Discovery of benzothiazole derivatives as novel non-sulfamide NEDD8 activating enzyme inhibitors by target-based virtual screening

Article abstract of DOI:10.1016/j.ejmech.2017.03.076

NEDD8 activating enzyme (NAE) plays a critical role in various cellular functions in cancers. In this study, a target-based virtual screening was applied to discover benzothiazoles to be potent non-covalent NAE inhibitors. Further two round optimizations

Full text of DOI:10.1016/j.ejmech.2017.03.076

Accepted Manuscript
Discovery of benzothiazole derivatives as novel non-sulfamide NEDD8 activating
enzyme inhibitors by target-based virtual screening
Hao Ma, Chunlin Zhuang, Xiguo Xu, Jiao Li, Juan Wang, Xiao Min, Wannian Zhang,
Huojun Zhang, Zhenyuan Miao
PII:
S0223-5234(17)30244-1
10.1016/j.ejmech.2017.03.076
EJMECH 9336
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 January 2017
Revised Date: 28 March 2017
Accepted Date: 30 March 2017
Please cite this article as: H. Ma, C. Zhuang, X. Xu, J. Li, J. Wang, X. Min, W. Zhang, H. Zhang, Z. Miao,
Discovery of benzothiazole derivatives as novel non-sulfamide NEDD8 activating enzyme inhibitors
by target-based virtual screening, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.076.
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Graphic Abstract
Discovery of Benzothiazole Derivatives as Novel Non-Sulfamide NEDD8 Activating
Enzyme Inhibitors by Target-Based Virtual Screening
Hao Ma,ab† Chunlin Zhuang,b† Xiguo Xu,a Jiao Li,b Juan Wang,d Xiao Min,b Wannian
Zhang,ab* Huojun Zhang,c* and Zhenyuan Miaob*
Novel non-sulfamide NAE inhibitors were discovered by target-based virtual screening. The
compounds showed excellent anticancer activity and represent a promising lead for further
exploration.

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Discovery of Benzothiazole Derivatives as Novel Non-Sulfamide NEDD8 Activating Enzyme
Inhibitors by Target-Based Virtual Screening
Hao Ma,ab† Chunlin Zhuang,b† Xiguo Xu,a Jiao Li,b Juan Wang,d Xiao Min,b Wannian Zhang,ab*
Huojun Zhang,c* and Zhenyuan Miaob*
a School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China.
b
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
China.
c
Shanghai Changhai Hospital, Second Military Medical University, 168 Changhai Road,
Shanghai 200433, China.
d
Center for Pharmacological Evaluation and Research, Shanghai Institute of Pharmaceutical
Industry, 1111 Zhongshanbeiyi Road, Shanghai 200437, China
Corresponding authors:
Email:
miaozhenyuan@hotmail.com
(Z.M.);
Email:
chyyzhj@163.com
(H.Z.);
zhangwnk@hotmail.com (W.Z.).
†These two authors contributed equally to this work.
Abstract
NEDD8 activating enzyme (NAE) plays a critical role in various cellular functions in cancers. In
this study, a target-based virtual screening was applied to discover benzothiazoles to be potent
non-covalent NAE inhibitors. Further two round optimizations concluded a preliminary structure-
activity relationship (SAR) of their derivatives. Three compounds (6k, 7b, ZM223) exhibited
antitumor activities in nanomolar range. ZM223 showed excellent anticancer activity against
HCT116 colon cancer cells with an IC50 value of 100 nM. Mechanistically, compounds 6k, 7b,
and ZM223 caused a dose-response decrease in the level of NEDD8 and an increase in the
downstream UBC12 protein. This scaffold represents a promising lead for developing non-
sulfamide NAE inhibitors.
Keywords: Benzothiazoles, Virtual screening, NAE inhibitors

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1. Introduction
The ubiquitin proteasome system (UPS) controls the degradation of intracellular proteins [1]. In
cancer, aberrant protein degradation contributes to disease progression, metastasis, and drug
resistance [2-4]. Thus, targeting this protein degradation is a reasonable strategy for cancer
therapy. The first proteasome inhibitor bortezomib (Velcade) has been approved by FDA in 2003
for the treatment of multiplemyeloma (MM) and relapsed mantle cell lymphoma (MCL) [5]. The
success of bortezomib attracts much attention for developing other agents targeting the UPS
system.
In UPS system, proteins are mediated by the coordination of three classes of enzymes with
different functions: E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin
ligases) [6]. Neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8)
has been identified as covalently modification of the cullin-RING ubiquitin E3 ligases (CRLs),
playing a critical role in various cellular functions in cancers [7-9]. As shown in Fig. 1, an acyl
adenylate intermediate (NEDD8-AMP) is formed and a transthiolation reaction was followed to
form NAE-NEDD8. Then, a second NEDD8-AMP is generated by a second round of NEDD8
binding. A ternary complex is obtained. NEDD8-specific E2s (UBC12) is then transferred
through transthiolation reaction. At the end, NEDD8 from E2-NEDD8 covalently binds to the
specific substrates (CLRs) in the presence of E3 [10, 11]. Therefore, targeting NAE can modulate
the rate of ubiquitination and subsequent degradation of proteins regulated by CRLs and lead to
cancer cell death. This therapeutic strategy has attracted many interests for the treatment of
cancer in recent years [8, 12-16].
MLN4924 (Fig. 2), developed by Millennium Pharmaceuticals, is a first-in-class sulfamide NAE
inhibitor. This compound is designed based on the mechanism of NEDD8 pathway [16]. The
phase І clinical trial of MLN4924 for the treatment of both solid tumours and leukaemia has been
completed. The X-ray co-crystal structure (PDB entry: 3GZN) with NEDD8 revels that this
compound smoothly mimics the binding mode of the natural substrate AMP (The structure is

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shown in Fig. 1.) of NEDD8 [17]. The sulfamide of MLN4924 is recognized to be an essential
group for the inhibitory activity as it could form covalent bond with NAE [13, 17]. Although
great success has been made for this compound, there are still urgent needs for discovering more
small-molecule inhibitors of NEDD8 pathway [12, 18-20].
In this study, a target-based virtual screening was applied to discover new agents for NEDD8
pathway. A non-covalent docking strategy was used to identify the hits in order to evaluate the
importance of the sulfamide for the binding activity (Fig. 2).
Fig. 1. NEDD8 pathway and structure of AMP.
2. Results and Discussion
2.1 Virtual screening
A schematic of the overall target-based virtual screening procedure following our previous
protocols [21, 22] is presented in Fig. 2. After analysing the binding mode of MLN4924-NEDD8
complex, two critical binding parts were obtained (Fig. S1): 1) a covalent binding site with the
sulfamide; and 2) a non-covalent binding site with three key residues forming hydrogen bonds.
The covalent binding pocket was not taken into consideration for the subsequent molecular
docking. The three hydrogen-bond sites (Asp100, Ile148 and Gln149) were used as the

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constraints for the virtual screening, which was performed by Glide docking in standard precision
(SP). The compound library contained 150K compounds cleaned from Specs database (~300K)
using standard procedure [22]. After SP docking, 2910 compounds can match well with the
constraints of hydrogen bonds and the top 500 of these compounds were selected for the visual
inspection. Then the 38 compounds with reasonable poses were selected to the next examination
of chemical diversity in order to limit redundancy in our initial testing. Finally, after synthetic
feasibility evaluation, 15 compounds (Table S1) were purchased from the commercial source for
biological evaluation.
Fig. 2. The workflow of the target-based virtual screening and the structures of NAE
inhibitors.
2.2 Biological evaluation of the purchased compounds
The antitumor activities of these 15 compounds (Structures are shown in Table S1) were first
evaluated using HCT116 colon cancer cells and U-2OS osteosarcoma cells based on the reference

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[16]. MLN4924 was used as a positive control. To our delight, nine compounds (H1, H2, H8,
H10-15, hit rate: 60%) showed promising antitumor activities (Table 1). Except H8 and H12, the
other seven compounds had good potency against these two cell lines. Compound H10 was
sensitive to U-2OS with a good IC50 value of 5.14
µM. Especially, compound H11 (The
structures is shown in Fig. 3) bearing benzothiazole scaffold exhibited excellent antitumor
activities against HCT116 and U-2OS in submicromolar range, leading to further structural
optimization.
Table 1. The antitumor activities of 15 selected compounds (IC50, µM)a
Entry
H1
HCT116
15.34
39.49
>100
U-2OS
79.28
63.47
>100
>100
>100
>100
>100
>100
Entry
H9
HCT116
>100
U-2OS
93.08
5.14
11.12
2.12
H2
H10
3.19
H3
H11
>100
66.62
38.14
47.36
20.14
0.19
>100
40.39
88.79
21.71
0.16
H4
H12
>100
H5
H13
>100
H6
H14
91.38
68.33
H7
H15
H8
MLN4924
aAll the experiments were duplicated and measured with the MTT method. Data are
presented as the average values.
2.3 Chemistry
Compound H11 with best antitumor activities was selected for structural optimization. As
shown in Scheme 1, the target compounds were synthesized by a multi-step substitution
reaction using benzothiazole (1) as a starting material. First, the benzyl substituted

