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

Article abstract of DOI:10.1016/j.bmc.2012.12.025

Vascular adhesion protein-1 (VAP-1), an amine oxidase that is also known as a semicarbazide-sensitive amine oxidase (SSAO), is present in particularly high levels in human plasma, and is considered a potential therapeutic target for various inflammatory diseases, including diabetes complications such as macular edema. In our VAP-1 inhibitor program, structural modifications following high-throughput screening (HTS) of our compound library resulted in the discovery that thiazole derivative 10, which includes a guanidine group, shows potent human VAP-1 inhibitory activity (IC₅₀ of 230 nM; rat IC ₅₀ of 14 nM). Moreover, compound 10 exhibited significant inhibitory effects on ocular permeability in STZ-induced diabetic rats.

Full text of DOI:10.1016/j.bmc.2012.12.025

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
Takayuki Inoue, Masataka Morita, Takashi Tojo, Kousei Yoshihara, Akira
Nagashima, Ayako Moritomo, Mitsuru Ohkubo, Hiroshi Miyake
PII:
S0968-0896(12)00998-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.12.025
BMC 10491
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 December 2012
19 December 2012
20 December 2012
Please cite this article as: Inoue, T., Morita, M., Tojo, T., Yoshihara, K., Nagashima, A., Moritomo, A., Ohkubo,
M., 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, Bioorganic & Medicinal Chemistry (2012), doi: http://
dx.doi.org/10.1016/j.bmc.2012.12.025
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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
Takayuki Inouea *, Masataka Moritaa, Takashi Tojoa, Kousei Yoshiharaa, Akira Nagashimaa, Ayako
Moritomoa, Mitsuru Ohkubob and Hiroshi Miyakeb
a Drug Discovery Research, Astellas Pharma Inc.,21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
bAstellas Research Technology Inc.,21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
* Corresponding author.
Tel.: +81-29-829-6227;
fax: +81-29-854-1519;
e-mail: takayuki.inoue@astellas.com

Abstract
Vascular adhesion protein-1 (VAP-1), an amine oxidase that is also known as a semicarbazide-sensitive
amine oxidase (SSAO), is present in particularly high levels in human plasma, and is considered a potential
therapeutic target for various inflammatory diseases, including diabetes complications such as macular
edema.
In our VAP-1 inhibitor program, structural modifications following high-throughput screening (HTS) of
our compound library resulted in the discovery that thiazole derivative 10, which includes a guanidine
group, shows potent human VAP-1 inhibitory activity (IC50 of 230 nM; rat IC50 of 14 nM). Moreover,
compound 10 exhibited significant inhibitory effects on ocular permeability in STZ-induced diabetic rats.
Keywords
VAP-1; inhibitor; macular edema; thiazole; guanidine

1. Introduction
Amine oxidases (AOs) are classified by their active site cofactor. One group includes well-known flavin
adenine dinucleotide (FAD)-containing AOs such as monoamine oxidase (MAO)-A/MAO-B and
polyamine oxidase (PAO). The second group consists of copper-containing semicarbazide-sensitive amine
oxidases (SSAOs), which include diamine oxidase (DAO), lysyl oxidase (LO), and SSAO proteins as
soluble or membrane-associated forms. Vascular adhesion protein-1 (VAP-1) belongs to the SSAOs. There
are two types of VAP-1, a membrane binding type present in the vascular endothelium and a soluble type
present in the serum. The former serves as a molecule which adheres to white blood cells and lymphocytes
and is involved in inflammation,1 while the latter detoxifies amines in vivo by its amine oxidase activity. In
other words, VAP-1 has two different functions.
The 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
aminoacetone)2,3 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
(eq. 1).4
RCH2NH2 + H2O + O2 → RCHO + NH3 + H2O2 (eq. 1)
The reaction products are known to be cytotoxic and might therefore be associated with a number of
vasculopathies.5,6 Consistent with these results, an increase in plasma and/or membrane-associated VAP-1
derived from adipocytes and vascular tissue has been observed in many inflammation-associated diseases,
such as type 1 and type 2 diabetes mellitus,7-9 rheumatoid arthritis,1,10 inflammatory bowel disease,
atherosclerosis, chronic heart failure,11 and Alzheimer’s disease.12 These results suggest that VAP-1 may be
a promising therapeutic target for these conditions.
Additionally, it was recently reported that VAP-1 is expressed on the retinal vascular endothelium and
plays an important role in the recruitment of leukocytes on retinal vessels.13 These events are thought to
mark a breakdown in the blood–retinal barrier, leading to the edema seen in diabetic retinopathy. On this
basis, toxic products and leukocyte adhesion activity might be a cause of the pathological processes

occurring in 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.
Furthermore, untreated edema may lead to irreversible change in the macular region, and may exacerbate
the retinopathy.
Although therapies able to inhibit VAP-1 activity are not yet available, a number of compounds are under
development.14 While many compounds are still at the preclinical phase, Biotie Therapies (in partnership
with Roche) has developed a VAP-1 antibody, BTT-1023, for the treatment of chronic inflammatory
diseases.15 BTT-1023 was reported to be generally well tolerated without serious adverse events in multiple
dose studies for the treatment patients with psoriasis and rheumatoid arthritis.16 In addition, Biotie
Therapies and Proximagen are developing small-molecule SSAO-inhibitors for the treatment of vascular
inflammation.
The recently published X-ray structure of human VAP-1 may accelerate the drug discovery of small
molecules. For example, docking analyses of various hydrazine derivatives were recently reported using 3D
crystal structure of VAP-1 with bound inhibitor 2-hydrazinopyridine (PDB code 2C1117).18,19
Here, we describe the synthesis of novel VAP-1 inhibitors derived from high-throughput screening
(HTS) hit compound 9a, as well as their structure-activity relationships (SARs) for VAP-1 inhibition. In
addition, we also conducted a docking study of 10 based on the crystal structure of VAP-1, which
reinforced our SARs result. Finally, we confirmed the in vivo activity of compound 10 toward diabetic
macular edema.
2. Chemistry
The syntheses of N-(4-phenylethyl-1,3-thiazol-2-yl)acetamide derivatives 9a‒ c, 10, 11, 15a‒ c are
shown in Scheme 1. Cyclization of 3-chloro-2-oxopropyl acetate (1) with thiourea gave
(2-amino-1,3-thiazol-4-yl)methyl acetate hydrochloride (2). Acylation of 2-aminothiazole derivative 2 with

acetyl chloride gave acetamide derivative 3. Deprotection of the acetate group in compound 3 with
potassium carbonate in methanol gave hydroxymethyl derivative 4. The aldehyde derivative 5 was obtained
by oxidation of compound 4 with manganese (IV) oxide. Wittig reaction of compound 5 with
(4-nitrobenzyl)triphenylphosphonium bromide and subsequent hydrogenation in the presence of palladium
on carbon gave the aniline intermediate 8. Substitution reaction of compound 8 with appropriate
electrophiles afforded N-(4-phenylethyl-1,3-thiazol-2-yl)acetamide derivatives 9a‒ c. Guanidine derivative
10 was prepared by substitution reaction of the aniline 8 with cyanamide. Hydrolysis of compound 10 with
6 M hydrochloric acid at 90 ˚C gave the corresponding amine 11. Compound 8 was coupled with the
benzoyl isothiocyanate, followed by deprotection of the benzoyl group to afford the thiourea 13.
Methylation of 13 and subsequent substitution reaction with various amines yielded the desired compounds
15a‒ c.
The synthesis of N-(4-{2-[4-(2-aminoethyl)phenyl]ethyl}-1,3-thiazol-2-yl)acetamide (22) is outlined in
Scheme 2. Compound 16 was treated with triphenylphosphine to afford the phosphonium salt 17.
Compound 17 was coupled with the aldehyde 19, which was synthesized from aldehyde 18 and phthalimide,
followed by hydrogenation to provide phthalimide derivative 21. Deprotection of the phthalimide group of
compound 21 by hydrazine afforded the phenethylamine derivative 22.
N-(5-phenylethyl-1,3-thiazol-2-yl)acetamide derivative 31 and N-(phenethylphenyl)acetamide
derivatives 35a‒ b were synthesized as illustrated in Scheme 3. Bromination of
1,1,3,3-tetramethoxypropane (23) with bromine gave 2-bromomalonaldehyde (24), followed by cyclization
with N’-[(E)-ethanoyl]carbamimidothioic acid (25) in refluxing acetone to afford the aldehyde derivative
26. Compound 28 was synthesized from aldehyde 26 by the same method as that for the synthesis of
compound 8. Substitution of the aniline derivative 28 by
N,N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (29) provided the desired compound 30,
followed by deprotection of the tert-butoxycarbonyl (Boc) group to afford the guanidine derivative 31.
Guanidines 35a‒ b were obtained from commercially available benzaldehydes 32a‒ b by the same
procedure as that of compound 31.

