Phenylimidazole derivatives of 4-pyridone as dual inhibitors of bacterial enoyl-acyl carrier protein reductases FabI and FabK

Article abstract of DOI:10.1021/jm0705354

FabI and FabK are bacterial enoyl-acyl carrier protein (ACP) reductases that catalyze the final and rate-limiting step of bacterial fatty acid biosynthesis (FAS) and are potential targets of novel antibacterial agents. We have reported 4-pyridone derivati

Full text of DOI:10.1021/jm0705354

4710
J. Med. Chem. 2007, 50, 4710-4720
Phenylimidazole Derivatives of 4-Pyridone as Dual Inhibitors of Bacterial Enoyl-Acyl Carrier
Protein Reductases FabI and FabK
Hideo Kitagawa,*,† Tomohiro Ozawa,† Sho Takahata,‡ Maiko Iida,† Jun Saito,‡ and Mototsugu Yamada†
Medicinal Chemistry Research Laboratories and Pharmacology Research Laboratories, Pharmaceutical Research Center, Meiji Seika Kaisha,
Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
ReceiVed May 7, 2007
FabI and FabK are bacterial enoyl-acyl carrier protein (ACP) reductases that catalyze the final and rate-
limiting step of bacterial fatty acid biosynthesis (FAS) and are potential targets of novel antibacterial agents.
We have reported 4-pyridone derivative 3 as a FabI inhibitor and phenylimidazole derivative 5 as a FabK
inhibitor. Here, we will report phenylimidazole derivatives of 4-pyridone as FabI and FabK dual inhibitors
based on an iterative medicinal chemistry and crystallographic study of FabK from Streptococcus pneumoniae/
compound 26. A representative compound 6 showed strong FabI inhibitory (IC50 ) 0.38 µM) and FabK
inhibitory (IC50 ) 0.0045 µM) activities with potent antibacterial activity against S. pneumoniae (MIC )
0.5 µg/mL). Since elevated MIC value was observed against S. pneumoniae mutant possessing one amino
acid substitution in FabK, the antibacterial activity of the compound was considered to be due to the inhibition
of FabK. Moreover, this compound showed no significant cytotoxicity (IC50 > 69 µM). These results support
compound 6 as a novel agent for the treatment of bacterial infections.
Introduction
NADPH-dependent â-ketoacyl-ACP reductase.14,15 Subsequent
dehydration by FabA or FabZ,16,17 which are â-hydroxyacyl-
ACP dehydrases, leads to trans-2-enoyl-ACP. FabI is an enoyl-
ACP reductase that catalyzes the conjugate reduction of an
enoyl-ACP to the corresponding acyl-ACP using the cofactor
NADPH or NADH as a hydride source.18-20 Further rounds of
this cycle, successively adding two carbon atoms per cycle,
finally lead to palmitoyl-ACP. Then, the cycle is stopped due
to feedback inhibition of FabH and FabI by palmitoyl-ACP.
Therefore, FabI and FabH are rate-determining for the overall
biosynthetic pathway21(Figure 1).
The emergence of bacterial resistance to most of all antibiotics
poses a threat to health care, and novel therapeutics are needed.
In particular, the increase of methicillin-resistant Staphylococcus
aureus (MRSA)a,1 and penicillin-resistant Streptococcus pneu-
moniae (PRSP)a is of major concern, and two strains of
vancomycin-resistant S. aureus have been isolated recently.2-4
A key strategy to overcome antibiotic-resistant pathogens is the
discovery of antibacterial agents with novel mechanisms of
action and no cross-resistance. However, only three novel classes
of antibiotics, oxazolidinones,5 lipopeptides,6 and glycylcy-
clines,7 have reached the market.
Bacterial FASa (type II)8-10 provides essential fatty acids for
use in the assembly of key cellular components such as cell
envelope, phospholipids, lipoproteins, lipopolysaccharides, and
mycolic acids. In bacteria, different monofunctional enzymes
catalyze each of the reactions, and reaction intermediates are
carried through the cytosol as a thioester of the small ACPa.
On the other hand, mammalians use a single large multifunc-
tional protein in which the growing chain is covalently attached
to the protein to make fatty acids. This difference of fatty acid
biosynthesis between bacteria and mammalian offers an attrac-
tive opportunity for selective inhibition of bacterial FASa.
Therefore, inhibitors of the bacterial enzymes are expected to
be the candidates for novel antibacterial agents.
In Escherichia coli, there is a single NADH-dependent
isoform of this enzyme, FabI, which is essential for bacterial
viability.22,23 Various compounds including isoniazid,24 diaz-
aborines,25,26 triclosan,27-32 indole naphthyridinones,33-35 and
thiopyridine36 have been reported as inhibitors of bacterial enoyl-
ACP reductase, and a FabI-targeting approach to antibacterial
drug therapy appears feasible (Figure 2).
However, recent studies have shown that other bacterial
enoyl-ACP reductases exist in addition to FabI.37 A triclosan-
resistant flavoprotein, termed FabK, has been shown to be the
sole enoyl-ACP reductase in S. pneumoniae and to exist together
with FabI in key pathogens such as Enterococcus faecalis and
Pseudomonas aeruginosa.38 Inhibitors of both FabI and FabK
are expected to have broad-spectrum antibacterial activity. The
small-molecular FabI and FabK inhibitors 1 and 2 have been
reported,33 but they showed weaker inhibition of S. pneumoniae
FabK than of S. aureus FabI.39
The first step in the bacterial biosynthetic cycle is the
condensation of malonyl-ACP with acetyl-CoA by FabH.11-13
In subsequent rounds, malonyl-ACP is condensed with the
growing-chain acyl-ACP (FabB and FabF). In the elongation
cycle of the second step, FabG mediates ketoester reduction by
Therefore, we are interested in finding novel small-molecular
FabI and FabK dual inhibitors. We have previously reported a
potent FabI inhibitor (3)40 with potent antibacterial activity
against S. aureus and a FabK inhibitor (5) with potent
antibacterial activity against S. pneumoniae41, 42(Figure 3). The
macromolecular synthesis assay demonstrated that a compound
related to 3 and compound 4 selectively inhibited lipid biosyn-
thesis.42,45 We considered that it might be possible to combine
the pharmacophores of these inhibitors to produce specific dual
inhibitors.
* Corresponding author. Tel: +81-45-541-2521. Fax: +81-45-541-0667.
E-mail: hideo_kitagawa@meiji.co.jp.
† Medicinal Chemistry Research Laboratories.
‡ Pharmacology Research Laboratories.
a Abbreviation: ACP, acyl carrier protein; FAS, fatty acid biosynthesis;
MRSA, methicillin-resistant Staphylococcus aureus; PRSP, penicillin-
resistant Streptococcus pneumoniae; SAR, structure-activity relationship;
FMN, flavin mononucleotide; MOA, mode of action.
10.1021/jm0705354 CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/22/2007

Inhibitors of Enoyl-ACP Reductases FabI and FabK
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4711
Figure 1. The bacterial biosynthetic cycle of fatty acids.
The 4-pyridone derivative 17 was prepared as illustrated in
Scheme 2.40,44,45 Commercially available 4-methoxypyridine was
treated with methyl magnesium bromide and carbobenzyloxy
chloride in THF at -25 °C to afford compound 13 in 94% yield.
The reaction of 13 with 2,6-dichlorobenzaldehyde was carried
out in THF at -78 °C in the presence of lithium hexamethyl-
disilazide, and the corresponding aldol adduct 14 was obtained
in 98% yield. The hydroxyl group of compound 14 was
converted to the methanesulfonate 15 by treatment with meth-
anesulfonyl chloride in 94% yield. Elimination of the meth-
anesulfonyloxy group and isomerization of the exo-olefin by
using potassium tert-butoxide gave compound 16 in 87% yield.
Treatment of 16 with tetravinyltin in the presence of Cu(OAc)2
in MeCN/DMF under an O2 atmosphere at room temperature
afforded compound 17.
Figure 2. The reported FabI inhibitors.
Through iterative medicinal chemistry and X-ray crystal
structure-based design,43 we have developed novel phenylimi-
dazole derivatives having a 4-pyridone moiety as FabI and FabK
dual inhibitors. Among them, compound 6 exhibited strong FabI
and FabK inhibitory activities, and it showed potent antibacterial
activity against S. pneumoniae. Here, we would like to present
details of the development of novel FabI and FabK dual
inhibitors.
