Phenylimidazole derivatives as specific inhibitors of bacterial enoyl-acyl carrier protein reductase FabK

Article abstract of DOI:10.1016/j.bmc.2007.08.050

Bacterial enoyl-acyl carrier protein (ACP) reductases (FabI and FabK) catalyze the final step in each cycle of bacterial fatty acid biosynthesis and are attractive targets for the development of new antibacterial agents. Here, we report the development of novel FabK inhibitors with antibacterial activity against Streptococcus pneumoniae. Based on structure-activity relationship (SAR) studies of our screening hits, we have developed novel phenylimidazole derivatives as potent FabK inhibitors.

Full text of DOI:10.1016/j.bmc.2007.08.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7325–7336
Phenylimidazole derivatives as specific inhibitors of bacterial
enoyl-acyl carrier protein reductase FabK
Tomohiro Ozawa,a Hideo Kitagawa,a,* Yasuo Yamamoto,a Sho Takahata,b
Maiko Iida,a Yumi Osakib and Keiko Yamadab
aMedicinal Chemistry Research Laboratories, Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd,
760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
bPharmacology Research Laboratories, Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd,
760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
Received 27 July 2007; revised 13 August 2007; accepted 14 August 2007
Available online 4 September 2007
Abstract—Bacterial enoyl-acyl carrier protein (ACP) reductases (FabI and FabK) catalyze the final step in each cycle of bacterial
fatty acid biosynthesis and are attractive targets for the development of new antibacterial agents. Here, we report the development of
novel FabK inhibitors with antibacterial activity against Streptococcus pneumoniae. Based on structure–activity relationship (SAR)
studies of our screening hits, we have developed novel phenylimidazole derivatives as potent FabK inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
a single large multifunctional protein to which the grow-
ing chain remains covalently attached. The difference be-
tween bacterial and mammalian fatty acid biosynthesis
offers an attractive opportunity for selective inhibition
of type II FAS. Therefore, inhibitors of the bacterial en-
zymes are expected to be candidates antibacterial agents.
Streptococcus pneumoniae is the main causative patho-
gen of respiratory tract infections such as community-
acquired pneumonia and acute otitis media. The
increase of penicillin-resistant S. pneumoniae (PRSP)
and macrolide-resistant S. pneumoniae is of great
concern worldwide. In addition, the emergence of
quinolone-resistant S. pneumoniae has been reported
recently.1–3 A key strategy to overcome drug-resistant
pathogens is the discovery of antibacterial agents with
novel mechanisms of action.4
FabI (enoyl-ACP reductase) is an enzyme which cata-
lyzes the final and rate-limiting step of bacterial FAS.
The reaction involves the conjugate reduction of an en-
oyl-ACP to the corresponding acyl-ACP using the
cofactor NADH or NADPH as a hydride source.8,9
Various compounds, including isoniazid,10 diazabo-
rines,11,12 triclosan,13–18 indole naphthyridinones 1,
219–22 and thiopyridine 323 have been reported as inhib-
itors of bacterial enoyl-ACP reductase FabI. We have
also reported a series of 4-pyridone derivatives, such
as compound 4, as small-molecular FabI inhibitors with
potent antibacterial activity against Staphylococcus aur-
eus.24–26 A FabI-targeting approach to antibacterial
drug therapy appears feasible (Fig. 1).
In this regard, fatty acid biosynthesis is an attractive tar-
get.5–7 Bacterial fatty acid biosynthesis (type II FAS)
provides fatty acids which are used in the assembly of
essential cellular components of bacteria such as cell
envelopes, phospholipids, lipoproteins, lipopolysaccha-
rides or mycolic acids. In bacteria, different monofunc-
tional enzymes catalyze each of the reactions and
reaction intermediates are carried through the cytosol
as thioesters of the small acyl carrier protein (ACP).
On the other hand, mammals synthesize fatty acids on
However, recent studies have shown that other bacterial
enoyl-ACP reductases exist in addition to FabI.27,28
A
triclosan-resistant flavoprotein, termed FabK, has been
shown to be the sole enoyl-ACP reductase in S. pneumo-
niae. Consequently, a selective inhibitor of FabK is ex-
pected to be a novel narrow-spectrum antibacterial
Keywords: FabK; Enoyl-ACP reductase; Phenylimidazole; Streptococ-
cus pneumoniae.
*
Corresponding author. Tel.: +81 45 541 2521; fax: +81 45 541
0667; e-mail: hideokitagawa@coda.ocn.ne.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.050

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T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
Figure 1. Structures of conventional FabI or FabK inhibitors.
agent against S. pneumoniae including PRSP and macro-
lide-resistant S. pneumoniae. Only a few FabK inhibitors
have been identified so far. The reported FabK inhibi-
tors 1 and 2 had weak inhibitory activity and failed to
show selective inhibition of bacterial FAS.19,22 Atro-
mentin and leucomelone were also reported as FabK
inhibitors, but they did not show any antibacterial activ-
ity against S. pneumoniae.29
these compounds,31 we found the ureido compound
47, which has strong FabK-inhibitory activity
(IC50 = 0.0024 lM) and potent antibacterial activity
against S. pneumoniae (MIC = 0.25 lg/ml). In this pa-
per, we would like to report our development of specific
inhibitors of enoyl-ACP reductase FabK in detail. All
the compounds in Tables 4 and 5 (compounds 41–49)
and several compounds in Tables 1–3 (compounds 16,
33, 34, 37, 40) are novel compounds which we have
never reported before.
To discover small-molecular FabK inhibitors, we
screened our compound library for inhibitory activity
toward FabK of S. pneumoniae. Among the hits, the
amide 5 and the oxime 6 were selected as lead com-
pounds because these compounds exhibited moderate
and selective inhibitory activity toward FabK of S.
pneumoniae (compound 5: S. pneumoniae FabK
IC50 = 1.5 lM, Escherichia coli FabI IC50 > 32 lM;
compound 6: S. pneumoniae FabK IC50 = 1.1 lM,
E. coli FabI IC50 > 32 lM).30 Based on SAR studies of
2. Chemistry
Amide-type derivatives were prepared by means of stan-
dard procedures.32–35 For example, compound 11 was
prepared as illustrated in Scheme 1. Commercially avail-
able 2-aminobenzo[d]thiazole 7 reacted with chloroace-
tyl chloride 8 in toluene at 60 ꢁC to give the
Table 1. Effects of various structural units on enzyme-inhibitory and antibacterial activities
Compound
FabK IC50 (lM)
S. pneumoniae
MIC (lg/ml)
Fabl IC50 (lM)
E. coli
S. pneumoniaea
6
1.1 0.2
>32
>32
>32
>32
11
>32
16
28
>32
>32
>32
>32
>32
32
>32
>32
29
6.9 1.5
a S. pneumoniae KU197.

T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
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Table 2. Effects of substitution on the benzothiazole ring on enzyme-inhibitory and antibacterial activities
Compound
R1
FabK IC50 (lM)
S. pneumoniae
MIC (lg/ml)
Fabl IC50 (lM)
E. coli
S. pneumoniaea
29
30
31
32
33
34
35
36
H
6.9 1.5
>32
>32
32
>32
>32
>32
>32
>32
>32
>32
>32
4-OMe
5-OMe
6-OMe
6-Me
NTb
NT
16
2.0 0.5
2.6 0.4
6.4 1.3
4.5 0.6
0.74 015
16
6-F
6-CN
6-SO2Me
NT
8
4
a S. pneumoniae KU197.
b NT, not tested.
