Design and synthesis of aminothiazole based Hepatitis B Virus (HBV) capsid inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.01.059

The capsid assembly is an essential step for Hepatitis B Virus (HBV) life cycle and is an important target for anti-HBV drug development. In this report, we identified a hit compound with aminothiazole structure by the high throughput screening (HTS) which inhibited the interaction of HBV capsid protein within the cells. The structure hopping and SAR studies of the hit compound afforded compound 79 with potent anti-HBV replication activity and good basic drug-like properties. The working mechanism studies showed that compound 79 could bind to the similar binding site of known HBV capsid inhibitor with heteroaryldihydropyrimidine (HAP) scaffold, through similar hydrophobic interactions but with a different hydrogen bond. This compound exerted potent inhibitory effect upon HBV production, either in cell culture or in mice with no obvious acute toxicity. We propose that further development of this compound could lead to novel potent anti-HBV inhibitors that target HBV capsid assembly.

Full text of DOI:10.1016/j.ejmech.2019.01.059

Accepted Manuscript
Design and synthesis of aminothiazole based Hepatitis B Virus (HBV) capsid
inhibitors
Ting Pan, Yanchao Ding, Liyang Wu, Liting Liang, Xin He, Qianwen Li, Chuan Bai,
Hui Zhang
PII:
S0223-5234(19)30058-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.059
EJMECH 11066
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2018
Revised Date: 30 December 2018
Accepted Date: 18 January 2019
Please cite this article as: T. Pan, Y. Ding, L. Wu, L. Liang, X. He, Q. Li, C. Bai, H. Zhang, Design
and synthesis of aminothiazole based Hepatitis B Virus (HBV) capsid inhibitors, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.059.
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ACCEPTED MANUSCRIPT

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Design and Synthesis of Aminothiazole Based Hepatitis B Virus
(HBV) Capsid Inhibitors
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†
, ‡, #
†, ‡, #
†, ‡, #
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†, ‡,
†, ‡,
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Ting Pan
, Yanchao Ding
, Liyang Wu
, Liting Liang , Xin He , Qianwen Li
,
†
, ‡,
†, ‡,
Chuan Bai
*
，and Hui Zhang
*
†
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Institute of Human Virology,
‡
Key Laboratory of Tropical Disease Control of Ministry of Education,
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Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology
Zhongshan School of Medicine,
Sun Yat-sen University,
Guangzhou, Guangdong, China, 510080
* To whom correspondence should be addressed:
Sun Yat-sen University
nd
74 Zhongshan 2
Road
Guangzhou 510080, China
Phone: +86-20-8733-2588
Fax: +86-20-8733-2588
Dr. Chuan Bai or Dr. Hui Zhang
Email: baichuan@mail.sysu.edu.cn or zhangh92@mail.sysu.edu.cn
# These authors contributed equally to this work.
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Abstract
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33
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The capsid assembly is an essential step for Hepatitis B Virus (HBV) life cycle and is an important
target for anti-HBV drug development. In this report, we identified a hit compound with aminothiazole
structure by the high throughput screening (HTS) which inhibited the interaction of HBV capsid protein
within the cells. The structure hopping and SAR studies of the hit compound afforded compound 79
with potent anti-HBV replication activity and good basic drug-like properties. The working mechanism
studies showed that compound 79 could bind to the similar binding site of known HBV capsid inhibitor
with heteroaryldihydropyrimidine (HAP) scaffold, through similar hydrophobic interactions but with a
different hydrogen bond. This compound exerted potent inhibitory effect upon HBV production, either
in cell culture or in mice with no obvious acute toxicity. We propose that further development of this
compound could lead to novel potent anti-HBV inhibitors that target HBV capsid assembly.
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Introduction
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HBV has been a severe public health threat because it has infected approximate 257 million people
worldwide in 2017[1]. The progression of HBV infection is highly correlated with cirrhosis and
hepatocellular carcinoma. However, the currently available therapeutics including IFN-α and
nucleot(s)ides have not been able to afford satisfying effects in clinical practice because of the high cost,
severe side effects, or the development of viral drug resistance[2-4]. Moreover, neither IFN-α nor
nucleot(s)ides could eradicate HBV infection because of the stable covalently-closed circular DNA
(cccDNA) of HBV. Therefore, further development of new anti-HBV drugs targeting other vital process
in the viral life cycle has been an urgent requirement [5, 6]. The HBV capsid assembly is one of the key
steps for HBV replication and plays important roles in stabilizing cccDNA. Previous studies showed
that blocking HBV capsid assembly led to effective inhibition of HBV replication and impaired the
cccDNA stability [7-14]. Since the first HBV capsid assembly inhibitor BisANS was discovered [15],
many HBV capsid assembly inhibitors with different scaffolds have been studied [16-21]. Among these
HBV capsid inhibitors, the compounds with heteroaryldihydropyrimidine (HAP) structure including
Bay 41-4109[10], GLS4 [22, 23], and NVR-010–001-E2 [24] showed potent anti-HBV activity in vitro
and in vivo, and their binding models and working mechanisms against HBV capsid assembly have been
elucidated [22, 25, 26].
55
56
57
58
59
60
61
Most of the HBV capsid inhibitors were first discovered with the HTS system in which the expressed
capsid proteins assembled in a non-cellular environment [10, 27, 28]. Therefore, this HTS system may
not be able to investigate some important factors involved in capsid assembly in the cellular
environment. To overcome this problem, we developed a capsid-GFP assay by using capsid protein
fused with GFP fragments that was expressed in 293T cells. This screening method was designed to be
more correlated with the real conditions that was required for HBV capsid assembly in viral replication
and therefore, could identify hit compounds more efficiently and accurately. With our HTS system, we
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identified a hit compound targeting HBV capsid assembly. Based on the hit compound, we discovered a
new potent HBV capsid assembly inhibitor (79) and it was able to inhibit HBV replication inhibitor in
vivo and in vitro. The aminothiazole structure of this active compound may have hydrogen bond
interactions with Ser121 of HBV capsid protein. Our discoveries afforded a new scaffold for the
development of new HBV capsid assembly inhibitor and shed light on the design of more potent
structure.
6
8
9
Results
6
Section 1. High throughput screening of HBV capsid inhibitors
7
7
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7
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7
7
7
7
8
0
1
2
3
4
5
6
7
8
9
0
To explore new HBV capsid inhibitors, we performed a cell-based high-throughput screening by
using a bimolecular fluorescence complementation (BiFC) assay to determine the binding states of HBV
capsid proteins in 293T cells. In brief, we first constructed plasmids of two nonfluorescent fragments of
VFP (Venus fluorescent protein) fused with HBV capsid protein, which were HBVcAg-VFP-N1-154
(HBc-VN) and HBVcAg-VFP-C155-239 (HBc-VC). These two fragments could reconstitute the
fluorophore only when the HBV capsid proteins get close enough during the assembly of HBV capsids.