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thioether was retained and phenyl substitutions were introduced on the amide side.
Compounds 6a-6n and H11 were synthesized with the yields of 25-70.9%.
Scheme 1. (a): Et3N, DCM, room temperature, 3 h; (b): Pd/C, H2, CH3OH, room temperature, 4 h;
(c): Et3N, DCM, room temperature, 2.5 h; (d): 3-mercaptophenol or 4-chlorobenzenethiol,
NaHCO3, CH3COCH3, reflux, 5 h.
Second round optimization was carried out to introduce various cyclic groups on the other
side of the lead compound H11. As shown in Scheme 2, compounds 7a-7h were
synthesized by substitution with trifluoromethylphenyl intermediate 5f
.
Scheme 2. (a): K2CO3, DMF, RSH, room temperature, 4 h.
2.4 Biological evaluation of the synthetic compounds

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The binding mode of compound H11 with NEDD8 was re-evaluated to guide further
structural optimization. As shown in Fig. 3A, besides the three constrained hydrogen
bonds, an additional hydrogen bond with Asp167 and a π-cation interaction between the
chlorophenyl with the Lys124 were observed (Table S1). It was demonstrated that both
sides of the compound H11 highlighted in red and blue did not form hydrogen bonds with
the enzyme. Thus, different substituents were introduced into these two positions.
Fig. 3. Docking modes of compounds H11 (A) and 7g (B) with NAE and the optimization
strategy. Hydrogen bonds were highlighted in red and green dash lines. The key residues
for the binding were labelled.
As illustrated in Table 2, the antitumor activities showed that the para-methyl (6b) and -
fluoro phenyl (6d) compounds had similar activities as the chlorophenyl compound H11
against the two cell lines. The trifluoromethylphenyl compound (6c) exhibited better
activity with an IC50 value of 0.93 µM against HCT116 colon cancer cells and comparable
antitumor activity towards U-2OS with that of H11. Napthyl group (6a) was introduced
and decreased the activity and the di-substituted group (6e) was unfavorable to the activity.

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There was not decent difference to the activity if changing the 4-chlorophenyl group (6a
-
6e, H11) to 3-hydroxylphenyl group (6f-6n). A similar structure-activity relationship
(SAR) was concluded. Phenyl (6g) and thiophenyl (6h) were unfavorable to the antitumor
activity against the HCT-116 and loss the potency towards the U-2OS osteosarcoma cells.
The napthyl (6i) and di-chlorophenyl groups (6j) were sensitive to U-2OS with IC50 values
of 1.77 and 1.53 µM, respectively. The trifluoromethylphenyl compound (6k) was the
most potent derivative of this series towards the two cell lines in nanomolar range (0.51
µM and 0.63 µM, respectively).
Table 2. The antitumor activities of H11 analogues (IC50, µM)a
Entry
6a
6b
6c
HCT116
4.04
U-2OS
14.48
3.19
Entry
HCT116
7.73
U-2OS
12.40
2.82
6m
2.12
5.13
6n
0.93
2.89
0.32
1.00
7a
1.36
2.51
0.30
3.94
6d
6e
7b
65.05
10.59
7.02
>100
10.12
62.96
>100
1.77
2.22
28.10
18.76
7.11
7c
2.69
6f
7d
3.76
6g
6h
6i
7e
7f
40.98
4.25
21.04
0.10
12.34
1.22
7g (ZM223)
7h
5.44
1.53
2.30
13.56
3.19
6j
0.51
0.63
2.12
6k
6l
H11
6.17
5.12
0.19
0.16
MLN4924
aAll the experiments were duplicated and measured with the MTT method. Data are
presented as the average values.

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In the second-round optimization, the obtained compounds possessed good to excellent
antitumor activities, except compound 7f with cyclohexyl group. This result indicated that
the aromatic group was favourable to the activities. These compounds were more sensitive
towards HCT116 with better IC50 values than U-2OS cells. The heterocycle substituted
compounds reserved the activity of compound H11, such as pyrimidinyl (7c), imidazolyl
(
7d) and nicotinic acid (7h). Compound 7a, 7b and 7g with phenyl or napthyl groups had
excellent IC50 values in nanomolar range. Compounds 7a and 7g exhibited increased
activities against both two cell lines. Especially, compound 7g with 4-aminophenyl group
showed better antitumor activity compared with the positive MLN4924 and enhanced 20-
fold compared with the lead compound H11. Molecular docking (Fig. 3B) demonstrated
that compound 7g had similar binding mode with the lead compound H11 and formed
three key hydrogen bonds with Asp100, Ile148 and Gln149 and a hydrogen bond with
Asp167. The amino group on the phenyl ring did not form additional hydrogen bond,
indicating other reasons for the elevation of the antitumor activity.
2.5 Western blotting assay
In order to examine the mechanism in HCT116 cells, the most potent three compounds (6k, 7b,
and 7g) in nanomolar range were selected for western blotting assay (Fig. 4). The three
compounds caused a dose-response decrease in the level of NEDD8 consistent with the prediction
of the docking study. Compounds 7b and 7g with better IC50s showed higher inhibitory potency
at 1 µM. As mentioned in the introduction section, the downstream NEDD8-UBC12 complex will
be inhibited following the NEDD8 deactivation. The accumulation of the UBC12 protein was
then evaluated and showed dose-response manner, indicating the decrease of the subsequent
NEDD8-UBC12 complex.

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Fig. 4. Western blotting assay of compound 6k
,
7b and 7g (HCT-116 cells, 4h treatment):
Lane 1, DMSO control with no treatment; Lane 2, compound 6k (0.1 µM); Lane 3,
compound 6k (1 µM); Lane 4, compound 7b (0.1 µM); Lane 5, compound 7b (1 µM);
Lane 6, compound 7g (0.1 µM); Lane 7, compound 7g (1 µM).
3. Conclusion
In conclusion, we applied a hydrogen-bonding constrained target-based virtual
screening for searching a novel non-sulfamide NAE inhibitor. Nine compounds were
found to show promising antitumor activities. Compound H11 bearing benzothiazole
scaffold exhibited best activity. Subsequent two round structural optimizations focusing
on the non-hydrogen bonding sides of compound H11 led to a preliminary SAR of the
benzothiazole derivatives. Most of the compounds showed good antitumor activities and
the most potent compounds 6k, 7b, and 7g (ZM223) exhibited antitumor activities with
IC50 values in nanomolar range. Mechanistically, these compounds inhibited NEDD8
protein and subsequently elevated UBC12 level. The benzothiazole scaffold represents a
promising non-covalent lead compound as NEDD8 inhibitor for drug discovery. Further
exploration of the SAR and biological properties of these derivatives are under
investigation in our laboratory.

