Synthesis and SAR study of new thiazole derivatives as vascular adhesion protein-1 (VAP-1) inhibitors for the treatment of diabetic macular edema: Part 2

Article abstract of DOI:10.1016/j.bmc.2013.02.048

Novel thiazole derivatives were synthesized and evaluated as vascular adhesion protein-1 (VAP-1) inhibitors. Although our previous compound 1 showed potent VAP-1 inhibitory activity, the activity differed between humans and rats. This issue was overcome by a hybrid design using human VAP-1 specific inhibitor 2, which was found by high-throughput screening (HTS), a docking study of a human VAP-1 homology model, and an analysis of sequence information for humans and rats. As a result, we identified compound 35c, which showed strong VAP-1 inhibitory activity (human IC₅₀ of 20 nM; rat IC₅₀ of 72 nM) and significant inhibitory effects in the ex vivo test.

Full text of DOI:10.1016/j.bmc.2013.02.048

Accepted Manuscript
Synthesis and SAR study of new thiazole derivatives as vascular adhesion pro‐
tein-1 (VAP-1) inhibitors for the treatment of diabetic macular edema: part 2
Takayuki Inoue, Masataka Morita, Takashi Tojo, Akira Nagashima, Ayako
Moritomo, Keisuke Imai, Hiroshi Miyake
PII:
S0968-0896(13)00193-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.02.048
BMC 10643
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 February 2013
27 February 2013
28 February 2013
Please cite this article as: Inoue, T., Morita, M., Tojo, T., Nagashima, A., Moritomo, A., Imai, K., Miyake, H.,
Synthesis and SAR study of new thiazole derivatives as vascular adhesion protein-1 (VAP-1) inhibitors for the
treatment of diabetic macular edema: part 2, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/
1
0.1016/j.bmc.2013.02.048
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Synthesis and SAR study of new thiazole derivatives as vascular adhesion protein-1 (VAP-1)
inhibitors for the treatment of diabetic macular edema: part 2
a *
a
a
a
a
b
Takayuki Inoue , Masataka Morita , Takashi Tojo , Akira Nagashima , Ayako Moritomo , Keisuke Imai
c
and Hiroshi Miyake
a
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b
Astellas Business Service Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
c
Astellas Research Technology Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
*
Corresponding author.
Tel.: +81-29-829-6227;
fax: +81-29-854-1519;
e-mail: takayuki.inoue@astellas.com

Abstract
Novel thiazole derivatives were synthesized and evaluated as vascular adhesion protein-1 (VAP-1)
inhibitors. Although our previous compound 1 showed potent VAP-1 inhibitory activity, the activity
differed between humans and rats. This issue was overcome by a hybrid design using human VAP-1
specific inhibitor 2, which was found by high-throughput screening (HTS), a docking study of a human
VAP-1 homology model, and an analysis of sequence information for humans and rats. As a result, we
identified compound 35c, which showed strong VAP-1 inhibitory activity (human IC of 20 nM; rat IC
50
5
0
of 72 nM) and significant inhibitory effects in the ex vivo test.
Keywords
VAP-1; inhibitor; guanidine; species difference; hybrid; homology model; sequence information

1
. Introduction
Vascular adhesion protein-1 (VAP-1) belongs to the semicarbazide-sensitive amine oxidases (SSAOs).
Two types of VAP-1 have been identified—a membrane-binding type present in the vascular endothelium
and a soluble type present in the serum—both with different functions, as the former adheres to white blood
1
cells and lymphocytes and is involved in inflammation, while the latter detoxifies amines in vivo by its
amine oxidase activity.
Expression of VAP-1 increases markedly in vascular endothelial cells within an inflammatory area,
while VAP-1 itself is responsible for the metabolism of primary amines (e.g., methylamine and
2
,3
aminoacetone) and leukocyte trafficking. VAP-1 catalyzes the oxidative deamination of primary amines
to produce corresponding aldehydes with the concomitant production of ammonia and hydrogen peroxide.
The reaction products are known to be cytotoxic and might therefore be associated with a number of
4
5
,6
vasculopathies. Consistent with these previous findings, an increase in plasma and/or
membrane-associated VAP-1 derived from adipocytes and vascular tissue has been observed in many
7
inflammation-associated diseases. VAP-1 was recently reported to be expressed on the retinal vascular
8
endothelium, playing an important role in the recruitment of leukocytes on retinal vessels. These events
are thought to mark a breakdown in the blood-retinal barrier, leading to the edema seen in diabetic
retinopathy.
The above findings suggest that toxic products and leukocyte adhesion activity might be responsible for
the vascular injury seen in diabetic retinopathy, particularly diabetic macular edema. Diabetic retinopathy
stands as the leading cause of blindness in humans, while diabetic macular edema results in serious visual
dysfunction. The macular area is the most important region of the retina and has a central role in vision.
Therefore, once edema occurs here, even minimal disruption can cause severe visual loss. Further,
untreated edema may lead to irreversible changes in the macular region, which may exacerbate the
retinopathy.
We recently reported a novel VAP-1 inhibitor, compound 1, which was found by optimizing a
9
high-throughput screening (HTS) hit compound. However, compound 1 showed approximately 20-fold
lower VAP-1 inhibitory activity in humans than in rats (human IC of 230 nM; rat IC of 14 nM). We
5
0
50

have also identified another HTS hit compound 2, which showed specific human VAP-1 inhibitory activity
and no inhibitory effect against rat VAP-1. Because compound 2 has no guanidyl group, which is important
for the inhibitory action of compound 1, we sought a new pharmacophore site using a docking study of 2
and human VAP-1. Further, we also considered whether or not human VAP-1 inhibitory activity might be
improved by a molecular hybridization approach using the pharmacophores of 1 and 2. As expected, our
initial designed compound, 35a, demonstrated improved human VAP-1 inhibitory activity over 1. However,
a decrease in rat VAP-1 inhibitory activity was also observed. To enhance the rat VAP-1 inhibitory activity,
we used a VAP-1 human homology model and sequence information for humans and rats. These efforts
helped us to identify compound 35c, which showed no species difference in VAP-1 inhibitory activity (Fig.
1
).
Here, we describe efforts to minimize species differences in VAP-1 inhibitory activity. We also discuss
the synthesis and structure-activity relationships (SARs) of a series of novel 5-substituted thiazole
derivatives.
2
. Chemistry
Compound 13 was synthesized as illustrated in Scheme 1. The 3-amino-1H-pyrazole derivative 5 was
prepared from the corresponding ketone 3 by bromination, substitution with sodium cyanide and
cyclization with hydrazine in refluxing ethanol. Acetylation of 5, followed by alkylation of the pyrazole
ring afforded a mixture of 8a and 8b (8a:8b = 2:3). A regioisomer mixture of 8a and 8b was converted to
9
a and 9b without separation. The deprotection of the tert-butoxycarbonyl (Boc) group of a mixture of 9a
and 9b, followed by separation using column chromatography gave the desired product 10a. Compound
0a was coupled with N,N'-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (11), followed by
1
deprotection of the Boc groups to provide the desired compound 13.
The syntheses of guanidine derivatives 35a‒ e were accomplished as outlined in Schemes 2 and 3.
Treatment of 4-(methylsulfanyl)benzaldehyde (14) with (acetylamino)acetic acid and acetic anhydride in
the presence of sodium acetate under reflux gave

2
-methyl-4-[4-(methylsulfanyl)benzylidene]-1,3-oxazol-5(4H)-one (15). The 2-oxoester derivative 17 was
prepared from compound 15 by hydrolysis with 4 M HCl and esterification with iodomethane (MeI) in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Bromination of 17 and subsequent cyclization of
1
2
1
8 with thiourea provided intermediate 19a. Compound 21, which was prepared from the esterification of
0, was treated with diethyl oxalate and sodium methoxide in MeOH at 65 ˚C, followed by treatment with
5% aqueous H SO under reflux to provide a 2-oxocarboxylic acid derivative. This compound was
2
4
esterified with concentrated H SO in refluxing EtOH to give the 2-oxoester derivative 27a. Treatment of
2
4
2
2
2 with N,O-dimethylhydroxylamine hydrochloride afforded N-methoxy-N-methylcarboxamide derivative
3. Reduction of compound 23 with diisobutylaluminum hydride followed by a Wittig reaction with
phosphorane compound 24 in refluxing THF gave phenylacrylate derivative 25. Hydrogenation of 25
furnished the ester derivative 26, which was converted into the 2-oxoester derivative 27b by the same
method as that for the synthesis of 27a. Bromination of 27a‒ c with CuBr in refluxing AcOEt-CHCl and
2
3
subsequent cyclization of 28a‒ c with thiourea provided compounds 19b‒ d. Acetylation of 19a‒ d and
subsequent reduction with lithium aluminum hydride, followed by oxidation with manganese (IV) oxide
gave the aldehyde products 30a‒ d. A Wittig reaction of 30a‒ d with
(4-nitrobenzyl)-triphenylphosphonium bromide gave the desired 7H-pyrrolo[2,3-d]pyrimidine derivatives
®
3
2a,c,e,g. Treatment of 32a,c,e with Oxone afforded the corresponding sulfone derivatives 32b,d,f.
Hydrogenation of 32b,c,d,f,g furnished the aniline derivatives 33a‒ e. Compounds 33a‒ e were coupled
with 11, and subsequent deprotection of the Boc groups gave the desired compounds 35a‒ e.
Compound 49 was prepared as shown in Scheme 4. Cyclization of commercially available ethyl
4
-chloro-3-oxobutanoate (36) with N’-((E)-ethanoyl)carbamimidothioic acid (37) gave the chloromethyl
product 38. Compound 38 was treated with triphenylphosphine and aldehyde 39 to afford the styryl
derivative 40. Hydrogenation of 40 furnished the aniline derivative 41, and subsequent reaction of 41 with
bis(tert-butyl)dicarbonate gave the Boc-protected derivative 42. Hydrolysis of compound 42 with 1 M
NaOH in refluxing EtOH, and subsequent acetylation gave the corresponding carboxylic acid 43.
Condensation of the carboxylic acid 43 using N,O-dimethylhydroxylamine hydrochloride, followed by
reduction with lithium aluminum hydride gave the aldehyde 44. A Wittig reaction of compound 44 with