The HBc-VN and HBc-VC plasmids were co-transfected into 293T cells and the cells were treated with
a commercial drug-like chemical library composed of 40155 compounds at 50 µM (Figure 1A). After
duplicated screening, 439 hit compounds were identified and they were further screened by anti-HBV
replication assay in which the amount of HBV DNA was quantified by qPCR after the treatment. 125
compounds showed activity to inhibit HBV replication at 50
µ
M and 9 hits at 5
µ
M in HepG2.2.15 cells
M, Figure
µM in anti-HBV replication assay, Figure 1D), we chose it for further
81
82
83
(Figure 1B). Because one the hit compound (Figure 1B) showed low cytotoxicity (CC >500µ
5
0
1C) and high potency (IC50= 6.15
studies.
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Section 2. SAR of hit compound and the structure hopping
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We first conducted the structure-activity relationship (SAR) studies of the hit compound, with the
8
synthesis methods showed in scheme 1 [29-31].
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8
Scheme 1. The syntheses of derivatives of hit compound (22). Reagents and conditions: (a) THF, rt; (b)
89
3
n-BuLi, THF, -78℃; (c) Et SiH, TFA, DCM, rt; (d) Pyridine, DCM, rt.
90
91
92
93
In the SAR studies for this project, the compounds were first tested in the Capsid-GFP assay (CGA in
Tables) and in the cytotoxicity assay. Only when the tested compound was active in Capsid-GFP assay
at 50µM concentration and also showed no obvious cytotoxicity (CC50>50 µM), it would be tested in
HBV replication assay.
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96
First, the alky-, aromatic or hetero cyclic modifications on the amide group (14-20, Table 1) did not
afford better activity in Capsid-GFP assay. Replacement of p-fluorobenzene of the hit compound with
p-chlorobenzene (21) afforded improvement on the anti-HBV replication activity (IC from 6.15
µM to
5
0
9
7
8
3.62
µ
M). Therefore, in the further SAR studies, we focused on o-chlorobenzene as the substitution
9
group.
99
1
1
00
01
Table1. The SAR studies of the hit compound
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b
c
Compd.
No.
X
R
CGA
CC50
IC50
clog
P
a
d
(%)
14
15
16
17
18
-F
-F
-F
-F
-F
N
>50
µ
M
/
/
/
/
/
4.26
3.76
4.10
4.52
5.07
0.60
0.53
N
N
N
>50µM
Cl
N
>50µM
Cl
1
9
0
-F
-F
N
N
N
/
/
4.04
5.93
2
>50µM
21
22
23
-Cl
-Br
-Br
0.59 >50
µM
µM
µM
3.62µM 4.34
N
N
>50
>50
/
/
4.45
5.54
1
1
02
03
a: CGA, Capsid-GFP Assay. The percentage of inhibition with compounds at 50
assay. N: test result did not show activity.
µM concentration in Capsid-GFP
1
1
04
05
b: CC (µM), median (50%) cytotoxic concentration; N: CC value lower than 5µM and too toxic for further
50 50
test.
1
1
06
07
c: IC (
µ
M), half maximal (50%) inhibitory concentration to inhibit of HBV replication. This test was performed
50
only when capsid-GFP assay showed positive result and on obvious cytotoxicity was observed (CC >50 ).
µM
50
1
08
d: clogP was calculated by LigandScout 4.1
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09
10
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12
13
14
15
16
17
18
19
20
21
These results prompted us to design compounds by referring with the successful HAP structure. After
we compared the structures of hit compound with NVR-010-001-E2 (Scheme 2), we noticed that our hit
compound possessed two hydrophobic groups: benzene (green) and thiophene (blue), which were
similar with the ethyl (green) and thiazole groups (blue) in NVR-010-001-E2. The previous working
mechanism studies of NVR-010-001-E2 already elucidated that these two groups had strong
hydrophobic or aromatic interactions with the hydrophobic pockets of HBV capsid protein, and thus
they were important for its activity [24]. Moreover, HAP structure had the halogenated benzene ring
(cyan) which bound with the third hydrophobic pocket in HBV capsid protein. However, our hit
compound lack of the third hydrophobic group. The docking studies also showed that the hit compound
could bind in the same site as NVR-010-001-E2, but it was lack of a hydrophobic group to bind with the
hydrophobic cavity that was occupied by the halogenated benzene of NVR-010-001-E2 (Figure S1.).
Thus, we proposed that the methylene group on hit compound could be substituted with a hydrophobic
group (cyan) to afford the necessary hydrophobic interaction (Scheme 2).
1
22
1
1
1
1
23
24
25
26
Scheme 2. The design and structure hopping of hit compound. The hit compound has two
hydrophobic groups (green and blue). Our design added one more hydrophobic group (cyan)
enlightened by the capsid inhibitors in clinical trials with HAP structure (BAY41-4109 and
NVR-010-001-E2).
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27
Section 2. SAR studies of the compounds with the third hydrophobic groups
1
1
28
29
According to the analysis on the possible binding model of the hit compound described above, we
designed and synthesized its derivatives with the methods described in scheme 3.
A
R1
Y
OH
Cl
S
S
a
S
b
NHBoc
NHBoc
NHBoc
+
R YH
1
N
N
N
X
X
X
9
8
4
R1
Y
R1
Y
O
O
S
d
S
+
c
NH2
Cl
R2
NH
R2
N
X
N
X
1
0
11
12
B
R1
R1
Y
Y
S
e
S
+
R X
3
NH2
NH R3
N
N
X
X
10
1
3
1
1
30
31
1
1
1
32
33
34
Scheme 3. The syntheses of compounds with three hydrophobic groups. Reagents and conditions: (a)
SOCl , DCM, Reflux; (b) K CO , DCM, Reflux; (c) TFA, DCM, rt; (d) Pyridine, DCM, rt; (e) Cs CO
DMF, rt.
2
2
3
2
3
,
1
1
1
1
1
1
35
36
37
38
39
40
To explore the chemical diversity in the third hydrophobic groups on the methylene group, we
synthesized the intermediates with chloro-subtituted methylene group (compound 8, Scheme 3A) to
conveniently construct an ether link to install the third hydrophobic groups (compound 9, Scheme 3A).
With the p-chloride substitution on benzene ring and with Boc groups in the amide position, different
hydrophobic or aromatic rings were installed (24-33, Table 2). Among these tested compounds, the
α-bromide-naphthalene groups (31) afforded good activities both in Capsid-GFP assay and anti-HBV
8

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1
1
1
1
1
1
1
41
42
43
44
45
46
47
48
replication test. Next, we optimized compound 31 by replacing the Boc group with thiophene group and
found this modification (33) afforded good activity in Capsid-GFP assay (CGA) but its cell toxicity was
too high (CC <5µΜ) to test its anti-HBV replication activity. It was also interesting that other aromatic
5
0
groups tested in our studies (34-41) did not affect the activity in Capsid-GFP assay but led to the loss of
activity to inhibit HBV replication. These data indicated that the size of the aromatic groups may not be
important to interrupt the assembly of capsid protein, but the p-chloride benzene was important to
inhibit HBV replication in cells. The SAR studies in this series of compounds afforded support on our
hypothesis that the third hydrophobic groups could be a good position for further design.