The synthesis of the guanidine derivatives 43a‒ b was accomplished as outlined in Scheme 4.
Cyclization of commercially available ketoester 36 with thiourea in refluxing ethanol gave the
aminothiazole 37. Subsequent acylation of compound 37 with various acyl chlorides gave the amide
derivatives 38a‒ b. The ester groups of 38a‒ b were reduced with lithium borohydride to provide 39a‒ b,
and oxidation of 39a‒ b with manganese (IV) oxide gave 40a‒ b. Wittig reaction of 40a‒ b with
(4-nitrobenzyl)triphenylphosphonium bromide in N,N-dimethylformamide gave the olefin derivatives
41a‒ b. Compound 41b was converted to 41d via deprotection of the benzyloxycarbonyl (Cbz) group with
hydrochloric acid and subsequent acylation with benzoyl chloride. The guanidine derivatives 43a‒ b were
synthesized from compound 41a and 41d by the same method as that for the synthesis of compound 10.
Scheme 5 shows the synthesis of compounds 49 and 54. 2-Aminothiazole derivative (45) was prepared
from 5-(4-nitrophenyl)pentan-2-one (44) by bromination and subsequent cyclization. Compound 45 was
treated with acetyl chloride to afford the acetamide derivative 46. Hydrogenation of the nitro group of 46
furnished the amino derivative 47. The guanidine derivative 49 was synthesized from compound 47 by the
same method as that for the synthesis of compound 31. Bromination of compound 50 with bromine and
hydrobromic acid in acetic acid followed by treatment with thiourea in refluxing ethanol gave
2-aminothiazole derivative 51. The guanidine derivative 54 was synthesized from compound 51 by
acylation, hydrogenation of the nitro group and substitution with cyanamide.
The N-(4-phenyl-1,3-thiazol-2-yl)acetamide derivative 62 was prepared as shown in Scheme 6.
Friedel-Crafts reaction of N-(2-phenylethyl)acetamide (55) with chloroacetylchloride in the presence of an
aluminum chloride afforded chloroketone 56. Cyclization of compound 56 with thiourea gave the
2-aminothiazol derivative 57. Compound 57 had a protecting group (acetyl group) replaced with a Boc
group to give compound 59. Acylation of 2-aminothiazole derivative 59 with acetic anhydride and
4-dimethylaminopyridine gave acetamide derivative 60, followed by deprotection with 4 M hydrogen
chloride in ethyl acetate to give N-{4-[4-(2-aminoethyl)phenyl]-1,3-thiazol-2-yl}acetamide hydrochloride
(61). The guanidine derivative 62 was synthesized from compound 61 by the same method as that for the
synthesis of compound 31.

3. Results and discussion
The synthesized compounds were evaluated for in vitro inhibitory activity against human VAP-1. VAP-1
activity was determined by a radiochemical-enzyme assay using 14C-benzylamine as an artificial substrate.
The enzyme was prepared from human blood plasma as the source of VAP-1 for routine potency
measurements.
In our search for novel VAP-1 inhibitors, HTS of our compound library resulted in the discovery of a
novel series of thiazole derivatives. Among them, compound 9a showed inhibitory activity against human
VAP-1 with an IC50 value of 3.5 μM.
The SARs of the thiazoline moiety seen in compound 9a are summarized in Table1. The aniline
compound 8, which does not have a thiazoline ring, showed no activity, suggesting that the thiazoline
group plays an important role in VAP-1 inhibitory activity. Whereas a loss of activity was observed in
thiourea 13, S-methylisothiourea 14 showed 3-fold improved activity compared to 9a, indicating the
importance of the basicity of the amino group. Imidazoline 9b, in which the sulfur atom of thiazoline is
converted into a nitrogen atom, was equipotent to 9a. Ethylguanidine 15a, which is a ring opening analog
of 9b, showed an approximately 4-fold increase in in vitro human VAP-1 activity compared to 9b. Slightly
less activity was seen when the ethyl group of 15a was converted into a slightly bulky isopropyl group
(15b). On the other hand, a dramatic decrease in activity was observed with dimethylguanidine 15c. The
most potent of all compounds, though, was unsubstituted guanidine 10 (0.23 μM), which showed an
approximately 15-fold improvement in inhibitory activity compared to 9a. Replacement of the guanidine
moiety such as amidine 9c and aminoethyl 22, led to lower activity than guanidine 10. Accordingly, these
results indicate that the steric tolerance of the guanidine moiety is narrow, and that guanidine is important
for the VAP-1 inhibitory activity. Considering other reports,18,19 we propose that the guanidine compound
mimics a substrate Schiff base intermediate complex with the TPQ at the VAP-1 enzyme active center.

The SARs of the acetylaminothiazole moiety are shown in Table 2. The regioisomer of the thiazole ring
in compound 31 had less activity than compound 10. Because this decreased activity was also seen in
benzene derivatives 35a and 35b, this result suggests that the hetero atom on the thiazole ring might
interact with the VAP-1 enzyme.
Next, we investigated the effect of substituents on the amino group at the 2-position of the thiazole ring.
The results are summarized in Table 3. Replacement of acetyl group 10 with isopropionyl group 43a was
well tolerated. However, introduction of a more bulky group such as benzoyl 43b led to a severe loss of in
vitro human VAP-1 activity. Deletion of acetyl group 11 yielded a significant drop in activity. Therefore,
the amide group appears necessary and bulkiness is not tolerated at the 2-position of the thiazole ring.
Finally, we considered the linker unit connecting the thiazole ring and guanidine component. The results
are summarized in Table 4. First, we varied the distance between the thiazole ring and benzene ring.
Replacement of the ethylene linker 10 with propylene 49 or methylene 54 decreased the activity. On the
other hand, compound 62, which has the reverse phenetyl moiety of compound 10, caused minimal activity
change. These data suggest the importance of the distance between the thiazole ring and the guanidine
component. Consequently, it was suggested that this part served as a linker.
The crystal structure of human VAP-1 showed a homodimetric, with the active site located near the
dimerization interface buried deep within the protein and included a trihydroxyphenylalanine quinone
(TPQ) cofactor.17,20,21 The accessibility of the active site was restricted by Leu469, which blocked the inlet
to the ligand-binding site. In the crystal structure of VAP-1 with the bound inhibitor 2-hydrazinopyridine,
which was available from the PDB (code 2C1117), the primary amine group of the hydrazine bound
covalently to the TPQ cofactor. In addition, it was reported that in the case of inhibitors larger than
2-hydrazinopyridine, the side chain of Leu469 rotated and unblocked the ligand-binding cavity, providing
the ligands free access from the solvent side to the ligand-binding pocket.22,23 Based on this information, we
constructed a structure model of human VAP-1 with the side chain of Leu469 rotated. Compound 10 was
docked using GOLD version 5.0 (Figure 1). As a starting structure, the primary amine group of the
guanidine formed a Schiff base intermediate to the TPQ. In this model, the guanidine moiety formed a tight
hydrogen bond network with Asp386 except where it bound covalently to the TPQ 471, indicating the

importance of the guanidine moiety for activity. Furthermore, the sulfur atom in the thiazole ring formed an
S-O interaction with the Thr210 backbone carbonyl oxygen24 and the amide moiety formed a proton-π
interaction with Tyr176. These results are consistent with findings from SARs that the guanidine group,
thiazole ring, and amido group are all important parts for the VAP-1 inhibitory activity.
The inhibitory properties of 10, the most potent compound, were further evaluated against rat VAP-1 and
other AOs such as human/rat DAO and human MAO-B with a radiochemical-enzyme assay (Table 5).
Compound 10 showed a 16-fold improvement in rat VAP-1 inhibitory activity and more than 435-fold
selectivity for VAP-1 inhibitory activity over other AOs.
Compound 10 was also evaluated by an in vivo test. Its plasma concentration profile after subcutaneous
administration at doses of 0.1‒ 10 mg/kg in normal rats was confirmed (Figure 2A), and consequently it
was exposed dose-dependently in rat plasma. Inhibitory activity against plasma VAP-1 activity in
streptozotocin (STZ)-induced diabetic rats was sufficient at doses ≥ 0.1 mg/kg (Figure 2B). Based on the
PK/PD data, we considered that the efficacy in ex vivo is reasonable because the concentration of the
inhibitor in blood plasma is 21.8 ng/mL at a dose of 0.1 mg/kg, which is 5.1 times as high as the IC50 value
of 10. Additionally, the VAP-1 enzyme has narrow activity center because its intrinsic ligands are small
molecular amines. Therefore, once compound 10 forms a Schiff base with TPQ at the activity center, it
continues to stay inside the enzyme and the inhibitory activity lasts longer than the duration of its plasma
concentration. Finally, we evaluated ocular permeability in STZ-induced diabetic rats as a pathological
model of macular edema (Figure 2C). An inhibitory effect on ocular permeability was observed on
subcutaneous administration at 1 and 10 mg/kg, but no significant effects were observed on the animal’s
condition after repeated dose. The results confirm the success of this proof of concept of 10 as a VAP-1
inhibitor for macular edema.
4. Conclusion
We report the SARs of a series of thiazole derivatives and related analogues in the inhibition of VAP-1.
A SAR study based on the HTS hit compound 9a identified the key pharmacophore, and showed that a
sterically compact basic moiety is important for the strong inhibition of VAP-1. By optimizing compound

9a, we identified the importance of the guanidine moiety in compound 10. Molecular modeling showed that
the guanidine moiety forms a tight hydrogen bond network, that the sulfur atom in the thiazole ring forms
an S-O interaction, and that the amide moiety forms a proton-π interaction. Because these docking results
were closely related to our SAR data, this model should help advance further drug design. Finally, the
results for compound 10 in our pathological model of diabetic retinopathy demonstrate that VAP-1
inhibitors may be useful in the treatment of macular edema.
5. Experimental
5.1. Chemistry
1H NMR spectra were measured with a JEOL EX400 or GX500 spectrometer; chemical shifts are
expressed in  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 by Wakogel C-200 or
Merck Silica gel 60.
5.1.1. (2-Amino-1,3-thiazol-4-yl)methyl acetate hydrochloride (2).
A mixture of 3-chloro-2-oxopropyl acetate (1) (5.0 g) and thiourea (2.5 g) in EtOH (25 mL) was refluxed
for 4 h. The reaction mixture was cooled to room temperature and the resulting crystalline precipitate was
collected by filtration and washed with EtOH (20 mL) to give 2 (3.5 g, 51%) as a white solid. 1H NMR
(DMSO-d6) δ 2.07 (3H, s), 4.92 (2H, s), 6.87 (1H, s); FAB MS m/e (M+H)+ 173.
5.1.2. [2-(Acetylamino)-1,3-thiazol-4-yl]methyl acetate (3).
To a mixture of 2 (56 g) and pyridine (45 g) in CH2Cl2 (560 mL) was added acetyl chloride (23 g) over a
period of 30 min at 5 ˚C, and the reaction mixture was stirred for 10 min at the same temperature. The
reaction mixture was poured into H2O (500 mL) and extracted with CHCl3 (1 L). The organic layer was
dried over anhydrous Na2SO4 and concentrated in vacuo. The residual solid was collected by filtration with
iPr2O to give 3 (47 g, 82%) as a white solid. 1H NMR (DMSO-d6) δ 2.12 (3H, s), 2.29 (3H, s), 5.08 (2H, s),