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4. Experimental sections
4.1. General methods
All reagents and solvents were analytically pure and purchased from the vendors and used
without further purification. TLC analysis was carried out on silica gel plates GF254 (Qindao
Haiyang Chemical, China). Silica gel chromatography was carried out on 300-400 mesh gel.
Anhydrous solvents and reagents were dried by routine protocols. 1H NMR spectra were recorded
on Bruker Avance 300, 500 or 600 spectrometers (Bruker Company, Germany), using TMS as an
internal standard and CDCl3 as solvents. Chemical shifts (δ values) and coupling constants (J
values) are given in ppm and Hz, respectively. The mass spectra were recorded on an API-3000
LC-MS mass spectrometer. HPLC purity was tested using Agilent 1100 instrument with YMC
Pack ODS-A column (10*250 mm, 5 µm).
4.2. Synthesis of intermediates 3a-3i
4.2.1. N-(6-nitrobenzo[d]thiazol-2-yl)butyramide (3a): To
nitrobenzothiazole (200 mg, 1.0 mmol) in CH2Cl2 (20 mL) was slowly added triethylamine (280
uL). Then the butyric anhydride (120 L, 1.0 mmol) in CH2Cl2 was added dropwise. The reaction
a
solution of 2-amino-6-
µ
mixture was stirred at room temperature for 3 hours. The reaction mixture was then diluted with
CH2Cl2 and washed with water. The organic layer was separated and washed with brine, dried
over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash
chromatography on silica gel to give yellow solid, yield 90.4%. 1H NMR (300 MHz, DMSO-d6)
δ:
12.73 (s, 1 H, NH), 9.03 (d, J = 2.3 Hz, 1 H, Ar-H), 8.26 (dd, J = 2.4, 9.0 Hz, 1 H, Ar-H), 7.86 (d,
J = 8.9 Hz, 1 H, Ar-H), 2.49 (t, 2 H, COCH2), 1.59-1.71 (m, 2 H, CH2), 0.91 (t, 3 H, CH3); ESI-
MS (m/z): 264.22 [M-H]-.
1
4.2.2. N-(6-nitrobenzo[d]thiazol-2-yl)benzamide (3c): Yellow solid, yield 88.2%. H NMR (300
MHz, DMSO-d6) δ: 13.32 (s, 1 H, NH), 9.10 (d, J = 2.3 Hz, 1 H, Ar-H), 8.31 (dd, J = 2.4, 8.9 Hz,
1 H, Ar-H), 8.12-8.18 (m, 2 H, Ar-H), 7.93 (d, J = 8.9 Hz, 1 H, Ar-H), 7.69 (t, 1 H, Ar-H), 7.58 (t,
2 H, Ar-H); ESI-MS (m/z): 298.30 [M-H]-.

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1
4.2.3. N-(6-nitrobenzo[d]thiazol-2-yl)-1-naphthamide (3d): Yellow solid, yield 66.4%. H NMR
(300 MHz, DMSO-d6) : 13.42 (s, 1 H, NH), 9.11 (s, 1 H, Ar-H), 8.67 (s, 1 H, Ar-H), 8.32 (dd, J
δ
= 2.3, 8.9 Hz, 1 H, Ar-H), 8.17 (dd, J = 1.5, 8.7 Hz, 1 H, Ar-H), 8.08-8.12 (m, 2 H, Ar-H), 8.04 (d,
J = 8.0 Hz, 1 H, Ar-H), 7.91-7.97 (m, 1 H, Ar-H), 7.70 (t, 1 H, Ar-H), 7.65 (t, 1 H, Ar-H); ESI-
MS (m/z): 438.30 [M-H]-.
4.2.4. 3,5-Dichloro-N-(6-nitrobenzo[d]thiazol-2-yl)benzamide (3f): Yellow solid, yield 62.1%. 1H
NMR (300 MHz, DMSO-d6) δ: 12.63 (s, 1 H, NH), 8.12 (s, 2 H, Ar-H), 7.90-7.91 (t, 1 H, Ar-H),
7.43-7.45 (d, J = 8.5 Hz, 1 H, Ar-H), 7.03 (s, 1 H, Ar-H), 6.74-6.76 (d, J = 8.6 Hz, 1 H, Ar-H);
ESI-MS (m/z): 366.32 [M-H]-.
4.2.5. N-(6-nitrobenzo[d]thiazol-2-yl)-4-(trifluoromethyl)benzamide (3g): Yellow solid, yield
1
70.5%. H NMR (300 MHz, DMSO-d6)
δ: 13.54 (s, 1 H, NH), 9.12 (d, J = 2.3 Hz, 1 H, Ar-H),
8.31-8.35 (m, 2 H, Ar-H), 8.13 (d, J = 8.0 Hz, 1 H, Ar-H), 7.97 (d, J = 8.1 Hz, 2 H, Ar-H), 7.88
(d, J = 8.3 Hz, 1 H, Ar-H); ESI-MS (m/z): 368.20 [M+H]+, 366.30 [M-H]-.
1
4.2.6. 4-Fluoro-N-(6-nitrobenzo[d]thiazol-2-yl)benzamide (3h): Orange solid, yield 74.4%. H
NMR (300 MHz, DMSO-d6) δ: 12.82 (s, 1 H, NH), 8.27 (d, J = 8.2 Hz, 2 H, Ar-H), 7.92 (d, J =
8.2 Hz, 2 H, Ar-H), 7.45 (d, J = 8.5 Hz, 1 H, Ar-H), 7.04 (d, J = 2.1 Hz, 1 H, Ar-H), 6.74 (dd, J =
2.2, 8.5 Hz, 1 H, Ar-H); ESI-MS (m/z): 316.30 [M-H]-.
4.2.7. 3,5-Dimethyl-N-(6-nitrobenzo[d]thiazol-2-yl)benzamide (3i): Yellow solid, yield 66.7%.
1H NMR (300 MHz, DMSO-d6)
δ: 13.13 (s, 1 H, NH), 9.08 (d, J = 2.4 Hz, 1 H, Ar-H), 8.30 (dd, J
= 2.4, 8.9 Hz, 1 H, Ar-H), 7.92 (d, J = 8.7 Hz, 1 H, Ar-H), 7.78 (s, 1 H, Ar-H), 7.54 (s, 1 H, Ar-
H), 7.31 (s, 1 H, Ar-H), 2.36 (s, 3 H, CH3), 2.06 (s, 3 H, CH3); ESI-MS (m/z): 362.28 [M-H]-.
4.3. Synthesis of intermediates 4a-4i
4.3.1. N-(6-aminobenzo[d]thiazol-2-yl)butyramide (4a): One drop of acetic acid was added into a
solution of 3a (53 mg, 0.2 mmol) and Pd/C (10%, 6 mg) in methanol (10 mL).Then the mixture
was stirred at room temperature under hydrogen atmosphere for 4 h. After completion monitoring
by TLC, the reaction mixture was evaporated under vacuum to dryness. The residue was purified

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1
by flash chromatography to give yellow solid, yield 98.7%. H NMR (300 MHz, DMSO-d6)
δ
:
11.94 (s, 1 H, NHCO), 7.35 (d, J = 8.5 Hz, 1 H, Ar-H), 6.95 (d, J = 2.0 Hz, 1 H, Ar-H), 6.65 (dd,
J = 2.1, 8.5 Hz, 1 H, Ar-H), 5.12 (s, 2 H, NH2), 2.37 (t, 2 H, COCH2), 1.51-1.66 (m, 2 H, CH2),
0.87 (t, 3 H, CH3); ESI-MS (m/z): 234.38 [M+H]+, 236.25 [M-H]-.
4.3.2. N-(6-aminobenzo[d]thiazol-2-yl)benzamide (4c): Yellow solid, yield 93.4%. 1H NMR (300
MHz, DMSO-d6) δ: 12.53 (s, 1 H, CONH), 8.08 (s, 1 H, Ar-H), 8.06 (s, 1 H, Ar-H), 7.61 (t, 1 H,
Ar-H), 7.52 (t, 2 H, Ar-H), 7.42 (d, J = 8.6 Hz, 1 H, Ar-H), 7.01 (d, J = 2.0 Hz, 1 H, Ar-H), 6.70
(dd, J = 2.1, 8.5 Hz, 1 H, Ar-H), 5.20 (s, 2 H, NH2); ESI-MS (m/z): 270.36 [M+H]+, 268.32 [M-
H]-.
4.3.3. N-(6-aminobenzo[d]thiazol-2-yl)-1-naphthamide (4d): Yellow solid, yield 95.4%. 1H NMR
(300 MHz, DMSO-d6) δ: 12.51 (s, 1H, CONH), 8.78 (d, J = 1.0 Hz, 1 H, Ar-H), 8.12 (dd, J = 1.8,
8.5 Hz, 1 H, Ar-H), 8.06 (t, 2 H), 8.01 (d, J = 8.0 Hz, 1 H, Ar-H), 7.60-7.68 (m, 2 H, Ar-H), 7.45
(d, J = 8.6 Hz, 1 H, Ar-H), 7.05 (d, J = 2.2 Hz, 1 H, Ar-H), 6.74 (dd, J = 2.2, 8.5 Hz, 1 H, Ar-H),
5.20 (s, 2 H, NH2); ESI-MS (m/z): 320.27 [M+H]+, 318.30 [M-H]-.
1
4.3.4. N-(6-aminobenzo[d]thiazol-2-yl)-4-methylbenzamide (4e): Yellow solid, yield 97.2%. H
NMR (300 MHz, DMSO-d6) δ: 12.31 (s, 1 H, CONH), 8.01 (s, 2 H, Ar-H), 7.42-7.47 (m, 1 H,
Ar-H), 7.35 (s, 2 H, Ar-H), 7.02-7.05 (m, 1 H, Ar-H), 6.71-6.76 (m, 1 H, Ar-H), 5.20 (s, 2 H,
NH2), 2.38 (s, 3 H, Ar- CH3); ESI-MS (m/z): 284.25 [M+H]+, 282.33 [M-H]-.
4.3.5. N-(6-aminobenzo[d]thiazol-2-yl)-3,5-dichlorobenzamide (4f): Yellow solid, yield 90.4%.
1H NMR (300 MHz, DMSO-d6)
δ: 12.75 (s, 1 H, CONH), 8.12 (d, J = 1.9 Hz, 2 H, Ar-H), 7.91 (t,
1 H, Ar-H), 7.40-7.48 (m, 1 H, Ar-H), 7.03 (d, J = 2.0 Hz, 1 H, Ar-H), 6.75 (dd, J = 2.2, 8.6 Hz, 1
H, Ar-H), 5.26 (s, 2 H, NH2); ESI-MS (m/z): 338.24 [M+H]+, 336.26 [M-H]-.
4.3.6. N-(6-aminobenzo[d]thiazol-2-yl)-4-(trifluoromethyl)benzamide (4g): Yellow solid, yield
92.4%. 1H NMR (300 MHz, DMSO-d6)
δ: 12.83 (s, 1 H, CONH), 8.27 (d, J = 8.0 Hz, 2 H, Ar-H),
7.93 (d, J = 8.1 Hz, 2 H, Ar-H), 7.46 (d, J = 8.6 Hz, 1 H, Ar-H), 7.05 (s, 1 H, Ar-H), 6.75 (d, J =
8.9 Hz, 1 H, Ar-H), 5.26 (s, 2 H, NH2); ESI-MS (m/z): 338.27 [M+H]+, 336.33 [M-H]-.