1
49
1
1
50
51
Table 2. The SAR studies of the compounds with the third hydrophobic groups
Compd
No.
X
R
1
R
2
CGA
CC50
IC50
clogP
7.50
a
(%)
24
-Cl
N
>50µM
/
25
26
27
-Cl
-Cl
-Cl
N
N
N
>50
>50
>50
µM
µM
µM
/
/
/
6.83
6.20
5.94
28
-Cl
N
>50µM
/
6.25
9

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Compd
No.
X
R
1
R
2
CGA
CC50
IC50
clogP
7.13
a
(%)
29
-Cl
N
>50µM
/
3
0
-Cl
-Cl
N
>50
µ
M
/
7.36
7.00
31
0.55 >50
µ
M
M
2.27µM
3
2
3
-Cl
-Cl
N
>50µ
/
/
7.52
7.71
3
0.47
N
3
4
5
H
H
0.64
0.59
N
N
/
/
5.76
5.65
3
3
6
7
H
H
0.64
0.64
N
N
/
/
5.31
5.01
3
3
8
9
H
H
0.26
0.27
N
N
/
/
5.28
5.90
3
1
0

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Compd
No.
X
H
R
1
R
2
CGA
CC50
IC50
clogP
5.77
a
(%)
40
0.45
0.47
N
/
41
H
N
/
6.16
1
52
Section 3. The SAR studies at thiophene group and its linker
1
1
1
1
1
53
54
55
56
57
Based on the results of section 2, we first tested whether it would afford better activity to replace the
amide group with alky-substituted amine groups. The Capsid-GFP assay showed that this type of
modification totally abolished the activity to inhibit HBV replication (42-48, Table 3). It indicated that
the aromatic ring or/and the amide structure were necessary for the activity, which fit our hypothesis
that the large hydrophobic or aromatic groups were required in this position.
1
1
58
59
Table 3. The SAR studies of alkyl-substituted amine
a
Compd.
No.
R
3
CGA (%)
CC50
IC50
clogP
42
43
44
0.55
0.61
0.37
N
N
N
/
/
/
9.26
8.62
6.69
11

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4
5
6
0.38
N
/
7.08
7.39
4
0.35
N
/
4
7
8
0.40
0.43
N
N
/
/
7.39
7.47
4
1
1
1
1
1
1
1
1
1
1
1
1
1
60
61
62
63
64
65
66
67
68
69
70
71
72
We then synthesized amide structure with alky or aromatic ring substitutions (Table 4). First, the small
alkyl groups, such as cyclopropane (49) and neo-butyl (51), could maintain the activity in Capsid-GFP
assay but did not afford any activity in HBV replication assay. The similar results were observed in the
compounds with substitution groups (52-54) with hetero atoms. Second, among the compounds with
substituted benzene ring (55), or benzene rings with electron donating groups (56, 57) or electron
withdrawing groups (57-63), most of them were active in Capsid-GFP assay. Among these compounds,
only compounds 57 and 61 showed potent anti-HBV replication activity but they had a too high LogP
value for good DMPK properties. Therefore, we tried to install heterocycles (64-65) or carbohydrate
precursor (66) in this position. However, these structures did not afford higher anti-HBV replication
activity. Meanwhile, based on compound 57, we studied the effects of different linkage atoms. It turned
out that the compound with nitrogen atoms (67) was still active in both assays, although less active than
compound 57. However, the sulfur atom (68) led to the loss of the activity to inhibit HBV replication.
1
1
73
74
Table 4. The SAR studies of alkyl- or aromatic-substituted amide
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2

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Compd.
No.
Y
R
4
CGA
CC50
IC50
clogP
6.92
a
(%)
4
9
-O
0.67
N
/
5
5
0
1
-O
-O
N
>50
µM
/
/
8.15
7.62
0.47
N
5
5
2
3
-O
-O
0.67
0.35
N
N
/
/
7.51
6.75
5
5
4
5
-O
-O
0.66
0.58
N
N
/
/
7.00
7.65
5
5
5
5
6
6
7
8
9
0
-O
-O
-O
-O
-O
0.71
0.54
0.50
N
N
/
7.96
7.66
7.79
8.42
7.93
>50
µ
M
1.45µM
N
/
/
/
>50
µ
M
0.51
N
6
6
1
2
-O
-O
0.65
0.44
>50
µM
3.55
/
µ
M
8.55
8.07
N
1
3

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Compd.
No.
Y
R
4
CGA
CC50
IC50
clogP
a
(%)
6
3
-O
-O
0.70
0.64
>50
µM
/
/
7.53
7.26
6
4
N
6
6
5
6
-O
-O
0.45
0.40
N
N
/
/
7.70
5.37
6
6
7
8
-NH
-S
0.69
0.49
>50
µ
M
6.85
µ
M
6.93
7.61
N
/
1
75
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
To further optimize compound 57, we then tested if sulfonamide bond would better than amide
(Table 5). Compound 69 showed that the sulfonamide bond would not decrease the activity in
Capsid-GFP assay, but it was inactivity in anti-HBV test. We then synthesized derivatives with benzene
(70) or substituted benzene rings (71-74). It turned out that only compound 70 afforded good activity in
both Capsid-GFP assay and anti-HBV replication assay. We next try to install larger hydrophobic
groups (75-78) and proposed that they could occupy the hydrophobic pocket better in the active site of
capsid protein. However, these compounds did not show good activity in either assay. Finally, the
quinoline (79) group showed good activities to abolish capsid assembly and to inhibit HBV replication.
We then isolated the two enantiomers of compound 79 with chiral chromatography (Supplemental
information). Both of the enantiomers showed similar activities in Capsid-GFP assay and anti-HBV
replication assay (Figure S2). These data indicated that the relatively large hydrophobic groups, such as
quinoline in compound 79, would afford good binding affinity with the hydrophobic pocket in HBV
capsid protein. The different activities between compounds 76 and compound 79 indicated that the
nitrogen atom of quinoline on compound 79 may have strong interactions, such as hydrogen bonds, with
certain amino acid residues in the binding pocket.
1
4

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1
1
91
92
Table 5. The SAR studies of compounds with sulfonamide bonds
a
Compd.
No.
R5
CGA (%)
CC50
IC50
clogP
6
7
7
9
0
1
0.44
0.63
N
N
/
8.58
8.51
9.13
>50µM
>50µM
2.32µM
/
7
7
7
2
3
4
N
>50µM
/
/
/
9.08
9.17
8.56
0.50
0.30
N
N
7
7
5
6
N
N
>50µM
>50µM
/
/
8.45
9.67
7
7
N
>50µM
/
9.98
1
5

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a
Compd.
No.
R
5
CGA (%)
CC50
IC50
clogP
8.52
7
8
0.50
N
/
7
9
0.63
>50µM
0.18µM
9.06
1
1
93
94
Section 4. SAR studies at the 4-Chlorobenzene group
1
1
1
1
1
2
95
96
97
98
99
00
In section 2, we observed that the chloride atom on benzene ring was required for the activity. So we
used compound 57 as standard to further study whether the substitution groups in this position would
increase the activity (Table 6). The compounds with halogen atoms (80-83) were active in Capsid-GFP
assay, but they did not afford better activity in HBV replication assay. Both nitrile and methylsulfonyl
groups totally abolished both of the activities. These results, together with the results from section 2,
indicated that the chloride group in this position is important for the anti-HBV activity.