Table 3. Effects of other heterocyclic groups on enzyme-inhibitory and antibacterial activities
Compound
R1
FabK IC50 (lM)
S. pneumoniae
MIC (lg/ml)
Fabl IC50 (lM)
E. coli
S. pneumoniaea
36
0.74 0.15
3.4 0.9
4
>32
>32
37
>32
38
39
23
30
2
9
>32
>32
>32
>32
24
40
0.088 0.014
0.5
>32
>32
>32
NTb
a S. pneumoniae KU197.
b NT, not tested.
chloroacetamide 9 in 70% yield. Compound 9 was trea-
ted with 2-mercaptobenzimidazole 10 in the presence of
sodium methoxide to give the amide compound 11 in
73% yield.
Compound 16 was prepared as illustrated in Scheme 2.
Commercially available 2-hydroxymethylbenzo[d]thia-
zole 12 reacted with phthalimide 13 under Mitsunobu
reaction conditions to give the phthalimide derivative,
which was converted to the amine 14 by using hydrazine
in 46% yield (2 steps).36,37 The resulting amine 14 re-
acted with chloroacetyl chloride 8 in toluene at 80 ꢁC
Scheme 1. Synthesis of compound 11. Reagents and conditions:
(a) toluene, 60 ꢁC (70%); (b) NaOMe (1.5 equiv), DMF, rt (73%).

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T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
available 2-amino-6-methylsulfonylbenzo[d]thiazole 22
and 1,10-carbonyldiimidazole (CDI), to give the ureido
derivative 24 in 33% yield. Other ureido compounds
were also obtained according to the procedures illustrated
in Scheme 3. Intermediates such as 6-substituted
benzo[d]thiazoles and 5-substituted thiazoles were pre-
pared according to standard procedures.41–43
3. Biological methods
3.1. Preparation of S. pneumoniae FabK
The fabK genes were amplified by PCR from S. pneumo-
niae 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 b-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.
Scheme 2. Synthesis of compound 16. Reagents and conditions:
(a) DEAD (2 equiv), Ph3P (2 equiv), THF, rt; (b) NH2NH2 ꢁ H2O
(1.5 equiv), EtOH, reflux (46% for two steps); (c) toluene, 80 ꢁC (68%);
(d) NaOMe (1.5 equiv), DMF, rt (19%).
to give the chloroacetamide 15 in 68% yield. Compound
15 was treated with 2-mercaptobenzimidazole 10 in the
presence of sodium methoxide to give the amide com-
pound 16 in 19% yield.
The ureido compound 24 was prepared as illustrated in
Scheme 3. Commercially available benzyl cyanomethylc-
arbamate 17 was allowed to react with a catalytic
amount of sodium methoxide in MeOH, then converted
to the amidine 18 by treatment with ammonium chloride
in 97% yield (2 steps).38 Compound 18 was coupled with
bromoacetophenone 19 in the presence of potassium
carbonate to give benzyl (4-phenyl-1H-imidazol-2-
yl)methylcarbamate 20 in 29% yield.39,40 Deprotection
of the benzyloxycarbonyl group under a hydrogen
atmosphere at room temperature in MeOH/HCl pro-
vided (4-phenyl-1H-imidazol-2-yl)methylamine hydro-
chloride 21 in 76% yield. The resulting methylamine
hydrochloride 21 was allowed to react with crude
imidazolide 23, which was prepared from commercially
3.2. Preparation of His-tagged FabI
The fabI gene from E. coli DH5a 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 pel-
lets 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.
Scheme 3. Synthesis of compound 24. Reagents and conditions: (a) NaOMe (0.1 equiv), MeOH, rt; (b) NH4Cl (1 equiv), MeOH, rt (97% for two
i
steps); (c) K2CO3 (1 equiv), DMF, rt (29%); (d) 10 wt% Pd/C (30 w/w%), H2, MeOH, HClaq (76%); (e) THF, rt; (f) Pr2NEt (2.2 equiv), THF, rt
(33%).

T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
7329
3.3. Enzyme inhibition assay
measurement conditions by means of liquid chromatog-
raphy-mass spectrometry indicated that compound 5
readily decomposed to 26 and 27, which lack FabK-
inhibitory activity (Fig. 2).
Enzymatic activity of FabI and FabK was measured as
the reduction of NADH and monitored the change in
absorbance at 340 nm. Assays were performed in 96
half-area plates in a final assay volume of 100 ll. For
FabI inhibition assay, reaction mixture consisted of
100 mM sodium phosphate (pH 7.4), 0.25 mM croto-
noyl-CoA, 0.4 mM NADH, and 50 lg/ml of His-tagged
E. coli FabI. For FabK inhibition assay, reaction was
performed in 100 mM 2-(N-morpholino)ethanesulfonic
acid (pH 7.0), 100 mM NH4Cl, 0.2 mM crotonoyl-
CoA, 0.4 mM NADH, and 2 lg/ml of purified FabK.
The reaction was initiated by addition of the enzyme,
and measured absorption at 340 nm for 10 min at room
temperature. Concentration giving 50% reduction in the
enzymatic activity was determined as IC50.
To improve the chemical stability of the amide bond, we
prepared methylene amide 16, carbamate 28, and ureido
type 29 derivatives. Compounds 16 and 28 did not show
FabK-inhibitory or antibacterial activity, while 29 pos-
sessed weak FabK-inhibitory activity (IC50 = 6.9 lM).
Moreover, compound 29 showed antibacterial activity
against S. pneumoniae, probably due to its improved
chemical stability in the MIC assay medium
(MIC = 32 lg/ml). On the other hand, the oxime com-
pound 6 possessing moderate FabK-inhibitory activity
did not show any antibacterial activity, presumably
due to its poor solubility (Table 1). Compounds 28
and 29 have been reported in our previous paper.31
3.4. MIC testing
We therefore selected an ureido group as a spacer. The
SAR of benzothiazole derivatives is shown in Table 2.
Antibacterial activity was determined by the broth mic-
rodilution 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 the visible growth. S. pneumo-
niae KU197 (PRSP) and S. pneumoniae KU197 mutant
with FabK (A141S)30 were used routinely. Twenty-nine
clinical isolates of S. pneumoniae were used for the MIC
testing of compound 47. They consisted of 11 strains of
PRSP, 6 strains of PISP, and 12 strains of PSSP. While 5
out of 29 strains were sensitive to macrolides, remaining
24 strains were resistant to macrolides (ermB positive: 9
strains, mefA: 9 strains, mefA and ermB positive: 6
strains).
Compounds 32 having a methoxy group at the 6-posi-
tion of benzothiazole showed greater FabK-inhibitory
activity than compound 29 (R1 = H), while compounds
30 and 31 (R1 = 4-OMe or 5-OMe) showed no FabK-
inhibitory activity. These results indicate that some kind
of functional group at the 6-position is essential to im-
prove FabK-inhibitory activity. Next, the substituent ef-
fects at the 6-position on the FabK-inhibitory and
antibacterial activities were examined. Compounds 33–
36, having various functional group at the 6-position
of benzothiazole, showed increased FabK-inhibitory
activity compared with compound 29. In particular,
compound 36 having a methylsulfonyl group showed
good FabK-inhibitory activity (IC50 = 0.74 lM). Com-
pounds 30–32, 35, and 36 have been reported in our pre-
vious paper.31
3.5. Cytotoxicity
Cytotoxicities of the test compounds were assessed by
using human leukemia cells K562. K562 was maintained
in RPMI 1640 medium supplemented with 10% fetal bo-
vine serum (Moregate Biotec). The assays were per-
formed in 96-well microtiter plates (Greiner). The cells
(5 · 103 cells/150 ll) were suspended in the appropriate
assay medium and it was added to each well followed
by the testing compounds. The cells were incubated with
the drug for 2 days at 37 ꢁC in the presence of 5% CO2.
On day 3, Cell Counting Kit-8 (Dojindo Lab.) was
added to each well, and the suspension was reincubated
for 4 h under the same conditions. Cell proliferaion was
determined by recording the absorbance change at
450 nm. IC50 values were defined as the concentration
of each test sample that reduced absorbance to 50% of
vehicle-treated controls.
