2-Substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)acetamides as BACE1 inhibitors: Synthesis, biological evaluation and?docking studies

Article abstract of DOI:10.1016/j.ejmech.2017.06.020

In this work, a series of 2-substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)acetamide derivatives were developed as β-secretase (BACE-1) inhibitors. Supported by docking study, a small library of derivatives were designed, synthesized and biologically evaluated in vitro. In addition, the selected compounds were tested with affinity (K_D) towards BACE-1, blood brain barrier (BBB) permeability and cytotoxicity. The studies revealed that the most potent analog 41 (IC₅₀ = 4.6 μM) with high predicted BBB permeability and low cellular cytotoxicity, could serve as a good lead structure for further optimization.

Full text of DOI:10.1016/j.ejmech.2017.06.020

Accepted Manuscript
2-Substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)acetamides as BACE1
inhibitors: Synthesis, biological evaluation and docking studies
Gang Yan, Lina Hao, Yan Niu, Wenjie Huang, Wei Wang, Fengrong Xu, Lei Liang,
Chao Wang, Hongwei Jin, Ping Xu
PII:
S0223-5234(17)30466-X
10.1016/j.ejmech.2017.06.020
EJMECH 9515
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 March 2017
Revised Date: 7 June 2017
Accepted Date: 10 June 2017
Please cite this article as: G. Yan, L. Hao, Y. Niu, W. Huang, W. Wang, F. Xu, L. Liang, C. Wang, H.
Jin, P. Xu, 2-Substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)acetamides as BACE1 inhibitors:
Synthesis, biological evaluation and docking studies, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.06.020.
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European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com/locate/ejmech
Original artical
2-Substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)acetamides as
BACE1 inhibitors: Synthesis, biological evaluation and docking studies
Gang Yan a, 1, Lina Hao a, 1, Yan Niu a, *, Wenjie Huang a, Wei Wang a, Fengrong Xu a, Lei Liang a,
Chao Wang a, Hongwei Jin b, Ping Xu a, *
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing
100191, China
b State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, 38
Xueyuan Road, Beijing 100191, China
ARTICLE INFO
ABSTRACT
Article history:
In
this
work,
a
series
of
2-substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)acetamide derivatives were
developed as β-secretase (BACE-1) inhibitors. Supported by docking study, a small
library of derivatives were designed, synthesized and biologically evaluated in vitro. In
addition, the selected compounds were tested with affinity (KD) towards BACE-1, blood
brain barrier (BBB) permeability and cytotoxicity. The studies revealed that the most
potent analog 41 (IC50 = 4.6 µM) with high predicted BBB permeability and low cellular
cytotoxicity, could serve as a good lead structure for further optimization.
Keywords:
Alzheimer’s disease
BACE-1 inhibitors
Docking study
BBB
.
Permeability
Surface Plasmon Resonance (SPR)
PAMPA
1. Introduction
as hydroxyethylamine (HEA) and hydroxyethylene (HE)[13]
in the early years. These compounds had very good
potency in vitro, but most of them suffered from poor blood
Alzheimer’s disease (AD) is
a
neurodegenerative
disorder disease characterized by dementia and
neurofunctional impairment, which will finally lead to
death[1, 2]. The two pathological hallmarks which were
found generally in the brains of AD patients include
extracellular amyloid plaque, which is the deposition of
insoluble β-amyloid peptides (Aβ) derived from β-amyloid
precursor protein (APP)[3], and intracellular neurofibrillay
tangle (NFT) related with hyperphosphorylated tau
protein[4]. Based on the "Amyloid Hypothesis"[5, 6], the
production and aggregation of Aβ are highly related with
the pathology of AD. Aβ42, which is produced from APP
through successive cleavages by β-secretase (BACE-1)
and γ-secretase[7], is the major component of Aβ in
amyloid plaques[8]. BACE-1 catalyzes the initial step of Aβ
generation, therefore, has been regarded as a potential
target for the treatment of AD[9].
brain barrier (BBB) permeability and thus poor in vivo
activity[14]. In recent years, more researches were
dedicated to developing non-peptidomimetic inhibitors
targeting BACE-1, which had good in vitro activity, much
smaller
molecular
weight
and
better
BBB
permeability[15-17], and therefore became more druglike
agents for the treatment of AD. Among those inhibitors,
only a few of compounds had been advanced into the
clinic[17-20]. However, none of them had been approved
by FDA.
In this paper, a series of non-peptide BACE-1 inhibitors
bearing
the
scaffold
of
2-substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)
acetamide were designed and synthesized. The
bioactivities of these compounds including enzyme
inhibition, affinity (KD) towards BACE1, BBB permeability
and cytotoxicity towards HEK293 cell line were also
evaluated.
BACE-1 is an aspartyl protease located in the central
nervous system (CNS)[10-12], which makes it hard to
develop a drug towards it. In the last decade, scientists had
developed many inhibitors towards BACE-1. Some of the
programs were focused on peptidomimetic inhibitors such
* Corresponding author:
E-mail addresses: pingxu@bjmu.edu.cn (P. Xu), yanniu@bjmu.edu.cn (Y. Niu).
1 These two authors contributed equally.

ACCEPTED MANUSCRIPT
carbonyl group formed another H-bond with Thr72. The S1
pocket of BACE-1 was occupied by the phenyl group at
4-position of thiazole ring of 12. And the substituted
pyrimidine ring, which form three H-bonds with Tyr197 and
Arg128, was extended to S2’ pocket.
According to the above docking studies, the potency
towards BACE-1 might be achieved by the introduction of
proper substituents at phenyl ring in S1 pockets, which
might have key interaction with nearby residues and/or
extend into S3 pockets[24, 25]. A series of compounds with
different substituted phenyl ring at 4-position of pyrimidine
ring were also designed to find the most favorable
substituent. Asp32 and Asp 228 are two key amino acids in
the catalytic site.[26] However, 12 only formed one H-bond
with Asp228, while with no interaction with Asp32. Docking
studies showed that the distance between S atom of the
thiazole ring and O atom of Asp32 was 3.07 Å. It might form
another H-bond [27] by replacing –S– with a hydrogen
bond donor –NH–, which might increase the activity.
Fig. 1 Predicted binding mode of compound 12 in the active site
of BACE-1 (protein PDB ID: 3VF3)
2. Design
Based on in silico screening and biological evaluation, a
series of compounds had been found with some extent
activity towards BACE-1 in our previous work[21, 22].
Among them, compound 12 (IC50 = 37.4 µM) was chosen
as the hit compound for further optimization in this work.
Molecular docking studies are powerful tools of
Computer-Aided Drug Design (CADD) for designing new
analogues.[23] The docking studies of compound 12 (Fig.
1) showed the interactions mode between BACE-1 and 12.
The nitrogen atom outside the thiazole ring formed an
H-bond with catalytic Asp228, and nearby oxygen of the
3. Chemistry
As
shown
in
Scheme
1,
2-substituted-thio-N-(4-substituted thiazol-2-yl)acetamide
analogs 12-39 were prepared in 3-6 steps through
convergent synthetic strategy. Starting from three
commercially available materials thiourea 1, substituted
aldehydes 2a-2h and ethyl cyanacetate 3, compounds
Scheme 1. Synthesis of 2-thio-N-(thiazol-2-yl)acetamides derivatives. (Reagents and conditions: (a) K2CO3, EtOH, 80 °C, 6h; (b) Br2, CHCl3,
reflux, 15min; (c) HOP(OC2H5)2, TEA, THF, 0 °C to R.T., 10min; (d) thiourea, H2O, R.T., 2h; (e) ClCH2COCl, TEA or DIPEA, CH2Cl2, 0 °C to
R.T., 1-2h; (f) (2-fluoropyridin- 3-yl)boronic acid, Pd(PPh3)4 (5 mol%), Na2CO3 (2 equiv.), DMF, 120 °C, 24h; (g) CH3COCl, AcOH, 90 °C, 20min;
(h) KOH, EtOH, 70 °C, 3h.)
* Only 6g were prepared from 5g. Other chemicals from 6a to 6f and 6i are commercial available.

ACCEPTED MANUSCRIPT
reacting with chloroacetyl chloride. 8g was reacted with
acetyl chloride to obtain 8h. 7i was reacted with
(2-fluoropyridinyl-3-yl)boronic acid to obtain 7j, which was
later converted to 8j as mentioned above. The final
compounds 12-36 were generated through a nucleophilic
substitution between compounds 8a-8j and 4a-4h. In
addition, compound 8a could directly react with three
commercially available materials 4g, 4h or 4i to form three
derivitives 37-39, respectively.
The synthesis of 2-substituted-thio-N-(4-substituted-1H-
imidazol-2-yl)acetamide derivatives was shown in Scheme
2. Substituted bromoethanones 6a and 6g were
transformed into N-(1H-imidazol-2-yl)acetamides 9a and
9g by heating with N-acetylguanidine[32]. The
intermediates 2-chloro-N-(1H-imidazol -2-yl)acetamides
10a and 10g were prepared through hydrolysis reaction of
compounds 9a and 9g under acidic condition[33], followed
by reacting with chloroacetyl chloride. After that,
compounds 10a or 10g reacted with 4a or 4d to form the
final products 40-42.
Scheme 2. Synthesis of 2-thio-N-(1H-imidazol-2-yl)acetamides
derivatives. (Reagents and conditions: (a) N-acetylguanidine,
CH3CN, 100 °C, 3h; (b) HCl (40%) or H2SO4 (20%) in MeOH/H2O
(1:1), 100 °C, 1h; (c) ClCH2COCl, TEA, CH2Cl2, 0 °C, 1h; (d) KOH,
EtOH, 70 °C, 3h.)
4a-4h were prepared under base condition[28, 29]. Another
individual
1-(4-amino-3,5-dichlorophenyl) ethanone 5g, it was reacted
with Br2 to form
1-(4-amino-3,5-dichlorophenyl)-2-bromoethanone[30] 6g,
route
was
starting
from
4. Results and discussion
other
commercially
substituted
phenyl
bromoethanones
The substituted
were
phenyl
All
synthesized
available.
2-substituted-thio-N-(4-substituted-thiazol-2- yl)acetamide
derivatives were tested for BACE-1 inhibitory potencies
with data given in Table 1. As for the R1 substitutions on
2-substituted-thio-N-(4-substituted-thiazol-2-yl)acetamide
bromoethanone 6a - 6g and 6i were reacted with thiourea
to form 4-substituted-2-aminothiazole[31] 7a - 7g and 7j,
which
were
subsequently
converted
to
2-chloro-N-(thiazol-2-yl)acetamide 8a – 8g and 8i by
Table 1. BACE-1 inhibitory activities of 2-substituted-thio-N-(4-substituted-thiazol-2-yl)acetamide derivatives
Inhibition%
Inhibition%
BACE-1
Inhibition%
Inhibition%
BACE-1
NO.
R1
R2
NO.
R1
R2
(10 µM) a
(50 µM) a
IC50/µM a
(10 µM) a
(50 µM) a
IC50/µM a
12
13
14
15
16
17
18
19
20
21
22
Ph
m-MeOPh
o-MeOPh
p-MeOPh
o-MeOPh
m-MeOPh
p-MeOPh
o-MeOPh
m-MeOPh
p-MeOPh
m-MeOPh
m-EtOPh
10.8±5.5
11.7±1.7
6.3±0.1
50.7±3.7
25.9±0.8
10.7±4.1
77.5±8.5
83.9±1.5
2.1±13.6
62.4±7.1
78.5±3.7
7.5±2.0
37.4±1.2
n.d.b
26
27
28
29
30
31
32
33
34
35
36
p-MePh
p-MeOPh
m-EtOPh
m-EtOPh
m-EtOPh
m-MeOPh
m-EtOPh
m-CF3Ph
m-CF3Ph
m-CF3OPh
m-CF3OPh
m-MeOPh
m-MeOPh
-0.3±0.8
-3.0±0.6
24.5±8.7
0.3±3.6
2.5±0.4
16.8±1.1
13.0±1.8
6.8±1.6
6.1±2.7
14.3±1.4
26.5±0.3
11.5±1.0
9.4±2.5
n.d.
n.d.
Ph
Ph
n.d.
3,5-Cl2-4-NH2Ph
3,5-Cl2-4-AcNHPh
3,5-Cl2-4-AcNHPh
Ph
79.9±2.3
1.4±2.9
13.5±4.6
n.d.
p-NO2Ph
p-NO2Ph
p-NO2Ph
m-NO2Ph
m-NO2Ph
m-NO2Ph
o-NO2Ph
p-NO2Ph
19.3±0.7
31.1±1.9
5.9±2.9
27.6±2.2
26.1±3.9
n.d.
4.7±3.5
n.d.
82.5±9.4
86.1±0.6
85.8±2.3
39.6±1.7
90.4±3.1
88.7±2.4
19.1±5.9
13.4±0.6
29.6±6.6
n.d.
16.8±3.9
4.4±1.0
38.6±0.8
24.3±2.1
n.d.
3,5-Cl2-4-NH2Ph
Ph
12.3±2.6
-13.2±4.5
22.7±4.1
3,5-Cl2-4-NH2Ph
m-BrPh
6.5±5.0
n.d.
17.5±3.6
15.0±0.8
85.6±9.6
15.0±3.4
(2-F-Py- 3-yl)Ph
23
24
25
3,5-Cl2-4-NH2Ph
p-MePh
m-MeOPh
m-MeOPh
m-MeOPh
58.9±6.6
5.6±3.4
7.7±2.2
91.8±3.7
8.1±1.5
7.70±0.5
n.d.
37
38
39
1.7±2.5
3.6±2.5
-0.4±7.1
22.5±2.7
17.8±1.2
21.8±3.2
n.d.
n.d.
n.d.
p-MeOPh
16.5±2.7
n.d.
a Values are mean ± SD for BACE-1 inhibition (n ≥ 2).
b n.d. = not determined