2
2
01
02
Table 6 The SAR studies at the 4-Chlorobenzene group
Compd.
No.
X
CGA
CC50
IC50
clogP
a
(%)
8
8
8
0
1
2
Br
F
0.78
0.76
N
>50
>50
µ
M
/
/
/
7.77
7.15
6.89
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-CN
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CGA
CC50
IC50
clogP
a
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3
N
N
/
7.50
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03
04
Section 5. Compound 79 inhibited the HBV capsid assembly
2
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SAR studies afforded compound 79 as the best active compound. Then we performed further studies
on how it disrupted the assembly of HBV capsid. We first co-transfected HBc-VN and
HBc-VC-expressing plasmids into 293T cells and incubated for 48 h. Then we treated the cells with
compound 79 at 0, 0.5, 5, or 50 µM respectively. The MFI (mean fluorescent intensity) of Envision
showed that the GFP protein level was inhibited by compound 79 in a dose-dependent manner (Figure
2A). Second, we confirmed that the interactions of HBV capsid protein was interrupted by compound
79 with co-immunoprecipitation assay. The HA-tagged-HBc-VN-expressing plasmid was co-transfected
with HA-tagged-HBc-VC-expressing plasmid into 293T cells. Treatment with compound 79 (50
µM,
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100 M) obviously disrupted the dimerization of HBV capsid protein (Figure 2B, C). These results
µ
showed that compound 79 inhibited the HBV by disrupting the HBV capsid interactions. To further
identify whether treatment of compound 79 had effect on capsid morphology, recombinant HBV capsid
protein (HBV capsid protein 1-149, Cp149) was expressed in E.coli and purified, and then subjected to
negative staining and examined with electronic microscopy (EM) [15, 26, 32]. After the treatment
with compound 79, almost all capsids did not display a normal morphology (Figure 2D). These results
showed that 79 inhibits the aggregation of HBVcAg and consequently disrupts the formation of HBV
capsid.
2
21
Section 6. The binding model of compound 79 with HBV capsid proteins.
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To further elucidate the working mechanism of compound 79, we performed the molecular docking
studies of compound 79 with the crystal structure of HBV capsid protein which included HAP ligand
NVR-010-001-E2 (PDB: 5E0I). Frist, the optimized conformation of compound 79 was prepared. Then,
the crystal structure of HBV capsid protein was also prepared by removing ligand and unrelated water
molecules. We did not specify the HAP ligand site for compound 79 but instead we selected the whole
protein for docking studies. The top 5 poses of compound 79 were just in the same binding pocket as the
ligand (NVR-010-001-E2). Moreover, compound 79 and NVR-010-001-E2 showed quite similar
binding models (Figure 3A). The bromonaphthalene group of 79 had strong hydrophobic or aromatic
interactions with the hydrophobic pocket composed by Phe23, Phe24, Trp102, Tyr118, Leu140 of chain
B, and Ala132 of chain C (Figure 3A). This was the largest hydrophobic pocket and it was the thiazole
group of NVR-010-001-E2 that had bound in this pocket. The quinoline group of compound 79 bound
with Thr109 and Val120 of Chain C and it was the similar position that 2-Bromo-4-Fluorophenyl of
NVR-010-001-E2 had bound in (Figure 3A). The 4-cholorophenyl group of compound 79 had
hydrophobic interactions with Val124 and Trp125 of Chain C (Figure 3B) and the chloride atom pointed
to the solvent environment. Compound 79 had three important hydrophobic/aromatic interactions that
were also important hydrophobic interactions for HAP inhibitor NVR-010-001-E2. Besides hydrophobic
interactions, the nitrogen atoms of the thiazole group and the quinoline group of compound 79 may
form hydrogen bound with Ser121 (Figure 3C). The hydrogen bonds between quinoline and Ser121
afforded support on the SAR results of section 3 where we found that the naphthalene group (76, 77) or
other aromatic groups could not good activity but only quinoline showed best activities.
2
2
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2
42
43
44
45
46
To verify the possible hydrogen bonds and the hydrophobic interaction between compound 79 and
Ser121 and Tyr118 would be important for its activity, we constructed two mutant HBV capsid proteins
Ser121A and Tyr118A. The surface plasmon resonance (SPR) experiments showed that the binding
-6
affinity of compound 79 with wildtype HBV capsid protein was high (K =5.93*10 , Figure 4A).
D
However, the HBV capsid mutants S121A and Y118A showed low binding affinity with compound 79
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(Figure. 4B and 4C). These results showed that the hydrogen bond and hydrophobic interaction were
vital for the binding affinity and activities of compound 79.
2
49
Section 7. Compound 79 inhibits the HBV replication in vitro and in vivo.
4
2
2
2
2
50
51
52
53
Compound 79 was applied to treat HepG2.2.15 cells (5×10 ) for 7 days, the HBV replication was
detected by the quantitation of HBV DNA in the supernatant with real-time PCR. Compound 79 showed
potent anti-HBV replication activity in a dose-dependent way (Figure 5A) with the IC50 value of 182.9
nM (Figure 5B) in HepG2.2.15 cells.
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The in vivo efficacy of compound 79 was evaluated in BALB/c transgenic mice which were injected
with replication-competent HBV DNA plasmid HBV-1.2mer and they could produce HBV DNA,
HBeAg, HBsAg and HBcAg [33]. These mice were randomly divided into two groups and each group
included 3 male BALB/c transgenic mice. We injected by tail vein with 30mg/kg compound 79 and
DMSO, respectively. After 2 weeks, mice were scarified and the blood samples were collected to
determine the antiviral activity. The data displayed here showed that compound 79 had a significant
inhibition of HBV DNA level (Figure. 5C) and HBsAg level (Figure. 5D). Alternatively, to confirm the
anti-HBV replication activity of compound 79 in vivo, the nude mice were subcutaneously inoculated
with HepAD38 cells and then treated with or without compound 79 every 3 days. After 4 weeks, the
HBV DNA level in the blood in the treated groups significantly lower than control and it showed that
compound 79 had potent antiviral activity in vivo (Figure. 5E). Meanwhile, the HBVcAg level in the
tumor tissue detected by immunofluorescence assay also decreased in the compound 79 treated group
(Figure. 5F).
2
67
Section 8. Compound 79 did not show obvious toxicity in acute toxicity test.
2
2
68
69
The DMPK and toxic properties were the major concerns for the HBV capsid inhibitors [7, 9].
Compound 79 was tested in 5 male Balb/c mice with intraperitoneal injection in the dose of 0 mg/kg,
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500 mg/kg, and 1000 mg/kg, respectively. These mice did not show significant change of body weight
th
two weeks after the injections (Figure 6A). In the 14 day, the blood samples and the tissues of heart,
liver, lung, spleen, and kidney were examined and no significant abnormity was found. The hepatic and
renal functions including the levels of AST, ALT, BUN, and CRE were normal (Figure 6B-D). The
histological sections of these organs were also normal (Figure 6E). These preliminary studies on the
toxic properties of compound 79 showed that compound 79 could be a good start point to design new
HBV capsid inhibitors for lead discoveries.
