6 Hz, 1H), 7.57 (d, J )
9.9 Hz, 2H), 7.77 (d, 1H), 7.96 (d, J ) 2.1 Hz, 1H). EI-MS m/z
526 (M+) 292 (100%). HRMS (EI) m/z calcd for C26H23ClN2O6S
(M+) 526.0965, found 526.0972.

Inhibitors Against the FabZ of Helicobacter pylori
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2479
1
min, mp 230-231 °C. H NMR (DMSO-d6): δ 3.36 (s, 3H), 3.81
(M+) 292 (100%). HRMS (EI) m/z calcd for C26H23ClN2O6S (M+)
526.0965, found 526.0958.
(t, J ) 5.1 Hz, 2H), 4.24 (t, 2H), 7.10 (d, J ) 3.6 Hz, 1H), 7.23 (d,
1H), 7.28 (d, J ) 3.9 Hz, 1H), 7.47-7.61 (m, 6H), 7.80-7.88 (m,
2H), 7.98 (t, 2H). ESI-MS m/z 532 [M + H]+. HRMS (ESI) m/z
calcd for C28H21ClN2O5SNa [M + Na]+ 555.0732, found 555.0757.
2-Chloro-5-{5-[2-(4-fluoro-phenylimino)-3-(2-methoxy-ethyl)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4p). In the same manner as described in the preparation
of 4c, 4p was prepared from 2-(4-fluoro-phenylimino)-3-(2-
methoxy-ethyl)-thiazolidin-4-one (16p), 2-chloro-5-(5-formyl-furan-
2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
60.2%. HPLC: 96.94%, tR ) 9.26 min, mp 119-120 °C. 1H NMR
(DMSO-d6): δ 3.29 (s, 3H), 3.68 (t, J ) 10.8 Hz, 2H), 3.83 (s,
3H), 4.05 (t, 2H), 7.10-7.13 (m, 2H), 7.17 (dd, J ) 1.2 Hz, J )
1.2 Hz, 1H), 7.25-7.29 (m, 2H), 7.36 (dd, J ) 1.5 Hz, J ) 1.5
Hz, 1H), 7.60-7.63 (m, 2H), 7.81 (d, 1H), 7.96 (d, J ) 1.8 Hz,
1H). EI-MS m/z 514 (M+) 292 (100%). HRMS (EI) m/z calcd for
C25H20ClFN2O5S (M+) 514.0762, found 514.0765.
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-4-oxo-2-(4-trifluorometh-
yl-phenylimino)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-ben-
zoic Acid Methyl Ester (4k). In the same manner as described in
the preparation of 4c, 4k was prepared from 3-(2-methoxy-ethyl)-
2-(4-trifluoromethyl-phenylimino)-thiazolidin-4-one (16e), 2-chloro-
5-(5-formyl-furan-2-yl)-benzoic acid methyl ester (19a), and hexahy-
dropyridine. Yield: 64.1%. HPLC: 98.08%, tR ) 14.52 min, mp
1
155-157 °C. H NMR (DMSO-d6): δ 3.30 (s, 3H), 3.69 (t, J )
11.4 Hz, 2H), 3.79 (s, 2H), 4.08 (t, J ) 11.7 Hz 2H), 7.18 (d, J )
3.6 Hz 1H), 7.28 (d, J ) 4.8 Hz, 2H), 7.35 (d, J ) 2.4 Hz 1H),
7.50 (d, 1H), 7.63 (s, 1H), 7.73 (dd, J ) 1.8 Hz and J ) 2.4 Hz
1H), 7.82 (d, J ) 8.7 Hz, 2H), 8.02 (d, J ) 1.8 Hz, 1H). EI-MS
m/z 564 (M+) 292 (100%). HRMS (EI) m/z calcd for
C26H20ClF3N2O5S (M+) 564.0739, found 564.0734.