Introduction of other heterocyclic rings (compounds 37,
38, 39, and 40) instead of the benzimidazole ring did not
improve either of the activities, but the phenylimidazole
derivative (compound 24) showed strong FabK-inhibi-
tory activity (IC50 = 0.088 lM) and antibacterial activity
(MIC = 0.5 lg/ml) (Table 3). Thus, we selected the
phenylimidazole moiety as a basic structure. Since ben-
zothiazole derivatives tended to show worse solubility in
aqueous 5% DMSO, we examined other heterocycles in-
stead of benzothiazoles and found that the substituted
thiazoles could be employed (Table 4). Compounds 38,
39, and 24 have been reported in our previous paper.31
Compound 41, having a phenyl group at the 5-position
of thiazole, showed stronger FabK-inhibitory activity
than compound 42 having a phenyl group at the 4-posi-
tion. This result indicates that substitution at the 5-posi-
tion is favorable for FabK-inhibitory activity. Next, the
substituent effects at the 5-position were examined.
Compounds 43–46, having a thiophenyl or thiopyridyl
group at the 5-position of thiazole, showed increased
FabK-inhibitory activity. In particular, compound 44,
having a thiopyridyl group, showed strong FabK-inhib-
itory activity (IC50 = 0.042 lM) and potent antibacterial
4. Results and discussion
Although several of the amide derivatives, including 5,
showed moderate FabK-inhibitory activity, they showed
no antibacterial activity against S. pneumoniae in stan-
dard broth media (cation adjusted Mueller-Hinton
broth supplemented with 2% lysed horse blood). Exam-
ination of the stability of the compound under the MIC

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T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
Figure 2. Expected compounds derived from compound 5.
Table 4. Effects of substituted thiazoles on enzyme-inhibitory and antibacterial activities
Compound
R1
R2
FabK IC50 (lM)
S. pneumoniae
MIC (lg/ml)
Fabl IC50 (lM)
E. coli
S. pneumoniaea
41
42
H
Ph
Ph
H
0.38 0.02
>32
4
NTb
>32
>32
43
44
H
H
0.037 0.006
0.042 0.005
2
2
>33
>32
45
46
H
H
0.037 0.004
0.098 0.015
2
8
>32
>32
a S. pneumoniae KU197.
b NT, not tested.
activity against S. pneumoniae (MIC = 2 lg/ml) with
better solubility in aqueous 5% DMSO, as compared
with compound 43.
141 of FabK is replaced by serine,30 its mode of action is
considered to involve inhibition of FabK. Moreover,
compound 47 showed no significant cytotoxicity
(IC50 > 20 lM in human erythroleukemia cells K562).
These results indicate that FabK inhibitor 47 is a candi-
date antibacterial agent.
We therefore selected the thiopyridyl group as a basic
structure and the substituent effects of phenylimidazole
derivatives were examined (Table 5).
Compounds 47–49 having a halogen atom (chlorine or
bromine) on the phenyl group of phenylimidazole
5. Conclusion
showed excellent FabK-inhibitory activity (IC50
=
We have described the discovery of novel small-molecu-
lar inhibitors of bacterial enoyl-ACP reductase FabK.
Through the SAR studies of our screening hits, we iden-
tified phenylimidazole derivatives as potent FabK-inhib-
itors. A representative compound 47 showed excellent
FabK-inhibitory activity and potent in vitro antibacte-
rial activity against S. pneumoniae. An elevated MIC va-
lue was observed with a FabK mutant of S. pneumoniae.
Compound 47 showed no significant cytotoxicity. These
results indicate that the phenylimidazole derivative 47 is
a specific inhibitor of bacterial enoyl-ACP reductase
FabK with antibacterial activity against S. pneumoniae,
and we consider that it is an interesting candidate drug
for the treatment of bacterial infections.
0.0024–0.0053 lM). In particular, compound 47 showed
potent antibacterial activity against S. pneumoniae
(MIC = 0.25 lg/ml).
The antibacterial activity of compound 47 was evaluated
for 29 clinical isolates of S. pneumoniae, including drug-
resistant pathogens (11 PRSP strains, 6 penicillin-inter-
mediate S. pneumoniae (PISP) strains, and 12 penicil-
lin-susceptible (PSSP) strains). MIC50 and MIC90 of
compound 47 were 1 and 4 lg/ml, respectively (MIC
range of 0.125–4 lg/ml). Since compound 47 showed
an elevated MIC value (>16-fold) against S. pneunomiae
KU197 mutant, in which the alanine residue at position

T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
7331
Table 5. Effects of phenylimidazole substitution on enzyme-inhibitory and antibacterial activities
Compound
R1
Ph
FabK IC50 (lM)
S. pneumoniae
MIC (lg/ml)
Fabl IC50 (lM)
E. coli
S. pneumoniaea
44
47
0.042 0.005
2
>32
0.0024 0.0002
0.25
>32
48
0.0053 0.0005
16
>32
49
0.0037 0.0001
>32
1
>32
Triclosan
NTb
0.90 0.36
a S. pneumoniae KU197.
b NT, not tested.
6. Experimental
was diluted with EtOH, then the solid was collected by
suction filtration. The filter cake was washed with
EtOH. Drying in high vacuum at 50 ꢁC gave the title
6.1. Chemistry
1
compound as a white solid (160 mg, 70%). H NMR
1H NMR (400 MHz) and 13C NMR (100 MHz or
125 MHz) spectra were recorded on a JEOL Lambda
400 spectrometer or BRUKER Avance 500 spectrome-
ter. Chemical shifts were reported in d 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 spectrometer.
High-resolution mass spectra (HRMS) were obtained
on a JMS-FABmate spectrometer. LC–MS analyses
were performed using the following methods. Method
A: Capcellpak C18 MG (Shiseido) 3 mm · 150 mm col-
umn; 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 ammo-
nium acetate) at a flow rate 0.4 ml/min. Elemental anal-
yses were performed by Toray Research Center, Inc.
Melting points were recorded with a Yanako MICRO
MELTINGPOINT APPARATUS. Column chroma-
tography was performed with silica gel (Kanto Chemi-
cal: 60N spherical, neutral). Preparative thin layer
chromatography (Preparative TLC) was performed with
silica gel (Merck: TLC plates Silica gel 60 F254). All the
reagents and solvents were from commercial suppliers,
and were used without further purification.
(DMSO-d6): d 8.01 (1H, dd, J = 8.0 Hz, J = 0.5 Hz),
7.78 (1H, d, J = 8.0 Hz), 7.46 (1H, dt, J = 8.0 Hz,
J = 1.2 Hz), 7.34 (1H, dt, J = 8.1 Hz, J = 1.2 Hz), 4.48
(2H, s). 13C NMR (DMSO-d6): d 165.9, 157.5, 148.3,
131.3, 126.2, 123.7, 121.8, 120.6, 42.4. HRMS calcd
for C9H7ClN2OS (M+H) 227.0046, found 227.0041.
6.1.2. 2-(1H-Benz[d]imidazol-2-ylthio)-N-(benzo[d]thia-
zol-2-yl)acetamide (11).31 To a solution of chloroaceta-
mide 9 (113 mg, 0.5 mmol) in DMF (1 ml) was added
1H-benz[d]imidazole-2-thiol 10 (113 mg, 0.75 mmol), so-
dium methoxide (40.5 mg, 0.75 mmol) and stirred at
room temperature for 24 h. The mixture was diluted
with H2O and CH2Cl2, then the solid was collected by
suction filtration. The filter cake was washed with
H2O. Drying in high vacuum at 50 ꢁC gave the title com-
pound as a white solid (124.5 mg, 73%). 1H NMR
(DMSO-d6): d 7.97 (1H, m), 7.77 (1H, m), 7.50 (2H,
dd, J = 5.9 Hz, J = 2.7 Hz), 7.45 (1H, m), 7.31 (1H,
m), 7.20 (2H, dd, J = 6.1 Hz, J = 2.9 Hz), 4.49 (2H, s).