6.93 (1H, s); FAB MS m/e (M+H)+ 215.
5.1.3. N-[4-(Hydroxymethyl)-1,3-thiazol-2-yl]acetamide (4).
A mixture of 3 (46 g) and K2CO3 (30 g) in MeOH (640 mL) was stirred for 3 h at room temperature. The
reaction mixture was concentrated in vacuo. The residue was diluted with CHCl3, and the insoluble
material was filtered off. The resulting solution was purified by flash column chromatography on silica-gel
with MeOH-CHCl3 (1:99) as an eluent and triturated with iPr2O to give 4 (35 g, 95%) as a white solid. 1H
NMR (DMSO-d6) δ 2.12 (3H, s), 4.44 (2H, d, J = 5.0 Hz), 5.20 (1H, t, J = 5.0 Hz), 6.88 (1H, s), 12.02 (1H,
brs); FAB MS m/e (M+H)+ 173.
5.1.4. N-(4-Formyl-1,3-thiazol-2-yl)acetamide (5).
Compound 4 (2.8 g) was dissolved in MeOH (10 mL) and CHCl3 (200 mL). Then manganese (IV) oxide
(28.3 g) was added to the solution under a nitrogen atmosphere. The reaction mixture was stirred at room
temperature for 7 h and filtered through a calcite® pad. The filtrate was concentrated in vacuo. The
resulting solid was washed with Et2O to give 5 (2.0 g, 73%) as an off-white solid. Mp 195.5–199 ˚C; 1H
NMR (DMSO-d6) δ 2.17 (3H, s), 8.28 (1H, s), 9.79 (1H, s), 12.47 (1H, brs).
5.1.5. N-{4-[(Z)-2-(4-Nitrophenyl)ethenyl]-1,3-thiazol-2-yl}acetamide (7).
To a mixture of 4-nitrobenzyl bromide (6) (1.9 g) in DMF (20 mL) was added triphenylphosphine (2.3 g) at
0 ˚C under a nitrogen atmosphere, and the reaction mixture was stirred at room temperature for 2.5 h. Then
potassium tert-butoxide (1.2 g) and 5 (1.5 g) were added at 0 ˚C, and the mixture was stirred at room
temperature for 14 h. The reaction mixture was poured into ice-H2O and extracted with AcOEt. The organic
layer was washed with 1 M HCl, H2O and brine, dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was purified by flash column chromatography over silica gel with Hexane-AcOEt (1:1–1:2) as
an eluent and triturated with Et2O to give 7 (1.6 g, 62%) as a yellow solid. Mp 155–157 ˚C; 1H NMR
(DMSO-d6) δ 2.13 (3H, s), 6.64 (1H, d, J = 12.5 Hz), 6.71 (1H, d, J = 12.5 Hz), 7.18 (1H, s), 7.79 (2H, d, J
= 9.0 Hz), 8.17 (2H, d, J = 9.0 Hz), 12.02 (1H, brs); FAB MS m/e (M+H)+ 290.

5.1.6. N-(4-(2-(4-Aminophenyl)ethyl)-1,3-thiazol-2-yl)acetamide (8).
A mixture of 7 (2.0 g) and 10% palladium on carbon (400 mg) in MeOH (25 mL), THF (25 mL) and AcOH
(18 mL) was stirred under hydrogen atmosphere (4 atm) at room temperature for 5 h. The reaction mixture
was filtered through a celite® pad, and the filtrate was concentrated in vacuo. The residue was dissolved in
AcOEt. The organic solution was washed with saturated aqueous NaHCO3 and brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography over silica
gel with Hexane-AcOEt (1:2–0:1) as an eluent and triturated with EtOH-Et2O to give 8 (540 mg, 30%) as
an off-white solid. Mp 102.5–104 ˚C; 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.75 (4H, brs), 4.82 (2H, s), 6.46
(2H, d, J = 8.5 Hz), 6.69 (1H, s), 6.83 (2H, d, J = 8.5 Hz), 12.07 (1H, brs); FAB MS m/e (M+H)+ 262;
HRMS (ESI) Calcd for C13H16N3OS (M+H)+: 262.1014, found: 262.1019.
5.1.7. N-(4-(2-(4-(4,5-Dihydro-1,3-thiazol-2-ylamino)phenyl)ethyl)-1,3-thiazol-2-yl)acetamide (9a).
A mixture of 8 (1.8 g), 2-(methylsulfanyl)-4,5-dihydro-1,3-thiazole (918 mg), concentrated HCl (0.57 mL)
and 2-methoxyethanol (28 mL) was stirred at 120 ˚C for 10 h. After cooling to room temperature, the
reaction mixture was concentrated in vacuo. The residue was dissolved in THF-H2O and made basic with
aqueous K2CO3. The mixture was extracted with AcOEt. The organic layer was dried over anhydrous
MgSO4, and evaporated in vacuo. The residue was purified by flash column chromatography over silica gel
with CHCl3-MeOH (30:1–20:1) as an eluent and triturated with AcOEt to give 9a (485 mg, 40%) as an
off-white solid. Mp 218–219.5 ˚C; 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.84 (4H, s), 3.26 (2H, t, J = 7.5
Hz), 3.35 (2H, t, J = 7.5 Hz), 4.02 (1H, brs), 6.71 (1H, brs), 7.05 (2H, d, J = 8.5 Hz), 7.51 (1H, brs), 9.25
(1H, brs), 12.10 (1H, brs); FAB MS m/e (M+H)+ 347; HRMS (ESI) Calcd for C16H19N4OS2 (M+H)+:
347.1000, found: 347.1003; Anal. Calcd for C16H18N4OS2: C, 55.47; H, 5.24; N, 16.17; S, 18.51. Found: C,
55.47; H, 5.28; N, 15.89; S, 18.03.
5.1.8. N-(4-{2-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)phenyl]-ethyl}-1,3-thiazol-2-yl)acetamide
(9b).

A mixture of 8 (65 mg), ethyl 2-(methylsulfanyl)-4,5-dihydro-1H-imidazole-1-carboxylate (56 mg), AcOH
(0.1 mL) and EtOH (0.9 mL) was stirred at 65 ˚C for 6 h and then refluxed for 5 h. The reaction mixture
was poured into AcOEt (5 mL) and saturated aqueous NaHCO3. The precipitated solid was filtered, and the
solid was dissolved in 50% MeOH-CHCl3. Insoluble materials were filtered, and the filtrate was
concentrated in vacuo. The resulting solid was collected and washed with AcOEt to give 9b (40 mg, 49%)
as an off-white solid. 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.72 (4H, s), 3.33 (4H, s), 6.73 (1H, s),
6.85–7.08 (4H, m); FAB MS m/e (M+H)+ 330; HRMS (ESI) Calcd for C16H20N5OS (M+H)+: 330.1389,
found: 330.1390.
5.1.9. N-[4-(2-{4-[(Iminomethyl)amino]phenyl}ethyl)-1,3-thiazol-2-yl]acetamide (9c).
Compound 8 (150 mg) was dissolved in MeOH (3 mL) under a nitrogen atmosphere. Then isopropyl
formimidate hydrochloride (567 mg) was added to the solution at room temperature, and the reaction
mixture was stirred at room temperature for 66 h. The precipitate was filtered off, and the filtrate was
concentrated in vacuo. The residue was purified by preparative thin-layer chromatography (NH silicagel,
Fuji Silysia Chemical Ltd.) with CHCl3-MeOH (20:1) as an eluent to give 9c (17 mg, 10%) as an off-white
solid. Mp 225–227 ˚C; 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.86 (4H, s), 6.71 (1H, s), 7.10 (1H, d, J = 2.0
Hz), 7.13 (2H, d, J = 8.5 Hz), 7.47 (2H, d, J = 8.5 Hz), 8.23 (1H, d, J = 2.0 Hz), 10.09 (1H, brs), 12.07 (1H,
brs); FAB MS m/e (M+H)+ 289; HRMS (ESI) Calcd for C14H17N4OS (M+H)+: 289.1123, found: 289.1105.
5.1.10. N-{4-[2-(4-{[Amino(imino)methyl]-amino}phenyl)ethyl]-1,3-thiazol-2-yl}acetamide (10).
To a suspension of 8 (26.0 g) in EtOH (500 mL) was added 4 M HCl-AcOEt (25 mL) and cyanamide (6.3
g). The mixture was refluxed for 26 h. The reaction mixture was cooled to room temperature and poured
into a mixture of AcOEt (500 mL) and saturated aqueous NaHCO3 (500 mL). The resulting precipitate was
collected by filtration and washed with H2O (300 mL) and EtOH (300 mL) to give 10 (18.0 g, 60%) as a
white solid. 1H NMR (DMSO-d6) δ 2.10 (3H, s), 2.85 (4H, s), 6.79 (1H, s), 6.83 (2H, d, J = 7 Hz), 7.10 (2H,
d, J = 7 Hz); FAB MS m/e (M+H)+ 304; HRMS (ESI) Calcd for C14H18N5OS (M+H)+: 304.1232, found:
304.1230.