2-Chloro-5-{5-[2-(3-fluoro-phenylimino)-3-(2-methoxy-ethyl)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4q). In the same manner as described in the preparation
of 4c, 4q was prepared from 2-(3-fluoro-phenylimino)-3-(2-
methoxy-ethyl)-thiazolidin-4-one (16q), 2-chloro-5-(5-formyl-furan-
2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
65.7%. HPLC: 95.15%, tR ) 9.54 min, mp 126-128 °C. 1H NMR
(DMSO-d6): δ 3.29 (s, 3H), 3.68 (t, J ) 11.7 Hz, 2H), 3.82 (s,
3H), 4.06 (t, J ) 11.7 Hz, 2H), 6.92-6.96 (m, 2H), 7.05-7.12
(m, 1H), 7.18 (d, J ) 3.9 Hz, 1H), 7.36 (d, J ) 3.9 Hz, 1H),
7.50-7.53 (m, 1H), 7.59-7.63 (m, 2H), 7.81 (dd, J ) 2.1 Hz and
J ) 2.1 Hz, 1H), 7.97 (d, J ) 2.4 Hz,1H). EI-MS m/z 514 (M+)
292 (100%). HRMS (EI) m/z calcd for C25H20ClFN2O5S (M+)
514.0759, found 514.0765.
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-4-oxo-2-(2-trifluorometh-
yl-phenylimino)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-ben-
zoic Acid Methyl Ester (4l). In the same manner as described in
the preparation of 4c, 4l was prepared from 3-(2-methoxy-ethyl)-
2-(2-trifluoromethyl-phenylimino)-thiazolidin-4-one (16k), 2-chloro-
5-(5-formyl-furan-2-yl)-benzoic acid methyl ester (19a), and hexahy-
dropyridine. Yield: 65.6%. HPLC: 98.23%, tR ) 10.40 min, mp
1
141-143 °C. H NMR (DMSO-d6): δ 3.27 (s, 3H), 3.70 (t, J )
11.1 Hz, 2H), 3.81 (s, 3H), 4.07 (t, J ) 11.7 Hz, 2H), 7.19(d, J )
3.6 Hz, 1H), 7.27 (d, 1H), 7.35 (d, J ) 3.9 Hz, 1H), 7.45 (m, 1H),
7.58 (d, 1H), 7.66 (s, 1H), 7.72-7.82 (m, 3H), 7.93 (d, J ) 1.8
Hz, 1H). EI-MS m/z 564 (M+) 292 (100%). HRMS (EI) m/z calcd
for C26H20ClF3N2O5S (M+) 564.0737, found 564.0734.
2-Chloro-5-{5-[2-(2-fluoro-phenylimino)-3-(2-methoxy-ethyl)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4r). In the same manner as described in the preparation
of 4c, 4r was prepared from 2-(2-fluoro-phenylimino)-3-(2-meth-
oxy-ethyl)-thiazolidin-4-one (16o), 2-chloro-5-(5-formyl-furan-2-
yl)-benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-4-oxo-2-(3-trifluorometh-
yl-phenylimino)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-ben-
zoic Acid Methyl Ester (4m). In the same manner as described in
the preparation of 4c, 4m was prepared from 3-(2-methoxy-ethyl)-
2-(3-trifluoromethyl-phenylimino)-thiazolidin-4-one (16l), 2-chloro-
5-(5-formyl-furan-2-yl)-benzoic acid methyl ester (19a), and hexahy-
dropyridine. Yield: 60.3%. HPLC: 96.60%, tR ) 13.76 min, mp
92-95 °C. 1H NMR (DMSO-d6): δ 3.30 (s, 3H), 3.69 (t, J ) 11.1
Hz, 2H), 3.78 (s, 3H), 4.07 (t, J ) 11.1 Hz, 2H), 7.15 (d, J ) 3.6
Hz, 1H), 7.32 (d, J ) 3.6 Hz, 1H), 7.40-7.42 (m, 2H), 7.47-7.49
(m, 1H), 7.60-7.62 (m, 2H), 7.66-7.69 (m, 2H), 7.96 (d, J ) 2.1
Hz, 1H). EI-MS m/z 564 (M+) 292 (100%). HRMS (EI) m/z calcd
for C26H20ClF3N2O5S (M+) 564.0741, found 564.0734.