13C NMR (DMSO-d6): d 167.2, 157.7, 149.4, 148.4,
138.0, 131.4, 126.1, 123.6, 122.3, 121.7, 120.6, 113.7,
35.2. HRMS calcd for C16H12N4OS2 (M+H) 341.0531,
found 341.0529.
6.1.3. Benzo[d]thiazol-2-ylmethanamine (14). To a solu-
tion of 2-hydroxymethylbenzothiazole 12 (ACROS,
800 mg, 4.8 mmol) in THF (45 ml) was added triphenyl-
phosphine (2.5 g, 9.7 mmol), phthalimide 13 (1.4 g,
9.7 mmol) and diethyl azodicarboxylate (1.7 g,
9.7 mmol) and stirred at room temperature for 7 h.
The mixture was diluted with H2O and EtOAc, then ex-
tracted with EtOAc, washed with brine, dried over
MgSO4 and concentrated under reduced pressure. The
6.1.1. N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide (9).31
To a solution of 2-amino-benzo[d]thiazole 7 (150 mg,
1 mmol) in toluene (3 ml) was added 2-chloroacetyl
chloride 8 (112.9 mg, 1 mmol) and stirred at 60 ꢁC for
6 h. It was allowed to cool to room temperature, then
it was concentrated under reduced pressure. The residue

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T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
resulting residue was diluted with Et2O and stirred at
room temperature. The generated solid was collected
by suction filtration. Drying in high vacuum at 50 ꢁC
gave 2-(benzo[d]thiazol-2-ylmethyl)isoindoline-1,3-dione
as a white solid (953 mg). To a solution of 2-(benzo[d]-
thiazol-2-ylmethyl)isoindoline-1,3-dione (936 mg, 3.18
mmol) in EtOH (30 ml) was added hydrazine monohy-
drate (2.39 g, 4.77 mmol) and stirred at reflux for
1.5 h. It was allowed to cool to room temperature, then
the generated solid was collected by suction filtration
and washed with CHCl3. The filtrated solution was con-
centrated under reduced pressure, and the resulting res-
idue diluted with H2O, acidified with 1 N HClaq to pH
1.6. The water layer was washed with EtOAc, and
turned into pH8 with 1 N NaOH aq, extracted with
EtOAc, washed with H2O, brine, dried over MgSO4,
and concentrated under reduced pressure. The title com-
pound was obtained as a white solid (361 mg, 46% in 2
steps). 1H NMR (CDCl3): d 7.97 (1H, m), 7.89 (1H, m),
7.47 (1H, m), 7.37 (1H, m), 4.31 (2H, s). MS (FAB+):
165 (M+H).
mixture was stirred at room temperature for overnight,
then it was added ammonium chloride (3.4 g, 63 mmol).
The mixture was stirred at room temperature for 2 more
days, then it was cooled in ice-bath, added HCl/EtOAc,
and stirred for 2 h more without ice-bath. The mixture
was concentrated under reduced pressure, and the
resulting solid was dissolved to Hexane/EtOAc = 1/1
(60 ml), stirred at room temperature for 20 min, then it
was filtered to obtain the title compound (15 g, 97%)
1
as a dark white powder. H NMR (CD3OD): d 7.39–
7.31 (5H, m), 5.14 (2H, s), 4.10 (2H, s). HRMS calcd
for C10H13N3O2 (M+H) 208.1086, found 208.1084.
6.1.7. Benzyl (4-phenyl-1H-imidazol-2-yl)methylcarba-
mate (20).31 To a solution of amidine hydrochloride
18 (3.0 g, 12 mmol) in DMF (90 ml) was added phen-
acylbromide 19 (2.5 g, 12 mmol) and potassium carbon-
ate (1.7 g, 12 mmol). The mixture was stirred at room
temperature for 2.5 h, then it was filtered and concen-
trated under reduced pressure. The resulting residue
was dissolved to CHCl3, washed with brine, dried over
Na2SO4, and concentrated under reduced pressure.
The resulting residue was purified by chromatography
(CHCl3 to CHCl3/MeOH = 20/1) to obtain the title
6.1.4. N-(Benzo[d]thiazol-2-ylmethyl)2-chloroacetamide
(15). To a solution of amine 14 (327 mg, 2 mmol) in tol-
uene (20 ml) was added 2-chloroacetyl chloride 8
(247 mg, 2.19 mmol) and stirred at 80 ꢁC for 2 h. It
was allowed to cool to room temperature, then concen-
trated under reduced pressure. The resulting residue was
purified by chromatography (CHCl3/MeOH) to obtain
the title compound as a white powder (325 mg, 68%).
1H NMR (CDCl3): d 8.02 (1H, m), 7.88 (1H, m), 7.50
(1H, m), 7.41 (1H, m), 4.92 (2H, d, J = 5.6 Hz), 4.17
(2H, s). 13C NMR (CDCl3): d 166.7, 166.2, 152.7,
135.2, 126.3, 125.5, 123.1, 121.8, 42.5, 42.0. MS
(FAB+): 241 (M+H).
1
compound as a brownish white solid (1.1 g, 29%). H
NMR (CDCl3): d 7.39–7.34 (8H, m), 7.24–7.22 (3H,
m), 5.15 (2H, s), 4.43 (2H, d, J = 6.4 Hz). HRMS calcd
for C18H17N3O2 (M+H) 308.1399, found 308.1394.
6.1.8. 2-Aminomethyl-4-phenyl-1H-imidazol hydrochlo-
ride (21).31 To a solution of carbamate 20 (310 mg,
1.0 mmol) in MeOH/HCl (9.9 ml) was added 10 wt%
Pd/C (92 mg, 30 wt%). The mixture was stirred at room
temperature for 4 h under hydrogen atmosphere, then it
was filtered through Celite and concentrated under re-
duced pressure to obtain the title compound (190 mg,
1
6.1.5. 2-(1H-Benz[d]imidazol-2-ylthio)-N-(benzo[d]thia-
zol-2-ylmethyl)acetamide (16). To a solution of chloroac-
etamide 15 (100 mg, 0.42 mmol) in DMF (3 ml) was
added 1H-benz[d]imidazole-2-thiol 10 (93.6 mg,
0.62 mmol) and sodium methoxide (33.7 mg,
76%). H NMR (CD3OD): d 7.70 (2H, d, J = 7.6 Hz),
7.44 (1H, s), 7.37 (2H, dd, J = 8.0 Hz, J = 7.6 Hz),
7.25 (1H, t, J = 7.6 Hz), 4.14 (2H, s). HRMS calcd for
C10H11N3 (M+H) 174.1031, found 174.1027.
0.62 mmol) and the mixture was stirred at room temper-
ature for 5 days. The mixture was diluted with H2O and
CH2Cl2, then extracted with CH2Cl2, washed with H2O,
brine, dried over MgSO4 and concentrated under re-
duced pressure. The resulting residue was purified by
chromatography (CHCl3/MeOH) to obtain the title
compound as a white powder (28.4 mg, 19%). 1H
NMR (DMSO-d6): d 12.62 (1H, br s), 9.31 (1H, t,
J = 5.8 Hz), 7.94 (2H, m), 7.50 (2H, m), 7.39 (2H, m),
7.13 (2H, m), 4.71 (2H, d, J = 6.1 Hz), 4.17 (2H, s).