5.1.11. 1-{4-[2-(2-Amino-1,3-thiazol-4-yl)ethyl]phenyl}guanidine dihydrochloride (11).
A mixture of 10 (200 mg) and 6 M HCl (2.2 mL) was heated at 90 ˚C for 6 h. After cooling to room
temperature, the mixture was concentrated in vacuo. The residual solid was washed with MeCN to give 11
(153 mg, 70%) as an off-white solid. 1H NMR (DMSO-d6) δ 2.79–2.97 (4H, m), 6.51 (1H, s), 7.16 (2H, d, J
= 8.5 Hz), 7.32 (2H, d, J = 8.5 Hz), 7.53 (3H, s), 9.23 (2H, s), 10.09 (1H, s); FAB MS m/e (M+H)+ 262;
HRMS (ESI) Calcd for C12H16N5S (M+H)+: 262.1126, found: 262.1128; Anal. Calcd for C12H15N5S·2HCl:
C, 43.12; H, 5.13; N, 20.95; S, 9.59; Cl, 21.21. Found: C, 42.60; H, 5.15; N, 20.56; S, 9.33; Cl, 21.39.
5.1.12. N-{4-[2-(4-{[(Benzoylamino)carbonothioyl]amino}phenyl)ethyl]-1,3-thiazol-2-yl}acetamide
(12).
To a mixture of 8 (300 mg) and acetone (5 mL) was added benzoyl isothiocyanate (187 mg) in an ice bath,
and the mixture was refluxed for 2 h and cooled to 0 ˚C. The precipitated solid was filtered and washed
with ice-cold acetone to give 12 (359 mg, 74%) as a yellow solid. 1H NMR (CDCl3) δ 2.25 (3H, s),
2.90–3.05 (4H, m), 6.51 (1H, s), 7.21 (2H, d, J = 7 Hz), 7.50–7.70 (5H, m), 7.89 (2H, d, J = 7 Hz), 9.03
(1H, s), 9.12 (1H, s); FAB MS m/e (M+H)+ 425.
5.1.13. N-[4-(2-{4-[(Aminocarbonothioyl)amino]phenyl}ethyl)-1,3-thiazol-2-yl]acetamide (13).
A mixture of 12 (200 mg), 6 M NaOH (0.19 mL) and EtOH (2 mL) was stirred at 60 ˚C for 2 h. The
reaction mixture was cooled to room temperature and neutralized with 1 M HCl (1.2 mL). The precipitated
solid was filtered and washed with water to give 13 (120 mg, 49%) as an off-white solid. Mp 191.5–192.5
˚C; 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.88 (4H, s), 6.75 (1H, s), 7.15 (2H, d, J = 7 Hz), 7.27 (2H, d, J =
7 Hz), 9.60 (1H, s); FAB MS m/e (M+H)+ 321; HRMS (ESI) Calcd for C14H17N4OS2 (M+H)+: 321.0844,
found: 321.0841.
5.1.14. Methyl N-{4-[2-(2-acetamido-1,3-thiazol-4-yl)ethyl]phenyl}carbamimidothioate hydroiodide
(14).

A mixture of 13 (123 mg), MeI (0.029 mL) and MeOH (2.5 mL) was refluxed for 3 h. The reaction mixture
was concentrated in vacuo. The residue was diluted with AcOEt and stirred for 30 min. The precipitated
solid was filtered and washed with AcOEt to give 14 (148 mg, 83%) as a pale yellow solid. 1H NMR
(DMSO-d6) δ 2.13 (3H, s), 2.68 (3H, s), 2.87–3.05 (4H, m), 6.75 (1H, s), 7.24 (2H, d, J = 7 Hz), 7.35 (2H,
d, J = 7 Hz); FAB MS m/e (M+H)+ 335; HRMS (ESI) Calcd for C15H19N4OS2 (M+H)+: 335.1000, found:
335.1001; Anal. Calcd for C15H18N4OS2·HI: C, 38.96; H, 4.14; N, 12.12; S, 13.87; I, 27.45. Found: C,
38.72; H, 4.22; N, 11.84; S, 13.81; I, 26.85.
5.1.15. N-{4-[2-(4-{[(Ethylamino)(imino)methyl]amino}phenyl)ethyl]-1,3-thiazol-2-yl}acetamide
(15a).
A mixture of 14 (50 mg), ethylamine (0.056 mL) and EtOH (1 mL) was stirred at room temperature for 20
h. The precipitated solid was filtered and washed with EtOH to give 15a (15 mg, 42%) as an off-white solid.
1H NMR (DMSO-d6) δ 1.13 (3H, t, J = 6 Hz), 2.11(3H, s), 2.70–3.00 (6H, m), 6.70 (1H, s), 6.77 (2H, d, J =
7 Hz), 7.17 (2H, d, J = 7 Hz); FAB MS m/e (M+H)+ 332.
5.1.16. N-(4-{2-[4-(N'-Isopropylcarbamimidamido)phenyl]ethyl}-1,3-thiazol-2-yl)acetamide (15b).
A mixture of compound 14 (47 mg), EtOH (0.93 mL) and 2-aminopropane (119 mg) was refluxed for 16 h
under a nitrogen atmosphere. After cooling to room temperature, the mixture was concentrated in vacuo.
The crude oil was purified by flash column chromatography over NH2-silica gel (Fuji Silysia Chemical
Ltd.) with CHCl3-MeOH (50:1-30:1) as an eluent. The yellow oil was solidified with MeCN to give 15b
(15 mg, 42%) as an off-white solid. 1H NMR (DMSO-d6) δ 1.08 (3H, s), 1.10 (3H, s), 2.11 (3H, s), 2.83
(4H, s), 3.77–3.88 (1H, m), 6.65 (2H, d, J = 8.5 Hz), 6.72 (1H, s), 7.00 (2H, d, J = 8.5 Hz); FAB MS m/e
(M+H)+ 346; HRMS (ESI) Calcd for C17H24N5OS (M+H)+: 346.1702, found: 346.1701.
5.1.17. N-(4-{2-[4-(N,N-Dimethylcarbamimidamido)phenyl]ethyl}-1,3-thiazol-2-yl)acetamide (15c).
Compound 15c was prepared from 14 and 2 M dimethylamine in THF solution according to the same
procedure as that of compound 15b. Compound 15c was obtained as an off-white solid (59% yield). 1H

NMR (DMSO-d6) δ 2.11 (3H, s), 2.84 (4H, s), 2.85 (6H, s), 6.61 (2H, d, J = 8.5 Hz), 6.72 (1H, s), 7.02 (2H,
d, J = 8.5 Hz); FAB MS m/e (M+H)+ 332; HRMS (ESI) Calcd for C16H22N5OS (M+H)+: 332.1545, found:
332.1544.
5.1.17. {[2-(Acetylamino)-1,3-thiazol-4-yl]methyl}(triphenyl)phosphonium chloride (17).
To a solution of N-[4-(chloromethyl)-1,3-thiazol-2-yl]acetamide (16) (23.6 g) in toluene (200 mL) and
MeCN (80 mL) was added triphenylphosphine (35.7 g) at 25 ˚C. The mixture was stirred at 130 ˚C for 12 h.
The resulting precipitate was collected by filtration and washed with iPr2O to give 17 (35.7 g, 64%) as a
colorless powder. 1H NMR (DMSO-d6) δ 2.11 (3H, s), 5.25 (2H, d, J = 15.3 Hz), 6.86 (1H, d, J = 3.8 Hz),
7.68-7.92 (15H, m), 12.06 (1H, s).
5.1.18. 4-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]benzaldehyde (19).
To a solution of phthalimide potassium salt (46.2 g) in DMF (300 mL) was added
4-(2-bromoethyl)benzaldehyde (18) (40.9 g) in DMF (50 mL) dropwise at 60 ˚C, and the mixture was
stirred for 2 h. The reaction mixture was cooled to 20 ˚C and then poured into H2O (1.5 L). The resulting
precipitate was collected by filtration to give a yellow solid. The solid was dissolved in CHCl3 (250 mL)
and insoluble material was removed by filtration. The filtrate was concentrated in vacuo. The residue was
washed with Et2O and collected by filtration to give 19 (19.7 g, 37%) as an off-white solid. 1H NMR
(DMSO-d6) δ 3.04 (2H, t, J = 7Hz), 3.88 (2H, t, J = 7 Hz), 7.44 (2H, d, J = 8.5 Hz), 7.75–7.89 (6H, m),
9.94 (1H, s); FAB MS m/e (M+H)+ 280.
5.1.19.
N-[4-((E)-2-{4-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]phenyl}vinyl)-1,3-thiazol-2-yl]acetam
ide (20).
Potassium tert-butoxide (12.8 g) was added to a mixture of compound 17 (46.9 g) and DMF (190 mL) at 0
˚C under a nitrogen atmosphere. The reaction mixture was stirred at 0 ˚C for 15 min, and 19 (19.28 g) was
added to the mixture at 0 ˚C. The reaction mixture was stirred at room temperature for 2 h and poured into