1
68.3%; mp 129-132 °C. H NMR (DMSO-d6): δ 3.28 (s, 3H),
3.70 (t, J ) 10.8 Hz, 2H), 3.82 (s, 3H), 4.09 (t, J ) 11.1 Hz, 2H),
7.19 (d, J ) 3.6 Hz, 2H), 7.26-7.37 (m, 4H), 7.60 (d, J ) 8.1 Hz,
1H), 7.65 (s, 1H), 7.78 (dd, J ) 2.1 Hz and J ) 2.1 Hz, 1H), 7.96
(d, J ) 2.1 Hz, 1H). EI-MS m/z 514 (M+) 292 (100%). HRMS
(EI) m/z calcd for (M+) 514.0775, found 514.0765. Anal.
(C25H20ClFN2O5S) calcd, C 58.31, H 3.91, N 5.44; found C 58.15,
H 3.83, N 5.28.
2-Chloro-5-{5-[3-(3-dimethylamino-propyl)-4-oxo-2-phenylimi-
no-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4s). In the same manner as described in the preparation
of 4c, 4s was prepared from 3-(3-dimethylamino-propyl)-2-phe-
nylimino-thiazolidin-4-one (16f), 2-chloro-5-(5-formyl-furan-2-yl)-
benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
67.5%. HPLC: 96.26%, tR ) 2.15 min, mp 134-136 °C. 1H NMR
(DMSO-d6): δ 1.83 (t, 2H), 2.13 (s, 6H), 2.29 (t, J ) 6.3 Hz, 2H),
3.81 (s, 3H), 3.90 (t, J ) 7.5 Hz, 2H), 7.06 (d, J ) 7.2 Hz, 2H),
7.10 (d, J ) 3.6 Hz, 1H), 7.24 (m, 1H), 7.31 (d, J ) 3.6 Hz, 1H),
7.45 (m, 2H), 7.55 (t, 2H), 7.71 (d, J ) 2.1 Hz, 2H), 7.93 (d, J )
2.4 Hz, 1H). EI-MS m/z 523 (M+) 84 (100%). HRMS (EI) m/z
calcd for C27H26ClN3O4S (M+) 523.1337, found 523.1333.
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-2-(2-methoxy-phenylimino)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4n). In the same manner as described in the preparation
of 4c, 4n was prepared from 3-(2-methoxy-ethyl)-2-(2-methoxy-
phenylimino)-thiazolidin-4-one (16i), 2-chloro-5-(5-formyl-furan-
2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
63.4%. HPLC: 97.24%, tR ) 6.50 min, mp 150-152 °C. 1H NMR
(DMSO-d6): δ 3.29 (s, 3H), 3.71 (t, J ) 11.4 Hz, 2H), 3.76 (s,
3H), 3.83 (s, 3H), 4.08 (t, J ) 11.4 Hz, 2H), 6.97-7.00 (m, 2H),
7.11-7.14 (m, 2H), 7.25 (m, 1H), 7.33 (d, J ) 3.6 Hz, 1H),
7.56-7.59 (m, 2H), 7.74 (m, 1H), 7.94 (d, J ) 1.8 Hz, 1H). EI-
MS m/z 526 (M+) 292 (100%). HRMS (EI) m/z calcd for
C26H23ClN2O6S (M+) 526.0968, found 526.0965.
2-Chloro-5-{5-[3-(2-diethylamino-ethyl)-4-oxo-2-phenylimino-
thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Methyl
Ester (4t). In the same manner as described in the preparation of
4c, 4t was prepared from 3-(2-diethylamino-ethyl)-2-phenylimino-
thiazolidin-4-one (16g), 2-chloro-5-(5-formyl-furan-2-yl)-benzoic
acid methyl ester (19a), and hexahydropyridine. Yield: 64.9%.
HPLC: 95.32%, tR ) 2.06 min, mp 103-105 °C. 1H NMR (DMSO-
d6): δ 0.99 (t, J ) 14.1 Hz, 6H), 2.59 (q, 4H), 2.87 (t, 2H), 3.82 (s,
3H), 3.99 (t, J ) 12.6 Hz, 2H), 7.10 (d, J ) 7.8 Hz, 3H), 7.14 (d,
J ) 3.9 Hz, 1H), 7.25 (t, 1H), 7.33 (d, J ) 3.6 Hz, 2H), 7.46 (t,
2H), 7.56-7.59 (m, 2H), 7.76 (dd, J ) 2.1 Hz and J ) 2.1 Hz,
1H), 7.95 (d, J ) 2.1 Hz, 1H). EI-MS m/z 537 (M+) 99 (100%).