Chemistry
Compound 6, a FabI and FabK dual inhibitor, was prepared
as illustrated in Scheme 3. Compound 11 was coupled with 17
in the presence of Pd2(dba)3, P(tBu)3, and Cy2NMe to afford
compound 18 in 23% yield. Deprotection of the benzyloxycar-
bonyl and 2-(trimethylsilyl)ethoxymethyl groups under acidic
condition afforded compound 19. Hydrogenation of compound
19 gave the corresponding saturated amine (compound 20).
Coupling 20 with 22, which is obtained from 21 as a crude
intermediate, in the presence of N,N′-diisopropylethylamine
The phenylimidazole derivatives were prepared as follows.
As an example, the synthesis of compound 11 is illustrated in
Scheme 1. Commercially available benzyl cyanomethylcarbam-
ate 7 was treated with sodium methoxide followed by am-
monium bromide to give compound 8. Compound 8 was treated
with 4-bromophenacyl bromide 9 to give phenylimidazole 10
in 28% and then the amino group of the imidazole was protected
with 2-(trimethylsilyl)ethoxymethyl to afford compound 11 in
60% yield.
Figure 3. Our developed FabI and/or FabK inhibitors.

4712 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Scheme 1. Synthesis of Phenylimidazole Derivativesa
Kitagawa et al.
a Reagents and conditions: (a) NaOMe (0.1 equiv), MeOH, rt; (b) NH4Br (1.05 equiv), rt (87%, two steps); (c) K2CO3 (1 equiv), THF/H2O ) 10/1, 80
°C (28%); (d) NaH (1 equiv), SEMCl (1 equiv), THF, rt to 40 °C (60%).
Scheme 2. Synthesis of 4-Pyridone Derivativesa
activity toward FabK of S. pneumoniae. Among them, com-
pound 5 showed strong FabK-inhibitory activity (IC50 ) 0.14
µM) and potent antibacterial activity (MIC ) 0.5 µg/mL) against
S. pneumoniae.41 However, it did not show either E. coli FabI-
inhibitory activity (IC50>32 µM) or antibacterial activity against
S. aureus (MIC > 64 µg/mL).
Evaluation of several derivatives structurally related to 5
revealed the importance of the phenylimidazole structure for
FabK-inhibitory activity. Structure-activity relationship (SAR)
studies concerning the benzothiazole ring in compound 5
revealed that compound 23, having a pyridylthio group at the
5-position of thiazole, showed strong FabK inhibitory activity
a Reagents and conditions: (a) BnOCOCl (1 equiv), CH3MgBr (1.2
equiv), THF, -25 °C; (b) 3 N HCl, rt (94% for two steps); (c)
2,6-dichlorobenzaldehyde (1.3 equiv), LiHMDS (1.1 equiv), THF, -78 °C
(98%); (d) MsCl (2 equiv), pyridine, 0 °C to rt (94%); (e) tBuOK (3 equiv),
THF, rt (87%); (f) tetravinyltin (2 equiv), Cu(OAc)2 (2 equiv), O2, MeCN/
DMF, rt (76%).
and potent antibacterial activity against S. pneumoniae (IC50
)
0.042 µM, MIC ) 2 µg/mL). Therefore, we selected the thiazole
moiety with a 5-pyridylthio group as a basic structure and
prepared a range of phenylimidazole derivatives. SAR of these
derivatives is shown in Table 1.
afforded compound 6. The other related compounds were
Compounds 24 and 25, having a bromo substituent, and
compound 26, having a carboxylic acid group on the phenyl
ring, showed strong FabK inhibitory activity. However, com-
pound 25 showed decreased antibacterial activity, and compound
26 lacked antibacterial activity against S. pneumoniae. The
reason for these results is not known, but several factors, such
similarly prepared.
Results and Discussion
We have developed a series of small-molecule, phenylimi-
dazole-based FabK inhibitors by optimizing a lead compound
4 discovered by screening our compound library for inhibitory
Scheme 3. Synthesis of Compound 6a
a Reagents and conditions: (a) Pd2(dba)3 (0.2 equiv), P(tBu)3 (0.4 equiv), Cy2NMe (2 equiv), DMF, 80 °C (23%); (b) 4 N HCl/1,4-dioxane, 100 °C
(25%); (c) 10% Pd/C (10 w/w%), MeOH/HCl, H2 atmosphere (44%); (d) CDI (2 equiv), THF, rt; (e) N,N′-diisopropylethylamine (4 equiv), DMF, rt (21%,
two steps).

Inhibitors of Enoyl-ACP Reductases FabI and FabK
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4713
Table 1. Antibacterial Activities for Phenylimidazole Derivatives as
FabK Inhibitors
Figure 6. Schematic model of the active site of FabK.
a E. coli FabI. b S. pneumoniae FabK. c S. pneumoniae KU197. d S.
aureus ATCC29213 MIC ) 0.25 µg/mL. e S. aureus ATCC29213 MIC )
0.063 µg/mL.
Figure 7. Expanded view of the complex of compound 26 and FabK
around site II.
Figure 4. Structure of compound 26 used for the crystallographic study.
The phenylimidazole moiety is buried in a hydrophobic cleft.
In addition, the hydrophobic amino acids Leu122 and Pro118
are located near the phenyl ring of compound 26 (Figure 7).
The bromo substituent on the phenyl ring in compounds 24 and
25 may occupy the gap and reinforce the hydrophobic interaction
with Leu122. Although Pro118 has a hydrophobic interaction
with the phenyl ring of compounds, there is an additional
shallow, hydrophobic hollow, called site II, extending to the
plane of Pro118. Site III on the left side of site I is a hydrophobic
cleft. The pyridine ring faces the empty hydrophobic pocket in
site III. A detailed analysis of this crystal structure will be
reported elsewhere.
We have reported that novel 4-pyridone derivatives having
a hydrophobic group at the 1-position showed good E. coli FabI
inhibitory activity and strong antibacterial activity against S.
aureus.40 In particular, compound 3, having a cyclohexylmethyl
group at the 1-position, exhibited strong activities.
Figure 5. Crystal structure of compound 26 bound to the active site
We have also shown that it is possible to incorporate various
bulky hydrophobic groups at the 1-position of 4-pyridones.40
Therefore, we assumed that the substituent at the 1-position of
4-pyridones was recognized as an alkyl side chain of fatty acid.
On the basis of this hypothesis, we speculated that introduction
of a hydrophobic FabK inhibitory moiety at the 1-position of
4-pyridones might yield FabI and FabK dual inhibitors.
There are two possible strategies to hybridize FabI and FabK
inhibitors (Figure 8). One is to connect the phenylimidazole
side of the FabK inhibitory moiety to the FabI inhibitory moiety
(type I), and the other is to connect the thiazole side of the
FabK inhibitory moiety to the FabI inhibitory moiety (type II).
From the perspective of a FabI inhibitor, the hydrophobic FabK
inhibitory moiety expected to be recognized by the same site
of the FabI active pocket in both types. On the other hand, from
the perspective of a FabK inhibitor, the FabI inhibitory moiety
or 4-pyridone added at either side of the FabK inhibitory moiety
is expected to be recognized by different sites of the FabK active
pocket. The 4-pyridone substructure in the case of types I and
of FabK.
as poor penetration through the bacterial cell membrane or
recognition by an efflux transporter of the bacteria, might be
involved.
We have already crystallized FabK from S. pneumoniae.43
Although compound 26 showed no antibacterial activity against
S. pneumoniae, it was the only compound that had good
solubility in aqueous 5% DMSO, and it was suitable for a
crystallographic study. Therefore, to allow further optimization
of inhibitors by structure-based drug design, we solved the
crystal structure of the compound 26/FabK complex (Figure 4).
The active pocket of FabK can be divided into three sites
(Figures 5 and 6). The thiazole ring and a part of the ureido
moiety of 26 are involved in a face-to-face π-π stacking
interaction with the isoalloxazine ring of flavin mononucleotide
(FMN), with additional hydrophobic interactions between the
thiazole ring and the side chains of hydrophobic amino acids.

4714 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Kitagawa et al.
Figure 8. Synthetic strategy for FabI and FabK inhibitors.
Table 2. The Effect of Chain Length between 4-Pyridone and
Table 3. Antibacterial Activities for Thiazole-Side Derivatives as FabI
Phenylimidazole
and FabK Inhibitors
a E. coli FabI. b S. pneumoniae FabK. c S. aureus ATCC29213. d S.
pneumoniae KU197.
a E. coli FabI. b S. pneumoniae FabK. c S. aureus ATCC29213. d S.
pneumoniae KU197.