13C NMR (DMSO-d6): d 170.8, 168.1, 152.7, 149.5,
143.5, 135.5, 134.6, 126.1, 125.0, 122.3, 122.1, 121.7,
121.2, 117.4, 110.4, 41.6, 34.8. MS (FAB+): 355
(M+H). HRMS calcd for C17H14N4OS2 (M+H)
355.0687, found 355.0682. Anal. Calcd for
C17H14N4OS2: C, 57.6; H, 4.0; N, 15.8. Found: C,
57.5; H, 4.0; N, 15.8.
6.1.9. 1-((4-Phenyl-1H-imidazol-2-yl)methyl)-3-(6-(meth-
ylsulfonyl)benzo[d]thiazol-2-yl)urea (24).31 To a solution
of N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)-1H-imid-
azole-1-carboxamide 23 (113 mg, 0.35 mmol) in THF
(4 ml) was added (4-phenyl-1H-imidazol-2-yl)methyl-
amine hydrochloride 21 (95 mg, 0.39 mmol) and N,N0-
diisopropylethylamine (149 ll, 0.88 mmol). The mixture
was stirred at room temperature for 3 days, then it was
poured into brine, extracted with CHCl3, dried over
Na2SO4, and concentrated under reduced pressure.
The resulting residue was purified by chromatography
(CHCl3/MeOH) to obtain the title compound. Recrys-
tallization from MeOH afforded completely pure prod-
uct as a white powder (50 mg, 33%). 1H NMR
(DMSO-d6): d 8.52–8.51 (1H, m), 7.86 (1H, dd,
J = 8.0 Hz, J = 1.6 Hz), 7.80–7.74 (3H, m), 7.55 (1H,
br s), 7.44–7.32 (3H, m), 7.19–7.15 (1H, m), 4.46 (2H,
d, J = 5.4 Hz), 3.22 (3H, s), 3.17 (1H, d, J = 5.4 Hz).
13C NMR (DMSO-d6): d 163.9, 153.9, 152.7, 145.0,
139.7, 134.7, 134.3, 132.0, 128.3, 125.9, 124.5, 124.0,
121.4, 119.6, 112.7, 44.0, 37.4. MS (ESI+): 428
(M+H). HRMS calcd for C19H17N5O3S2 (M+H)
6.1.6. Benzyl 2-amino-2-iminoethylcarbamate hydrochlo-
ride (18).31 To a solution of benzyl cyanomethylcarba-
mate 17 (Aldrich, 12 g, 63 mmol) in MeOH (240 ml)
was added sodium methoxide (0.34 g, 6.3 mmol). The

T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
7333
428.0851, found 428.0847. Anal. Calcd for C19H17N5O3-
S2Æ0.5H2O: C, 52.3; H, 4.2; N, 16.0. Found: C, 52.3; H,
4.3; N, 15.8. Mp: 259 ꢁC.
161.0, 158.5, 154.0, 152.2, 150.3, 123.0, 121.7, 121.5,
118.4, 111.5, 111.2, 103.7, 55.4, 38.0. HRMS calcd for
C17H15N5O2S (M+H) 354.1025, found 354.1019.
6.1.10. N-(Benzo[d]thiazol-2-yl)-2-(3-nitrobenzylidenea-
minoxy)acetamide (6). Purchased from SPECS.
6.1.15. 1-(6-Methoxybenzo[d]thiazol-2-yl)-3-((1H-benz[d]imi-
dazol-2-yl)methyl)urea (32).31 The title compound was
obtained from N-(6-methoxybenzo[d]thiazol-2-yl)-1H-
imidazole-1-carboxamide and (1H-benz[d]imidazol-2-
yl)methanamine hydrochloride according to the similar
6.1.11. (1H-Benz[d]imidazol-2-yl)methyl benzo[d]thiazol-2-
ylcarbamate (28).31 To a solution of N-(benzo[d]thiazol-
2-yl)-1H-imidazole-1-carboxamide (20 mg, 0.082 mmol)
in THF (0.6 ml) was added (1H-benz[d]imidazol-2-
yl)methanol (12 mg, 0.082 mmol). The mixture was
stirred at 40 ꢁC for overnight, then it was poured into
brine, extracted with CHCl3, dried over Na2SO4, and
concentrated under reduced pressure. The resulting
residue was purified by Preparative TLC (hexane/ace-
tone = 1/1) to obtain the title compound as a white pow-
1
procedure used to prepare 24. H NMR (DMSO-d6): d
7.54–7.49 (5H, m), 7.15 (2H, dd, J = 6.1 Hz, 2.6 Hz),
6.95 (1H, dd, J = 8.9 Hz, J = 2.6 Hz), 4.62 (2H, d,
J = 5.6 Hz), 3.78 (3H, s). 13C NMR (DMSO-d6): d
157.8, 155.6, 154.1, 152.2, 143.1, 132.6, 121.6, 120.3,
114.7, 114.2, 104.9, 55.6, 38.0. HRMS calcd for
C17H15N5O2S (M+H) 354.1025, found 354.1019.
1
der (6.8 mg, 26%). H NMR (CD3OD): d 7.82 (1H, d,
6.1.16. 1-(6-Methylbenzo[d]thiazol-2-yl)-3-((1H-benz[d]imi-
dazol-2-yl)methyl)urea (33). The title compound was
obtained from N-(6-methylbenzo[d]thiazol-2-yl)-1H-
imidazole-1-carboxamide and (1H-benz[d]imidazol-2-
yl)methanamine hydrochloride according to the similar
J = 8.4 Hz), 7.67 (1H, d, J = 8.0 Hz), 7.59 (2H, dd,
J = 5.6 Hz, J = 3.2 Hz), 7.39 (1H, dd, J = 8.4 Hz,
J = 8.0 Hz), 7.29–7.25 (3H, m), 5.51 (2H, s). MS
(FAB+): 325 (M+H). HRMS calcd for C16H12N4O2S
(M+H) 325.0759, found 325.0764.
1
procedure used to prepare 24. H NMR (DMSO-d6): d
12.37 (1H, br s), 10.92 (1H, br), 7.67 (1H, s), 7.56–7.47
(4H, m), 7.18–7.15 (3H, m), 4.62 (2H, d, J = 5.3 Hz),
2.38 (3H, s). 13C NMR (DMSO-d6): d 158.9, 154.1,
152.2, 147.0, 143.0, 134.3, 132.0, 131.5, 127.0, 121.9,
121.14, 121.09, 119.3, 118.3, 111.2, 38.0, 20.9. MS
(FAB+): 338 (M+H). HRMS calcd for C17H15N5OS
(M+H) 338.1076, found 338.1080. Anal. Calcd for
C17H15N5OSÆ0.25H2O: C, 59.7; H, 4.6; N, 20.5. Found:
C, 59.4; H, 4.5; N, 20.8.
6.1.12. 1-((1H-Benz[d]imidazol-2-yl)methyl)-3-(benzo[d]thia-
zol-2-yl)urea (29).31 To a solution of N-(benzo[d]thiazol-
2-yl)-1H-imidazole-1-carboxamide (32 mg, 0.13 mmol)
in THF (0.9 ml) was added (1H-benz[d]imidazol-2-
yl)methanamine hydrochloride (29 mg, 0.13 mmol) and
N,N0-diisopropylethylamine (45 ll, 0.26 mmol). The
mixture was stirred at room temperature for 1 h, then
it was poured into brine, extracted with CHCl3. The
generated solid was filtered to obtain the title compound
1
as a white powder (33 mg, 78%). H NMR (CD3OD): d
6.1.17. 1-(6-Fluorobenzo[d]thiazol-2-yl)-3-((1H-benz[d]imi-
dazol-2-yl)methyl)urea (34). The title compound was
obtained from N-(6-fluorobenzo[d]thiazol-2-yl)-1H-
imidazole-1-carboxamide and (1H-benz[d]imidazol-2-
yl)methanamine hydrochloride according to the similar
7.78 (1H, d, J = 7.2 Hz), 7.65 (1H, d, J = 8.8 Hz), 7.53
(2H, br), 7.38 (1H, dd, J = 8.8 Hz, J = 7.2 Hz), 7.25–
7.20 (3H, m), 4.75 (2H, s). MS (ESI+): 324 (M+H).