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Table 2. BACE-1 inhibitory activities of 2-substituted-thio-
N-(4-substituted-1H-imidazol-2-yl)acetamide derivatives
Inhibition%
(1 µM) a
Inhibition%
BACE-1
IC50/µM a
4.2±0.5
4.6±1.1
NO.
R1
R2
(10 µM) a
78.6±1.8
84.5±0.7
40
41
Ph
Ph
m-MeOPh
m-EtOPh
16.8±1.1
17.9±1.8
42
3,5-Cl2-4-NH2Ph
m-MeOPh
26.4±3.2
87.7±3.5
3.2±0.6
a Values are mean ± SD for BACE-1 inhibition (n ≥ 2).
substituents at meta- position were designed and
synthesized, including –OCH3 (12, 16, 23), -OC2H5 (22, 28),
-CF3 (31, 32), -OCF3 (33, 34). But based on the enzyme
activities, there was no significant difference among these
substituents, except that compound 34 almost lose the
activity. Relatively, when R1 were non-substituted phenyl
rings, compound with –CF3 (31) was more favorable; while
when R1 were 3,5-Cl2-4-NH2Ph, R2 with m-OCH3Ph was
better. From the docking study of compound 12 (Fig. 1), we
could find that R2, -CN group and pyrimidine ring all had
important interactions with adjacent amino acid. From the
data in scheme 1, if R2 was replaced by -H (37) or -NH2
(38), or the -CN group was replaced by -H, or the
pyrimidine ring was replaced by 1,3,4-thiodiazole (39), they
would lose activities.
Fig. 2 Predicted binding mode of compound 23 (A) and 36 (B)
in the active site of BACE-1 (protein PDB ID: 3VF3)
Three
synthesized
derivatives, compounds with para- (16) or meta- (19)
substitution on the phenyl ring appeared to be more potent
than ortho- (21) position. As for the various R2, phenyl ring
with meta- position (12, 16, 19) substitution showed better
potency than ortho- position (13, 15, 18), which was more
potent than para- position (14, 17, 20). With this conclusion,
the following optimization of the hit compound 12 was
focused on p- and m- position of the phenyl ring of R1 and
m- position of the phenyl ring of R2. Specifically for R1
group, The compounds with p-NO2Ph (16, 22),
3,5-Cl2-4-NH2Ph (23, 28) substituents were more potent
than 12. While compounds with p-MeOPh (25, 27) and
p-MePh (24, 26) substituents almost had no activity
towards BACE-1. Docking result of 23 (Fig. 2A) suggested
that it had similar interaction mode to 12, while the phenyl
ring was rotated so that the chlorine atom could partly
extend into the S3 pocket, and the amino group had an
extra H-bond with Phe108 at S1 pocket, which might
contribute to the enhanced activity most. However, when
the amino group was acetylated (29, 30), the molecule
might be too large to occupy the pocket, this might be the
reason why these two compounds had no activity.
Meanwhile, the docking study of compound 23 (Fig. 2A)
suggested that the meta- position of phenyl ring could
tolerate bulkier group, so Br atom (35) and another
aromatic ring (36) at meta- site were designed and
synthesized, and their activities were increased compared
with hit compound 12. Docking of compound 36 (Fig. 2B)
showed that 36 had similar binding pose to 12, while the
meta- substituted aromatic ring was extended into S3
pocket, which might have more hydrophobic interaction
with BACE-1, and this could explain the enhancement of
activity. As for the phenyl ring of R2 group, different
2-substituted-thio-N-(4-substituted-1H-imidazol-2-yl)aceta
mide compounds were tested for BACE-1 inhibitory
potencies with data given in Table 2. The activities of
imidazole compounds 40 and 41 were 10 times more
potent than the similar thiazole series compounds 12.
Compound 42 was also more favorable compared with
compound 23. This significant activity enhancement could
be explained by docking study (Fig. 3). We could find out
that compound 40 had similar interaction mode to 12 with
BACE-1 when changing -S- to -NH-. While compound 40
had an additional H-bond between -NH- of the imidazole
ring and Asp32. Combined with the other H-bond between
amide proton and Asp228, compound 40 formed a stronger
Fig. 3 Predicted binding mode of compound 12 (yellow) and 40
(green) in the active site of BACE-1 (protein PDB ID: 3VF3)

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From the results in Table 3, Pe values of control drugs
Table 3. KD values, predicted BBB permeabilities and Cytotoxic
are in accord with the reference[35, 36], which indicates
that the assay used by us is reliable. From the results in
Table 3 and the ranges above, it could be concluded that
compounds that had larger polarity group such as NO2 (15,
16) or NH2 (23, 28, 32, 42) might have low BBB
permeability. In addition, we could find out that compound
12, 31 and 41 possessed great predicted BBB permeability.
However, the predicted permeabilities of other compounds
were uncertain or low.
profiles of selected compounds.
NO.
MW
KD/µM a
Pe/10-6cm s-1 b Predicition CC50/µM
Verapamil
-
n.d.c
16.2±7.1
5.9±0.37
2.8±2.8
CNS+
CNS+
CNS±
CNS-
n.d.
n.d.
n.d.
Clonidine
-
n.d.
Hydrocortisone
-
n.d.
n.d.
Theophylline
-
n.d.
0.13±0.001
n.d.
n.d.
Staurosporine
-
n.d.
0.00354
44.47
>50
The cytotoxicities of the sixteen active compounds and
positive control (Staurosporine) towards HEK293 cells were
measured, and the results were listed in Table 3. Almost all
the CC50 values were higher than 50 µM, suggesting these
BACE-1 inhibitors had low significant cytotoxic effects.
12
15
16
18
19
22
23
28
31
32
33
35
36
40
41
42
475.5
520.5
520.5
520.5
520.5
534.6
2.14±1.7
n.d.
9.1±1.5
CNS+
CNS-
CNS-
CNS±
CNS-
CNS-
CNS-
CNS-
CNS+
CNS-
CNS±
CNS-
CNS-
CNS±
CNS+
CNS-
1.7±0.01
0.42±0.11
3.1±1.2
1.84±0.62
1.43±1.2
1.75±0.74
1.23±0.96
>50
From all the data above (Table 3.), 42 had the most
favorable activity towards BACE-1 and low cytotoxicity, but
it showed poor predicted BBB permeability and high
molecular weight. Compound 41 had similar high activity
(IC50 = 4.6 µM) and affinity (KD = 0.213 µM), low cytotoxicity
(CC50 < 50 µM) compared with 42, it also showed greater
BBB permeability and lower molecular weight (MW =
472.52), which made it the most potent compound.
>50
0.46±0.03
0.73±0.10
0.11±0.01
0.07±0.02
4.6±0.17
0.88±0.19
3.2±0.61
0.94±0.10
0.04±0.02
2.8±0.47
7.1±2.7
>50
>50
559.5 0.861±0.37
>50
573.5
513.5
597.4
529.5
554.4
570.6
n.d.
>50
1.03±0.19
1.33±0.63
1.88±0.54
1.74±0.43
1.44±0.25
>50
47.73
>50
5. Conclusions
Guided
by
docking
study,
a
series
of
>50
2-substituted-thio-N-(4-substituted-thiazol/1H-imidazol-2-yl)
acetamide derivatives were designed, synthesized and
evaluated as BACE-1 inhibitors in enzymatic assay, SPR
affinity assay, in vitro BBB permeability assay and
cytotoxicity assay. Sixteen compounds were found to have
IC50s at µM level and KDs at µM to sub-µM level, and
compound 41 exhibited optimized drug-like profiles with the
relatively low IC50 of 4.6 µM, low molecular weight of 472,
high predicted BBB permeability and low toxicity towards
HEK293 cells, making it a good lead structure for further
modification.
>50
458.5 0.554±0.24
472.5 0.213±0.072
542.4 0.169±0.068
>50
>50
0.34±0.05
>50
a Values are mean ± SE for BACE-1 Affinity Assay (SPR assay, n ≥ 2).
b Values are mean ± SD for PAMPA Assay (n ≥ 2).
c n.d. = not determined
bidentate interaction with catalytic center of BACE1 than
compound 12. Compound 42 was the most potent
compound in BACE-1 inhibitory assay.
6. Experimental section
6.1. General chemical methods
The affinity of compounds towards BACE1 was
measured by Surface Plasmon Resonance (SPR) assay
(Table 3.). The affinity (KD) trends of these compounds
were following the same rule as that in the
Melting points were measured with an X4 apparatus and
1
were uncorrected. H NMR and 13C NMR spectra were
recorded on a Bruker (Bruker BioSpin AG, Fällanden,
Switzerland) Avance III 400 MHz system. Chemical shifts
were reported in parts permillion (ppm) relative to
tetramethylsilane (TMS) as internal standard. The spin
multiplicities were given as s (singlet), d (doublet), t (triplet),
enzyme-inhibitory assay above. The best affinity (KD
=
0.169 µM) was also obserbed from compound 42. The
affinities of other active compounds are at µM to sub-µM
level.
q
(quartet),
m (multiplet) or br (broad). MS and
BBB penetration capacity plays a key role in CNS
drugs[34]. The parallel artificial membrane permeability
assay (PAMPA)[35] was used to study the permeability of
target compounds through BBB. The in vitro permeability
(Pe) of sixteen active compounds and control drugs
(verapamil, hydrocortisone, clonidine and theophylline)
were detected. According to the reference[35], the
compounds can be classified:
high-resolution mass spectra (HRMS) were obtained using
Electrospray Ionization (ESI) technique on a Bruker’s
Fourier Transform Ion Cyclotron resonance Mass
Spectrometer. Thin layer chromatography (TLC) analysis
was performed on silica gel GF254 purchased from
Qingdao Haiyang Chemical Co. (Qingdao, Shandong
Province, China) or Merck (Darmstadt, Germany).
“CNS+” (high BBB permeation): Pe (10−6 cm s−1) > 4.0;
“CNS−” (low BBB permeation): Pe (10−6 cm s−1) < 2.0;
6.1.1. 6-(3-methoxyphenyl)-4-oxo-2-thioxo-1,2,3
,4-tetrahydropyrimidine-5-carbonitrile (4a)
To a vial was added thiourea (0.83 g, 11 mmol), ethyl
cyanoacetate (1.13 g, 11 mmol), 3-methoxybenzaldehyde
(1.36 g, 10 mmol), K2CO3 (1.52 g, 11 mmol) and EtOH (10
mL). The mixture was stirring at 80 °C for 6 h. After that, the
“CNS±” (BBB permeation uncertain): 2.0 < Pe (10−6 cm
s−1) < 4.0.