ACCEPTED MANUSCRIPT
4.3.7. N-(6-aminobenzo[d]thiazol-2-yl)-4-fluorobenzamide (4h): Yellow solid, yield 88.7%. 1H
NMR (300 MHz, DMSO-d6) δ: 12.48 (s, 1 H, CONH), 8.15-8.21 (m, 2 H, Ar-H), 7.44 (d, J = 8.5
Hz, 1 H, Ar-H), 7.36-7.41 (m, 2 H, Ar-H), 7.04 (d, J = 2.2 Hz, 1 H, Ar-H), 6.74 (dd, J = 2.2, 8.6
Hz, 1 H, Ar-H), 5.22 (s, 2 H, NH2); ESI-MS (m/z): 288.20 [M+H]+, 286.27 [M-H]-.
4.3.8. N-(6-aminobenzo[d]thiazol-2-yl)-3,5-dimethylbenzamide (4i): Yellow solid, yield 89.7%.
1H NMR (300 MHz, DMSO-d6)
δ: 12.22 (s, 1 H, CONH), 7.72 (s, 2 H, Ar-H), 7.43 (d, J = 8.5 Hz,
1 H, Ar-H), 7.25 (s, 1 H, Ar-H), 7.03 (d, J = 2.1 Hz, 1 H, Ar-H), 6.73 (dd, J = 2.1, 8.5 Hz, 1 H,
Ar-H), 5.18 (s, 2 H, NH2), 2.34 (s, 6 H, 2CH3); ESI-MS (m/z): 298.17 [M+H]+, 296.30 [M-H]-.4.4.
Synthesis of intermediate 5a-5i
4.4.1. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)butyramide (5a): To a solution of 4a (85 mg,
0.36 mmol) in dry CH2Cl2 was added 1 mL of triethylamine. Then the solution of 2-chloroacetyl
chloride (36 µL) in dry CH2Cl2 was added slowly. The reaction mixture was stirred at room
temperature for 2.5 hours and then diluted with CH2Cl2. The organic layer was separated washed
with brine, dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was then
1
purified by flash chromatography on silica gel to give yellow solid, yield 73.6%. H NMR (300
MHz, DMSO-d6) δ: 12.24 (s, 1 H, NHCO), 10.42 (s, 1 H, CONH), 8.26 (s, 1 H, Ar-H), 7.67 (d, J
= 8.7 Hz, 1 H, Ar-H), 7.51 (d, J = 8.7 Hz, 1 H, Ar-H), 4.26 (s, 2 H, CH2CO), 2.45 (t, 2 H,
COCH2), 1.59-1.67 (m, 2 H, CH2), 0.91 (t, 3 H, CH3); ESI-MS (m/z): 312.35 [M+H]+, 310.21 [M-
H]-.
4.4.2. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)thiophene-2-carboxamide (5b): Yellow
solid, yield 65.2%. 1H NMR (300 MHz, DMSO-d6)
δ: 12.93 (s, 1 H, NHCO), 10.42 (d, J = 8.9 Hz,
1 H, CONH), 8.28-8.33 (m, 1 H, Ar-H), 7.96-8.04 (m, 1 H, Ar-H), 7.85 (d, J = 5.0 Hz, 1 H, Ar-
H), 7.71-7.74 (m, 1 H, Ar-H), 7.56 (dd, J = 2.0, 8.7 Hz, 1 H, Ar-H), 7.22-7.29 (m, 1 H, Ar-H),
4.28 (s, 2 H, CH2CO); ESI-MS (m/z): 352.31 [M+H]+, 350.18 [M-H]-.
4.4.3. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)benzamide (5c): Yellow solid, yield 80.2%.
1H NMR (300 MHz, DMSO-d6)
δ: 12.83 (s, 1 H, NHCO), 10.48 (s, 1 H, CONH), 8.33 (s, 1 H,

ACCEPTED MANUSCRIPT
Ar-H), 8.12 (d, J = 7.0 Hz, 2 H, Ar-H), 7.72-7.78 (m, 1 H, Ar-H), 7.62-7.67 (m, 1 H, Ar-H), 7.52-
7.59 (m, 3 H, Ar-H, Ar-H), 4.28 (s, 2 H, CH2CO); ESI-MS (m/z): 346.39 [M+H]+, 344.25 [M-H]-.
4.4.4. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)-1-naphthamide (5d): Yellow solid, yield
68.2%. 1H NMR (300 MHz, DMSO-d6)
δ: 12.99 (s, 1 H, NHCO), 10.49 (s, 1 H,CONH), 8.83 (s,
1 H, Ar-H), 8.47 (s, 1 H, Ar-H), 8.35 (s, 1 H, Ar-H), 8.14 (t, 1 H, Ar-H), 8.08 (t, 1 H, Ar-H),
7.98-8.04 (m, 1 H, Ar-H), 7.93-7.97 (m, 1 H, Ar-H), 7.70 (t, 1 H, Ar-H), 7.65 (t, 1 H, Ar-H),
7.56-7.61 (m, 1 H, Ar-H), 4.29 (s, 2 H, CH2CO); ESI-MS (m/z): 396.14 [M+H]+, 394.14 [M-H]-.
4.4.5. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)-4-methylbenzamide (5e): Yellow solid,
1
yield 74.8%. H NMR (300 MHz, DMSO-d6)
δ: 12.75 (s, 1 H, NHCO), 10.46 (s, 1 H, CONH),
8.32 (d, J = 1.9 Hz, 1 H, Ar-H), 7.99-8.06 (m, 2 H, Ar-H), 7.73 (d, J = 8.6 Hz, 1 H, Ar-H), 7.56
(dd, J = 2.2, 8.8 Hz, 1 H, Ar-H), 7.32-7.38 (m, 2 H, Ar-H), 4.28 (s, 2 H, CH2CO), 2.39 (s, 3 H,
Ar-CH3); ESI-MS (m/z): 360.19 [M+H]+, 358.15 [M-H]-.
4.4.6. 3,5-Dichloro-N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)benzamide (5f): Crude product
was used for the next step directly. ESI-MS (m/z): 412.10 [M-H]-.
4.4.7. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)benzamide (5g): Yellow
1
solid, yield 55%. H NMR (300 MHz, DMSO-d6)
δ
: 13.08 (s, 1 H, NHCO), 10.47 (s, 1 H,
CONH), 8.33 (s, 1 H, Ar-H), 8.29 (d, J = 8.2 Hz, 2 H, Ar-H), 7.92 (d, J = 8.5 Hz, 2 H, Ar-H),
7.74 (d, J = 5.2 Hz, 1 H, Ar-H), 7.56 (dd, J = 2.0, 8.7 Hz, 1 H, Ar-H), 4.27 (s, 2 H, CH2CO); ESI-
MS (m/z): 412.23 [M+H]+.
4.4.8. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)-4-fluorobenzamide (5h): Yellow solid,
1
yield 69.7%. H NMR (300 MHz, DMSO-d6)
δ: 12.85 (s, 1 H, NHCO), 10.12 (s, 1 H, CONH),
8.34 (s, 1 H, Ar-H), 8.19-8.23 (m, 2 H, Ar-H), 7.71 (d, J = 8.5 Hz, 1 H, Ar-H), 7.52 (dd, J = 2.1,
8.7 Hz, 1 H, Ar-H), 7.38-7.44 (m, 2 H, Ar-H), 4.29 (s, 2 H, CH2CO); ESI-MS (m/z): 364.16
[M+H]+, 362.17 [M-H]-.
4.4.9. N-(6-(2-chloroacetamido)benzo[d]thiazol-2-yl)-3,5-dimethylbenzamide (5i): Yellow solid,
1
yield 75.4%. H NMR (300 MHz, DMSO-d6)
δ: 12.70 (s, 1 H, NHCO), 10.49 (s, 1 H, CONH),