II is expected to be recognized by sites II and III of the FabK
active pocket, respectively.
we considered that the 4-pyridone could not interact with the
hydrophobic hollow on Pro118 of FabK because of the long
linkers. These results indicated that our strategy for producing
dual FabI/FabK inhibitors might be valid. Therefore, we chose
a saturated two-carbon chain for the basic structure. Finally,
we optimized the thiazole substitution. The SAR of substituted
thiazole is shown in Table 3.
Since site II of the FabK active pocket is a hydrophobic
hollow on Pro118, adding a plane structure such as 4-pyridone
to the phenylimidazole moiety of a FabK inhibitor with an
appropriate linker might improve the binding affinity to FabK.
On the other hand, site III of FabK has larger space than site
II, so it might be more difficult to introduce favorable interac-
tions. Therefore, we first focused on type I inhibitors. The SAR
of spacer connecting phenylimidazole and 4-pyridone is shown
in Table 2.
Compound 27 having a one-carbon chain length showed FabI
inhibitory activity, but weak FabK inhibitory activity. Although
compound 28, which has a trans-ethenyl fragment showed a
decrease in both activities, the saturated compound 29 showed
strong FabI and FabK inhibitory activities and moderate
antibacterial activity against S. pneumoniae. Compounds 30 and
31, having a three-carbon chain length, showed increased FabI
inhibitory activity but decreased FabK inhibitory activity and
lacked antibacterial activity against S. pneumoniae. In com-
pounds 27 and 28, the 4-pyridone may interfere sterically with
binding to FabK. On the other hand, in compounds 30 and 31,
We previously found that 6-substituted benzo[d]thiazole41 or
5-substituted thiazole derivatives showed potent FabK inhibitory
activity and suggested that steric effects of those substituent
groups were important for FabK inhibitory activity. More details
of the SAR studies of our FabK inhibitors will be reported
elsewhere. On the basis of these results, compound 32 showed
strong FabI and FabK inhibitory activities. Compound 33,
having imidazole instead of thiazole, showed strong FabI
inhibitory activity but lacked FabK inhibitory activity, indicating
that the thiazole ring was essential for FabK inhibition, and 33
did not show antibacterial activity against S. pneumoniae.
Compound 6 substituted at the 6-position on benzothiazole and
compound 34 substituted at the 5-position of thiazole showed
strong FabI and FabK inhibitory activities (FabI IC50 ) 0.38

Inhibitors of Enoyl-ACP Reductases FabI and FabK
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4715
spectrometer. LC-MS analyses were performed using the following
methods. Method A: Capcellpak C18 MG (Shiseido) 3 mm × 150
mm column; linear gradient from 10% to 90% CH3CN in H2O over
10 min (0.01% trifluoroacetic acid) at a flow rate 0.4 mL/min.
Method B: Capcellpak C18 MGII (Shiseido) 3 mm × 150 mm
column; linear gradient from 10% to 90% CH3CN in H2O over 10
min (5 mM ammonium acetate) at a flow rate 0.4 mL/min.
Elemental analyses were performed by Toray Research Center, Inc.
Column chromatography was performed with silica gel (Kanto
Chemical, 60N spherical, neutral). Preparative thin layer chroma-
tography (preparative TLC) was performed with silica gel (Merck
PLC plates, silica gel 60 F254). All the reagents and solvents were
from commercial suppliers and were used without further purifica-
tion.
Benzyl 2-Amino-2-iminoethylcarbamate Hydrobromide (8).
To a solution of benzyl cyanomethylcarbamate (7) (Aldrich, 12.5
g, 66 mmol) in MeOH (190 mL) was added sodium methoxide
(0.36 g, 6.6 mmol). The mixture was stirred at room temperature
overnight and then to it was added ammonium bromide (6.8 g, 69
mmol). The mixture was stirred at room temperature for 1 h more,
and then it was concentrated under reduced pressure. The resulting
solid was dissolved in hexane/EtOAc ) 1/1 (300 mL), stirred
atroom temperature for 1.5 h, and filtered to obtain the title
compound as an off-white powder (16.5 g, 87%). 1H NMR (CD3-
OD): δ 7.39-7.31 (5H, m), 5.14 (2H, s), 4.10 (2H, s). MS
(ESI+): 208 (M + H). HRMS: calcd for C10H13N3O2 (M + H)
208.1086, found 208.1085.
µM, 0.31 µM, respectively; FabK IC50 ) 0.0045 µM, 0.0094
µM, respectively) and potent antibacterial activity against S.
pneumoniae (MIC ) 0.5 µg/mL, 1 µg/mL, respectively). FabK
inhibitory activities of these compounds tended to follow the
SAR of our FabK inhibitors. Although the compounds in Table
3 showed the potent FabI inhibitory activity, they lacked
antibacterial activity against S. aureus. We have reported that
the 4-pyridone derivatives having longer alkyl chain at the
1-position did not show antibacterial activity against S. aureus,
despite of strong FabI inhibitory activity.40 Thus, we concluded
that a 4-pyridone structure with a strongly hydrophobic side
chain was unfavorable for antibacterial activity. The reason for
these results is not clear now, but it might involve poor
membrane permeability or exclusion of the compounds.
MICs of the representative compound 6 against other bacteria
were evaluated. Compound 6 showed no antibacterial activities
(MICs>64 µg/mL) against not only wild type strains (E. coli,
Haemophilus influenzae, Enterococcus faecalis, and Pseudomo-
nas aeruginosa) but also efflux pump deficient mutant (E. coli
and H. influenzae). These results support the idea that compound
6 might have poor penetration.
On the other hand, compound 6 and other phenylimidazole
derivatives in Tables 2 and 3 were evaluated against S.
pneumoniae KU197 mutant with the alanine residue at position
141 in FabK replaced by serine,42 and elevated MICs (>4-fold)
versus a parent strain were observed. Furthermore, the phe-
nylimidazole derivatives in Tables 2 and 3 showed no significant
cytotoxicity (IC50 > 69 µM, in human K562-erythroleukemia
cells). These studies support that the mode of action (MOA) of
compound 6 is the inhibition of fatty acid synthesis.
Benzyl (4-(4-Bromophenyl)-1H-imidazol-2-yl)methylcarbam-
ate (10). To a solution of benzyl 2-amino-2-iminoethylcarbamate
hydrobromide (8) (8.0 g, 28 mmol) in THF/H2O ) 10/1 (330 mL)
were added potassium carbonate (3.8 g, 28 mmol) and 2, 4′-
dibromoacetophenone (9) (Acros, 5.4 g, 19 mmol). The mixture
was stirred at 80 °C for 30 min and then it was poured into brine,
extracted with CH2Cl2, dried over Na2SO4, and concentrated under
reduced pressure. To the resulting residue was added hexane/EtOAc
to obtain the crude solid. Recrystallization from MeOH/CHCl3
Conclusion
We have discovered phenylimidazole derivatives of 4-pyri-
done as novel dual inhibitors of bacterial enoyl-ACP reductases
FabI and FabK. On the basis of the iterative medicinal chemistry
and crystallographic study of FabK from S. pneumoniae/
compound 26, we designed novel FabI and FabK dual inhibitors,
where a FabK inhibitory moiety (phenylimidazoles) was com-
bined with a FabI inhibitory moiety (4-pyridones). Since a
saturated two-carbon chain was identified as a suitable spacer,
related compounds were synthesized and evaluated. Among
them, compound 6 showed strong FabI and FabK inhibitory
activities with potent antibacterial activity against S. pneumo-
niae. This compound showed elevated MICs against FabK
mutant of S. pneumoniae versus wild type strains. The macro-
molecular synthesis assay demonstrated that the 4-pyridone
derivative and imidazole derivative selectively inhibited the lipid
biosynthesis. Additionally, these compounds showed no sig-
nificant cytotoxicity. These studies support a MOA for com-
pound 6 as fatty acid synthesis inhibitor. Small-molecule FabI
and FabK dual inhibitors could be candidates for systematically
active agents for the treatment of clinically important bacterial
infections. Further design and chemical modifications including
the preparation of type II compounds to overcome clinically
important bacterial infections are in progress.