H2O. The resulting precipitate was collected by filtration to give a crude brown solid. The brown solid was
washed with iPr2O-MeCN (1:1) and then MeCN to give 20 (24.9 g, 86%) as a beige solid. 1H NMR
(DMSO-d6) δ 2.15 (3H, s), 2.94 (2H, t, J = 7.1 Hz), 3.83 (2H, t, J = 7.1 Hz), 7.12 (1H, d, J = 15.8 Hz), 7.14
(1H, d, J = 15.8 Hz), 7.16 (1H, s), 7.19 (2H, d, J = 8 Hz), 7.44 (2H, d, J = 8.4 Hz), 7.8–7.88 (4H, m), 12.22
(1H, s); FAB MS m/e (M+H)+ 418.
5.1.20.
N-[4-(2-{4-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]phenyl}ethyl)-1,3-thiazol-2-yl]acetamide
(21).
A mixture of 20 (24.9 g) and 10% palladium on carbon (50% wet) (24.4 g) in MeOH (80 mL), DMF (800
mL) and AcOH (8 mL) was stirred under hydrogen atmosphere (4 atm) at room temperature for 16 h. The
reaction mixture was filtered through a celite® pad, and the filtrate was concentrated in vacuo. The residue
was washed with iPr2O (200 mL) and purified by flash column chromatography over silica gel with
CHCl3-AcOEt (1:1) as an eluent, and triturated with iPr2O (200 mL) to give 21 (17.9 g, 71%) as an
off-white solid. 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.78–2.92 (6H, m), 3.79 (2H, t, J = 7.3 Hz), 6.66 (1H,
s), 7.08 (2H, d, J = 8.9 Hz), 7.1 (2H, d, J = 8.8 Hz), 7.79–7.89 (4H, m), 12.08 (1H, s); FAB MS m/e
(M+H)+ 420, (M+Na)+ 442.
5.1.21. N-(4-{2-[4-(2-Aminoethyl)phenyl]ethyl}-1,3-thiazol-2-yl)acetamide (22)
To a solution of 21 (2.1 g) in MeCN (20 mL) was added hydrazine monohydrate (2.4 mL), and the
mixture was stirred at 50 ˚C for 2 h. Volatiles were evaporated. To the mixture was added CHCl3 (10 mL),
and an insoluble material was removed by filtration. The residue was purified by flash column
chromatography over NH2-silica gel with CHCl3-MeOH (10:0–10:2) as an eluent to give 22 (1.1 g, 80%) as
a pale yellow solid. 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.58 (2H, t, J = 7.3 Hz), 2.72 (2H, t, J = 7.1 Hz),
2.81–2.94 (4H, m), 6.73 (1H, s), 7.08 (2H, d, J = 8.4 Hz), 7.11 (2H, d, J = 8.4 Hz); FAB MS m/e (M+H)+
290.

5.1.22. 2-Bromomalonaldehyde (24).
A solution of 1,1,3,3-tetramethoxypropane (23) (10 g) and concentrated HCl (0.43 mL) in H2O (11 mL)
was stirred at room temperature for 10 min. Br2 (3.1 mL) was added dropwise to the solution at room
temperature for more than 50 min. The reaction mixture was stirred at room temperature for 20 min and
concentrated in vacuo. The residual solid was washed with H2O to give 24 (3.6 g, 39%) as a yellow solid.
Mp 147–148 ˚C; 1H NMR (CDCl3) δ 4.73–4.80 (1H, m), 8.47 (2H, brs); FAB MS m/e (M-H)- 149.
5.1.23. N-(5-Formyl-1,3-thiazol-2-yl)acetamide (26).
To a solution of N’-[(E)-ethanoyl]carbamimidothioic acid (25) (2.7 g) in acetone (20 mL) was added 24
(3.5 g) under reflux. The reaction mixture was refluxed for an hour under a nitrogen atmosphere and cooled
to room temperature. The precipitate was collected in vacuo. The solid was washed with H2O and acetone,
and purified by flash column chromatography over silica gel with CHCl3-MeOH (20:1) as an eluent to give
26 (1.2 g, 31%) as an off-white solid. Mp 235–235.5 ˚C; 1H NMR (DMSO-d6) δ 2.21 (3H, s), 8.41 (1H, s),
9.95 (1H, s), 12.71 (1H, brs); FAB MS m/e (M-H)- 169.
5.1.24. N-{5-[(Z)-2-(4-Nitrophenyl)ethenyl]-1,3-thiazol-2-yl}acetamide (27).
Compound 27 was prepared from 26 and 4-nitrobenzyl bromide (6) according to the same procedure as that
of compound 7. Compound 27 was obtained as a yellow solid (24% yield). Mp 221–223 ˚C; 1H NMR
(DMSO-d6) δ 2.07 (3H, s), 6.63 (1H, d, J = 12.0 Hz), 6.92 (1H, d, J = 12.0 Hz), 7.55 (1H, s), 7.62 (2H, d, J
= 9.0 Hz), 8.24 (2H, d, J = 9.0 Hz), 12.16 (1H, brs); FAB MS m/e (M+H)+ 290.
5.1.25. N-{5-[2-(4-Aminophenyl)ethyl]-1,3-thiazol-2-yl}acetamide (28).
A mixture of 27 (1.0 g) and 10% palladium on carbon (1.0 g) in AcOEt (100 mL) and DMF (20 mL) was
stirred under hydrogen atmosphere (4 atm) at room temperature for 4 h. The reaction mixture was filtered
through a celite® pad, and the filtrate was concentrated in vacuo. The residue was purified by flash column
chromatography over silica gel with CHCl3-MeOH (30:1–20:1) as an eluent and triturated with Et2O to
give 28 (241 mg, 27%) as an off-white solid. Mp 218–219.5 ˚C; 1H NMR (DMSO-d6) δ 2.09 (3H, s), 2.70

(2H, t, J = 7.5 Hz), 2.92 (2H, t, J = 7.5 Hz), 4.85 (2H, s), 6.47 (2H, d, J = 8.5 Hz), 6.86 (2H, d, J = 8.5 Hz),
7.08 (1H, s), 11.86 (1H, brs); FAB MS m/e (M+H)+ 262.
5.1.26. Di-tert-butyl
{[(4-{2-[2-(Acetylamino)-1,3-thiazol-5-yl]ethyl}phenyl)amino]-methylidene}biscarbamate (30).
To a solution of 28 (100 mg) in DMF (1 mL) and THF (2 mL) was added
N,N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (29) (119 mg) under a nitrogen atmosphere,
and the mixture was stirred at 50 ˚C for 5.5 h. After cooling to room temperature, the reaction mixture was
concentrated in vacuo. The residue was purified by preparative silica gel column chromatography with
hexane-AcOEt (1:2) as an eluent to give 30 (94 mg, 49%) as a colorless solid. Mp 203–205 ˚C; 1H NMR
(DMSO-d6) δ 1.40 (9H, s), 1.51 (9H, s), 2.10 (3H, s), 2.87 (2H, t, J = 7.5 Hz), 3.02 (2H, t, J = 7.5 Hz), 7.11
(1H, s), 7.21 (2H, d, J = 8.5 Hz), 7.45 (2H, d, J = 8.5 Hz), 9.96 (1H, brs), 11.43 (1H, brs), 11.88 (1H, brs);
FAB MS m/e (M+H)+ 504.
5.1.27. N-{5-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-1,3-thiazol-2-yl}acetamide
hydrochloride (31).
A mixture of 30 (86 mg) and 4 M HCl-dioxane (2 mL) was stirred at room temperature for 8 h under a
nitrogen atmosphere. The solvent was removed in vacuo. The residue was washed with AcOEt to give 31
(60 mg, 103%) as a colorless solid. Mp 105–107 ˚C; 1H NMR (DMSO-d6) δ 2.11 (3H, s), 2.91 (2H, t, J =
7.5 Hz), 3.04 (2H, t, J = 7.5 Hz), 7.14 (1H, s), 7.14 (2H, d, J = 8.5 Hz), 7.32 (2H, d, J = 8.5 Hz), 7.46 (3H,
brs), 9.89 (1H, s), 11.95 (1H, brs); FAB MS m/e (M+H)+ 304; HRMS (ESI) Calcd for C14H18N5OS
(M+H)+ : 304.1232, found: 304.1233.
5.1.28. A mixture of N-{4-[(E)-2-(4-Nitrophenyl)vinyl]phenyl}acetamide and
N-{4-[(Z)-2-(4-Nitrophenyl)vinyl]phenyl}acetamide (33a).
Compound 33a was prepared from 32a and 4-nitrobenzyl bromide (6) according to the same procedure as
that of compound 7. Compound 33a was obtained as a pale brown wax (50% yield, E : Z =2 : 5). 1H NMR