2
77
Discussion and Conclusion
2
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78
79
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In this report, we developed a cell-based HTS system and screened a commercial library, resulting one
of the hit compounds with aminothiazole structure that could block HBV capsid assembly and inhibit
HBV replication. In the light of the known working mechanism and structure biology data of active
HAP compounds (NVR-010–001-E2 and GLS4), we proposed that our hit compound may have a
similar binding model with HAP compounds. Based on this hypothesis, we predicted that the activity of
the hit compound may be promoted if it possessed three hydrophobic groups to bind in the binding
pocket composed by two HBV capsid proteins, as HAP compounds did. Among the compounds that we
deigned and synthesized according to this hypothesis, compound 79 exhibited potent activity to block
HBV capsid assembly in the Capsid-GFP assay and it led to the abnormal size and shape of HBV capsid.
Compound 79 also inhibited HBV replication both in vitro and in vivo with no obvious acute toxicity. To
elucidate the working mechanism of our effective compounds, we performed docking studies and it
showed that compound 79 had similar binding model with ligand NVR-010–001-E2 and the third
hydrophobic groups (bromo napthalene) showed strong hydrophobic interactions with pocket composed
by Thr128, Tyr118 etc. The quinoline group had hydrophobic interaction with Val120 of Chain C and
Thr109 of Chain B. Moreover, the docking studies showed that the nitrogen atoms of aminothiazole and
quinoline groups were close to amino acid Ser121 and they could form hydrogen bonds. To acquire
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better understand of the hydrogen bond interactions, we tested the binding affinity of compound 79 with
WT HBV capsid protein and S121A mutant with SPR. Compound 79 showed high binding affinity with
WT capsid protein but did not bind with the mutant S121A.
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09
Since the HBV capsid assembly process has been proved to be a novel anti-HBV target, both
academia and industry have developed HBV capsid inhibitor with different scaffolds. The HAP structure
has afforded some promising lead compounds or NCEs in clinical trials. However, the working
mechanism of HAP compounds required that the active compounds must have three relatively large
hydrophobic/aromatic substitution groups, thus bringing problems in their DMPK properties. Recently,
the optimization of HAP structure has focused on the 6-C of dihydropyrimidine by installing various
hydrophilic groups to afford better water solubility [7, 9]. The molecular docking data also suggested
that this position may point to the solvent environment. Therefore, it could be useful to install
hydrophilic groups to acquire both high anti-HBV replication activity and good water solubility. In
addition, our work showed that compound 79 probably has a key hydrogen bond with Ser121, which has
not been identified before. This hydrogen bond, together with other possible water participated
hydrogen bonds, could afford strong binding affinity and also shed light on the design of new HBV
capsid inhibitors to bind efficiently to Ser121.
3
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3
10
11
12
13
14
In conclusion, our work has identified 2-aminothiazole based HBV capsid inhibitors with potent
anti-HBV replication activity. They may bind with HBV capsid protein by hydrophobic interactions and
a hydrogen bond with Ser121. These active compounds, especially compound 79, could be a good start
to develop new anti-HBV lead compounds or could be used as chemical tools to study the roles of HBV
capsid in viral life cycle.
3
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16
Experimental methods
General methods for chemistry. All starting materials were commercially available and used without
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further purification. All reactions were carried out with the use of standard techniques under an inert
atmosphere (Ar or N ). NMR spectra were generated on a Bruker 500 or 400MHz instrument and
2
obtained as CDCl or DMSO-d6 solutions (reported in ppm), using CDCl as the reference standard
3
3
(7.26 ppm) or DMSO-d6 (2.50 ppm). Mass spectral data (ESI) were gathered on Thermofisher LCQ or
QE Mass spectrometry. HPLC (Agilent Prostar 218 or 1200) was employed for purity determination.
3
22
Synthesis of derivatives of the hit compound
3
23
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24
25
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2
A suspension of 1, 3-Thiazol-2-amine (1) (1g, 9.98 mmol), (Boc) O (1.45g, 6.65 mmol) in anhydrous
THF was stirred at room temperature for 16h. Upon completion, the reaction mixture was diluted with
EtOAc and washed with brine. The organic layer was evaporated to dryness under reduced pressure.
The crude product was purified by flash column chromatography on a silica column using
c-hexane/acetone (6:1) as the eluent to get while powder compound 2.
3
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3
3
3
29
30
31
32
33
n-BuLi (1.5 mL 2.5 M in THF, 4 equiv) was added drop wise to compound 2 (1 mmol) in THF (5
mL ) at -78℃ for 1 h and then benzaldehyde derivatives (3) (1.2 mmol) were added at -78℃ with stirring
3-4h. After completion, the reaction system was quenched by adding 1M HCl, extracted with EtOAc
and washed with brine. The organic phase was concentrated, and the residue was purified by flash
column chromatography on a silica column using c-hexane/acetone (0-50%) to get compound 4.
3
3
34
35
A mixture of compound 4 (1 equiv), triethylsilane (8 equiv) and TFA (14 equiv) in DCM (10 mL)
was stirred overnight at room temperature. After removal of the solvent, the residue was dissolved with
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3
DCM and washed with a saturated solution of aqueous NaHCO The organic layer was collected and
purified using cyclohexane/acetone (0-30%) to get compound 5
3
3
3
3
38
39
40
41
To a solution of compound 5 in pyridine (3 mL was added acyl chloride derivatives, the reaction
mixture was stirred at room temperature for 2h. After concentration, the residue was dissolved in
EtOAc, acidified with 1N HCl and washed with brine The organic phase was purified by flash column
chromatography on a silica column using cyclohexane/acetone (15%-25%) to get compound 6
3
42
General Procedures for the Synthesis of 24-41, 49-83 analogues for SAR studies
3
3
3
43
44
45
To a solution of compound 4 (0.08 mmol) in 5 mL DCM was added SOCl (1 mL). The mixture was
2
refluxed overnight at 80 ℃ . Then the reaction mixture was concentrated in vacuo to get compound 7. It
was used for the next reaction directly.
3
3
3
3
46
47
48
49
To a solution of compound 7 in DCM was added compound 8 (0.12 mmol) and K CO (0.24 mmol)
2 3
The resulting mixture was refluxed at 80 ℃ for 3h before it was quenched with 5 mL of brine The
organic layer was collected and purified by flash column chromatography on a silica column using
cyclohexane/acetone (0-50%) to get compound 9.
3
3
3
3
50
51
52
53
A mixture of compound 9 (1 equiv) and TFA (14 equiv) in DCM (10 mL) was stirred overnight at
room temperature. Then the solvent was evaporated. The residue was washed with a saturated solution
3
of aqueous NaHCO mand extracted with DCM The organic layer was collected and purified using
cyclohexane/acetone (0-30%) to get compound 10.
3
3
3
3
54
55
56
57
To a solution of compound 10 in pyridine was added acyl chloride or sulfuryl chloride derivatives
(11), the reaction mixture was stirred at room temperature for 2h. After completion, the reaction system
was quenched by adding 1M HCl, extracted with EtOAc and washed with brine. The residue was
purified by flash column chromatography on a silica column using cyclohexane/acetone (15%-25%) to
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get compound 12
General procedures for the synthesis of 42-48 analogues for SAR studies.
A mixture of compound 10, Cs CO and halogen substituted alkanes in DMF was stirred overnight at
3
3
3
3
60
61
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63
2
3
room temperature .The resulting mixture was diluted with EtOAc and washed with brine. The organic
layer was collected and purified by flash column chromatography on a silica column using
cyclohexane/acetone (0-25%) to get compound 13.
3
64
N-(5-(4-fluorobenzyl) thiazol-2-yl) cyclohexanecarboxamide (14)
1
3
3
3
3
65
66
67
68
H NMR (500 MHz, CDCl ) δ 7.21 – 7.16 (m, 2H), 7.06 (s, 1H), 7.00 (ddd, J = 8.7, 4.5, 2.6 Hz, 2H),
3
4.03 (s, 2H), 2.48 – 2.39 (m, 1H), 2.34 (tt, J = 11.2, 3.6 Hz, 2H), 1.95 (d, J = 13.3 Hz, 6H), 1.84 – 1.73
(m, 6H), 1.72 – 1.62 (m, 3H), 1.52 (dtd, J = 24.2, 12.4, 3.1 Hz, 7H), 1.39 – 1.17 (m, 11H). ESI-MS m/z:
+
319.42 [M+H] .
1
3
3
3
3
69
70
71
C NMR (101 MHz, DMSO) δ 175.79 (s), 163.94 (s), 161.53 (s), 158.95 (s), 138.38 (s), 136.36 (s),
132.69 (s), 131.99 (d, J = 8.0 Hz), 117.43 – 117.22 (m), 117.04 (d, J = 21.2 Hz), 45.09 (s), 32.87 (s),
30.60 (s), 26.97 (d, J = 23.4 Hz), 26.73 – 26.48 (m).
3
72
N-(5-(4-fluorobenzyl) thiazol-2-yl) benzamide (15)
1
3
3
3
73
74
75
H NMR (500 MHz, CDCl ) δ 8.24 (dt, J = 8.7, 1.7 Hz, 1H), 8.17 – 8.13 (m, 2H), 7.66 – 7.62 (m, 1H),
3
7.59 – 7.52 (m, 3H), 7.50 – 7.45 (m, 1H), 7.24 – 7.19 (m, 3H), 7.09 (s, 1H), 7.07 – 7.01 (m, 3H), 4.18
+
(s, 1H), 4.08 (s, 2H), 2.17 (s, 1H), 1.26 (s, 1H). ESI-MS m/z: 313.36 [M+H] .
1
3
3
3
3
76
77
78
C NMR (101 MHz, DMSO) δ 166.85 (s), 163.98 (s), 161.57 (s), 159.66 (s), 138.28 (s), 135.28 (d, J =
203.1 Hz), 134.08 (s), 133.28 (s), 132.07 (d, J = 8.0 Hz), 130.36 (s), 129.91 (s), 117.09 (d, J = 21.2 Hz),
116.94 – 116.70 (m), 32.90 (s).
3
79
N-(5-(4-fluorobenzyl) thiazol-2-yl)-4-methylbenzamide (16)
1
3
3
80
81
H NMR (500 MHz, CDCl ) δ 7.91 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.0 Hz, 3H), 7.22 – 7.17 (m, 2H),
3
+
7.04 – 6.98 (m, 2H), 6.92 (s, 1H), 4.05 (s, 2H), 2.44 (s, 3H). ESI-MS m/z: 327.39 [M+H] .
1
3
3
3
82
83
C NMR (101 MHz, DMSO) δ 166.66 (s), 163.97 (s), 161.57 (s), 159.71 (s), 144.52 (s), 138.29 (s),
136.34 (s), 133.18 (s), 132.06 (d, J = 8.0 Hz), 131.23 (s), 130.91 (s), 129.94 (s), 117.22 (s), 117.08 (d, J
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= 21.2 Hz), 32.89 (s), 22.89 (s).
4-Bromo-N-(5-(4-fluorobenzyl) thiazol-2-yl) benzamide (17)
1
3
3
3
86
87
88
H NMR (500 MHz, CDCl ) δ 8.12 – 8.07 (m, 1H), 7.93 – 7.88 (m, 2H), 7.66 – 7.61 (m, 2H), 7.60 (dd,
3
J = 6.1, 3.6 Hz, 1H), 7.22 – 7.17 (m, 2H), 7.07 – 7.01 (m, 2H), 6.90 (s, 1H), 4.18 (s, 1H), 4.06 (s, 2H),
+
1.26 (s, 1H). ESI-MS m/z: 392.26 [M+H] .
1
3
3
3
89
90
C NMR (101 MHz, DMSO) δ 166.25 (s), 163.98 (s), 161.58 (s), 159.76 (s), 138.22 (s), 133.40 (s),
132.06 (d, J = 10.4 Hz), 128.16 (s), 117.20 (s), 116.99 (s), 32.91 (s).
3
91
2,4-Dichloro-N-(5-(4-fluorobenzyl) thiazol-2-yl) benzamide (18)
1
3
3
3
92
93
94
H NMR (500 MHz, CDCl ) δ 7.66 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.36 (dd, J = 8.3, 2.0
3
Hz, 1H), 7.20 – 7.15 (m, 2H), 7.03 (ddd, J = 8.7, 5.4, 2.1 Hz, 2H), 6.51 (s, 1H), 4.01 (s, 2H). ESI-MS
+
m/z: 382.25 [M+H] .
1
3
3
3
3
95
96
97
C NMR (101 MHz, DMSO) δ 165.66 (s), 164.00 (s), 161.59 (s), 158.56 (s), 138.26 (s), 137.44 (s),
136.50 (s), 135.22 (s), 133.85 (s), 133.40 (s), 132.60 (s), 132.06 (d, J = 8.0 Hz), 131.15 (s), 129.25 (s),
117.23 (s), 117.02 (s), 32.90 (s).
3
98
N-(5-(4-chlorobenzyl) thiazol-2-yl)-2,4-difluorobenzamide (19)
1
3
4
4
99
00
01
H NMR (400 MHz, CDCl ) δ 8.17 (dd, J = 15.3, 8.8 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 8.3
3
Hz, 3H), 7.10 – 7.02 (m, 1H), 6.99 – 6.90 (m, 1H), 4.07 (s, 2H), 1.26 (s, 3H). ESI-MS m/z:365.80
+
[M+H] .