1
afforded the title compound as a white powder (1.13 g, 28%). H
NMR (CDCl3): δ 7.60 (2H, d, J ) 8.5 Hz), 7.47 (2H, d, J ) 8.5
Hz), 7.37-7.32 (5H, m), 7.22 (1H, s), 5.63 (1H, br), 5.15 (2H, s),
4.41 (2H, d, J ) 6.1 Hz). 13C NMR (DMSO-d6): δ 156.2, 145.8,
138.4, 136.9, 134.1, 131.1, 128.2, 127.70, 127.66, 126.0, 118.4,
113.3, 65.4, 38.2. MS (FAB+): 386 (M + H). HRMS: calcd for
C18H16BrN3O2 (M + H) 386.0504, found 386.0496.
Benzyl 4-(4-Bromophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-imidazol-2-yl)methylcarbamate (11). To a solution of benzyl
(4-(4-bromophenyl)-1H-imidazol-2-yl)methylcarbamate (10) (386
mg, 1.0 mmol) in THF (8 mL) were added sodium hydride (24
mg, 1.0 mmol) and (2-(chloromethoxy)ethyl)trimethylsilane (175
µL, 1.0 mmol). The mixture was stirred at room temperature for 2
h and at 40 °C for another 1 h and then it was poured into brine,
extracted with CH2Cl2, dried over Na2SO4, and concentrated under
reduced pressure. The resulting residue was purified by chroma-
tography (hexane/EtOAc ) 2/1) to obtain the title compound as a
1
pale yellow powder (312 mg, 60%). H NMR (CDCl3): δ 7.62
(2H, d, J ) 8.3 Hz), 7.49 (2H, d, J ) 8.3 Hz), 7.38-7.33 (5H, m),
5.34 (2H, brs), 5.15 (2H, brs), 4.54 (2H, d, J ) 5.6 Hz), 3.55 (2H,
t, J ) 8.2 Hz), 0.93 (2H, t, J ) 8.2 Hz). 13C NMR (CDCl3): δ
156.2, 145.4, 139.5, 136.3, 132.6, 131.7, 128.5, 128.2, 126.4, 120.6,
116.2, 75.2, 67.0, 66.5, 37.3, 17.8, -1.4. MS (FAB+): 516 (M +
H). HRMS: calcd for C24H30BrN3O3Si (M + H) 516.1318, found
516.1331.
1-Benzyloxycarbonyl-2,3-dihydro-2-methylpyridin-4(1H)-
one (13). To a solution of methylmagnesium bromide (178 mmol)
and 4-methoxypyridine (12) (16.1 g, 148 mmol) in THF (300 mL)
at -25 °C was added benzyl chloroformate over a period of 30
min. The resulting mixture was stirred at -25 °C for 4 h and then
poured into 3 N HCl (300 mL). After stirring for 10 min at room
temperature, the mixture was extracted with EtOAc twice and the
combined organic extract was washed with 5% aqueous NaHCO3,
dried over Na2SO4, filtered, and concentrated under reduced
pressure. The resulting residue was purified by chromatography
Experimental Section
1
Chemistry. H NMR (400 MHz) and 13C NMR (100 MHz or
125 MHz) spectra were recorded on a JEOL Lambda 400
spectrometer or BRUKER Avance 500 spectrometer. Chemical
shifts were reported in δ value (ppm) with tetramethylsilane (TMS)
as the internal standard (NMR peak designations: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak). Mass
spectra were recorded with a JEOL JMS-700 spectorometer. High-
resolution mass spectra (HRMS) were obtained on a JMS-FABmate

4716 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Kitagawa et al.
(hexane/EtOAc ) 3/1) to obtain the title compound (34.1 g, 94%).
1H NMR (CDCl3) δ 7.74 (1H, d, J ) 8.0 Hz), 7.45-7.28 (5H, m),
5.40-5.22 (3H, m), 4.73 (1H, br), 2.85 (1H, dd, J ) 16.6 Hz, J )
6.8 Hz), 2.31 (1H, d, J ) 16.6 Hz), 1.26 (3H, t, J ) 6.8 Hz). MS
(FAB+): 246 (M + H).
294 (M + H). HRMS: calcd for C15H13Cl2NO (M + H) 294.0452,
found 294.0449.
Benzyl (4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyri-
din-1(4H)-yl)vinyl)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-imidazol-2-yl)methylcarbamate (18). To a solution of benzyl
(4-(4-bromophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imida-
zol-2-yl)methylcarbamate (11) (400 mg, 0.77 mmol) and 3-(2,6-
dichlorobenzyl)-2-methyl-1-vinylpyridin-4(1H)-one (17) (216 mg,
0.74 mmol) in DMF (15 mL) were added tris(dibenzylideneac-
etone)dipalladium (142 mg, 0.16 mmol), tri-tert-butylphosphine (77
µL, 0.31 mmol), and dicyclohexylmethylamine (329 µL, 1.6 mmol).
The mixture was stirred at 80 °C for 2 h, and then it was poured
into brine, extracted with CH2Cl2, dried over Na2SO4, and concen-
trated under reduced pressure. The resulting residue was purified
by preparative TLC (CHCl3/MeOH ) 20/1). Further purification
by LH-20 chromatography (Sephadex, CH2Cl2/MeOH ) 1/1)
provided the title compound as a yellow foam, which was a
diastereomixture (121 mg, 23%, E/Z ) ca. 7/1). 1H NMR (E isomer,
CDCl3): δ 7.74 (2H, d, J ) 8.4 Hz), 7.55 (1H, d, J ) 7.8 Hz),
7.36-7.27 (10H, m), 7.17 (1H, d, J ) 14 Hz), 7.07 (1H, t, J ) 7.6
Hz), 6.63 (1H, d, J ) 14 Hz), 6.38 (1H, d, J ) 7.8 Hz), 5.35 (2H,
s), 5.13 (2H, s), 4.54 (2H, d, J ) 5.6 Hz), 4.38 (2H, s), 3.54 (2H,
t, J ) 8.0 Hz), 2.16 (3H, s), 0.92 (2H, t, J ) 8.0 Hz), 0.01 (9H,
m). 13C NMR (E isomer, CDCl3): δ 178.1, 156.2, 145.5, 143.9,
139.9, 137.7, 137.1, 136.3, 136.0, 134.3, 132.2, 128.6, 128.4, 128.2,
128.1, 128.0, 127.6, 126.9, 126.9, 126.4, 125.3, 116.6, 115.7, 75.3,
67.1, 66.6, 37.5, 27.4, 17.8, 17.1, -1.4. MS (FAB+): 729 (M +
H). HRMS: calcd for C39H42Cl2N4OSi (M + H) 729.2431, found
729.2427.
1-Benzyloxycarbonyl-3-((2,6-dichlorophenyl)hydroxymethyl)-
2,3-dihydro-2-methylpyridin-4(1H)-one (14). To a solution of
1-benzyloxycarbonyl-2,3-dihydro-2-methylpyridin-4(1H)-one (13)
(5.0 g, 20.4 mmol) in THF (60 mL) at -78 °C was added 1 M
lithium bis(trimethylsilyl)amide in THF (22.4 mL), and the resulting
mixture was stirred at 0 °C for 30 min and cooled to -78 °C. To
this solution was added 2,6-dichlorobenzaldehyde (4.64 g, 26.5
mmol). The resulting solution was stirred at -78 °C for 1 h and
then poured into saturated, aqueous NH4Cl (200 mL). The mixture
was extracted with EtOAc twice and the combined organic extract
was washed with brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The resulting residue was purified
by chromatography (hexane/EtOAc ) 3/1) to obtain the title
compound (8.4 g, 98%). 1H NMR (CDCl3) δ 7.70-8.00 (1H, br),
7.50-7.05 (8H, br), 5.63-5.10 (4H, m), 4.15-3.90 (1H, br), 3.30-
3.17 (1H, br), 3.12 (1H, d, J ) 9.0 Hz), 1.20 (3H, d, J ) 6.8 Hz).
MS (FAB+): 420 (M + H).
1-Benzyloxycarbonyl-3-((2,6-dichlorophenyl)(methylsulfony-
loxy)methyl)-2,3-dihydro-2-methylpyridin-4(1H)-one (15). To a
solution of 1-benzyloxycarbonyl-3-((2,6-dichlorophenyl)hydroxym-
ethyl)-2,3-dihydro-2-methylpyridin-4(1H)-one (14) (1.61 g, 3.83
mmol) in pyridine (10 mL) at 0 °C was added mesyl chloride (0.593
mL, 7.66 mmol). The resulting mixture was stirred at room
temperature for 3 h and then poured into water (150 mL). The
mixture was extracted with EtOAc (150 mL) and the organic extract
was washed with water, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The resulting residue was purified
by chromatography (hexane/EtOAc ) 3/1) to obtain the title
compound (1.79 g, 94%). 1H NMR (CDCl3) δ 8.05-7.80 (1H, br),
7.52-7.05 (8H, br), 6.40 (1H, d, J ) 10.7 Hz), 5.59-5.02 (3H,
m), 4.15-3.91 (1H, br), 3.61-3.40 (1H, br), 2.89 (3H, s), 1.22
(3H, d, J ) 6.8 Hz). MS (FAB): 498 (M + H).
