N-(7-Cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[ d]thiazol-2-yl)cyclopropanecarboxamide (TAK-632) Analogues as Novel Necroptosis Inhibitors by Targeting Receptor-Interacting Protein Kinase 3 (RIPK3): Synthesis, Structure-Activity Relationships, and in Vivo Efficacy

Article abstract of DOI:10.1021/acs.jmedchem.9b00611

Necroptosis, a form of programmed cell death, plays a critical role in various diseases, including inflammatory, infectious, and degenerative diseases. We previously identified N-(7-cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK-632) (6) as a potent inhibitor of necroptosis by targeting both receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) kinases. Herein, we performed three rounds of structural optimizations of TAK-632 and elucidated structure-activity relationships to generate more potent inhibitors by targeting RIPK3. The analogues with carbamide groups exhibited great antinecroptotic activities, and compound 42 showed >60-fold selectivity for RIPK3 than RIPK1. It blocked necrosome formation by specifically inhibiting the phosphorylation of RIPK3 in necroptotic cells. In a tumor necrosis factor-induced systemic inflammatory response syndrome model, it significantly protected mice from hypothermia and death at a dose of 5 mg/kg, which was much more effective than TAK-632. Moreover, it showed favorable and druglike pharmacokinetic properties in rats with an oral bioavailability of 25.2%. Thus, these RIPK3-targeting small molecules represent promising lead structures for further development.

Full text of DOI:10.1021/acs.jmedchem.9b00611

Subscriber access provided by University Library of Southern Denmark
Article
N-(7-Cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)
benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK-632) Analogues as Novel
Necroptosis Inhibitors by Targeting Receptor-Interacting Protein Kinase 3
(RIPK3): Synthesis, Structure-Activity Relationships and In Vivo Efficacy
Hao Zhang, Lijuan Xu, Xia Qin, Xiaofei Chen, Hui Cong, Longmiao Hu, Long
Chen, Zhenyuan Miao, Wannian Zhang, Zhenyu Cai, and Chunlin Zhuang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00611 • Publication Date (Web): 16 May 2019
Downloaded from http://pubs.acs.org on May 16, 2019
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Journal of Medicinal Chemistry
N-(7-Cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)
benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK-632) Analogues as Novel
Necroptosis Inhibitors by Targeting Receptor-Interacting Protein Kinase 3
(RIPK3): Synthesis, Structure-Activity Relationships and In Vivo Efficacy
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Hao Zhang,1† Lijuan Xu,1† Xia Qin,3† Xiaofei Chen,2 Hui Cong,1,2 Longmiao Hu,3 Long Chen,2 Zhenyuan
Miao,2 Wannian Zhang,1,2* Zhenyu Cai,3, 4* Chunlin Zhuang1,2,5*
1 School of Pharmacy, Ningxia Medical University, 1160 Shengli Street, Yinchuan 750004, China
2 School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
3 National Center for Liver Cancer, Second Military Medical University, 225 Changhai Road, Shanghai 200438,
China
4 Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai 200032, China
5 Department of Chemistry, Fudan University, Shanghai 200433, China
ABSTRACT
Necroptosis, a form of programmed cell death, plays a critical role in various diseases, including inflammatory,
infectious and degenerative diseases. We previously identified TAK-632 (6) as a potent inhibitor of necroptosis
by targeting both RIPK1 and RIPK3 kinases. Herein, we performed three rounds of structural optimizations of
TAK-632 and elucidated structure-activity relationships to generate more potent inhibitors by targeting RIPK3.
The analogues with carbamide groups exhibited great anti-necroptotic activities and compound 42 showed >60-
fold selectivity for RIPK3 than RIPK1. It blocked necrosome formation by specifically inhibiting the
phosphorylation of RIPK3 in necroptotic cells. In a TNF-induced systemic inflammatory response syndrome
model, it significantly protected mice from hypothermia and death at a dose of 5 mg/kg, which was much more
effective than TAK-632. Moreover, it showed favorable and drug-like pharmacokinetic properties in rats with
an oral bioavailability of 25.2%. Thus, these RIPK3-targeting small molecules represent promising lead
structures for further development.
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INTRODUCTION
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Necroptosis is a caspase-independent, programmed necrosis.1 It plays a critical role in various lethal diseases
such as atherosclerosis, myocardial infarction, pancreatitis, ischemic brain damage, kidney damage, and
systemic inflammatory response syndrome (SIRS).2-9 Thus, targeting the pathologic necroptosis pathway has
broad therapeutic relevance. Necroptosis can be induced by the activation of death receptors, such as TNF
receptor 1 (TNFR1), CD95 (FAS), toll-like receptors 3/4 (TLR3/4), and interferons (IFNs).10 Then, downstream
receptor-interacting protein kinase 1 (RIPK1) interacts with RIPK3, initiating the formation of necrosomes and
activating RIPK3 through phosphorylation.11, 12 Subsequently, activated RIPK3 is recruited, and mixed lineage
kinase domain-like protein (MLKL) is phosphorylated.13, 14 Phosphorylated MLKL oligomerizes and then
translocates into the plasma membrane to trigger membrane rupture, initiating necroptosis.15-18 To date, RIPK1,
RIPK3, and MLKL have been recognized as critical therapeutic targets in the machinery of necroptosis.19
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O
O
HN
HN
O
N
O
N
N
H
N
NH
O
N
S
O
N
N
N
H
H
Cl
3
GSK2982772 ( )
1
Nec-1 ( )
2
Nec-1s ( )
F
N
O
O
H
F
O
S
S
N
F
N
S
N
O
NH
O
O S
NH
NO2
S
N
NH
O
F3C
N
H
O
N
F
CN
N
H2N
4
NSA-1 ( )
5
6
TAK-632 ( )
Dabrafenib ( )
Figure 1. Representative known necroptosis inhibitors targeting RIPK1, RIPK3, or MLKL.
The first RIPK1 inhibitor, Nec-1 (1, Figure 1), was identified by the Yuan group in 2005.6 However, its poor
metabolic stability (t1/2 < 5 min) limited further drug discovery efforts based on this compound. A subsequent
analogue, Nec-1s (2), offered improved metabolic stability, but its potency remained moderate.20-22
GlaxoSmithKline has made great advances in the discovery of necroptosis inhibitors. Compound 3
(GSK2982772), an RIPK1-specific inhibitor, has been advanced into phase II trials in patients with active
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ulcerative colitis (UC) (NCT02903966), psoriasis (NCT02776033) or moderate to severe rheumatoid arthritis
(NCT02858492).23-26 A BBB permeable RIPK1 inhibitor, DNL747, with the chemical structure undisclosed,
recently has been advanced into phase I trials in patients with amyotrophic lateral sclerosis (NCT03757351) and
Alzheimer’s disease (NCT03757325).9, 27
1
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3
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5
6
7
8
9
However, since RIPK3 can signal necroptosis independent of RIPK1, Nec-1 and these highly potent RIPK1
inhibitors cannot block RIPK3-independent necroptosis.28 In addition, accumulating evidence suggests that
RIPK1 is also involved in other signaling pathways independent of necroptosis such as apoptosis and
inflammation.29 Thus, identification of small molecules specifically targeting RIPK3 or MLKL is important for
the development of new drug associated with this pathway. A few RIPK3 inhibitors, such as GSK’840,
GSK’843, GSK’872 and GW’39B, have been reported.30 However, these RIPK3 inhibitors have been shown to
induce RIPK1-dependent apoptosis.31, 32 Recently, dabrafenib (5), an FDA-approved drug for patients with
BrafV600E melanoma, was identified as a RIPK3 kinase inhibitor to block necroptosis 33, 34 . However, so far no
RIPK3 inhibitors have been used for clinical trials in necroptosis-associated diseases. In addition, although
NSA-1 (4) is reported to be an MLKL inhibitor by covalent modification of the protein, it shows high
cytotoxicity.13, 35 Thus, identification of novel necroptosis inhibitors with lower cytotoxicity will undoubtedly
has broad clinical relevance.10
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Very recently, we identified the pan-Raf inhibitor TAK-632 (6) as novel necroptosis inhibitor by targeting both
RIPK1 and RIPK3 kinases from an in-house library of fluorine-containing compounds.36 In this study, we
aimed to comprehensively analyze the structure-activity relationship (SAR) of TAK-632 and generate more
potent analogues to inhibit necroptosis by targeting RIPK3.
RESULTS AND DISCUSSION
Chemistry.
Structural variations were introduced at three different positions of TAK-632. First, the amide-linked
substituents at R1 and R2 were changed, but the cyano group (R3) was left intact. Second, the cyano moiety was
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removed, R1 was fixed as a cyclopropyl group, and substituents on the phenylamide (R2) were varied. Third, a
carbamide was introduced at the R2 position. All compounds in this study were prepared following the general
strategies illustrated in Scheme 1 and Scheme 2. Key intermediates m11-m16 were prepared following the
procedure established in the literature.37 Then, intermediates m17-m22 were obtained by deprotection of the
trifluoroacetyl group. Target compounds (7-34) were then obtained using HATU as the coupling reagent.
1
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Scheme 1a
O
O
O
R1
R1
F
F
R1
N
S
N
S
F
N
S
O
NH
NH
R2
b
NH
a
H2N
O
N
H
O
F3C
N
H
O
R3
R3
R3
m17-m22
m11-m16
7-34
aReagents and conditions: (a) NaBH4, EtOH/MeOH (20:1), 0 °C to room temperature, 3 h, 95%-98%; (b)
R2COOH, HATU, dry pyridine, 85 °C, 8 h, 19%-71%. Details of the synthetic route and procedures for
accessing intermediates m11-m16 are presented in the Supporting Information.
Scheme 2a
O
F
N
S
O
R2
O
NH
S
N
H
O
35
a
O
F
N
S
O
NH
F
R2
N
H
O
N
S
NH
b
36-37
H2N
O
O
m21
F
N
S
O
NH
R4
N
H
N
H
O
c
38-42
Reagents and conditions: (a) R2SO2Cl, dry pyridine, 0 °C to room temperature, 12 h, 45%; (b) R2CHO,
NaBH3CN, AcOH, room temperature, 24 h, 26%-32%; (c) R4NCO, DMF, MeCN or DMSO, room temperature,
12 h, 18%-54%.
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Journal of Medicinal Chemistry
The preparation of compounds 35-42 is shown in Scheme 2. Compounds 36 and 37 were obtained by reductive
amination via treating intermediate m21 with the appropriate benzaldehyde derivatives. Similarly, compound 35
was easily prepared by a substitution reaction. Compounds 38-42 were directly synthesized using different
isocyanates. The procedures and characterization data of the key intermediates are presented in the Supporting
1
2
3
4
5
6
7
8
9
1
Information. The final structures were fully characterized by H NMR, 13C NMR, and HRMS. All target
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11
12
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16
17
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22
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compounds were purified to >95%, as determined by high-performance liquid chromatography (HPLC)
analysis, prior to use in subsequent experiments.
Structure optimization, biological evaluation and SAR analysis identify necroptosis inhibitors by
targeting RIPK3.
The anti-necroptosis activity of each compound was measured by a chemiluminescence assay using a model
based on treating human HT-29 cells with TNF-α, Smac mimetic (SM-164) and a caspase inhibitor (z-VAD-
FMK, abbreviated as TSZ).36 The first round of optimizations was performed by modifying the substituents at
R1 and R2 and retaining the cyano group (R3) (Table 1). Initially, the cyclopropyl group of TAK-632 was
replaced by ethyl (7), phenyl (8), nonyl (9) or benzyloxy (10) groups. The ability to inhibit TSZ-induced
necroptosis at a concentration of 20 μM was completely lost; for comparison, parent compound TAK-632
exhibited an EC50 values of 1.44 ± 0.62 μM. Then, the cyclopropyl group was restored, and the substituents on
the phenylamide (R2) were changed. Compounds with para-nitrobenzyl (11), benzyl (12), meta-fluorobenzyl
(13), and meta-trifluoromethylphenyl (14) groups, showed no inhibitory activities against necroptosis at 20 μM,
indicating the importance of meta-trifluoromethyl group. At ortho-position of the benzyl moiety, an additional
fluoro group was introduced to obtain compound 15. It showed a similar activity (EC50 = 2.03±0.86 μM) with
that of TAK-632. Thus, these results suggested a narrow window in which the structure of TAK-632 could be
further modified.
Table 1. First round of optimization of TAK-632.
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R1
NH
F
N
S
O
1
2
R2
N
O
H
CN
3
4
5
6
7
8
Compound
7
R1
R2
EC50, (μM)a
O
9
>20
>20
F3C
F3C
10
11
12
13
14
15
16
17
18
19
20
21
22
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O
8
9
>20
O
F3C
O
10
11
12
13
14
>20
>20
>20
>20
>20
O
F3C
O2N
O
O
O
O
F
F3C
O
O
15
2.03±0.86
1.44±0.62
F3C
F3C
F
6 (TAK-632)
a
Human HT-29 cells were pretreated with DMSO or the test compound and then stimulated with TNF-α (20
ng/mL), Smac mimetic (10 nM), and z-VAD-fmk (20 μM) (TSZ) for 16 h. The inhibition of TSZ-induced
necroptosis in HT-29 cells is presented as the EC50 ± SD. All experiments were repeated independently at least
3 times.
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TAK-632 is a pan-Raf inhibitor;38 however, Raf kinase is not involved in the necroptosis signaling pathway.33
In a previous study, we experimentally showed that TAK-632 protects cells from necroptosis by targeting
RIPK1 (Kd = 480 nM) and RIPK3 (Kd = 105 nM).36 Thus, a structural optimization study was performed to
improve the anti-necroptotic activity and the inhibition of RIPK1 and/or RIPK3. The first potential position for
structural optimization was the cyano group of TAK-632, which may slightly improve Raf kinase inhibition but
it is not critical to the activity.37 To our delight, compound 16, lacking the cyano group, exhibited
approximately 8-fold better anti-necroptotic activity, ~4-fold and ~2-fold better in vitro RIPK1/3 kinase
inhibition (Table 2) and ~2-fold lower inhibition of Raf kinase.36 However, it showed slight cytotoxicity with a
CC50 value of 36.50 µM. Encouraged by this cyano-free analogue of TAK-632, we then used 16 as a lead
compound for further optimization. The meta-trifluoromethyl group on the benzylamine of 16 was critical to the
anti-necroptotic activity. Compound 17, with an ortho-trifluoromethyl group, had an EC50 value of 11.35 µM,
which is a ~66-fold decrease relative to that of 16. Introducing an additional fluorine atom at the ortho- (18) or
para-position (19) resulted in a ~3-fold lower effects on necroptosis and RIPK1/3 kinase activity. However, a
chlorine atom at the ortho-position (20) completely eliminated the activity. An additional trifluoromethyl group
(21) in the meta-position was detrimental to the anti-necroptotic activity (EC50 = 9.7 µM). The presence of two
adjacent methyl groups (22) decreased the anti-necroptotic effect to 1.67±0.83 µM and the RIPK1 kinase
activity to 1.2 µM, while the RIPK3 kinase activity (Kd = 72 nM) was similar to that of 16. However, more
active compounds (18, 19 and 22) showed unfavorable RIPK1 binding, suggesting small substituents on the
phenyl ring are tolerated and can preserve RIPK3 inhibition ability.
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60
Then, we focused on mono-substituted benzylamines to improve the anti-necroptotic potency. The nitro- (23),
methyl- (24), and methoxyl- (25) substituted analogues showed anti-necroptotic potencies that were 2~4-fold
better than that of TAK-632, and they had cell potencies and kinase inhibitory activities similar to those of 16.
Compounds 24 and 25 had better RIPK3 inhibitory activities (Kd = 33 and 55 nM) than 16. Introducing a
trifluoromethoxyl group (26) resulted in the best anti-necroptosis potency seen in this series (EC50 = 60±30
nM). In addition, this compound showed increased the inhibitory activity for RIPK1 while retaining the
inhibitory activity for RIPK3. Replacing the trifluoromethyl group of 16 with a hydroxyl group (27) completely
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removed the activity, and the installation of a cyano group (28) was also detrimental to its inhibitory potency,
resulting in an EC50 value of 1.29±0.47 µM. The RIPK1 inhibitory activity of compound 28 was 8-fold lower
and its RIPK3 inhibitory ability was comparable to that of compound 16. Then, the phenyl ring was replaced by
a pyridinyl group (29), and the resulting compound possessed an EC50 of 0.32±0.05 μM against cell necroptosis.
However, removing the trifluoromethyl group from compound 29 to give 30 was detrimental to the activity,
which decreased to 8.3 μM. Introducing heterocycles (31 and 32) negatively impacted cell necroptosis activity,
and 31 and 32 showed 10~20-fold lower activities than 16. Nevertheless, the selectivity for RIPK3 over RIPK1
was maintained. The binding affinity of compound 31 for RIPK3 was Kd = 81 nM, and its affinity for RIPK1
was 13-fold lower. Indolyl analogue 32 had Kd values of 4.8 and 1.4 µM for RIPK1 and RIPK3, respectively.
Moreover, it is also reported that the linker in lead molecules might be useful to the selectivity.39 Therefore, an
additional methylene (33) unit was added in the linker between the trifluoromethylphenyl and amide moieties,
but this decreased the anti-necroptotic activity approximately 4-fold compared with that of 16; however, the
activity of 33 was 2-fold higher than that of TAK-632. Introducing cyclopropyl (34) or sulfamide (35) moieties
as the linker destroyed the activity. When the carbonyl of the amide was removed to afford benzyl-substituted
compounds 36 and 37, the resulting necroptosis inhibitory activities were undetectable at 20 μM. These results
suggest the importance of the amide linker for further optimization. Moreover, the cytotoxicities of these
analogues that showed anti-necroptotic activities were significantly lower (CC50 > 50 µM).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Second round of optimization of TAK-632.
O
F
N
S
NH
R2
N
H
O
R3
EC50,
Cytotoxicity
(CC50, µM)
RIPK1 (Kd,
nM)b
RIPK3 (Kd,
nM)
Compound
R2
R3
(μM)a
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O
16c
H
H
0.17±0.03
11.35
36.50
N.D.
97
77
1
2
3
4
5
6
7
8
F3C
O
N.D. d
N.D.
17
18
CF3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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36
37
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40
41
42
43
44
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
H
H
H
0.49±0.07
0.40±0.04
>20
>50
>50
510
590
99
F3C
F
F
O
O
130
N.D.
19
20
21
F3C
N.D.
N.D.
F3C
Cl
CF3
O
H
9.7
>50
N.D.
N.D.
F3C
H3C
H3C
O
O
O
H
H
H
H
H
H
1.67±0.83
0.61±0.05
0.38±0.05
0.33±0.05
0.06±0.03
>20
>50
>50
>50
>50
>50
N.D.
1200
770
320
240
49
72
110
33
22
23
24
25
26
27
O2N
H3C
O
55
H3CO
O
120
N.D.
F3CO
O
N.D.
HO
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O
O
1
2
3
4
5
6
7
8
H
H
H
H
1.29±0.47
0.32±0.05
8.3
>50
>50
N.D.
>50
780
220
120
280
N.D.
81
28
29
30
31
NC
F3C
N
9
O
N.D.
1100
10
11
12
13
14
15
16
17
18
19
20
21
22
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
O
O
O
2.32±0.94
N
O
H
3.81±0.92
>50
4800
1400
32
O
F3C
H
H
H
0.65±0.11
>20
>50
>50
510
N.D.
N.D.
790
N.D.
N.D.
33c
34c
O
O
F3C
>20
N.D.
35c
36
F3C
S
O
O
O
H
H
>20
>20
N.D.
N.D.
>50
N.D.
N.D.
480
N.D.
N.D.
105
F3C
37c
O
6 (TAK-632)
CN 1.44±0.62
F3C
a Inhibition of TSZ-induced necroptosis in HT-29 cells.
b
The inhibitory effects of the compounds on RIPK1 and RIPK3 kinases were detected by a KINOMEscan™
assay. The experiments were conducted in duplicate, and the Kd values are presented as the average.
c These compounds have been included in our previous publication.36
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d N.D. = not determined if the anti-necroptotic activity was higher than 5 μM.
1
2
3
4
5
6
7
8
Next, the third round of optimization focused on the substituents on the amide fragment in order to improve the
selectivity. We first introduced a carbamide group into compound 16, which is a potential functional group for
kinase binding with additional hydrogen bond. To our delight, this series of compounds showed great selectivity
for RIPK3 over RIPK1; their selectivities were much higher than those of TAK-632 (selectivity = 4.6) and 16
(no selectivity). Then, different substituents were introduced at the meta-position due to the substantial
influence of groups at this position on the anti-necroptotic activity. The methoxyl (38) and methyl (39)
analogues showed anti-necroptosis EC50 values of 1.37±0.08 and 0.58±0.16 μM, respectively. They possessed
binding affinities for RIPK3 with Kd values of 69 and 93 nM and for RIPK1 with Kd values of 3.6 and 2.2 μM,
respectively. Compound 40, with an ethoxycarbonyl group, showed an RIPK3/1 selectivity of >69.4; however,
its EC50 value was 7.74±1.87 μM, making it less potent than TAK-632. Compound 41, with an acetyl group,
exhibited the best RIPK3 binding affinity (Kd = 53 nM) and the highest RIPK3/1 selectivity (> 94.3). Moreover,
its anti-necroptotic activity was 7-fold higher than that of compound 40. Bromo-substituted compound 42 had a
Kd value of 81 nM toward RIPK3 and no binding for RIPK1 at 5 μM (selectivity > 61.7). 42 also exhibited
good anti-necroptotic activity with an EC50 value of 0.44±0.10 μM, which is comparable to that of 16, and low
cytotoxicity (CC50 > 50 μM).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Structural optimization of compound 16.
O
F
N
S
O
NH
R4
N
N
H
O
H
H
RIPK1
(Kd,
EC50,
Cytotoxicity
(CC50, µM)
RIPK3 (Kd, Selectivity
nM) RIPK3/1
Compound
R4
X
(μM)a
nM)b
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1
2
3
4
5
6
7
8
38
39
40
O
O
O
1.37±0.08
0.58±0.16
7.74±1.87
>50
>50
>50
3600
2200
69
93
72
52.1
H3CO
23.6
H3C
9
O
>5000
>69.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
41
42
O
O
1.0±0.28
>50
>50
>5000
>5000
53
81
>94.3
>61.7
O
0.44±0.10
Br
6 (TAK-
632)
/
/
1.44±0.62
>50
480
105
4.6
a Inhibition of TSZ-induced necroptosis in HT-29 cells.
b
The inhibitory effects of the test compounds on RIPK1 and RIPK3 kinases were detected by a
KINOMEscan™ assay. The experiments were conducted in duplicate, and the Kd values are presented as the
average.
With the above structural and biological information, SARs can be derived for further structural optimization
(Figure 2). (1) A narrow SAR of TAK-632 analogues with cyano groups is observed. (2) The removal of the
cyano group has a positive effect on the anti-necroptotic activity. (3) Small substituents at the para- and ortho-
positions are tolerated. The linker between the trifluoromethylphenyl and amide moieties is of great importance
to the anti-necroptotic activity. (4) The carbamide group has a positive effect on the anti-necroptosis activity
and the selectivity for binding RIPK3 over RIPK1.
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The linker between the trifluoromethylphenyl and amide moieties is
of great importance to the anti-necroptotic activity.
1
2
3
4
5
6
F
N
S
O
The carbamide group has a positive effect on
the anti-necroptotic activity and RIPK3 selective
binding affinity.
NH
O
Cyclopropyl may not be replaced
by alkane and benzene ring.
F3C
N
H
O
CN
7
8
9
Removing the cyano group has a positive
effect on the anti-necroptotic activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Systematic summary of the SAR.
Kinase Inhibitory Profiles of Compound 42 and Molecular Docking Analysis.
In our screen, we found that compound 26 showed the highest anti-necroptotic activity in HT-29 cells by
inhibiting the kinase activities of both RIPK1 and RIPK3. The dual RIPK1 and RIPK3 kinase inhibitors may
have off-target effects since RIPK1 is also involved in other signaling pathways independent of necroptosis
such as apoptosis and inflammation.29 In this study, we aimed to comprehensively analyze the structure-activity
relationship (SAR) of TAK-632 and generate more potent analogues to inhibit necroptosis by targeting RIPK3.
Thus, we selected compound 42, a compound with imporved anti-necroptotic activity by specifically targeting
RIPK3, for further evaluation.
The kinase inhibitory profile against 25 different kinases including Raf family kinases was performed based on
the previous study37 (Table 4). Similar to TAK-632 and compound 16,36 compound 42 targeted additional 6
kinases other than RIPK3 in vitro (% Ctrl < 10). In cells, the anti-necroptotic activity of compound 42 cannot be
explained by these additional targets since vemurafenib (a Raf inhibitor) and vandetanib (a VEGFR2 inhibitor)
with similar target profiles did not inhibit necroptosis.33, 40 Besides, the identified targets of compound 42,
including Raf, VEGFR, PDGFR and SRC, were not involved in necroptotic signaling pathway.33, 41
Table 4. Kinase selectivity of compound 42
Kinase
%Ctrl @ 1000 Kinase
nM
%Ctrl @ 1000 Kinase
nM
%Ctrl @ 1000
nM (Kd, nM)
79
Braf (wt)
3.6
CDK2
93
JNK1
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Braf (V600E)
EGFR
1.8
33
CDK3
96
p38α
23
100
97
5.3
0
1
2
3
4
5
6
7
8
CDK7
81
IKKα
EGFR (L858R)
ERK1
61
GSK3B
MAP3K4
MAPKAPK2
MEK1
100
100
100
100
83
IKKβ
100
22
PDGFRA
PDGFRB
SRC
9
FGFR2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
FGFR3
62
0
Raf1
13
MEK2
MET
14
VEGFR2
2.8
a The inhibitory effects were detected by a KINOMEscan™ assay and reported as %Ctrl (DMSO = 100% Ctrl)
The molecular docking studies were performed in order to understand the structural basis of the protein target
specificity. Two compounds (16 and 42) biochemically targets RIPK1 and/or RIPK3 were selected. Compound
16 could bind well with RIPK1 (Figure 3A) and RIPK3 (Figure 3C). Compound 42 could bind well with RIPK3
(Figure 3D) but not RIPK1 (Figure 3B). A key hydrogen-bonding interaction between the thiazole ring and
protein residue D156 was observed in the predicted model of compound 16-RIPK1 complex. Besides, the
benzothiazole of 16 formed strong π-π stacking interactions with F162. The aniline group was located in the
gatekeeper region (I43, K45 and M92 residues) and the trifluorophenyl group was inserted through the
gatekeeper pointing to the solvent exposed region. However, in the predicted model of 42-RIPK1 complex, the
key hydrogen bond with D156 was missed as the benzothiazole ring was flipped (Figure 3B and Figure 3E).
Although the compound showed a similar conformation as compound 16, the repulsion of carbamide group and
amide group of K45 made 42 slightly moved (Figure 3E). These might disrupt its interaction with RIPK1 and
probably made 42 inactive toward RIPK1. For RIPK3, we generated a homology model of human RIPK3 based
on murine RIPK3 (PDB entry: 4M69), which shares 69% sequence identity and 77% sequence similarity with
each other.42 Both 16 and 42 showed similar binding conformations in a “U” mode with RIPK3 (Figure 3F), and
a key hydrogen bond between the nitrogen of thiazole ring and T94 was observed. The trifluorophenyl or
bromophenyl group was inserted into the region of G31, D142 and N147. Besides, the carbamide formed
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additional hydrogen bonds with S146 and a halogen bond was formed by the bromo group with G31 and N147,
which were probably useful to maintain the “U” conformation and kinase inhibitory activity.
1
2
3
4
5
6
7
8
9
As compound 42 also inhibited the kinase activity of B-raf, the binding mode of compound 42 with B-raf kinase
was predicted (Figure S1). Compound 42 showed similar binding conformation with TAK-632 and kept all the
key interactions of TAK-632 with B-raf, including three hydrogen-bonding interactions with D593, E500 and
C531, π-π stacking interactions with W530 and F594, and π-catin interaction with K482. Additionally, 42
formed a hydrogen-bonding interaction with E500 as the introduction of carbamide group. In future, it would be
of importance to generate more derivatives of TAK-632 to segregate the specificity for RIPK3 from its other
targets. Collectively, the molecular docking analysis further support the experimental results that compound 42
targets RIPK3 but not RIPK1.
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12
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14
15
16
17
18
19
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23
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41
42
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1
2
A
B
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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19
20
21
22
23
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25
26
27
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30
31
32
33
34
35
36
37
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39
40
41
42
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
D
C
E
F
Move
Figure 3. Binding modes of compound 16 (carbon in green) or 42 (carbon in yellow) with RIPK1 (PDB entry:
6HHO) and RIPK3 (A homology model based on PDB 4M66). (A) compound 16 with RIPK1; (B) compound
42 with RIPK1; (C) compound 16 with RIPK3; (D) compound 42 with RIPK3. Superposition of the binding
modes of compound 16 (carbon in green) or 42 (carbon in yellow) with RIPK1 or RIPK3. (E) compounds 16
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and 42 with RIPK1. (F) compounds 16 and 42 with RIPK3. Residues of enzymes are depicted as grey sticks.
1
2
3
Hydrogen bonds and halogen bonds are depicted as green and purple dash lines.
4
5
6
7
8
Compound 42 specifically inhibits necroptosis.
9
As compound 42 has been shown to inhibit the kinase activity of RIPK3 in vitro, we then further evaluate its
anti-necroptotic ability in cells. It also protected against TNF-α, cycloheximide and z-VAD-FMK (TCZ)-
induced necroptosis in human HT-29 cells (Figure 4A), but it did not protected cells from TS- or TC-induced
apoptosis (Figure 4B). In murine L929 cells, we found that compound 42 protected against necroptosis induced
by TNF-α and z-VAD-fmk (TZ) in a dose-dependent manner (Figure 4C). The above results indicate that
compound 42 specifically inhibits the necroptosis pathway.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
B
HT-29
ns
HT-29
***
***
ns
120
100
80
60
40
20
0
120
100
80
60
40
20
0
***
DMSO
TCZ
DMSO
TS
TC
***
0
42 (3 μM)
TAK-632 (3 μM)
0
42 (3 μM) TAK-632 (3 μM)
C
L929
120
100
80
DMSO
TZ
***
60
***
40
**
20
ns
ns
0
0
1
5
1
5
.
1
.
0
0
.
.
0
0
0
0
42 (μM)
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Figure 4. Effects of TAK-632 analogues on necroptosis and apoptosis. (A) The HT-29 cells were pretreated
with DMSO, compound 42 (3 μM) or TAK-632 (3 μM), and then stimulated with TNF-α (20 ng/mL),
cycloheximide (5 μg/mL) and z-VAD-fmk (20 μM) (TCZ) for 12 h. (B) The HT-29 cells were pretreated with
DMSO, compound 42 or TAK-632 (3 μM), and then stimulated with TNF-α (20 ng/mL) plus cycloheximide (5
μg/mL) (TC) or TNF-α (20 ng/mL) plus Smac mimetic (10 nM) (TS) for 20 h. (C) The L929 cells were
pretreated with DMSO or compound 42 at the indicated concentrations and then stimulated with mouse TNF-α
(20 ng/mL) and z-VAD-fmk (20 μM) (TZ) for 4 h. **p < 0.005, ***p < 0.0005, compounds 42 or TAK632
versus no treatment.
1
2
3
4
5
6
7
8
9
10
11
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13
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15
16
17
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33
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35
36
37
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40
41
42
43
44
45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 42 blocks necrosome formation by inhibiting the phosphorylation of RIPK3.
To investigate the inhibitory mechanism of compound 42 against necroptosis, we examined phosphorylation of
RIPK1, RIPK3, and MLKL in TSZ-treated HT-29 cells with or without compound 42. Consistent with the in
vitro kinase assay, compound 42 did not influence the phosphorylation of RIPK1 (Figure 5A). However, it
selectively inhibited the phosphorylation of RIPK3 and MLKL. Since the phosphorylation of RIPK1 and RIPK3
are required for RIPK1-RIPK3 necrosome formation,12 we then explored the formation of necrosome in HT-29
cells after pretreated with compound 42 and we found that compound 42 blocked TSZ-induced necrosome
formation (Figure 5B). Thus, compound 42 blocks necrosome formation by inhibiting TSZ-induced
phosphorylation of RIPK3.
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Figure 5. Compound 42 blocks necrosome formation by inhibiting the phosphorylation of RIPK3. (A) The HT-
29 cells were pretreated with compound 42 (1 µM) and then stimulated with TSZ at the indicated time points.
The cells were lysed and immunoblotted with the indicated antibodies. (B) The HT-29 cells were treated with
DMSO or compound 42 (1 µM) for 4 h. The cell lysates were immunoprecipitated with an anti-RIPK1 antibody
(IP: RIPK1) and analyzed by immunoblotting with the indicated antibodies.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
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39
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41
42
43
44
45
46
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48
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50
51
52
53
54
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58
59
60
Compound 42 protects mice from TNF-induced systemic inflammatory response syndrome (SIRS).
To explore whether compound 42 protects against RIPK-driven inflammation in vivo, we tested this compound
in a TNF-induced SIRS model.5, 36 The compound was intragastrically administered 2 h before intravenous
injection of mTNF-α. We found that compound 42 significantly protected mice from hypothermia and death at
the doses of 5, 25, or 50 mg/kg (Figure 6A and B), which was much more effective than TAK-632 and its
previous reported analogue compound 16 to protect mice from death and hypothermia.36
A
B
mTNFα +Z-VAD-fmk
mTNFα +Z-VAD-fmk
100
80
60
40
20
0
40
35
30
25
20
***
***
vehicle (n=10)
42 (50 mg/kg,n=10)
42 (25 mg/kg,n=10)
42 (5 mg/kg,n=10)
0
10
20
30
40
50
0
10
20
30
40
50
Time after mTNFα (h)
Time after mTNFα (h)
Figure 6. Compound 42 protected mice from SIRS. (A) The body temperature and (B) survival curve of
C57BL/6 female mice (n=10 per group) injected with mTNF-α (400 μg/kg) after pretreatment with different
doses of compound 42 (5, 25 and 50 mg/kg). ***p < 0.0005, treatment group versus vehicle.
Compound 42 has favorable and drug-like pharmacokinetic properties in rats.
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The pharmacokinetic (PK) characteristics of compound 42 in rats was evaluated. The key p.o. and i.v.
administration PK parameters are summarized in Table 5. After a single i.v. administration with 1 mg/kg
compound 42, the half time (t1/2), the clearance rate (CL) and apparent distribution volume (Vss) of 42 are 15.4 h,
2.6 mL/min/kg, and 3.49 L/kg, respectively. When 42 was administrated by oral route at 5 mg/kg, its maximum
plasma concentration (Cmax) is 345 ng/mL. Compound 42 had a favorable oral bioavailability (F) of 25.2%,
suggesting a drug-like property. In addition, no apparent adverse effects were observed in rat.
Table 5. PK Profile of compound 42a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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16
17
18
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26
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40
41
42
43
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60
Parameter
T1/2 (h)
1 mg/kg (i.v.) 5 mg/kg (p.o.)
15.4 ± 1.0
0.083
15.2 ± 2.8
8.67 ± 1.15
345 ± 101
7014 ± 2528
9317 ± 4081
-
Tmax (h)
Cmax (ng/mL)
4603 ± 554
AUC0-t (h*ng/mL) 5563 ± 894
AUC0-∞ (h*ng/mL) 6498 ± 959
CL (mL/min/kg) 2.6 ± 0.38
VSS (L/kg)
F (%)
3.49 ± 0.74
-
-
25.2 ± 9.1
a PK parameters (mean ± SD, n = 3)
CONCLUSION
In summary, novel RIPK3-selective inhibitors were successfully identified by three rounds of structural
optimization starting from TAK-632 as the lead compound. An obvious SAR was derived to guide further
optimizations. Most analogues significantly suppressed necroptotic cell death in both murine and human cells
with low cytotoxicity. Compounds with carbamide groups exhibited promising selectivities for RIPK3 over
RIPK1 kinase. Of the prepared compounds, compound 42 showed more than 60-fold selectivity for RIPK3/1
kinase and exhibited good anti-necroptotic activity with an EC50 value of 0.44±0.10 μM in human HT-29 cells.
Molecular docking analysis explained the target selectivity of compound 42 and provided structural basis for
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further optimization. Consistent with the in vitro kinase assays, compound 42 showed no effect on RIPK1
phosphorylation and selectively inhibited RIPK3 and MLKL phosphorylation in cells. Remarkably, compound
42 significantly protected mice from hypothermia and death at a dose of 5 mg/kg, which was much more
effective than its parent chemical TAK-632. Thus, the carbamide small molecules represent promising leads for
the development of novel anti-necropototic therapeutic agents.
1
2
3
4
5
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13
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16
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42
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EXPERIMENTAL SECTION
Chemistry. All starting materials were obtained from commercial sources and were analytically pure. TAK-632
1
(CAS#1228591-30-7) was purchased from Targetmol with a purity of 98% (Targetmol, USA). The H NMR
and 13C NMR spectra were recorded on Bruker Avance 300 and 600 spectrometers (Bruker Company,
Germany) using TMS as an internal standard and CDCl3 or DMSO-d6 as solvents. Chemical shifts (δ values)
and coupling constants (J values) are given in ppm and hertz (Hz), respectively. The mass spectra were
recorded on an Esquire 3000 LC-MS mass spectrometer. High-resolution mass spectrometry data were acquired
using a Q-TOF micro mass spectrometer. TLC analyses were carried out on silica gel plates GF254 (Qindao
Haiyang Chemical, China). Column chromatography separations were carried out on silica gel 200-300 mesh.
The purities of the compounds were analyzed by HPLC (Agilent 1260) using a C18 column (Elite Hypersil
ODS2, 4.6*250 mm, 5 μm) with 85% methanol/15% water as the mobile phase at a flow rate of 0.6 mL/min,
and all final compounds exhibited purities greater than 95%.
General procedure for the preparation of compounds 7-34.
To a solution of NaBH4 (20 equiv.) in EtOH (20 mL) was added MeOH (1 mL), and the intermediate (m11-
m16) (1 equiv.) was added to this suspension at 0 °C. The mixture was stirred at 0 °C for 1 h and then warmed
to room temperature for 2 h. After the addition was completed, TLC analysis indicated the reaction was
complete. The solvent was removed under reduced pressure, and the residue was re-extracted with ethyl acetate.
The combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure to give a crude intermediate (m17-m22), which was used directly in the next step
without purification. A solution of the corresponding intermediate (m17-m22) (1 equiv.), carboxylic acid
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Page 22 of 45
derivative (1.2 equiv.) and HATU (2 equiv.) in anhydrous pyridine was stirred at 85 °C for 8 h under a nitrogen
atmosphere, at which time TLC analysis indicated the reaction was completed. The solvent was removed under
reduced pressure, and the residue was extracted with ethyl acetate. The organic phase was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product,
which was then purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 1:1). The
product was recrystallized using petroleum ether/ethyl acetate to give the target compounds.
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N-(7-cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)propionamide (7). White solid (28 mg, yield: 64%). 1H NMR (600 MHz, DMSO-d6) δ 12.62 (s, 1H), 10.22
(s, 1H), 7.97 (d, 1H, J = 9.0 Hz), 7.84 (s, 1H), 7.68 (s, 1H), 7.60-7.61 (m, 2H), 7.53-7.56 (m, 1H), 7.37 (t, 1H, J
= 9.6 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.96-6.97 (m, 1H), 3.88 (s, 2H), 2.54 (q, 2H, J = 7.2 Hz), 1.12 (t, 3H, J =
13
7.8 Hz). C NMR (150 MHz, DMSO-d6) δ 173.61, 169.36, 157.64, 155.65, 151.37, 150.64, 149.84, 144.63,
136.93, 135.92, 133.49, 129.27, 128.94, 127.54, 126.56, 125.87, 125.14, 123.38, 116.72, 115.19, 114.39, 113.70,
95.44, 41.98, 28.41, 8.78. HRMS (ESI, positive) m/z calcd for C26H18F4N4O3S [M+H]+ 543.1109, found
543.1105. HPLC analysis: retention time = 12.2 min; peak area, >95% (210, 254 nm).
N-(7-cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)benzamide (8). White solid (23 mg, yield: 53%). 1H NMR (300 MHz, DMSO-d6) δ 12.21 (s, 1H), 10.25 (s,
1H), 8.14 (d, 2H, J = 6.6 Hz), 8.02 (d, 1H, J = 8.7 Hz), 7.87 (s, 1H), 7.57-7.68 (m, 7H), 7.38 (t, 1H, J = 9.6 Hz),
6.99-7.11 (m, 2H), 3.89 (s, 2H). HRMS (ESI, positive) m/z calcd for C30H18F4N4O3S [M+H]+ 591.1109, found
591.1116. HPLC analysis: retention time = 7.9 min; peak area, >95% (210, 254 nm).
N-(7-cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)nonanamide (9). White solid (30 mg, yield: 71%). 1H NMR (300 MHz, DMSO-d6) δ 12.66 (s, 1H), 10.23 (s,
1H), 7.98 (d, 1H, J = 9.0 Hz), 7.82-7.85 (m, 1H), 7.68 (s, 1H), 7.52-7.62 (m, 3H), 7.38 (t, 1H, J = 9.3 Hz), 7.06
(d, 1H, J = 9.0 Hz), 6.94-7.00 (m, 1H), 3.88 (s, 2H), 2.50-2.53 (m, 2H), 1.60-1.62 (m, 2H), 1.25-1.27 (m, 10H),
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0.83-0.87 (m, 3H). C NMR (75 MHz, DMSO-d6) δ 172.92, 169.39, 157.50, 155.72, 151.78, 144.59, 136.94,
135.91, 133.52, 129.30, 128.73, 127.55, 126.65, 125.91, 123.37, 116.80, 116.53,115.25, 114.39, 113.69, 41.98,
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Journal of Medicinal Chemistry
35.03, 31.23, 28.65, 28.52, 28.48, 24.42, 22.07, 13.95. HRMS (ESI, positive) m/z calcd for C32H30F4N4O3S
[M+H]+ 627.2048, found 627.2055. HPLC analysis: retention time = 16.3 min; peak area, >95% (210, 254 nm).
Benzyl(7-cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)carbamate (10). White solid (41 mg, 19%). 1H NMR (300 MHz, DMSO-d6) δ 12.54 (s, 1H), 10.22 (s, 1H),
7.94 (d, 1H, J = 9.3 Hz), 7.83 (s, 1H), 7.57-7.68 (m, 4H), 7.37-7.46 (m, 6H), 6.95-7.07 (m, 2H), 5.30 (s, 2H),
3.88 (s, 2H). HRMS (ESI, positive) m/z calcd for C31H20F4N4O4S [M+H]+ 621.1214, found 621.1219. HPLC
analysis: retention time = 10.1 min; peak area, >95% (210, 254 nm).
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N-(7-cyano-6-(4-fluoro-3-(2-(4-nitrophenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (11)
White solid (39 mg, yield: 93%). 1H NMR (300 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.27 (s, 1H), 8.18 (d, 2H, J
= 8.1 Hz), 7.98(d, 1H, J = 9 Hz), 7.83-7.84 (m, 1H), 7.58 (d, 2H, J = 8.4 Hz), 7.37 (t, 1H, J = 9.6 Hz), 7.07 (d,
1H, J = 9.0 Hz), 6.95-6.98 (m, 1H), 3.94 (s, 2H), 1.98-2.04 (m, 1H), 0.99-1.01 (m, 4H). HRMS (ESI, positive)
m/z calcd for C26H18FN5O5S [M+H]+ 532.1085, found 532.1095. HPLC analysis: retention time = 6.9 min; peak
area, >95% (210, 254 nm).
N-(7-cyano-6-(4-fluoro-3-(2-phenylacetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide
(12). White solid (20 mg, yield: 53%). 1H NMR (300 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.15 (s, 1H), 7.98 (d,
1H, J = 9.0 Hz), 7.84-7.87 (m, 1H), 7.30-7.39 (m, 6H), 7.06 (d, 1H, J = 9.0 Hz), 6.94-6.98 (m, 1H), 1.98-2.03
(m, 1H), 0.99-1.02 (m, 4H). HRMS (ESI, positive) m/z calcd for C26H19FN4O3S [M+H]+ 487.1235, found
487.1235. HPLC analysis: retention time = 7.8 min; peak area, >95% (210, 254 nm).
N-(7-cyano-6-(4-fluoro-3-(2-(3-fluorophenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (13). White solid (22 mg, yield: 55%). 1H NMR (300 MHz, DMSO-d6) δ 12.99
(s, 1H), 10.18 (s, 1H), 7.99 (d, 1H, J = 9.0 Hz), 7.83-7.86 (m, 1H), 7.31-7.40 (m, 2H), 7.04-7.15 (m, 4H), 6.95-
6.99 (m, 1H), 3.78 (s, 2H), 1.98-2.03 (m, 1H), 0.99-1.02 (m, 4H); HRMS (ESI, positive) m/z calcd for
C26H18F2N4O3S [M+H]+ 505.1140, found 505.1148. HPLC analysis: retention time = 7.7 min; peak area, >95%
(210, 254 nm).
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N-(5-((7-cyano-2-(cyclopropanecarboxamido)benzo[d]thiazol-6-yl)oxy)-2-fluorophenyl)-3-
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6
7
8
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(trifluoromethyl)benzamide (14). White solid (20 mg, yield: 50%). 1H NMR (300 MHz, DMSO-d6) δ 12.99 (s,
1H), 10.50 (s, 1H), 8.23-8.28 (m, 2H), 7.97-8.06 (m, 2H), 7.78 (t, 1H, J = 7.8 Hz), 7.50-7.53 (m, 1H), 7.43 (t,
1H, J = 9.6 Hz), 7.11-7.18 (m, 2H), 1.99-2.04 (m, 1H), 0.99-1.02 (m, 4H). HRMS (ESI, positive) m/z calcd for
C26H16F4N4O3S [M+H]+ 541.0952, found 541.0945. HPLC analysis: retention time = 8.1 min; peak area, >95%
(210, 254 nm).
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N-(7-cyano-6-(4-fluoro-3-(2-(2-fluoro-3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (15). White solid (33 mg, yield: 55%). 1H NMR (300 MHz, DMSO-d6) δ 12.95
(s, 1H), 10.28 (s, 1H), 7.98 (d, 1H, J = 9 Hz), 7.82-7.85 (m, 1H), 7.65-7.74 (m, 2H), 7.34-7.41 (m, 2H), 7.08 (d,
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1H, J = 9.0 Hz), 6.95-7.00 (m, 1H), 3.96 (s, 2H), 1.98-2.03 (m, 1H), 0.98-1.01 (m, 4H). F NMR (282 MHz,
DMSO-d6) δ -59.91 (d, J = 12.4 Hz, CF3), -119.86 (Ar-F), -129.81 (Ar-F). HRMS (ESI, positive) m/z calcd for
C27H17F5N4O3S [M+H]+ 573.1014, found 573.1024. HPLC analysis: retention time = 7.1 min; peak area, >95%
(210, 254 nm).
N-(6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (16). White solid (100 mg, yield: 56%). 1H NMR (600 MHz, DMSO-d6) δ 12.61
(s, 1H), 10.11 (s, 1H), 7.67-7.72 (m, 3H), 7.63 (d, 1H, J = 2.4 Hz), 7.59-7.61 (m, 2H), 7.53-7.56 (m, 1H), 7.26-
7.29 (m, 1H), 7.10 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 6.78-6.81 (m, 1H), 3.86 (s, 2H), 1.97-2.01 (m, 1H), 0.93-
0.96 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 172.71, 169.17, 157.56, 153.07, 152.78, 149.08, 144.93,
137.00, 133.45, 132.87, 129.26, 128.94, 127.15, 125.78, 125.13, 123.33, 121.45, 118.02, 116.24, 114.34, 112.99,
111.56, 41.99, 13.75, 8.61. HRMS (ESI, positive) m/z calcd for C26H19F4N3O3S [M+H]+ 530.1156, found
530.1169. HPLC analysis: retention time = 9.0 min; peak area, >95% (210, 254 nm).
N-(6-(4-fluoro-3-(2-(2-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (17). White solid (20 mg, yield: 32%). 1H NMR (600 MHz, DMSO-d6) δ 12.60
(s, 1H), 10.10 (s, 1H), 7.70 (d, 1H, J = 8.4 Hz), 7.67-7.68 (m, 2H), 7.63 (d, 1H, J = 2.4 Hz), 7.61 (t, 1H, J = 7.8
Hz), 7.44-7.49 (m, 2H), 7.28 (t, 1H, J = 9.6 Hz), 7.10 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 6.78-6.80 (m, 1H), 4.00
(s, 2H), 1.97-2.01 (m, 1H), 0.94-0.95 (m, 4H); 13C NMR (150 MHz, DMSO-d6) δ 172.71, 168.76, 157.57,
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Journal of Medicinal Chemistry
153.11, 152.77, 149.02, 144.94, 133.64, 133.39, 132.87, 132.18, 127.46, 127.28, 125.57, 125.33, 123.52, 121.44,
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118.03, 116.21, 114.18, 112.89, 111.58, 13.75, 8.59. F NMR (282 MHz, DMSO-d6) δ -58.65 (CF3), -131.94
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(Ar-F). HRMS (ESI, positive) m/z calcd for C26H19F4N3O3S [M+H]+ 530.1156, found 530.1173. HPLC analysis:
retention time = 8.7 min; peak area, >95% (210, 254 nm).
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N-(6-(4-fluoro-3-(2-(2-fluoro-3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (18). White solid (25 mg, yield: 38%). 1H NMR (600 MHz, DMSO-d6) δ 12.60
(s, 1H), 10.17 (s, 1H), 7.66-7.72 (m, 4H), 7.63 (d, 1H, J = 2.4 Hz), 7.36 (t, 1H, J = 7.8 Hz), 7.27-7.30 (m, 1H),
7.09 (dd, 1H, J1 = 2.4 Hz, J2 = 9.0 Hz), 6.79-6.82 (m, 1H), 3.93 (s, 2H), 1.97-2.01 (m, 1H), 0.93-0.96 (m, 4H).
13C NMR (150 MHz, DMSO-d6) δ 172.70, 168.64, 157.57, 153.09, 152.81, 149.16, 144.95, 138.92, 132.89,
130.35, 129.88, 127.06, 126.06, 124.26, 122.45, 121.44, 120.36, 117.97, 116.24, 114.48, 113.14, 111.51, 41.51,
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13.74, 8.57. F NMR (282 MHz, DMSO-d6) δ -59.91 (d, J = 12.4 Hz, CF3), -119.87 (Ar-F), -131.79 (Ar-F).
HRMS (ESI, positive) m/z calcd for C26H18F5N3O3S [M+H]+ 548.1056, found 548.1074. HPLC analysis:
retention time = 8.8 min; peak area, >95% (210, 254 nm).
N-(6-(4-fluoro-3-(2-(4-fluoro-3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (19). White solid (26 mg, yield: 40%). 1H NMR (600 MHz, DMSO-d6) δ 12.60
(s, 1H), 10.09 (s, 1H), 7.64-7.72 (m, 4H), 7.62 (d, 1H, J = 2.4 Hz), 7.42-7.48 (m, 1H), 7.26-7.29 (m, 1H), 7.09
13
(dd, 1H, J1 = 2.4 Hz, J2 = 9.0 Hz), 6.78-6.81 (m, 1H), 3.84 (s, 2H), 1.98-2.00 (m, 1H), 0.94-0.95 (m, 4H). C
NMR (150 MHz, DMSO-d6) δ 172.72, 169.13, 157.79, 157.58, 153.10, 152.79, 149.08, 144.95, 136.04, 132.88,
132.74, 127.88, 127.15, 123.56, 121.75, 121.46, 118.02, 116.96, 116.20, 114.37, 112.98, 111.56, 41.11, 13.76,
19
8.60. F NMR (282 MHz, DMSO-d6) δ -59.98 (d, J = 12.1 Hz, CF3), -119.23 (Ar-F), -131.83 (Ar-F). HRMS
(ESI, positive) m/z calcd for C26H18F5N3O3S [M+H]+ 548.1062, found 548.1069. HPLC analysis: retention time
= 8.9 min; peak area, >95% (210, 254 nm).
N-(6-(3-(2-(2-chloro-3-(trifluoromethyl)phenyl)acetamido)-4-fluorophenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (20). White solid (21 mg, yield: 31%). 1H NMR (600 MHz, DMSO-d6) δ 12.60
(s, 1H), 10.19 (s, 1H), 7.75 (d, 1H, J = 7.8 Hz), 7.70-7.72 (m, 3H), 7.62 (d, 1H, J = 3 Hz), 7.50 (t, 1H, J = 7.8
Hz), 7.26-7.30 (m, 1H), 7.10 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 6.78-6.81 (m, 1H), 4.06 (s, 2H), 1.97-2.00 (m,
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13
1H), 0.94-0.95 (m, 4H). C NMR (150 MHz, DMSO-d6) δ 172.72, 168.19, 157.59, 153.14, 152.78, 149.00,
144.96, 136.73, 136.45, 132.89, 131.31, 127.26, 126.93, 126.43, 123.91, 122.10, 121.45, 118.03, 116.24, 114.23,
112.89, 111.59, 40.49, 13.76, 8.60. HRMS (ESI, positive) m/z calcd for C26H18ClF4N3O3S [M+H]+ 564.0766,
found 564.0767. HPLC analysis: retention time = 9.3 min; peak area, >95% (210, 254 nm).
N-(6-(3-(2-(3,5-bis(trifluoromethyl)phenyl)acetamido)-4-fluorophenoxy)benzo[d]thiazol-2-
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yl)cyclopropanecarboxamide (21). White solid (32 mg, yield: 46%). 1H NMR (600 MHz, DMSO-d6) δ 12.59
(s, 1H), 10.15 (s, 1H), 8.01 (s, 2H), 7.98 (s, 1H), 7.71 (d, 1H, J = 9.0 Hz), 7.65-7.67 (m, 1H), 7.62 (d, 1H, J =
2.4 Hz), 7.27-7.30 (m, 1H), 7.09 (dd, 1H, J1 = 3.0 Hz, J2 = 9.0 Hz), 6.79-6.82 (m, 1H), 3.31 (s, 2H), 1.97-2.00
(m, 1H), 0.93-0.96 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 172.71, 168.12, 157.58, 157.39, 153.11, 152.79,
149.54, 144.95, 136.93, 132.89, 127.12, 125.78, 124.72, 124.55, 123.64, 121.84, 121.44, 118.01, 116.24, 114.37,
19
112.96, 111.56, 35.46, 13.75, 8.59. F NMR (282 MHz, DMSO-d6) δ -61.24 (CF3), -131.72 (Ar-F). HRMS
(ESI, positive) m/z calcd for C27H18F7N3O3S [M+H]+ 598.1024, found 598.1032. HPLC analysis: retention time
= 11.4 min; peak area, >95% (210, 254 nm).
N-(6-(3-(2-(3,4-dimethylphenyl)acetamido)-4-fluorophenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (22).
White solid (26 mg, yield: 45%). 1H NMR (300 MHz, DMSO-d6) δ 12.63 (s, 1H), 9.96 (s, 1H), 7.70 (d, 1H, J =
8.7 Hz), 7.62 (d, 1H, J = 2.1 Hz), 7.26 (t, 1H, J = 9.6 Hz), 6.99-7.11 (m, 4H), 6.76-6.80 (m, 1H), 3.63 (s, 2H),
2.15-2.16 (m, 6H), 1.98-2.02 (m, 1H), 0.94-0.95 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 172.75, 170.02,
157.60, 153.10, 152.82, 148.99, 144.96, 135.90, 134.28, 132.92, 132.91, 130.27, 129.36, 127.41, 126.47, 121.48,
118.06, 116.18, 114.08, 112.86, 111.59, 42.26, 19.35, 18.95, 13.80, 8.67. HRMS (ESI, positive) m/z calcd for
C27H24FN3O3S [M+H]+ 490.1595, found 490.1597. HPLC analysis: retention time = 7.2 min; peak area, >95%
(210, 254 nm).
N-(6-(4-fluoro-3-(2-(3-nitrophenyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide
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(23). White solid (22 mg, yield: 33%). H NMR (600 MHz, DMSO-d6) δ 12.59 (s, 1H), 10.14 (s, 1H), 8.20 (s,
1H), 8.09-8.11 (m, 1H), 7.75 (d, 1H, J = 7.8 Hz), 7.70-7.71 (m, 2H), 7.59-7.62 (m, 2H), 7.28 (t, 1H, J = 10.2
Hz), 7.09 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 6.78-6.81 (m, 1H), 3.92 (s, 2H), 1.97-2.00 (m, 1H), 0.94-0.95 (m,
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Journal of Medicinal Chemistry
4H). 13C NMR (150 MHz, DMSO-d6) δ 172.71, 168.93, 157.57, 153.11, 152.76, 149.02, 147.64, 144.94,
137.76, 136.17, 132.87, 129.65, 127.13, 123.97, 121.63, 121.45, 118.03, 116.23, 114.32, 112.88, 111.57, 41.75,
13.75, 8.60. HRMS (ESI, positive) m/z calcd for C25H19FN4O5S [M+H]+ 507.1127, found 507.1151. HPLC
analysis: retention time = 8.2 min; peak area, >95% (210, 254 nm).
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N-(6-(4-fluoro-3-(2-(m-tolyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide
(24).
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White solid (21 mg, yield: 38%). 1H NMR (600 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.98 (s, 1H), 7.71 (d, 1H, J =
8.4 Hz), 7.62 (d, 1H, J = 2.4 Hz), 7.24-7.28 (m, 1H), 7.18 (t, 1H, J = 7.8 Hz), 7.08-7.11 (m, 3H), 7.02 (d, 1H, J
= 7.2 Hz), 6.77-6.79 (m, 1H), 3.67 (s, 2H), 2.26 (s, 3H), 1.98-2.00 (m, 1H), 0.94-0.95 (m, 4H). 13C NMR (150
MHz, DMSO-d6) δ 172.70, 169.78, 157.56, 153.05, 152.79, 148.99, 144.92, 137.28, 135.53, 132.87, 129.75,
128.14, 127.33, 127.14, 126.17, 121.44, 118.02, 116.16, 114.16, 112.89, 111.55, 42.53, 20.92, 13.75, 8.60.
HRMS (ESI, positive) m/z calcd for C26H22FN3O3S [M+H]+ 476.1439, found 476.1449. HPLC analysis:
retention time = 9.3 min; peak area, >95% (210, 254 nm).
N-(6-(4-fluoro-3-(2-(3-methoxyphenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (25). White solid (30 mg, yield: 52%). 1H NMR (300 MHz, DMSO-d6) δ 12.62
(s, 1H), 10.01 (s, 1H), 7.68-7.73 (m, 2H), 7.63 (d, 1H, J = 2.4 Hz), 7.18-7.30 (m, 2H), 7.10 (dd, 1H, J1 = 2.4 Hz,
J2 = 8.7 Hz), 6.85-6.88 (m, 2H), 6.76-6.81 (m, 2H), 3.72 (s, 3H), 3.68 (s, 2H), 1.97-2.01 (m, 1H), 0.94-0.96 (m,
4H). 13C NMR (75 MHz, DMSO-d6) δ 172.75, 169.64, 159.18, 157.59, 153.07, 152.80, 149.04, 144.95, 137.12,
132.89, 129.30, 127.32, 121.49, 121.38, 118.06, 116.23, 114.90, 114.21, 112.92, 111.98, 111.60, 54.94, 42.66,
13.79, 8.66. HRMS (ESI, positive) m/z calcd for C26H22FN3O4S [M+H]+ 492.1388, found 492.1399. HPLC
analysis: retention time = 8.4 min; peak area, >95% (210, 254 nm).
N-(6-(4-fluoro-3-(2-(3-(trifluoromethoxy)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (26). White solid (32 mg, yield: 49%). 1H NMR (300 MHz, DMSO-d6) δ 12.63
(s, 1H), 10.11 (s, 1H), 7.63-7.73 (m, 3H), 7.44 (t, 1H, J = 7.8 Hz), 7.22-7.34 (m, 4H), 7.10 (d, 1H, J = 8.4 Hz),
6.78-6.81 (m, 1H), 3.80 (s, 1H), 1.99-2.01 (m, 1H), 0.93-0.95 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ
172.75, 169.16, 157.61, 153.13, 152.79, 149.09, 148.30, 144.97, 138.32, 132.91, 130.12, 128.44, 127.19,
121.66, 121.49, 119.12, 118.39, 118.07, 116.27, 114.40, 112.99, 111.62, 42.01, 13.79, 8.66. 19F NMR (282
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Journal of Medicinal Chemistry
Page 28 of 45
MHz, DMSO-d6) δ -56.66 (OCF3), -131.82 (Ar-F). HRMS (ESI, positive) m/z calcd for C26H19F4N3O4S
[M+H]+ 546.1105, found 546.1121. HPLC analysis: retention time = 9.3 min; peak area, >95% (210, 254 nm).
N-(6-(4-fluoro-3-(2-(3-hydroxyphenyl)acetamido)phenoxy)benzo[d]thiazol-2-
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yl)cyclopropanecarboxamide (27). White solid (26 mg, yield: 46%). 1H NMR (600 MHz, DMSO-d6) δ 12.61
(s, 1H), 9.97 (s, 1H), 9.31 (s, 1H), 7.71-7.73 (m, 2H), 7.63 (d, 1H, J = 2.4 Hz), 7.24-7.28 (m, 1H), 7.06-7.11 (m,
2H), 6.76-6.79 (m, 1H), 6.71-6.73 (m, 2H), 6.61-6.63 (m, 1H), 3.62 (s, 2H) 1.98-2.00 (m, 1H), 0.94-0.96 (m,
4H). 13C NMR (150 MHz, DMSO-d6) δ 172.73, 169.76, 157.58, 157.25, 153.07, 152.81, 148.98, 144.95, 136.91,
132.89, 129.19, 127.36, 121.47, 119.75, 118.05, 116.18, 116.00, 114.12, 113.56, 112.85, 111.58, 42.65, 13.78,
8.63. HRMS (ESI, positive) m/z calcd for C25H20FN3O4S [M+H]+ 478.1231, found 478.1244. HPLC analysis:
retention time = 7.2 min; peak area, >95% (210, 254 nm).
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N-(6-(3-(2-(3-cyanophenyl)acetamido)-4-fluorophenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide
(28). White solid (32 mg, yield: 55%). 1H NMR (300 MHz, DMSO-d6) δ 12.62 (s, 1H), 10.12 (s, 1H), 7.70-7.74
(m, 4H), 7.62-7.64 (m, 2H), 7.51 (t, 1H, J = 7.8 Hz), 7.28 (t, 1H, J = 9.3 Hz), 7.09 (dd, 1H, J1 = 2.4 Hz, J2 = 8.7
13
Hz), 6.77-6.82 (m, 1H), 3.83 (s, 2H), 1.98-2.01 (m, 1H), 0.93-0.95 (m, 4H). C NMR (75 MHz, DMSO-d6) δ
172.77, 169.04, 157.61, 153.12, 152.80, 149.06, 144.97, 137.24, 134.42, 132.92, 130.48, 129.49, 127.19, 121.50,
118.78, 118.08, 116.27, 114.36, 112.96, 111.62, 111.20, 41.76, 13.81, 8.68. HRMS (ESI, positive) m/z calcd for
C26H19FN4O3S [M+H]+ 487.1352, found 487.1244. HPLC analysis: retention time = 7.1 min; peak area, >95%
(210, 254 nm).
N-(6-(4-fluoro-3-(2-(6-(trifluoromethyl)pyridin-2-yl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (29). White solid (30 mg, yield: 41%). 1H NMR (300 MHz, DMSO-d6) δ 12.62
(s, 1H), 10.21 (s, 1H), 8.04 (t, 1H, J = 7.8 Hz), 7.63-7.78 (m, 5H), 7.29 (t, 1H, J = 9.6 Hz), 7.10 (dd, 1H, J1 =
2.1 Hz, J2 = 8.7 Hz), 6.79-6.83 (m, 1H), 4.08 (s, 2H), 1.98-2.01 (m, 1H), 0.93-0.95 (m, 4H). 13C NMR (75 MHz,
DMSO-d6) δ 172.75, 168.21, 157.62, 156.77, 153.16, 152.78, 148.99, 145.86, 144.98, 138.75, 132.91, 128.05,
127.14, 121.58, 121.49, 118.90, 118.09, 116.28, 114.23, 112.84, 111.64, 44.68, 13.79, 8.66. 19F NMR (282
MHz, DMSO-d6) δ -66.37 (CF3), -131.96 (Ar-F). HRMS (ESI, positive) m/z calcd for C25H18F4N4O3S [M+H]+
531.1109, found 531.1120. HPLC analysis: retention time = 8.5 min; peak area, >95% (210, 254 nm).
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Journal of Medicinal Chemistry
N-(6-(4-fluoro-3-(2-(pyridin-2-yl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide (30).
White solid (23 mg, yield: 42%). 1H NMR (600 MHz, DMSO-d6) δ 12.60 (s, 1H), 10.21 (s, 1H), 8.49 (d, 1H, J
= 3.6 Hz), 7.70-7.76 (m, 3H), 7.63 (d, 1H, J = 2.4 Hz), 7.36 (d, 1H, J = 7.8 Hz), 7.25-7.29 (m, 2H), 7.10 (dd, 1H,
J1 = 2.4 Hz, J2 = 9.0 Hz), 6.77-6.80 (m, 1H), 3.93 (s, 2H), 1.97-2.01 (m, 1H), 0.94-0.95 (m, 4H). 13C NMR (150
MHz, DMSO-d6) δ 172.71, 168.67, 157.56, 155.65, 153.09, 152.79, 148.88, 148.83, 144.93, 136.66, 132.87,
127.31, 124.03, 121.93, 121.46, 118.04, 116.15, 114.06, 112.60, 111.57, 45.05, 13.75, 8.61. HRMS (ESI,
positive) m/z calcd for C24H19FN4O3S [M+H]+ 463.1325, found 463.1248. HPLC analysis: retention time = 7.7
min; peak area, >95% (210, 254 nm).
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N-(6-(3-(2-(benzo[d][1,3]dioxol-5-yl)acetamido)-4-fluorophenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (31). White solid (30 mg, yield: 50%). 1H NMR (300 MHz, DMSO-d6) δ 12.62
(s, 1H), 9.96 (s, 1H), 7.70-7.73 (m, 2H), 7.62 (d, 1H, J = 2.1 Hz), 7.26 (t, 1H, J = 10.2 Hz), 7.10 (dd, 1H, J1 =
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2.4 Hz, J2 = 8.7 Hz), 6.74-6.86 (m, 4H), 5.96 (s, 2H), 3.62 (s, 2H), 1.98-2.01 (m, 1H), 0.93-0.95 (m, 4H). C
NMR (75 MHz, DMSO-d6) δ 172.75, 169.90, 157.59, 153.10, 152.81, 148.99, 147.12, 145.94, 144.95, 132.89,
129.26, 127.35, 122.21, 121.49, 118.07, 116.21, 114.21, 112.85, 111.61, 109.59, 108.06, 100.79, 42.18, 13.79,
8.66. HRMS (ESI, positive) m/z calcd for C26H20FN3O5S [M+H]+ 506.1180, found 506.1189. HPLC analysis:
retention time = 8.0 min; peak area, >95% (210, 254 nm).
N-(6-(4-fluoro-3-(2-(1-methyl-1H-indol-3-yl)acetamido)phenoxy)benzo[d]thiazol-2-
yl)cyclopropanecarboxamide (32). White solid (25 mg,, yield: 41%). 1H NMR (600 MHz, DMSO-d6) δ 12.59
(s, 1H), 9.90 (s, 1H), 7.69-7.72 (m, 2H), 7.62 (d, 1H, J = 2.4 Hz), 7.58 (d, 1H, J = 7.8 Hz), 7.37 (d, 1H, J = 8.4
Hz), 7.24-7.27 (m, 1H), 7.21 (s, 1H), 7.11-7.14 (m, 1H), 7.09 (dd, 1H, J1 = 3.0 Hz, J2 = 9.0 Hz), 6.99-7.01 (m,
1H), 6.75-6.78 (m, 1H), 3.80 (s, 2H), 3.74 (s, 3H), 1.98-2.01 (m, 1H), 0.93-0.96 (m, 4H). 13C NMR (150 MHz,
DMSO-d6) δ 172.70, 170.17, 157.54, 153.04, 152.79, 148.97, 144.90, 136.47, 132.85, 128.24, 127.47, 121.44,
121.07, 118.80, 118.47, 118.01, 116.12, 114.00, 112.82, 111.53, 109.50, 107.45, 32.87, 32.22, 13.74, 8.59.
HRMS (ESI, positive) m/z calcd for C28H23FN4O3S [M+H]+ 515.1548, found 515.1560. HPLC analysis:
retention time = 7.7 min; peak area, >95% (210, 254 nm).
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