[
4-(methylsulfonyl)benzyl]triphenylphosphonium chloride and subsequent hydrogenation in the presence of
0% palladium on carbon gave compound 47. Deprotection of the Boc group gave the aniline 48, which
1
was converted to the desired molecule 49 by the same method as that for synthesis of 13.
Compound 60 was synthesized as shown in Scheme 5. Compound 56 was synthesized from ethyl ester
5
0 by the same method as that for the synthesis of 32c. A methoxycarbonyl group was introduced to the
iodo analogue 56 using 1,3-bis(diphenylphosphino)propane and palladium (II) acetate under a carbon
monoxide atmosphere in MeOH/DMF to give the methyl ester 57. Hydrogenation of 57 followed by
coupling of 58 with 11 and subsequent deprotection of the Boc groups afforded the desired product 60.
Syntheses of the sulfonamide derivatives 64a,b were carried out as detailed in Scheme 6. Compound 32g
was treated with chlorosulfonic acid to afford the benzenesulfonyl chloride derivative 61. The
sulfonylamidation of 61 with the appropriate amines, followed by hydrogenation afforded the desired
products 62a,b. Compounds 64a,b were synthesized from the aniline derivatives 62a,b by the same method
as that for the synthesis of 13.
3
. Results and discussion
We initially performed docking studies of compound 2 and the human VAP-1 model using a previously
9
-14
reported method, with results showing that the amide oxygen formed a hydrogen bond with the Thr212
backbone amide hydrogen, the methanesulfonyl moiety formed a proton-π interaction with Tyr394, and the
4
-fluoro benzene moiety formed a π-proton interaction with Leu469 (Fig. 2A). These findings
demonstrated that the docking mode of compound 2 is characteristically different from that of compound 1
Fig. 2B). Therefore, to improve the human VAP-1 inhibitory activity of compound 1, we considered
(
whether or not the novel pharmacophore of compound 2 could be incorporated into compound 1.
On evaluation of hybrid-type compounds 35a and 13, compound 35a showed a 7-fold improvement in
human VAP-1 inhibitory activity but a 250-fold loss in rat inhibitory potency compared to compound 1
(Table 1). Compound 13 showed reduced human and rat VAP-1 inhibitory activities compared to 35a.
Consequently, the thiazole ring showed better VAP-1 inhibitory activity than the pyrazole ring, because the
9
,15
sulfur atom in the thiazole ring contributed to the requisite S-O interaction for the enzyme. We analyzed

the reason for the attenuation of the rat VAP-1 inhibitory activity of 35a using sequence information for
humans and rats.
We investigated the sequence homology between human and rat VAP-1 enzymes, finding two residual
differences (F173/T, L447/F) around the ligand binding pocket of the VAP-1 models. From the docking
mode of 35a and human VAP-1, it became clear that the methanesulfonylphenyl moiety of 35a existed
beside Leu447 (Fig. 3). The corresponding residue in rat was Phe447, which is more sterically bulky than
Leu. We supposed that the methanesulfonyl moiety of 35a could not exist at this optimum position in rats
due to the large steric repulsion. However, this steric repulsion was comparatively small in humans. We
therefore reduced the steric repulsion in rats by enhancing the flexibility of the phenyl moiety. Converting
the phenyl group (35a) into a benzyl group (35c) caused a 50-fold increase in rat VAP-1 inhibitory activity
(Table 2). In addition, human VAP-1 inhibitory activity also improved approximately 2-fold. These results
strongly support our hypothesis regarding steric hindrance and confirm the utility of this approach using
sequence information for humans and rats.
The results for compound 35c derivatives are shown in Table 2. Unsubstituted benzyl derivative 35e
showed 10-fold-less potent human VAP-1 inhibitory activity compared to 35c, clarifying the importance of
the methanesulfonyl group. In addition, the benzyl was found to be the most suitable unit for the linker, as
phenethyl group (49) replacement of the benzyl group reduced the human activity by 8-fold. Substitution of
the methanesulfonyl group at the 3-position (35d) resulted in 2-fold lower activity than 35c. Human VAP-1
inhibitory activity was reduced 50-fold by converting sulfonyl into sulfinyl (35b), and methoxycarbonyl
group (60) replacement of the sulfone reduced the human activity more than 10-fold. Sulfonylamide
derivatives (64a, 64b) as sulfonyl group bioisosteres did not improve VAP-1 inhibitory activity of 35c.
Consequently, the 4-position methanesulfonyl group was found to be the most suitable substituent for the
benzyl group and important for VAP-1 inhibitory activity.
Compound 35c was docked to the human VAP-1 model (Fig. 4). The human VAP-1 binding mode of
3
5c was found to be similar to that of 35a even if an extra methylene linker was inserted. In addition to its
solid interaction of the guanidine, the sulfonyl moiety of 35c formed tight hydrogen bonds with Leu447 and
Asp446, the sulfonylphenyl moiety formed a π-proton interaction with Leu447, and the amide nitrogen

formed a proton-π interaction with Tyr176. These results suggest that the reason for the improved human
VAP-1 inhibitory activity of 35c compared to compound 1 was the two strong hydrogen bonds and two
π-proton interactions between the methanesulfonylphenyl moiety and enzyme.
The inhibitory properties of 35c were further evaluated against other amine oxidases (AOs) such as
human/rat diamine oxidase (DAO) and human monoamine oxidase (MAO)-B with a radiochemical-enzyme
assay (Table 3). Compound 35c showed more than 1400-fold selectivity for VAP-1 inhibitory activity over
other AOs. We also examined the inhibitory effect of 35c on plasma VAP-1 activity in streptozotocin
9
(
STZ)-induced diabetic rats (Fig. 5). Sufficient inhibitory activity at doses ≥ 0.32 mg/kg after
subcutaneous (sc) administration of 0.32‒ 3.2 mg/kg was observed. These results suggest that 35c might
show promise for use in treating diabetic macular edema.
4
. Conclusion
Here, we reported the SARs for VAP-1 inhibition of a series of thiazole derivatives and related
analogues. First, to enhance the human VAP-1 inhibitory activity of compound 1, we considered a hybrid
design based on the human VAP-1 docking model using compound 1 and HTS hit 2. The hybrid compound
3
5a showed 7-fold improvement in human VAP-1 inhibitory activity compared to 1. Next, to enhance the
rat VAP-1 inhibitory activity of compound 35a, we considered a design based on sequence information for
humans and rats. Consequently, we succeeded in finding compound 35c (human IC of 20 nM; rat IC of
5
0
50
7
2 nM), which proved to be highly potent, selective, and effective in the ex vivo test. Further studies to
examine the possibility of 35c as a candidate for clinical investigation are underway.
5
. Experimental
.1. Chemistry
5
1
H NMR spectra were measured with a JEOL EX400 or GX500 spectrometer; chemical shifts are
expressed in δ units using tetramethylsilane as the standard (in NMR description: s, singlet; d, doublet; t,
triplet; q, quartet, m, multiplet, and br, broad peak). Mass spectra were recorded with a Hitachi M-80 or
JEOL JMS-DX300 spectrometer. Silica gel column chromatography was performed using a Wakogel

C-200 or Merck Silica gel 60. Unless otherwise noted, all commercial reagents and solvents were used
without further purification.
5
.1.1. 3-[4-(Methylsulfanyl)phenyl]-3-oxopropanenitrile (4).
Compound 3 (5.2 g) was dissolved in AcOH (52 mL), and then 90% pyridinium bromide perbromide (11.1
g) and 30% HBr/AcOH (5.2 mL) were added to the solution at 0 ˚C. The reaction mixture was stirred at
room temperature for an hour and then poured into H O and extracted with AcOEt. The organic layer was
2
washed with saturated aqueous NaHCO and brine, dried over anhydrous MgSO , and concentrated in
3
4
vacuo.
A solution of sodium cyanide (4.6 g) in H O (39 mL) was added dropwise to the suspension of the
2
residual yellow solid (8.2 g) in EtOH (52 mL) at 0 ˚C over 10 min. The reaction mixture was stirred at
room temperature for 4 h and then poured into H O. The mixture was neutralized with 1 M HCl and
2
extracted with AcOEt (twice). The combined organic layer was washed with brine, dried over anhydrous
i
MgSO , and concentrated in vacuo. The residual solid was recrystallized from AcOEt/ Pr O to give 4 (2.6 g,
4
2
1
4
8
3%) as a brown solid. Mp 97‒ 97.5 ˚C; H NMR (DMSO-d ) δ 2.55 (3H, s), 4.71 (2H, s), 7.40 (2H, d, J =
6
-
.5 Hz), 7.85 (2H, d, J = 8.5 Hz); FAB MS m/e (M-H) 190.
5
.1.2. 3-[4-(Methylsulfanyl)phenyl]-1H-pyrazol-5-amine (5)
To a solution of 4 (2.0 g) in EtOH (10 mL) and H O (10 mL) were added hydrazine hydrate (0.66 mL) and
2
concentrated HCl (0.2 mL), and the reaction mixture was refluxed for 2.5 h. After cooling to room
temperature, the mixture was poured into AcOEt/H O. The mixture was made basic with saturated aqueous
2
NaHCO and extracted. The organic layer was dried over anhydrous MgSO and concentrated in vacuo.
3
4
The resulting solid was washed with EtOH/Et O to give 5 (1.7 g, 77%) as a pale brown solid. Mp 164‒ 165
2
1
˚
C; H NMR (DMSO-d ) δ 2.48 (3H, s), 5.73 (1H, br s), 7.25 (2H, d, J = 8.5 Hz), 7.58 (2H, d, J = 8.5 Hz);
6
+
FAB MS m/e (M+H) 206.
5
.1.3. N-{3-[4-(Methylsulfanyl)phenyl]-1H-pyrazol-5-yl}acetamide (6).
Compound 5 (1.6 g) was dissolved in pyridine (16 mL) under a nitrogen atmosphere, and then Ac O (1.5
2