ACCEPTED MANUSCRIPT
8.32 (d, J = 1.0 Hz, 1 H, Ar-H), 7.74-7.77 (m, 2 H, Ar-H), 7.72-7.73 (m, 1 H, Ar-H), 7.56 (dd, J =
2.1, 8.7 Hz, 1 H, Ar-H), 7.28 (s, 1 H, Ar-H), 4.28 (s, 2 H, CH2CO), 2.35 (s, 6 H, 2CH3); ESI-MS
(m/z): 372.17 [M+H]+.
4.5. Synthesis of intermediates 6a-6n and H11
4.5.1. N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)butyramide (6f): To a
solution of 5a (70 mg, 0.19 mmol) in dry DMF (5 mL) was added K2CO3 (140 mg) and 3-
hydroxythiophenol (28 mg, 0.22 mmol) slowly. The reaction mixture was stirred at room
temperature for 3.5 h. Water (5 mL) was added into the solution and extracted with EtOAc. The
organic layer was separated and washed with brine, dried over anhydrous Na2SO4 and evaporated
to dryness. The crude product was purified by flash chromatography on silica gel to give yellow
1
solid, yield 69.1%. H NMR (300 MHz, DMSO-d6)
δ
: 12.26 (s, 1 H, NHCO), 10.34 (s, 1 H,
CONH), 9.58 (s, 1 H, OH), 8.26 (s, 1 H, Ar-H), 7.65 (d, J = 8.7 Hz, 1 H, Ar-H), 7.48 (d, J = 8.7
Hz, 1 H, Ar-H), 7.10 (t, 1 H, Ar-H), 6.74-6.84 (m, 2 H, Ar-H), 6.59 (d, J = 8.3 Hz, 1 H, Ar-H),
3.83 (s, 2 H, SCH2), 2.44 (t, 2 H, COCH2), 1.55-1.70 (m, 2 H, CH2), 0.90 (t, 3 H, CH3); ESI-MS
(m/z): 402.30 [M+H]+, 400.26 [M-H]-.
4.5.2. N-(6-(2-((4-chlorophenyl)thio)acetamido)benzo[d]thiazol-2-yl)butyramide (H11): Yellow
1
solid, yield 60.1%. H NMR (300 MHz, DMSO-d6)
δ
: 12.24 (s, 1 H, NHCO), 10.34 (s, 1 H,
CONH), 8.23 (d, J = 2.0 Hz, 1 H, Ar-H), 7.64 (d, J = 8.7 Hz, 1 H, Ar-H), 7.45 (dd, J = 2.1, 8.8 Hz,
1 H, Ar-H), 7.40-7.44 (m, 2 H, Ar-H), 7.36-7.39 (m, 2 H, Ar-H), 3.88 (s, 2 H, SCH2), 2.43 (t, 2 H,
COCH2), 1.59-1.64 (m, 2 H, CH2), 0.89 (t, 3 H, CH3); ESI-MS (m/z): 420.22 [M+H]+, 418.21 [M-
H]-.
4.5.3.
N-(6-(2-((4-chlorophenyl)thio)acetamido)benzo[d]thiazol-2-yl)-1-naphthamide
(6a):
1
Yellow solid, yield 65.3%. H NMR (300 MHz, DMSO-d6)
δ
: 12.67 (s, 1 H, NHCO), 10.39 (s, 1
H, CONH), 8.81 (s, 1 H, Ar-H), 8.31 (s, 1 H, Ar-H), 8.13 (d, J = 8.6 Hz, 1 H, Ar-H), 8.07 (t, 2 H,
Ar-H), 8.02 (d, J = 8.0 Hz, 1 H, Ar-H), 7.73 (d, J = 8.2 Hz, 1 H, Ar-H), 7.68 (t, 1 H, Ar-H), 7.64

ACCEPTED MANUSCRIPT
(t, 1 H, Ar-H), 7.52 (dd, J = 2.0, 8.7 Hz, 1 H, Ar-H), 7.44 (d, J = 8.6 Hz, 2 H, Ar-H), 7.39 (d, J =
8.6 Hz, 2 H, Ar-H), 3.91 (s, 2 H, SCH2); ESI-MS (m/z): 504.11 [M+H]+, 502.29 [M-H]-.
4.5.4. N-(6-(2-((4-chlorophenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-methylbenzamide (6b):
1
Yellow solid, yield 63.8%. H NMR (300 MHz, DMSO-d6)
δ
: 12.74 (s, 1 H, NHCO), 10.39 (s, 1
H, CONH), 8.31 (s, 1 H, Ar-H), 8.05 (d, J = 8.1 Hz, 2 H, Ar-H), 7.73 (d, J = 8.6 Hz, 1 H, Ar-H),
7.52 (dd, J = 1.8, 8.7 Hz, 1 H, Ar-H), 7.44-7.49 (m, 2 H, Ar-H), 7.39-7.43 (m, 2 H, Ar-H), 7.35-
7.39 (m, 2 H, Ar-H), 3.91 (s, 2 H, SCH2), 2.41 (s, 3 H, Ar-CH3); ESI-MS (m/z): 468.35 [M+H]+,
466.26 [M-H]-.
4.5.5. N-(6-(2-((4-chlorophenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)benzam-
1
ide (6c): Yellow solid, yield 58.7%. H NMR (300 MHz, DMSO-d6)
δ: 13.06 (s, 1 H, NHCO),
10.38 (s, 1 H, CONH), 8.31 (s, 1 H, Ar-H), 8.28 (d, J = 8.1 Hz, 2 H, Ar-H), 7.93 (d, J = 8.3 Hz, 2
H, Ar-H), 7.70-7.74 (m, 1 H, Ar-H), 7.51 (dd, J = 2.0, 8.7 Hz, 1 H, Ar-H), 7.42-7.46 (m, 2 H, pH),
7.37-7.40 (m, 2 H, Ar-H), 3.89 (s, 2 H, SCH2); ESI-MS (m/z): 522.40 [M+H]+, 520.26 [M-H]-.
4.5.6. N-(6-(2-((4-chlorophenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-fluorobenzamide (6d):
1
Yellow solid, yield 25%. H NMR (300 MHz, CD3OD)
δ: 13.69 (s, 1 H, NHCO), 11.23 (s, 1 H,
CONH), 9.13 (s, 1 H, Ar-H), 9.02 (q, 2 H, Ar-H), 8.55 (d, J = 8.6 Hz, 1 H, Ar-H), 8.33 (dd, J =
2.0, 8.7 Hz, 1 H, Ar-H), 8.25-8.29 (m, 2 H, Ar-H), 8.19-8.24 (m, 4 H, Ar-H), 4.15 (s, 2 H, SCH2);
ESI-MS (m/z): 470.24 [M-H]-.
4.5.7.
N-(6-(2-((4-chlorophenyl)thio)acetamido)benzo[d]thiazol-2-yl)-3,5-dimethylbenzamide
(6e): Yellow solid, yield 70.6%. 1H NMR (300 MHz, DMSO-d6)
δ
: 12.69 (s, 1 H, NHCO), 10.40
(s, 1 H, CONH), 8.31 (d, J = 1.8 Hz, 1 H, Ar-H), 7.76 (s, 2 H, Ar-H), 7.72 (d, J = 8.7 Hz, 1 H,
Ar-H), 7.53 (dd, J = 1.9, 8.7 Hz, 1 H, Ar-H), 7.46 (d, J = 8.7 Hz, 2 H, Ar-H), 7.40 (d, J = 8.7 Hz,
2 H, Ar-H), 7.28 (s, 1 H, Ar-H), 3.91 (s, 2 H, SCH2), 2.36 (s, 6 H, 2CH3); ESI-MS (m/z): 480.26
[M-H]-.
4.5.8. N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)thiophene-2-carboxamide
(6g): Yellow solid, yield 67.4%. 1H NMR (300 MHz, DMSO-d6)
δ: 12.94 (s, 1 H, NHCO), 10.38