(E)-(4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)vinyl)phenyl)-1H-imidazol-2-yl)methylamine Hydro-
chloride (19). Benzyl (4-(4-(2-(3-(2,6-dichlorobenzyl)-2-
methyl-4-oxopyridin-1(4H)-yl)vinyl)phenyl)-1-((2-(trimethylsilyl)-
ethoxy)methyl)-1H-imidazol-2-yl) methylcarbamate (18) (21 mg,
0.029 mmol) was dissolved to 4 N HCl/dioxane/H2O (0.9 mL).
The mixture was stirred at 100 °C for 1.5 h, and then it was washed
with ether. The water layer was concentrated under reduced
pressure. The resulting residue was recrystalyzed from CHCl3/
MeOH to obtain the title compound as a white powder (4.1 mg,
3-(2,6-Dichlorobenzyl)-2-methylpyridin-4-(1H)-one (16). To
a solution of 1-benzyloxycarbonyl-3-((2,6-dichlorophenyl)(meth-
ylsulfonyloxy)methyl)-2,3-dihydro-2-methylpyridin-4(1H)-one (15)
(1.00 g, 2.01 mmol) in THF (35 mL) was added potassium tert-
butoxide (675.5 mg 6.02 mmol). The resulting solution was stirred
at room temperature for 10 min and then poured into 5% aqueous
NH4Cl (100 mL). The mixture was extracted with chloroform/2-
propanol (5:1, 100 mL) twice and the combined organic phase was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. The resulting residue was purified by chromatography
(CHCl3/MeOH ) 9/1) to obtain the title compound as a white
powder (469 mg, 87%). IR (KBr) 3333, 1624, 1503, 1159, 842,
1
25%). H NMR (CD3OD): δ 8.63 (1H, d, J ) 7.2 Hz), 8.01 (1H,
s), 7.94-7.89 (3H, m), 7.80 (2H, d, J ) 8.4 Hz), 7.41 (2H, d, J )
8.0 Hz), 7.29-7.19 (3H, m), 4.54 (4H, m), 2.68 (3H, m). 13C NMR
(CD3OD): δ 172.1, 154.6, 144.6, 142.1, 137.1, 136.8, 136.7, 135.4,
133.9, 133.3, 130.2, 130.0, 129.8, 129.3, 126.5, 126.4, 118.0, 112.8,
36.7, 29.1, 18.3. MS (FAB+): 465 (M + H). HRMS: calcd for
C25H22Cl2N4O (M + H) 465.1249, found 465.1258.
(4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-1(4H)-
yl)ethyl)phenyl)-1H-imidazole-2-yl)methylamine Hydrochloride
(20). To a solution of (E)-(4-(4-(2-(3-(2,6-dichlorobenzyl)-2-methyl-
4-oxopyridin-1(4H)-yl)vinyl)phenyl)-1H-imidazol-2-yl)methy-
lamine hydrochloride (19) (25 mg, 0.44 mmol) in MeOH/HCl (1.1
mL) was added 10% Pd/C (2.5 mg, 10 w/w%). The reaction mixture
was stirred at room temperature for 4.5 h under hydrogen
atmosphere, and then it was filtered through Celite and concentrated
under reduced pressure. The resulting residue was purified by
preparative TLC (CHCl3/MeOH/NH3 aq ) 5/1/0.1), and then it was
acidified by HCl/EtOAc and concentrated under reduced pressure
to obtain the title compound as pale yellow powder (11 mg, 44%).
1H NMR (CD3OD): δ 8.21 (2H, d, J ) 7.2 Hz), 7.65 (2H, d, J )
8.2 Hz), 7.59 (1H, s), 7.35 (2H, d, J ) 8.2 Hz), 7.18-7.12 (3H,
m), 6.97 (2H, d, J ) 7.2 Hz), 4.68 (2H, t, J ) 6.8 Hz), 4.44 (2H,
s), 4.32 (2H, s), 3.18 (2H, t, J ) 6.8 Hz), 2.43 (3H, s). 13C NMR
(CD3OD): δ 170.7, 155.1, 146.0, 141.6, 139.0, 137.0, 137.0, 135.4,
131.5, 130.8, 130.1, 130.0, 127.2, 126.6, 117.7, 112.4, 58.8, 36.9,
36.5, 29.0, 17.2. MS (FAB+): 467 (M + H). HRMS: calcd for
C25H24Cl2N4O (M + H) 467.1405, found 467.1410.
1
768, 543 cm-1. H NMR (CDCl3) δ 7.26 (1H, br), 7.22 (2H, d, J
) 8.1 Hz), 7.05 (1H, t, J ) 8.1 Hz), 6.23 (1H, d, J ) 7.1 Hz), 4.24
(2H, s), 2.10 (3H, s). 13C NMR (CD3OD): δ 180.4, 147.2, 137.63,
137.57, 137.2, 129.7, 129.3, 126.4, 115.3, 28.0, 17.5. HRMS: calcd
for C13H12Cl2NO (M + H) 268.0296, found: 268.0294.
3-(2,6-Dichlorobenzyl)-2-methyl-1-vinylpyridin-4(1H)-one (17).
To a solution of 3-(2,6-dichlorobenzyl)-2-methylpyridin-4(1H)-one
(16) (804 mg, 3.0 mmol) in DMF/CH3CN ) 1/1 (30 mL) were
added tetravinyltin (1.09 mL, 6.0 mmol) and copper(II) acetate (1.09
g, 6.0 mmol). The mixture was stirred at room temperature
overnight under oxygen atmosphere, and then it was poured into
aqueous AcONH4, extracted with EtOAc, washed with brine, dried
over Na2SO4, and concentrated under reduced pressure. The
resulting residue was purified by chromatography (CHCl3/MeOH
) 10/1) to obtain the title compound as a white powder (669 mg,
1
76%). H NMR (CDCl3): δ 7.48 (1H, d, J ) 7.7 Hz), 7.30-7.25
(2H, m), 7.07 (1H, t, J ) 8.0 Hz), 6.82 (1H, dd, J ) 15 Hz, J )
7.3 Hz), 6.36 (1H, d, J ) 7.7 Hz), 5.28 (1H, dd, J ) 15 Hz, J )
1.7 Hz), 5.16 (1H, dd, J ) 7.3 Hz, J ) 1.7 Hz), 4.35 (2H, s), 2.10
(3H, s). 13C NMR (CDCl3): δ 178.2, 143.4, 136.98, 136.95, 135.9,
135.5, 128.4, 127.6, 126.7, 115.7, 109.2, 27.3, 16.8. MS (FAB+):
1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(6-(methyl-
sulfonyl)benzo[d]thiazol-2-yl)urea (6). To a solution of 2-amino-
6-(methylsulfonyl)benzo[d]thiazole (21) (2.28 g, 10 mmol) in THF
was added 1,1-carbonyldiimidazole (3.24 g, 20 mmol). The mixture

Inhibitors of Enoyl-ACP Reductases FabI and FabK
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4717
was stirred at room temperature overnight, and then it was filtered.