mL) was added to the solution at 0 ˚C. The reaction mixture was stirred at 0 ˚C for 30 min and concentrated
in vacuo. The residue was suspended in EtOH (30 mL), and then 1 M NaOH (7.8 mL) was added. The
mixture was stirred at room temperature for an hour, and the precipitate was collected. The solid was
1
washed with H O and EtOH to give 6 (1.4 g, 73%) as an off-white solid. Mp 231.5‒ 232 ˚C; H NMR
2
(
DMSO-d ) δ 2.02 (3H, s), 2.50 (3H, s), 6.84 (1H, br s), 7.31 (2H, d, J = 8.5 Hz), 7.65 (2H, d, J = 8.5 Hz),
6
+
1
0.40 (1H, br s), 12.75 (1H, br s); FAB MS m/e (M+H) 248.
5
.1.4. A mixture of tert-butyl
[
4-(2-{3-acetamido-5-[4-(methylsulfanyl)phenyl]-1H-pyrazol-1-yl}ethyl)phenyl]carbamate (8a) and
tert-butyl [4-(2-{5-acetamido-3-[4-(methylsulfanyl)phenyl]-1H-pyrazol-1-yl}ethyl)phenyl]carbamate
8b).
Compound 6 (700 mg) was dissolved in DMF (14 mL) under a nitrogen atmosphere. K CO (469 mg) and
(
2
3
tert-butyl [4-(2-iodoethyl)phenyl]carbamate (7) (1.1 g) were then added to the solution at 0 ˚C. The
reaction mixture was stirred at 55 ˚C for 18 h. After cooling to room temperature, the mixture was poured
into H O and extracted with AcOEt (twice). The combined organic layer was washed with H O and brine,
2
2
dried over anhydrous MgSO , and concentrated in vacuo. The residual solid was washed with
4
MeOH/CHCl , and the filtrate was concentrated in vacuo. The residue was purified by flash column
3
chromatography over silica gel with hexane/AcOEt (3:1) as an eluent to give a mixture of 8a and 8b (696
1
mg, 53%; 8a:8b=2:3) as a brown oil. H NMR (DMSO-d ) δ 1.49 (9H, s), 2.01 (3H×2/5, s), 2.07 (3H×3/5,
6
s), 2.50 (3H, s), 2.91‒ 3.03 (2H, m), 4.07‒ 4.23 (2H, m), 6.49 (1H×2/5, s), 6.60 (1H×3/5, s), 6.86 (2H×2/5,
d, J=8.5Hz), 7.06‒ 7.14 (2H, m), 7.23‒ 7.29 (2H×8/5, m), 7.36 (2H×2/5, d, J = 8.5 Hz), 7.70 (2H×3/5, d, J
=
8.5 Hz), 9.24 (1H×2/5, br s), 9.26 (1H×3/5, br s), 9.89 (1H×3/5, br s), 10.52 (1H×2/5, br s); FAB MS m/e
+
(
M+H) 467.
5
.1.5. A mixture of tert-butyl
4-(2-{3-acetamido-5-[4-(methylsulfonyl)phenyl]-1H-pyrazol-1-yl}ethyl)phenyl]carbamate (9a) and
tert-butyl [4-(2-{5-acetamido-3-[4-(methylsulfonyl)phenyl]-1H-pyrazol-1-yl}ethyl)phenyl]carbamate
[

(9b).
®
Potassium peroxymonosulfate (Oxone ) (1.1 g) was suspended in H O (3 mL) and THF (14 mL), and then
2
a mixture of 8a and 8b (652 mg) was added portionwise to the suspension at 0 ˚C. The reaction mixture
was stirred at room temperature for 1.5 h, and then H O was added to the suspension. The solution was
2
made basic with saturated aqueous NaHCO and extracted with CHCl (twice). The combined organic layer
3
3
was washed with brine, dried over anhydrous MgSO , and concentrated in vacuo to give a mixture of 9a
4
+
and 9b (570 mg, 82%) as pale brown amorphous. FAB MS m/e (M+H) 499.
5
.1.6. N-{1-[2-(4-Aminophenyl)ethyl]-5-[4-(methylsulfonyl)phenyl]-1H-pyrazol-3-yl}acetamide
(10a) and N-{1-[2-(4-aminophenyl)ethyl]-3-[4-(methylsulfonyl)phenyl]-1H-pyrazol-5-yl}acetamide
(10b).
A mixture of 9a and 9b (550 mg) was dissolved in CH Cl (2 mL), and then TFA (2 mL) was added to the
2
2
solution at 0 ˚C. The reaction mixture was stirred at room temperature for an hour, and concentrated in
vacuo. The residue was dissolved in CHCl , washed with 1 M NaOH, H O and brine, dried over anhydrous
3
2
MgSO , and concentrated in vacuo. The residue was purified by preparative silica gel column
4
chromatography with CHCl /MeOH (10:1) as an eluent to give 10a (126 mg, 29%) as an off-white solid
3
and 10b (170 mg, 39%) as an off-white solid.
1
1
0a : Mp 203‒ 204 ˚C; H NMR (DMSO-d ) δ 2.02 (3H, s), 2.87 (2H, t, J = 7.0 Hz), 3.27 (3H, s), 4.10 (2H,
6
t, J = 7.0 Hz), 4.89 (2H, s), 6.40 (2H, d, J = 8.5 Hz), 6.61 (2H, d, J = 8.5 Hz), 6.62 (1H, s), 7.42 (2H, d, J =
+
8
.5 Hz), 7.92 (2H, d, J = 8.5 Hz), 10.61 (1H, br s); FAB MS m/e (M+H) 399.
1
1
0b : Mp 188‒ 189°C; H NMR (DMSO-d ) δ 2.09 (3H, s), 2.89 (2H, t, J = 7.5 Hz), 3.23 (3H, s), 4.18 (2H,
6
t, J = 7.5 Hz), 4.90 (2H, s), 6.48 (2H, d, J = 8.5 Hz), 6.80 (1H, s), 6.88 (2H, d, J = 8.5 Hz), 7.93 (2H, d, J =
+
8
.5 Hz), 8.03 (2H, d, J = 8.5 Hz), 9.95 (1H, br s); FAB MS m/e (M+H) 399.
5
.1.7. tert-Butyl
{(Z)-{[4-(2-{3-acetamido-5-[4-(methylsulfonyl)phenyl]-1H-pyrazol-1-yl}ethyl)phenyl]amino}-
[(tert-butoxycarbonyl)amino]methylene}carbamate (12).

A mixture of 10a (105 mg), N,N'-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (11) (82 mg),
DMF (0.5 mL) and THF (2 mL) was stirred at 50 ˚C for 10 h and at room temperature for 75 h under a
nitrogen atmosphere. The solvent was removed in vacuo, and the residue was purified by preparative silica
gel column chromatography with CHCl /MeOH (20:1) as an eluent to give 12 (62 mg, 36%) as an off-white
3
1
wax. H NMR (DMSO-d ) δ 1.38 (9H, s), 1.51 (9H, s), 2.02 (3H, s), 3.00 (2H, t, J = 7.0 Hz), 3.25 (3H, s),
6
4
.24 (2H, t, J = 7.0 Hz), 6.60 (1H, s), 6.90 (2H, d, J = 8.5 Hz), 7.32 (2H, d, J = 8.5 Hz), 7.48 (2H, d, J = 8.5
+
Hz), 7.92 (2H, d, J = 8.5 Hz), 9.90 (1H, br s), 10.61 (1H, br s), 11.43 (1H, br s); FAB MS m/e (M+H) 641.
5
.1.8.
N-{1-[2-(4-Carbamimidamidophenyl)ethyl]-5-[4-(methylsulfonyl)phenyl]-1H-pyrazol-3-yl}acetamide
hydrochloride (13).
Compound 12 (50 mg) was dissolved in 4 M HCl/dioxane (4 mL). The mixture was stirred at room
temperature for 7 h and then evaporated in vacuo. The residual solid was washed with Et O to give 13 (37
2
1
mg, 98%) as an off-white solid. Mp 155‒ 156 ˚C; H NMR (DMSO-d ) δ 2.03 (3H, s), 3.05 (2H, t, J = 7.5
6
Hz), 3.28 (3H, s), 4.25 (2H, t, J = 7.5 Hz), 6.64 (1H, s), 7.03 (2H, d, J = 8.5 Hz), 7.07 (2H, d, J = 8.5 Hz),
7
.37 (4H, br s), 7.50 (2H, d, J = 8.5 Hz), 7.97 (2H, d, J = 8.5 Hz), 9.77 (1H, br s), 10.61 (1H, br s); FAB
+
MS m/e (M+H) 441.
5
.1.9. 2-Methyl-4-[4-(methylsulfanyl)benzylidene]-1,3-oxazol-5(4H)-one (15).
To a mixture of 4-(methylsulfanyl)benzaldehyde (14) (31.8 g), (acetylamino)acetic acid (24.5 g) and Ac O
2
(35 mL) was added sodium acetate (8.6 g) at room temperature under a nitrogen atmosphere. The reaction
mixture was refluxed for 3.5 h. After cooling to room temperature, the mixture was poured into ice-H O
2
and AcOEt with stirring, and filtered in vacuo. The filtrate was separated, and the organic layer was washed
with brine, dried over anhydrous MgSO , and concentrated in vacuo. The residue and the previously
4
obtained solid were combined, and the mixture was purified by flash column chromatography over silica
i
gel with CHCl -AcOEt (30:1) as an eluent and triturated with Pr O to give 15 (17.8 g, 37%) as a brown
3
2
1
solid. Mp 154‒ 155 ˚C; H NMR (DMSO-d ) δ 2.38 (3H, s), 2.53 (3H, s), 7.19 (1H, s), 7.36 (2H, d, J = 8.5
6