ACCEPTED MANUSCRIPT
(s, 1 H, CONH), 9.58 (s, 1 H, OH), 8.32 (s, 2H, Ar-H), 8.01 (d, J = 4.3 Hz, 1 H, Ar-H), 7.72 (d, J
= 8.3 Hz, 1 H, Ar-H), 7.54 (dd, J = 2.0, 8.7 Hz, 1 H, Ar-H), 7.28 (t, 1 H, Ar-H), 7.12 (t, 1 H, Ar-
H), 6.79-6.85 (m, 2 H, Ar-H), 6.58-6.63 (m, 1 H, Ar-H), 3.86 (s, 2 H, SCH2); ESI-MS (m/z):
440.21 [M-H]-.
4.5.9. N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)benzamide (6h): Yellow
solid, yield 58.6%, m.p. >200 oC 1H NMR (300 MHz, DMSO-d6)
δ: 12.81 (s, 1 H, NHCO), 10.35
(s, 1 H, CONH), 9.54 (s, 1 H, OH), 8.32 (s, 1 H, Ar-H), 8.12 (d, J = 7.4 Hz, 2 H, Ar-H), 7.65 (t, 1
H, Ar-H), 7.56 (t, 2 H, Ar-H), 7.52 (dd, J = 1.9, 8.5 Hz, 1 H, Ar-H), 7.11 (t, 1 H, Ar-H), 6.79-6.83
(m, 2 H, Ar-H), 6.59 (dd, J = 2.2, 8.1 Hz, 1 H, Ar-H), 3.84 (s, 2 H, SCH2); 13C NMR (125 MHz,
CD3OD-d4) δ: 169.97, 168.14, 166.39, 159.09, 137.44, 135.89, 134.03, 133.73, 132.93, 131.03,
129.89, 129.15, 128.64, 122.13, 121.79, 120.68, 117.85, 115.27, 113.82, 40.42. ESI-MS (m/z):
436.36 [M+H]+, 434.32 [M-H]-; HRMS (m/z): 436.0790 [M+H]+, 434.0640 [M-H]-.
4.5.10. N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)-1-naphthamide (6i):
o
1
Yellow solid, yield 26.0%, m.p. >200 C H NMR (300 MHz, DMSO-d6) δ: 13.00 (s, 1 H,
NHCO), 10.40 (s, 1 H, CONH), 9.59 (s, 1 H, OH), 8.84-8.87 (m, 1 H, Ar-H), 8.36 (s, 1 H, Ar-H),
8.18 (d, J = 8.4 Hz, 1 H, Ar-H), 8.11 (t, 1 H, Ar-H), 8.06 (d, J = 8.3 Hz, 1 H, Ar-H), 7.71-7.73 (m,
1 H, Ar-H), 7.67-7.69 (m, 1 H, Ar-H), 7.57 (dd, J = 2.1, 8.7 Hz, 1 H, Ar-H), 7.20 (t, 1 H, Ar-H),
7.15 (t, 1 H, Ar-H), 6.93-6.96 (m, 1 H, Ar-H), 6.83-6.86 (m, 1 H, Ar-H), 6.67-6.70 (m, 1 H, Ar-
H), 6.61-6.64 (m, 1 H, Ar-H), 3.88 (s, 2 H, SCH2); 13C NMR (125 MHz, CD3OD-d4)
δ: 169.96,
168.91, 164.45, 159.20, 137.51, 136.87, 135.89, 133.97, 131.03, 130.87, 130.40, 130.24, 129.73,
129.61, 128.90, 128.17, 125.07, 122.13, 120.69, 119.19, 117.85, 115.45, 115.27, 114.68, 113.83,
40.42. ESI-MS (m/z): 486.24 [M+H]+, 484.27 [M-H]-; HRMS (m/z): 486.0945 [M+H]+.
4.5.11. 3,5-Dichloro-N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)benzamide
o
1
(6j): Yellow solid, yield 48.0%, m.p. 199 C. H NMR (600 MHz, CD3OD-d4) δ: 13.01 (s, 1H,
NHCO), 10.37 (s, 1H, CONH), 9.55 (s, 1H, OH), 8.18 (d, J = 1.9 Hz, 1 H, Ar-H), 8.01 (d, J = 1.7
Hz, 2 H, Ar-H), 7.72 (t, J = 1.8 Hz, 1 H, Ar-H), 7.66 (d, J = 8.7 Hz, 1 H, Ar-H), 7.45-7.46 (m, 1

ACCEPTED MANUSCRIPT
H, Ar-H), 7.10-7.13 (m, 2 H, Ar-H), 6.63-6.67 (m, 2 H, Ar-H), 3.76 (s, 2 H, SCH2); 13C NMR
(125 MHz, CD3OD-d4) δ: 169.99, 168.55, 166.93, 157.00, 137.46, 136.73, 136.09, 133.37, 133.30,
131.03, 127.95, 122.13, 120.83, 119.20, 117.86, 115.45, 115.27, 114.69, 113.93, 40.42. ESI-MS
(m/z): 502.19 [M-H]-; HRMS (m/z): 504.0007 [M+H]+, 501.9866 [M-H]-. HPLC purity: >98%,
Rt = 19.79 min, UV 254 nm, 70% Methanol, flow rate: 2 ml/min.
4.5.12. N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)ben-
1
zamide (6k): Yellow solid, yield 52.1%. H NMR (300 MHz, DMSO-d6)
δ: 13.08 (s, 1 H,
NHCO), 10.28 (s, 1 H, CONH), 9.75 (s, 1 H, OH), 8.30 (d, J = 8.3 Hz, 2 H, Ar-H, Ar-H), 7.94 (d,
J = 8.3 Hz, 2 H, Ar-H), 7.62 (dd, J = 2.1, 8.7 Hz, 1 H, Ar-H), 7.34 (t, 1 H, Ar-H), 7.20 (t, 1 H, Ar-
H), 7.10-7.15 (m, 1 H, Ar-H), 6.90-6.94 (m, 1 H, Ar-H), 6.64-6.67 (m, 1 H, Ar-H), 4.74 (s, 2 H,
SCH2); ESI-MS (m/z): 502.24 [M-H]-; HRMS (m/z): 504.0659 [M+H]+.
4.5.13. 4-Fluoro-N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)benzamide (6l):
White solid, yield 44.7%, m.p. >200 oC. 1H NMR (300 MHz, DMSO-d6)
δ: 12.88 (s, 1 H, NHCO),
10.39 (s, 1 H, CONH), 9.59 (s, 1 H, OH), 8.35 (d, J = 1.6 Hz, 1 H, Ar-H),8.21-8.26 (m, 2 H, Ar-
H), 7.75 (d, J = 8.7 Hz, 1 H, Ar-H), 7.56 (dd, J = 2.1, 8.7 Hz, 1 H, Ar-H), 7.40-7.46 (m, 2 H, Ar-
H), 7.14 (t, 1 H, Ar-H), 6.83-6.87 (m, 2 H, Ar-H), 6.61-6.64 (m, 1 H, Ar-H), 3.88 (s, 2 H, SCH2);
13C NMR (125 MHz, CD3OD-d4)
δ: 169.98, 168.01, 166.01, 159.10, 139.45, 137.43, 136.73,
135.90, 132.03, 131.96, 131.03, 122.13, 121.87, 121.78, 121.67, 120.69, 117.86, 116.91, 116.73,
115.27, 113.83, 40.42. ESI-MS (m/z): 454.23 [M+H]+, 452.25[M-H]-; HRMS (m/z): 454.0695
[M+H]+, 452.0546 [M-H]-.. HPLC purity: >98%, Rt = 10.81 min, UV 254 nm, 80% Methanol,
flow rate: 2 ml/min.
4.5.14.
N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-methylbenzamide
1
(6m): Yellow solid, yield 46.1%, m.p. >200 oC H NMR (300 MHz, DMSO-d6)
δ
: 12.76 (s, 1 H,
NHCO), 10.39 (s, 1 H, CONH), 9.59 (s, 1 H, OH), 8.35 (d, J = 1.8 Hz, 1 H, Ar-H), 8.07 (d, J =
8.1 Hz, 2 H, Ar-H), 7.75 (d, J = 8.8 Hz, 1 H, Ar-H), 7.55 (dd, J = 2.1, 8.7 Hz, 1 H, Ar-H), 7.39 (d,
J = 8.1 Hz, 2 H, Ar-H), 7.14 (t, 1 H, Ar-H), 6.81-6.86 (m, 2 H, Ar-H), 6.61-6.64 (m, 1 H, Ar-H),