1
3
4
4
4
4
02
03
04
05
C NMR (101 MHz, DMSO) δ 167.87 (d, J = 12.2 Hz), 165.97 (d, J = 2.9 Hz), 165.24 (dd, J = 23.1,
12.6 Hz), 162.54 (d, J = 13.0 Hz), 136.37 (s), 135.81 (d, J = 9.5 Hz), 132.04 (d, J = 7.9 Hz), 117.93 (d, J
= 6.8 Hz), 117.20 (s), 116.99 (s), 113.73 (dd, J = 21.6, 3.3 Hz), 107.41 (s), 107.15 (s), 106.89 (s), 32.88
(s).
1
6
4
06
N , N -bis(5-(4-fluorobenzyl) thiazol-2-yl) adipamide (20)
1
4
4
4
07
08
09
H NMR (500 MHz, DMSO-d6) δ 7.27 (dd, J = 8.6, 5.6 Hz, 2H), 7.19 (s, 1H), 7.13 – 7.08 (m, 2H), 4.04
(s, 6H), 3.10 (dd, J = 3.2, 1.6 Hz, 1H), 2.36 (d, J = 5.6 Hz, 3H), 1.53 (s, 3H). ESI-MS m/z: 527.20
+
[M+H] .
1
3
4
10
C NMR (101 MHz, DMSO) δ 172.71 (s), 163.92 (s), 161.52 (s), 158.77 (s), 138.34 (s), 136.37 (s),
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132.72 (s), 132.01 (d, J = 8.0 Hz), 117.04 (d, J = 21.2 Hz), 36.36 (s), 32.86 (s), 26.11 (s).
N-(5-(4-chlorobenzyl) thiazol-2-yl) thiophene-2-carboxamide (21)
1
4
4
4
13
14
15
H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 8.16 (s, 1H), 7.91 (d, J = 4.9 Hz, 1H), 7.38 (d, J = 8.4
Hz, 2H), 7.34 – 7.28 (m, 3H), 7.24 – 7.19 (m, 1H), 4.11 (s, 2H), 2.08 (s, 1H). ESI-MS m/z:335.84
+
[M+H] .
1
3
4
4
16
17
C NMR (101 MHz, DMSO) δ 141.05 (s), 135.12 (s), 132.96 (s), 132.51 (s), 132.11 (s), 130.33 (s),
33.02 (s).
4
18
N-(5-(4-bromobenzyl) thiazol-2-yl) thiophene-2-carboxamide (22)
1
4
4
4
19
20
21
H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 8.16 (s, 1H), 7.91 (d, J = 4.9 Hz, 1H), 7.51 (d, J = 8.3
Hz, 2H), 7.31 (s, 1H), 7.28 – 7.19 (m, 3H), 4.09 (s, 2H), 2.08 (s, 1H), 1.23 (s, 1H). ESI-MS m/z: 380.29
+
[M+H] .
1
3
4
4
22
23
C NMR (101 MHz, DMSO) δ 141.46 (s), 134.99 (s), 133.24 (s), 132.45 (s), 130.23 (s), 121.39 (s),
119.73 (s), 33.08 (s).
4
24
N-(5-(4-bromobenzyl) thiazol-2-yl)-2-naphthamide (23)
1
4
4
4
25
26
27
H NMR (500 MHz, CDCl3) δ 11.99 (s, 1H), 8.49 (s, 1H), 8.02 (dd, J = 8.6, 1.7 Hz, 1H), 7.94 (dd, J =
8.4, 3.3 Hz, 2H), 7.89 (d, J = 8.1 Hz, 1H), 7.65 (dd, J = 11.1, 4.0 Hz, 1H), 7.57 (t, J = 7.1 Hz, 1H), 7.38
+
(d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.3 Hz, 2H), 6.76 (s, 1H), 3.95 (s, 2H). ESI-MS m/z:424.33 [M+H] .
1
3
4
4
4
28
29
30
C NMR (101 MHz, DMSO) δ 166.96 (s), 159.90 (s), 141.60 (s), 136.48 (s), 133.84 (s), 133.44 –
132.31 (m), 131.08 (t, J = 24.6 Hz), 130.86 – 130.59 (m), 130.41 (s), 130.05 (d, J = 13.8 Hz), 129.51 (s),
128.79 (s), 126.14 (s), 121.40 (s), 33.09 (s).
4
4
31
32
tert-Butyl (5-((4-chlorophenyl) (4-isopropyl-2-methylphenoxy) methyl) thiazol-2-yl) carbamate
(24)
1
4
4
4
4
33
34
35
36
H NMR (500 MHz, Chloroform-d) δ 11.53 (s, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H),
7.07 – 7.01 (m, 2H), 6.73 (d, J = 7.5 Hz, 1H), 6.60 (s, 1H), 6.32 (s, 1H), 2.74 (dt, J = 13.7, 6.9 Hz, 1H),
2.26 (s, 3H), 1.59 (s, 4H), 1.47 (s, 9H), 1.34 (d, J = 2.6 Hz, 1H), 1.26 (s, 1H), 1.15 – 1.10 (m, 6H).
+
ESI-MS m/z: 474.03 [M+H] .
1
3
4
37
C NMR (101 MHz, DMSO) δ 148.90 (s), 138.22 (s), 134.21 (s), 132.19 (s), 130.54 (d, J = 9.3 Hz),
2
6

ACCEPTED MANUSCRIPT
130.02 (s), 129.65 (d, J = 22.3 Hz), 125.82 (s), 120.60 (s), 113.87 (s), 86.52 (s), 83.05 (s), 75.18 (s),
35.06 (s), 29.69 (s), 29.22 (s), 25.61 (s), 17.63 (s).
4
4
38
39
4
40
tert-Butyl (5-((4-bromophenoxy) (4-chlorophenyl) methyl) thiazol-2-yl) carbamate (25)
1
4
4
4
41
42
43
H NMR (400 MHz, CDCl ) δ 11.32 (d, J = 17.4 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.36 (s, 1H), 7.33 (t,
3
J = 1.8 Hz, 2H), 7.31 (d, J = 2.2 Hz, 1H), 6.98 (s, 1H), 6.82 – 6.77 (m, 2H), 6.26 (s, 1H), 1.47 (s, 9H).
+
ESI-MS m/z: 496.82 [M+H] .
1
3
4
4
4
4
44
45
46
47
C NMR (101 MHz, DMSO) δ 162.48 (s), 158.60 (s), 157.76 (s), 154.61 (s), 141.13 (s), 138.61 (s),
134.77 – 134.64 (m), 134.64 – 133.56 (m), 132.74 (s), 130.64 (d, J = 9.3 Hz), 129.97 (d, J = 17.5 Hz),
129.55 (s), 120.34 (d, J = 13.6 Hz), 119.38 (s), 114.85 (s), 111.73 (s), 83.11 (s), 75.91 (s), 29.79 (s),
29.43 (d, J = 47.8 Hz).
4
48
tert-Butyl (5-((3-chloro-4-fluorophenoxy) (4-chlorophenyl) methyl) thiazol-2-yl) carbamate (26)
1
4
4
4
49
50
51
3
H NMR (500 MHz, CDCl ) δ 7.29 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.0 Hz,
1H), 6.84 (d, J = 11.3 Hz, 1H), 6.74 (d, J = 1.0 Hz, 1H), 5.82 (s, 1H), 1.44 (s, 9H). ESI-MS m/z: 470.36
+
[M+H] .