ACCEPTED MANUSCRIPT
reaction mixtures were poured into ice-water, then was
mmol), 3-(trifluoromethoxy)benzaldehyde (1.90 g, 10
adjusted to pH = 3~5 with 1 mol/L HCl. The precipitate was
filtered off, dried and then purified by column
chromatography on silica gel to give 4a as a yellowish solid
mmol) and K2CO3 (1.52 g, 11 mmol), and 4f was obtained
as a yellow solid (1.28 g, 41% yield). m.p. 189-190 °C; H
1
NMR (400 MHz, DMSO-d6) δ 13.44 (s, 1H), 13.23 (s, 1H),
7.77 – 7.69 (m, 3H), 7.69 – 7.61 (m, 1H); 13C NMR (100
MHz, DMSO-d6): δ 176.59, 159.73, 158.73, 148.28, 131.77,
131.23, 128.46, 124.92, 124.32, 122.04, 120.49 (q, J = 256
Hz), 114.77, 91.97; MS (ESI) m/z calcd for C12H6F3N3O2S-
[M - H+] 312.01, found 312.03.
1
(0.91 g, 35% yield). m.p. 232-233 °C; H NMR (400 MHz,
DMSO-d6): δ 13.29 (s, 1H), 13.18 (s, 1H), 7.49 (t, J = 7.5
Hz, 1H), 7.19 – 7.26 (m, 3H), 3.83 (s, 3H); HRMS (ESI) m/z
calcd for C12H8N3O2S- [M - H+] 258.0343, found 258.0390.
6.1.2. 6-(2-methoxyphenyl)-4-oxo-2-thioxo-1,2,3
,4-tetrahydropyrimidine-5-carbonitrile (4b)
6.1.7. 1-(4-amino-3,5-dichlorophenyl)-2-bromoe
thanone (6g)
The procedure described for 4a was used, starting from
thiourea (0.83 g, 11 mmol), ethyl cyanoacetate (1.13 g, 11
mmol), 2-methoxybenzaldehyde (1.36 g, 10 mmol) and
K2CO3 (1.52 g, 11 mmol), and 4b was obtained as a
To a vial was added chloroform (120 mL) and
1-(4-amino-3,5-dichlorophenyl)ethanone (6.12 g, 30 mmol).
The mixtures were heated to reflux and then bromine (2.04
ml, 39 mmol) was slowly added. The mixture was stirred for
15 min and then cooled to room temperature. The solvent
was evaporated to afford the crude product. Then crude
product was dissolved in THF (120 ml), and cooled to 0 °C.
1
yellowish solid (0.54g, 21% yield). m.p. 241-242 °C; H
NMR (400 MHz, DMSO-d6) δ 13.26 (s, 1H), 13.17 (s, 1H),
7.58 (ddd, J = 8.5, 7.4, 1.7 Hz, 1H), 7.47 (dd, J = 7.6, 1.7
Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.10 (td, J = 7.5, 0.9 Hz,
1H), 3.85 (s, 3H). MS (ESI) m/z calcd for C12H8N3O2S- [M -
H+] 258.03, found 258.18.
To the solution was slowly added
a
solution of
diethylphosphite (0.51 ml, 4 mmol) and triethylamine (TEA,
3.3 ml, 24 mmol) in THF (30 ml). The mixture was warmed
to room temperature and stirred for 10 min. After
completion of the reaction, the mixture was concentrated in
vacuo and poured into crushed ice. The precipitate was
filtered out, washed with water and air-dried to give the
pure product 6g as light brown solid (6.78 g, 80% yield).
6.1.3. 6-(4-methoxyphenyl)-4-oxo-2-thioxo-1,2,3
,4-tetrahydropyrimidine-5-carbonitrile (4c)
The procedure described for 4a was used, starting from
thiourea (0.83 g, 11 mmol), ethyl cyanoacetate (1.13 g, 11
mmol), 4-methoxybenzaldehyde (1.36 g, 10 mmol) and
K2CO3 (1.52 g, 11 mmol), and 4c was obtained as a
1
m.p. 140-141 °C; H NMR (400 MHz, DMSO-d6) δ 7.88 (s,
1
yellowish solid (0.90 g, 35% yield). m.p. 254-255 °C; H
2H), 6.60 (s, 2H), 4.79 (s, 2H); MS (ESI) m/z calcd for
C8H7BrCl2NO+ [M + H+] 281.91, found 281.88.
NMR (400 MHz, DMSO-d6) δ 13.19 (s, 1H), 13.11 (s, 1H),
7.66 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H), 3.85 (s,
3H). 13C NMR (100MHz, DMSO-d6): δ 176.64, 162.82,
160.92, 159.11, 131.34, 121.51, 115.53, 114.34, 90.26,
56.05. MS (ESI) m/z calcd for C12H8N3O2S- [M - H+] 258.03,
found 258.10.
6.1.8. 4-phenylthiazol-2-amine (7a)
To a solution of thiourea (1.67 g, 22 mmol) in water (100
ml) was added 2-bromo-1-phenylethanone (3.98 g, 20
mmol). The mixture was stirred at room temperature for 2h.
After the completion of the reaction, the mixture was filtered
to obtain the crude solid product. The crude product was
further purified by recrystalization using EtOH/H2O to afford
the pure product 7a as white acicular crystal (3.20 g, 91%
yield). m.p. 150-151 °C; 1H NMR (400 MHz, DMSO-d6) δ
8.61 (s, 2H), 7.82 – 7.57 (m, 2H), 7.57 – 7.28 (m, 3H), 7.22
(s, 1H); HRMS (ESI) m/z calcd for C9H9N2S+ [M + H+]
177.0481, found 177.1929.
6.1.4. 6-(3-ethoxyphenyl)-4-oxo-2-thioxo-1,2,3,
4-tetrahydropyrimidine-5-carbonitrile (4d)
The procedure described for 4a was used, starting from
thiourea (0.83 g, 11 mmol), ethyl cyanoacetate (1.13 g, 11
mmol), 3-ethoxybenzaldehyde (1.50 g, 10 mmol) and
K2CO3 (1.52 g, 11 mmol), and 4d was obtained as a
1
yellowish solid (0.93 g, 34% yield). m.p. 210-211 °C; H
NMR (400 MHz, DMSO-d6): δ 13.27 (s, 1H), 13.18 (s, 1H),
7.47 (t, J = 7.5 Hz, 1H), 7.17 – 7.24 (m, 3H), 4.11 (q, J = 7.5
Hz, 2H), 1.37 (t, J = 7.5 Hz, 3H); MS (ESI) m/z calcd for
C13H10N3O2S- [M - H+] 272.05, found 272.13.
6.1.9. 4-(4-nitrophenyl)thiazol-2-amine (7b)
The procedure described for 7a was used, starting from
2-bromo-1-(4-nitrophenyl)ethanone (4.88 g, 20 mmol) and
thiourea (1.67 g, 22 mmol), the recrystalization condition
was DMF/H2O, and 7b was obtained as an orange solid
(4.11 g, 93% yield). m.p. 285-286 °C; 1H NMR (400 MHz,
DMSO-d6) δ 8.23 (d, J = 9.0 Hz, 2H), 8.05 (d, J = 8.9 Hz,
2H), 7.41 (s, 1H), 7.22 (s, 2H); MS (ESI) m/z calcd for
C9H8N3O2S+ [M + H+] 222.03, found 222.10.
6.1.5. 4-oxo-2-thioxo-6-(3-(trifluoromethyl)phen
yl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(4e)
The procedure described for 4a was used, starting from
thiourea (0.83 g, 11 mmol), ethyl cyanoacetate (1.13 g, 11
mmol), 3-(trifluoromethyl)benzaldehyde (1.74 g, 10 mmol)
and K2CO3 (1.52 g, 11 mmol), and 4e was obtained as a
6.1.10. 4-(3-nitrophenyl)thiazol-2-amine (7c)
1
yellow solid (1.66 g, 56% yield). m.p. 224-225 °C; H NMR
The procedure described for 7b was used, starting from
2-bromo-1-(3-nitrophenyl)ethanone (4.88 g, 20 mmol) and
thiourea (1.67 g, 22 mmol), and 7c was obtained as yellow
acicular crystal (3.71 g, 84% yield). m.p. 189-190 °C; 1H
NMR (400 MHz, DMSO-d6) δ 8.61 (t, J = 2.0 Hz, 1H), 8.24
(ddd, J = 7.8, 1.7, 1.0 Hz, 1H), 8.11 (ddd, J = 8.2, 2.4, 1.0
Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.34 (s, 1H), 7.23 (s, 2H);
13CNMR (100 MHz, DMSO-d6): δ 168.58, 148.21, 147.37,
136.38, 131.49, 130.03, 121.64, 119.92, 104.23; MS (ESI)
m/z calcd for C9H8N3O2S+ [M + H+] 222.03, found 222.10.
(400 MHz, DMSO-d6) δ 13.47 (s, 1H), 13.26 (s, 1H), 8.08
(s, 1H), 8.04 – 7.91 (m, 2H), 7.82 (t, J = 7.9 Hz, 1H); MS
(ESI) m/z calcd for C12H6F3N3OS- [M - H+] 296.01, found
296.06.
6.1.6. 4-oxo-2-thioxo-6-(3-(trifluoromethoxy)phe
nyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(4f)
The procedure described for 4a was used, starting from
thiourea (0.83 g, 11 mmol), ethyl cyanoacetate (1.13 g, 11