The filter cake was washed with THF to give N-(5-(6-(methylsul-
fonyl)benzo[d]thiazol-2-yl)-1H-imidazole-1-carboxamide (22) as a
crude intermediate (3.01 g). To a solution of 22 (22.4 mg, 0.069
mmol) in DMF (1 mL) were added (4-(4-(2-(3-(2,6-dichlorobenzyl)-
2-methyl-4-oxopyridin-1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)-
methylamine hydrochloride (20) (40 mg, 0.069 mmol) and N,N′-
diisopropylethylamine (47 µL, 0.28 mmol). The mixture was stirred
at room temperature for 3 h, and then it was poured into water,
extracted with CHCl3, dried over Na2SO4, and concentrated under
reduced pressure. The resulting residue was purified by preparative
TLC (CHCl3/MeOH/NH3 aq ) 5/1/0.1) to obtain the title compound
as a white powder (12 mg, 21%). 1H NMR (CD3OD): δ 8.43 (1H,
d, J ) 1.8 Hz), 7.92 (1H, dd, J ) 8.6 Hz, J ) 1.8 Hz), 7.80 (1H,
d, J ) 8.6 Hz), 7.57-7.51 (3H, m), 7.30 (1H, s), 7.22 (2H, d, J )
8.0 Hz), 7.01 (2H, d, J ) 8.4 Hz), 6.94 (1H, t, J ) 8.4 Hz), 6.29
(1H, d, J ) 7.2 Hz), 4.58 (2H, s), 4.26-4.22 (4H, m), 3.14 (3H,
m), 2.99 (2H, t, J ) 6.4 Hz), 1.86 (3H, s). 13C NMR (CD3OD): δ
178.5, 165.4, 155.7, 153.9, 148.5, 146.8, 142.4, 138.1, 137.3, 136.7,
135.7, 133.5, 131.6, 130.3, 129.5, 128.8, 128.0, 126.1, 125.9, 122.4,
121.1, 116.8, 115.3, 56.0, 44.9, 37.9, 37.4, 27.9, 16.3. MS
(FAB+): 721 (M + H). HRMS: calcd for C34H30Cl2N6O4S2 (M
+ H) 721.1225, found 721.1227. Anal. (C34H30Cl2N6O4S2‚3H2O)
C, H, N.
1-((4-(4-Bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyri-
din-2-ylthio)thiazol-2-yl)urea (24). To a solution of N-(5-(pyridin-
2-ylthio)thiazol-2-yl)-1H-imidazole-1-carboxamide (30 mg, 0.10
mmol) in THF (1 mL) were added (4-(4-bromophenyl)-1H-
imidazol-2-yl)methylamine hydrochloride (36 mg, 0.11 mmol) and
N,N′-diisopropylethylamine (37 µL, 0.22 mmol). The mixture was
stirred at room temperature overnight, and then it was poured into
brine, extracted with CHCl3, dried over Na2SO4, and concentrated
under reduced pressure. The resulting solid was filtered and the
filter cake was washed with CHCl3/MeOH to obtain the title
compound as a white powder (17 mg, 34%). Recrystallization from
CHCl3/MeOH afforded completely pure compound. 1H NMR
(CDCl3/CD3OD): δ 8.38-8.36 (1H, m), 7.58-7.49 (6H, m), 7.23
(1H, s), 7.09 (1H, ddd, J ) 8.2 Hz, J ) 7.6 Hz, J ) 0.6 Hz), 7.01
(1H, dd, J ) 8.2 Hz, J ) 0.6 Hz), 4.47 (2H, s). 13C NMR (DMSO-
d6): δ 164.4, 160.0, 153.7, 149.3, 146.8, 145.5, 138.5, 137.6, 134.0,
131.2, 126.0, 120.6, 119.6, 118.5, 113.3, 112.8, 37.2. MS
(FAB+): 487 (M + H). HRMS: calcd for C19H15BrN6OS2 (M +
H) 487.0010, found 487.0017. Anal. (C19H15BrN6OS2) C, H, N.
BrN6OS2 (M + H) 487.0010, found 487.019. Anal. (C19H15BrN6-
OS2‚0.1H2O) C, H, N.
Sodium 2-(4-(2-((3-(5-(Pyridin-2-ylthio)thiazol-2-yl)ureido)-
methyl)-1H-imidazol-4-yl)phenoxy)acetate (26). To a solution of
1-((4-(4-((ethoxycarbonyl)methyoxy)phenyl)-1H-imidazol-2-yl)-
methyl)-3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea (99 mg, 0.19 mmol),
which was obtained according to the similar procedure used to
prepare 24, in EtOH (3 mL) was added 5 N aqueous NaOH (0.39
mL, 1.9 mmol). The mixture was stirred at room temperature for
30 min, and then it was concentrated under reduced pressure. The
resulting residue was purified by Dianion HP-20 (Mitsubishi
Chemical) to obtain the title compound. Recrystallization from
MeOH afforded completely pure compound as a white powder (28
1
mg, 28%). H NMR (CD3OD): δ 8.36-8.33 (1H, m), 7.67 (1H,
ddd, J ) 8.0 Hz, J ) 7.8 Hz, J ) 1.7 Hz), 7.57-7.54 (3H, m),
7.19-7.13 (2H, m), 7.07 (1H, d, J ) 8.0 Hz), 6.94 (2H, d, J ) 8.8
Hz) 4.52 (2H, s), 4.39 (2H, s). 13C NMR (CD3OD): δ 176.7, 167.4,
162.4, 159.3, 156.4, 150.2, 147.8, 146.9, 139.1, 127.0, 121.9, 116.0,
115.6, 68.5, 38.4. MS (ESI+): 483 (M + H). HRMS: calcd for
C21H18N6O4S2 (M + H) 483.0909, found 483.0914. Anal. (C21H17N6-
NaO4S2‚3.4H2O) C, H, N.
1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-bromothi-
azol-2-yl)urea (29). To a solution of N-(5-bromothiazol-2-yl)-1H-
imidazole-1-carboxamide (19 mg, 0.069 mmol) in DMF (1 mL)
were added (4-(4-(2-(3-(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methylamine hydrochlo-
ride (20) (40 mg, 0.069 mmol) and N,N′-diisopropylethylamine (47
µL, 0.28 mmol). The mixture was stirred at room temperature for
3.5 h, and then it was poured into water, extracted with CHCl3,
dried over Na2SO4, and concentrated under reduced pressure. The
resulting residue was purified by preparative TLC (CHCl3/MeOH/
NH3 aq ) 7/1/0.1) to obtain the title compound. Recrystallization
from MeOH/CHCl3/DMSO afforded completely pure compound
as an off-white powder (23 mg, 49%). 1H NMR (CD3OD): δ 7.56
(2H, d, J ) 7.6 Hz), 7.51 (2H, d, J ) 8.0 Hz), 7.30-7.21 (4H, m),
7.01 (2H, d, J ) 8.2 Hz), 6.94 (1H, t, J ) 8.2 Hz), 6.29 (1H, d, J
) 7.6 Hz), 4.52 (2H, s), 4.27-4.22 (4H, m), 2.99 (2H, t, J ) 6.2
Hz), 1.86 (3H, s). 13C NMR (DMSO-d6): δ 175.7, 160.3, 153.8,
145.1, 145.0, 140.9, 139.6, 138.4, 136.8, 134.9, 134.8, 133.2, 129.0,
128.5, 127.9, 125.6, 124.2, 113.7, 112.6, 99.6, 54.0, 37.3, 35.9,
27.4, 15.4. MS (FAB+): 671 (M + H). HRMS: calcd for C29H25-
BrCl2N6O2S (M + H) 671.0398, found 671.0404. LC-MS: method
A, tR ) 8.41 min, 671 (M + H), purity 100%; method B, tR
10.37 min, 671 (M + H), purity 99.3%.
)
1-((4-Phenyl-1H-imidazol-2-yl)methyl)-3-(5-(pyridin-2-ylthio)-
thiazol-2-yl)urea (23). The title compound was obtained from N-(5-
(pyridin-2-ylthio)thiazol-2-yl)-1H-imidazole-1-carboxamide and (4-
phenyl-1H-imidazol-2-yl)methylamine hydrochloride according to
the similar procedure used to prepare 24. Recrystallization from
MeOH/CHCl3/diisopropyl ether afforded completely pure com-
pound as a white powder (17 mg, 13%). 1H NMR (DMSO-d6): δ
8.41 (1H, m), 7.77-7.68 (3H, m), 7.64 (1H, s), 7.55 (1H, br), 7.36-
7.32 (2H, m), 7.19-7.10 (3H, m), 7.03 (1H, dd, J ) 8.0 Hz, J )
0.7 Hz), 4.41 (2H, d, J ) 5.4 Hz). 13C NMR (DMSO-d6): δ 164.4,
160.0, 153.6, 149.6, 149.3, 146.8, 145.2, 137.6, 128.3, 125.9, 124.0,
120.6, 119.6, 115.5, 112.8, 112.6, 37.2. MS (FAB+): 409 (M +
H). HRMS: calcd for C19H16N6OS2 (M + H) 409.0905, found
409.0905. Anal. (C19H16N6OS2‚0.25H2O) C, H, N.
1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)methyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-bromothi-
azol-2-yl)urea (27). The title compound was obtained from N-(5-
bromothiazol-2-yl)-1H-imidazole-1-carboxamide and (4-(4-(2-(3-
(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-1(4H)-yl)methyl)phenyl)-
1H-imidazol-2-yl)methylamine hydrochloride according to the
similar procedure used to prepare 29 as an off-white powder (5.6
mg, 21%). 1H NMR (CD3 OD): δ 7.85 (1H, d, J ) 7.6 Hz), 7.67
(2H, d, J ) 8.0 Hz), 7.37 (1H, s), 7.28 (2H, d, J ) 7.8 Hz), 7.25
(1H, s), 7.12 (1H, t, J ) 7.8 Hz), 7.03 (2H, d, J ) 8.0 Hz), 6.43
(2H, d, J ) 7.6 Hz), 5.26 (2H, s), 4.51 (2H, s), 4.33 (2H, s), 2.06
(3H, s). 13C NMR (CD3OD): δ 179.6, 162.5, 156.1, 149.1, 147.6,
143.9, 139.1, 137.7, 136.9, 135.8, 129.8, 129.3, 129.0, 127.4, 126.4,
115.7, 102.1, 58.5, 38.3, 28.5, 16.8. MS (FAB+): 657 (M + H).