ACCEPTED MANUSCRIPT
3.88 (s, 2 H, SCH2), 2.42 (s, 3 H, Ar -CH3); 13C NMR (125 MHz, DMSO-d6)
δ: 166.84, 165.55,
164.73, 157.74, 143.23, 136.84, 135.01, 132.11, 129.90, 129.24, 129.08, 128.34, 120.51, 118.68,
118.57, 114.72, 113.24, 111.62, 40.43, 21.14. ESI-MS (m/z): 450.33 [M+H]+, 448.27 [M-H]-;
HRMS (m/z): 450.0947 [M+H]+, 448.0790 [M-H]-. HPLC purity: >98%, Rt = 23.67 min, UV 254
nm, 70% Methanol, flow rate: 2 ml/min.
4.5.15. N-(6-(2-((3-hydroxyphenyl)thio)acetamido)benzo[d]thiazol-2-yl)-3,5-dimethylbenzamide
o
1
(6n): White solid, yield 49.6%, m.p. >200 C. H NMR (300 MHz, DMSO-d6) δ: 12.67 (s, 1 H,
NHCO), 10.35 (s, 1 H, CONH), 9.55 (s, 1 H, OH), 8.31 (d, J = 1.9 Hz, 1 H, Ar-H), 7.75 (s, 2 H,
Ar-H), 7.71 (d, J = 8.6 Hz, 1 H, Ar-H), 7.53 (dd, J = 2.1, 8.7 Hz, 1 H, Ar-H), 7.28 (s, 1 H, Ar-H),
7.11 (t, 1 H, Ar-H), 6.79-6.83 (m, 2 H, Ar-H), 6.58-6.61 (m, 1 H, Ar-H), 3.84 (s, 2 H, SCH2),
2.35 (s, 6 H, 2CH3); 13C NMR (125 MHz, CD3OD-d4)
δ: 169.97, 168.49, 167.45, 159.18, 139.90,
137.44, 135.86, 135.53, 133.98, 133.60, 131.03, 126.81, 122.14, 121.77, 120.66, 119.20, 117.86,
115.27, 114.68, 113.80, 40.42, 21.27. ESI-MS (m/z): 464.40 [M+H]+, 462.26 [M-H]-. HRMS
(m/z): 464.1100 [M+H]+, 462.0946 [M-H]-. HPLC purity: 94.3%, Rt = 11.25 min, UV 254 nm, 70%
Methanol, flow rate: 2 ml/min.
4.6. Synthesis of compounds 7a-7h
4.6.1. N-(6-(2-((2,4-dimethylphenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)be-
nzamide (7a): To a solution of 2,4-dimethylbenzenethiol (35 mg, 0.25 mmol) and 5f (105 mg,
0.25 mmol) in DMF was added K2CO3 (150 mg). The reaction mixture was stirred at room
temperature for 4 hours. The solution was then diluted with water and extracted with EtOAc. The
organic layer was separated, washed with brine and dried over anhydrous Na2SO4. The combined
organic layers were evaporated under vacuum and the residue was purified by flash
1
chromatography to give yellow solid (96 mg, yield 73.3%). H NMR (300 MHz, DMSO-d6)
δ:
13.09 (s, 1 H, NHCO), 10.35 (s, 1 H, CONH), 8.32 (s, 2 H, Ar-H), 8.30 (s, 1 H, Ar-H), 7.95 (d, J
= 8.3 Hz, 2 H, Ar-H), 7.74 (d, J = 8.7 Hz, 1 H, Ar-H), 7.53 (dd, J = 2.0, 8.8 Hz, 1 H, Ar-H), 7.33

ACCEPTED MANUSCRIPT
(d, J = 7.9 Hz, 1 H, Ar-H), 7.05 (s, 1 H, Ar-H), 7.01 (d, J = 8.1 Hz, 1 H, Ar-H), 3.79 (s, 2 H,
SCH2), 2.32 (s, 3 H, Ar- CH3), 2.24 (s, 3 H, Ar-CH3); ESI-MS (m/z): 514.31 [M-H]-.
4.6.2.
N-(6-(2-(naphthalen-2-ylthio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)be-
1
nzamide (7b): Yellow solid, yield 63.0%. H NMR (300 MHz, DMSO-d6)
δ: 13.09 (s, 1 H,
NHCO), 10.48 (s, 1 H, CONH), 8.35 (s, 1 H, Ar-H), 8.32 (s, 1 H, Ar-H), 8.30 (s, 1 H, Ar-H),
7.91-7.80 (m, 3 H, Ar-H), 7.88 (d, J = 8.2 Hz, 2 H, Ar-H), 7.83 (d, J = 7.9 Hz, 1 H, Ar-H), 7.74
(d, J = 8.3 Hz, 1 H, Ar-H), 7.56 (d, J = 8.1 Hz, 2 H, Ar-H), 7.50-7.53 (m, 1 H, Ar-H), 7.45-7.50
(m, 1 H, Ar-H), 4.03 (s, 2 H, SCH2); ESI-MS (m/z): 538.40 [M+H]+, 536.27 [M-H]-.
4.6.3. N-(6-(2-(pyrimidin-2-ylthio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)benzamide
(7c): Yellow solid, yield 76.2%. 1H NMR (300 MHz, DMSO-d6)
δ: 13.09 (s, 1 H, NHCO), 10.47
(s, 1 H, CONH), 8.66 (d, J = 4.9 Hz, 2 H, Ar-H), 8.35 (s, 1 H, Ar-H), 8.31 (d, J = 8.2 Hz, 2 H,
Ar-H), 7.95 (d, J = 8.2 Hz, 2 H, Ar-H), 7.75 (d, J = 9.0 Hz, 1 H, Ar-H), 7.59 (dd, J = 1.8, 8.8 Hz,
1 H, Ar-H), 7.24 (t, 1 H, Ar-H), 4.15 (s, 2 H, SCH2); ESI-MS (m/z): 490.30 [M+H]+, 488.28 [M-
H]-.
4.6.4. N-(6-(2-((1H-imidazol-2-yl)thio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)ben-
1
zamide (7d): Yellow solid, yield 66.1%. H NMR (300 MHz, DMSO-d6)
δ: 13.11 (s, 1 H,
NHCO), 12.36 (s, 1 H, NH), 10.64 (s, 1 H, CONH), 8.34 (d, J = 1.7 Hz, 1 H, Ar-H), 8.31 (d, J =
8.3 Hz, 2 H, Ar-H), 7.96 (d, J = 8.3 Hz, 2 H, Ar-H), 7.75 (d, J = 8.6 Hz, 1 H, Ar-H), 7.55 (dd, J =
1.8, 8.8 Hz, 1 H, Ar-H), 7.09 (s, 2 H, Ar-H), 3.94 (s, 2 H, SCH2); 13C NMR (125 MHz, DMSO-d6)
δ
: 166.04, 159.44, 156.34, 149.80, 135.12, 129.23, 128.29, 126.39, 125.58, 125.56, 125.53,
122.30, 121.79, 118.71, 111.63, 40.43. ESI-MS (m/z): 478.20 [M+H]+, 476.17 [M-H]-. HRMS
(m/z): 478.0619 [M+H]+, 476.0464 [M-H]-.
4.6.5.
N-(6-(2-((1H-benzo[d]imidazol-2-yl)thio)acetamido)benzo[d]thiazol-2-yl)-4-
1
(trifluoromethyl)benzamide (7e): Yellow solid, yield 45.7%. H NMR (300 MHz, DMSO-d6)
δ
:
13.12 (s, 1 H, NHCO), 12.68 (s, 1 H, NH), 10.72 (s, 1 H, CONH), 8.37 (d, J = 1.7 Hz, 1 H, Ar-H),
8.31 (d, J = 8.2 Hz, 2 H, Ar-H), 7.95 (d, J = 8.4 Hz, 2 H, Ar-H), 7.75 (d, J = 8.7 Hz, 1 H, Ar-H),