HRMS (EI) m/z calcd for C28H28ClN3O4S (M+) 537.1471, found
537.1489.
2-Chloro-5-{5-[3-(2-methoxy-ethyl)-2-(3-methoxy-phenylimino)-
4-oxo-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4o). In the same manner as described in the preparation
of 4c, 4o was prepared from 3-(2-methoxy-ethyl)-2-(3-methoxy-
phenylimino)-thiazolidin-4-one (16j), 2-chloro-5-(5-formyl-furan-
2-yl)-benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
66.7%. HPLC: 97.50%, tR ) 8.56 min, mp 131-132 °C. 1H NMR
(DMSO-d6): δ 3.29 (s, 3H), 3.68 (t, J ) 11.4 Hz, 2H), 3.76 (s,
3H), 3.81 (s, 3H), 4.06 (t, J ) 11.4 Hz, 2H), 6.64 (dt, J ) 3.9 Hz
and J ) 1.2 Hz, 2H), 6.80-6.83 (m, 1H), 7.13 (d, J ) 3.6 Hz,
1H), 7.33-7.38 (m, 2H), 7.57-7.58 (m, 2H), 7.77 (dd, J ) 2.1 Hz
and J ) 2.1 Hz, 1H), 7.97 (d, J ) 2.1 Hz, 1H). EI-MS m/z 526

2480 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
He et al.
2-Chloro-5-{5-[4-oxo-2-phenylimino-3-(2-pyrrolidin-1-yl-eth-
yl)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Meth-
yl Ester (4u). In the same manner as described in the preparation
of 4c, 4u was prepared from 2-phenylimino-3-(2-pyrrolidin-1-yl-
ethyl)-thiazolidin-4-one (16h), 2-chloro-5-(5-formyl-furan-2-yl)-
benzoic acid methyl ester (19a), and hexahydropyridine. Yield:
55.2%. HPLC: 95.98%, tR ) 2.07 min, mp 110-112 °C. 1H NMR
(DMSO-d6): δ 1.90 (m, 4H), 2.90 (m, 4H), 3.11 (t, 2H), 3.87 (s,
3H), 4.10 (t, J ) 6.9 Hz, 2H), 6.92 (d, J ) 3.6 Hz, 1H), 7.05-7.11
(m, 2H), 7.25 (m, 1H), 7.40-7.44 (m, 4H), 7.47 (s, 1H), 7.62 (d,
J ) 2.4 Hz, 1H), 7.97 (d, J ) 2.1 Hz, 1H). EI-MS m/z 535 (M+)
97 (100%). HRMS (EI) m/z calcd for C28H26ClN3O4S (M+)
535.1314, found 535.1333.
4-Chloro-3-{5-[3-(2-methoxy-ethyl)-4-oxo-2-phenylimino-thia-
zolidin-5-ylidenemethyl]-furan-2-yl}-benzoic Acid Methyl Ester
(4a). In the same manner as described in the preparation of 4c, 4a
was prepared from 3-(2-methoxy-ethyl)-2-phenylimino-thiazolidin-
4-one (16a), 4-chloro-3-(5-formyl-furan-2-yl)-benzoic acid methyl
ester (19b), and hexahydropyridine. Yield: 66.3%. HPLC: 100.00%,
tR ) 14.19 min, mp 111-113 °C. 1H NMR (DMSO-d6): δ 3.30 (s,
3H), 3.69 (t, J ) 11.7 Hz, 2H), 3.79 (s, 3H), 4.07 (t, 2H), 7.06 (d,
J ) 7.5 Hz, 2H), 7.17-7.19 (m, 2H), 7.38-7.43 (m, 3H), 7.62 (s,
1H), 7.69-7.71 (d, J ) 8.4 Hz, 1H), 7.84-7.88 (m, 1H), 8.28 (d,
J ) 2.1 Hz, 1H). EI-MS m/z 496 (M+) 292 (100%). HRMS (EI)
m/z calcd for C25H21ClN2O5S (M+) 496.0858, found 496.0860.