HRMS calcd for C16H13N5OS (M+H) 324.0919, found
324.0914.
1
procedure used to prepare 24. H NMR (DMSO-d6): d
12.38 (1H, br s), 11.03 (1H, br), 7.81 (1H, dd,
J = 8.8 Hz, J = 2.7 Hz), 7.64 (1H, dd, J = 8.8 Hz,
J = 4.7 Hz), 7.56 (1H, br) 7.51–7.45 (2H, m), 7.24–7.14
(3H, m), 4.62 (2H, d, J = 5.6 Hz). 13C NMR (DMSO-
d6, 125 Hz): d 159.6, 158.1 (d, J = 237 Hz), 153.9,
152.0, 145.7, 142.9, 134.2, 132.6 (d, J = 13 Hz), 121.8,
121.1, 120.6, 118.2, 113.5 (d, J = 24 Hz), 111.1,
107.8 (d, J = 27 Hz), 37.9. MS (FAB+): 342 (M+H).
HRMS calcd for C16H12FN5OS (M+H) 342.0825,
found 342.0828. Anal. Calcd for C16H12FN5OSÆ0.5H2O:
C, 54.9; H, 3.7; N, 20.0. Found: C, 55.3; H, 3.7; N,
20.4.
6.1.13. 1-(4-Methoxybenzo[d]thiazol-2-yl)-3-((1H-benz[d]imi-
dazol-2-yl)methyl)urea (30).31 The title compound was
obtained from N-(4-methoxybenzo[d]thiazol-2-yl)-1H-
imidazole-1-carboxamide and (1H-benz[d]imidazol-2-
yl)methanamine hydrochloride according to the similar
1
procedure used to prepare 24. H NMR (DMSO-d6): d
12.42 (1H, br s), 11.01 (1H, br s), 7.52 (2H, m), 7.44
(1H, d, J = 7.3 Hz), 7.39 (2H, m), 7.17 (1H, m), 7.15
(1H, m), 6.94 (1H, d, J = 7.5 Hz), 4.61 (2H, d,
J = 5.6 Hz), 3.89 (3H, s). 13C NMR (DMF-d6): d
159.3, 155.2, 153.0, 152.5, 140.0, 134.2, 124.2, 122.1,
113.9, 113.8, 108.9, 59.5, 39.1. HRMS calcd for
C17H15N5O2S (M+H) 354.1025, found 354.1024.
6.1.18. 1-(6-Cyanobenzo[d]thiazol-2-yl)-3-((1H-benz[d]imi-
dazol-2-yl)methyl)urea (35).31 The title compound was
obtained from N-(6-cyanobenzo[d]thiazol-2-yl)-1H-imid-
azole-1-carboxamide and (1H-benz[d]imidazol-2-yl)-
methanamine hydrochloride according to the similar
procedure used to prepare 24. 1H NMR (CDCl3/
CD3OD): d 8.59 (1H, br s), 7.73 (1H, d, J = 8.3 Hz),
7.63 (1H, dd, J = 8.3 Hz, J = 1.5 Hz), 7.55 (2H, dd,
J = 6.0 Hz, J = 3.2 Hz), 7.25 (2H, dd, J = 6.0 Hz,
J = 3.2 Hz), 4.72 (2H, s). HRMS calcd for C17H12N6OS
(M+H) 349.0872, found 349.0860.
6.1.14. 1-(5-Methoxybenzo[d]thiazol-2-yl)-3-((1H-benz[d]imi-
dazol-2-yl)methyl)urea (31).31 The title compound was
obtained from N-(5-methoxybenzo[d]thiazol-2-yl)-1H-
imidazole-1-carboxamide and (1H-benz[d]imidazol-2-
yl)methanamine hydrochloride according to the similar
1
procedure used to prepare 24. H NMR (DMSO-d6): d
7.72–7.70 (2H, m), 7.51 (2H, br), 7.18–7.14 (3H, m),
6.81 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 4.62 (2H, d,
J = 5.6 Hz), 3.78 (3H, s). 13C NMR (DMSO-d6): d

7334
T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
6.1.19. 1-(6-(Methylsulfonyl)benzo[d]thiazol-2-yl)-3-((1H-
benz[d]imidazol-2-yl)methyl)urea (36).31
The title
dazol-4-yl)ethylamine hydrochloride according to
the similar procedure used to prepare 24. 1H NMR
(DMSO-d6): d 11.88 (1H, br), 11.14 (1H, br), 8.52 (1H,
d, J = 2.0 Hz), 7.86 (1H, dd, J = 8.5 Hz, J = 2.0 Hz),
7.78 (1H, d, J = 8.5 Hz), 7.57 (1H, br s), 6.90–6.86 (2H,
m), 3.46–3.41 (2H, m), 3.22 (3H, s), 2.73–2.70 (2H, m).
13C NMR (DMSO-d6): d 163.8, 153.6, 152.7, 134.8,
134.4, 132.0, 124.6, 121.5, 119.7, 44.1, 39.3, 27.2. MS
(FAB+): 366 (M+H). HRMS calcd for C14H15N5O3S2
(M+H) 366.0695, found 366.0693. LC–MS: Method A;
tR = 6.53 min, 366 (M+H), purity 100%, Method B;
tR = 7.35 min, 366 (M+H), purity 99.8%.
compound was obtained from N-(6-(methylsulfonyl)
benzo[d]thiazol-2-yl)-1H-imidazole-1-carboxamide and
(1H-benz[d]imidazol-2-yl)methanamine hydrochloride
according to the similar procedure used to prepare 24.
1H NMR (DMSO-d6): d 8.50 (1H, s), 7.85 (1H, d,
J = 8.4 Hz), 7.78 (1H, d, J = 8.4 Hz), 7.57 (2H, br),
7.46 (1H, br), 7.16 (2H, br), 4.64 (2H, d, J = 5.2 Hz),
3.22 (3H, s). 13C NMR (DMSO-d6): d 163.7, 153.9,
152.6, 151.9, 142.9, 134.4, 134.2, 131.9, 124.6, 121.8,
121.5, 121.1, 119.7, 118.2, 111.2, 44.0, 38.0. HRMS
calcd for C17H15N5O3S2 (M+H) 402.0695, found
402.0691.
6.1.24. 1-((4-Phenyl-1H-imidazol-2-yl)methyl)-3-(5-phen-
ylthiazol-2-yl)urea (41). The title compound was
obtained from N-(5-phenylthiazol-2-yl)-1H-imidazole-
1-carboxamide and (4-phenyl-1H-imidazol-2-yl)methyl-
amine hydrochloride according to the similar proce-
dure used to prepare 24. 1H NMR (DMSO-d6): d
12.08 (1H, br s), 10.69 (1H, br s) 7.75–7.73 (3H, m),
7.57–7.55 (3H, m), 7.41–7.32 (4H, m), 7.26 (1H, t,
J = 7.1 Hz), 7.18 (1H, t, J = 7.1 Hz), 7.10 (1H, br), 4.42
(2H, d, J = 5.6 Hz). 13C NMR (DMSO-d6): d 159.0,
153.6, 145.3, 139.6, 134.7, 133.6, 131.8, 129.5, 129.0,
128.3, 127.0, 125.8, 125.2, 124.1, 112.7, 37.3. MS
(FAB+): 376 (M+H). HRMS calcd for C20H17N5OS
376.1232, found 376.1226. LC–MS: Method A;
tR = 9.07 min, 376 (M+H), purity 99.2%, Method B;
tR = 10.97 min, 376 (M+H), purity 98.8%. Mp: 221 ꢁC.