1
3
4
4
4
52
53
54
C NMR (101 MHz, CD CN) δ 169.81 (s), 154.21 (s), 143.28 (s), 138.71 (s), 132.71 (d, J = 12.2 Hz),
3
132.06 (d, J = 15.3 Hz), 130.74 (s), 130.34 (s), 130.06 (s), 129.49 (s), 129.25 (s), 127.49 (s), 121.96 (s),
120.84 (s), 117.31 (s), 62.82 (s), 40.89 (s), 31.25 (s), 15.19 (s).
4
55
tert-Butyl (5-((4-chlorophenyl) (3-cyanophenoxy) methyl) thiazol-2-yl) carbamate (27)
1
4
4
4
56
57
58
H NMR (500 MHz, Chloroform-d) δ 11.40 (s, 1H), 7.39 (q, J = 8.4 Hz, 5H), 7.33 (d, J = 7.6 Hz, 1H),
7.24 (s, 1H), 7.16 (d, J = 7.4 Hz, 2H), 6.99 (s, 1H), 6.32 (s, 1H), 2.17 (d, J = 0.9 Hz, 2H), 1.47 (s, 9H),
+
1.42 (s, 3H), 1.34 (s, 2H), 1.25 (s, 2H). ESI-MS m/z: 442.93 [M+H] .
1
3
4
4
4
59
60
61
C NMR (101 MHz, DMSO) δ 163.45 (s), 158.38 (s), 157.17 (s), 154.64 (s), 153.65 (s), 140.79 (s),
136.05 (s), 132.10 – 129.83 (m), 129.56 (s), 121.33 (s), 118.76 (s), 118.24 (s), 102.84 (s), 65.10 (s),
63.06 (s), 62.29 (s), 29.69 (s), 29.18 (s), 16.30 (d, J = 9.9 Hz), 15.83 (s).
4
62
tert-Butyl (5-((4-chlorophenyl) (4-(2-methoxyethyl) phenoxy) methyl) thiazol-2-yl) carbamate (28)
1
4
4
63
64
H NMR (500 MHz, CDCl ) δ 7.41 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.5 Hz,
3
2H), 6.98 (s, 1H), 6.83 (d, J = 8.6 Hz, 2H), 6.26 (s, 1H), 3.54 (t, J = 7.0 Hz, 2H), 3.33 (s, 3H), 2.79 (t, J
2
7

ACCEPTED MANUSCRIPT
+
4
65
= 7.0 Hz, 2H), 1.47 (s, 9H), 1.43 (s, 5H), 1.34 (s, 2H), 1.26 (s, 3H). ESI-MS m/z: 476 [M+H] .
1
3
4
4
66
67
C NMR (101 MHz, DMSO) δ 162.27 (s), 156.84 (s), 131.58 (s), 130.53 (s), 129.89 (s), 117.82 (s),
83.07 (s), 75.64 (s), 74.67 (s), 59.58 (s), 36.25 (s), 29.68 (s).
4
68
tert-Butyl (5-((3-bromo-5-methylphenoxy) (4-chlorophenyl) methyl) thiazol-2-yl) carbamate (29)
1
4
4
69
70
H NMR (400 MHz, CDCl ) δ 11.51 (s, 1H), 7.39 (s, 2H), 7.36 (s, 2H), 6.99 (s, 1H), 6.93 (s, 1H), 6.87
3
+
(s, 1H), 6.67 (s, 1H), 6.27 (s, 1H), 2.25 (s, 3H), 1.48 (s, 9H). ESI-MS m/z: 510.84 [M+H] .
1
3
4
4
4
4
71
72
73
74
C NMR (101 MHz, DMSO) δ 162.47 (s), 160.22 (s), 159.22 (s), 154.61 (s), 143.08 (d, J = 27.2 Hz),
141.14 (s), 138.55 (s), 134.50 (s), 132.76 (s), 130.60 (s), 129.85 (s), 129.56 (s), 126.79 (s), 124.14 (s),
123.44 (d, J = 13.0 Hz), 118.19 (s), 118.17 – 117.04 (m), 117.04 – 116.77 (m), 83.11 (s), 75.65 (s),
29.67 (s), 29.20 (s), 22.52 (s).
4
75
tert-Butyl (5-((4-(benzyloxy) phenoxy) (4-chlorophenyl) methyl) thiazol-2-yl) carbamate (30)
1
4
4
4
76
77
78
H NMR (500 MHz, Chloroform-d) δ 11.35 (s, 1H), 7.42 (s, 1H), 7.41 (s, 2H), 7.38 (d, J = 3.7 Hz, 2H),
7.37 (s, 1H), 7.35 (s, 1H), 7.32 (d, J = 10.6 Hz, 2H), 6.96 (s, 1H), 6.83 (s, 4H), 6.18 (s, 1H), 4.97 (s,
+
2H), 1.58 (s, 4H), 1.47 (s, 9H), 1.34 (s, 1H), 1.26 (s, 1H). ESI-MS m/z: 524.04 [M+H] .
1
3
4
4
4
79
80
81
C NMR (101 MHz, DMSO) δ 162.32 (s), 154.96 (s), 154.77 (d, J = 32.1 Hz), 152.52 (s), 141.73 (s),
139.07 (s), 138.27 (s), 134.30 (s), 133.45 (s), 130.48 (s), 130.20 (s), 129.94 (s), 129.52 (d, J = 9.6 Hz),
119.35 (s), 117.39 (s), 83.05 (s), 76.42 (s), 71.45 (s), 29.68 (s).
4
4
82
83
tert-Butyl (5-(((6-bromonaphthalen-2-yl) oxy) (4-chlorophenyl) methyl) thiazol-2-yl) carbamate
(31)
1
4
4
4
84
85
86
H NMR (500 MHz, MeOD) δ 7.94 (d, J = 2.1 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.67 (d, J = 8.9 Hz,
1H), 7.36 (dd, J = 9.2, 1.8 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.23 (t, J = 3.7 Hz, 3H), 7.19 (d, J = 9.0
-
Hz, 1H), 7.06 (s, 1H), 6.61 (s, 1H), 2.15 (s, 1H), 1.47 (s, 9H). FTMS-c ESI m/z: 545.01319 [M-H] .
1
3
4
4
4
4
87
88
89
90
3
C NMR (101 MHz, CD CN) δ 162.23 (s), 158.11 (s), 154.33 (s), 154.00 (s), 142.83 (s), 136.97 (s),
133.83 (s), 133.62 (s), 133.46 – 132.72 (m), 132.65 (s), 132.11 (d, J = 9.4 Hz), 130.90 (s), 130.25 (dd, J
= 28.2, 18.3 Hz), 129.59 (s), 127.36 (s), 121.64 (s), 120.84 (s), 117.34 (s), 83.00 (s), 62.84 (s), 40.66 (s),
28.69 (s), 15.20 (s).
4
91
tert-Butyl (5-((4-benzylphenoxy) (4-chlorophenyl) methyl) thiazol-2-yl) carbamate (32)
2
8