5-[5-(4-Chloro-phenyl)-furan-2-ylmethylene]-3-(2-methoxy-
ethyl)-2-phenylimino-thiazolidin-4-one (4b). In the same manner
as described in the preparation of 4c, 4b was prepared from 3-(2-
methoxy-ethyl)-2-phenylimino-thiazolidin-4-one (16a), 5-(4-chloro-
phenyl)-furan-2-carbaldehyde (19c), and hexahydropyridine. Yield:
64.9%. HPLC: 96.29%, tR ) 13.03 min, mp 176-178 °C. 1H NMR
(DMSO-d6): δ 3.30 (s, 3H), 3.70 (t, J ) 5.7 Hz, 2H), 4.07 (t, J )
6.3 Hz, 2H), 7.08-7.14 (m, 3H), 7.26-7.28 (m, 2H), 7.43-7.51
(m, 4H), 7.59-7.64 (m, 3H). EI-MS m/z 438 (M+) 234 (100%).
HRMS (EI) m/z calcd for C23H19ClN2O3S (M+) 438.0811, found
438.0805.
Bioassay. Enzymatic Inhibition Assay. The enzymatic inhibi-
tion assay of HpFabZ was monitored by the spectrophotomeric
method as described previously.21,25 In brief, the activity of HpFabZ
was measured by detection of the decrease in absorbance at 260
nm for the conversion of crotonoyl-CoA to ꢀ-hydroxybutyryl-CoA.
The compounds dissolved in 1% DMSO were incubated with the
enzyme for 1 h before the assays were started. The 50% inhibitory
concentration (IC50) of each inhibitor was estimated by fitting the
inhibition data to a dose-dependent curve using a logistic derivative
equation.41
Anti-H. pylori Activity Assay. The bacterial growth inhibition
activity for the compounds was evaluated by using Paper Discus
Method. Dimethyl sulfoxide and ampicillin paper were used as
negative and positive control respectively. The minimum inhibitory
concentrations (MIC) values were determined by the standard agar
dilution method42 using Columbia agar supplemented with 10%
sheep blood containing 2-fold serial dilutions of agents. Bacterial
suspension of H. pylori was made by swabbing and suspending
the cells in sterile saline with a turbidity equivalent to a 2.0
McFarland standard. Compound-free Columbia agar media were
used as controls. Inoculated plates were incubated at 37 °C under
microaerobic conditions (85% N2, 10% CO2 and 5% O2) and
examined after 3 days. The MIC was generally defined as the lowest
concentration of antimicrobial agent that completely inhibits visible
bacterial growth.
S4 and S5 in Supporting Information). The structures were solved
by molecular replacement method with the crystal structure of
HpFabZ as the search model (PDB code 2GLL) and refined by
using the program CNS.49 Electron density interpretation and model
building were performed using the computer graphics program
“coot”.50
Acknowledgment. We thank the Shanghai Supercomputing
Center and Computer Network Information Center of Chinese
Academy of Sciences for allocation of computing time. This
work was supported by the National Natural Science Foundation
of China (grants 20721003 and 20872153), International Col-
laboration Projects (grants 2007DFB30370 and 20720102040)
and the 863 Hi-Tech Program of China (grants 2006AA020602).
Supporting Information Available: Figure S1 and Table S1
showing the binding energies (in kcal/mol) and actual ranks of the
280 compounds in focused combinatorial library; Figure S2 showing
the stereo view of the omit electron density map contoured at 1.0
σ around the inhibitors; Tables S2 and S3 listing the element
analysis and the HPLC reports for the purity check of the active
compounds 1-5, as measured in two different mobile phases;
Tables S4 and S5 listing the summary of diffraction data and
structure refinement statistics. This material is available free of
charge via the Internet at http://pubs.acs.org.
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a final concentration of ∼20 mM inhibitors for 24 h. Diffraction
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Inhibitors Against the FabZ of Helicobacter pylori
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