Hz), 8.12 (2H, d, J = 8.5 Hz).
5
.1.10. 3-(4-(Methylsulfanyl)phenyl)-2-oxopropanoic acid (16).
To a solution of 15 (17.5 g) in dioxane (100 mL) was added 4 M HCl (27 mL), and the reaction mixture
was refluxed for 3 h. After cooling to room temperature, the mixture was concentrated in vacuo. AcOEt and
H O were added to the residue, and the precipitate was filtered in vacuo to give 16 (16.4g, 104%) as a pale
2
1
brown solid. Mp 165‒ 167 ˚C; H NMR (DMSO-d ) δ 2.48 (3H, s), 6.37 (1H, s), 7.23 (2H, d, J = 8.5 Hz),
6
-
7
.70 (2H, d, J = 8.5 Hz), 9.44 (1H, s); FAB MS m/e (M-H) 209.
5
.1.11. Methyl 3-(4-(methylsulfanyl)phenyl)-2-oxopropanoate (17).
To a solution of 16 (16.2 g) in DMF (81 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (11.5 mL) at 0
˚C under a nitrogen atmosphere. The mixture was stirred at the same temperature for an hour, and then MeI
(9.6 mL) was added to the solution at the same temperature. The reaction mixture was stirred at room
temperature for 4 h, poured into 1 M HCl, and extracted with AcOEt (twice). The combined organic layer
was washed with brine, dried over anhydrous MgSO , and concentrated in vacuo. The residue was purified
4
by flash column chromatography over silica gel with CHCl /AcOEt (30:1) as an eluent, and triturated with
3
i
1
hexane/ Pr O to give 17 (10.2 g, 59%) as a dark yellow solid. Mp 112‒ 113 ˚C; H NMR (DMSO-d ) δ 2.48
2
6
(3H, s), 3.79 (3H, s), 6.41 (1H, s), 7.24 (2H, d, J = 8.5 Hz), 7.72 (2H, d, J = 8.5 Hz), 9.52 (1H, br s); FAB
-
MS m/e (M-H) 223.
5
.1.12. Methyl 3-bromo-3-[4-(methylsulfanyl)phenyl]-2-oxopropanoate (18).
To a solution of 17 (2.8 g) in CH Cl (140 mL) and AcOH (0.5 mL) was added pyridinium tribromide (5.0
2
2
g) at 0 ˚C under a nitrogen atmosphere. The reaction mixture was stirred at the same temperature for 2 h,
and poured into H O. The mixture was extracted with AcOEt (twice). The combined organic layer was
2
dried over anhydrous MgSO and concentrated in vacuo to give 18 (4.1 g, 105%) as a brown oil.
4
5
.1.13. Methyl 3-[4-(methylthio)phenyl]propanoate (21).

To a mixture of 3-(4-mercaptophenyl)propanoic acid (20) (5.0 g) and K CO (11.4 g) in DMF (30 mL) was
2
3
added MeI (5.1 mL) at 0 ˚C under a nitrogen atmosphere. The reaction mixture was stirred at room
temperature for 13 h and poured into ice-H O. The mixture was extracted with AcOEt. The organic layer
2
was washed with H O (twice) and brine, dried over anhydrous MgSO , and concentrated in vacuo to give
2
4
1
2
1 (4.2 g, 73%) as a pale yellow oil. H NMR (CDCl ) δ 2.47 (3H, s), 2.61 (2H, t, J = 8.0 Hz), 2.91 (2H, t,
3
J = 8.0 Hz), 3.67 (3H, s), 7.12 (2H, d, J = 8.5 Hz), 7.20 (2H, d, J = 8.5 Hz).
5
.1.14. N-Methoxy-N-methyl-3-(methylsulfanyl)benzamide (23).
1
-Hydroxybenzotriazole (HOBt) (3.7 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (4.1 g)
were added to a solution of 3-(methylsulfanyl)benzoic acid (22) (15.0 g) in DMF (150 mL) at 0 ˚C. After
stirring for 30 min at room temperature, N,O-dimethylhydroxylamine hydrochloride (8.7 g) was added at 0
˚C. The reaction mixture was stirred at room temperature for 13 h and poured into saturated aqueous
NaHCO at 0 ˚C. The mixture was extracted with AcOEt (twice). The combined organic layer was washed
3
with brine, dried over anhydrous MgSO , and concentrated in vacuo to give 23 (18.3 g, 97%) as a yellow
4
1
oil. H NMR (CDCl ) δ 2.50 (3H, s), 3.36 (3H, s), 3.56 (3H, s), 7.28‒ 7.45 (3H, m), 7.54 (1H, s); FAB MS
3
+
m/e (M+H) 212.
5
.1.15. Methyl (2E)-3-[3-(methylsulfanyl)phenyl]acrylate (25).
To a stirred solution of 23 (18.0 g) in THF (360 mL) was added diisobutylaluminum hydride (170 mL)
dropwise at -78 ˚C over 40 min under a nitrogen atmosphere. The reaction mixture was let warm to room
temperature over 2.5 h and then quenched with MeOH. 1 M HCl was added and the mixture was extracted
with AcOEt. The organic layer was washed with brine, dried over anhydrous MgSO , and concentrated in
4
vacuo. A mixture of the residual yellow oil (12.9 g), methyl (triphenylphosphoranylidene)acetate (24) (28.5
g) and THF (260 mL) was refluxed for 2 h. The solvent was removed in vacuo, and the residue was
suspended in AcOEt. The solid was filtered off, and the filtrate was concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel with hexane/AcOEt (3:1) as an eluent to give 25
1
(
14.4 g, 81%) as a colorless wax. H NMR (DMSO-d ) δ 2.51 (3H, s), 3.81 (3H, s), 6.44 (1H, d, J = 16.0
6

Hz), 7.24‒ 7.32 (3H, m), 7.38 (1H, m), 7.65 (1H, d, J = 16.0 Hz).
5
.1.16. Methyl 3-[3-(methylthio)phenyl]propanoate (26).
A mixture of 25 (14.0 g) and 10% palladium on carbon (6.7 g) in MeOH (140 mL) and AcOH (70 mL) was
®
stirred at room temperature for 9 h under a hydrogen atmosphere (4 atm) and filtered through a celite pad.
The filtrate was concentrated in vacuo. The residue was purified by flash column chromatography over
1
silica gel with hexane/AcOEt (3:1) as an eluent to give 26 (12.5 g, 88%) as a colorless oil. H NMR
(
DMSO-d ) δ 2.48 (3H, s), 2.62 (2H, t, J = 8.0 Hz), 2.92 (2H, t, J = 8.0 Hz), 3.68 (3H, s), 6.94‒ 7.00 (1H,
6
m), 7.07‒ 7.14 (2H, m), 7.15‒ 7.24 (1H, m).
5
.1.17. Ethyl 4-[4-(methylthio)phenyl]-2-oxobutanoate (27a).
Sodium methoxide, 28% solution in MeOH (3.7 mL), was added dropwise to a mixture of 21 (4.0 g) and
diethyl oxalate (5.2 mL) at 0 ˚C with stirring. The reaction mixture was stirred at 65 ˚C for 30 min under
reduced pressure. 15% aqueous H SO (35 mL) was added to the mixture, and the mixture was refluxed for
2
4
1
5 h. After cooling to room temperature, the mixture was extracted with AcOEt. The organic layer was
washed with H O and brine, dried over anhydrous MgSO , and concentrated in vacuo. The residual oil was
2
4
dissolved in EtOH (20 mL), and concentrated H SO (0.4 mL) was added dropwise to the solution. The
2
4
reaction mixture was refluxed for 2 h. After cooling to room temperature, EtOH was removed in vacuo.
AcOEt and H O were added to the residue, and extracted. The organic layer was washed with H O and
2
2
brine, dried over anhydrous MgSO , and concentrated in vacuo. The residue was purified by flash column
4
chromatography over silica gel with hexane/AcOEt (6:1) as an eluent to give 27a (2.4 g, 51%) as a yellow
1
oil. H NMR (CDCl ) δ 1.35 (3H, t, J = 7.0 Hz), 2.46 (3H, s), 2.92 (2H, t, J = 7.0 Hz), 3.16 (2H, t, J = 7.0
3
Hz), 4.31 (2H, q, J = 7.0 Hz), 7.13 (2H, d, J = 8.5 Hz), 7.20 (2H, d, J = 8.5 Hz).
5
.1.18. Ethyl 4-[3-(methylthio)phenyl]-2-oxobutanoate (27b).
Compound 27b was prepared from 26 according to the same procedure as that of compound 27a.
1
Compound 27b was obtained as a pale yellow oil (49% yield). H NMR (CDCl ) δ 1.36 (3H, t, J = 7.5 Hz),
3

2
7
.48 (3H, s), 2.92 (2H, t, J = 7.0 Hz), 3.17 (2H, t, J = 7.0 Hz), 4.32 (2H, q, J = 7.5 Hz), 6.94‒ 7.01 (1H, m),
.05‒ 7.13 (2H, m), 7.17‒ 7.26 (1H, m).
5
.1.19. Ethyl 3-bromo-4-[4-(methylthio)phenyl]-2-oxobutanoate (28a).
To a suspension of copper (II) bromide (212 g) in AcOEt (1600 mL) was added a solution of 27a (80 g) in
00 mL of CHCl . The reaction mixture was refluxed for 13 h, cooled to room temperature, and filtered in
8
3
vacuo. The filtrate was concentrated in vacuo, and the residue was purified by flash column
chromatography over silica gel with hexane/AcOEt (1:1) as an eluent to give 28a (102 g, 97%) as a yellow
1
oil. H NMR (CDCl ) δ 1.37 (3H, t, J = 7.0 Hz), 2.47 (3H, s), 3.20 (1H, dd, J = 14.5, 7.5 Hz), 3.49 (1H, dd,
3
J = 14.5, 7.5 Hz), 4.35 (2H, q, J = 7.0 Hz), 5.22 (1H, d, J = 7.5 Hz), 7.17 (2H, d, J = 8.5 Hz), 7.20 (2H, d, J
=
8.5 Hz).
5
.1.20. Ethyl 3-bromo-4-[3-(methylthio)phenyl]-2-oxobutanoate (28b).
Compound 28b was prepared from 27b and copper (II) bromide according to the same procedure as that of
1
compound 28a. Compound 28b was obtained as a pale brown oil (96% yield). H NMR (CDCl ) δ 1.38 (3H,
3
t, J = 7.5 Hz), 2.47 (3H, s), 3.21 (1H, dd, J = 14.5, 7.5 Hz), 3.50 (1H, dd, J = 14.5, 7.5 Hz), 4.36 (2H, q, J =
7
.5 Hz), 5.21 (1H, t, J = 7.5 Hz), 6.98‒ 7.05 (1H, m), 7.11‒ 7.29 (3H, m).
5
.1.21. Ethyl 3-bromo-2-oxo-4-phenylbutanoate (28c).
Compound 28c was prepared from 27c and copper (II) bromide according to the same procedure as that of
1
compound 28a. Compound 28c was obtained as a yellow oil (84% yield). H NMR (CDCl ) δ 1.37 (3H, t, J
3
=
7.0 Hz), 3.25 (1H, dd, J = 14.5, 7.5 Hz), 3.54 (1H, dd, J = 14.5, 7.5 Hz), 4.35 (2H, q, J = 7.0 Hz), 5.27
(1H, d, J = 7.5 Hz), 7.18‒ 7.41 (5H, m).
5
.1.22. Methyl 2-amino-5-[4-(methylthio)phenyl]-1,3-thiazole-4-carboxylate (19a).
Compound 18 (4.1 g) was dissolved in EtOH (55 mL), and then thiourea (1.3 g) was added to the solution.
The reaction mixture was refluxed for an hour under a nitrogen atmosphere. The cooled reaction mixture

was evaporated in vacuo. The residual solid was suspended in saturated aqueous NaHCO and H O. The
3
2
solid was collected by filtration, and purified by flash column chromatography over silica gel with
1
CHCl /MeOH (10:1) as an eluent to give 19a (2.7 g, 75%) as a brown solid. Mp 184‒ 185 ˚C; H NMR
3
(
DMSO-d ) δ 2.50 (3H, s), 3.64 (3H, s), 7.25 (2H, d, J = 8.5 Hz), 7.34 (2H, d, J = 8.5 Hz); FAB MS m/e
6
+
(
M+H) 281.
5
.1.23. Ethyl 2-amino-5-[4-(methylthio)benzyl]-1,3-thiazole-4-carboxylate (19b).
Compound 28a (39.0 g) was dissolved in EtOH (600 mL), and then thiourea (17.9 g) was added to the
solution. The reaction mixture was refluxed for 2 h under a nitrogen atmosphere. The cooled reaction
mixture was evaporated in vacuo. Saturated aqueous NaHCO and H O were added to the residue, and the
3
2
mixture was extracted with AcOEt. The organic layer was washed with H O and brine, dried over
2
anhydrous MgSO , and concentrated in vacuo. The residue was purified by flash column chromatography
4
over silica gel with CHCl /MeOH (40:1‒ 10:1) as an eluent to give 19b (30.9 g, 85%) as a yellow solid. Mp
3
1
1
11‒ 112.5 ˚C; H NMR (DMSO-d ) δ 1.25 (3H, t, J = 7.0 Hz), 2.44 (3H, s), 4.20 (2H, q, J = 7.0 Hz), 4.28
6
+
(
2H, s), 7.02 (2H, s), 7.19 (4H, s); FAB MS m/e (M+H) 309; HRMS (ESI) Calcd for C H N O S
1
4
17
2
2
2
+
(
M+H) : 309.0731, found: 309.0738.
5
.1.24. Ethyl 2-amino-5-[3-(methylthio)benzyl]-1,3-thiazole-4-carboxylate (19c).
Compound 19c was prepared from 28b and thiourea according to the same procedure as that of compound
1
1
9b. Compound 19c was obtained as a yellow amorphous (90% yield). H NMR (DMSO-d ) δ 1.25 (3H, t,
6
+
J = 7.0 Hz), 2.45 (3H, s), 4.21 (2H, q, J = 7.0 Hz), 4.30 (2H, s), 6.96-7.29 (4H, m); FAB MS m/e (M+H)
3
09.
5
.1.25. Ethyl 2-amino-5-benzyl-1,3-thiazole-4-carboxylate (19d).
Compound 19d was prepared from 28c and thiourea according to the same procedure as that of compound
1
1
9b. Compound 19d was obtained as an off-white solid (75% yield). Mp 86‒ 88 ˚C; H NMR (DMSO-d ) δ
6
1
.25 (3H, t, J = 7.0 Hz), 4.21 (2H, q, J = 7.0 Hz), 4.33 (2H, s), 7.02 (2H, s), 7.11‒ 7.39 (5H, m); FAB MS

+
m/e (M+H) 263.
5
.1.26. Methyl 2-(acetylamino)-5-[4-(methylthio)phenyl]-1,3-thiazole-4-carboxylate (29a).
Compound 19a (8.8 g) was dissolved in pyridine (88 mL), and then acetyl chloride (6.7 mL) was added
dropwise to the solution at 0 ˚C under a nitrogen atmosphere. The reaction mixture was stirred at room
temperature for 30 min and at 50 ˚C for 2 h. After cooling to 0 ˚C, H O was added to the solution. The
2
precipitate was filtered in vacuo, and the solid was washed with Et O to give 29a (9.3 g, 92%) as an
2
1
off-white solid. Mp 253‒ 254.5 ˚C; H NMR (DMSO-d ) δ 2.16 (3H, s), 2.52 (3H, s), 3.70 (3H, s), 7.30
6
+
(
2H, d, J = 8.5 Hz), 7.44 (2H, d, J = 8.5 Hz); FAB MS m/e (M+H) 323.
5
.1.27. Ethyl 2-(acetylamino)-5-[4-(methylthio)benzyl]-1,3-thiazole-4-carboxylate (29b).
Compound 29b was prepared from 19b and acetyl chloride according to the same procedure as that of
1
compound 29a. Compound 29b was obtained as an off-white solid (93% yield). Mp 205‒ 206 ˚C; H NMR
(DMSO-d ) δ 1.28 (3H, t, J = 7.0 Hz), 2.09 (3H, s), 2.45 (3H, s), 4.27 (2H, q, J = 7.0 Hz), 4.43 (2H, s), 7.22
6
+
+
(
4H, s), 12.41 (1H, s); FAB MS m/e (M+H) 351; HRMS (ESI) Calcd for C H N O S (M+H) : 351.0837,
16
19
2
3 2
found: 351.0851; Anal. Calcd for C H N O S ·0.1H O: C, 54.55; H, 5.21; N, 7.95; S, 18.21. Found: C,
1
6
18
2
3
2
2
5
4.24; H, 5.19; N, 8.04; S, 18.37.
5
.1.28. Ethyl 2-(acetylamino)-5-[3-(methylthio)benzyl]-1,3-thiazole-4-carboxylate (29c).
Compound 29c was prepared from 19c and acetyl chloride according to the same procedure as that of
1
compound 29a. Compound 29c was obtained as a colorless solid (60% yield). Mp 187.5‒ 188.5 ˚C; H
NMR (DMSO-d ) δ 1.28 (3H, t, J = 7.0 Hz), 2.09 (3H, s), 2.45 (3H, s), 4.28 (2H, q, J = 7.0 Hz), 4.45 (2H,
6
+
s), 7.00‒ 7.23 (3H, m), 7.26 (1H, t, J = 7.5 Hz), 12.43 (1H, s); FAB MS m/e (M+H) 351.
5
.1.29. Ethyl 2-(acetylamino)-5-benzyl-1,3-thiazole-4-carboxylate (29d).
Compound 29d was prepared from 19d and acetyl chloride according to the same procedure as that of
1
compound 29a. Compound 29d was obtained as a brown solid (79% yield). Mp 178‒ 180 ˚C; H NMR

(
DMSO-d ) δ 1.28 (3H, t, J = 7.0 Hz), 2.09 (3H, s), 4.28 (2H, q, J = 7.0 Hz), 4.48 (2H, s), 7.19‒ 7.39 (5H,
6
+
m), 12.41 (1H, s); FAB MS m/e (M+H) 305.
5
.1.30. N-{4-Formyl-5-[4-(methylsulfanyl)phenyl]-1,3-thiazol-2-yl}acetamide (30a).
Compound 29a (200 mg) was dissolved in THF (2 mL), and then lithium aluminum hydride (35 mg) was
added portionwise to the solution at 0 ˚C under a nitrogen atmosphere. The reaction mixture was stirred at 0
˚C for 30 min, at room temperature for 30 min, and then quenched with MeOH. 1 M HCl was added and
the mixture was extracted with AcOEt. The aqueous layer was extracted with AcOEt (twice). The
combined organic layer was washed with brine, dried over anhydrous MgSO , and concentrated in vacuo.
4
The residual solid was dissolved in MeOH (0.4 mL) and CHCl (7 mL). Manganese (IV) oxide (1.1 g) was
3
then added to the solution under a nitrogen atmosphere. The reaction mixture was stirred at room
®
temperature for 13 h and filtered through a celite pad. The filtrate was concentrated in vacuo. The residue
was purified by flash column chromatography over silica gel with CHCl /MeOH (20:1) as an eluent to give
3
1
3
0a (154 mg, 85%) as a pale brown amorphous. H NMR (DMSO-d ) δ 2.18 (3H, s), 2.54 (3H, s), 7.38 (2H,
6
+
d, J = 8.5 Hz), 7.58 (2H, d, J = 8.5 Hz), 9.77 (1H, s), 12.59 (1H, br s); FAB MS m/e (M+H) 293.
5
.1.31. N-{4-Formyl-5-[4-(methylthio)benzyl]-1,3-thiazol-2-yl}acetamide (30b).
Compound 30b was prepared from 29b according to the same procedure as that of compound 30a.
1
Compound 30b was obtained as a yellow wax (71% yield). H NMR (DMSO-d ) δ 2.12 (3H, s), 2.45 (3H,
6
+
s), 4.48 (2H, s), 7.23 (4H, s), 10.03 (1H, s), 12.33 (1H, s); FAB MS m/e (M+H) 307.
5
.1.32. N-{4-Formyl-5-[3-(methylthio)benzyl]-1,3-thiazol-2-yl}acetamide (30c).
Compound 30c was prepared from 29c according to the same procedure as that of compound 30a.
1
Compound 30c was obtained as an off-white solid (71% yield). Mp 148‒ 149.5 ˚C; H NMR (DMSO-d ) δ
6
2
.12 (3H, s), 2.45 (3H, s), 4.50 (2H, s), 6.99‒ 7.25 (3H, m), 7.27 (1H, t, J = 7.5 Hz), 10.04 (1H, s), 12.35
+
(
1H, br s); FAB MS m/e (M+H) 307.

5
.1.33. N-(5-benzyl-4-formyl-1,3-thiazol-2-yl)acetamide (30d).
Compound 30d was prepared from 29d according to the same procedure as that of compound 30a.
1
Compound 30d was obtained as a pale yellow solid (67% yield). Mp 191‒ 192.5 ˚C; H NMR (DMSO-d )
6
+
δ 2.12 (3H, s), 4.53 (2H, s), 7.19‒ 7.40 (5H, m), 10.04 (1H, s), 12.34 (1H, s); FAB MS m/e (M+H) 261.
5
.1.34. A mixture of
N-{5-[4-(methylthio)phenyl]-4-[(E)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-yl}acetamide and
N-{5-[4-(methylthio)phenyl]-4-[(Z)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-yl}acetamide (32a).
To a mixture of 4-nitrobenzyl bromide (31) (182 mg) in DMF (2 mL) was added triphenylphosphine (221
mg) at 0 ˚C under a nitrogen atmosphere, and the reaction mixture was stirred at room temperature for 3 h.
Potassium tert-butoxide (111 mg) and 30a (145mg) were then added to the mixture at 0 ˚C, which was
stirred at room temperature for 15 h. The reaction mixture was poured into ice-H O and extracted with
2
AcOEt. The organic layer was washed with 1 M HCl, H O and brine, dried over anhydrous MgSO , and
2
4
concentrated in vacuo. The residue was purified by flash column chromatography over silica gel with
CHCl /MeOH (40:1) as an eluent to give 32a (E : Z = 2 : 1) (177 mg, 87%) as a brown wax. FAB MS m/e
3
+
(
M+H) 412.
5
.1.35. A mixture of
N-{5-[4-(methylsulfonyl)phenyl]-4-[(E)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-yl}acetamide and
N-{5-[4-(methylsulfonyl)phenyl]-4-[(Z)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-yl}acetamide (32b).
®
Oxone (408 mg) was suspended in H O (1 mL) and THF (1 mL), and then 32a (182 mg) in THF (3 mL)
2
was added dropwise to the suspension at 0 ˚C. The reaction mixture was stirred at room temperature for 2 h,
and then H O was added to the suspension. The precipitate was filtered in vacuo. The solid was washed
2
with H O and AcOEt to give 32b (83 mg, 42%, E : Z = 2 : 1) as a yellow solid. Mp 294‒ 295 ˚C; FAB MS
2
+
m/e (M+H) 442.
5
.1.36. A mixture of

N-{5-[4-(methylthio)benzyl]-4-[(E)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide and
N-{5-[4-(methylthio)benzyl]-4-[(Z)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide (32c).
Compound 32c was prepared from 30b according to the same procedure as that of compound 32a.
+
Compound 32c was obtained as a yellow amorphous (97% yield, E : Z = 1 : 2 ). FAB MS m/e (M+H) 426.
5
.1.37. A mixture of
N-{5-[4-(methylsulfonyl)benzyl]-4-[(E)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide and
N-{5-[4-(methylsulfonyl)benzyl]-4-[(Z)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide (32d).
Compound 32d was prepared from 32c according to the same procedure as that of compound 32b.
+
Compound 32d was obtained as a yellow solid (99% yield, E : Z = 1 : 2 ). FAB MS m/e (M+H) 458.
5
.1.38. A mixture of
N-{5-[3-(methylthio)benzyl]-4-[(E)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide and
N-{5-[3-(methylthio)benzyl]-4-[(Z)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide (32e).
Compound 32e was prepared from 30c according to the same procedure as that of compound 32a.
+
Compound 32e was obtained as a yellow amorphous (97% yield, E : Z = 1 : 2 ). FAB MS m/e (M+H) 426.
5
.1.39. A mixture of
N-{5-[3-(methylsulfonyl)benzyl]-4-[(E)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide and
N-{5-[3-(methylsulfonyl)benzyl]-4-[(Z)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide (32f).
Compound 32f was prepared from 32e according to the same procedure as that of compound 32b.
+
Compound 32f was obtained as a brown amorphous (100% yield, E : Z = 1 : 2 ). FAB MS m/e (M+H) 458.
5
.1.40. A mixture of N-{5-benzyl-4-[(E)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide and
N-{5-benzyl-4-[(Z)-2-(4-nitrophenyl)vinyl]-1,3-thiazol-2-yl}acetamide (32g).
Compound 32g was prepared from 30d according to the same procedure as that of compound 32a.
Compound 32g was obtained as a yellow amorphous (107% yield, E : Z = 1 : 2). FAB MS m/e (M+H)
+

4
26.
5
.1.41. N-{4-[2-(4-Aminophenyl)ethyl]-5-[4-(methylsulfonyl)phenyl]-1,3-thiazol-2-yl}acetamide
(33a).
A mixture of 32b (600 mg), 10% palladium on carbon (906 mg), MeOH (6 mL), THF (6 mL) and AcOH (1
mL) was stirred at room temperature for 3 h under a hydrogen atmosphere (3 atm), and filtered through a
®
celite pad. 1 M NaOH was added to the residue, and the mixture was extracted with AcOEt. The organic
layer was washed with H O and brine, dried over anhydrous MgSO , and concentrated in vacuo. The
2
4
residual solid was washed with Et O to give 33a (366 mg, 65%) as a pale yellow solid. Mp 202‒ 204 ˚C;
2
1
H NMR (DMSO-d ) δ 2.17 (3H, s), 2.77‒ 2.88 (4H, m), 3.24 (3H, s), 6.84 (2H, br s), 6.45 (2H, d, J = 8.5
6
Hz), 6.77 (2H, d, J = 8.5 Hz), 7.49 (2H, d, J = 8.5 Hz), 7.91 (2H, d, J = 8.5 Hz), 12.34 (1H, br s); FAB MS
+
m/e (M+H) 416.
5
.1.42. N-{4-[2-(4-Aminophenyl)ethyl]-5-[4-(methylsulfanyl)benzyl]-1,3-thiazol-2-yl}acetamide
(33b).
Compound 33b was prepared from 32c according to the same procedure as that of compound 33a.
1
Compound 33b was obtained as a yellow solid (51% yield). H NMR (DMSO-d ) δ 2.07 (3H, s), 2.43 (3H,
6
s), 2.64‒ 2.79 (4H, m), 3.80 (2H, s), 4.83 (2H, s), 6.46 (2H, d, J = 8.5 Hz), 6.77 (2H, d, J = 8.5 Hz), 6.97
+
(
2H, d, J = 8.5 Hz), 7.16 (2H, d, J = 8.5 Hz), 11.94 (1H, br s); FAB MS m/e (M+H) 398; HRMS (ESI)
+
calcd for C H N OS (M+H) : 398.1361, found: 398.1359.
2
1
24
3
2
5
.1.43. N-{4-[2-(4-Aminophenyl)ethyl]-5-[4-(methylsulfonyl)benzyl]-1,3-thiazol-2-yl}acetamide
(33c).
Compound 33c was prepared from 32d according to the same procedure as that of compound 33a.
1
Compound 33c was obtained as a brown oil (90% yield). H NMR (DMSO-d ) δ 2.08 (3H, s), 2.58‒ 2.87
6
(4H, m), 3.18 (3H, s), 3.98 (2H, s), 4.85 (2H, s), 6.46 (2H, d, J = 8.5 Hz), 6.77 (2H, d, J = 8.5 Hz), 7.27 (2H,
+
d, J = 8.5 Hz), 7.82 (2H, d, J = 8.5 Hz), 12.02 (1H, s); FAB MS m/e (M+H) 430.

5
.1.44. N-{4-[2-(4-Aminophenyl)ethyl]-5-[3-(methylsulfonyl)benzyl]-1,3-thiazol-2-yl}acetamide
(33d).
Compound 33d was prepared from 32f according to the same procedure as that of compound 33a.
1
Compound 33d was obtained as a yellow amorphous (51% yield). H NMR (DMSO-d ) δ 2.08 (3H, s),
6
2
.59‒ 2.86 (4H, m), 3.18 (3H, s), 4.02 (2H, s), 4.84 (2H, br s), 6.46 (2H, d, J = 8.5 Hz), 6.78 (2H, d, J = 8.5
+
Hz), 7.25‒ 7.88 (4H, m), 12.03 (1H, s); FAB MS m/e (M+H) 430.
5
.1.45. N-{4-[2-(4-Aminophenyl)ethyl]-5-benzyl-1,3-thiazol-2-yl}acetamide (33e).
Compound 33e was prepared from 32g according to the same procedure as that of compound 33a.
1
Compound 33e was obtained as an off-white amorphous (61% yield). H NMR (DMSO-d ) δ 2.07 (3H, s),
6
2
.59‒ 2.85 (4H, m), 3.85 (2H, s), 4.84 (2H, s), 6.46 (2H, d, J = 8.5 Hz), 6.78 (2H, d, J = 8.5 Hz), 7.07 (2H,
+
d, J = 8.0 Hz), 7.16‒ 7.31 (3H, m), 11.96 (1H, s); FAB MS m/e (M+H) 352.
5
.1.46. Di-tert-butyl
{[(4-{2-[2-(acetylamino)-5-(4-(methylsulfonyl)phenyl)-1,3-thiazol-4-yl]ethyl}phenyl)amino]-
methylidene}biscarbamate (34a).
A mixture of 33a (360 mg), N,N'-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (11) (296 mg)
and THF (7 mL) was stirred at room temperature for 12 h, and then concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel with CHCl /AcOEt (1:1) as an eluent to give 34a
3
1
(
451 mg, 79%) as a colorless amorphous. H NMR (DMSO-d ) δ 1.39 (9H, s), 1.51 (9H, s), 2.17 (3H, s),
6
2
.97 (4H, s), 3.24 (3H, s), 7.11 (2H, d, J = 8.5 Hz), 7.38 (2H, d, J = 8.5 Hz), 7.56 (2H, d, J = 8.5 Hz), 7.92
+
(
2H, d, J = 8.5 Hz), 9.92 (1H, s), 11.43 (1H, br s), 12.34 (1H, br s); FAB MS m/e (M+H) 658.
5
.1.47. tert-Butyl
(Z)-{[4-(2-{2-acetamido-5-[4-(methylsulfanyl)benzyl]-1,3-thiazol-4-yl}ethyl)phenyl]amino}-
(tert-butoxycarbonyl)amino]methylene}carbamate (34b).
{
[

Compound 34b was prepared from 33b and 11 according to the same procedure as that of compound 34a.
1
Compound 34b was obtained as a pale yellow oil (73% yield). H NMR (DMSO-d ) δ 1.39 (9H, s), 1.51
6
(9H, s), 2.08 (3H, s), 2.42 (3H, s), 2.85 (4H, s), 3.83 (2H, s), 6.99 (2H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.5
Hz), 7.14 (2H, d, J = 8.5 Hz), 7.43 (2H, d, J = 8.5 Hz), 9.69 (1H, s), 11.44 (1H, s), 11.97 (1H, s); FAB MS
+
m/e (M+H) 640.
5
.1.48. Di-tert-butyl
((Z)-{[4-(2-{2-(acetylamino)-5-[4-(methylsulfonyl)benzyl]-1,3-thiazol-4-yl}ethyl)phenyl]amino}-
methylidene)biscarbamate (34c).
Compound 34c was prepared from 33c and 11 according to the same procedure as that of compound 34a.
1
Compound 34c was obtained as an off-white amorphous (73% yield). H NMR (DMSO-d ) δ 1.39 (9H, s),
6
1
8
.51 (9H, s), 2.08 (3H, s), 2.86 (4H, s), 3.16 (3H, s), 4.03 (2H, s), 7.13 (2H, d, J = 8.5 Hz), 7.33 (2H, d, J =
.5 Hz), 7.43 (2H, d, J = 8.5 Hz), 7.81 (2H, d, J = 8.5 Hz), 9.97 (1H, s), 11.45 (1H, s), 12.05 (1H, s); FAB
+
MS m/e (M+H) 672.
5
.1.49. Di-tert-butyl ({[4-(2-{2-(acetylamino)-5-[3-(methylsulfonyl)benzyl]-1,3-thiazol-4-
yl}ethyl)phenyl]amino}methylidene)biscarbamate (34d).
Compound 34d was prepared from 33d and 11 according to the same procedure as that of compound 34a.
1
Compound 34d was obtained as a yellow amorphous (68% yield). H NMR (DMSO-d ) δ 1.39 (9H, s),
6
1
.51 (9H, s), 2.09 (3H, s), 2.85 (4H, s), 3.18 (3H, s), 4.06 (2H, s), 7.13 (2H, d, J = 8.0 Hz), 7.37‒ 7.45 (1H,
m), 7.41 (2H, d, J = 8.0 Hz), 7.56 (1H, t, J = 8.0 Hz), 7.74‒ 7.80 (2H, m), 9.94 (1H, s), 11.44 (1H, s), 12.05
+
(
1H, s); FAB MS m/e (M+H) 672.
5
.1.50. Di-tert-butyl
(Z)-[(4-{2-[2-(acetylamino)-5-benzyl-1,3-thiazol-4-yl]ethyl}phenyl)amino]methylidene}biscarbamate
34e).
Compound 34e was prepared from 33e and 11 according to the same procedure as that of compound 34a.
{
(

1
Compound 34e was obtained as an off-white amorphous (85% yield). H NMR (DMSO-d ) δ 1.39 (9H, s),
6
1
.51 (9H, s), 2.07 (3H, s), 2.85 (4H, s), 3.89 (2H, s), 7.05‒ 7.33 (7H, m), 7.42 (2H, d, J = 8.5 Hz), 9.95 (1H,
+
s), 11.44 (1H, s), 11.99 (1H, s); FAB MS m/e (M+H) 594.
5
.1.51.
N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-5-[4-(methylsulfonyl)phenyl]-1,3-thiazol-2-yl}
acetamide hydrochloride (35a).
A mixture of 34a (130 mg) and 4 M HCl/dioxane (2 mL) was stirred at room temperature for 13 h under a
nitrogen atmosphere. The solvent was removed in vacuo. The residue was solidified with AcOEt to give
1
3
5a (83 mg, 85%) as an off-white solid. Mp 228‒ 229.5 ˚C; H NMR (DMSO-d ) δ 2.18 (3H, s), 2.99 (4H,
6
br s), 3.25 (3H, s), 7.11 (2H, d, J = 8.0 Hz), 7.22 (2H, d, J = 8.0 Hz), 7.38 (3H, br s), 7.57 (2H, d, J = 8.0
+
Hz), 7.94 (2H, d, J = 8.0 Hz), 9.79 (1H, s), 12.36 (1H, br s); FAB MS m/e (M+H) 458.
5
.1.52.
N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-5-[4-(methylsulfanyl)benzyl]-1,3-thiazol-2-yl}
acetamide (35b).
A mixture of 34b (200 mg) and 4 M HCl/dioxane (8 mL) was stirred at room temperature for 2 h under a
nitrogen atmosphere. The solvent was removed in vacuo, and the residue was dissolved in H O and AcOEt.
2
The solution was made basic (pH=8) by saturated aqueous NaHCO . The precipitate was filtered in vacuo
3
1
to give 35b (31 mg, 23%) as a colorless solid. H NMR (DMSO-d ) δ 2.07 (3H, s), 2.44 (3H, s), 2.72‒ 2.84
6
(4H, m), 3.85 (2H, s), 6.73 (2H, d, J = 8.5 Hz), 6.99 (2H, d, J = 8.5 Hz), 7.05 (2H, d, J = 8.5 Hz), 7.17 (2H,
+
+
d, J = 8.5 Hz); FAB MS m/e (M+H) 440; HRMS (ESI). calcd for C H N OS (M+H) : 440.1579, found:
2
2
26
5
2
4
40.1581.
5
.1.53.
N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-5-[4-(methylsulfonyl)benzyl]-1,3-thiazol-2-yl}
acetamide (35c).

Compound 35c was prepared from 34c according to the same procedure as that of compound 35b.
1
Compound 35c was obtained as a pale yellow solid (100% yield). Mp 228‒ 229.5 ˚C; H NMR (DMSO-d )
6
δ 2.08 (3H, s), 2.79 (4H, m), 3.18 (3H, s), 4.05 (2H, s), 6.72 (2H, d, J = 8.0 Hz), 6.99 (2H, d, J = 8.0 Hz),
+
7
.37 (2H, d, J = 8.5 Hz), 7.84 (2H, d, J = 8.5 Hz); FAB MS m/e (M+H) 472; HRMS (ESI) calcd for
+
C H N O S (M+H) : 472.1477, found: 472.1471; Anal. Calcd for C H N O S ·0.4H O: C, 55.19; H,
22
26
5
3
2
22 25
5
3
2
2
5
.43; N, 14.63; S, 13.39. Found: C, 55.19; H, 5.49; N, 14.71; S, 13.34.
5
.1.54.
N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-5-[3-(methylsulfonyl)benzyl]-1,3-thiazol-2-yl}
acetamide (35d).
Compound 35d was prepared from 34d according to the same procedure as that of compound 35b.
1
Compound 35d was obtained as an off-white solid (47% yield). Mp 212‒ 213.5 ˚C; H NMR (DMSO-d ) δ
6
2
.08 (3H, s), 2.67‒ 2.91 (4H, m), 3.19 (3H, s), 4.08 (2H, s), 6.80 (2H, d, J = 8.0 Hz), 7.04 (2H, d, J = 8.0
Hz), 7.42 (1H, d, J = 8.0 Hz), 7.57 (1H, t, J = 8.0 Hz), 7.77 (1H, s), 7.79 (1H, d, J = 8.0 Hz); FAB MS m/e
+
+
(
M+H) 472; HRMS (ESI) calcd for C H N O S (M+H) : 472.1477, found: 472.1481.
22 26 5 3 2
5
.1.55. N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-5-benzyl-1,3-thiazol-2-yl}acetamide
hydrochloride (35e).
Compound 35e was prepared from 34e according to the same procedure as that of compound 35a.
1
Compound 35e was obtained as a pale green solid (96% yield). Mp 97‒ 99 ˚C; H NMR (DMSO-d ) δ 2.09
6
(3H, s), 2.86 (4H, s), 3.93 (2H, s), 7.05‒ 7.37 (9H, m), 7.47 (3H, s), 9.98 (1H, s), 12.01 (1H, br s); FAB MS
+
m/e (M+H) 394.
5
.1.56. Ethyl 2-(acetylamino)-4-(chloromethyl)-1,3-thiazole-5-carboxylate (38).
Ethyl 4-chloro-3-oxobutanoate (36) (35.0 g) was dissolved in CH Cl (70 mL), and then sulfuryl chloride
2
2
(
17.1 mL) in CH Cl (20 mL) was added dropwise to the solution at 0 ˚C over 15 min under a nitrogen
2 2
atmosphere. The reaction mixture was stirred at room temperature for 3 h and concentrated in vacuo. A

mixture of the residual oil, N’-((E)-ethanoyl)carbamimidothioic acid (37) (25.1 g) and acetone (600 mL)
was refluxed for 2.5 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo.
i
The residual solid was washed with H O and Pr O to give 38 (21.2 g, 38%) as a pale yellow solid. Mp
2
2
1
1
64‒ 165 ˚C; H NMR (DMSO-d ) δ 1.30 (3H, t, J = 7.0 Hz), 2.19 (3H, s), 4.29 (2H, q, J = 7.0 Hz), 5.00
6
+
(
2H, s), 12.72 (1H, s); FAB MS m/e (M+H) 263.
5
.1.57. Ethyl 2-(acetylamino)-4-[(E)-2-(4-nitrophenyl)ethenyl]-1,3-thiazole-5-carboxylate (40).
To a stirring solution of 38 (1.0 g) in DMF (20 mL) was added triphenylphosphine (1.2 g) at room
temperature. The resultant mixture was stirred at 65 ˚C for 5 h. To the mixture was added potassium
tert-butoxide (555 mg) at 0 ˚C, and the resultant mixture was stirred at 0 ˚C for 30 min.
p-Nitrobenzaldehyde (39) (805 mg) was added at 0 ˚C. After stirring for an hour at room temperature, the
reaction mixture was quenched with H O, and the precipitate was collected to give 40 (1.0 g, 73%) as a
2
1
yellow solid. H-NMR (CDCl ), δ (ppm): 1.40 (3H, t, J = 7.2 Hz), 2.33 (3H, s), 4.38 (2H, q, J = 7.2 Hz),
3
7
.59 (1H, d, J = 16.0 Hz), 7.70 (2H, d, J = 8.8 Hz), 8.18 (1H, d, J = 16.0 Hz), 8.22 (2H, d, J = 8.8 Hz), 8.90
(1H, m).
5
.1.58. Ethyl 2-(acetylamino)-4-[2-(4-aminophenyl)ethyl]-1,3-thiazole-5-carboxylate (41).
Compound 40 (394 mg) was suspended in EtOH (4 mL) and THF (4 mL), and then 10% palladium on
carbon (305 mg) was added to the mixture under a nitrogen atmosphere. The reaction mixture was stirred at
®
room temperature for 3 h under a hydrogen atmosphere (4 atm), filtered through a celite pad, and the
filtrate was concentrated in vacuo. The residual amorphous was crystallized from Et O to give 41 (266 mg,
2
1
7
2
8
3%) as an off-white solid. Mp 184‒ 185 ˚C; H NMR (DMSO-d ) δ 1.27 (3H, t, J = 7.0 Hz), 2.17 (3H, s),
6
.73 (2H, t, J = 7.5 Hz), 3.17 (2H, t, J = 7.5 Hz), 4.23 (2H, q, J = 7.0 Hz), 4.84 (2H, br s), 6.47 (2H, d, J =
+
.5 Hz), 6.82 (2H, d, J = 8.5 Hz), 12.51 (1H, br s); FAB MS m/e (M+H) 334.
5
2
.1.59. Ethyl
-(acetylamino)-4-(2-{4-[(tert-butoxycarbonyl)amino]phenyl}-ethyl)-1,3-thiazole-5-carboxylate (42).

Compound 41 (310 mg) was dissolved in THF (6 mL) under a nitrogen atmosphere.
bis(tert-Butyl)dicarbonate (223 mg) in THF (1 mL) was then added to the solution at room temperature.
The reaction mixture was refluxed for 2 h. After cooling to room temperature, the mixture was concentrated
in vacuo. The residual solid was washed with Et O to give 42 (371 mg, 92%) as an off-white solid. Mp
2
1
2
13‒ 214 ˚C; H NMR (DMSO-d ) δ 1.26 (3H, t, J = 7.0 Hz), 1.46 (9H, s), 2.17 (3H, s), 2.85 (2H, t, J = 7.5
6
Hz), 3.23 (2H, t, J = 7.5 Hz), 4.22 (2H, q, J = 7.0 Hz), 7.04 (2H, d, J = 8.5 Hz), 7.33 (2H, d, J = 8.5 Hz),
+
9
.23 (1H, br s), 12.55 (1H, br s); FAB MS m/e (M+H) 434.
5
2
.1.60.
-(Acetylamino)-4-(2-{4-[(tert-butoxycarbonyl)-amino]phenyl}ethyl)-1,3-thiazole-5-carboxylic acid
(43)
1
M NaOH (17.5 mL) was added to a solution of 42 (3.0 g) in EtOH (30 mL) and the mixture was refluxed
for 5 h. After cooling to room temperature, the organic solvent was removed in vacuo. The aqueous
solution was acidified (pH=4) with 1 M HCl and extracted with AcOEt (twice). The combined organic
layer was dried over anhydrous MgSO and concentrated in vacuo. The residual solid (2.4 g) was dissolved
4
in pyridine (45 mL), and then acetyl chloride (1.5 mL) was added dropwise to the solution at 0 ˚C under a
nitrogen atmosphere. The reaction mixture was stirred at room temperature for 13 h, and pyridine was
removed in vacuo. H O was added to the residue, and acidified with 1 M HCl. The precipitate was
2
collected in vacuo. The solid was washed with H O and Et O to give 43 (2.7 g, 93%) as an off-white solid.
2
2
1
Mp 237‒ 238 ˚C; H NMR (DMSO-d ) δ 1.46 (9H, s), 2.16 (3H, s), 2.85 (2H, m), 3.23 (2H, m), 7.04 (2H, d,
6
-
J = 8.5 Hz), 7.33 (2H, d, J = 8.5 Hz), 9.24 (1H, s), 12.46 (1H, s); FAB MS m/e (M-H) 404.
5
.1.61. tert-Butyl (4-{2-[2-(acetylamino)-5-formyl-1,3-thiazol-4-yl]ethyl}phenyl)carbamate (44).
To a solution of 43 (8.3 g) in 44 mL of CH Cl and 44 mL of DMF was added N,O-dimethylhdroxylamine
2
2
hydrochloride (2.6 g), EDC (9.5 g) and HOBt (4.1 g). The solution was then stirred at room temperature for
3
1
h under a nitrogen atmosphere. The reaction mixture was diluted with 400 mL of AcOEt and washed with
00 mL×3 of H O. The organic layer was dried over anhydrous MgSO and evaporated in vacuo. The
2
4

i
residual solid was triturated with Pr O and collected by filtration to give tert-butyl
2
{
4-[2-(2-(acetylamino)-5-{[methoxy(methyl)amino]carbonyl}-1,3-thiazol-4-yl)ethyl]phenyl}carbamate
(7.0 g, 76%) as a pale yellow solid.
Lithium aluminum hydride (499 mg) was slowly added (over 15 min) at 0 °C under a nitrogen atmosphere
to a solution of the compound obtained in Step 1 (3.9 g) in THF (80 mL). The mixture was stirred at 0 ˚C
for an hour. 1 M aqueous potassium sodium tartrate solution (30 mL) was added slowly under ice-cooled
conditions, and then the mixture was stirred at room temperature for another 30 min. The mixture was
extracted with AcOEt, and the organic layer was dried over anhydrous MgSO , and then concentrated in
4
i
vacuo. This pale yellow oil was triturated with Pr O/AcOEt to give 44 (2.7 g, 78%, 2 steps yield:59%) as a
2
1
pale yellow powder. H NMR (DMSO-d ) δ 1.46 (9H, s), 2.19 (3H, s), 2.90 (2H, t, J = 7.3 Hz), 3.22 (2H, t,
6
J = 7.3 Hz), 7.01(2H, d, J = 8.5 Hz), 7.32 (2H, d, J = 8.5 Hz), 9.22 (1H, s), 9.77 (1H, s), 12.68 (1H, s); FAB
+
MS m/e (M+H) 390.
5
.1.62. A mixture of tert-butyl {4-[2-(2-acetamido-5-{(E)-2-[4-(methylsulfonyl)phenyl]vinyl}-1,3-
thiazol-4-yl)ethyl]phenyl}carbamate and tert-butyl {4-[2-(2-acetamido-5-{(Z)-2-[4-(methylsulfonyl)-
phenyl]vinyl}-1,3-thiazol-4-yl)ethyl]phenyl}carbamate (46).
To a mixture of 1-(chloromethyl)-4-(methylsulfonyl)benzene (45) (768 mg) in DMF (10 mL) was added
triphenylphosphine (943 mg) at 0 ˚C under a nitrogen atmosphere, and the reaction mixture was stirred at
6
5 ˚C for 2 h. Potassium tert-butoxide (461 mg) and 44 (400 mg) were then added to the solution at 0 ˚C,
and the mixture was stirred at room temperature for 30 h. The reaction mixture was poured into 1 M HCl
under ice-cooled conditions and extracted with AcOEt. The organic layer was washed with H O and brine,
2
dried over anhydrous MgSO , and concentrated in vacuo. The residue was purified by flash column
4
chromatography over silica gel with hexane/AcOEt (1:1‒ 1:2) as an eluent to give 46 (697 mg, 125%) as a
+
pale brown oil. FAB MS m/e (M+H) 542.
5
.1.63. tert-Butyl
N-{4-[2-(2-(acetylamino)-5-{2-[4-(methylsulfonyl)phenyl]ethyl}-1,3-thiazol-4-yl)ethyl]phenyl}carbam