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7.58 (dd, J = 2.0, 8.8 Hz, 1 H, Ar-H), 7.53 (s, 1 H, Ar-H), 7.39 (d, J = 4.5 Hz, 1 H, Ar-H), 7.10-
7.17 (m, 2 H, Ar-H), 4.32 (s, 2 H, SCH2); ESI-MS (m/z): 528.20 [M+H]+, 526.15 [M-H]-; HRMS
(m/z): 528.0773 [M+H]+, 526.0623 [M-H]-.
4.6.6.
N-(6-(2-(cyclohexylthio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)benzamide
(7f): Yellow solid, yield 34%. 1H NMR (300 MHz, DMSO-d6)
δ: 13.10 (s, 1 H, NHCO), 10.25 (s,
1 H, CONH), 8.37 (d, J = 1.5 Hz, 1 H, Ar-H), 8.31 (d, J = 8.1 Hz, 2 H, Ar-H), 7.95 (d, J = 8.2 Hz,
2 H, Ar-H), 7.74 (d, J = 8.7 Hz, 1 H, Ar-H), 7.55 (dd, J = 2.0, 8.7 Hz, 1 H, Ar-H), 3.36 (s, 2 H,
SCH2), 2.54-2.92 (m, 1 H, SCH), 1.94-2.03 (m, 2 H, cyclohexyl-H), 1.66-1.73 (m, 2 H,
cyclohexyl-H), 1.54-1.59 (m, 1 H, cyclohexyl-H), 1.20-1.31 (m, 5 H, cyclohexyl-H); 13C NMR
(125 MHz, DMSO-d6) δ: 168.33, 164.01, 135.36, 129.21, 128.27, 126.77, 125.57, 125.54, 125.51,
122.75, 118.75, 111.58, 43.29, 40.43, 34.10, 32.87, 25.37, 25.29. ESI-MS (m/z): 494.51 [M+H]+,
492.35 [M-H]-; HRMS (m/z): 494.1184 [M+H]+, 492.1035 [M-H]-.
4.6.7.
N-(6-(2-((4-aminophenyl)thio)acetamido)benzo[d]thiazol-2-yl)-4-(trifluoromethyl)ben-
zamide (7g): Yellow solid, yield 70.4%. 1H NMR (300 MHz, DMSO-d6)
δ
: 13.09 (s, 1 H, NHCO),
10.17 (s, 1 H, CONH), 8.33 (s, 2 H, Ar-H), 8.30 (s, 1 H, Ar-H), 7.95 (d, J = 8.4 Hz, 2 H, Ar-H),
7.73 (d, J = 8.7 Hz, 1 H, Ar-H), 7.52 (dd, J = 1.9, 8.7 Hz, 1 H, Ar-H), 7.17 (d, J = 8.4 Hz, 2 H,
13
Ar-H), 6.51 (d, J = 8.4 Hz, 2 H, Ar-H), 5.32 (s, 2 H, NH2), 3.57 (s, 2 H, SCH2); C NMR (150
MHz, CD3OD-d4) δ: 170.71, 168.65, 164.60, 149.92, 136.27, 136.08, 135.19, 134.97, 130.00,
126.82, 126.80, 126.78, 126.12, 121.50, 120.78, 116.70, 113.82, 40.44. ESI-MS (m/z): 503.50
[M+H]+, 501.27 [M-H]-; HRMS (m/z): 503.0826 [M+H]+, 501.0667 [M-H]-. HPLC purity: 95.6%,
Rt = 10.38 min, UV 254 nm, 85% Methanol, flow rate: 2 ml/min.
4.6.8.
2-((2-Oxo-2-((2-(4-(trifluoromethyl)benzamido)benzo[d]thiazol-6-yl)amino)ethyl)thio)
1
nicotinic acid (7h): Purple solid, yield 64.9%. H NMR (300 MHz, DMSO-d6)
δ:10.33 (s, 1 H,
CONH), 8.45 (dd, J = 2.0, 4.8 Hz, 1 H, Ar-H), 8.31 (d, J = 8.0 Hz, 2 H, Ar-H), 8.26 (d, J = 1.5 Hz,
1 H, Ar-H), 8.12 (dd, J = 1.7, 7.4 Hz, 1 H, Ar-H), 7.90 (d, J = 8.1 Hz, 2 H, Ar-H), 7.64 (d, J = 8.6

ACCEPTED MANUSCRIPT
Hz, 1 H, Ar-H), 7.52 (dd, J = 1.7, 7 8.2 Hz, 1 H, Ar-H), 7.13 (q, 1 H, Ar-H), 3.90 (s, 2 H, SCH2).
ESI-MS (m/z): 531.09 [M-H]-; HRMS (m/z): 533.0570 [M+H]+, 531.0402 [M-H]-.
4.7. In vitro cytotoxicity assay
Cells were plated in 96-well microtiter plates at a density of 5,000/well and incubated in a
o
humidified atmosphere with 5% CO2 at 37 C for 24 h. Test compounds were added to
triplicate wells with different concentrations and 0.1% DMSO for control. After
incubation for 72 h, 20 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) solution (5 mg/mL) was added to each well and the plate was incubated for
additional 4 h. The formazan was dissolved in 100 mL of DMSO. The absorbance (OD)
was read on a WellscanMK-2 microplate reader (Labsystems) at 570 nm. The
concentration causing 50% inhibition of cell growth (IC50) was determined by the Logit
method.
4.8. Western blotting assay
HCT116 cells were plated to medium plates at 2 x 106 cells confluence and treated with
test compounds at various concentrations (0.1 µM and 1 µM) for 4 hours. The protein
concentration was determined by bicinchoninic acid (BCA) assay (Pierce,
ThermoScientific, Rockford, IL). Following quantification, 40 µg protein were mixed with
loading dye and sample reducing agent (NuPAGE, Invitrogen, Carlsbad, CA), boiled for 5
minutes, and separated by gel electrophoresis at 150 V on a NuPAGE 10% Bis-Tris Gel
(Invitrogen, Carlsbad, CA). Proteins were then transferred at 40 V to a polyvinylidene
difluoride (PVDF) membrane from Millipore (Billerica, MA). Membranes were blocked
in 5% milk in tris buffered saline Tween-20 (TBST) for 1 hour at room temperature
followed by incubation with primary antibody (1: 1000) overnight at 4 °C. Membranes
were washed 3 times in TBST for 5 minutes and then incubated in the appropriate HRP
conjugated secondary antibody (1:2000) for 1 hour at room temperature. Detection was
performed using super signal chemiluminescence system from Pierce (Rockford, IL). β-

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actin and secondary antibodies were purchased from Sigma-Aldrich. NEDD8 and UBC12
antibodies were from Cell Signaling Technology.
Abbreviations
CRLs: cullin-RING ubiquitin E3 ligases
MCL: mantle cell lymphoma
MM: multiplemyeloma
NAE: NEDD8 activating enzyme
NEDD8: neural precursor cell-expressed developmentally downregulated protein 8
SAR: structure-activity relationship
SP: standard precision
UBC12: NEDD8-specific E2s
UPS: ubiquitin proteasome system
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation
of China (81502978 to C.Z. and 81673352 to Z.M.), the Bio-Pharmaceutical Project of
Science and Technology of Shanghai (15431901700 to Z.M.), and Shanghai
‘‘ChenGuang’’ Project (16CG42 to C.Z.).
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Figure Caption
Fig. 1. NEDD8 pathway and structure of AMP.
Fig. 2. The workflow of the target-based virtual screening and the structures of NAE
inhibitors.
Fig. 3. Docking modes of compounds H11 (A) and 7g (B) with NAE and the optimization
strategy. Hydrogen bonds were highlighted in red and green dash lines. The key residues
for the binding were labelled.
Fig. 4. Western blotting assay of compound 6k, 7b and 7g (HCT-116 cells, 4h treatment):
Lane 1, DMSO control with no treatment; Lane 2, compound 6k (0.1 µM); Lane 3,
compound 6k (1 µM); Lane 4, compound 7b (0.1 µM); Lane 5, compound 7b (1 µM);
Lane 6, compound 7g (0.1 µM); Lane 7, compound 7g (1 µM).
Table 1. The antitumor activities of 15 selected compounds (IC50, µM)a
Table 2. The antitumor activities of H11 analogues (IC50, µM)a
Scheme 1. (a): Et3N, DCM, room temperature, 3 h; (b): Pd/C, H2, CH3OH, room temperature, 4 h;
(c): Et3N, DCM, room temperature, 2.5 h; (d): 3-mercaptophenol or 4-chlorobenzenethiol,
NaHCO3, CH3COCH3, reflux, 5 h.
Scheme 2. (a): K2CO3, DMF, RSH, room temperature, 4 h.