6.1.20. 1-(6-(Methylsulfonyl)benzo[d]thiazol-2-yl)-3-((5-
(pyridin-2-yl)thiophen-2-yl)methyl)urea (37). The title
compound was obtained from N-(6-(methylsulfonyl)-
benzo[d]thiazol-2-yl)-1H-imidazole-1-carboxamide and
(5-(pyridin-2-yl)thiophen-2-yl)methylamine according
1
to the similar procedure used to prepare 24. H NMR
(DMSO-d6): d 8.54 (1H, d, J = 1.7 Hz), 8.48 (1H, dd,
J = 4.9 Hz, J = 1.7 Hz), 7.88–7.86 (2H, m), 7.82–7.79
(2H, m), 7.64 (1H, dd, J = 3.7 Hz, J = 1.2 Hz), 7.47
(1H, br), 7.24 (1H, m), 7.06 (1H, d, J = 3.7 Hz), 4.55
(2H, d, J = 5.8 Hz), 3.23 (3H, s). 13C NMR (DMSO-
d6): d 163.9, 153.8, 152.8, 151.8, 149.3, 144.8, 143.4,
137.0, 134.4, 132.1, 126.6, 124.8, 124.6, 122.2, 121.5,
119.8, 118.3, 44.1, 38.5. MS (FAB+): 445 (M+H).
HRMS calcd for C19H16N4O3S3 445.0463, found
445.0461. Anal. Calcd for C19H16N4O3S3 ꢁ H2O: C,
49.3; H, 3.9; N, 12.1. Found: C, 49.1; H, 3.5; N, 12.3.
6.1.25. 1-((4-Phenyl-1H-imidazol-2-yl)methyl)-3-(4-phen-
ylthiazol-2-yl)urea (42). The title compound was
obtained from N-(4-phenylthiazol-2-yl)-1H-imidazole-
1-carboxamide and (4-phenyl-1H-imidazol-2-yl)methyl-
amine hydrochloride according to the similar procedure
6.1.21. 1-(6-(Methylsulfonyl)benzo[d]thiazol-2-yl)-3-(pyri-
din-3-yl)methyl)urea (38).31 The title compound was
obtained from N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)-
1H-imidazole-1-carboxamide and (pyridin-3-yl)methyl-
amine according to the similar procedure used to
prepare 24. 1H NMR (CDCl3/CD3OD): d 8.52–8.48
(2H, m), 8.35 (1H, d, J = 5.1 Hz), 7.90 (1H, m), 7.79–
7.77 (2H, m), 7.36 (1H, m), 7.36 (1H, m), 4.53 (2H, d,
J = 6.6 Hz), 3.11 (3H, s). 13C NMR (DMSO-d6): d163.9,
154.1, 152.6, 148.8, 148.3, 135.2, 134.9, 134.5, 132.0,
124.7, 123.6, 121.6, 119.8, 44.1, 40.8. HRMS calcd for
C15H14N4O3S2 (M+H) 363.0586, found 363.0582.
1
used to prepare 24. H NMR (DMSO-d6): d 12.08 (1H,
br s), 10.78 (1H, br s), 7.87 (2H, d, J = 7.3 Hz), 7.77 (2H,
d, J = 7.3 Hz), 7.55 (1H, s), 7.47 (1H, s), 7.42–7.12 (6H,
m), 4.42 (2H, d, J = 5.4 Hz). 13C NMR (DMSO-d6): d
159.7, 153.8, 148.4, 145.3, 139.6, 134.7, 134.3, 128.5,
128.3, 127.5, 125.8, 125.4, 124.1, 112.7, 106.5, 37.2.
MS (FAB+): 376 (M+H). HRMS calcd for
C20H17N5OS (M+H) 376.1232, found 376.1237. LC–
MS: Method A; tR = 9.18 min, 376 (M+H), purity
99.1%, Method B; tR = 11.23 min, 376 (M+H), purity
99.3%. Mp: 189 ꢁC.
6.1.22. 1-(6-(Methylsulfonyl)benzo[d]thiazol-2-yl)-3-(thio-
phen-2-yl)methyl)urea (39).31 The title compound was
obtained from N-(6-(methylsulfonyl)benzo[d]thiazol-2-
6.1.26. 1-((4-Phenyl-1H-imidazol-2-yl)methyl)-3-(5-(phe-
nylthio)thiazol-2-yl)urea (43). The title compound was
obtained from N-(5-(phenylthio)thiazol-2-yl)-1H-imid-
azole-1-carboxamide and (4-phenyl-1H-imidazol-2-
yl)methylamine hydrochloride according to the similar
yl)-1H-imidazole-1-carboxamide
yl)methylamine according to the similar procedure used
and
(thiophen-2-
1
to prepare 24. H NMR (CDCl3/CD3OD): d 8.36 (1H,
1
s), 7.90 (1H, m), 7.79–7.77 (1H, m), 7.27 (1H, d,
J = 4.9 Hz), 7.05 (1H, m), 6.98 (1H, m), 4.68 (2H, s),
3.14 (3H, s). 13C NMR (DMSO-d6): d 163.9, 153.8,
152.7, 142.1, 134.5, 132.0, 126.9, 125.8, 125.3, 124.7,
121.6, 119.8, 44.1, 38.1. HRMS calcd for C14H13N3O3S3
(M+H) 368.0197, found 368.0190.
procedure used to prepare 24. H NMR (DMSO-d6): d
12.05 (1H, br), 10.97 (1H, br), 7.75 (2H, d,
J = 7.6 Hz), 7.62 (1H, s), 7.53 (1H, s), 7.35–7.13 (8H,
m), 4.40 (2H, d, J = 5.4 Hz). 13C NMR (DMSO-d6): d
163.9, 153.6, 146.2, 145.1, 139.6, 137.5, 134.7, 129.2,
128.3, 126.3, 126.2, 125.8, 124.0, 115.1, 112.6, 37.2.
MS (FAB+): 408 (M+H). HRMS calcd for
C20H17N5OS2 (M+H) 408.0953, found 408.0954. LC–
MS: Method A; tR = 9.64 min, 408 (M+H), purity
99.0%, Method B; tR = 11.77 min, 408 (M+H), purity
98.5%. Mp: 210 ꢁC.
6.1.23. 1-(2-(1H-Imidazol-4-yl)ethyl)-3-(6-(methylsulfo-
nyl)benzo[d]thiazol-2-yl)urea (40). The title compound
was obtained from N-(6-(methylsulfonyl)benzo[d]thia-
zol-2-yl)-1H-imidazole-1-carboxamide and 2-(1H-imi-

T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
7335
6.1.27. 1-((4-Phenyl-1H-imidazol-2-yl)methyl)-3-(5-(pyri-
din-2-ylthio)thiazol-2-yl)urea (44). The title compound
was obtained from N-(5-(pyridin-2-ylthio)thiazol-2-yl)-
1H-imidazole-1-carboxamide and (4-phenyl-1H-imi-
dazol-2-yl)methylamine hydrochloride according to the
similar procedure used to prepare 24. 1H NMR
(DMSO-d6): d 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): d 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.