ACCEPTED MANUSCRIPT
1
6.1.11. 4-(2-nitrophenyl)thiazol-2-amine (7d)
yield). m.p. 153-154 °C; H NMR (400 MHz, DMSO-d6) δ
8.27 (dt, J = 4.7, 1.5 Hz, 1H), 8.21 – 8.10 (m, 1H), 8.05 (s,
1H), 7.93 – 7.83 (m, 1H), 7.54 – 7.43 (m, 3H), 7.14 (s, 1H),
7.12 (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ 168.76,
160.12 (d, J = 235 Hz), 149.73, 147.00 (d, J = 15 Hz),
141.93 (d, J = 4 Hz), 135.86, 134.15 (d, J = 5 Hz), 129.44,
127.97, 126.44, 125.99, 123.47 (d, J = 28 Hz), 123.16 (d, J
= 4 Hz), 102.77; MS (ESI) m/z calcd for C14H11FN3S+ [M +
H+] 272.07, found 272.21.
The procedure described for 7a was used, starting from
2-bromo-1-(2-nitrophenyl)ethanone (4.88 g, 20 mmol) and
thiourea (1.67 g, 22 mmol), and 7d was obtained as orange
1
acicular crystal (4.07 g, 92% yield). m.p. 105-106 °C; H
NMR (400 MHz, DMSO-d6): δ 7.78 (dd, J = 7.5, 1.6 Hz,
1H), 7.73 (dd, J = 7.5, 1.6 Hz, 1H), 7.66 (dt, J = 7.5, 1.6 Hz,
1H), 7.53 (dt, J = 7.5, 1.6 Hz, 1H), 7.07 (s, 2H), 6.91 (s,
1H); MS (ESI) m/z calcd for C9H8N3O2S+ [M + H+] 222.03,
found 222.10.
6.1.17. 2-chloro-N-(4-phenylthiazol-2-yl)acetam
ide (8a)
6.1.12. 4-(p-tolyl)thiazol-2-amine (7e)
To a vial was added 7a (1.76 g, 10 mmol) and DIPEA
(2.3 ml, 13 mmol). The mixture was stirred at 0 °C in CH2Cl2
(50 ml), then a solution of chloroacetic chloride (0.83 ml, 11
mmol) in CH2Cl2 (10 mL) was added to the mixture slowly.
The mixture was then warm to room temperature and
stirred for 1 h. After completion of the reaction, the mixture
was diluted with CH2Cl2 and washed thrice with saturated
NaCl. The organic layer was dried over anhydrous sodium
sulfate, the solvent was removed under reduced pressure
and the residual mass was purified by column
chromatography to obtain 8a as white solid (2.25 g, 89%
yield). m.p. 143-144 °C; 1H NMR (400 MHz, DMSO-d6) δ
12.66 (s, 1H), 7.94 – 7.87 (m, 2H), 7.70 (s, 1H), 7.48 – 7.39
(m, 2H), 7.37 – 7.29 (m, 1H), 4.42 (s, 2H); MS (ESI) m/z
calcd for C11H10ClN2OS+ [M + H+] 253.02, found 253.00.
The procedure described for 7a was used, starting from
2-bromo-1-(p-tolyl)ethanone (4.26 g, 20 mmol) and
thiourea (1.67 g, 22 mmol), and 7e was obtained as white
1
acicular crystal (3.43 g, 90% yield). m.p. 124-125 °C; H
NMR (400 MHz, DMSO-d6) δ 8.70 (s, 2H), 7.63 (d, J = 8.2
Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.15 (s, 1H), 2.35 (s, 3H);
MS (ESI) m/z calcd for C10H11N2S+ [M + H+] 191.06, found
191.11.
6.1.13. 4-(4-methoxyphenyl)thiazol-2-amine (7f)
The procedure described for 7a was used, starting from
2-bromo-1-(4-methoxyphenyl)ethanone (4.58 g, 20 mmol)
and thiourea (1.67 g, 22 mmol), and 7f was obtained as
white acicular crystal (2.64 g, 64% yield). m.p. 206-207 °C;
1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 2H), 7.67 (d, J =
8.8 Hz, 2H), 7.11 – 7.00 (m, 3H), 3.81 (s, 3H); MS (ESI) m/z
calcd for C10H11N2OS+ [M + H+] 207.06, found 207.10.
6.1.18. 2-chloro-N-(4-(4-nitrophenyl)thiazol-2-yl
)acetamide (8b)
6.1.14. 4-(4-amino-3,5-dichlorophenyl)thiazol-2-
amine (7g)
The procedure described for 8a was used, starting from
7b (2.21 g, 10 mmol) and TEA (2.8 ml, 15 mmol), and 8b
was obtained as yellow solid (1.67 g, 56% yield). m.p.
211-212 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H),
8.31 (d, J = 8.8 Hz, 2H), 8.16 (d, J = 8.8 Hz, 2H), 8.06 (s,
1H), 4.44 (s, 2H); MS (ESI) m/z calcd for C11H7ClN3O3S- [M
- H+] 295.99, found 296.03.
The procedure described for 7a was used, starting from
1-(4-amino-3,5-dichlorophenyl)-2-bromoethanone (6g, 5.66
g, 20 mmol) and thiourea (1.67 g, 22 mmol), and 7g was
obtained as a light brown solid (3.95 g, 76% yield). m.p.
1
192-193 °C; H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 2H),
7.68 (s, 2H), 7.14 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ
170.34, 142.39, 125.94, 118.56, 101.09; MS (ESI) m/z
calcd for C9H8Cl2N3S+ [M + H+] 259.98, found 259.98.
6.1.19. 2-chloro-N-(4-(3-nitrophenyl)thiazol-2-yl
)acetamide (8c)
The procedure described for 8a was used, starting from
7c (2.21 g, 10 mmol) and TEA (2.8 ml, 15 mmol), and 8c
was obtained as yellow solid (1.73 g, 58% yield). m.p.
215-217 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.77 (s, 1H),
8.74 (s, 1H), 8.36 (d, J = 7.5 Hz, 1H), 8.19 (d, J = 7.5 Hz,
1H), 8.02 (s, 1H), 7.75 (t, J = 7.5 Hz, 1H), 4.44 (s, 2H); MS
(ESI) m/z calcd for C11H7ClN3O3S- [M - H+] 295.99, found
296.03.
6.1.15. 4-(3-bromophenyl)thiazol-2-amine (7i)
The procedure described for 7a was used, starting from
2-bromo-1-(3-bromophenyl)ethanone (5.66 g, 20 mmol)
and thiourea (1.67 g, 22 mmol), and 7i was obtained as
white acicular crystal (4.59 g, 90% yield). m.p. 116-118 °C;
1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 2H), 7.97 (s, 1H),
7.76 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.44 (t, J =
7.9 Hz, 1H), 7.38 (s, 1H); MS (ESI) m/z calcd for
C9H8BrN2S+ [M + H+] 254.96, found 254.95.
6.1.20. 2-chloro-N-(4-(2-nitrophenyl)thiazol-2-yl
)acetamide (8d)
6.1.16. 4-(3-(2-fluoropyridin-3-yl)phenyl)thiazol-
2-amine (7j)
The procedure described for 8a was used, starting from
7d (2.21 g, 10 mmol) and TEA (2.8 ml, 15 mmol), and 8d
was obtained as yellowish solid (1.52 g, 51% yield). m.p.
140-141 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H),
7.89 (dd, J = 8.0, 1.3 Hz, 1H), 7.80 (dd, J = 7.7, 1.5 Hz, 1H),
7.74 (td, J = 7.5, 1.3 Hz, 1H), 7.63 (s, 1H), 7.63 – 7.58 (m,
1H), 4.42 (s, 2H); MS (ESI) m/z calcd for C11H9ClN3O3S+ [M
+ H+] 298.00, found 298.05.
To a schlenk flask was added 7i (2.55 g, 10 mmol),
(2-fluoropyridin-3-yl)boronic acid (1.97 g, 14 mmol),
Pd(PPh3)4 (1.16 g, 1 mmol) and Na2CO3 (2.12 g, 20 mmol).
The flask was evacuated and backfilled with argon (×3).
Then DMF (50 ml) and H2O (2 ml) was added afterwaards.
The mixture was stirred at 120 °C for 24 h. After completion
of the reaction, the mixture was diluted with ethyl acetate
and then washed with saturated Na2CO3 and brine. The
organic layer was collected and dried over anhydrous
sodium sulfate. The solvent was removed under reduced
pressure and the residual mass was purified by column
chromatography to obtain 7j as white solid (1.36 g, 50%
6.1.21. 2-chloro-N-(4-(p-tolyl)thiazol-2-yl)aceta
mide (8e)
The procedure described for 8a was used, starting from
7e (1.90 g, 10 mmol) and DIPEA(2.3 ml, 13 mmol), and 8e
was obtained as white solid (1.68 g, 63% yield). m.p.
157-158 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H),

ACCEPTED MANUSCRIPT
6.1.27. N-(4-phenyl-1H-imidazol-2-yl)acetamide
(9a)
To a vial was added 2-bromo-1-phenylethanone (3.98 g,
7.79 (d, J = 8.1 Hz, 2H), 7.61 (s, 1H), 7.25 (d, J = 7.9 Hz,
2H), 4.41 (s, 2H), 2.33 (s, 3H); MS (ESI) m/z calcd for
C12H12ClN2OS+ [M + H+] 267.04, found 267.03.
20 mmol), N-acetylguanidine (6.06 g, 60 mmol) and CH3CN
(500 ml). The mixture was stirred at 100 °C for 3h. After
completion of the reaction, the solution was cooled to room
temperature and the solvent was removed to obtain the
crude product. The pure product 9a was obtained through
column chromatography as white solid (2.57 g, 64% yield).
m.p. 218-219 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.63 (s,
1H), 11.23 (s, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.32 (t, J = 7.6
Hz, 2H), 7.25 (s, 1H), 7.16 (t, J = 7.3 Hz, 1H), 2.07 (s, 3H);
MS (ESI) m/z calcd for C11H10N3O- [M - H+] 200.08, found
200.19.
6.1.22. 2-chloro-N-(4-(4-methoxyphenyl)thiazol-
2-yl)acetamide (8f)
The procedure described for 8a was used, starting from
7f (2.06 g, 10 mmol) and DIPEA (2.3 ml, 13 mmol), and 8f
was obtained as white solid (1.70 g, 60% yield). m.p.
161-162 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H),
7.83 (d, J = 8.8 Hz, 2H), 7.53 (s, 1H), 7.00 (d, J = 8.8 Hz,
2H), 4.41 (s, 2H), 3.79 (s, 3H); MS (ESI) m/z calcd for
C12H10ClN2O2S- [M - H+] 281.02, found 281.16.
6.1.23. N-(4-(4-amino-3,5-dichlorophenyl)thiazo
l-2-yl)-2-chloroacetamide (8g)
6.1.28. N-(4-(4-amino-3,5-dichlorophenyl)-1H-i
The procedure described for 8a was used, starting from
7g (2.60 g, 10 mmol) and DIPEA (2.3 ml, 13 mmol), and 8g
was obtained as light brown solid (2.05 g, 61% yield). m.p.
208-209 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H),
7.78 (s, 2H), 7.60 (s, 1H), 5.68 (s, 2H), 4.40 (s, 2H); MS
(ESI) m/z calcd for C11H7Cl3N3OS- [M - H+] 333.94, found
333.90.
midazol-2-yl)acetamide (9g)
The procedure described for 9a was used, starting from
6g (5.66 g, 20 mmol) and N-acetylguanidine (6.06 g, 60
mmol), 9g was obtained as yellow solid (4.62 g, 81% yield).
m.p. 188-189 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.62 (s,
1H), 11.18 (s, 1H), 7.59 (s, 2H), 7.21 (s, 1H), 5.40 (s, 2H),
2.06 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ =168.94,
141.54, 139.52, 134.26, 124.76, 124.04, 118.95, 109.45,
23.25; MS (ESI) m/z calcd for C11H11Cl2N4O+ [M + H+]
285.03, found 285.11.
6.1.24. N-(4-(4-acetamido-3,5-dichlorophenyl)th
iazol-2-yl)-2-chloroacetamide (8h)
To a solution of 8g (1.68 g, 5 mmol) in AcOH (5 ml) was
added acetyl chloride (430 mg, 5.5 mmol). The mixture was
stirred at 90 °C for 20min. After completion of the reaction,
the mixture was pured into ice water, and the crude product
was precipitated. Then the solid was filtered out and
air-dried. The crude product was purified by column
chromatography to obtain 8h as light brown solid (1.65 g,
87% yield). m.p. 258-259 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.69 (s, 1H), 9.87 (s, 1H), 8.03 (s, 2H), 7.97
(s, 1H), 4.43 (s, 2H), 2.09 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 168.72, 165.79, 158.31, 146.28, 135.14,
134.55, 132.81, 125.64, 111.73, 42.74, 22.89; MS (ESI)
m/z calcd for C13H9Cl3N3O2S- [M - H+] 375.95, found
375.96.
6.1.29. 2-chloro-N-(4-phenyl-1H-imidazol-2-yl)a
cetamide (10a)
To a 25 mL mixture of a 1:1 MeOH:H2O containing 20%
H2SO4 was added 9a (1.01 g, 5 mmol). The mixture was
heated at 100 °C for 1 h. The mixture was then
concentrated under reduced pressure, and the white
sulfate salt was crystallized from solution, then the solid
was filtered out, washed with MeOH and air-dried. After
that, the solid was added into a mixture of CH2Cl2 (20 ml)
and TEA (2.8 ml, 15 mmol). After that, a solution of
chloroacetic chloride (0.83 ml, 5 mmol) in CH2Cl2 (5 mL)
was added into the above mixture slowly under ice bath.
The mixture was stirred for 1 h under ice bath. After the
reaction, the mixture was diluted with CH2Cl2 and then
washed thrice with saturated NaCl. The organic layer was
dried over anhydrous sodium sulfate, the solvent was
removed under reduced pressure and the residual mass
was purified by column chromatography to obtain 10a as
white solid (200 mg, 17% yield in total). m.p. 171-172 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.81 (s, 1H), 11.62 (s,
1H,), 7.72 (d, J = 7.5 Hz, 2H), 7.34 (d, J = 7.5 Hz, 2H), 7.32
(s, 1H), 7.18 (t, J = 7.5 Hz, 1H), 4.30 (s, 2H); MS (ESI) m/z
calcd for C11H11ClN3O+ [M + H+] 236.06, found 235.97.
6.1.25. N-(4-(3-bromophenyl)thiazol-2-yl)-2-chl
oroacetamide (8i)
The procedure described for 8a was used, starting from
7i (2.55 g, 10 mmol) and DIPEA (2.3 ml, 13 mmol), and 8i
was obtained as white solid (2.69 g, 81% yield). m.p.
147-148 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H),
8.10 (t, J = 1.9 Hz, 1H), 7.90 (dt, J = 7.8, 1.3 Hz, 1H), 7.83
(s, 1H), 7.52 (ddd, J = 8.1, 2.1, 1.1 Hz, 1H), 7.40 (t, J = 7.9
Hz, 1H), 4.42 (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ
165.68, 158.10, 147.79, 136.81, 131.41, 130.95, 128.78,
125.02, 122.66, 110.52, 42.75; MS (ESI) m/z calcd for
C11H9BrClN2OS+ [M + H+] 330.93, found 331.00.
6.1.30. N-(4-(4-amino-3,5-dichlorophenyl)-1H-i
midazol-2-yl)-2-chloroacetamide (10g)
The procedure described for 10a was used, starting from
9g (1.43 g, 5 mmol), except that 40% HCl in 1:1 MeOH/H2O
was used as the solvent instead of 20% H2SO4, the 10g
was obtained as yellow solid (160 mg, 10% yield). m.p.
232-233 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H),
11.56 (s, 1H), 7.60 (s, 2H), 7.28 (s, 1H), 5.43 (s, 2H), 4.30
(s, 2H); MS (ESI) m/z calcd for C11H10Cl3N4O+ [M + H+]
318.99, found 318.98.
6.1.26. 2-chloro-N-(4-(3-(2-fluoropyridin-3-yl)ph
enyl)thiazol-2-yl)acetamide (8j)
The procedure described for 8a was used, starting from
7j (2.71 g, 10 mmol) and DIPEA (2.3 ml, 13 mmol), and 8j
was obtained as white solid (2.47 g, 71% yield). m.p.
187-188 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H),
8.28 (d, J = 4.7 Hz, 1H), 8.24 – 8.10 (m, 2H), 7.99 (t, J = 3.5
Hz, 1H), 7.83 (s, 1H), 7.64 – 7.55 (m, 2H), 7.55 – 7.46 (m,
1H), 4.43 (s, 2H); MS (ESI) m/z calcd for C16H12ClFN3OS+
[M + H+] 348.04, found 348.01.
6.1.31. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-phenylthiaz
ol-2-yl)acetamide (12)

ACCEPTED MANUSCRIPT
To a vial was added 4a (52 mg, 0.2 mmol), 8a (50 mg,
mg, 0.22 mmol), 15 was obtained as yellowish solid (90 mg,
86% yield). m.p. 206-207 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.59 (s, 1H), 8.32 (d, J = 8.5 Hz, 2H), 8.15 (d,
J = 8.6 Hz, 2H), 8.02 (s, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.17
(d, J = 6.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.78 (t, J = 7.5
Hz, 1H), 4.04 (s, 2H), 3.76 (s, 3H); 13C NMR (100MHz,
DMSO-d6): δ 166.76, 166.72, 158.79, 156.77, 147.29,
146.98, 140.69, 132.75, 130.40, 127.00, 124.71, 120.39,
115.44, 113.14, 112.30, 97.04, 55.81, 34.78; HRMS (ESI)
0.2 mmol) and KOH (13 mg, 0.22 mmol). The mixture was
stirred in EtOH (5 mL) at 70 °C for 3 h. After the reaction,
the mixture was purified by column chromatography and 12
was obtained as white solid (87 mg, 91% yield). m.p.
205-206 °C, 1H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H),
12.61 (s, 1H), 7.91 (d, J = 7.6 Hz, 2H), 7.64 (s, 1H), 7.45 (t,
J = 7.6 Hz, 2H), 7.34 (d, J = 4.2 Hz, 3H), 7.20 (t, J = 7.9 Hz,
1H), 7.02 (d, J = 8.2 Hz, 1H), 4.32 (s, 2H), 3.64 (s, 3H); 13
C
+
NMR (100 MHz, DMSO-d6): δ 167.43, 166.34, 166.01,
161.73, 159.46, 158.18, 149.44, 136.81, 134.74, 129.89,
129.22, 128.28, 126.13, 121.42, 117.62, 116.26, 114.40,
108.72, 93.82, 55.60, 34.82; HRMS (ESI) m/z calcd for
m/z calcd for C23H16KN6O5S2 [M + K+] 559.02552, found
+
559.01728; C23H16N6NaO5S2 [M + Na+] 543.05158, found
543.05260; C23H17N6O5S2 [M + H+] 521.06964, found
+
521.07068. HPLC 99.2% (Rt = 5.36 min, 50-80% CH3OH in
water for 7 min and 80% CH3OH in water for 3 min, 10 min
in total).
+
C23H17KN5O3S2 [M + K+] 514.04044, found 514.04064;
C23H17N5NaO3S2 [M + Na+] 498.06650, found 498.06647;
+
+
C23H18N5O3S2 [M + H+] 476.08456, found 476.08431;
6.1.35. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(4-nitrophe
nyl)thiazol-2-yl)acetamide (16)
HPLC 97.5% (Rt = 6.02 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
6.1.32. 2-((5-cyano-4-(2-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-phenylthiaz
ol-2-yl)acetamide (13)
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8b (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 16 was obtained as yellowish solid (94 mg,
90% yield). m.p. 187-188 °C; 1H NMR (400 MHz,
DMSO-d6): δ 14.00 (s, 1H) , 12.73 (s, 1H), 8.33 (d, J = 7.5
Hz, 2H) , 8.18 (d, J = 7.5 Hz, 2H), 8.02 (s, 1H) , 7.30-7.35
(m, 2H), 7.19 (t, J = 7.5 Hz, 1H), 7.01 (dd, J = 7.5, 1.5 Hz,
1H), 4.33 (s, 2H), 3.62 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 167.44, 167.02, 159.42, 158.79, 147.25,
146.96, 140.70, 137.24, 129.84, 126.99, 124.73, 121.28,
117.20, 116.97, 114.37, 113.17, 93.20, 55.59, 34.76;
The procedure described for 12 was used, starting from
4b (52 mg, 0.2 mmol), 8a (50 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 13 was obtained as white solid (84 mg,
88% yield). m.p. 209-210 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.46 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.64 (s,
1H), 7.44 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.7 Hz, 2H), 7.19
(d, J = 7.5 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.79 (t, J = 7.4
Hz, 1H), 4.03 (s, 2H), 3.76 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 170.78, 169.09, 168.22, 166.98, 158.22,
156.73, 149.35, 134.75, 131.08, 130.12, 129.18, 128.24,
127.38, 126.15, 120.44, 119.07, 112.03, 108.49, 93.86,
55.77, 34.42; HRMS (ESI) m/z calcd for C23H17KN5O3S2+ [M
+
HRMS (ESI) m/z calcd for C23H16KN6O5S2 [M + K+]
+
559.02552, found 559.01886; C23H16N6NaO5S2 [M + Na+]
+
543.05158, found 543.05286; C23H17N6O5S2 [M + H+]
521.06964, found 521.07087. HPLC 95.2% (Rt = 5.88 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
+
+ K+] 514.04044, found 514.04096; C23H17N5NaO3S2 [M +
Na+] 498.06650, found 498.06669; C23H18N5O3S2+ [M + H+]
476.08456, found 476.08444; HPLC 98.2% (Rt = 4.98 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
6.1.36. 2-((5-cyano-4-(4-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(4-nitrophe
nyl)thiazol-2-yl)acetamide (17)
6.1.33. 2-((5-cyano-4-(4-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-phenylthiaz
ol-2-yl)acetamide (14)
The procedure described for 12 was used, starting from
4c (52 mg, 0.2 mmol), 8b (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 17 was obtained as yellowish solid (95 mg,
91% yield). m.p. 245-246 °C; 1H NMR (400 MHz,
DMSO-d6): δ 13.90 (s, 1H), 12.82 (s, 1H) , 8.34 (d, J = 7.5
Hz, 2H), 8.19 (d, J = 7.5 Hz, 2H), 8.05 (s, 1H) 7.83 (t, J =
7.5 Hz, 2H), 6.76 (d, J = 7.5 Hz, 2H), 4.32 (s, 2H), 3.55 (s,
3H); 13C NMR (100 MHz, DMSO-d6): δ 166.80, 165.18,
162.43, 158.90, 147.24, 146.99, 140.63, 131.19, 127.48,
126.99, 124.72, 116.57, 114.09, 113.28, 92.18, 55.57,
The procedure described for 12 was used, starting from
4c (52 mg, 0.2 mmol), 8a (50 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 14 was obtained as white solid (88 mg,
93% yield). m.p. 216-217 °C; 1H NMR (400 MHz,
DMSO-d6) δ 13.80 (s, 1H), 12.63 (s, 1H), 7.99 – 7.86 (m,
2H), 7.82 (d, J = 8.9 Hz, 2H), 7.67 (s, 1H), 7.45 (t, J = 7.6
Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 9.0 Hz, 2H),
4.26 (s, 2H), 3.55 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ
166.97, 166.71, 163.89, 162.10, 158.33, 149.42, 134.69,
130.99, 129.21, 128.32, 128.13, 126.13, 117.54, 114.03,
108.71, 91.42, 55.49, 34.75; HRMS (ESI) m/z calcd for
+
34.83; HRMS (ESI) m/z calcd for C23H16KN6O5S2 [M + K+]
559.02552, found 559.02596. HPLC 95.5% (Rt = 5.96 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
+
C23H17KN5O3S2 [M + K+] 514.04044, found 514.04092;
6.1.37. 2-((5-cyano-4-(2-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(3-nitrophe
nyl)thiazol-2-yl)acetamide (18)
The procedure described for 12 was used, starting from
4b (52 mg, 0.2 mmol), 8c (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 18 was obtained as yellowish solid (91 mg,
87% yield). m.p. 192-193 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.56 (s, 1H), 8.72 (t, J = 2.0 Hz, 1H), 8.35
(dd, J = 7.9, 1.3 Hz, 1H), 8.19 (ddd, J = 8.2, 2.4, 1.0 Hz,
1H), 7.97 (s, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.31 (td, J = 8.1,
+
C23H17N5NaO3S2 [M + Na+] 498.06650, found 498.06668;
+
C23H18N5O3S2 [M + H+] 476.08456, found 476.08440;
HPLC 98.2% (Rt = 5.47 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
6.1.34. 2-((5-cyano-4-(2-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(4-nitrophe
nyl)thiazol-2-yl)acetamide (15)
The procedure described for 12 was used, starting from
4b (52 mg, 0.2 mmol), 8b (60 mg, 0.2 mmol) and KOH (13

ACCEPTED MANUSCRIPT
7.4, 1.8 Hz, 1H), 7.15 (dd, J = 7.5, 1.8 Hz, 1H), 7.06 (d, J =
136.71, 132.98, 131.27, 130.00, 129.77, 128.63, 124.45,
121.42, 117.89, 116.13, 114.22, 112.70, 93.86, 55.61,
8.3 Hz, 1H), 6.78 (t, J = 7.4 Hz, 1H), 4.02 (s, 2H), 3.75 (s,
3H); 13C NMR (100MHz, DMSO-d6): δ 166.68, 166.07,
158.70, 156.77, 148.83, 147.02, 136.26, 132.76, 132.23,
130.89, 130.41, 122.83, 120.58, 120.41, 115.41, 112.29,
111.26, 97.09, 55.81, 34.78; HRMS (ESI) m/z calcd for
34.81; HRMS (ESI) m/z calcd for C23H16KN6O5S2 [M + K+]
+
+
559.02552, found 559.02704; C23H16N6NaO5S2 [M + Na+]
+
543.05158, found 543.05289; C23H17N6O5S2 [M + H+]
521.06964, found 521.07087; HPLC 92.0% (Rt = 4.11 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
+
C23H16KN6O5S2 [M + K+] 559.02552, found 559.02312;
+
C23H16N6NaO5S2 [M + Na+] 543.05158, found 543.05288;
C23H17N6O5S2 [M + H+] 521.06964, found 521.07094.
+
6.1.41. 2-((5-cyano-4-(3-ethoxyphenyl)-6-oxo-1,
6-dihydropyrimidin-2-yl)thio)-N-(4-(4-nitropheny
l)thiazol-2-yl)acetamide (22)
HPLC 96.2% (Rt = 4.94 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
6.1.38. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(3-nitrophe
nyl)thiazol-2-yl)acetamide (19)
The procedure described for 12 was used, starting from
4d (55 mg, 0.2 mmol), 8b (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 22 was obtained as yellowish solid (92 mg,
86% yield). m.p. 220-221 °C; 1H NMR (400 MHz,
DMSO-d6) δ 14.02 (s, 1H), 12.74 (s, 1H), 8.33 (d, J = 8.7
Hz, 2H), 8.17 (d, J = 8.7 Hz, 2H), 8.04 (s, 1H), 7.32 (d, J =
7.9 Hz, 1H), 7.28 (t, J = 2.1 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H),
7.00 (dd, J = 8.3, 2.5 Hz, 1H), 4.33 (s, 2H), 3.89 (q, J = 6.9
Hz, 2H), 1.17 (t, J = 6.9 Hz, 3H); 13C NMR (100MHz,
DMSO-d6): δ 167.60, 166.60, 165.91, 161.64, 158.82,
158.71, 147.30, 147.00, 140.69, 136.82, 129.89, 127.00,
124.75, 121.34, 118.44, 116.20, 114.40, 113.25, 93.92,
63.60, 34.82, 14.91; HRMS (ESI) m/z calcd for
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8c (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 19 was obtained as yellowish solid (96 mg,
92% yield). m.p. 226-227 °C; 1H NMR (400 MHz,
DMSO-d6) δ 13.97 (s, 1H), 12.72 (s, 1H), 8.75 (t, J = 2.0
Hz, 1H), 8.37 (dt, J = 8.0, 1.3 Hz, 1H), 8.25 – 8.15 (m, 1H),
7.97 (s, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.39 – 7.28 (m, 2H),
7.19 (t, J = 7.9 Hz, 1H), 7.05 – 6.95 (m, 1H), 4.33 (s, 2H),
3.63 (s, 3H); 13C NMR (100MHz, DMSO-d6): δ 167.53,
166.54, 159.44, 158.68, 148.85, 146.99, 136.75, 136.23,
132.20, 130.91, 129.91, 122.83, 121.38, 120.56, 117.56,
116.14, 114.46, 111.30, 93.99, 55.60, 34.83; HRMS (ESI)
+
C24H19N6O5S2 [M + H+] 535.08529, found 535.08372;
HPLC 96.2% (Rt = 7.30 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
+
m/z calcd for C23H16KN6O5S2 [M + K+] 559.02552, found
+
559.02623; C23H17N6O5S2 [M + H+] 521.06964, found
6.1.42. N-(4-(4-amino-3,5-dichlorophenyl)thiazo
l-2-yl)-2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-1
,6-dihydropyrimidin-2-yl)thio)acetamide (23)
521.07068.
6.1.39. 2-((5-cyano-4-(4-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(3-nitrophe
nyl)thiazol-2-yl)acetamide (20)
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8g (67 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 23 was obtained as light orange solid (96
mg, 86% yield). m.p. 190-191 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.48 (s, 1H), 7.79 (s, 2H), 7.54 (s, 1H), 7.36 –
7.28 (m, 2H), 7.22 (t, J = 7.9 Hz, 1H), 7.05 – 6.99 (m, 1H),
5.68 (s, 2H), 4.19 (s, 2H), 3.68 (s, 3H); 13C NMR (100MHz,
DMSO-d6): δ 167.39, 167.08, 159.42, 158.11, 147.35,
141.14, 137.78, 129.75, 125.73, 124.28, 121.22, 118.82,
117.79, 116.91, 114.26, 106.77, 92.34, 55.60, 34.66;
The procedure described for 12 was used, starting from
4c (52 mg, 0.2 mmol), 8c (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 20 was obtained as yellowish solid (98 mg,
94% yield). m.p. 224-225 °C; 1H NMR (400 MHz, DMSO-d6)
δ 13.86 (s, 1H), 12.77 (s, 1H), 8.76 (t, J = 2.0 Hz, 1H), 8.38
(d, J = 7.9 Hz, 1H), 8.27 – 8.13 (m, 1H), 7.98 (s, 1H), 7.82
(d, J = 8.9 Hz, 2H), 7.76 (t, J = 8.0 Hz, 1H), 6.76 (d, J = 8.7
Hz, 2H), 4.30 (s, 2H), 3.53 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 166.71, 165.17, 162.41, 161.51, 158.81,
148.83, 146.96, 136.17, 132.20, 131.18, 130.92, 127.50,
122.86, 120.53, 116.55, 114.09, 111.39, 92.23, 55.50,
+
HRMS (ESI) m/z calcd for C23H16Cl2KN6O3S2 [M + K+]
+
596.97339, found 596.97199; C23H16Cl2N6NaO3S2 [M +
+
Na+] 580.99946, found 580.99964; C23H17Cl2N6O3S2 [M +
+
34.83; HRMS (ESI) m/z calcd for C23H16KN6O5S2 [M + K+]
H+] 559.01751, found 559.01645; HPLC 95.8% (Rt = 6.14
min, 50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
+
559.02552, found 559.02678; C23H16N6NaO5S2 [M + Na+]
+
543.05158, found 543.05237; C23H17N6O5S2 [M + H+]
521.06964, found 521.07053. HPLC 96.6% (Rt = 5.72 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
6.1.43. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(p-tolyl)thia
zol-2-yl)acetamide (24)
6.1.40. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(2-nitrophe
nyl)thiazol-2-yl)acetamide (21)
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8e (58 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 24 was obtained as white solid (90 mg,
92% yield). m.p. 234-235 °C; 1H NMR (400 MHz,
DMSO-d6) δ 13.99 (s, 1H), 12.59 (s, 1H), 7.80 (d, J = 7.7
Hz, 2H), 7.56 (s, 1H), 7.44 – 7.29 (m, 2H), 7.25 (d, J = 7.8
Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H),
4.32 (s, 2H), 3.63 (s, 3H), 2.34 (s, 3H); 13C NMR (100MHz,
DMSO-d6): δ 167.48, 166.20, 165.81, 161.39, 159.46,
158.06, 149.53, 137.60, 136.73, 132.10, 129.90, 129.79,
126.07, 121.43, 117.68, 116.11, 114.42, 107.84, 93.95,
55.60, 34.82, 21.27; HRMS (ESI) m/z calcd for
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8d (60 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 21 was obtained as yellowish solid (84 mg,
81% yield). m.p. 231-232 °C; 1H NMR (400 MHz,
DMSO-d6) δ 13.96 (s, 1H), 12.54 (s, 1H), 7.90 (d, J = 8.0
Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H),
7.62 (t, J = 7.9 Hz, 1H), 7.58 (s, 1H), 7.41 – 7.28 (m, 2H),
7.23 (t, J = 8.0 Hz, 1H), 7.09 – 7.00 (m, 1H), 4.32 (s, 2H),
3.67 (s, 3H); 13C NMR (100MHz, DMSO-d6): δ 167.41,
166.45, 165.76, 161.45, 159.44, 158.24, 149.07, 145.50,
+
C24H19KN5O3S2 [M + K+] 528.05609, found 528.05718;

ACCEPTED MANUSCRIPT
+
C24H19N5NaO3S2 [M + Na+] 512.08215, found 512.08155;
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
C24H20N5O3S2 [M + H+] 490.10021, found 490.09982;
+
HPLC 98.1% (Rt = 6.98 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
6.1.47. N-(4-(4-amino-3,5-dichlorophenyl)thiazo
l-2-yl)-2-((5-cyano-4-(3-ethoxyphenyl)-6-oxo-1,
6-dihydropyrimidin-2-yl)thio)acetamide (28)
6.1.44. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(4-methoxy
phenyl)thiazol-2-yl)acetamide (25)
The procedure described for 12 was used, starting from
4d (55 mg, 0.2 mmol), 8g (67 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 28 was obtained as yellowish solid (94 mg,
82% yield). m.p. 221-222 °C; 1H NMR (400 MHz,
DMSO-d6) δ 14.00 (s, 1H), 12.53 (s, 1H), 7.79 (s, 2H), 7.56
(s, 1H), 7.39 – 7.25 (m, 2H), 7.17 (t, J = 7.9 Hz, 1H), 7.01
(dd, J = 8.1, 2.5 Hz, 1H), 5.70 (s, 2H), 4.31 (s, 2H), 3.90 (q,
J = 6.9 Hz, 2H), 1.20 (t, J = 6.9 Hz, 3H); 13C NMR (100MHz,
DMSO-d6): δ 167.55, 167.55, 166.20, 158.73, 158.09,
147.40, 141.18, 136.76, 129.87, 125.73, 124.20, 121.36,
118.79, 118.51, 116.15, 114.36, 106.86, 93.90, 63.62,
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8f (62 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 26 was obtained as white solid (78 mg,
77% yield). m.p. 214-215 °C; 1H NMR (400 MHz,
DMSO-d6) δ 13.99 (s, 1H), 12.57 (s, 1H), 7.84 (d, J = 8.2
Hz, 2H), 7.48 (s, 1H), 7.41 – 7.27 (m, 2H), 7.19 (t, J = 8.1
Hz, 1H), 7.11 – 6.89 (m, 3H), 4.32 (s, 2H), 3.80 (s, 3H),
3.64 (s, 3H); 13C NMR (100MHz, DMSO-d6): δ 166.16,
165.83, 159.48, 158.02, 149.35, 136.75, 129.92, 127.61,
127.47, 121.44, 117.73, 116.10, 114.61, 114.41, 106.69,
93.92, 55.63, 34.82; HRMS (ESI) m/z calcd for
+
34.79, 14.92; HRMS (ESI) m/z calcd for C24H18Cl2KN6O3S2+
[M + K+] 610.98904, found 610.98743; C24H18Cl2N6NaO3S2+
[M + Na+] 595.01511, found 595.01430; C24H19Cl2N6O3S2
[M + H+] 573.03316, found 573.03197; HPLC 99.0% (Rt =
7.23 min, 50-80% CH3OH in water for 7 min and 80%
CH3OH in water for 3 min, 10 min in total).
+
C24H19KN5O4S2 [M + K+] 544.05100, found 544.05057;
C24H19N5NaO4S2 [M + Na+] 528.07707, found 528.07617;
+
+
C24H20N5O4S2 [M + H+] 506.09512, found 506.09427;
HPLC 99.0% (Rt = 5.73 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
6.1.48. N-(4-(4-acetamido-3,5-dichlorophenyl)th
iazol-2-yl)-2-((5-cyano-4-(3-methoxyphenyl)-6-o
xo-1,6-dihydropyrimidin-2-yl)thio)acetamide
(29)
6.1.45. 2-((5-cyano-4-(3-ethoxyphenyl)-6-oxo-1,
6-dihydropyrimidin-2-yl)thio)-N-(4-(p-tolyl)thiaz
ol-2-yl)acetamide (26)
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8h (76 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 29 was obtained as white solid (103 mg,
86% yield). m.p. 191-193 °C; 1H NMR (400 MHz,
DMSO-d6) δ 14.00 (s, 1H), 12.65 (s, 1H), 9.89 (s, 1H), 8.04
(s, 2H), 7.94 (s, 1H), 7.38 – 7.28 (m, 2H), 7.17 (t, J = 8.2
Hz, 1H), 7.09 – 6.99 (m, 1H), 4.32 (s, 2H), 3.65 (s, 3H),
2.09 (s, 3H); 13C NMR (100MHz, DMSO-d6): δ 168.75,
167.49, 166.50, 165.79, 161.38, 159.46, 158.57, 146.16,
136.73, 135.28, 134.57, 132.76, 129.86, 125.60, 121.39,
117.63, 116.08, 114.46, 111.43, 94.00, 55.63, 34.83,
22.90; HRMS (ESI) m/z calcd for C25H19Cl2N6O4S2+ [M + H+]
601.02808, found 601.02756; HPLC 95.0% (Rt = 3.37 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
The procedure described for 12 was used, starting from
4d (55 mg, 0.2 mmol), 8e (58 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 26 was obtained as white solid (88 mg,
87% yield). m.p. 238-239 °C; 1H NMR (400 MHz,
DMSO-d6) δ 14.05 (s, 1H), 12.60 (s, 1H), 7.79 (d, J = 7.9
Hz, 2H), 7.57 (s, 1H), 7.36 – 7.32 (m, 1H), 7.31 (t, J = 2.1
Hz, 1H), 7.25 (d, J = 7.9 Hz, 2H), 7.17 (t, J = 8.0 Hz, 1H),
7.01 (dd, J = 8.4, 2.5 Hz, 1H), 4.32 (s, 2H), 3.90 (q, J = 6.9
Hz, 2H), 2.34 (s, 3H), 1.19 (t, J = 6.9 Hz, 3H); 13C NMR
(100MHz, DMSO-d6): δ 167.53, 166.22, 165.86, 161.42,
158.72, 158.09, 149.54, 137.61, 136.75, 132.07, 129.89,
129.78, 126.07, 121.40, 118.59, 116.18, 114.28, 107.86,
93.89, 63.60, 34.81, 21.28, 14.91; HRMS (ESI) m/z calcd
+
for C25H21KN5+O3S2 [M + K+] 542.07174, found 542.07020;
C25H22N5O3S2 [M + H+] 504.11586, found 504.11451;
HPLC 96.7% (Rt = 7.58 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
6.1.49. N-(4-(4-acetamido-3,5-dichlorophenyl)th
iazol-2-yl)-2-((5-cyano-4-(3-ethoxyphenyl)-6-ox
o-1,6-dihydropyrimidin-2-yl)thio)acetamide (30)
6.1.46. 2-((5-cyano-4-(3-ethoxyphenyl)-6-oxo-1,
6-dihydropyrimidin-2-yl)thio)-N-(4-(4-methoxyph
enyl)thiazol-2-yl)acetamide (27)
The procedure described for 12 was used, starting from
4d (55 mg, 0.2 mmol), 8h (76 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 30 was obtained as white solid (104 mg,
86% yield). m.p. 203-205 °C; 1H NMR (400 MHz,
DMSO-d6) δ 14.04 (s, 1H), 12.65 (s, 1H), 9.88 (s, 1H), 8.04
(s, 2H), 7.94 (s, 1H), 7.39 – 7.24 (m, 2H), 7.15 (t, J = 7.9
Hz, 1H), 7.07 – 6.94 (m, 1H), 4.32 (s, 2H), 3.91 (q, J = 6.9
Hz, 2H), 2.09 (s, 3H), 1.21 (t, J = 7.0 Hz, 3H).; 13C NMR
(100MHz, DMSO-d6): δ 168.74, 167.56, 166.46, 165.76,
161.40, 158.73, 158.60, 146.17, 136.74, 135.26, 134.55,
132.75, 129.84, 125.59, 121.34, 118.49, 116.10, 114.41,
111.44, 93.95, 63.63, 34.81, 22.90, 14.92; HRMS (ESI) m/z
The procedure described for 12 was used, starting from
4d (55 mg, 0.2 mmol), 8f (62 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 27 was obtained as white solid (80 mg,
77% yield). m.p. 231-232 °C; 1H NMR (400 MHz,
DMSO-d6): δ 13.94 (s, 1H), 12.57 (s, 1H) , 7.84 (d, J = 7.5
Hz, 2H), 7.48 (s, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.18 (t, J =
7.5 Hz, 1H), 7.01 (d, J = 7.5 Hz, 3H), 4.32 (s, 2H), 3.90 (s,
3H), 3.80 (q, J = 6.9 Hz, 2H), 1.19 (t, J = 6.9 Hz, 3H); 13C
NMR (100MHz, DMSO-d6): δ 166.12, 159.49, 158.72,
158.05, 149.36, 129.90, 127.57, 127.45, 121.40, 118.60,
116.12, 114.58, 114.30, 106.70, 93.92, 63.61, 55.62,
34.79, 14.90; HRMS (ESI) m/z calcd for C25H21KN5O4S2+ [M
+
calcd for C26H21Cl2N6O4S2 [M + H+] 615.04373, found
615.04309; HPLC 97.0% (Rt = 4.64 min, 50-80% CH3OH in
water for 7 min and 80% CH3OH in water for 3 min, 10 min
in total).
+
+ K+] 558.06665, found 558.06594; C25H21N5NaO4S2 [M +
Na+] 542.09272, found 542.09167; C25H22N5O4S2+ [M + H+]
520.11077, found 520.10980; HPLC 95.8% (Rt = 6.70 min,

ACCEPTED MANUSCRIPT
6.1.50. 2-((5-cyano-6-oxo-4-(3-(trifluoromethyl)
phenyl)-1,6-dihydropyrimidin-2-yl)thio)-N-(4-phe
nylthiazol-2-yl)acetamide (31)
phenyl)-1,6-dihydropyrimidin-2-yl)thio)acetamid
e (34)
The procedure described for 12 was used, starting from
The procedure described for 12 was used, starting from
4e (59 mg, 0.2 mmol), 8a (50 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 31 was obtained as yellowish solid (91 mg,
89% yield). m.p. 149-150 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.51 (s, 1H), 8.07 (s, 1H), 8.04 (d, J = 7.9 Hz,
1H), 7.89 (d, J = 7.3 Hz, 2H), 7.81 (d, J = 7.8 Hz, 1H), 7.63
(s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H),
7.33 (t, J = 7.3 Hz, 1H), 4.13 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 170.51, 167.75, 166.01, 158.21, 149.37,
138.20, 134.74, 132.78, 129.84, 129.66, 129.35, 129.16,
128.22, 127.22, 126.13, 125.14 (q, J = 3.8 Hz), 124.36 (q, J
= 271 Hz), 118.89, 108.53, 91.30, 34.58; HRMS (ESI) m/z
4f (63 mg, 0.2 mmol), 8g (67 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 34 was obtained as yellowish solid (104
mg, 85% yield). m.p. 139-140 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.51 (s, 1H), 7.79 (s, 2H), 7.79 – 7.76 (m,
1H), 7.73 (s, 1H), 7.54 (s, 1H), 7.50 – 7.45 (m, 1H), 7.41 (t,
J = 8.0 Hz, 1H), 5.69 (s, 2H).; 13C NMR (100 MHz,
DMSO-d6): δ 166.36, 166.24, 165.95, 161.22, 157.94,
148.53, 147.44, 141.16, 137.57, 130.85, 128.21, 125.73,
124.49, 124.22, 120.38 (q, J = 255 Hz), 118.76, 115.79,
106.77, 94.71, 34.85; HRMS (ESI) m/z calcd for
C23H14Cl2F3N6O3S2+ [M + H+] 612.98925, found 612.98761;
HPLC 97.2% (Rt = 7.94 min, 50-80% CH3OH in water for 7
min and 80% CH3OH in water for 3 min, 10 min in total).
+
calcd for C23H14F3KN5O2S2 [M + K+] 552.01726, found
552.01705; C23H14F3N5NaO2S2 [M + Na+] 536.04332,
+
6.1.54. N-(4-(3-bromophenyl)thiazol-2-yl)-2-((5-
cyano-4-(3-methoxyphenyl)-6-oxo-1,6-dihydropy
rimidin-2-yl)thio)acetamide (35)
+
found 536.04245; C23H15F3N5O2S2 [M + H+] 514.06138,
found 514.06083; HPLC 97.3% (Rt = 6.90 min, 50-80%
CH3OH in water for 7 min and 80% CH3OH in water for 3
min, 10 min in total).
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8i (66 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 35 was obtained as white solid (103 mg,
93% yield). m.p. 212-213 °C; 1H NMR (400 MHz,
DMSO-d6) δ 13.99 (s, 1H), 12.63 (s, 1H), 8.12 (t, J = 1.8
Hz, 1H), 7.92 (dt, J = 7.8, 1.3 Hz, 1H), 7.80 (s, 1H), 7.54 (d,
J = 8.1 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.37 – 7.30 (m,
2H), 7.19 (t, J = 7.9 Hz, 1H), 7.03 (dd, J = 8.5, 2.0 Hz, 1H),
4.33 (s, 2H), 3.64 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ
167.49, 166.41, 165.79, 161.40, 159.45, 158.36, 147.68,
136.96, 136.74, 131.45, 130.90, 129.90, 128.77, 125.00,
122.69, 121.41, 117.59, 116.12, 114.46, 110.23, 94.01,
6.1.51. N-(4-(4-amino-3,5-dichlorophenyl)thiazo
l-2-yl)-2-((5-cyano-6-oxo-4-(3-(trifluoromethyl)p
henyl)-1,6-dihydropyrimidin-2-yl)thio)acetamide
(32)
The procedure described for 12 was used, starting from
4e (59 mg, 0.2 mmol), 8g (67 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 32 was obtained as yellowish solid (103
mg, 86% yield). m.p. 202-203 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.55 (s, 1H), 8.12 (s, 1H), 8.01 (d, J = 8.0 Hz,
1H), 7.83 (d, J = 8.0 Hz, 1H), 7.78 (s, 2H), 7.54 (s, 1H),
7.48 (t, J = 7.9 Hz, 1H), 6.07 (s, 2H), 4.32 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 166.45, 166.25, 161.14, 157.97,
147.41, 141.16, 136.43, 133.12, 129.98, 129.87, 129.55,
128.44, 125.74, 125.50 (q, J = 4 Hz), 124.20, 124.08 (q, J =
271 Hz), 118.79, 115.83, 106.82, 94.85, 34.87; HRMS
55.60,
34.83;
HRMS
(ESI)
+
Na+] 575.97701, found
+
m/z
calcd
for
C23H1681BrKN5O3S2++ [M + K+] 593.94891, found 593.94716;
C23H16BrN5NaO3S2
[M
575.97594; C23H17BrN5O3S2 [M + H+] 553.99507, found
553.99384; HPLC 99.4% (Rt = 7.31 min, 50-80% CH3OH in
water for 7 min and 80% CH3OH in water for 3 min, 10 min
in total).
+
(ESI) m/z calcd for C23H13Cl2F3KN6O2S2 [M + K+]
634.95021, found 634.95043; C23H14Cl2F3N6O2S2+ [M + H+]
596.99433, found 596.99382; HPLC 94.8% (Rt = 7.70 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
6.1.55. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-(3-(2-fluoro
pyridin-3-yl)phenyl)thiazol-2-yl)acetamide (36)
6.1.52. 2-((5-cyano-6-oxo-4-(3-(trifluoromethox
y)phenyl)-1,6-dihydropyrimidin-2-yl)thio)-N-(4-p
henylthiazol-2-yl)acetamide (33)
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 8j (70 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 36 was obtained as white solid (81 mg,
71% yield). m.p. 146-147 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.64 (s, 1H), 8.29 (dt, J = 4.8, 1.5 Hz, 1H),
8.23 – 8.18 (m, 1H), 8.18 – 8.12 (m, 1H), 8.04 – 7.94 (m,
1H), 7.78 (s, 1H), 7.66 – 7.55 (m, 2H), 7.52 (ddd, J = 7.1,
4.8, 1.8 Hz, 1H), 7.41 – 7.30 (m, 2H), 7.20 (t, J = 7.9 Hz,
1H), 7.06 – 6.95 (m, 1H), 4.34 (s, 2H), 3.64 (s, 3H); 13C
NMR (100 MHz, DMSO-d6): δ 167.47, 166.37, 165.83,
161.41, 160.11 (d, J = 236 Hz), 159.46, 158.30, 148.86,
147.13 (d, J = 14 Hz), 141.94 (d, J = 4 Hz), 136.74, 135.20,
134.43 (d, J = 5 Hz), 129.90, 129.77, 128.62, 126.55,
126.15, 123.44, 123.16 (d, J = 4 Hz), 121.44, 117.67,
116.12, 114.41, 109.51, 93.94, 55.60, 34.84; HRMS (ESI)
The procedure described for 12 was used, starting from
4f (63 mg, 0.2 mmol), 8a (50 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 33 was obtained as yellowish solid (96 mg,
91% yield). m.p. 149-150 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.52 (s, 1H), 7.90 (d, J = 7.7 Hz, 2H), 7.80 (d,
J = 7.6 Hz, 1H), 7.71 (s, 1H), 7.63 (s, 1H), 7.46 (dt, J =
14.6, 7.8 Hz, 4H), 7.34 (q, J = 7.4, 6.1 Hz, 1H), 4.14 (s, 2H);
13C NMR (100 MHz, DMSO-d6): δ 170.34, 167.69, 165.74,
158.18, 149.40, 148.51, 139.33, 134.77, 130.68, 129.14,
128.89, 128.21, 127.88, 126.13, 123.22, 121.20, 120.48 (q,
J = 255 Hz), 118.68, 108.49, 91.39, 34.60; HRMS (ESI) m/z
+
calcd for C23H15F3N5O3S2 [M + H+] 530.05629, found
+
m/z calcd for C28H19FKN6O3S2 [M + K+] 609.05757, found
530.05563; HPLC 96.4% (Rt = 7.45 min, 50-80% CH3OH in
water for 7 min and 80% CH3OH in water for 3 min, 10 min
in total).
609.04987; C28H19FN6NaO3S2+ [M + Na+] 593.08363, found
+
593.08289; C28H20FN6O3S2 [M + H+] 571.10168, found
571.10112; HPLC 98.4% (Rt = 6.33 min, 50-80% CH3OH in
water for 7 min and 80% CH3OH in water for 3 min, 10 min
in total).
6.1.53. N-(4-(4-amino-3,5-dichlorophenyl)thiazo
l-2-yl)-2-((5-cyano-6-oxo-4-(3-(trifluoromethoxy)

ACCEPTED MANUSCRIPT
6.1.56. 2-((6-oxo-1,6-dihydropyrimidin-2-yl)thio)
-N-(4-phenylthiazol-2-yl)acetamide (37)
Hz, 1H) , 7.00 (d, J = 7.5 Hz, 1H), 3.93 (s, 2H), 3.75 (s, 3H);
13C NMR (100 MHz, DMSO-d6): δ 171.59, 170.63, 168.11,
167.32, 159.32, 141.40, 139.38, 136.80, 135.14, 129.64,
128.88, 126.37, 124.48, 121.00, 120.41, 115.95, 113.88,
109.92, 89.83, 55.57, 34.64; HRMS (ESI) m/z calcd for
C23H18KN6O3S+ [M + K+] 497.07927, found 497.07838;
C23H19N6O3S+ [M + H+] 459.12339, found 459.12291; HPLC
98.1% (Rt = 8.11 min, 40-60% CH3OH in water for 10 min).
The procedure described for 12 was used, starting from
2-thioxo-2,3-dihydropyrimidin-4(1H)-one (26 mg, 0.2
mmol), 8a (50 mg, 0.2 mmol) and KOH (13 mg, 0.22 mmol),
37 was obtained as white solid (61 mg, 89% yield). m.p.
194-195 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.58 (s, 1H),
7.91 (d, J = 7.2 Hz, 2H), 7.88 (d, J = 6.6 Hz, 1H), 7.64 (s,
1H), 7.44 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 6.16
(d, J = 6.5 Hz, 1H), 4.24 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 166.86, 163.97, 163.11, 158.26, 154.36,
149.37, 134.67, 129.22, 128.31, 126.14, 109.90, 108.69,
6.1.60. 2-((5-cyano-4-(3-ethoxyphenyl)-6-oxo-1,
6-dihydropyrimidin-2-yl)thio)-N-(4-phenyl-1H-im
idazol-2-yl)acetamide (41)
The procedure described for 12 was used, starting from
4d (55 mg, 0.2 mmol), 10a (47 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 41 was obtained as white solid (81 mg,
86% yield). m.p. 213-214 °C; 1H NMR (400 MHz,
DMSO-d6) δ 11.78 (s, 1H), 11.45 (s, 1H), 7.72 (d, J = 7.5
Hz, 2H), 7.41 – 7.23 (m, 6H), 7.17 (t, J = 7.2 Hz, 1H), 7.00
(d, J = 8.0 Hz, 1H), 4.00 (t, J = 7.1 Hz, 4H), 1.28 (t, J = 6.0
Hz, 4H); 13C NMR (100 MHz, DMSO-d6): δ 167.91, 167.70,
158.62, 141.46, 138.85, 136.27, 134.80, 129.71, 128.90,
126.42, 124.48, 121.04, 119.62, 117.01, 114.18, 110.85,
90.66, 63.56, 34.76, 15.05; HRMS (ESI) m/z calcd for
C24H20KN6O3S+ [M + K+] 511.09492, found 511.09466;
C24H20N6NaO3S+ [M + Na+] 495.12098, found 495.12022;
C24H21N6O3S+ [M + H+] 473.13904, found 473.13818; HPLC
97.9% (Rt = 10.01 min, 40-60% CH3OH in water for 10 min
and 60% CH3OH in water for 5 min, 15min in total).
34.22; HRMS (ESI) m/z calcd for C15H12KN4O2S2 [M + K+]
+
+
383.00333, found 382.99370; C15H12N4NaO2S2 [M + Na+]
+
367.02939, found 367.02913; C15H13N4O2S2 [M + H+]
345.04744, found 345.04717; HPLC 97.2% (Rt = 6.07 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
6.1.57. 2-((4-amino-6-oxo-1,6-dihydropyrimidin-
2-yl)thio)-N-(4-phenylthiazol-2-yl)acetamide
(38)
The procedure described for 12 was used, starting from
6-amino-4-hydroxypyrimidine-2(1H)-thione (29 mg, 0.2
mmol), 8a (50 mg, 0.2 mmol) and KOH (13 mg, 0.22 mmol),
38 was obtained as light brown solid (62 mg, 86% yield).
m.p. 239-240 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.43 (s,
1H), 11.54 (s, 1H), 7.90 (d, J = 7.6 Hz, 2H), 7.63 (s, 1H),
7.43 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H), 6.48 (s,
2H), 5.06 (s, 1H), 4.14 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 167.48, 165.91, 164.30, 158.26, 149.38,
134.70, 129.20, 128.28, 126.15, 109.05, 108.69, 81.94,
6.1.61. N-(4-(4-amino-3,5-dichlorophenyl)-1H-i
midazol-2-yl)-2-((5-cyano-4-(3-methoxyphenyl)-
6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetamide
(42)
+
33.82; HRMS (ESI) m/z calcd for C15H13KN5O2S2 [M + K+]
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 10g (64 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 42 was obtained as yellow solid (80 mg,
74% yield). m.p. 210-211 °C; 1H NMR (400 MHz,
DMSO-d6): δ 11.93 (s, 3H), 7.63 (s, 2H), 7.43 (d, J = 7.5
Hz, 2H), 7.41 (s, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.09 (d, J =
7.5 Hz, 1H), 5.45 (s, 2H), 4.26 (s, 2H), 3.69 (s, 3H); 13C
NMR (100MHz, DMSO-d6): δ 167.49, 166.11, 165.87,
161.68, 159.48, 140.88, 139.75, 136.78, 134.17, 130.05,
124.17, 121.56, 118.95, 117.96, 116.28, 114.25, 110.11,
93.69, 55.62, 35.00; HRMS (ESI) m/z calcd for
C23H17Cl2KN7O3S+ [M + K+] 580.01222, found 580.01111;
C23H18Cl2N7O3S+ [M + H+] 542.05634, found 542.05512;
HPLC 95.7% (Rt = 9.59 min, 40-60% CH3OH in water for 10
min and 60% CH3OH in water for 2 min, 12min in total).
+
398.01422, found 398.00396; C15H13N5NaO2S2 [M + Na+]
+
382.04029, found 382.03961; C15H14N5O2S2 [M + H+]
360.05834, found 360.05781; HPLC 95.4% (Rt = 5.48 min,
50-80% CH3OH in water for 7 min and 80% CH3OH in
water for 3 min, 10 min in total).
6.1.58. 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N
-(4-phenylthiazol-2-yl)acetamide (39)
The procedure described for 12 was used, starting from
5-amino-1,3,4-thiadiazole-2-thiol (27 mg, 0.2 mmol), 8a (50
mg, 0.2 mmol) and KOH (13 mg, 0.22 mmol), 39 was
obtained as yellowish solid (57 mg, 82% yield). m.p.
224-225 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H),
7.90 (d, J = 7.7 Hz, 2H), 7.66 (s, 1H), 7.44 (t, J = 7.6 Hz,
2H), 7.41 – 7.23 (m, 3H), 4.12 (s, 2H).; 13C NMR (100 MHz,
DMSO-d6): δ 170.61, 166.99, 158.09, 149.44, 149.30,
134.65, 129.21, 128.31, 126.15, 108.82, 37.81; HRMS
6.2. In Vitro BACE-1 inhibition assays.
+
(ESI) m/z calcd for C13H11KN5OS3 [M + K+] 387.97573;
BACE-1 inhibition assays were carried out using a
Fluorescense Resonance Energy Transfer (FRET) assay
kit purchased from invitrogen (P2985) in the 384-well black
round-bottomed microplate (Corning 3677) with a final
found 387.97517; C13H12N5OS3+ [M + H+] 350.01985; found
350.01943; HPLC 99.0% (Rt = 6.29 min, 50-80% CH3OH in
water for 7 min and 80% CH3OH in water for 3 min, 10 min
in total).
volume
of
18
µL/well.
A
statin
peptide
(H-Lys-Thr-Glu-Glu-Ile-Ser-Glu-
Val-Asn-Sta-Val-Ala-Glu-Phe-OH,
6.1.59. 2-((5-cyano-4-(3-methoxyphenyl)-6-oxo-
1,6-dihydropyrimidin-2-yl)thio)-N-(4-phenyl-1H-i
midazol-2-yl)acetamide (40)
The procedure described for 12 was used, starting from
4a (52 mg, 0.2 mmol), 10a (47 mg, 0.2 mmol) and KOH (13
mg, 0.22 mmol), 40 was obtained as white solid (88 mg,
96% yield). m.p. 269-270 °C; 1H NMR (400 MHz,
DMSO-d6): δ11.68 (s, 1H), 11.45 (s, 1H) , 7.71 (d, J = 7.5
Hz, 2H), 7.32 (m, PhH, 5H) , 7.26 (s, 1H), 7.16 (t, J = 7.5
AnaSpec
23959)
derivative was used in the assay as a positive control.The
assays were run under the following step: 1. Add 6 µL 3X
BACE1 Substrate (750 nM) to 10 µL 3X Test Compound (or
BACE1 Assay Buffer if preparing a control well) and mix
gently; 2. Add 6 µL of 3X BACE1 Enzyme (1 Units/mL); 3.
Incubate 60 minutes at room temperature and read the
fluorescence. Compounds 12-39 were tested at
concentration of 10 µM and 50µM, and compounds 40-42
a

ACCEPTED MANUSCRIPT
were tested at a concentration of 1 µM and 10 µM. The IC50
donor plate was removed after incubation. The
concentrations of tested compounds in the acceptor and
reference solutions were determined by HPLC. Reference
solutions were prepared by diluting the sample secondary
stock solution to the same concentration as that with no
membrane barrier. Every sample was analyzed at three
wavelengths, in three wells, and in two independent runs.
The Pe was obtained by the following equation:
values were then determined on all the compounds that
displayed 50% or higher inhibition rate at 50 µM or 10 µM.
IC50 values were calculated using Graph Pad Prism
Software. All the IC50 measurements were done in
duplicate.
6.3. Molecular docking
The molecular docking procedure of compound 12, 23, 36
and 40 was performed with GOLD 3.0 (Cambridge
Crystallographic Data Centre, Chembridge, UK) using the
default parameter settings and GOLDScore was used as
the fitness function. The three-dimensional (3D) structure of
BACE1 (PDB ID: 3VF3) was downloaded from Protein Data
Bank (PDB). The ligand was built by ChemDraw
(ChembridgeSoft Inc, Chembridge, MA) and initial 3D
conformation was constructed by Chem3D. Next, the ligand
was prepared for docking using Discovery Studio 2.5
(Accelrys Software Inc, San Diego, CA). The binding
modes were visually inspected by PyMol 0.99 (DeLano
Scientific LLC Inc, San Carlos, CA).
where Vdn (mL) = volume of the donor compartment, Vac
(mL) = volume of the acceptor compartment, [drug]ac
=
compound concentration of the solution of the acceptor
compartment, [drug]ref = compound concentration of the
reference solution, s (cm2) = membrane area (0.3 cm2), and
t (s) = incubation time.
6.6. In Vitro Cytotoxicity assay.
The assay was performed with the Human Embryonic
Kidney 293 cells (HEK293) which were purchased from the
cell bank of the Shanghai Institute of Cell Biology. The
HEK293 cell line was cultured in the DMEM medium. The
media was supplemented with 10% fetal bovine serum
(FBS), 100 units/ ml penicillin, and 100 units/ ml
streptomycin (Invitrogen). The cells were maintained at 37
°C in a humidified environment with 5% CO2. The cell
viability was determined by using the CellTiter GloTM
luminescent cell viability assay (Promega). Briefly, the cells
were seeded into 384-well plates at an initial density of
1000 cells/well in 45 mlof medium. Then the cells were
treated with compounds at varying concentrations. The first
compound work concentration is 100 mM, and then
three-fold dilution ten more times. Staurosporine
(SigmaeAldrich, catalog No. S4400) was used as a positive
control. After incubation for 72 h, 10% of CellTiter GloTM
reagent was added and luminescent signals were read on a
VeriScan reader (Thermo Fisher Scientific). The IC50 value
was calculated from the curves generated by plotting the
percentage of the viable cells versus test concentrations on
a logarithmic scale using Sigma Plot 10.0 software.
6.4. SPR based assays and binding Affinity of BACE-1
BACE-1 was immobilized on a CM5 sensor chip using
standard amine coupling procedure at a concentration of 20
µg mL-1 (in 10 mM acetic acid, pH 4.5) to a level of
7500-8000 response units (RU). Typically, 2 flow cells of
the sensor were used: flow cell 3 served as a reference cell
was activated and deactivated, flow cell 4 contained
BACE-1.
Affinity experiments were performed at 25 °C using
Biacore T200 instrument by initial injection of 20 buffer
blanks to equilibrate the surfaces followed by the injection
of analyte solutions at different concentrations in 1.5-fold
dilutions over all flow cells. The running buffer for the
binding experiments was PBS, pH 7.4 with 5% DMSO with
a flow rate of 30 µL min-1. Running buffer samples
containing 4.5-5.8% DMSO were also injected to create a
DMSO calibration plot. Affinity constants (KD) were
determined using the Biacore T200 evaluation software
V.2.0 by curve fitting using steady state model.
Acknowledgments
6.5. In Vitro Blood−Brain Barrier Permeability Assay.
The ability to cross the BBB was predicted and
evaluated using PAMPA. The 96-well filter plate (catalog
no. MAIPN4550) and the donor plate (catalog no.
MATRNPS50) were both purchased from Millipore. The
porcine polar brain lipid (PBL) was purchased from Avanti
Polar Lipids. Dodecane was obtained from J&K®.
Verapamil, clonidine, hydrocortisone and theophylline were
purchased from Energy Chemical and used as positive
control. Tested compounds were dissolved in DMSO at 5
mg ml−1 as stock solutions, which were diluted in PBS to
make a final solution (final concentration 25 µg ml−1). The
PBL was dissolved in dodecane at 20 mg ml−1. A 300 µL
final solution was added to the donor well, the filter
membrane was coated with 4 µL of PBL solution, and the
acceptor well was filled with 150 µL of PBS. The acceptor
filter plate was carefully put on the donor plate to form a
“sandwich” which was composed of the donor with tested
compounds on the bottom, artificial lipid membrane in the
middle, and the acceptor on the top. The sandwich was
incubated undisturbed at room temperature for 18 h. The
This work was supported by the National Natural
Science Foundation of China (21002002) and the Beijing
Natural Science Foundation (7162110).
Appendix And. Supplementary data
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ò
ò
ò
ò
Guided by docking studies, a series of compounds were designed and synthesized
Enzyme activity, affinity, permeability and cytotoxicity have been evaluated
Sixteen compounds were found to have µM to sub-µM level activities towards BACE1
Compound 41 exhibited high activities and the most optimized drug-like profiles