(DMSO-d6) δ 2.03 (3Hx5/7, s), 2.07 (3Hx2/7, s), 6.65 (1Hx5/7, d, J = 12.5 Hz), 6.79 (1Hx5/7, d, J = 12.5
Hz), 7.16 (2Hx5/7, d, J = 9.0 Hz), 7.30 (1Hx2/7, d, J = 16.5 Hz), 7.47 (1Hx2/7, d, J = 16.5 Hz), 7.49
(2Hx5/7, d, J = 9.0 Hz), 7.61 (2Hx2/7, d, J = 9.0 Hz), 7.64 (2Hx2/7, d, J = 9.0 Hz), 7.83 (2Hx2/7, d, J = 9.0
Hz), 7.85 (2Hx5/7, d, J = 9.0 Hz), 8.13 (2Hx5/7, d, J = 9.0 Hz), 8.22 (2Hx2/7, d, J = 9.0 Hz); FAB MS m/e
(M+H)+ 283.
5.1.29. A mixture of N-{3-[(E)-2-(4-Nitrophenyl)vinyl]phenyl}acetamide and
N-{3-[(Z)-2-(4-Nitrophenyl)vinyl]phenyl}acetamide (33b).
Compound 33b was prepared from 32b and 4-nitrobenzyl bromide (6) according to the same procedure as
that of compound 7. Compound 33b was obtained as a yellow oil (74% yield, E : Z =2 : 3). 1H NMR
(DMSO-d6) δ 1.99 (3Hx3/5, s), 2.07 (3Hx2/5, s), 6.74 (1Hx3/5, d, J = 12.5 Hz), 6.85 (1Hx3/5, d, J = 12.5
Hz), 6.86 (1Hx3/5, d, J = 7.5 Hz), 7.21 (1Hx3/5, t, J = 7.5 Hz), 7.27 (1Hx2/5, d, J = 16.5 Hz), 7.34 (1Hx2/5,
t, J = 7.5 Hz), 7.37 (1Hx2/5, t, J = 7.5 Hz), 7.47 (2Hx3/5, d, J = 8.5 Hz), 7.47–7.90 (2H, m), 7.51 (1Hx2/5,
d, J = 16.5 Hz), 7.90 (2Hx2/5, d, J = 8.5 Hz), 8.12 (2Hx3/5, d, J = 8.5 Hz), 8.23 (2Hx2/5, d, J = 8.5 Hz),
9.87 (1Hx3/5, s), 10.01 (1Hx2/5, s); FAB MS m/e (M+H)+ 283.
5.1.30. N-{4-[2-(4-Aminophenyl)ethyl]phenyl}acetamide (34a).
A mixture of 33a (1.7 g) and 10% palladium on carbon (641 mg) in MeOH (34 mL) and THF (17 mL) was
stirred at room temperature for 4 h under hydrogen atmosphere (3 atm). The mixture was filtered through a
celite® pad, and the filtrate was concentrated in vacuo. The residual solid was washed with MeCN to give
34a (743 mg, 49%) as a brown solid. 1H NMR (DMSO-d6) δ 2.01 (3H, s), 2.75 (4H, s), 6.80 (2H, d, J = 8.5
Hz), 7.03 (2H, d, J = 8.5 Hz), 7.09 (2H, d, J = 8.5 Hz), 7.46 (2H, d, J = 8.5 Hz), 7.53 (2H, brs), 9.88 (1H,
s); FAB MS m/e (M+H)+ 255.
5.1.31. N-{3-[2-(4-Aminophenyl)ethyl]phenyl}acetamide (34b).
Compound 34b was prepared from 33b according to the same procedure as that of compound 34a.
Compound 34b was obtained as an off-white amorphous (93% yield). 1H NMR (DMSO-d6) δ 2.02 (3H, s),

2.65‒ 2.78 (4H, m), 5.38 (2H, brs), 6.5 3(2H, d, J = 8.5 Hz), 6.86 (1H, d, J = 7.5 Hz), 6.89 (2H, d, J = 8.5
Hz), 7.16 (1H, t, J = 7.5 Hz), 7.38–7.44 (2H, m), 9.85 (1H, s); FAB MS m/e (M+H)+ 255.
5.1.32. N-{4-[2-(4-Carbamimidamidophenyl)ethyl]phenyl}acetamide hydrochloride (35a).
To a solution of 34a (200 mg) in DMF (2 mL) and THF (4 mL) was added
N,N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (29) (366 mg) under a nitrogen atmosphere,
and the mixture was stirred at 60 ˚C for 7 h. After cooling to room temperature, the mixture was
concentrated in vacuo. The residue was purified by flash column chromatography over silica gel with
CHCl3-MeOH (40:1–20:1) as an eluent to give tert-butyl
{(Z)-({4-[2-(4-acetamidophenyl)ethyl]phenyl}amino)[(tert-butoxycarbonyl)amino]methylene}carbamate
(276 mg) as a colorless amorphous. A mixture of di-Boc compound (276 mg) and 4 M HCl-dioxane (2.8
mL) was stirred at room temperature for 14 h under a nitrogen atmosphere. The solvent was removed in
vacuo. The residue was washed with MeCN to give 35a (82 mg, 45%) as a colorless solid. 1H NMR
(DMSO-d6) δ 2.02 (3H, s), 2.78‒ 2.91 (4H, m), 7.13 (2H, d, J = 8.5 Hz), 7.14 (2H, d, J = 8.5 Hz), 7.29 (2H,
d, J = 8.5 Hz), 7.41 (3H, brs), 7.48 (2H, d, J = 8.5 Hz), 9.82 (1H, s), 9.90 (1H, s); FAB MS m/e (M+H)+
297; HRMS (ESI) Calcd for C17H21N4O (M+H)+: 297.1715, found: 297.1714; Anal. Calcd for
C17H20N4O·1.3HCl·H2O·0.8C2H3N: C, 56.61; H, 6.56; N, 17.04; Cl, 11.68.
Found: C, 56.61; H, 6.37; N, 17.37; Cl, 11.75.
5.1.33. N-{3-[2-(4-carbamimidamidophenyl)ethyl]phenyl}acetamide hydrochloride (35b).
Compound 35b was prepared from 34b and N,N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine
(29) according to the same procedure as that of compound 35a. Compound 35b was obtained as a colorless
solid (48% yield). 1H NMR (DMSO-d6) δ 2.02 (3H, s), 2.79‒ 2.93 (4H, m), 6.92 (1H, d, J = 7.5 Hz), 7.14
(2H, d, J = 8.5 Hz), 7.18 (1H, t, J = 7.5 Hz), 7.30 (2H, d, J = 8.5 Hz), 7.36 (1H, d, J = 7.5 Hz), 4.42 (3H,
brs), 7.47 (1H, s), 9.82 (1H, s), 9.92 (1H, s); FAB MS m/e (M+H)+ 297; HRMS (ESI) Calcd for C17H21N4O
(M+H)+: 297.1715, found: 297.1711; Anal. Calcd for C17H20N4O·1.1HCl·0.4H2O: C, 59.41; H, 6.42; N,
16.30; Cl, 11.35. Found: C, 59.57; H, 6.40; N, 16.67; Cl, 11.04.

5.1.34. Ethyl 2-amino-1,3-thiazole-4-carboxylate hydrobromide (37).
A mixture of 36 (100 g), thiourea (39 g) and EtOH (500 mL) was refluxed for 2 h. The reaction mixture
was concentrated in vacuo. The crystalline residue was collected and washed with AcOEt to give 37 (116 g,
131%) as a pale yellow solid. 1H NMR (DMSO-d6) δ 1.28 (3H, t, J = 7 Hz), 4.26 (2H, q, J = 7 Hz), 7.60
(1H, s).
5.1.35. Ethyl 2-(iso-butyrylamino)-1,3-thiazole-4-carboxylate (38a).
To a mixture of 37 (2.0 g), pyridine (1.3 mL) and CH2Cl2 (20 mL) was added i-butyryl chloride (0.91 mL)
in an ice-water bath, and the mixture was stirred for 30 min. To the mixture was then added saturated
aqueous NaHCO3 (30 mL) and the organic layer was separated, dried over Na2SO4 and concentrated in
vacuo. The crystalline residue was collected and washed with AcOEt to give 38a (1.3 g, 70%) as an
off-white solid. 1H NMR (CDCl3) δ 1.30 (6H, d, J = 7 Hz), 1.40 (3H, t, J = 7 Hz), 2.57‒ 2.73 (1H, m), 4.41
(2H, q, J = 7 Hz), 7.83 (1H, s), 8.98 (1H, s); FAB MS m/e (M+H)+ 243.
5.1.36. Ethyl 2-{[(benzyloxy)carbonyl]amino}-1,3-thiazole-4-carboxylate (38b).
To a mixture of 37 (5.0 g), pyridine (3.4 mL) and CH2Cl2 (50 mL) was added benzyloxycarbonyl chloride
(3.1 mL) in an ice-water bath, and the reaction mixture was stirred at room temperature for an hour. The
mixture was washed with saturated aqueous NaHCO3 (30 mL), dried over anhydrous Na2SO4 and
concentrated in vacuo. The crystalline residue was collected and washed with iPr2O to give 38b (5.1 g,
84%) as an off-white solid. 1H NMR (CDCl3) δ 1.48 (3H, t, J = 7 Hz), 4.38 (2H, q, J = 7 Hz), 5.27 (2H, s),
7.36‒ 7.44 (5H, m), 7.82 (1H, s); FAB MS m/e (M+H)+ 307.
5.1.37. N-[4-(Hydroxymethyl)-1,3-thiazol-2-yl]-2-methylpropanamide (39a).
To a mixture of 38a (1.4 g) and THF (28 mL) was added lithium borohydride (252 mg) portionwise, and
the mixture was refluxed for 6 h. The reaction mixture was cooled to 0 ˚C, quenched with MeOH (5 mL)
and concentrated in vacuo. The residue was suspended with 10% MeOH-CHCl3 (100 mL), and the

insoluble materials were filtered. The filtrate was purified by flash column chromatography on silica-gel
with 5% MeOH-CHCl3 as an eluent. The crystalline residue was collected and washed with iPr2O to give
39a (1.0 g, 86%) as an off-white solid. 1H NMR (CDCl3) δ 1.32(6H, d, J = 5 Hz), 2.58‒ 2.73 (1H, m), 4.68
(2H, s), 6.82 (1H, s); FAB MS m/e (M+H)+ 200.
5.1.38. Benzyl 4-(hydroxymethyl)-1,3-thiazol-2-ylcarbamate (39b).
Compound 39b was prepared from 38b and lithium borohydride according to the same procedure as that of
compound 39a. Compound 39b was obtained as an off-white solid (93% yield). 1H NMR (CDCl3) δ 4.56
(2H, s), 5.27 (2H, s), 6.80 (1H, s), 7.30‒ 7.46 (5H, m); FAB MS m/e (M+H)+ 265.
5.1.39. N-(4-Formyl-1,3-thiazol-2-yl)-2-methylpropanamide (40a).
A mixture of 39a (520 mg), manganese (IV) oxide (2.26 g), MeOH (0.5 mL) and CHCl3 (5 mL) was stirred
at room temperature for 18 h. The reaction mixture was filtered through a celite® pad, and the filtrate was
concentrated in vacuo. The crystalline residue was collected and washed with iPr2O to give 40a (365 mg,
71%) as a yellow solid. 1H NMR (CDCl3) δ 1.13 (6H, d, J = 5 Hz), 2.60‒ 2.77 (1H, m), 7.86 (1H, s); FAB
MS m/e (M+H)+ 199.
5.1.40. Benzyl 4-formyl-1,3-thiazol-2-ylcarbamate (40b).
Compound 40b was prepared from 39b and manganese (IV) oxide according to the same procedure as that
of compound 40a and obtained as an off-white solid (73% yield). 1H NMR (CDCl3) δ 5.29 (2H, s),
7.35‒ 7.45 (5H, m), 7.81 (1H, s), 9.80 (1H, s); FAB MS m/e (M+H)+ 263.
5.1.41. A mixture of 2-methyl-N-{4-[(E)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-yl}propanamide
and 2-methyl-N-{4-[(Z)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-yl}propanamide (41a).
Compound 41a was prepared from 40a and 4-nitrobenzyl bromide (6) according to the same procedure as
that of compound 7 and obtained as a yellow solid (64% yield, E : Z =1 : 2). 1H NMR (CDCl3) δ (ppm):
1.25 (6x2/3H, d, J = 5 Hz), 1.30 (6x1/3H, d, J = 5 Hz), 2.50‒ 5.70 (1H, m), 6.63 (2Hx2/3H, s), 6.79

(1Hx2/3H, s), 6.97 (1Hx1/3H, s), 7.14 (1x1/3H, d, J = 15 Hz), 7.33 (1x1/3H, d, J = 15 Hz), 7.53 (2x2/3H, d,
J = 7 Hz), 7.62 (2x1/3H, d, J = 7 Hz), 8.13 (2x2/3H, d, J = 7 Hz), 8.22 (2x1/3H, d, J = 7 Hz); FAB MS m/e
(M+H)+ 318.
5.1.42. Benzyl 4-[(E)-2-(4-nitrophenyl)ethenyl]-1,3-thiazol-2-ylcarbamate (41b)
Compound 41b was prepared from 40b and 4-nitrobenzyl bromide (6) according to the same procedure as
that of compound 7 and obtained as a yellow solid (76% yield). FAB MS m/e (M+H)+ 382.
5.1.43. 4-[(E)-2-(4-Nitrophenyl)ethenyl]-1,3-thiazol-2-amine (41c).
A mixture of 41b (2.7 g) and 6 M HCl (50 mL) was refluxed for 3 h. After cooling to room temperature,
the mixture was concentrated in vacuo. To the residue were added AcOEt and saturated aqueous NaHCO3.
The precipitated solid was filtered and washed with AcOEt and H2O to give 41c (1.3 g, 77%) as a yellow
solid. Mp 278‒ 278.5 ˚C; 1H NMR (DMSO-d6) δ 7.02 (1H, s), 7.33 (2H, s), 7.77 (2H, d, J = 8.5 Hz), 8.25
(2H, d, J = 8.5 Hz); FAB MS m/e (M+H)+ 248.
5.1.44. N-{4-[(E)-2-(4-Nitrophenyl)ethenyl]-1,3-thiazol-2-yl}benzamide (41d).
To a mixture of 41c (300 mg) and N,N-dimethylaniline (4 mL) was added benzoyl chloride (0.31 mL)
dropwise under a nitrogen atmosphere, and the reaction mixture was stirred at 110 ˚C for 2 h. After cooling
to room temperature, the mixture was diluted with AcOEt. The organic solution was washed with 1 M HCl,
H2O, saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, and concentrated in vacuo. The
residual solid was washed with Et2O to give 41d (299 mg, 70%) as a yellow solid. Mp 224.5-225 ˚C; 1H
NMR (DMSO-d6) δ 7.40 (1H, d, J = 16.0 Hz), 7.45 (1H, s), 7.53 (1H, d, J = 16.0 Hz), 7.56 (2H, t, J = 7.0
Hz), 7.66 (1H, t, J = 7.0 Hz), 7.84 (2H, d, J = 8.5 Hz), 8.13 (2H, d, J = 7.0 Hz), 8.23 (2H, d, J = 8.5 Hz),
12.80 (1H, brs); FAB MS m/e (M+H)+ 352.
5.1.45. N-{4-[2-(4-Aminophenyl)ethyl]-1,3-thiazol-2-yl}-2-methylpropanamide (42a).
A mixture of 41a (333 mg), 10% palladium on carbon (33 mg), AcOH (1 mL), MeOH (2 mL) and THF (2

mL) was stirred under hydrogen atmosphere (4 atm) at room temperature for 5 h. The catalyst was filtered,
and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography on
silica gel with 5% MeOH-AcOEt as an eluent. The residual solid was collected and washed with iPr2O to
give 42a (260 mg, 86%) as an off-white amorphous. 1H NMR (CDCl3) δ 1.38 (6H, d, J = 5 Hz), 2.57‒ 2.73
(1H, m), 2.39‒ 2.43 (4H, m), 6.45 (1H, s), 6.62 (2H, d, J = 7 Hz), 6.97 (2H, d, J = 7 Hz); FAB MS m/e
(M+H)+ 290.
5.1.46. N-{4-[2-(4-Aminophenyl)ethyl]-1,3-thiazol-2-yl}benzamide (42b).
A mixture of 41d (285 mg), 10% palladium on carbon (162 mg), AcOH (1 mL), MeOH (3 mL) and THF (3
mL) was stirred under hydrogen atmosphere (4 atm) at room temperature for 4 h. The reaction mixture was
filtered through a celite® pad, and the filtrate was concentrated in vacuo. A mixture of the residual oil, 10%
palladium on carbon (162 mg), AcOH (1 mL), MeOH (3 mL) and THF (3 mL) was stirred under hydrogen
atmosphere (4 atm) at room temperature for 3 h. The reaction mixture was filtered through a celite® pad,
and the filtrate was concentrated in vacuo. The residue was dissolved in AcOEt. The organic solution was
washed with saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4 and concentrated in
vacuo to give 42b (238 mg, 91%) as an off-white amorphous. 1H NMR (CDCl3) δ 2.82 (4H, s), 3.57 (2H,
brs), 6.53 (1H, s), 6.61 (2H, d, J = 8.0 Hz), 6.92 (2H, d, J = 8.0 Hz), 7.50 (2H, t, J = 7.0 Hz), 7.60 (1H, t, J
= 7.0 Hz), 7.93 (2H, d, J = 7.0 Hz), 10.15 (1H, brs); FAB MS m/e (M+H)+ 324.
5.1.47.
N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-1,3-thiazol-2-yl}-2-methylpropanamide (43a).
A mixture of 42a (125 mg), cyanamide (26.1 mg), 4 M HCl-AcOEt (0.1 mL) and EtOH (2 mL) was stirred
at 100 ˚C for 72 h. The reaction mixture was concentrated in vacuo. To the residue were added AcOEt and
saturated aqueous NaHCO3. The precipitated solid was filtered and washed with AcOEt and H2O to give
43a (45 mg, 33%) as an off-white solid. 1H NMR (DMSO-d6) δ 1.01 (6H, d, J = 5 Hz), 2.62‒ 2.78 (1H, m),
2.83 (4H, s), 6.72 (2H, d, J = 7 Hz), 6.75 (1H, s), 7.04 (2H, d, J = 7 Hz); FAB MS m/e (M+H)+ 332.

5.1.48. N-{4-[2-(4-{[Amino(imino)methyl]amino}phenyl)ethyl]-1,3-thiazol-2-yl}benzamide
hydrochloride (43b).
A mixture of 42a (180 mg), cyanamide (23.4 mg), 4 M HCl-AcOEt (0.14 mL) and EtOH (4 mL) was
stirred at 100 ˚C for 72 h. The reaction mixture was concentrated in vacuo. To the residue were added
AcOEt and saturated aqueous NaHCO3. The precipitated solid was filtered and washed with AcOEt and
H2O. A mixture of the solid and 4 M HCl-dioxane (2 mL) was concentrated in vacuo. The residue was
washed with AcOEt to give 43a (165 mg, 74%) as an off-white solid. Mp 229‒ 232 ˚C; 1H NMR
(DMSO-d6) δ 2.91‒ 3.05 (4H, m), 6.88 (1H, s), 7.15 (2H, d, J = 8.5 Hz), 7.32 (2H, d, J = 8.5 Hz), 7.44 (3H,
brs), 7.54 (2H, t, J = 7.5 Hz), 7.64 (1H, t, J = 7.5 Hz), 8.10 (2H, d, J = 7.5 Hz), 9.88 (1H, s); FAB MS m/e
(M+H)+ 366; HRMS (ESI) Calcd for C19H20N5OS (M+H)+: 366.1389, found: 366.1382.
5.1.49. 4-[3-(4-Nitrophenyl)propyl]-1,3-thiazol-2-amine (45).
To a solution of 5-(4-nitrophenyl)pentan-2-one (44) (4.0 g) in MeOH (35 mL) was added Br2 (1.0 mL), and
the mixture was stirred at room temperature for 2 h. Then to the reaction mixture were added thiourea (1.5
g) and K2CO3 (6.7 g), and the mixture was stirred at 50 ˚C for 2 h. Volatiles were concentrated in vacuo.
The residue was dissolved in H2O (50 mL) and THF (15 mL), extracted with AcOEt (50 mL, 3 times),
washed with brine (50 mL), dried over anhydrous MgSO4 and evaporated in vacuo. The residue was
purified by flash column chromatography over silica gel with CH2Cl2-MeOH (100:0–100:1.5) as an eluent
to give 45 (855 mg, 17%) as a pale brown solid. 1H NMR (DMSO-d6) δ 1.78‒ 2.01 (2H, m), 2.41 (2H, t, J
= 7.4 Hz), 2.73 (2H, t, J = 7.6 Hz), 6.13 (1H, s), 6.81 (2H, s), 7.49 (2H, d, J = 8.7 Hz), 8.15 (2H, d, J = 8.8
Hz); FAB MS m/e (M+H)+ 264.
5.1.50. N-{4-[3-(4-Nitrophenyl)propyl]-1,3-thiazol-2-yl}acetamide (46).
Compound 45 (847 mg) was dissolved in CH2Cl2 (8.5 mL) under a nitrogen atmosphere. Then pyridine
(0.83 mL) and acetyl chloride (0.37 mL) were added dropwise to the solution at 0 ˚C. The reaction mixture
was stirred at room temperature for 30 min. The organic solution was washed with 1 M HCl, H2O and brine,
dried over anhydrous MgSO4, and concentrated in vacuo. The solid was washed with AcOEt-Et2O (1:1) to

give 46 (704 mg, 72%) as a pale yellow solid. 1H NMR (DMSO-d6) δ 1.87‒ 2.06 (2H, m), 2.61 (2H, t, J =
7.4 Hz), 2.75 (2H, t, J = 7.6 Hz), 6.77 (1H, s), 7.5 (2H, d, J = 8.6 Hz), 8.16 (2H, d, J = 8.7 Hz), 12.03 (1H,
s); FAB MS m/e (M+H)+ 306.
.
5.1.51. N-{4-[3-(4-Aminophenyl)propyl]-1,3-thiazol-2-yl}acetamide (47).
Compound 46 (100 mg) and 10% palladium on carbon (50% wet) (98 mg) in MeOH (2 mL), THF (2 mL)
and AcOH (0.3 mL) were stirred under hydrogen atmosphere (3 atm) at room temperature for 5 h. The
reaction mixture was filtered through a celite® pad, and the filtrate was concentrated in vacuo. The residue
was dissolved in AcOEt. The organic solution was washed with saturated aqueous NaHCO3 and brine,
dried over anhydrous MgSO4, and concentrated in vacuo to give 47 (94 mg, 105%) as pale yellow oil. FAB
MS m/e (M+H)+ 276.
5.1.52. Di-tert-butyl
{(Z)-[(4-{3-[2-(acetylamino)-1,3-thiazol-4-yl]propyl}phenyl)amino]methylidene}biscarbamate (48).
To a solution of 47 (90 mg) in THF (1 mL) was added 29 (152 mg), and the mixture was stirred at room
temperature for 14 h. Volatiles were evaporated in vacuo, and the residue was purified by preparative TLC
(0.5 mm×2, CHCl3-MeOH (15:1), then 0.5 mm×1, AcOEt) to give 48 (142 mg, 84%) as an off-white
amorphous. 1H NMR (CDCl3) δ 1.50 (9H, s), 1.53 (9H, s), 1.86‒ 2.07 (2H, m), 2.22 (3H, s), 2.62 (2H, t, J =
8 Hz), 2.66 (2H, t, J = 8 Hz), 6.53 (1H, s), 7.12 (2H, d, J = 8.4 Hz), 7.48 (2H, d, J = 8.4 Hz), 10.26 (1H, s),
11.64 (1H, s); FAB MS m/e (M+H)+ 518, (M+Na)+ 540.
5.1.53. N-{4-[3-(4-{[Amino(imino)methyl]amino}phenyl)propyl]-1,3-thiazol-2-yl}acetamide
hydrochloride (49).
A mixture of 48 (140 mg) and 4 M HCl-dioxane (2 mL) was stirred at room temperature for 38 h under a
nitrogen atmosphere. The solvent was removed in vacuo to give 49 (81 mg, 85%) as a pale yellow solid. 1H
NMR (DMSO-d6) δ 1.86‒ 1.98 (2H, m), 2.11 (3H, s), 2.57‒ 2.65 (4H, m), 6.75 (1H, s), 7.15 (2H, d, J = 8.3
Hz), 7.27 (2H, d, J = 8.3 Hz), 7.41 (4H, s), 9.82 (1H, s), 12.03 (1H, s); FAB MS m/e (M+H)+ 318; HRMS

(ESI) Calcd for C15H20N5OS (M+H)+: 318.1389, found: 318.1387.
5.1.54. 4-(4-Nitrobenzyl)-1,3-thiazol-2-amine hydrobromide (51).
To a solution of 4-nitrophenylacetone (50) (5.0 g) in AcOH (10 mL) and 48% aqueous HBr (5 mL) was
added a solution of Br2 (9.0 g) in AcOH (8 mL) at 0 ˚C. The reaction mixture was stirred at room
temperature for 4 h. Then acetone (50 mL) was added to the solution, and the reaction mixture was stirred
at room temperature for 14 h. The mixture was concentrated in vacuo, diluted with brine, and extracted
with CH2Cl2 (twice). Anhydrous MgSO4 and SiO2 were added to the combined CH2Cl2 extracts, which
were then filtered in vacuo. The filtrate was concentrated in vacuo to give
1-bromo-3-(4-nitrophenyl)acetone (11.6 g) as a brown solid. To a solution of
1-bromo-3-(4-nitrophenyl)acetone (11.6 g) in EtOH (232 mL) was added thiourea (3.4 g). The reaction
mixture was refluxed for 3 h. After cooling to room temperature, the mixture was concentrated in vacuo.
The residual solid was washed with MeCN to give 51 (6.3 g, 2 steps yield:72%) as an off-white solid. 1H
NMR (DMSO-d6) δ 4.07 (2H,s), 6.53 (1H, s), 7.56 (2H, d, J = 9.0 Hz), 8.22 (2H, d, J = 9.0 Hz), 9.04 (2H,
brs); FAB MS m/e (M+H)+ 236.
5.1.55. N-[4-(4-Nitrobenzyl)-1,3-thiazol-2-yl]acetamide (52).
Compound 51 (2.0 g) was suspended in CH2Cl2 (20 mL) under a nitrogen atmosphere. Then pyridine (2.3
mL) and acetyl chloride (0.68 mL) were added dropwise to the solution at 0 ˚C. The reaction mixture was
stirred at room temperature for 18 h. The organic solution was washed with 1 M HCl, H2O, saturated
aqueous NaHCO3 and brine, dried over anhydrous MgSO4, and concentrated in vacuo. The residual solid
was washed with MeCN to give 52 (949 mg, 54%) as an off-white solid. 1H NMR (DMSO-d6) δ 2.08
(3H,s), 4.10 (2H,s), 6.91 (1H, s), 7.50 (2H, d, J = 9.0 Hz), 8.17 (2H, d, J = 9.0 Hz), 12.06 (1H, s); FAB MS
m/e (M+H)+ 278.
5.1.56. N-[4-(4-Aminobenzyl)-1,3-thiazol-2-yl]acetamide (53).
Compound 52 (500 mg) was suspended in MeOH (10 mL) and DMF (2.5 mL). Then 10% palladium on

carbon (360 mg) was added to the solution under a nitrogen atmosphere. The reaction mixture was stirred at
room temperature for 2 h under hydrogen atmosphere (3 atm) and filtered through a celite® pad. The
filtrate was concentrated in vacuo. The residue was solidified with MeCN-Et2O to give 53 (400 mg, 90%)
as an off-white solid. 1H NMR (DMSO-d6) δ 2.08 (3H,s), 3.73 (2H,s), 5.15 (2H, brs), 6.51 (2H, d, J = 8.5
Hz), 6.65 (1H, s), 6.89 (2H, d, J = 8.5 Hz), 12.00 (1H, s); FAB MS m/e (M+H)+ 248.
5.1.57. N-[4-(4-Carbamimidamidobenzyl)-1,3-thiazol-2-yl]acetamide (54).
Compound 54 was prepared from 53 and cyanamide according to the same procedure as that of compound
43a. Compound 54 was obtained as an off-white solid (77% yield). 1H NMR (DMSO-d6) δ 2.08 (3H,s),
3.83 (2H,s), 6.73 (1H, s), 6.77 (2H, d, J = 8.0 Hz), 7.09 (2H, d, J = 8.0 Hz); FAB MS m/e (M+H)+ 290;
HRMS (ESI) Calcd for C13H16N5OS (M+H)+: 290.1076, found: 290.1075.
5.1.58. N-{2-[4-(2-Chloroacetyl)phenyl]ethyl}-acetamide (56).
Aluminium chloride (1.6 g) was dissolved in 1,2-dichloroethane (15 mL). Chloroacetylchloride (0.73 mL)
was added to the mixture at 0 ˚C, and the mixture was stirred additionally for 20 min followed by the
dropwise addition of N-(2-phenylethyl)acetamide (55) (1.0 g) in 1,2-dichloroethane (5 mL). The mixture
was stirred at room temperature for an hour and then poured into ice-H2O. It was then extracted with CHCl3,
washed with H2O and brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The solid was
washed with AcOEt and Et2O, and dried in vacuo to give 56 (1.2 g, 80%) as a white powder. 1H NMR
(DMSO-d6) δ 1.98 (3H, s), 2.90‒ 2.94 (2H, m), 3.55‒ 3.60 (2H, m), 4.70 (2H, s), 5.66 (1H, br), 7.34 (2H, d,
J = 6 Hz), 7.92 (2H, d, J = 6 Hz).
5.1.59. N-{2-[4-(2-Amino-1,3-thiazol-4-yl)phenyl]ethyl}acetamide hydrochloride (57).
Compound 56 (1.1 g) and thiourea (505 mg) were dissolved in EtOH (20 mL). The mixture was refluxed
for an hour and cooling to room temperature. The white solid was collected with filtration and washed with
EtOH to give 57 (1.2 g, 90%) as a white solid. 1H NMR (DMSO-d6) δ 1.78 (3H, s), 2.70‒ 2.76 (2H, m),
3.23‒ 3.30 (2H, m), 7.16 (1H, s), 7.30 (2H, d, J = 6 Hz), 7.69 (2H, d, J = 6 Hz), 7.93‒ 7.96 (2H, m); FAB