Ring closure strategy leads to potent RIPK3 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113327

Necroptosis is a form of regulated necrotic cell death that is independent of caspases. Receptor-interacting protein kinase 3 (RIPK3) has been identified as a key regulator for necroptosis, and has been proposed as a potential therapeutic target for the treatment of diseases associated with necroptosis. In this report, we describe the design, synthesis, and evaluation of a series of novel RIPK3 inhibitors. The lead compound 38 exhibited potent activity (EC₅₀ = 0.42 μM) in blocking TNFα, Smac mimetic and z-VAD (TSZ) induced cell death in HT-29 cells. Mechanistic studies showed that compound 38 bound to RIPK3 with high affinity (K_d = 7.1 nM), and inhibited RIPK3 kinase activity in a ADP-Glo functional assay. In addition, compound 38 displayed good selectivity over another necroptosis regulator RIPK1 (K_d = 6000 nM). Furthermore, compound 38 demonstrated excellent in vitro safety profiles with minimal inhibition of CYP isozymes and hERG potassium channel. Lastly, compound 38 efficiently blocked hypothermia and death in mice in the TNFα-induced systemic inflammatory response syndrome model.

Full text of DOI:10.1016/j.ejmech.2021.113327

European Journal of Medicinal Chemistry 217 (2021) 113327
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Ring closure strategy leads to potent RIPK3 inhibitors
,
,
, c
Shuwei Wu a 1, Chen Xu a 1, Kaijiang Xia a, Yu Lin a, Sheng Tian a, Haikuo Ma a, Yuting Ji b
,
Fang Zhu b c, Sudan He b **, Xiaohu Zhang a
,
,
c
,
, *
a Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, 215123, PR China
b Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medial College, Beijing, Suzhou
Institute of Systems Medicine, Suzhou, 215123, Jiangsu, PR China
c Cyrus Tang Hematology Center, Jiangsu Institute of Hematology and Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215123,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Necroptosis is a form of regulated necrotic cell death that is independent of caspases. Receptor-
interacting protein kinase 3 (RIPK3) has been identified as a key regulator for necroptosis, and has
been proposed as a potential therapeutic target for the treatment of diseases associated with necroptosis.
In this report, we describe the design, synthesis, and evaluation of a series of novel RIPK3 inhibitors. The
Received 6 November 2020
Received in revised form
10 February 2021
Accepted 19 February 2021
Available online 9 March 2021
lead compound 38 exhibited potent activity (EC50 ¼ 0.42
mM) in blocking TNFa, Smac mimetic and z-VAD
(TSZ) induced cell death in HT-29 cells. Mechanistic studies showed that compound 38 bound to RIPK3
with high affinity (Kd ¼ 7.1 nM), and inhibited RIPK3 kinase activity in a ADP-Glo functional assay. In
addition, compound 38 displayed good selectivity over another necroptosis regulator RIPK1
(Kd ¼ 6000 nM). Furthermore, compound 38 demonstrated excellent in vitro safety profiles with min-
imal inhibition of CYP isozymes and hERG potassium channel. Lastly, compound 38 efficiently blocked
Keywords:
Cell death
Inflammation
Necroptosis
RIPK1
hypothermia and death in mice in the TNF
a-induced systemic inflammatory response syndrome model.
RIPK3
Kinase
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
the necrosome [3e5]. The necrosome serves as a molecular plat-
form to active RIPK3, resulting in recruitment and phosphorylation
of pseudokinase mixed lineage kinase domain-like protein (MLKL)
[6e9]. The activated MLKL subsequently executes necroptosis by
oligomerization and translocation to the plasma membrane, lead-
ing to the membrane rupture and the release of cellular contents
[10e13]. Necroptosis is a necrotic, pro-inflammatory form of cell
death that is involved in numerous human diseases including in-
flammatory diseases, degenerative diseases, ischemic infarction,
and cancer [14e21]. As such, the key regulators of necroptosis,
RIPK1, RIPK3, and MLKL, have been proposed as potential thera-
peutic targets for the treatment of the aforementioned diseases
[22,23].
Necroptosis is a form of regulated, non-apoptotic cell death [1,2].
Necroptosis is tightly regulated by receptor-interacting protein ki-
nase 1 (RIPK1) and RIPK3. Upon stimulation of necroptotic signals
to death receptors, RIPK1 recruits RIPK3 through their RIP homo-
typic interaction motif (RHIM) domains to initiate the formation of
Abbreviations: CYP, cytochrome P450; DCM, dichloromethane; DMF, N, N-
dimethylformamide; DMPK, Drug Metabolism and Pharmacokinetics; DMSO,
dimethyl sulfoxide; EA, ethyl acetate; HPLC, high performance liquid chromatog-
raphy; hERG, human ether-a-go-go-related gene; IPA, isopropanol; LAH, lithium
tetrahydroaluminate; m-CPBA, 3-chloroperbenzoic acid; NBS, N-bromosuccini-
RIPK1 is the master regulator downstream of the death re-
ceptors, which is involved in determination of cell fate such as
survival/inflammation or death (apoptosis and necroptosis) [24,25].
The multi-functional nature of RIPK1 complicates the interpreta-
tion of its role in necroptosis [26]. MLKL, on the other hand, is the
executioner of necroptosis and represents an ideal target for nec-
roptosis. However, as a pseudokinase, the enzyme activity (ATP
binding and phosphorylation) and overall function (oligomeriza-
tion and cell puncture) of MLKL is less tractable compares to RIPK3
mide; rt, room temperature; THF, tetrahydrofuran; TNF
TsCl, p-Toluenesulfonyl chloride; Xphos, 2-(Dicyclohexylphosphino)-2,4,6-
a, Tumor necrosis factor a;
Triisopropylbiphenyl;
xanthene.
XantPhos,
9,9-Dimethyl-4,5-bis(diphenylphosphino)
* Corresponding author.
** Corresponding author. Center of Systems Medicine, Institute of Basic Medical
Sciences, Chinese Academy of Medical Sciences & Peking Union Medial College,
Beijing, Suzhou Institute of Systems Medicine, Suzhou, 215123, Jiangsu, PR China.
E-mail addresses: hesudan2018@163.com (S. He), xiaohuzhang@suda.edu.cn
(X. Zhang).
1
these authors contributed equally to this paper.
https://doi.org/10.1016/j.ejmech.2021.113327
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
[8,27,28]. Moreover, it has been reported that necroptosis can
proceed independent of RIPK1, leading to the assumption that
RIPK3 may protect cell from a broader range of necroptotic pa-
thologies than RIPK1 [29]. A number of RIPK3 inhibitors have been
reported including the FDA approved drugs dabrafenib and pona-
tinib [30e32](Fig.1). Dabrafenib and ponatinib are potent BRAF and
BCR-ABL inhibitors, respectively. Their inhibition of RIPK3 was
discovered as off-target activity, making them difficult starting
points to selectively optimize RIPK3 activity. Very recently, two
type-II RIPK3 inhibitors were reported [33,34](Fig. 1). Both com-
pounds may need improvement for kinome-wide selectivity. For
example, compound 2 is a potent RIPK3 inhibitor which is highly
selective for RIPK3 over RIPK1 and RIPK2, but broader kinome scan
showed compound 2 inhibited roughly 40 kinases out of 246 tested
the 6-(isopropylsulfonyl)quinoline group of GSK0872 was solvent-
exposed and in close range of residues including Val28, Gly31
and Ser147 in the binding site of RIPK3. Based on these observa-
tions, we hypothesized that the structural optimization on the
quinoline group of GSK0872 for engagement of favorable in-
teractions with these nearby residues may improve the inhibitory
activity of rational-designed compounds. For the above-mentioned
reasons, we formulated chemical modification/optimization plan as
outlined in Fig. 3.
3. Chemistry
The synthesis of compounds 16, 18 was displayed in Scheme 1.
O-chloroaryl aldehyde was reacted with methyl 2-mercaptoacetate
under basic conditions to provide 7. Standard saponification of 7
resulted in carboxylic acid 8, which was decarboxylated with cop-
per powder to give 9. Bromination of 9 with NBS led to 10. In-
termediates 9 and 10 were then oxidized by m-CPBA to afford the
corresponding sulfones 11a-11b. Reduction with iron powder led to
the aromatic amines 12a-12b, which were reacted with 5-
with an IC50 < 1 m
M [34]. The seminal work on GSK0872 (Fig. 1)
established RIPK3 as a drug target. GSK0872 was reported to be a
potent and selective RIPK3 inhibitor which may induce apoptosis at
high concentrations [35].
In pursuit of novel RIPK3 inhibitor, we decided to focus on the
GSK0872 template since it was comprehensively characterized and
showed good kinome wide selectivity. Our primary goal is to
improve its moderate cellular potency. Ultimately, we want to un-
derstand the mechanism of RIPK3 inhibition induced apoptosis,
and develop compounds with low cytotoxicity.
(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione
fol-
lowed by cyclization in inert solvent at high temperature to provide
the tricyclic pyridine analogues 14a-14b. Treatment with POCl3
followed by amino-benzothiazole provided advanced in-
termediates 15a-15b. Final compound 16 was achieved by hydro-
genation of 15a. Final compound 18 was achieved by reacting 15b
with methanol followed by HCl hydrolysis. Compound 18 exists in
tautomers as shown by 1H NMR (experimental section).
The synthetic approach of compounds 26e33 was shown in
Scheme 2. Commercially available 19 was treated with sodium
ethyl sulfonate followed by cyclization reaction in the presence of
NaH, and then reacted with respective halogenated hydrocarbons
to yield intermediates 20a-20c. Reduction with iron powder
resulted in the aniline analogues 21a-21c. Bromination of com-
pounds 21a-21c with Oxone and NaBr afforded intermediates 22a-
22c, which were reacted with 5-(methoxymethylene)-2,2-
dimethyl-1,3-dioxane-4,6-dione followed by cyclization at high
temperature to yield tricyclic analogues 24a-24c. Debromination
was carried out in the presence of H2, Pd/C to yield intermediates
25a-25c, which were reacted with POCl3 followed by substitution
reactions with respective aromatic amines to form compounds
2. Design
In order to obtain promising RIPK3 inhibitors, structure-based
drug design strategy was employed to guide our structural modi-
fication efforts. Briefly, GSK0872 was docked into the binding site of
RIPK3 crystal complex (PDB ID: 4M69 [36]) from RCSB Protein Data
Bank [37] by using Glide extra precision (XP) scoring function of
€
Schrodinger 9.0 software package [38]. More detailed information
for molecular docking procedure can be seen in our previous
studies [39,40]. The binding conformation of GSK0872 predicted by
Glide docking and key interaction patterns between GSK0872 and
RIPK3 were depicted and analyzed. The computational results
indicated that GSK0872 located in the ATP-binding site was a typical
type I inhibitor, targeting the active DFG-in conformation of RIPK3
(Fig. 2a). Besides, GSK0872 had tight interactions with two key
residues (Val36 and Asp161, Fig. 2b). In addition, we also found that
Fig. 1. Representatives of RIPK3 inhibitors reported in the literature.
2

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Fig. 2. (a). The predicted binding pose of GSK0872 based on 4M69 as RIPK3 docking template from Glide docking, the carbon atoms of GSK0872 are colored in golden, the residues of
Hinge region are colored in blue, the DFG motif (Asp161-Phe162-Gly163) are colored in red and the favorable residues are labeled in green; (b). Interaction patterns between
GSK0872 and key residues in the binding site of RIPK3.
Fig. 3. Proposed SAR study plan.
26e33.
The synthesis of compounds 34e39 was performed as shown in
obtained by similar reactions presented in Scheme 1, which was
reacted with trifluoromethanesulfonic anhydride followed by
Buchwald coupling with 1-benzothiophen-5-amine to form the
target product 61.
Scheme 3. Nucleophilic reaction with ketone 26 was carried out by
hydride provided by NaBH4 or carbanions provided by respective
Grignards to yield corresponding secondary alcohol 34, or tertiary
alcohols 35e36. Wittig reaction with ketone 26 provided final
compound 37. A combination of Michael addition and substitution
reaction between 15b and ethylene glycol/methyl ethylene glycol
yielded compounds 38 and 39, respectively.
4. Results and discussion
4.1. Structure-activity-relationship of synthesized compounds
Compound 47 was prepared as described in Scheme 4. Palla-
dium catalyzed coupling of intermediate 10 with methyl acrylate
provided compound 40. The combination of NaBH4 and AlCl3
reduced both of the nitro and the ester to provide primary alcohol
41. Treatment of 41 with m-CPBA converted the amino to nitro
while oxidized the sulfur to sulfone to give intermediate 42. The
sulfone 42 facilitated intramolecular cyclization under basic con-
ditions to give 43. Following similar steps presented in Scheme 1,
intermediate 43 was converted to the target compound 47.
The synthesis of compound 61 was performed as shown in
Scheme 5. Commercially available 48 was reacted with dimethyl
carbonate under basic conditions to form compound 49, which was
reacted with ((chloromethoxy)methyl)benzene to achieve inter-
mediate 50. Reduction of 50 with LAH yielded 51. One of the hy-
droxyls was reacted with TsCl to form a leaving group, facilitating
an intramolecular cyclization to give intermediate 52. Debenzyla-
tion of 52 with H2, Pd/C provided 53, which was converted to 54 by
Appel reaction. Nitration reaction was accomplished with concen-
tration H2SO4 and HNO3 to afford 55, which was converted to 56 via
an intramolecular cyclization with Na2S.9H2O. Intermediate 60 was
We used a cell based assay to evaluate the anti-necroptosis ac-
tivity of synthesized compounds. Human colon cancer HT-29 cells
were treated with TNFa, Smac mimetic and z-VAD (a pan caspase
inhibitor) to induce necroptotic cell death (the TSZ induced cell
death). Cell viability was analyzed by monitoring ATP levels. The
anti-necroptosis activity of compound was evaluated by pre-
incubation of compound with the TSZ treated cells and its ability
to rescue cells from TSZ induced death was the readout. Com-
pounds were also run a concentration gradience without TSZ
treatment to assess their cellular toxicity. We excluded compounds
which showed significant cellular toxicity at 20 mM concentration
(>30% cell death). This is an efficient assay with stable readout and
good throughput. As compounds with enhanced potency are
identified, we would expand the necroptosis assay to include ro-
dent cells, and more mechanistic based assays such as kinase
binding and functional assays. As shown in Table 1, cyclization of
the sulfone group to form a simple 5-member ring led to compound
16 (2.28
to GSK0872 (1.51
ring with a hydrophilic carbonyl improved the novelty of the
mM), which showed anti-necroptosis activity comparable
m
M). Further functionalization of the 5-member
3

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Scheme 1. Reagents and conditions: (a) methyl 2-mercaptoacetate, K2CO3, DMF, r.t, overnight,; (b) NaOH, MeOH, H2O, 70 ꢀC, 4 h; (c) Cu, quinoline, 170 ꢀC, 4 h; (d) NBS, DMF, 60 ꢀC,
3 h; (e) m-CPBA, DCM, r.t, overnight; (f) Fe, NH4Cl, EtOH, H2O, 85 ꢀC, 2 h; (g) 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione, EtOH, r.t, 30 min; (h) Ph2O, 240 ꢀC, 5 min;
(i) POCl3, 110 ꢀC, 2 h; (j) benzo[d]thiazol-5-amine, EtOH, conc. HCl, reflux, 1 h; (k) H2, Pd/C, CH3OH, r.t, overnight; (l) Cs2CO3, CH3OH, r.t, 20 h; (m) CH3CN, conc. HCl, 80 ꢀC, overnight.
Scheme 2. Reagents and conditions: (a) 1) EtSO2Na, DMSO, r.t, overnight; 2) NaH, 0 ꢀC to r.t, 3 h; 3) R1-Br, r.t, 5 h; (b) Fe, NH4Cl, EtOH, H2O, 85 ꢀC, 2 h; (c) Oxone, NaBr, CH3OH/H2O,
r.t, 4 h; (d) 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione, EtOH, r.t, 30 min; (e) Ph2O, 240 ꢀC, 5 min; (f) H2, Pd/C, TEA, i-PrOH, H2O, r.t 1.5 h; (g) POCl3, 110 ꢀC, 2 h; (h)
R2-NH2, EtOH, conc. HCl, reflux, 1 h.
template (compound 18, 3.81
sulfone and the carbonyl is fairly acidic, which can be easily
m
M). The methylene between the
functionalized by alkylation reaction. Dimethyl analogue com-
pound 26 (2.23
M) displayed comparable activity to GSK0872,
m
4

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
active in our template (compounds 29 and 30, >20
nophenol fragments led to a number of potent compounds (com-
pounds 31, 32, 33; 0.52, 0.33, 1.03 M, respectively), however, these
compounds exhibited significant cellular toxicity (>30% cell death
at 20 M concentration) in the screen assay, making them unsuit-
mM). The ami-
m
m
able for further optimization. Overall, the amino-benzothiazole
remained to be the favorable back pocket substituent for addi-
tional structure-activity investigation.
Having established the amino-benzothiazole as the best struc-
tural element for the back pocket, we decided to further explore
modifications on the carbonyl of the cyclic sulfone ring. According
to our docking study described in the above section, this position
faces solvent, we therefore expected more structure diversity to be
tolerated. Reduction of the carbonyl gave compound 34 (2.19 mM),
which showed comparable activity to GSK0872. Nucleophilic addi-
tion of the carbonyl with carbanions led to compounds 35 and 36,
which unfortunately were inactive. Wittig reaction introduced
exocyclic ethene 37 (1.03
Conversion of the carbonyl into ketal with ethylene glycol led to
compound 38 (0.42 M), which was three times more potent than
m
M), which was as potent as GSK0872.
m
GSK0872 in blocking TSZ induced cell death in TH-29 cells. The
docking pose predicted by Glide docking of compound 38 and key
interactions between compound 38 and RIPK3 were analyzed. As
shown in Fig. 4, additional interactions, three hydrogen bonds were
formed between modified function group (2H-spiro[thieno[2,3-g]
quinoline-3,2’- [1,3]dioxolane] 1,1-dioxide) and Ser102 residue in the
solvent-exposed region of RIPK3. This finding may explain why
compound 38 has better inhibitory activity than GSK0872 to some
extent. More importantly, compound 38 did not exhibited any
Scheme 3. Reagents and conditions: (a) 1) NaBH4, CH3OH, CH2Cl2, 30 min for 34. 2) R1-
MgBr, THF, 0 ꢀC - r.t, 1 h for 35, 36; (b) Methyltriphenyl phosphonium bromide, n-BuLi,
THF, ꢁ78 ꢀC to r.t; (c) Cs2CO3, Ethylene glycol, 60 ꢀC, 5 h for 38; Cs2CO3, propane-1,2-
diol, 60 ꢀC, 5 h for 39.
cellular toxicity in HT-29 cell up to 20
the amino-phenol analogues. Addition of a methyl on the ketal
(compound 39, 1.07 M) did not improve activity. Removal of an
oxygen from the ketal was detrimental to activity (compound 47,
3.96 M). In an attempt to move oxygen to face solvent, compound
61 was proposed. Compound 61 was devoid of the potential
instability of 38 in strong acidic conditions and chirality associated
with 39 and 47. The synthesis of compound 61 proved challenging
and a dedicated synthetic sequence was developed (Scheme 5). To
mM. This is in stark contrast to
however, increased size (compounds 27 and 28, >20 mM) was not
well tolerated at this position. We chose compound 26 as the
prototype for the next round of exploration (see Table 2).
In order to investigate the back pocket interactions, we carefully
selected a small size of aromatic fragments based on our docking
study and structure-activity information reported in the literation.
5-fluoro-1H-indazol-3-amine and 3-amino-4,5-dimethyl pyrazole
were successfully applied to improve potency and reduce hERG
activity in related programs [41,42], but unfortunately were not
m
m
Scheme 4. Reagents and conditions: (a) methyl acrylate, Pd(OAc)2, XPhos, NaHCO3, DMF, 120 ꢀC, 4 h; (b) NaBH4, AlCl3, THF, 2 d; (c) m-CPBA, DCM, overnight; (d) CH3OH, Cs2CO3, r.t,
2 h; (e) Fe, NH4Cl, EtOH/H2O, 80 ꢀC, 1 h; (f) 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione, EtOH, r.t, 20 min; (g) Ph2O, 240 ꢀC, 5 min; (h) POCl3, 110 ꢀC, 2 h; (i) benzo[d]
thiazol-5-amine, EtOH, conc. HCl, reflux, 2 h.
5

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Scheme 5. Reagents and conditions: (a) dimethyl carbonate, NaH, THF, 70 ꢀC, overnight; (b) ((chloromethoxy)methyl)benzene, Cs2CO3, DMF, r.t, 1 h; (c)LAH, THF, 0 ꢀC to r.t, 2 h; (d)
1) n-BuLi, TsCl, THF, 0 ꢀC, 10 min; 2) n-BuLi, 55 ꢀC, overnight; (e) H2, Pd/C, CH3OH, r.t, 12 h; (f) CCl4, PPh3, 90 ꢀC, 24 h; (g) H2SO4, HNO3, 0 ꢀC to r.t, 5 min; (h) Na2S.9H2O, DMSO, r.t,
3 h; (i) m-CPBA, DCM, r.t overnight; (j) Fe, NH4Cl, EtOH/H2O, 80 ꢀC, 2 h; (k) 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane- 4,6-dione, EtOH, r.t, 20 min; (l) Ph2O, 240 ꢀC, 5 min;
(m) (CF3SO2)2O, DCM, Pyridine, r.t, 30 min; (n) benzo[d]thiazol-5-amine, Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 100 ꢀC, 1 h.
Table 1
SAR for R1, R2, R3, R.4.
Compd.
Structure
EC50HT-29 (
m
M)a
M.W.
367.4
clogPb
3.83
16
2.28 0.54
18
26
27
28
1
3.81 0.57
2.23 0.15
>20
381.4
409.5
422.5
449.5
3.17
3.98
4.48
4.75
>20
1.51 0.14
383.5
4.16
a Human HT-29 cells were pretreated with DMSO or the test compound and then stimulated with TNF
a
(20 ng/mL), Smac mimetic (100 nM), and z-VAD (20
mM) (TSZ) for 40 h.
The inhibition of TSZ-induced necroptosis in HT-29 cells is presented as geometric mean values of at least two runs the standard error measurement (SEM). b Calculated by
Molinspiration.
6

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Table 2
SAR for R.5.
Compd.
R5
EC50HT-29 (
m
M)a
M.W.
410.4
clogPb
2.55
29
>20
30
>20
370.4
2.67
31
32
33
0.43 0.09c
0.33 0.01c
1.03 0.31c
1.51 0.14
382.4
386.4
404.4
3.47
3.18
3.54
4.16
1
383.5
a
b
Inhibition of TSZ-induced necroptosis in HT-29 cells. Calculated by Molinspiration. c Significant cellular toxicity at 20
mM concentration (>30% cell death).
Fig. 4. (a). The predicted binding pose of compound 38 based on 4M69 as RIPK3 docking template from Glide docking, the carbon atoms of compound 38 are colored in golden, the
residues of Hinge region are colored in blue, the DFG motif (Asp161-Phe162-Gly163) are colored in red and the favorable residues are labeled in green; (b). Interaction patterns
between compound 38 and key residues in the binding site of RIPK3.
our disappointment, compound 61 was inactive in rescuing HT-
29 cells from TSZ necroptosis stimuli. Overall, the docking study
was helpful in the structure-activity relationship exploration.
However, some of the subtle difference, especially toward to
solvent-exposed region of the tricyclic template, could not be fully
rationalized.
4.2. Mechanistic evaluation of lead compound 38
The inhibition of TNF , Smac mimetic and z-VAD induced nec-
a
roptotic death in human colon cancer cells is a good indication of
anti-necroptosis activity of compounds in human (Fig. 5a, anti-
necroptosis effects of compound 38, 0.42 m mM in
M; GSK0872, 1.51
Collectively, structural modifications on the back pocket and the
newly formed sulfone ring led to numerous compounds with
improved anti-necroptosis activity. (e.g., compounds 31, 32, 33, and
HT-29 cells). We next tested compound 38 in mouse cells. Com-
pound 38 efficiently inhibited TNF-induced necroptosis in mouse
embryonic fibroblasts (MEFs) with an EC50 of 0.54
mM (Fig. 5b),
38, 0.52, 0.33, 1.03, and 0.42
mM, respectively). The significant
while GSK0872 displayed an EC50 of 2.51
m
M under the same
cellular toxicity displayed by compounds 31, 32, and 33 in the
screening assay deemed them unsuitable to be further optimized.
On the other hand, compound 38 was potent and exhibited no sign
experimental conditions. These results demonstrated that 38 is an
inhibitor of TNF-induced necroptosis in both human and mouse
cells. The consistent activity of compound 38 in human and mouse
cells deemed it suitable to be tested in preclinical species for effi-
cacy as a surrogate of human diseases. TNF-induced necroptosis
propagates through RIPK1/RIPK3/MLKL. Although compound 38
was derived from GSK0872, a well characterized RIPK3 inhibitor, we
sought to determine whether compound 38 directly targeted RIPK3
or RIPK1. Compound 38 was subjected to KINOMEscan to
of cellular toxicity up to 20 mM in the screening assay. Moreover,
compound 38 displayed moderate physicochemical properties
(M.W., 425; clogP, 3.5) and represented a very novel scaffold in the
RIPK3 patent space. Based on these merits, compound 38 was
selected for mechanistic testing (see Table 3).
7

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Fig. 5. Compound 38 is a potent RIPK3 inhibitor, but is inactive to RIPK1. HT-29 cells (a) and MEFs (b) were pretreated with DMSO, compound 38, or 1 and then stimulated with
TNF (20 ng/mL), Smac mimetic (100 nM) and z-VAD (20 M) (TSZ) for 40 h; (c) The binding affinity of the test compounds on RIPK1 and RIPK3 kinases was detected by a
KINOMEscan assay. (d) The functional kinase inhibition of test compounds was detected by the ADP-Glo kinase assay.
a
m
determine its binding to RIPK1 and RIPK3. As shown in Fig. 5c,
compound 38 displayed high binding affinity to RIPK3
(Kd ¼ 7.2 0.05 nM, one replicate curve was shown) while only
exhibited no inhibition of RIPK1 kinase function even at 10 mM
concentration (Fig. 5d). MLKL is the executioner of necroptosis and
is a potential molecular target of compound 38. We therefore used
enforced dimerization/polymerization of MLKL to test if compound
38 inhibited MLKL dimerization. Compound 38 did not block this
MLKL polymerization induced necroptosis, suggested that MLKL is
not the molecular target for compound 38 (data not shown). We
marginal binding affinity to RIPK1 (Kd ¼ 5950
1250 nM, one
replicate curve was shown). In consistent to the binding data,
compound 38 significant reduced the RIPK3 kinase functional ac-
tivity in the ADP-Glo kinase assay at 0.3 mM concentration, while
8

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Table 3
while showed moderate or no activity against other kinases. Both
SAR for R1, R2, R3, R.4.
compound 38 and GSK0872 displayed significant inhibition of RIPK2
kinase activity at 10 mM concentration (data not shown). RIPK2 is
involved in the NOD/RIPK2 signaling pathway and is not related to
necroptosis. Collectively, these results demonstrate that compound
38 is a potent necroptosis inhibitor in human and mouse cells, and
the inhibition is achieved by suppression of the RIPK3 kinase
activity.
Compd.
Structure
EC50HT-29 (
2.19 1.57
m
M)a
M.W.
411.5
clogPb
3.65
4.3. Preliminary in vitro safety and DMPK evaluation of lead
compound 38
34
We were concerned that the ketal functional group presented in
compound 38 might impose chemical instability under acidic
conditions. We therefore tested compound 38 in simulated gastric
fluid (pH 1.2). Compound 38 remained intact after 24 h. We next
evaluated compound 38 for its in vitro safety and DMPK profiles. As
shown in Table 5, compound 38 exhibited moderate inhibition of
35
36
>20
>20
425.5
451.6
4.23
4.72
CYP1A2 and 2C19 at 10 mM concentration (37% and 20%, respec-
tively), and minimal inhibition of CYP2C9, 2D6, and 3A4. This
favorable CYP isozyme inhibition profile suggested low drug/drug
interaction liability for compound 38. A standard patch clamp
(express) experiment was used to assess compound 38 for its in-
hibition of the human ether-a-go-go related gene (hERG) potas-
sium channel. Compound 38 exhibited very low inhibition
37
38
1.03 0.10
0.42 0.08
407.5
425.5
4.81
3.49
(IC50 > 30
mM) of hERG (positive control: cisapride, 0.03 mM),
suggesting low potential cardiotoxicity related to inhibition of
hERG. The DMPK profile of compound 38 was summarized in
Table 6. Compound 38 was found to be highly bound to plasma
proteins cross species (97.1, 98.7, and 97.3%; human, rat, and mouse,
respectively). Permeability of compound 38 was measured in a
Caco-2 assay. Compound 38 was highly permeable (35 ꢂ 10ꢁ6 cm/s)
with low efflux ratio (0.72), indicating good absorption in the
gastrointestinal track. Lastly, compound 38 displayed moderate to
high clearance in human (60 mL/min/kg) and mouse (270 mL/min/
kg) liver microsomes.
39
1.07 0.40
439.5
3.86
47
61
1
3.96 1.81
423.5
409.5
4.06
3.54
4.4. In vivo evaluation of lead compound 38 in the SIRS mouse
model
>20
Necroptosis is involved in many inflammatory disorders. One
acute model is the TNFa-induced systemic inflammatory response
syndrome (SIRS) [43,44]. Compound 38 was evaluated in the SIRS
model for its in vivo anti-necroptosis efficacy. C57BL/6 mice were
treated with vehicle or compound 38 (5 mg/kg, i.p.) for 15 min prior
1.51 0.14
383.5
4.16
a
b
Inhibition of TSZ-induced necroptosis in HT-29 cells.
Molinspiration.
Calculated by
to intravenous injection of mouse TNF
mouse). Treatment of compound 38 strongly reduced TNF
induced hypothermia and lethal shock in mice (Fig. 6a and b).
Moreover, treatment of compound 38 significantly reduced TNF
a (0.35 mg/g TNFa per
a-
further tested compound 38 in a brief kinase panel (Table 4).
Compound 38 showed inhibition of PDGFRa with an IC50 ¼ 152 nM,
a-
induced IL-6 production in serum (Fig. 6c). These results demon-
strate that inhibition of RIPK3 by compound 38 provides strong
protection against TNF-induced SIRS, highlighting the potential of
compound 38 as a lead for the development of anti-inflammatory
therapeutics.
Table 4
Kinase selectivity of compound 38.
Kinases
38 IC50 (nM)
Reference IC50 (nM)
EGFRa
>10000
152
>10000
3737
1362
>10000
7770
78
0.58
43
158
3.7
7.2
6.0
PDGFRaa
CDK4/CycD3a
ALK4b
5. Conclusion
BRAFc
Guided by molecular docking studies based on available RIPK3-
small molecule crystal complex, the current structural modification
identifies a number of compounds with improved anti-necroptosis
activity compared with GSK0872, exemplified by compound 38.
Mechanistic studies demonstrate that compound 38 potently binds
to RIPK3 (Kd ¼ 7.1 nM) with over 800-fold selectivity over RIPK1
(Kd ¼ 6000 nM). Consistently, compound 38 inhibits RIPK3 kinase
function in the ATP-Glo assay without affecting RIPK1 kinase
m TORd
PI3Kae
a: Detected by Caliper mobility shift assay and Staurosporine was used as reference
compound; b: Detected by ADP-Glo assay with ATP concentration at Km and
SB431542 was used as reference; c: Detected by Lantha Screen assay and GW5074
was used as reference; d: Detected by Lance Ultra assay and PI103 was used as
reference; e: Detected by ADP-Glo assay with ATP concentration at 25
was used as reference. These assays were performed by ChemPartner.
mM and PI103
9

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
Table 5
Preliminary in vitro safety evaluation of compound 38.
Compd.
hERG (
mM)
% of CYP inhibiton(10 mM)
1A2
2C9
ꢁ6.33
2C19
19.88
2D6
CYP3A4 (midazolam)
CYP3A4 (testosterone)
9.60
38
>30
37.04
ꢁ5.26
ꢁ13.67
Table 6
Preliminary in vitro DMPK evaluation of compound 38.
Compd.
Protein binding (%)
Caco-2 Permeability
Metabolic stability
T1/2(min)
A-B (10ꢁ6cm sꢁ1
35.24
)
B-A (10ꢁ6cm sꢁ1
)
B-A/A-B
Clint(mL/min/kg)
HLMd
MLMe
HLM
MLM
38
97.1(H)a
98.7(R)b
97.3(M)c
25.33
0.72
28.75
20.26
60.47
269.40
Ha: human; Rb: rat; Mc: mouse; HLMd: human liver microsomes; MLMe: mouse liver microsomes.
Fig. 6. Compound 38 protected mice from SIRS. Body temperature (a), survival curve (b), and serum level of IL-6 (c) of C57BL/6 mice injected with TNF
a (0.35 mg/g TNFa) after
pretreatment with compound 38 (5 mg/kg).
function. Moreover, compound 38 displays favorable in vitro safety
profiles with minimal inhibition of CYP enzymes and hERG. In
addition, compound 38 exhibits similar anti-necroptosis potency in
both mouse and human cells, making it suitable to be studied in
preclinical animal efficacy models. Compound 38 ameliorates hy-
phase A: 5 mM aqueous ammonium acetate. Mobile phase B:
MeOH. Temperature: 24 ꢀC. Gradient: 5e40% B over 1 min, 40e70%
B over 1 min, 70e95% B over 4 min, then a 1 min hold at 95% B.
Flow: 1.2 mL/min. Detection: UV at 214 and 254 nm.
pothermia and lethal shock in C57BL/6 mice in the TNFa-induced
6.1.1. Methyl 5-nitrobenzo[b]thiophene-2-carboxylate (7)
systemic inflammatory response syndrome model. The promising
in vitro and in vivo pharmacological profiles, combined with
moderate physicochemical properties and novel scaffold make
compound 38 an attractive lead for the development of RIPK3 in-
hibitors as potential therapeutics.
To a solution of 6 (64 g, 346 mmol) in DMF (600 mL) was added
K2CO3 (95.5 g, 692 mmol) and methyl 2-mercaptoacetate (34 mL,
381 mmol). The mixture was stirred at room temperature over-
night. The reaction was poured into water (3.5 L) and the resulting
solid was filtered, washed with water and dried in vacuum to give
the desired product 7 (78.5 g, 96%) as a white solid. 1H NMR
(400 MHz, CDCl3)
8.00 (d, J ¼ 8.8 Hz, 1H), 3.99 (s, 3H).
d
8.78 (s, 1H), 8.30 (d, J ¼ 8.8 Hz, 1H), 8.19 (s, 1H),
6. Experimental protocols
6.1. Chemistry
6.1.2. 5-nitrobenzo[b]thiophene-2-carboxylic acid (8)
To a solution of 7 (78.5 g, 331 mmol) in MeOH (400 mL) and H2O
(400 mL) was added NaOH (53.0 g,1.3 mol). The mixture was stirred
at 70 ꢀC for 4 h. After cooling to room temperature, the solution was
poured into water (4 L) and acidified with conc. HCl. The resulting
solid was filtered, washed with water, dried in vacuum to give the
desired product 8 (72.6 g, 98%) as a white solid. 1H NMR (400 MHz,
General reaction progress was monitored by analytical thin layer
chromatography performed on silica gel HSGF254 pre-coated
plates. Organic solutions were dried over anhydrous Na2SO4, and
the solvents were removed under reduced pressure. Final com-
pounds were purified with silica gel 100e200 mesh for column
chromatography. 1H NMR were obtained on 400 MHz (Varian)
spectrometer, and 13C NMR were obtained on 151 MHz or 101 MHz
(Varian) spectrometer. Chemical shifts were given in ppm using
tetramethylsilane as internal standard. Mass spectra were obtained
using an Agilent 1100 LC/MSD Trap SL version Mass Spectrometer.
HRMS analysis was recorded on an Agilent 6540 UHD Accurate-
Mass Q-TOF LC/MS. Values of optical rotation were measured on a
Rudolph Automatic Polarimeter A21101. HPLC method: Waters
DMSO‑d6)
d 8.97 (s, 1H), 8.49e8.19 (m, 3H).
6.1.3. 5-nitrobenzo[b]thiophene (9)
To a solution of 8 (65 g, 291 mmol) in quinoline (500 mL) was
added copper powder (18.6 g, 291 mmol). The reaction was stirred
at 170 ꢀC under N2 atmosphere for 4 h. The solid was removed via
filtration and washed with EA (400 mL). Another EA (2 L) was
added to the filtrate to dilute the solution and then the solution was
Acquity UPLC, BEH C18 2.1 mm ꢂ 50 mm, 1.7
mm particles. Mobile
10

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
acidified with conc. HCl with an ice bath. The organic layer was
separated, washed with 2N HCl (400 mL), saturate NaHCO3 aqueous
solution (400 mL), dried over Na2SO4, filtrated and concentrated.
The residue was rinsed with EA (200 mL) and dried in vacuum to
give the desired product 9 (48 g, 78%) as a white solid. 1H NMR
13b.
6.1.7.1. 5-(((1,1-dioxidobenzo[b]thiophen-5-yl)amino)methylene)-
2,2-dimethyl-1,3-dioxane-4,6-dione (13a). Compound 13a was ob-
tained as a yellow solid (1.4 g, 90%). 1H NMR (400 MHz, CDCl3)
(400 MHz, DMSO‑d6)
J ¼ 8.8 Hz, 1H), 8.05 (d, J ¼ 5.2 Hz, 1H), 7.72 (d, J ¼ 5.6 Hz, 1H).
d
8.84 (s, 1H), 8.29 (d, J ¼ 9.2 Hz, 1H), 8.17 (d,
d
11.35 (d, J ¼ 13.2 Hz, 1H), 8.66 (d, J ¼ 13.6 Hz, 1H), 7.78 (d,
J ¼ 8.0 Hz,1H), 7.36 (d, J ¼ 8.0 Hz,1H), 7.26 (s, 1H), 7.23 (d, J ¼ 7.2 Hz,
1H), 6.85 (d, J ¼ 6.8 Hz, 1H), 1.77 (s, 6H). LCMS (ESI/APCI) m/z: 333.7
[M - H]-.
6.1.4. 3-Bromo-5-nitrobenzo[b]thiophene (10)
To a solution of 9 (19.4 g, 108 mmol) in DMF (250 mL) was added
NBS (21.2 g, 119 mmol). The mixture was stirred at 60 ꢀC under
nitrogen atmosphere for 3 h. The solvent was removed in vacuum.
The remaining residue was washed with EA (250 mL) and water
(250 mL), dried in vacuum to give the desired product 10 (21.0 g,
6.1.7.2. 5-(((3-Bromo-1,1-dioxidobenzo[b]thiophen-5-yl)amino)
methylene)-2,2-dimethyl-1,3-dioxaneꢁ4,6-dione
Compound 13b was obtained as a yellow solid (18.0 g, 86%). 1H NMR
(400 MHz, DMSO‑d6)
(13b).
75%) as a white solid. 1H NMR (400 MHz, DMSO‑d6)
8.40 (d, J ¼ 8.8 Hz, 1H), 8.32e8.25 (m, 2H).
d8.49 (s, 1H),
d
11.42 (d, J ¼ 12.8 Hz, 1H), 8.68 (d, J ¼ 13.6 Hz,
1H), 8.13 (s, 1H), 7.99 (s, 1H), 7.90 (s, 2H), 1.69 (s, 6H). LCMS (ESI/
APCI) m/z: 411.5 [M - H]-.
6.1.5. General procedure for the synthesis of 11a-11b
To a solution of 9e10 (1 eq) in DCM (1 mmol/3 mL) with an ice
bath was added 85% m-CPBA (3 eq). The mixture was stirred at
room temperature overnight. The resulting solid was filtered off
and the filtrate was quenched with Na2SO3, washed with saturated
NaHCO3 aqueous solution and extracted with DCM. The combined
organic phase was dried over Na2SO4, filtrated and concentrated.
The residue was rinsed with EA and dried in vacuum to give the
desired product 11a-11b.
6.1.8. General procedure for the synthesis of 14a-14b
The diphenyl ether (1 mmol/10 mL) was added to a round-
bottomed flask and the solvent was heated to 240 ꢀC for 5 min.
Intermediate 13a-13b (1 eq) was added dropwise to the solution.
The mixture was stirred for 5 min. After cooling to room temper-
ature, the resulting solid was collected via filtration, washed with
ether and dried in vacuum to give the desired product 14a-14b.
6.1.8.1. 8-hydroxythieno[2,3-g]quinoline
Compound 14a was obtained as a grey solid (450 mg, 77%). 1H NMR
(400 MHz, DMSO‑d6)
1,1-dioxide
(14a).
6.1.5.1. 5-nitrobenzo[b]thiophene 1,1-dioxide (11a). Compound 11a
was obtained as a white solid (1.2 g, 73%). 1H NMR (400 MHz, CDCl3)
d
12.28 (s, 1H), 8.29 (s, 1H), 8.00 (d, J ¼ 7.2 Hz,
d
8.43 (d, J ¼ 8.0 Hz, 1H), 8.24 (s, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H), 7.33 (d,
1H), 7.80 (d, J ¼ 6.8 Hz, 1H), 7.69 (s, 1H), 7.56 (d, J ¼ 6.8 Hz, 1H), 6.18
J ¼ 7.2 Hz, 1H), 6.93 (d, J ¼ 6.8 Hz, 1H). LCMS (ESI/APCI) m/z: 212.9
þ
(d, J ¼ 7.2 Hz, 1H). LCMS (ESI/APCI) m/z: 233.8 [M þ H]
.
[M þ H] þ
.
6.1.8.2. 3-Bromo-8-hydroxythieno[2,3-g]quinoline 1,1-dioxide (14b).
Compound 14b was obtained as a grey solid (5.5 g, 41%). 1H NMR
(400 MHz, DMSO‑d6) d12.31 (s, 1H), 8.35 (s, 1H), 8.25 (s, 1H), 8.08 (d,
6.1.5.2. 3-Bromo-5-nitrobenzo[b]thiophene
Compound 11b was obtained as a white solid (20.8 g, 99%). 1H NMR
(400 MHz, DMSO‑d6)
1H), 8.30 (d, J ¼ 8.4 Hz, 1H), 8.20 (d, J ¼ 1.6 Hz, 1H).
1,1-dioxide
(11b).
d
8.56 (dd, J ¼ 8.4 Hz, J ¼ 1.2 Hz, 1H), 8.34 (s,
J ¼ 7.2 Hz, 1H), 7.77 (s, 1H), 6.23 (d, J ¼ 6.8 Hz, 1H). LCMS (ESI/APCI)
þ
m/z: 311.6 [M þ H]
.
6.1.6. General procedure for the synthesis of 12a-12b
To a solution of 11a-11b (1eq) in EtOH (1 mmol/4 mL) and H2O
(1 mmol/2 mL) was added iron powder (5 eq) and NH4Cl (5 eq). The
mixture was stirred at 85 ꢀC for 2 h. The reaction was filtered
through diatomaceous earth and the cake was washed with DCM.
The resulting filtrate was extracted with DCM. The combined
organic phase was dried over Na2SO4, filtrated and concentrated.
The residue was rinsed with EA, dried in vacuum to give the desired
product 12a-12b.
6.1.9. General procedure for the synthesis of 15a-15b
Intermediate 14a-14b (1 eq) was added to POCl3 (1 mmol/2 mL)
and then the mixture was stirred at reflux for 2 h to afford a light
brown solution. After cooling to room temperature, the excess
POCl3 was removed in vacuum. The residue was dissolved in EtOH
(1 mmol/4 mL) and benzo[d]thiazol-5-amine (1.1 eq) was added
subsequently. The mixture was stirred at reflux for 1 h. After
cooling to room temperature. The resulting solid was collected via
filtration, washed with EtOH and dried in vacuum to give the
desired product 15a-15b.
6.1.6.1. 5-aminobenzo[b]thiophene
Compound 12a was obtained as a yellow solid (0.8 g, 77%). 1H NMR
(400 MHz, CDCl3)
8.42 (d, J ¼ 8.4 Hz, 1H), 8.22 (s, 1H), 7.90 (d,
1,1-dioxide
(12a).
d
6.1.9.1. 8-(benzo[d]thiazol-5-ylamino)thieno[2,3-g]quinoline 1,1-
J ¼ 8.0 Hz, 1H), 7.33 (d, J ¼ 6.8 Hz, 1H), 6.92 (d, J ¼ 6.8 Hz, 1H). LCMS
dioxide (15a). Compound 15a was obtained as a hydrochloride
(ESI/APCI) m/z: 181.9 [M þ H] þ
.
salt (165 mg, 24%). 1H NMR (400 MHz, DMSO‑d6)
d 14.79 (br s, 1H),
11.15 (s, 1H), 9.54 (s, 1H), 9.31 (s, 1H), 8.56 (d, J ¼ 7.2 Hz, 1H), 8.39 (d,
J ¼ 8.4 Hz, 1H), 8.22 (s, 1H), 8.16 (s, 1H), 8.00 (d, J ¼ 7.6 Hz, 1H), 7.81
(d, J ¼ 6.8 Hz, 1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.00 (d, J ¼ 6.8 Hz, 1H).
6.1.6.2. 5-Amino-3-bromobenzo[b]thiophene
1,1-dioxide
(12b).
Compound 12b was obtained as a yellow solid (15 g, 81%). 1H NMR
(400 MHz, DMSO‑d6)
d 7.84 (s, 1H), 7.47 (s, 1H), 6.89e6.59 (m, 2H),
LCMS (ESI/APCI) m/z: 365.7 [M þ H] þ
.
6.44 (s, 2H). LCMS (ESI/APCI) m/z: 257.7 [M - H] -.
6.1.7. General procedure for the synthesis of 13a-13b
6.1.9.2. 8-(benzo[d]thiazol-5-ylamino)-3-bromothieno[2,3-g]quino-
To a solution of 12a-12b (1 eq) in EtOH (1 mmol/mL) was added
5-(methoxymethylene)-2,2- dimethyl-1,3-dioxane-4,6-dione (1.1
eq). The mixture was stirred at room temperature for 30 min. The
resulting solid was collected via filtration, washed with EtOH
(1 mmol/mL) and dried in vacuum to give the desired product 13a-
line 1,1-dioxide (15b). Compound 15b was obtained as a hydro-
chloride salt (6.0 g, 77%). 1H NMR (400 MHz, DMSO‑d6)
d 11.37 (s,
1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.63 (d, J ¼ 6.8 Hz, 1H), 8.50 (s, 1H),
8.40 (d, J ¼ 8.8 Hz, 1H), 8.27e8.21 (m, 2H), 7.62 (d, J ¼ 8.8 Hz, 1H),
þ
7.04 (d, J ¼ 7.2 Hz, 1H). LC-MS (ESI/APCI) m/z: 443.5 [M þ H]
.
11

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
-
6.1.10. 8-(benzo[d]thiazol-5-ylamino)-2,3-dihydrothieno[2,3-g]
quinoline 1,1-dioxide (16)
J ¼ 7.2 Hz, 3H). LCMS (ESI/APCI) m/z: 268.8 [M - H] .
To a solution of 15a (120 mg, 0.30 mmol) in MeOH (15 mL) was
added Pd/C (20 mg). the reaction was stirred at room temperature
under hydrogen atmosphere overnight. The solid was removed via
filtration. The filtrate was concentrated and purified by silica gel
column chromatography (DCM/MeOH ¼ 80/1) to give the desired
product 16 (40 mg, 36%) as a white solid. 1H NMR (400 MHz,
6.1.13.3. 2-(cyclopropylmethyl)-2-methyl-5-nitrobenzo[b]thiophen-
3(2H)-one 1,1-dioxide (20c). Compound 20c was obtained as a
yellow oil (400 mg, 6.8%).1H NMR (400 MHz, CDCl3)
d8.80 (s, 1H),
8.75 (d, J ¼ 8.0 Hz, 1H), 8.22 (d, J ¼ 8.0 Hz, 1H), 2.18e2.05 (m, 1H),
1.95e1.85 (m, 1H), 1.71 (s, 3H), 0.92e0.77 (m, 1H), 0.60e0.48 (m,
1H), 0.45e0.35þ(m, 1H), 0.16e0.14 (m, 2H). LCMS (ESI/APCI) m/z:
DMSO‑d6)
d
9.57 (s, 1H), 9.45 (s, 1H), 9.00 (s, 1H), 8.57 (d, J ¼ 5.6 Hz,
295.8 [M þ H]
.
1H), 8.22 (d, J ¼ 8.8 Hz, 1H), 8.06 (s, 1H), 7.98 (s, 1H), 7.56 (d,
J ¼ 8.4 Hz,1H), 7.05 (d, J ¼ 5.2 Hz,1H), 3.72 (t, J ¼ 6.8 Hz, 2H), 3.54 (t,
6.1.14. General procedure for the synthesis of 21a-21c
þ
J ¼ 6.4 Hz, 2H). HRMS (ESI) calcd for C18H13N3O2S2, [M þ H]
,
To a solution of 20a-20c (1.0 eq) in EtOH (1 mmol/10 mL) and
H2O (1 mml/1.5 mL) was added iron powder (5 eq) and NH4Cl (5
eq). The mixture was stirred at 85 ꢀC for 2 h. The reaction was
filtered through diatomaceous earth and the cake was washed with
DCM (1 mmol/50 mL). The resulting filtrate was washed with
saturated NaHCO3 aqueous solution, dried over Na2SO4, filtrated
and concentrated. The residue was rinsed with EA, dried in vacuum
to give the desired product 21a-21c.
368.0522, found, 368.0522. Purity: 94.5%.
6.1.11. 8-(benzo[d]thiazol-5-ylamino)-3-methoxythieno[2,3-g]
quinoline 1,1-dioxide (17)
To a solution of 15b (300 mg, 0.63 mmol) in MeOH (300 mL) was
added Cs2CO3 (462 mg, 1.42 mmol). The mixture was stirred at
room temperature for 20 h the solvent was concentrated and pu-
rified by silica gel column chromatography (DCM/MeOH ¼ 100/1)
to give the desired product 17 (105 mg, 42%) as a yellow solid. 1H
6.1.14.1. 5-Amino-2,2-dimethylbenzo[b]thiophen-3(2H)-one 1,1-
NMR (400 MHz, DMSO‑d6)
d
9.55 (s, 1H), 9.45 (s, 1H), 9.02 (s, 1H),
dioxide (21a). Compound 21a was obtained as a yellow solid
8.57 (d, J ¼ 4.8 Hz, 1H), 8.23 (d, J ¼ 8.4 Hz, 1H), 8.06 (s, 1H), 7.92 (s,
(8.7 g, 90%). 1H NMR (400 MHz, CDCl3)
d
7.75 (d, J ¼ 8.4 Hz, 1H),
1H), 7.55 (d, J ¼ 8.8 Hz, 1H), 7.10 (d, J ¼ 4.8 Hz, 1H), 6.91 (s, 1H), 4.04
7.12e7.07 (m, 1H), 7.07e7.05 (m, 1H), 4.77 (br s, 2H), 1.58 (s, 6H).
(s, 3H). LC-MS (ESI/APCI) m/z: 395.7 [M þ H] þ
.
LCMS (ESI/APCI) m/z: 225.9 [M þ H] þ
6.1.12. 8-(benzo[d]thiazol-5-ylamino) thieno[2,3-g] quinolin-
3(2H)-one 1,1-dioxide (18)
6.1.14.2. 5-Amino-2-ethyl-2-methylbenzo[b]thiophen-3(2H)-one 1,1-
dioxide (21b). Compound 21b was obtained as a yellow solid
To a solution of 17 (15 mg, 0.04 mmol) in CH3CN (2 mL) was
added conc. HCl (0.5 mL). The mixture was stirred at 80 ꢀC over-
night. The reaction was poured into water (20 mL). The resulting
solid was collected via filtration, washed with IPA (5 mL) and dried
in vacuum to give the desired product 18 (12 mg, 83%) as a hy-
(700 mg, 60%). 1H NMR (400 MHz, CDCl3)
d
7.74 (d, J ¼ 8.0 Hz, 1H),
7.10e7.00 (m, 2H), 4.38 (s, 2H), 2.20e1.98 (m, 2H), 1.56 (s, 3H), 1.06
(t, J ¼ 7.2 Hz, 3H). LCMS (ESI/APCI) m/z: 239.9 [M þ H] þ
.
6.1.14.3. 5-Amino-2-(cyclopropylmethyl)-2-methylbenzo[b]thiophen-
drochloride salt. 1H NMR (400 MHz, DMSO‑d6)
d
14.97 (s, 1H), 11.64
3(2H)-one 1,1-dioxide (21c). Compound 21c was obtained as a
(s, 0.67H), 11.30 (s, 0.33H), 9.70 (s, 0.67H), 9.55 (s, 1H), 9.30 (s,
0.33H), 8.72 (d, J ¼ 6.4 Hz, 0.67H), 8.63 (s, 0.67H), 8.59 (d, J ¼ 4.8 Hz,
0.33H), 8.43 (d, J ¼ 8.8 Hz, 1H), 8.28 (s, 0.67H), 8.24 (s, 0.33H), 8.20
(s, 0.33H), 7.70e7.58 (m, 1H), 7.07 (d, J ¼ 6.4 Hz, 0.67H), 7.00 (d,
J ¼ 4.4 Hz, 0.33H), 6.58 (s, 0.33H), 4.85 (s, 1.33H). HRMS (ESI) calcd
for C18H11N3O3S2, [M þ H] þ, 382.0315, found, 382.0314. Purity:
95.4%.
yellow oil (240 mg, 66%). 1H NMR (400 MHz, CDCl3)
d7.75 (d,
J ¼ 8.4 Hz, 1H), 7.14e7.00 (m, 2H), 4.37 (s, 2H), 2.10e1.98 (m, 1H),
1.90e1.80 (m, 1H), 1.64 (s, 3H), 1.10e0.80 (m, 1H), 0.65e0.35 (m,
2H), 0.16e0.05 (m, 2H). LCMS (ESI/APCI) m/z: 265.8 [M þ H] þ
6.1.15. General procedure for the synthesis of 22a-22c
.
To a solution of 21a-21c (1 eq) in MeOH (1 mmol/4 mL) and H2O
(1 mmol/2 mL) was added Oxone (1 eq). Then NaBr (1 eq) dissolved
in H2O (1 mmol/mL) was added dropwise. The mixture was stirred
at room temperature for 4 h. Saturated NaHCO3 (1 mmol/10 mL)
aqueous solution was added to quench the reaction. The aqueous
layer was extracted with DCM (1 mmol/10 mL * 3). The combined
organic phase was dried over Na2SO4, filtrated and concentrated.
The residue was purified by silica gel column chromatography
(DCM/MeOH ¼ 50/1) to give the desired product 22a-22c.
6.1.13. General procedure for the synthesis of 20a-20c
To a solution of 19 (1 eq) in DMSO (1 mmol/2.5 mL) was added
sodium ethyl sulfonate (1 eq). The mixture was stirred at room
temperature overnight. NaH (1 eq) was added slowly with an ice
bath. Recovering to room temperature. the mixture was stirred for
3 h. Halogenated hydrocarbon (3 eq) was added and the reaction
was stirred for another 5 h. EA (1 mmol/15 mL) was added to dilute
the solution and the reaction was acidified with 1 N HCl to adjust
pH to 5. The organic layer was separated, dried with Na2SO4, fil-
trated and concentrated. The residue was purified by silica gel
column chromatography (PE/EA ¼ 10/1) to give the desired product
20a-20c.
6.1.15.1. 5-Amino-4-bromo-2,2-dimethylbenzo[b]thiophen-3(2H)-
one 1,1-dioxide (22a). Compound 22a was obtained as a yellow
solid (10 g, 85%). 1H NMR (400 MHz, DMSO‑d6)
d
7.82 (d, J ¼ 8.8 Hz,
1H), 7.31 (d, J ¼ 8.4 Hz, 1H), 6.74 (br s, 2H), 1.45 (s, 6H). LCMS (ESI/
APCI) m/z: 303.6 [M þ H] þ
.
6.1.13.1. 2,2-Dimethyl-5-nitrobenzo[b]thiophen-3(2H)-one 1,1-
dioxide (20a). Compound 20a was obtained as a yellow solid
6.1.15.2. 5-Amino-4-bromo-2-ethyl-2-methylbenzo[b]thiophen-
(11 g, 43%)。1H NMR (400 MHz, DMSO‑d6)
d
8.83 (d, J ¼ 8.0 Hz, 1H),
3(2H)-one 1,1-dioxide (22b). Compound 22b was obtained as a
8.62 (s, 1H), 8.52 (d, J ¼ 8.8 Hz, 1H), 1.56 (s, 6H). LCMS (ESI/APCI) m/
yellow solid (500 mg, 66%). 1H NMR (400 MHz, DMSO‑d6)
d 7.80 (d,
z: 277.7 [M þ Na] þ
.
J ¼ 8.4 Hz, 1H), 7.30 (d, J ¼ 8.4 Hz, 1H), 6.73 (br s, 2H), 1.96e1.86 (m,
2H), 1.43 (s, 3H), 0.92 (t, J ¼ 7.2 Hz, 3H).
6.1.13.2. 2-Ethyl-2-methyl-5-nitrobenzo[b]thiophen-3(2H)-one 1,1-
dioxide (20b). Compound 20b was obtained (2.4 g, 45%) as a grey
6.1.15.3. 5-Amino-4-bromo-2-(cyclopropylmethyl)-2-methylbenzo[b]
solid. 1H NMR (400 MHz, CDCl3)
d8.80 (s,1H),8.75 (d, J ¼ 8.4 Hz,1H),
thiophen-3(2H)-one 1,1-dioxide (22c). Compound 22c was obtained
8.22 (d, J ¼ 8.4 Hz, 1H), 2.26e1.95 (m, 2H), 1.64 (s, 3H), 1.10 (t,
as a yellow oil (211 mg, 83%). 1H NMR (400 MHz, CDCl3)
d 7.72 (d,
12

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
þ
J ¼ 7.6 Hz,1H), 7.15 (d, J ¼ 8.0 Hz,1H), 4.94 (s, 2H), 2.10e1.98 (m,1H),
J ¼ 3.2 Hz, 2H). LCMS (ESI/APCI) m/z: 395.6 [M þ H]
6.1.18. General procedure for the synthesis of 25a-25c
.
1.90e1.75 (m, 1H), 1.58 (s, 3H), 0.90e0.85 (m, 1H), 0.55e0.35 (m,
þ
2H), 0.16e0.05 (m, 2H). LCMS (ESI/APCI) m/z: 343.7 [M þ H]
.
To a solution of 24a-24c (1 eq) in IPA (1 mmol/50 mL) and H2O (1
mmol/5 mL) was added 10% Pd/C (1 mmol/50 mg) and Et3N (1
mmol/3 mL). The reaction was stirred under H2 atmosphere for
1.5 h. Saturated Na2SO3 (1 mmol/50 mL) aqueous solution and DCM
(1 mmol/100 mL) were added. The organic layer was separated,
dried over Na2SO4, filtrated and concentrated to give the desired
product 25a-25c.
6.1.16. General procedure for the synthesis of 23a-23c
To a solution of 22a-22c (1 eq) in EtOH (1 mmol/2 mL) was
added
5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione (1.1 eq). The mixture was stirred at room temperature for
30 min. The resulting solid was collected via filtration, washed with
EtOH (1 mmol/1 mL) and dried in vacuum to give the desired
product 23a-23c.
6.1.18.1. 8-Hydroxy-2,2-dimethylthieno[2,3-g]quinolin-3(2H)-one
6.1.16.1. 5-(((4-Bromo-2,2-dimethyl-1,1-dioxido-3-oxo-2,3-
dihydrobenzo[b]thiophen-5-yl) amino) methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (23a). Compound 23a was obtained as a white
1,1-dioxide (25a). Compound 25a was obtained as a yellow solid
(4.0 g, 86%). 1H NMR (400 MHz, DMSO‑d6)
d 12.42 (s, 1H), 8.62 (s,
1H), 8.20e8.10 (m, 2H), 6.26 (d, J ¼ 7.2 Hz, 1H), 1.53 (s, 6H). LCMS
solid (13 g, 86%). 1H NMR (400 MHz, DMSO‑d6)
d 11.84 (d,
(ESI/APCI) m/z: 277.8 [M þ H] þ
.
J ¼ 13.6 Hz,1H), 8.92 (d, J ¼ 13.6 Hz,1H), 8.54 (d, J ¼ 8.4 Hz,1H), 8.31
(d, J ¼ 8.8 Hz, 1H), 1.71 (s, 6H), 1.53 (s, 6H).
6.1.18.2. 2-Ethyl-8-hydroxy-2-methylthieno[2,3-g]quinolin-3(2H)-
one 1,1-dioxide (25b). Compound 25b was obtained as a grey solid
6.1.16.2. 5-(((4-Bromo-2-ethyl-2-methyl-1,1-dioxido-3-oxo-2,3-
dihydrobenzo[b]thiophen-5-yl)amino) methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (23b). Compound 23b was obtained as a white
(50 mg, 61%)。1H NMR (400 MHz, DMSO‑d6)
d12.41 (br s, 1H), 8.62
(s, 1H), 8.22e8.10 (m, 2H), 6.27 (d, J ¼ 7.6 Hz,1H), 2.10e1.90 (m, 2H),
1.51 (s, 3H), 0.97 (t, J ¼ 7.6 Hz, 3H). LCMS (ESI/APCI) m/z: 291.7 [M þ
solid (673 mg, 90%). 1H NMR (400 MHz, DMSO‑d6)
d 11.83 (d,
þ
H]
.
J ¼ 13.6 Hz,1H), 8.91 (d, J ¼ 13.2 Hz,1H), 8.52 (d, J ¼ 8.8 Hz,1H), 8.29
(d, J ¼ 8.8 Hz,1H), 2.03e1.94 (m, 2H),1.71 (s, 6H),1.51 (s, 3H), 0.96 (t,
6.1.18.3. 2-(cyclopropylmethyl)-8-hydroxy-2-methylthieno[2,3-g]
-
J ¼ 7.2 Hz, 3H). LCMS (ESI/APCI) m/z: 469.6 [M - H] .
quinolin-3(2H)-one 1,1-dioxide (25c). Compound 25c was obtained
as a yellow solid (50 mg, 61%). 1H NMR (400 MHz, DMSO‑d6)
d12.41
6.1.16.3. 5-(((4-Bromo-2-(cyclopropylmethyl)-2-methyl-1,1-dioxido-
3-oxo-2,3-dihydrobenzo[b].
(s, 1H), 8.62 (s, 1H), 8.16 (s, 1H), 8.08e8.04 (m, 1H), 6.27 (d,
J ¼ 7.6 Hz, 1H), 2.04e1.95 (m, 1H), 1.96e1.80 (m, 1H), 1.59 (s, 3H),
0.80e0.70 (m, 1H), 0.44e0.38 (m, 1H), 0.34e0.28 (m, 1H),
thiophen-5-yl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione (23c).
-
0.12e0.05 (m, 2H). LCMS (ESI/APCI) m/z: 315.7 [M - H] .
Compound 23c was obtained as a white solid (290 mg, 93%). 1H
NMR (400 MHz, DMSO‑d6)
d
11.82 (d, J ¼ 11.6 Hz, 1H), 8.90 (d,
6.1.19. General procedure for the synthesis of 26e33
J ¼ 12.8 Hz, 1H), 8.51 (d, J ¼ 8.0 Hz, 1H), 8.29 (d, J ¼ 8.4 Hz, 1H),
2.04e1.98 (m, 1H), 1.83e1.77 (m, 1H), 1.71 (s, 6H), 1.57 (s, 3H),
0.69e0.62 (m, 1H), 0.44e0.38 (m, 1H), 0.31e0.25 (m, 1H), 0.05 (d,
J ¼ 4.4 Hz, 2H). LCMS (ESI/APCI) m/z: 495.5 [M - H] -.
Intermediate 25a-25c (1 eq) was added to POCl3 (1 mmol/4 mL)
and then the mixture was stirred at reflux for 2 h to afford a light
brown solution. After cooling to room temperature, the excess
POCl3 was removed in vacuum. The residue was dissolved in EtOH
(1 mmol/10 mL) and subsequently various aromatic amine (1.1 eq)
was added. The mixture was stirred at reflux for 1 h. After cooling to
room temperature. The resulting solid was collected via filtration,
washed with EtOH and dried in vacuum to give the desired product
26e33.
6.1.17. General procedure for the synthesis of 24a-24c
The diphenyl ether (1 mmol/10 mL) was added to a round-
bottomed flask and the solvent was heated to 240 ꢀC for 5 min.
Intermediate 23a-23c (1 eq) was added dropwise to the solution.
The mixture was stirred for 5 min. After cooling down to room
temperature, the resulting solid was collected via filtration, washed
with ether (5 mL) and dried in vacuum to give the desired product
24a-24c.
6.1.19.1. 8-(benzo[d]thiazol-5-ylamino)-2,2-dimethylthieno[2,3-g]
quinolin-3(2H)-one 1,1-dioxide (26). Compound 26 was obtained as
a yellow solid (450 mg, 50%)。1H NMR (400 MHz, DMSO‑d6)
d 11.39
(s, 1H), 9.71 (s, 1H), 9.55 (s, 1H), 8.73 (d, J ¼ 6.8 Hz, 1H), 8.63 (s, 1H),
6.1.17.1. 4-Bromo-8-hydroxy-2,2-dimethylthieno[2,3-g]quinolin-
8.41 (d, J ¼ 8.4 Hz, 1H), 8.25 (s, 1H), 7.64 (d, J ¼ 7.6 Hz, 1H), 7.10 (d,
3(2H)-one 1,1-dioxide (24a). Compound 24a was obtained as a grey
J ¼ 6.8 Hz, 1H), 1.61 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 194.4,
solid (6.0 g, 60%).1H NMR (400 MHz, DMSO‑d6)
d 11.78 (s, 1H), 8.60
157.6, 154.8, 154.1, 150.5, 149.3, 138.0, 137.0, 129.9, 129.3, 127.0,
124.5, 123.4,121.6,118.9,116.8,103.7, 64.1, 40.1, 39.9, 39.8, 39.7, 39.5,
(s, 1H), 8.06 (s, 1H), 6.34 (d, J ¼ 7.2 Hz, 1H), 1.54 (s, 6H). LCMS (ESI/
-
APCI) m/z: 353.6 [M - H] .
39.4, 39.2, 39.1, 19.5. HRMS (ESI) calcd for C20H15N3O3S2, [M þ H] þ
,
410.0628, found, 410.0628. Purity: 95.0%.
6.1.17.2. 4-Bromo-2-ethyl-8-hydroxy-2-methylthieno[2,3-g]quinolin-
3(2H)-one 1,1-dioxide (24b). Compound 24b was obtained as a
brown solid (100 mg, 19%). 1H NMR (400 MHz, DMSO‑d6)
d
11.77 (s,
6.1.19.2. 8-(benzo[d]thiazol-5-ylamino)-2-ethyl-2-methylthieno[2,3-
g]quinolin-3(2H)-one 1,1-dioxide (27). Compound 27 was obtained
as a yellow solid (15 mg, 30%)。1H NMR (400 MHz, DMSO‑d6)
1H), 8.59 (s, 1H), 8.05 (s, 1H), 6.35 (s, 1H), 2.10e1.98 (m, 2H), 1.53 (s,
3H), 0.96 (t, J ¼ 7.2 Hz, 3H). LCMS (ESI/APCI) m/z: 369.7 [M þ H] þ
.
d
11.46 (s, 1H), 9.72 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 8.66 (s, 1H), 8.41
6.1.17.3. 4-Bromo-2-(cyclopropylmethyl)-8-hydroxy-2-methylthieno
[2,3-g]quinolin-3(2H)-one 1,1-dioxide (24c). Compound 24c was
obtained as a white solid (60 mg, 26%). 1H NMR (400 MHz,
(d, J ¼ 8.4 Hz, 1H), 8.25 (s, 1H), 7.64 (d, J ¼ 8.0Hz, 1H), 7.09 (d,
J ¼ 6.4 Hz,1H), 2.20e1.99 (m, 2H),1.59 (s, 3H),1.02 (t, J ¼ 7.6 Hz, 3H).
13C NMR (151 MHz, DMSO‑d6)
d 193.5, 158.5, 155.4, 153.9, 146.1,
DMSO‑d6)
d
11.78 (s, 1H), 8.59 (s, 1H), 8.09e8.01 (m, 1H), 8.34 (d,
141.6, 139.1, 135.2, 133.0, 132.0, 124.1, 122.7, 122.4, 120.4, 119.4, 118.5,
102.2, 68.0, 26.8, 16.2, 8.3. HRMS (ESI) calcd for C21H17N3O3S2, [M þ
H] þ, 424.0784, found, 424.0785. Purity: 96.3%.
J ¼ 7.2 Hz, 1H), 2.01e1.95 (m, 1H), 1.88e1.81 (m, 1H), 1.59 (s, 3H),
0.73e0.63 (m, 1H), 0.42e0.37 (m, 1H), 0.30e0.24 (m, 1H), 0.04 (d,
13

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
6.1.20. 3.8-(benzo[d]thiazol-5-ylamino)-2-(cyclopropylmethyl)-2-
methylthieno[2,3-g]quinolin-3(2H)-one 1,1-dioxide (28)
118.9, 115.4, 105.8 (t, J ¼ 24.2 Hz), 103.9, 64.2, 19.5. HRMS (ESI) calcd
for C19H14F2N2O4S, [M þ H] þ, 405.0715, found, 405.0716. Purity:
95.0%.
Compound 28 was obtained as a yellow solid (12 mg, 44%)。1H
NMR (400 MHz, DMSO‑d6)
d 11.33 (s, 1H), 9.69 (s, 1H), 9.55 (s, 1H),
8.72 (d, J ¼ 6.8 Hz, 1H), 8.59 (s, 1H), 8.41 (d, J ¼ 8.4 Hz, 1H), 8.24 (s,
1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.11 (d, J ¼ 6.4 Hz, 1H), 2.15e2.07 (m,
1H), 1.96e1.85 (m, 1H), 1.66 (s, 3H), 0.82e0.70 (m, 1H), 0.50e0.40
(m, 1H), 0.34e0.26 (m, 1H), 0.20e0.05 (m, 2H). 13C NMR (151 MHz,
6.1.21. 8-(benzo[d]thiazol-5-ylamino)-3-hydroxy-2,2-dimethyl-2,3-
dihydrothieno[2,3-g] quinoline 1,1-dioxide (34)
To a solution of 26 (50 mg, 0.11 mmol) in MeOH (3 mL) and DCM
(5 mL) was added NaBH4 (11 mg, 0.11 mmol). The reaction was
stirred at room temperature for 30 min. Saturated NH4Cl (1 mL)
aqueous solution was added to quench the reaction. The reaction
was concentrated and purified by silica gel column chromatog-
raphy (DCM/MeOH ¼ 20/1) to give the desired product 34 (8 mg,
DMSO‑d6) d 194.3,157.6,154.5,154.1,150.3,149.5,138.0,137.4, 130.2,
129.9,126.3,124.3,123.4,121.6, 118.6, 116.9,103.6, 68.2,16.8, 6.2, 5.1,
4.78. HRMS (ESI) calcd for C23H19N3O3S2, [M þ H] þ, 450.0941,
found, 450.0942. Purity: 99.1%.
16%) as a yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 9.61 (s, 1H),
6.1.20.1. 8-((5-Fluoro-1H-indazol-3-yl)amino)-2,2-dimethylthieno
[2,3-g]quinolin-3(2H)-one 1,1-dioxide (29). Compound 29 was ob-
tained as a brown solid (20 mg, 16%). 1H NMR (400 MHz, DMSO‑d6)
9.45 (s, 1H), 9.04 (s, 1H), 8.58 (s, 1H), 8.23 (d, J ¼ 8.4 Hz,1H), 8.07 (s,
1H), 8.02 (s, 1H), 7.56 (d, J ¼ 8.0 Hz, 1H), 7.07 (s, 1H), 6.66 (d,
J ¼ 6.0 Hz, 1H), 5.12 (d, J ¼ 5.6 Hz, 1H),1.50 (s, 3H), 1.17 (s, 3H). 13
C
d
12.99 (s, 1H), 9.86 (s, 1H), 9.65 (s, 1H), 8.81 (d, J ¼ 5.2 Hz, 1H), 8.48
NMR (101 MHz, DMSO‑d6) d 157.5, 154.2, 153.4, 151.1, 149.1, 140.1,
(s, 1H), 7.82 (d, J ¼ 5.2 Hz, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 7.60 (dd,
138.5, 133.0, 129.4, 126.1, 123.2, 121.5, 119.6, 117.8, 116.4, 102.1, 73.þ9,
J ¼ 4.0Hz, 8.8 Hz, 1H),7.34 (t, J ¼ 8.4 Hz, 1H), 1.59 (s, 6H). 13C NMR
65.6, 17.6, 16.6. HRMS (ESI) calcd for C20H17N3O3S2, [M þ H]
,
(101 MHz, DMSO‑d6)
d
194.5, 156.56 (d, J ