Ligand-based optimization to identify novel 2-aminobenzo[d]thiazole derivatives as potent sEH inhibitors with anti-inflammatory effects

Article abstract of DOI:10.1016/j.ejmech.2020.113028

Inhibition of the soluble epoxide hydrolase (sEH) is a promising new therapeutic approach in the treatment of inflammation. Driven by the in-house database product lead 1, a hybridization strategy was utilized for the design of a series of novel benzo [d]thiazol derivatives. To our delight, D016, a byproduct of compound 9, was obtained with an extraordinarily low IC₅₀ value of 0.1 nM but poor physical and chemical properties. After removal of a non-essential urea moiety or replacement of the urea group by an amide group, compounds 15a, 17p, and 18d were identified as promising sEH inhibitors, and their molecular binding modes to sEH were constructed. Furthermore, compounds 15a and 18d exhibited more effective in vivo anti-inflammatory effect than t-AUCB in carrageenan-induced mouse paw edema. Compound 15a also showed moderate metabolic stability with a half-time of 34.7 min. Although 18d was unstable in rat liver microsomes, it might be a “prodrug”. In conclusion, this study could provide valuable insights into discovery of new sEH inhibitors, and compounds 15a and 18d were worthy of further development as potential drug candidates to treat inflammation.

Full text of DOI:10.1016/j.ejmech.2020.113028

Journal Pre-proof
Ligand-based optimization to identify novel 2-aminobenzo[d]thiazole derivatives as
potent sEH inhibitors with anti-inflammatory effects
Yufei Han, Desheng Huang, Sicong Xu, Lingling Li, Ye Tian, Shuo Li, Cong Chen,
Yingxiu Li, Yanping Sun, Yunlei Hou, Yongjun Sun, Mingze Qin, Ping Gong, Zibin
Gao, Yanfang Zhao
PII:
S0223-5234(20)31000-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.113028
EJMECH 113028
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2020
Revised Date: 11 November 2020
Accepted Date: 13 November 2020
Please cite this article as: Y. Han, D. Huang, S. Xu, L. Li, Y. Tian, S. Li, C. Chen, Y. Li, Y. Sun,
Y. Hou, Y. Sun, M. Qin, P. Gong, Z. Gao, Y. Zhao, Ligand-based optimization to identify novel 2-
aminobenzo[d]thiazole derivatives as potent sEH inhibitors with anti-inflammatory effects, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113028.
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Graphical Abstract
Three series of novel 2-aminobenzo[d]thiazole derivatives were designed, synthesized and
evaluated for their biological activities.

Ligand-based optimization to identify novel 2-aminobenzo[d]thiazole derivatives as
potent sEH inhibitors with anti-inflammatory effects
a
b
a
b
a
b
a
Yufei Han , Desheng Huang , Sicong Xu , Lingling Li , Ye Tian , Shuo Li , Cong Chen ,
a
b
a
b
a, c
a
Yingxiu Li , Yanping Sun , Yunlei Hou , Yongjun Sun , Mingze Qin , Ping Gong , Zibin Gao
b1*
a1*
,
Yanfang Zhao
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
b
State Key Laboratory Breeding Base
—Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei
University of Science and Technology，Shijiazhuang, China
c
Chinese People’s Liberation Army Logistics Support Force No.967 Hospital, Dalian 116021, PR China
Abstract:
Inhibition of the soluble epoxide hydrolase (sEH) is a promising new therapeutic
approach in the treatment of inflammation. Driven by the in-house database product
lead 1, a hybridization strategy was utilized for the design of a series of novel
benzo[d]thiazol derivatives. To our delight, D016, a byproduct of compound 9, was
obtained with an extraordinarily low IC50 value of 0.1 nM but poor physical and
chemical properties. After removal of a non-essential urea moiety or replacement of
the urea group by an amide group, compounds 15a, 17p, and 18d were identified as
promising sEH inhibitors, and their molecular binding modes to sEH were constructed.
Furthermore, compounds 15a and 18d exhibited more effective in vivo
anti-inflammatory effect than t-AUCB in carrageenan-induced mouse paw edema.
Compound 15a also showed moderate metabolic stability with a half-time of 34.7 min.
Although 18d was unstable in rat liver microsomes, it might be a “prodrug”. In
conclusion, this study could provide valuable insights into discovery of new sEH
inhibitors, and compounds 15a and 18d were worthy of further development as
potential drug candidates to treat inflammation.
1
Corresponding author
E-mail address: yanfangzhao@126.com (Yanfang Zhao), zbgao74@163.com (Zibin Gao).

Keywords: sEH, 2-aminobenzo[d]thiazole, structure-based design, anti-inflammation
1
. Introduction
Inflammation is a complex pathophysiological process, involving a myriad of
enzymes, mediators, and receptors [1]. The typical physiological characteristics of
inflammation are redness, swelling, fever, pain, and even loss of organ function [2]. In
the past decades, several studies have suggested that the arachidonic acid (AA)
pathway generates bioactive lipid mediators, which regulate a diverse range of
inflammatory processes. There are three major enzymatic pathways involved in
regulating eicosanoid synthesis, namely cyclooxygenase (COX), lipoxygenase (LOX),
and cytochrome P450 proteins. Recently, the cytochrome P450 pathway has garnered
great attention. Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived
eicosanoids that can produce vasodilation and inhibit the cytokine-induced
inflammatory response in the vasculature, heart, and kidney [3, 4]. EETs are known as
important signal components in an organism and produce biological effects such as
vasodilation, blood pressure, and anti-inflammation. EETs were reported to inhibit the
activity of IκB kinase (IKK) [5], thus inhibiting the cytokine-induced activation of
nuclear factor κB (NF-κB), an essential regulatory factor for endothelium activation,
and playing an anti-inflammation role [6]. Moreover, EETs can transcriptionally
downregulate cyclooxygenase 2 (COX-2), resulting in reduction of inflammatory
metabolites [7, 8]. Studies have shown that inhibition of soluble epoxide hydrolase
(sEH) in humans could increase the concentration of EETs [9]. Thus, inhibition of
sEH is considered a promising therapeutic approach for resolving inflammation.
sEH in humans is a bifunctional homodimeric enzyme in the cytosol and
peroxisomes with both epoxide hydrolase and phosphatase activities [10]. Notably,
the C-terminal domain of sEH transforms EETs to dihydroxyeicosatrienoic acids
(DHETs), thus exhibiting an epoxide hydrolase activity [11]. sEH inhibitors were
shown to increase the concentration of EETs in plasma and tissues [12-14] and exhibit
more potent anti-inflammation effect in vivo than NSAIDs and COXIBs, in a variety
of inflammation-based assays [15-17]. These results indicate that maintaining the in
vivo concentration of EETs through inhibiting sEH enzyme is a promising therapeutic

approach for inflammation. Thus, there is increasing interest in the development and
preclinical evaluation of novel sEH inhibitors.
Although several inhibitors of sEH have been identified, none is available on the
market to date. Only a few inhibitors have reached clinical trials. As shown in Fig.1,
di-substituted ureas and amides were found to be the most popular class of sEH
inhibitors [18-22]. Among these inhibitors, EC5026, a first-in-class and orally active
sEH inhibitor shows efficacy for inflammatory and neuropathic pain and for use as a
non-addictive opioid alternative [23]. GSK2256924 shows a protective effect against
cigarette smoke-induced pulmonary inflammation in mice [24]. Furthermore, X-ray
crystallographies of most sEH inhibitors, such as t-AUCB and TPPU, have
demonstrated that ureas or amides could form a strong net like hydrogen bonds with
residues Asp335, Tyr466, and Tyr383 in the catalytic domain of sEH [25-28].
Fig.1. Representative examples of sEH inhibitors.
Through a screening of the in-house database, lead compound 1,
2
-aminobenzo[d]thiazole, was identified with sEH inhibitory activity of 0.8 μM. To
identify novel scaffold entities as potent sEH inhibitors, a molecular hybridization
strategy between the urea group and lead 1 was applied. Thus, a series of
1
-(benzo[d]thiazol-2-yl)-3-phenylurea derivatives were designed and synthesized.
Interestingly, during the synthesis of compound 9, D016, a byproduct, exerted with
7-fold potency against sEH than t-AUCB (IC = 0.1 nM). Further modification
6
5
0

efforts were made through a structure-activity relationships (SARs) study of D016;
three other series of 1-(2-aminobenzo[d]thiazol-6-yl)urea derivatives (compounds
1
5a-h, 17a-v, and 18a-r) were obtained. Of these, compounds 15a, 17p, and 18d
exhibited significant enzymatic activities, and they were assayed using
carrageenan-induced mice models.
2
. Results and discussion
2
.1 Chemistry
The general synthetic routes of title compounds are illustrated in Scheme 1-5.
Commercially available starting material 2-amino-6-nitrobenzothiazole (1) was
reacted with phenyl (4-chlorophenyl) carbamate or phenyl (4-chlorobenzyl)
carbamate in a DBU/1,4-dioxane system to produce a satisfactory yield of 2a-2b,
.
followed by reduction of 2a-2b with 80% hydrazine hydrate and FeCl 6H O to yield
3
2
3
a-3b, and then a typical N-acetylation reaction to produce 4a-4b. The synthetic
routes of 9 and D016 are outlined in Scheme 2. 2-amino-6-nitrobenzothiazole (1) was
reacted with di-tert-butyl dicarbonate, then reduced with 80% hydrazine hydrate and
.
FeCl 6H O to yield 6. Reaction of intermediate 6 with 4-(2-chloroethyl) morpholine
3
2
followed by deprotection resulted in 8. During the preparation of compound 9, the
yield depended highly on the content of phenyl (4-chlorophenyl) carbamate. Lower
content of phenyl (4-chlorophenyl) carbamate led to a mixture of compound 9 and
D016. After optimization of the coupling conditions, D016 could be conveniently
prepared with 80% yield.
The synthetic route of compounds 12a-j is depicted in Scheme 3. Intermediate 6
was reacted with phenyl chloroformate in acetone at room temperature to generate 10,
which was further condensed with different anilines to produce the corresponding
compounds 11a-j. After treating intermediates 11a-j with trifluoroacetic acid in
dichloromethane, target compounds 12a-j were obtained in high yields. The syntheses
of target compounds 15a-h are depicted in Scheme 4. The substitution reaction of key
intermediate 6 with appropriate alkyl halide in the presence of K CO in 1,4-dioxane
2
3
at 90 °C afforded the corresponding compounds 13a-h. Target compounds 15a-h
were generated through substitution of 13a-h with phenyl (4-chlorophenyl) carbamate

and then deprotection with trifluoroacetic acid.
As shown in Scheme 5, intermediate 7 was condensed with different substituent
carbamates, followed by deprotection with trifluoroacetic acid at room temperature to
produce target compounds 17a-v. Compounds 18a-v were then obtained in moderate
yields through condensation reactions of 15a, 17e, and 17p with different acyl
chlorides.
Scheme 1 Reagents and conditions: (a) phenyl (4-chlorophenyl)carbamate or phenyl
o
.
.
(
4-chlorobenzyl)carbamate, DBU, 1,4-dioxane, 80 C, 12 h; (b) 80% N H H O, FeCl 6H O,
2 4 2 3 2
o
o
active carbon, 1,4-dioxane, 80 C, 12 h; (c) acetyl chloride, Et N, DCM, 0 C, 2 h.
3
N
N
Boc
NH
N
Boc
NH
N
Boc
NH
d
e
f
O
NH2
N
S
2
O N
S
2
H N
S
N
H
S
O2N
1
5
6
7
Cl
O
Cl
N
S
NH
O
NH
N
O
N
N
S
g
O
h
N
S
NH
NH2
O
O
NH
Cl
N
NH
+
N
H
N
N
H
8
9
D016
o
.
Scheme 2 Reagents and conditions: (d) (Boc) O, DMAP, DMF, 120 C, 12 h; (e) 80% N H H O,
2
o
2
4
2
.
FeCl 6H O, active carbon, 1,4-dioxane, 80 C, 12 h; (f) 4-(2-chloroethyl)morpholine, K CO ,
3
2
2
3
o
1
,4-dioxane, 90 C, 12 h; (g) CF COOH, DCM, rt, 3 h; (h) phenyl (4-chlorophenyl) carbamate,
3
o
DBU, 1,4-dioxane, 80 C, 12 h.

1
Scheme 3 Reagents and conditions: (i) phenyl chloroformate, acetone, rt, 1 h; (j) R -NH , Et N,
2
3
o
1
,4-dioxane, 60 C, 2 h; (k) CF COOH, DCM, rt, 3 h.
3
2
o
Scheme 4 Reagents and conditions: (l) R (CH ) Cl, alkyl halide, K CO , 1,4-dioxane, 90 C, 12 h;
2
n
2
3
o
(
m) phenyl (4-chlorophenyl) carbamate, Et N, 1,4-dioxane, 60 C, 12 h; (n) CF COOH, DCM, rt,
3
3
3
h.
Scheme 5 Reagents and conditions: (o) phenyl ester, sodium carbonate, THF/EA/H O, rt, 12 h; (p)
2

o
4
Et N, 1,4-dioxane, 60 C, 12h; (q) CF COOH, DCM, rt, 3 h. (r) R -COCl, K CO , EA, rt, 5 h.
3
3
2
3
2
2
2
.2 Biological activity and discussion
.2.1 In vitro sEH activity and SARs study
.2.1.1 Target compounds for hybridization strategy and D016
The urea group was reported as the active essential moiety of sEH inhibitors.
With the discovered lead compound 1, molecular hybridization strategy was
employed to introduce the 4-chlorophenyl or 4-chlorobenzyl urea group in the
2
-position or 6-position of benzo[d]thiazole. Therefore, nine different biaryl urea
compounds were synthesized (2a-2b, 3a-3b, 4a-4b, 9, and 12a-b).
As shown in Table 1, compounds with the urea group at 2-position of
benzo[d]thiazole (2a-2b, 3a-3b, 4a-4b and 9) exerted weak sEH inhibitory activities
(inhibition activity values < 20%). However, the 6-ureido derivatives showed higher
activity, of which, compound 12a displayed potent sEH inhibitory activity with an
1
IC value of 10.1 nM. Subsequently, compounds 12c-j with different R substituents
5
0
were prepared. Among them, compound 12c with 1-adamantyl group exhibited more
potent activity than other substituents but was still weaker than 12a.
Interestingly, during the preparation of compound 9 according to Scheme 2, a
byproduct (compound D016) was separated and obtained with 67-fold potency against
sEH than t-AUCB (IC = 0.1 nM). Then, by increasing the content of phenyl
5
0
(4-chlorophenyl) carbamate in the synthetic process, D016 could be obtained with 80%
yield. However, D016 showed poor physical and chemical properties (high ClogP
value and low solubility), which prompted us to conduct further modification.
Table 1. In vitro sEH activities assay of series A compounds.

a
sEH inhibitory activity
Inhibition @100nM
1
Compd.
R
IC50 (nM)
b
2
2
3
3
4
4
a
b
a
b
a
b
7.2%
10.9%
6.7%
ND
ND
ND
ND
ND
14.2%
12.5%
14.5%
16.2%
76.1%
ND
ND
9
1
2a
2b
10.1
1
47%
ND
822
ND
ND
ND
4.1
1
2c
77.3%
40.1%
39.3%
60.2%
59.9%
20.5%
34.1%
1
2d
1
2e
1
2f
1
2g
2h
1
ND
ND
1
2i
2j
1
H
-
25.3%
90.2%
87.8%
ND
0.1
6.7
D016
t-AUCB
a
b
All compounds were assayed at least twice, and the inhibitory values were averaged.
Not determined.
2
.2.1.2 Examination of the effect of morpholino moiety
Based on previous SARs, it was speculated that the urea group at 2-position of
benzo[d]thiazole is not essential for sEH inhibitory activities. Thus, to reduce ClogP

of D016, the urea group at the 2-position was removed to afford compound 15a.
Expectedly, compound 15a exerted good sEH inhibitory activity with an IC value of
50
5
.8 nM. Consequently, further modification was planned. The morpholino moiety was
first explored. Other hydrophilic substituents were introduced to obtain compounds
1
5a-h. As shown in Table 2, substituting the morpholino group for a
-methylpiperidinyl group (15 g) decreased the activity against sEH (%inhibition
4
range from 89.1% to 77.5%). Compounds 15b-f were synthesized to investigate the
effects of other hydrophilic substituents, such as N, N-diethylamino group (15b),
pyrrolidinyl group (15c), and piperidinyl group (15d). However, these compounds
2
showed weaker sEH inhibition than compound 15a. When the length of R was
increased, the sEH inhibition rate of 15h were decreased from 89.1% to 9.44%.
Regrettably, the expected results of modification in the morpholino group were not
obtained. However, a gap remains between the promising results with D016 and
t-AUCB. Further pharmacological evaluation shed more light on the importance of
the 4-chlorophenyl moiety.
Table 2. In vitro sEH enzymatic assay of target compounds(15a-15h).
a
sEH inhibitory activity
2
Compd.
n
2
2
2
2
2
2
2
R
Inhibition @100nM
IC50 (nM)
1
5a
5b
89.1%
2.8
1
86.3%
48.3%
84.6%
45.9%
39.1%
77.5%
24.5
N
b
1
5c
ND
1
5d
6.5
ND
ND
20.2
1
5e
1
5f
1
5g

1
5h
3
-
9.44%
87.8%
ND
6.7
t-AUCB
-
a
b
All compounds were assayed at least twice, and the inhibitory values were averaged.
Not determined.
2
.2.1.3 Examination of the effect of 4-chlorophenyl group
Based on the analysis described above, compounds 17a-17v were designed and
synthesized to investigate the influence of the phenyl group on potency. As shown in
Table 3, compounds containing electron-donating groups on the phenyl ring showed
more potent inhibitory activities against sEH than those with electron-withdrawing
groups (17b vs 17d and 17c vs 17j). Moreover, compounds 17f and 17g with
4
-fluorophenyl and 4-bromophenyl motifs, respectively, showed superior activity to
compound 17d. The halogen atom was essential for improved activity. In addition, the
phenyl ring terminal substituents exhibited a vital influence on sEH inhibition. As
expected, introducing 4-fluorophenyl and 4-methylphenyl groups yielded compounds
1
7f and 17d, respectively, which retained a high potency against sEH (%inhibition
activity values ranging from 73.1% to 74.3%). The sterically larger 4-ethylphenyl
17e) group as R moiety resulted in a pronounced increase in inhibitory activity
(
3
against sEH (%inhibition range from 84.7% to 85.8%). However, when larger
substituents were introduced, the inhibitory activities of related compounds 17h, 17i,
and 17j against sEH were significantly reduced. These phenomena indicated that the
size of the R group plays an essential role in the activity against sEH.
3
Table 3. In vitro sEH enzymatic assay of target compounds (17a-17v).
N
O
NH2
R3
S
N
N
H
N
O
a
sEH inhibitory activity
3
Cpd.
R
Inhibition @100nM
IC50 (nM)
b
1
7a
7b
40.4%
ND
1
19.2%
ND

1
7c
28.2%
ND
1
7d
73.1%
84.7%
74.3%
83.8%
8.3
1.9
1
7e
1
7f
ND
16.2
1
7g
7h
1
75.2%
11.2
1
7i
7j
62%
ND
122
ND
1
56.6%
59.7%
1
7k
1
7l
36.5%
26.3%
61.7%
ND
ND
1
7m
1
7n
101.3
1
7o
6.18%
90.5%
ND
1
1
7p
7q
0.25
58.4%
14.5%
61.3%
ND
ND
ND
1
7r
7s
1
1
7t
59.4%
ND

1
7u
7v
16.2%
39.0%
ND
1
150.8
D016
-
-
90.2%
87.8%
0.1
6.7
t-AUCB
a
b
All compounds were assayed at least twice, and the inhibitory values were averaged.
Not determined.
To investigate the influence of substituent positions at the phenyl ring,
compounds 17k-o were synthesized. Compound 15a with the para-substituent at the
phenyl ring showed the greatest potency against sEH, and a similar trend was found
among other analogues with their parent compounds. Then, di-substituted compounds
(17p and 17q) were synthesized. However, replacement of the phenyl ring with
heterocycle motifs (17u and 17v) reduced sEH potency. Surprisingly, compound 17p
with 3-fluro-4-bromo at the phenyl ring showed remarkably increased activity (IC =
50
0
.25 nM). We still have not found a compound with superior sEH inhibitory activity
than D016, indicating a certain cavity at the 2-position of benzo[d]thiazole.
2
.2.1.4 Further modification of the 2-position of benzo[d]thiazole
To improve the physical properties of D016, we employed
the principle of bioisosterism in substituting the urea group for an amide group to
obtain compounds 18a-n. First, the effect of the R substituent was considered (Table
4
4
); for 18a-e, compound 18d bearing phenylacetic acid motif showed the best potency.
Next, effects of various substituents with different electrostatic properties on the
phenyl ring were investigated. Unexpectedly, the introduction of substituents on the
phenyl ring seemed to be unfavorable to activity. However, compound 18h with an
ortho-fluoro phenyl group exerted comparable potency relative to t-AUCB.
Preliminary conclusions indicate that compound 18d bearing arylacetyl and
compound 18h bearing ortho-fluoroarylacetyl group at the 2-position of
benzo[d]thiazole showed more potent activities than the other substituents.
Table 4. In vitro sEH enzymatic assay of target compounds (18a-18r).

a
sEH inhibitory activity
3
4
Cpd.
R
R
Inhibition @100nM
IC50 (nM)
b
1
8a
8b
68.1%
ND
1
32.5%
48.8%
84.7%
77.7%
64%
ND
ND
0.082
ND
ND
ND
4.08
ND
ND
ND
ND
1
8c
1
8d
1
8e
1
8f
1
8g
8h
74.9%
82.5%
71.9%
70.6%
71.8%
65.8%
1
1
8i
8j
1
1
8k
1
8l

1
8m
58.1%
ND
1
8n
8o
8p
64%
76.8%
85.2%
ND
ND
5.4
1
1
1
8q
74.2%
73.8%
ND
ND
1
8r
Cl
O
D016
90.2%
87.8%
0.1 nM
6.7 nM
N
H
t-AUCB
-
-
a
b
All compounds were assayed at least twice, and the inhibitory values were averaged.
Not determined.
Subsequently, the well-tolerated para-ethylphenyl and 3-bromo-4-fluorophenyl
groups (compounds 18o-18r) were explored according to the SARs above. However,
they all showed lower sEH inhibition than 18d. Therefore, compound 18d was
selected as the most potent sEH inhibitor with the IC value of 0.082 nM.
5
0
2
.2.2 Enzyme inhibitory kinetics
To explore the mode of action of the representative compound 18d, enzyme
kinetic studies were conducted as shown in Fig. 2. The results showed that increasing
concentrations of 18d decreased Vmax but increased K , indicating that 18d is a
m
mixed-type inhibitor of sEH. Moreover, the values of K and K were calculated to be
i
is
4
.77 nM and 23.15 nM, respectively, which confirmed that 18d preferentially binds to
free enzyme rather than to the enzyme-substrate complex.

Fig 2. Lineweaver-Burk plot of fluorogenic substrate (PHOME) in the presence of compound 18d.
S]: the concentrations of PHOME (μM); V: initial reaction velocity (RFU/min).
[
2
.2.3 Evaluation of water solubility of potent compounds
Compound D016 showed potent sEH activity but poor solubility (30 μg/mL) and
high cLogP (5.96), possibly caused by the two urea groups. Therefore, optimal
compounds were selected to evaluate for solubility in water, and the results are shown
in Table 5. Compared with D016, compounds 15a and 17p, which were obtained by
removing the urea group at the 2- position of benzo[d]thiazole exhibited a decreased
cLogP; however, only compound 17p had an improved solubility (68 μg/mL).
Moreover, compound 18d obtained by bioisosterism exhibited an increased solubility
(61 μg/mL); this is considered a successful optimization strategy.
Table 5. Water solubility and cLogP of selected potent compounds
a
b
Compounds
5a
sEH IC (nM)
Solubility (μg/mL)
cLogP
3.38
3.69
5.28
5.96
5
0
1
2.8
0.25
0.082
0.1
39
68
61
30
1
1
7p
8d
D016
a
b
Tested using UV Spectrophotometer (UV-2600).
Calculated using instant JChem.

2
.2.4 Common properties of target compounds
Based on the in vitro data, compounds 15a, 17p, 18d, and D016 were chosen for
further studies. First, we focused on evaluating the drug-likeness score through an
empirical model [29]. With t-AUCB (drug-likeness score 0.38) and EC-5026
(
drug-likeness score 0.54) as the reference points, we present the prediction details of
these inhibitors in Fig. 3. Overall, all compounds were superior to t-AUCB (scored
.38) and EC-5026 (scored 0.54). Among them, compound 15a and 18d scored 1.13
0
and 1.17, respectively, indicating that they have promising druggability.
Fig 3. Representative drug-likeness model score results of t-AUCB, EC5026, 15a, 17p, 18d and
D016. (The green curve indicates that a compound is possibly not a drug, while the blue curve
suggests the compound may possess drug-likeness properties.). (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)
2
.2.5 In silico study
The inhibition profiles of the 2-aminobenzo[d]thiazole derivatives were
rationalized by studying the binding modes of compounds 12a, 15a, and 18d to sEH
PDB code: 5ALW). As shown in Fig. 4A, the oxygen atom of urea in compound 12a
(
formed an H-bond with Tyr 466, and the sulfur atom of 2-aminobenzo[d]thiazole
formed an H-bond with residue Trp 525.
The concrete binding mode of compound 15a to sEH is presented in Fig. 4B.
Except for the existing interactions between 12a and sEH, the newly introduced
2
-ethylmorpholine moiety in compound 15a forms an H-bond with Asp 335 and thus
may increase the inhibitory activity against sEH. The 2-aminobenzo[d]thiazole was

oriented towards a wide open and hydrophobic pocket, which may explain why
incorporating a benzoyl or phenylacetyl group into the 2-position of benzothiazole
was well-tolerated (see compounds D016 and 18b-r).
The binding pose of the most potent compound 18d with sEH is depicted in Fig.
4
C. As mentioned above, the terminal phenylacetyl group of 18d dovetailed in the
hydrophobic pocket, and an additional H-bond between the oxygen atom of amide
and Ser 415 was formed.
Fig. 4. The proposed binding mode of compound 12a, 15a and 18d to sEH hydrolase catalytic
domain (PDB code: 5ALW), hydrogen bonds were shown as green dashes.
2
.3 Assessment of in vitro cytotoxicity and metabolism
Based on their in vitro sEH inhibitory activities, 15a, 17p, and 18d were selected
for in vitro cytotoxicity and metabolic stability, and the results are outlined in Table 5.
All three compounds exhibited low cytotoxicity (IC values >25 μM) against HepG-2
50
cells, indicating their low toxicity against hepatic cells. Rat liver microsome studies
revealed that compound 17p exhibited an intrinsic liver clearance of 55.8 μL/min/mg
and was more stable than 15a with an intrinsic liver clearance of 71.9 μL/min/mg
However, compound 18d had a half-life of 1.7 min and intrinsic liver clearance of
476.7 μL/min/mg, which indicated that compound 18d was quite unstable in rat liver
.
1
microsomes. Compound 18d which had an amide group introduced at the 2-position
of compound 15a cleared more rapidly than 15a, indicating amide as a potential
metabolic site for 18d. Besides, 17p exhibited acceptable stability with a half-life of
4
4.7 min and showed moderate metabolism in rat liver microsomes.
Table 6. In vitro cytotoxicity and parameters of Rat Liver Microsomes Stability
HepG-2 cytotoxicity
μM
T
1/2
CLint(mic)
(μL/min/mg)
40.0
CLint(liver)
(μL/min/mg)
71.9
Remaining
(T=60min)
30.1%
Remaining
(NCF=60min)
99.5%
Cpd
a
b
c
IC50
(
)
(min)
34.7
1
5a
7p
>
>
25
25
1
44.7
31.0
55.8
36.2%
85.0%

1
8d
>25
1.7
1.3
820.4
1047.3
59.3
1476.7
1885.1
106.7
0%
115.5%
98%
d
Propafenone
Diclofenac
ND
0.4%
16.9%
d
ND
23.4
96.3%
a
b
c
d
CLint(mic): intrinsic clearance; CLint(mic)=0.693/T1/2/mg microsomal protein per mL
CLint(liver): CLint(mic) × mg microsomal protein/g liver weight × g liver weight/kg body weight
NCF (no co-factor): No NADPH regenerating system is added to NCF samples during the 60 min incubation.
Not determined.
2
.4 In vivo anti-inflammatory activity: carrageenan-induced mice paw edema test
Anti-inflammatory activity of 15a, 17p, and 18d were analyzed in vivo using the
carrageenan-induced mouse paw edema method reported by Sasso et al. [30].
t-AUCB served as the positive control at a dose of 100 mg/kg. The compounds
showed a rapid onset of action and sustained duration until the fourth hour after
administration. Compounds 15a and 18d exhibited more potent anti-inflammatory
activity than t-AUCB, and compound 17p exhibited equivalent in vivo activity to
t-AUCB (Table 6 and Fig. 5). However, compound 18d was unstable in rat liver
microsomes, so we speculated that it was metabolized to 15a. Meanwhile, 18d
exhibited a high drug-likeness score, though it was unstable in rat liver microsomes
and this warrants further research.
Table 7. In vivo anti-inflammatory activities of compounds 15a, 17d, 18d and t-AUCB
(100mg/kg) in BALB/c mice (n=6).
0
h
1h
2h
3h
4h
paw
thickness
paw
thickness
(mm)
paw
thickness
(mm)
paw
thickness
(mm)
paw
thickness
(mm)
a,b
Edema%
32.82
Edema%
Edema%
Edema%
(mm)
Control
2.64±0.09
2.57±0.08
2.40±0.06
2.74±0.09
2.64±0.13
3.51±0.19
3.11±0.17
3.15±0.04
3.33±0.16
3.30±0.15
3.56±0.29
3.06±0.09
34.58
3.61±0.36
3.13±0.21
36.49
3.60±0.12
3.17±0.12
36.24
c
1
1
1
5a
7p
8d
20.99*
19.06*
21.64*
23.39*
30.86
21.56*
25.01*
3.16±0.16
3.22±0.21
3.23±0.15
31.49
17.58*
22.61*
3.06±0.07
3.19±0.19
3.29±0.22
27.39*
16.48*
24.98*
3.06±0.10
3.30±0.11
3.28±0.15
27.12*
20.32*
24.71*
t-AUCB
a
Paw thickness were measured before (0 h) or 1, 2, 3, 4 h after injection of 50 μL of carrageenan in mice right hind paw and were
significantly different compared to vehicle-treated group.
b
Data are expressed as mean ± SEM. The data were compared using two-way analysis of variance (ANOVA) followed by Bonferroni’s
test for multiple comparisons.
c
*P＜0.05 vs. vehicle.

Fig.5. Graphical representation of in vivo anti-inflammatory activities of the selected compounds
in carrageenan-induced mice paws edema. *P＜0.05 vs.15a, #P＜0.05 vs.18d, $ P＜0.05 vs.17p,
§
P＜0.05 vs.t-AUCB.
3
. Conclusion
4
. Driven by the in-house database product lead 1, we employed a hybridization
strategy for the design of a series of novel benzo[d]thiazol derivatives as sEH
inhibitors. To our delight, D016, a by-product of compound 9, was obtained with
extraordinarily low IC value of 0.1 nM but poor physical and chemical properties.
5
0
Then, ligand-based optimization was implemented to design and synthesize three
series of compounds (15a-h, 17a-v, and 18a-r). Among them, compounds 15a, 17p,
and 18d were identified as potent sEH inhibitors with IC values of 2.8 nM, 0.25 nM,
5
0
and 0.082 nM, respectively. Moreover, compounds 15a and 17p exhibited acceptably
moderate metabolic stability in a metabolism assay of rat liver microsomes but not
compound 18d.. Compared to t-AUCB, compound 15a and 18d demonstrated a more
effective in vivo anti-inflammatory effect in carrageenan-induced mouse paw edema
models. Molecular binding models identified the critical interactions between 15a and
1
8d with sEH, which coincided with the SARs analysis. Altogether, 15a and 18d
warrant further study, to eventually confirm their potential application.
. Experimental section
4

4
.1 Chemistry
All melting points were acquired on a Mettler Melting Point MP70 apparatus
Mettler, Toledo, Switzerland) without calibration. Mass spectra (MS) was recorded in
(
electrospray ionization (ESI) mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA,
USA) with positive switching. Samples were analyzed from a 100 mM MeCN/H2O
(6:4) solution with a 3 mL injection volume. The reverse phase HPLC was conducted
on an Agilent 1260 Infinity chromatograph, which was equipped with ZORBAX
SB-C18 column (250 mm × 4.6 mm). The mobile phase A was methanol, and mobile
phase B was 30 mM NaH PO in water (pH 2.5). The gradient of 5−95% A was run at
2
4
a flow rate of 1.0 mL/min over 30 min. Reactions were monitored by thin-layer
1
chromatography (TLC) on silica plates (F-254) and visualized under UV light. H
1
3
NMR and C NMR spectra were performed using Bruker spectrometers (Bruker
Bioscience, respectively, Billerica, MA, USA) with TMS as an internal standard.
Column chromatography was run on silica gel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all materials were
obtained from commercially available sources and used without further purification.
4
.1.1 General procedure for compounds 2a-4a and 2b-4b
4
.1.1.1 1-(4-chlorophenyl)-3-(6-nitrobenzo[d]thiazol-2-yl)urea (2a)
To a solution of 2-amino-6-nitrobenzothiazole (1) (10 g, 51.2 mmol) and
,8-diazabicyclo[5.4.0]undec-7-ene (15.6 g, 102.4 mmol) in DMF (100 mL) was
1
added phenyl (4-chlorophenyl)carbamate (15.2 g, 61.4 mmol). The resulting solution
o
was allowed to stir 18 h at 120 C. The resulting mixture was poured into stirring 2M
HCl (500 mL), and then the yellow solid was filtered and dried under reduced
pressure. The crude product was further purified by flash column chromatography
using PE/EtOAc (5:1) as eluent to afford 14.5 g of 2a as a yellow solid; m.p.:156.2 –
1
56.6 °C. Yield: 82%; HPLC purity: 96.58%, retention time = 26.823 min. MS (ESI)
+
1
m/z: 349.3 [M+H] . H NMR (600 MHz, DMSO-d ) δ 8.98 (d, J = 2.4 Hz, 1H), 8.25
6
(dd, J = 8.9, 2.4 Hz, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.41 (d, J
1
3
=
8.8 Hz, 2H). C NMR (151 MHz, DMSO-d ) δ 164.96, 142.89, 137.65, 132.44,
6
1
29.25, 129.01, 127.18, 122.20, 120.74, 119.74, 119.09.

4
.1.1.2 1-(4-chlorobenzyl)-3-(6-nitrobenzo[d]thiazol-2-yl)urea (2b)
Synthesized using the procedure for 2a, 10 g (51.2 mmol) of 1 and 15,6 g (102.4
mmol) 1,8-Diazabicyclo[5.4.0]undec-7-ene in 100 mL DMF and 16.1 g (61.4 mmol)
of phenyl (4-chlorobenzyl)carbamate, and 13.3 g of 2b was obtained as yellow solid;
m.p.:146.1 – 146.3 °C. Yield: 72%; HPLC purity: 99.70%, retention time =
1
2
6.207min. MS (ESI) m/z: 363.4 [M+H]. H NMR (600 MHz, DMSO-d ) δ 8.22 (d, J
6
=
7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.41 (s, 2H), 7.37 (s, 2H), 7.28 (d, J = 7.0 Hz,
1
3
1
1
4
H), 4.40 (s, 2H). C NMR (151 MHz, DMSO-d ) δ 165.67, 142.68, 140.41, 138.66,
6
32.63, 131.95, 131.42, 129.50, 129.18, 128.72, 128.49, 42.80.
.1.1.3 1-(6-aminobenzo[d]thiazol-2-yl)-3-(4-chlorophenyl)urea (3a)
To a suspension of intermediate 2a (12.0 g, 34.5 mmol) in 120 mL of
.
1
8
1
,4-dioxane was added FeCl H O (1.4 g, 5.2 mmol), carbon (0.12 g, 10.4 mmol) and
3
2
0% hydrazine hydrate (32 mL, 517.5 mmol) in batches. The mixture was heated for
o
2 h at 90 C. After completion of the reaction as indicated by TLC, the mixture was
filtered through a celite bed and concentrated in a vacuum to afford 9.0 g of 3a as a
pale solid, m.p.: 158.2 – 160.0 °C. Yield: 73%; HPLC purity: 95.47%, retention time
+
1
=
20.800 min. MS (ESI) m/z: 319.2 [M+H] . H NMR (600 MHz, DMSO-d ) δ 7.54
6
(d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.2 Hz, 1H), 6.97 (s, 1H),
1
3
6
1
4
.67 (dd, J = 8.2, 2.0 Hz, 1H). C NMR (151 MHz, DMSO-d ) δ 168.55, 158.83,
6
52.57, 138.04, 135.37, 131.75, 129.18, 126.74, 120.48, 119.53, 118.63, 111.76.
.1.1.4 1-(6-aminobenzo[d]thiazol-2-yl)-3-(4-chlorobenzyl)urea (3b)
Synthesized using the procedure for 3a, 10g (27.7 mmol) of 2b and 1.1 g (4.2
.
mmol) of FeCl H O and 0.1 g (8.3 mmol) of carbon in 100 mL 1,4-dioxane, 28 mL
3
2
(415.5 mmol) of 80% hydrazine hydrate was added, and 7 g of 3b was obtained as a
pale solid, m.p.: 165.6 – 167.1 °C. Yield: 76%; HPLC purity: 97.25%, retention time
+
1
=
23.304 min. MS (ESI) m/z: 333.5 [M+H] . H NMR (600 MHz, DMSO-d ) δ 7.40
6
(d, J = 8.1 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.30 (s, 1H),
1
3
7
1
.26 (d, J = 8.0 Hz, 2H), 4.21 (d, J = 5.8 Hz, 2H). C NMR (151 MHz, DMSO-d ) δ
6
58.39, 145.42, 140.42, 139.14, 131.81, 131.41, 129.43, 129.19, 128.70, 128.50,
114.24, 104.78, 42.68.

4
.1.1.5 N-(2-(3-(4-chlorophenyl)ureido)benzo[d]thiazol-6-yl)acetamide (4a)
Compound 3a (6g, 18.9 mmol) was diluted in 60 mL DCM together with Et N
3
(2.1 g, 20.8 mmol). Acetyl chloride (1.6 g, 20.8 mmol) was added slowly dropwise at
room temperature. After completion of the reaction (thin-layer chromatography, TLC).
the reaction mixture was diluted with water (30 mL) and extracted with
dichloromethane (3 x 30 mL). The combined dichloromethane extracts were washed
successively with 10% sodium bicarbonate and H O, dried (Na SO ), and
2
2
4
concentrated under reduced pressure. The crude product was further purified by flash
column chromatography using PE/EtOAc (1:1) as eluent to afford 4.8 g of 4a as a
white solid; m.p.:165.2 – 166.8 °C. Yield: 71%; HPLC purity: 95.74%, retention time
+
1
=
23.658 min. MS (ESI) m/z: 361.4 [M+H] . H NMR (600 MHz, DMSO-d ) δ 10.12
6
(s, 1H), 9.99 (s, 1H), 8.26 (d, J = 1.1 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.56 (d, J =
1
3
8
.7 Hz, 2H), 7.47 (dd, J = 8.6, 1.1 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 2.07 (s, 3H). C
NMR (151 MHz, DMSO-d ) δ 168.55, 158.83, 152.57, 138.04, 135.37, 131.75,
6
1
29.18, 126.74, 120.48, 119.53, 118.63, 111.76, 24.33.
4
.1.1.6 N-(2-(3-(4-chlorobenzyl)ureido)benzo[d]thiazol-6-yl)acetamide (4b)
Synthesized using the procedure for 4a, 5 g (15.1 mmol) of 3b and 1.7 g (16.6
mmol) Et N in 50 mL DCM and 1.3 g (16.6 mmol) acetyl chloride, and 3g of 4b was
3
obtained as a white solid; m.p.:156.2 – 158.3 °C. Yield: 71%; HPLC purity: 99.27%,
+
1
retention time = 22.382 min. MS (ESI) m/z: 375.1 [M+H] . H NMR (600 MHz,
DMSO-d ) δ 8.22 (s, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.44 (dd, J = 8.7, 1.3 Hz, 1H),
6
7
.41 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.7 Hz, 1H), 4.37 (d, J
1
3
=
5.9 Hz, 2H), 4.22 (t, J = 6.1 Hz, 1H), 2.07 (s, 3H). C NMR (151 MHz, DMSO-d )
6
δ 168.49, 162.69, 140.42, 138.98, 135.07, 131.84, 131.40, 129.44, 129.18, 128.70,
1
4
4
28.49, 118.41, 111.62, 42.63, 24.31.
.1.2 Procedure for preparation of compound 9
.1.2.1 tert-butyl (6-nitrobenzo[d]thiazol-2-yl)carbamate (5)
To a solution of 2-amino-6-nitrobenzothiazole (1) (10 g, 51.2 mmol) and
-dimethylaminopyridine (3.2 g, 26.2 mmol) in DMF (100 mL) was added
4
di-tert-butyldicarbonate (16.8 g, 77.0 mmol) and the resulting solution was allowed to

o
stir 18 h at 90 C. The resulting mixture was poured into stirring ice-water (500 mL),
and then the yellow solid was filtered and dried under reduced pressure. The crude
product was further purified by flash column chromatography using PE/EtOAc (3:1)
as eluent to afford 15.3 g of 5 as a yellow solid; m.p.:125.2 – 126.6 °C. Yield: 85%;
+
1
HPLC purity: 96.20%, retention time = 25.703 min.MS (ESI) m/z: 296.3 [M+H] . H
NMR (600 MHz, DMSO-d ) δ 12.25 (s, 1H), 9.00 (d, J = 2.3 Hz, 1H), 8.24 (dd, J =
6
8
.9, 2.3 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 1.54 (s, 9H).
4
.1.2.2 tert-butyl (6-aminobenzo[d]thiazol-2-yl)carbamate (6)
To a suspension of intermediate 2 (12.0 g, 40.5 mmol) in 120 mL of 1,4-dioxane
.
was added FeCl H O (1.0 g, 6.2 mmol), active carbon (0.15 g, 12.5 mmol) and 80%
3
2
hydrazine hydrate (35 mL, 600 mmol) in batches. The mixture was heated for 12 h at
o
9
0 C. After completion of the reaction as indicated by TLC, the mixture was filtered
through a celite bed and concentrated in a vacuum to afford 9.3 g of 6 as a pale solid,
m.p.: 128.2 – 130.0 °C. Yield: 73.3%; HPLC purity: 90.17%, retention time = 17.117
+
min. MS (ESI) m/z: 266.2 [M+H] .
4
.1.2.3 tert-butyl (6-((2-morpholinoethyl)amino)benzo[d]thiazol-2-yl)carbamate (7)
-chloro-N, N-diethylethan-1-amine (2.1 g, 21.3 mmol) was added to a mixture
2
of tert-butyl (6-aminobenzo[d]thiazol-2-yl)carbamate (6) (1.3 g, 7.5 mmol) and
K CO (2.1 g, 22.4 mmol) in 1,4-dioxane (20 mL). The mixture was stirred at reflux
2
3
for 8 h. After the reaction was cooled to room temperature, it was poured into 80 mL
water and extracted with ethyl acetate (2×80 mL). The combined organic layers were
washed with brine (60 mL), dried over sodium sulfate, concentrated in vacuo, then
dried under vacuum to obtain 1.0 g of 7 as brown solid; m.p.: 131.2 – 133.5 °C. Yield:
5
6%; HPLC purity: 88.35%, retention time = 11.666 min. MS (ESI) m/z: 379.6
+
[
M+H] .
6
4
.1.2.4 N -(2-morpholinoethyl)benzo[d]thiazole-2,6-diamine (8)
The crude product (7) from the previous step was dissolved in 10 mL of
dichloromethane; 10 mL of trifluoroacetate was then slowly added. After stirring
overnight at room temperature, the mixture was evaporated under reduced pressure
and the resultant was added to 15 mL water, adjusting pH to 8 with saturated sodium

hydrogen carbonate, the precipitate was filtered off and washed with water, then the
crude product was dried in vacuum desiccator and further purified by column
chromatography on silica using mobile phase dichloromethane−methanol (30:1, v/v)
to furnish 8 as a pale yellow solid; yield: 77%; m.p.: 124.3 – 125.7 °C; HPLC purity:
+
9
9.42%, retention time = 9.070 min. MS (ESI) m/z: 279.4 [M+H] .
4
.1.2.5 1-(4-chlorophenyl)-3-(6-((2-morpholinoethyl)amino)benzo[d]thiazol-2-yl)urea
(9)
To a mixture of 8 (0.3 g, 1.1 mmol) and phenyl (4-chlorophenyl)carbamate (0.29
g, 1.2 mmol) in 5 mL DMF, triethylamine (0.13 g, 1.3 mmol) was slowly added, after
o
stirring at 60 C for 12 h, the mixture was poured into 10 mL water, the solution was
neutralized with 1N hydrochloric acid, then the precipitate was filtered and washed
with water, the crude product was dried in vacuum desiccator and further purified by
column chromatography on silica using mobile phase dichloromethane−methanol
(50:1, v/v) to furnish 9 as a pale yellow solid; m.p.: 133.1 – 135.6 °C Yield: 28%.
+
1
HPLC purity: 98.05%, retention time = 17.513 min. MS (ESI) m/z: 432.1 [M+H] . H
NMR (600 MHz, DMSO-d ) δ 7.71 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.22
6
(d, J = 8.7 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.69 (dd, J = 8.7, 2.0 Hz, 1H), 4.36 (t, J
1
3
=
6.7 Hz, 2H), 3.52 (s, 4H), 2.63 (t, J = 6.8 Hz, 2H), 2.50 (s, 4H). C NMR (151
MHz, DMSO-d ) δ 160.42, 145.83, 139.97, 128.72, 127.70, 126.92, 125.48, 120.06,
6
113.83, 112.63, 106.61, 66.58, 55.40, 53.89, 42.33.
4
.1.2.6 3-(4-chlorophenyl)-1-(2-(3-(4-chlorophenyl)ureido)benzo[d]thiazol-6-yl)-1-(2-
morpholinoethyl)urea (D016)
To a mixture of 8 (0.3 g, 1.1 mmol) and phenyl (4-chlorophenyl)carbamate (0.5 g,
2
.2 mmol) in 5 mL DMF, triethylamine (0.13 g, 1.3 mmol) was slowly added, after
o
stirring at 60 C for 12 h, the mixture was poured into 10 mL water, the solution was
neutralized with 1N hydrochloric acid, then the precipitate was filtered and washed
with water, the crude product was dried in vacuum desiccator and further purified by
column chromatography on silica using mobile phase dichloromethane−methanol
(50:1, v/v) to furnish 9 as a pale yellow solid; m.p.: 133.1 – 135.6 °C Yield: 80%.
+
1
HPLC purity: 96.87%, retention time = 27.044 min. MS (ESI) m/z: 432.1 [M+H] . H

NMR (600 MHz, DMSO-d ) δ 10.67 (s, 1H), 8.86 (s, 2H), 8.82 (s, 1H), 8.10 (s, 1H),
6
7
.65 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.4 Hz, 2H), 7.50 (d, J = 7.4 Hz, 2H), 7.45 (d, J
7.5 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 7.5 Hz, 2H), 4.44 (s, 2H), 3.54 (s,
=
1
3
4
1
1
4
4
H), 2.77 (s, 2H), 2.56 (s, 4H). C NMR (151 MHz, DMSO-d ) δ 160.21, 154.16,
6
52.93, 144.19, 139.13, 137.82, 135.73, 133.72, 129.97, 129.12, 129.01, 127.82,
25.69, 123.00, 120.84, 120.09, 118.13, 110.54, 66.42, 57.59, 55.30, 53.97.
.1.3 General procedure for preparation of compounds 12a-j.
.1.3.1 tert-butyl phenyl benzo[d]thiazole-2,6-diyldicarbamate (10)
To a stirred solution of intermediate 3 (10 g, 37.7 mmol) in acetone (100 mL)
was dropped phenyl chloroformate (9.0 g, 57.7 mmol). The mixture was stirred at
room temperature for 2 h. After completion of the reaction as indicated by TLC, the
mixture was filtered to obtain 9.4 g of 10 as a white solid, m.p.: 210.1 – 212.2 °C.
+
Yield: 83.6%; MS (ESI) m/z: 386.1 [M+H] .
4
4
.1.3.2 General procedure for preparation of intermediates 11a-j
.1.3.2.1 tert-butyl (6-(3-(3-chlorophenyl)ureido)benzo[d]thiazol-2-yl)carbamate
(11a)
tert-Butyl phenyl benzo[d]thiazole-2,6-diyldicarbamate (10) (0.5 g, 1.3 mmol) in
5
mL 1,4-dioxane was added cyclohexylamine (0.15 g, 1.5 mmol) followed by
o
triethylamine (0.13 g, 0.1 mL, 1.3 mmol). The mixture was stirred at 60 C for 3 h.
The resulting precipitate was filtered off, washed with diethyl ether, and dried to
+
afford 0.3 g of 5b as a white solid, yield: 85.6%; MS (ESI) m/z: 419.3 [M+H] .
4
.1.3.2.2 tert-butyl (6-(3-(4-chlorobenzyl)ureido)benzo[d]thiazol-2-yl)carbamate
11b)
Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.2 g (1.5
(
mmol) of 4-chlorobenzylamine in 5 mL of 1,4-dioxane and 0.1 mL of trimethylamine,
then 0.3 g of 11b was obtained as yellow solid; yield: 67.9%; MS (ESI) m/z: 433.1
+
[
M+H]
4
.1.3.2.3
tert-butyl(6-(3-((3s,5s,7s)-adamantane-1-yl)ureido)benzo[d]thiazol-2-yl)carbamate
11c)
(

Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.2 g (1.5
mmol) of amantadine in 5 mL 1,4-dioxane and 0.1 mL of trimethylamine, then 0.3 g
+
of 11c was obtained as yellow solid; yield: 89%; MS (ESI) m/z: 443.4 [M+H] .
4
.1.3.2.4 tert-butyl (6-(3-phenylureido)benzo[d]thiazol-2-yl)carbamate (11d)
Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.15 g (1.5
mmol) of aniline in 5 mL of 1,4-dioxane and 0.1 mL of trimethylamine, then 0.3 g of
+
1
1d was obtained as yellow solid; yield: 88.3%; MS (ESI) m/z: 385.1 [M+H] .
4
.1.3.2.5 tert-butyl (6-(3-(3-methoxyphenyl)ureido)benzo[d]thiazol-2-yl)carbamate
11e)
Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.2 g (1.5
(
mmol) of para-anisidine in 5 mL of 1,4-dioxane and 0.1 mL of trimethylamine, then
+
0
.3 g of 11e was obtained as yellow solid; yield: 85.4%; MS (ESI) m/z: 415.6 [M+H]
4
.1.3.2.6
tert-butyl-(6-(3-(3-(trifluoromethoxy)phenyl)ureido)benzo[d]thiazol-2-yl)carbamate
11f)
(
Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.2 g (1.5
mmol) of 4-(trifluoromethoxy)benzeneaime in 5 mL of 1,4-dioxane and 0.1 mL of
trimethylamine, then 0.3 g of 11f was obtained as yellow solid; yield: 78.5%; MS
+
(ESI) m/z: 469.4 [M+H]
4
.1.3.2.7 tert-butyl (6-(3-cyclohexylureido)benzo[d]thiazol-2-yl)carbamate (11g)
Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.2 g (1.5
mmol) of 4-(trifluoromethoxy)benzeneaime in 5 mL of 1,4-dioxane and 0.1 mL of
trimethylamine, then 0.3 g of 11g was obtained as yellow solid; yield: 86.1%; MS
+
(ESI) m/z: 391.2 [M+H]
4
.1.3.2.8 tert-butyl (6-(3-(pyridin-2-yl)ureido)benzo[d]thiazol-2-yl)carbamate (11h)
Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.15 g (1.5
mmol) of 2-aminopyridine in 5 mL of 1,4-dioxane and 0.1 mL of trimethylamine, then
+
0
.3 g of 11h was obtained as yellow solid; yield: 50.2%; MS (ESI) m/z: 386.4 [M+H]
4
.1.3.2.9 tert-butyl (6-(3-(naphthalen-2-yl)ureido)benzo[d]thiazol-2-yl)carbamate
(11i)

Synthesized using the procedure for 11a, 0.5 g (1.3 mmol) of 10 and 0.2 g (1.5
mmol) of 2-naphthylamine in 5 mL of 1,4-dioxane and 0.1 mL of trimethylamine,
then 0.3 g of 11i was obtained as yellow solid; yield: 50.2%; MS (ESI) m/z: 435.2
+
[
M+H]
4
.1.3.2.10 tert-butyl (6-ureidobenzo[d]thiazol-2-yl)carbamate (11j)
To a solution of intermediate 10 (0.5 g, 1.9 mmol) in acetic acid (5 mL) and
water (2.5 mL), the sodium cyanate (0.25 g, 3.8 mmol) in water (2.5 mL) was added
slowly at room temperature. The mixture was stirred at room temperature for 2 h and
monitored by TLC, and then was poured into water and adjusted to pH=7. After
stirring for 30 min, the precipitate was collected by filtration, and dried to provide
compound 0.3 g of compound 11j as white solid, yield: 83.6%; MS (ESI) m/z: 309.2
+
[
M+H] .
4
4
.1.3.3 General procedure for preparation of compounds 12a-j
.1.3.3.1 1-(2-aminobenzo[d]thiazol-6-yl)-3-(4-chlorophenyl)urea (12a)
The crude product (11a) from the previous step was dissolved in 3 mL of
dichloromethane; 3 mL of trifluoroacetate was then slowly added. After stirring
overnight at room temperature, the mixture was evaporated under reduced pressure
and the resultant was added to 5 mL water, adjusting pH to 8 with saturated sodium
hydrogen carbonate, the precipitate was filtered off and washed with water, then the
crude product was dried in vacuum desiccator and further purified by column
chromatography on silica using mobile phase dichloromethane−methanol (30:1, v/v)
to furnish 12a as a pale yellow solid; yield: 66.8%; m.p.: 195.5 – 197.0 °C; HPLC
+
1
purity: 96.56%, retention time = 20.379 min. MS (ESI) m/z: 319.3 [M+H] ; H NMR
(
600 MHz, DMSO-d ) δ 7.88 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 8.9 Hz,
6
13
2
H), 7.25 (s, 2H), 7.21 (d, J = 8.6 Hz, 1H). C NMR (151 MHz, DMSO-d ) δ 165.29,
6
1
53.56, 148.06, 140.20, 134.60), 131.60, 128.76, 124.79, 119.78, 117.86, 117.38,
111.04.
4
.1.3.3.2 1-(2-aminobenzo[d]thiazol-6-yl)-3-(4-chlorobenzyl)urea (12b)
Synthesized using the procedure for 12a, crude product was purified by column
chromatography on silica using mobile phase dichloromethane−methanol (30:1, v/v)

to furnish 12b as a pale yellow solid; yield: 50.7%; m.p.: 173.4 – 177.7 °C; HPLC
+
1
purity: 99.28%, retention time = 19.193 min. MS (ESI) m/z: 333.2 [M+H] ; H NMR
400 MHz, DMSO-d ) δ 8.69 (s, 1H), 7.83 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.32 (d, J
(
6
=
8.4 Hz, 2H), 7.24 (s, 2H), 7.19 (d, J = 8.6 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.83 (s,
1
3
1
1
4
4
H), 4.27 (d, J = 5.8 Hz, 2H). C NMR (151 MHz, DMSO-d ) δ 165.30, 155.99,
6
47.97, 140.22, 134.78, 131.75, 131.57, 129.42, 128.64, 117.95, 117.14, 110.90,
2.55.
.1.3.3.3 1-((3s,5s,7s)-adamantan-1-yl)-3-(2-aminobenzo[d]thiazol-6-yl)urea (12c)
Synthesized using the procedure for 12a, crude product was purified by column
chromatography on silica using mobile phase dichloromethane−methanol (30:1, v/v)
to furnish 12c as a pale yellow solid; yield: 89.2%; m.p.: 200.3 – 202.5 °C; HPLC
+
1
purity: 98.71%, retention time = 22.534 min. MS (ESI) m/z: 343.4 [M+H] ; H NMR
400 MHz, DMSO-d ) δ 8.45 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.23 (s, 2H), 7.18 (d, J
(
6
=
8.6 Hz, 1H), 7.06 (dd, J = 8.6, 2.2 Hz, 1H), 2.09 (s, 1H), 1.97 (s, 2H), 1.62 (d, J =
13
1
4.8 Hz, 2H), 1.57 (s, 3H), 1.53 (s, 2H), 1.50 (s, 1H), 1.48 (d, J = 2.3 Hz, 4H). C
NMR (101 MHz, DMSO-d ) δ 165.28, 156.66, 147.93, 134.88, 131.73, 117.94,
6
117.07, 110.84, 46.43, 45.72, 36.49, 31.60, 29.69.
4
.1.3.3.4 1-(2-aminobenzo[d]thiazol-6-yl)-3-phenylurea (12d)
Synthesized using the procedure for 12a, crude product was purified by column
chromatography on silica using mobile phase dichloromethane−methanol (30:1, v/v)
to furnish 12d as a pale yellow solid; yield: 75.3%; m.p.: 189.5 – 191.7 °C; HPLC
+
1
purity: 99.81%, retention time = 16.940 min. MS (ESI) m/z: 285.5 [M+H] ; H NMR
400 MHz, DMSO-d ) δ 9.04 (s, 1H), 8.99 (s, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.48 (d, J
(
6
=
7.9 Hz, 2H), 7.29 (d, J = 1.9 Hz, 2H), 7.26 (s, 1H), 7.24 (d, J = 3.6 Hz, 1H), 7.19
1
3
(
dd, J = 8.6, 1.7 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ
6
1
65.57, 153.32, 148.41, 140.59, 134.12, 131.82, 129.17, 121.95, 118.51, 118.02,
117.53, 111.35.
4
.1.3.3.5 1-(2-aminobenzo[d]thiazol-6-yl)-3-(4-methoxyphenyl)urea (12e)
Synthesized using the procedure for 12a, crude product was purified by column
chromatography on silica using mobile phase dichloromethane−methanol (30:1, v/v)