Calcd for C19H16N6OS2 ꢁ 0:25H2O: C, 55.3; H, 4.0; N,
20.4. Found: C, 55.4; H, 4.0; N, 20.6. Mp: 219 ꢁC.
azole-1-carboxamide (30 mg, 0.10 mmol) in THF (1 ml)
was added (4-(4-bromophenyl)-1H-imidazol-2-yl)methyl-
amine hydrochloride (36 mg, 0.11 mmol) and N,N0-
diisopropylethylamine (37 ll, 0.22 mmol). The mixture
was stirred at room temperature for overnight, 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. H
NMR (CDCl3/CD3OD): d 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): d
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. Calcd for C19H15BrN6OS2: C, 46.8; H, 3.1; N,
17.2. Found: C, 46.6; H, 3.2; N, 16.8. Mp: 246 ꢁC.
6.1.28. 4-(2-(3-((4-Phenyl-1H-imidazol-2-yl)methyl)ure-
ido)thiazol-5-ylthio)benzamide (45). To a solution of
4-(2-(3-((4-phenyl-1H-imidazol-2-yl)methyl)ureido)thiazol-
5-ylthio)benzoic acid 46 (24 mg, 0.053 mmol) in DMF
(0.6 ml) was added WSC. HCl (20 mg, 0.11 mmol),
1-hydroxybenzotriazole (14 mg, 0.11 mmol,) and 28%
NH3aq (9.6 ll, 0.16 mmol) under ice cooling. The mix-
ture was stirred at 0 ꢁC for 6 h, then it was poured into
water, extracted with CHCl3, dried over Na2SO4, and
concentrated under reduced pressure. The resulting res-
idue was purified by Preparative TLC (CHCl3/MeOH/
NH3aq = 5/1/0.1) to obtain the title compound as a
white powder (12 mg, 51%). 1H NMR (CDCl3/CD3OD):
6.1.31. 1-((4-(3-Bromophenyl)-1H-imidazol-2-yl)methyl)-
3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea (48). The title
compound was obtained from N-(5-(pyridin-2-ylthio)thia-
zol-2-yl)-1H-imidazole-1-carboxamide and (4-(3-bromo-
phenyl)-1H-imidazol-2-yl)methylamine hydrochloride
according to the similar procedure used to prepare 24.
1H NMR (CDCl3/CD3OD): d 8.39–8.37 (1H, m), 7.82
(1H, br s), 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): d
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 C19H15BrN6OS2 (M+H) 487.0010, found
487.019. Anal. Calcd for C19H15BrN6OS2 ꢁ 0:1H2O: C,
46.7; H, 3.1; N, 17.2. Found: C, 46.3; H, 3.3; N, 17.6.
Mp: 233 ꢁC.
d
7.76–7.71 (2H, m), 7.64 (2H, dd, J = 8.6 Hz,
J = 1.2 Hz), 7.52 (1H, s), 7.40–7.36 (2H, m), 7.28–7.18
(4H, m), 4.50 (2H, s). MS (FAB+): 451 (M+H). HRMS
calcd for C21H18N6O2S2 (M+H) 451.1011, found
451.1003. LC–MS: Method A; tR = 8.03 min, 451
(M+H), purity 100%, Method B; tR = 9.38 min, 451
(M+H), purity 100%. Mp: 223 ꢁC.
6.1.29. 4-(2-(3-((4-Phenyl-1H-imidazol-2-yl)methyl)ure-
ido)thiazol-5-ylthio)benzoic acid (46). To a solution of
1-((4-phenyl-1H-imidazol-2-yl)methyl)-3-(5-(4-methoxy-
carbonylphenylthio)thiazol-2-yl)urea (47 mg, 0.10 mmol)
in DMF (1.5 ml) was added 5 N-NaOHaq (200 ll,
1.0 mmol). The mixture was stirred at room temperature
for 4 h, then it was acidified by HCl/EtOAc and concen-
trated under reduced pressure. The resulting solid was
filtered and the filter cake was washed with H2O to ob-
tain the title compound as a white solid (29 mg, 64%).
1H NMR(DMSO-d6): d 7.85 (2H, dd, J = 8.3 Hz,
J = 1.2 Hz), 7.73 (2H, d, J = 8.0 Hz), 7.67 (1H, m),
7.49–7.48 (2H, m), 7.35–7.31 (2H, m), 7.21–7.15 (4H,
m), 4.40 (2H, d, J = 5.4 Hz). 13C NMR (CD3OD/
CDCl3): d 171.6, 165.3, 155.1, 146.2, 146.1, 143.1,
137.9, 132.4, 132.0, 130.4, 129.0, 127.4, 125.9, 125.0,
116.9, 116.6, 37.3. MS (ESI+): 452 (M+H). HRMS
calcd for C21H17N5O3S2 (M+H) 452.0851, found
452.0849. LC–MS: Method A; tR = 8.71 min, 452
(M+H), purity 99.2%, Method B; tR = 8.07 min, 452
(M+H), purity 98.3%.
6.1.32. 1-((4-(3,4-Dichlorophenyl)-1H-imidazol-2-yl)methyl)-
3-(5-(pyridin-2-ylthio)thiazol-2-yl) urea (49). The title
compound was obtained from N-(5-(pyridin-2-ylthio)thia-
zol-2-yl)-1H-imidazole-1-carboxamide and (4-(3,4-dichlor-
ophenyl)-1H-imidazol-2-yl)methylamine hydrochloride
according to the similar procedure used to prepare 24.
1H NMR (CDCl3/CD3OD): d 8.39–8.37 (1H, m), 7.77
(1H, m), 7.57–7.43 (4H, m), 7.24 (1H, s), 7.07 (1H, dd,
J = 7.3 Hz, J = 4.9 Hz), 7.00 (1H, d, J = 8.1 Hz), 4.46
(2H, s). 13C NMR (DMSO-d6, 125 MHz): d 164.8,
160.1, 154.1, 149.4, 146.8, 146.0, 137.7, 137.3, 135.6,
131.3, 130.7, 127.8, 125.6, 124.2, 120.7, 119.8, 114.5,
112.8, 37.3. MS (FAB+): 477 (M+H). HRMS calcd
for C19H14Cl2N6OS2 (M+H) 477.0126, found
477.0126. LC–MS: Method A; tR = 9.38 min, 477
(M+H), purity 97.2%, Method B; tR = 11.65 min, 477
(M+H), purity 97.9%. Mp: 246 ꢁC.
Acknowledgments
6.1.30. 1-((4-(4-Bromophenyl)-1H-imidazol-2-yl)methyl)-
3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea (47). To
solution of N-(5-(pyridin-2-ylthio)thiazol-2-yl)-1H-imid-
a
We are especially thankful to Dr. Keiichi Ajito Meiji
Seika Kaisha Ltd. for their stimulating discussion and

7336
T. Ozawa et al. / Bioorg. Med. Chem. 15 (2007) 7325–7336
DeWolf, W. E.; 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. Bioorg. Med.
Chem. Lett. 2001, 11(17), 2241.
strong encouragement of this work. We thank Miss Shi-
geko Miki, Mrs. Takako Miyara, and Mrs. Kazue Sasaki
of Meiji Seika Kaisha, Ltd. for their kindness and
assistance with mass spectrometry analysis. We also
thank Mr. Seigo Sato for his assistance with NMR study.
22. Payne, D. J.; Miller, W. H.; Berry, V.; Brosky, J.; Burgess,
W. J.; Chen, E.; DeWolf, W. E., Jr.; Fosberry, A. P.;
Greenwood, R.; Head, M. S.; Heerding, D. A.; Janson, C.
A.; Jaworski, D. D.; Keller, P. M.; Manley, P. J.; Moore,
T. D.; Newlander, K. A.; Pearson, S.; Polizzi, B. J.; Qiu,
X.; Rittenhouse, S. F.; Slater-Radosti, C.; Salyers, K. L.;
Seefeld, M. A.; Smyth, M. G.; Takata, D. T.; Uzinskas, I.
N.; Vaidya, K.; Wallis, N. G.; Winram, S. B.; Yuan, C. C.;
Huffman, W. F. Antimicrob. Agents Chemother. 2002,
46(10), 3118.
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