HRMS: calcd for C28H23BrCl2N6O2S (M + H) 657.0242, found
657.0229. LC-MS: method A, tR ) 8.47 min, 657 (M + H), purity
100%; method B, tR ) 10.33 min, 657 (M + H), purity 100%.
(E)-1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)vinyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-bromothi-
azol-2-yl)urea (28). The title compound was obtained from N-(5-
bromothiazol-2-yl)-1H-imidazole-1-carboxamide and (4-(4-(2-(3-
(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-1(4H)-yl)vinyl)phenyl)-
1H-imidazol-2-yl)methylamine hydrochloride (19) according to the
similar procedure used to prepare 29 as an off-white powder (4.4
mg, 40%). 1H NMR (DMSO-d6): δ 7.90 (1H, d, J ) 7.6 Hz), 7.76
(2H, d, J ) 8.0 Hz), 7.64-7.52 (4H, m), 7.41-7.38 (3H, m), 7.23
1-((4-(3-Bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyri-
din-2-ylthio)thiazol-2-yl)urea (25). The title compound was
obtained from N-(5-(pyridin-2-ylthio)thiazol-2-yl)-1H-imidazole-
1-carboxamide and (4-(3-bromophenyl)-1H-imidazol-2-yl)methy-
lamine hydrochloride according to the similar procedure used to
prepare 24. Recrystallization from MeOH/CHCl3/Et2O afforded
1
completely pure compound as a white powder (66 mg, 41%). H
NMR (CDCl3/CD3OD): δ 8.39-8.37 (1H, m), 7.82 (1H, brs),
7.58-7.52 (3H, m), 7.39-7.37 (1H, m), 7.27-7.23 (2H, m), 7.08-
7.06 (1H, m), 7.00 (1H, d, J ) 8.3 Hz), 4.46 (2H, s). 13C NMR
(DMSO-d6): δ 164.6, 160.1, 153.9, 149.5, 146.9, 145.8, 138.2,
137.8, 137.3, 130.7, 128.6, 126.6, 123.0, 122.1, 120.8, 119.8, 114.0,
113.0, 37.4. MS (FAB+): 487 (M + H). HRMS: calcd for C19H15-

4718 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Kitagawa et al.
1
(1H, t, J ) 8.0 Hz), 7.10 (1H, br), 6.82 (1H, d, J ) 13.6 Hz), 6.10
(1H, d, J ) 7.6 Hz), 4.39 (1H, d, J ) 4.8 Hz), 4.16 (2H, s), 2.23
(3H, s). 13C NMR (DMSO-d6): δ 176.4, 160.2, 153.8, 145.6, 144.5,
139.3, 138.39, 138.39, 136.6, 135.0, 134.7, 132.1, 128.6, 128.3,
128.2, 127.1, 125.2, 124.8, 124.3, 114.5, 113.4, 99.7, 37.3, 27.4,
16.7. MS (FAB+): 669 (M + H). HRMS: calcd for C29H23-
BrCl2N6O2S (M + H) 669.0242, found 669.0230. LC-MS: method
72%). H NMR (CD3OD): δ 7.56 (1H, d, J ) 7.6 Hz), 7.52 (2H,
d, J ) 8.0 Hz), 7.28 (1H, brs), 7.22 (2H, d, J ) 8.0 Hz), 7.01 (2H,
d, J ) 7.8 Hz), 6.94 (1H, t, J ) 7.8 Hz), 6.72 (2H, s), 6.30 (1H,
d, J ) 7.6 Hz), 4.51 (2H, s), 4.27-4.23 (4H, m), 2.07 (2H, t, J )
5.2 Hz), 1.87 (3H, s). 13C NMR (CDCl3/CD3OD): δ 177.9, 156.1,
146.5, 145.4, 142.8, 140.5, 137.3, 136.8, 135.9, 134.6, 131.9, 129.3,
128.6, 127.7, 127.6, 125.3, 117.3, 116.3, 115.0, 55.4, 37.3, 36.8,
27.4, 15.9. MS (FAB+): 576 (M + H). HRMS: calcd for C29H27-
Cl2N7O2 (M + H) 576.1682, found 576.1688. LC-MS: method
A, tR ) 9.07 min, 669 (M + H), purity 98.2%; method B, tR
11.14 min, 669 (M + H), purity 97.9%.
)
A, tR ) 7.21 min, 576 (M + H), purity 100%; method B, tR
8.97 min, 576 (M + H), purity 100%.
)
(E)-1-((4-(4-(3-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)prop-1-enyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-
bromothiazol-2-yl)urea (30). The title compound was obtained
from N-(5-bromothiazol-2-yl)-1H-imidazole-1-carboxamide and (4-
(4-(2-(3-(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-1(4H)-yl)prop-
1-enyl)phenyl)-1H-imidazol-2-yl)methylamine hydrochloride ac-
cording to the similar procedure used to prepare 29 as a white
powder (17 mg, 41%). 1H NMR (CD3OD): δ 7.76 (1H, d, J ) 7.6
Hz), 7.63 (2H, d, J ) 8.4 Hz), 7.36-7.31 (5H, m), 7.25 (1H, s),
7.14 (1H, t, J ) 7.6 Hz), 6.41 (1H, d, J ) 7.6 Hz), 6.34 (1H, dt,
J ) 16 Hz, J ) 4.8 Hz), 6.18 (1H, d, J ) 16 Hz), 4.80-4.77 (2H,
m), 4.51 (2H, s), 4.04 (2H, s), 2.20 (3H, s). 13C NMR (CD3OD):
δ 179.6, 162.5, 156.1, 149.0, 147.5, 143.2, 139.1, 137.9, 136.9,
135.7, 132.9, 129.8, 129.3, 128.7, 127.9, 126.0, 124.4, 115.9, 102.1,
56.9, 38.3, 28.5, 16.5. MS (FAB+): 683 (M + H). HRMS: calcd
for C30H25BrCl2N6O2S (M + H) 683.0398, found 683.0388. Anal.
(C30H25BrCl2N6O2S‚2.46H2O) C, H, N.
1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-(methyl-
sulfonyl)thiazol-2-yl)urea (34). The title compound was obtained
from N-(5-(methylsulfonyl)thiazol-2-yl)-1H-imidazole-1-carboxa-
mide and (4-(4-(2-(3-(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methylamine hydrochlo-
ride (20) according to the similar procedure used to prepare 29 as
1
a white powder (4.3 mg, 50%). H NMR (CD3OD): δ 7.88 (1H,
s), 7.56 (1H, d, J ) 7.6 Hz), 7.52 (1H, d, J ) 8.0 Hz), 7.29 (1H,
s), 7.23 (2H, d, J ) 8.0 Hz), 7.01 (2H, d, J ) 8.2 Hz), 6.94 (1H,
t, J ) 8.2 Hz), 6.29 (1H, d, J ) 7.6 Hz), 4.55 (2H, s), 4.26-4.22
(4H, m), 3.21 (3H, s), 2.99 (2H, t, J ) 6.4 Hz), 1.86 (3H, s). 13C
NMR (DMSO-d6): δ 169.2, 164.8, 154.3, 153.0, 145.9, 144.6,
144.5, 137.3, 135.1, 134.3, 132.3, 129.9, 129.0, 128.8, 128.6, 125.9,
125.2, 124.6, 115.0, 111.2, 56.6, 45.8, 35.6, 35.1, 28.1, 16.4. MS
(ESI+): 671 (M + H). HRMS: calcd for C30H28Cl2N6O4S2 (M +
H) 671.1069, found 671.1072. Anal. (C30H28Cl2N6O4S2) C, H, N.
1-((4-(4-(3-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)propyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-bromothi-
azol-2-yl)urea (31). The title compound was obtained from N-(5-
bromothiazol-2-yl)-1H-imidazole-1-carboxamide and (4-(4-(2-(3-
(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-1(4H)-yl)propyl)phenyl)-
1H-imidazol-2-yl)methylamine hydrochloride according to the
similar procedure used to prepare 29 as a white powder (19 mg,
Biology. Preparation of His-Tagged FabI. The fabI gene from
E. coli DH5R was amplified by PCR and cloned into pBAD/Myc-
His B vector (Invitrogen). The resulting plasmid was transformed
into E. coli TOP10. The expression of FabI protein fused with a
His-tag was induced with 0.2% arabinose. The cell pellets were
resuspended in lysis buffer (5 mM Tris-HCl, pH 8.0, 0.3 M NaCl,
containing 1 mg/mL of lysozyme) and lysed by sonication. Cell
lysates were applied to a Ni-NTA agarose column (QIAGEN) and
eluted with 250 mM imidazole. The solvent was exchanged to 20
mM Tris-HCl, pH 7.5, 10 mM EDTA, pH 8.0, 1 mM DTT by
dialysis, and the purified protein was stored at -80 °C until use.
Preparation of S. pneumoniae FabK. The fabK genes were
amplified by PCR from S. pneumoniae R6 and cloned into pET-
21b(+) expression vector (Novagen). The resulting plasmid was
transformed into E. coli BL21(DE3). The expression of FabK
protein fused with His-tag at C-terminal was induced by 1 mM
isopropyl â-D-thiogalactoside, and the cells cultivated in LB broth
were grown for a further 4 h before collecting by centrifugation.
Purification of the His-tagged FabK protein was performed as
above. Purified proteins were exchanged into 0.1 M sodium
phosphate buffer, pH 7.0, by dialysis and stored at -80 °C until
use.
Enzyme Inhibition Assay. Enzymatic activity of FabI and FabK
was measured as the reduction of NADH and monitored by the
change in absorbance at 340 nm. Assays were performed in 96-
half-area plates in a final assay volume of 100 µL. For FabI
inhibition assay, the reaction mixture consisted of 100 mM sodium
phosphate (pH 7.4), 0.25 mM crotonoyl-CoA, 0.4 mM NADH, and
50 µg/mL of His-tagged E. coli FabI. For FabK inhibition assay,
the reaction was performed in 100 mM 2-(N-morpholino)ethane-
sulfonic acid (pH 7.0), 100 mM NH4Cl, 0.2 mM crotonoyl-CoA,
0.4 mM NADH, and 2 µg/mL of purified FabK. The reaction was
initiated by addition of the enzyme and measured by absorption at
340 nm for 10 min at room temperature. Concentration giving 50%
reduction in the enzymatic activity was determined as IC50.
1
41%). H NMR (CD3OD): δ 7.90 (1H, s), 7.69 (1H, d, J ) 7.6
Hz), 7.59 (2H, d, J ) 8.4 Hz), 7.33-7.29 (3H, m), 7.25 (1H, s),
7.19-7.15 (3H, m), 6.35 (2H, d, J ) 7.6 Hz), 4.51 (2H, s), 4.32
(2H, s), 3.99 (2H, t, J ) 7.2 Hz), 2.62 (2H, t, J ) 7.2 Hz), 2.08
(3H, s), 2.05-1.97 (2H, m). 13C NMR (CD3OD): δ 179.2, 162.5,
156.0, 148.5, 147.2, 142.8, 140.5, 139.1, 137.8, 136.9, 129.8, 129.7,
129.3, 128.5, 126.1, 115.7, 102.1, 54.7, 38.3, 33.1, 32.9, 28.5, 16.3.
MS (FAB+): 685 (M + H). HRMS: calcd for C30H27BrCl2N6O2S
(M + H) 685.0555, found 685.0556. LC-MS: method A, tR
)
8.72 min, 685 (M + H), purity 100%; method B, tR ) 10.81 min,
685 (M + H), purity 99.7%.
1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyridin-
2-ylthio)thiazol-2-yl)urea (32). The title compound was obtained
from N-(5-(pyridin-2-ylthio)thiazol-2-yl)-1H-imidazole-1-carboxa-
mide and (4-(4-(2-(3-(2,6-dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methylamine hydrochlo-
ride (20) according to the similar procedure used to prepare 29 as
an off-white powder (13.4 mg, 37%). 1H NMR (CD3OD): δ 8.34-
8.32 (1H, m), 7.64 (1H, ddd, J ) 8.0 Hz, J ) 8.0 Hz, J ) 2.0 Hz),
7.58 (1H, d, J ) 7.4 Hz), 7.55 (1H, s), 7.28 (1H, s), 7.21 (2H, d,
J ) 8.0 Hz), 7.05-6.99 (3H, m), 6.91 (1H, t, J ) 8.0 Hz), 6.31
(1H, d, J ) 7.4 Hz), 4.53 (2H, s), 4.26-4.22 (4H, m), 2.99 (2H, d,
J ) 6.4 Hz), 1.83 (3H, s). 13C NMR (DMSO-d6): δ 175.6, 164.3,
160.0, 153.6, 149.3, 146.8, 145.0, 144.9, 140.9, 139.4, 137.6, 136.7,
134.81, 134.77, 133.0, 129.0, 128.4, 127.9, 125.5, 124.1, 120.6,
119.6, 113.7, 112.9, 112.5, 53.9, 37.2, 35.9, 27.3, 15.3. MS
(FAB+): 702 (M + H). HRMS: calcd for C34H29Cl2N7O2S2 (M
+ H) 702.1279, found 702.1281. LC-MS: method A, tR ) 8.27
min, 702 (M + H), purity 99.6%; method B, tR ) 10.18 min, 702
(M + H), purity 99.7%.
1-((4-(4-(2-(3-(2,6-Dichlorobenzyl)-2-methyl-4-oxopyridin-
1(4H)-yl)ethyl)phenyl)-1H-imidazol-2-yl)methyl)-3-(1H-imida-
zol-2-yl)urea (33). The title compound was obtained from N-(1H-
imidazol-2-y)-1H-imidazole-1-carboxamide and (4-(4-(2-(3-(2,6-
dichlorobenzyl)-2-methyl-4-oxopyridin-1(4H)-yl)ethyl)phenyl)-1H-
imidazol-2-yl)methylamine hydrochloride (20) according to the
similar procedure used to prepare 29 as a white powder (3.6 mg,
MIC Testing. Antibacterial activity was determined by the broth
microdilution method according to the NCCLS document M7-A6.
The minimum inhibitory concentration (MIC) was determined as
the lowest concentration of the test compound that prevented visible
growth. S. aureus ATCC29213, S. pneumoniae KU197 (PRSP), S.
pneumoniae KU197 (A141S) (PRSP, fabk mutant),42 H. influenzae
Rd, H. influenzae Rd∆acrB (efflux mutant), H. influenzae PRC44,

Inhibitors of Enoyl-ACP Reductases FabI and FabK
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4719
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(34) Miller, W. H.; Seefeld, M. A.; Newlander, K. A.; Uzinskas, I. N.;
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(35) Seefeld, M. A.; Miller, W. H.; Newlander, K. A.; Burgess, W. J.;
Payne, D. J.; Rittenhouse, S. F.; Moore, T. D.; DeWolf, W. E., Jr.;
Keller, P. M.; Qiu, X.; Janson, C. A.; Vaidya, K.; Fosberry, A. P.;
Smyth, M. G.; Jaworski, D. D.; Slater-Radosti, C.; Huffman, W. F.
Inhibitors of bacterial enoyl acyl carrier protein reductase (FabI):
2,9-Disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as potential
antibacterial agents. Bioorg. Med. Chem. Lett. 2001, 11 (17), 2241-
2244.
Acknowledgment. The authors wish to thank Miss Shigeko
Miki, Mrs. Takako Miyara, and Mrs. Kazue Sasaki of Meiji
Seika Kaisha, Ltd. (http://www.meiji.co.jp/home.html) for their
help with mass spectrometric analysis. The authors also wish
to thank Mr. Takashi Watanabe and Mr. Seigo Sato for their
help with computational or NMR study.
Supporting Information Available: Purity data including
HPLC and elemental analyses. This material is available free of
charge via the Internet at http://pubs.acs.org.
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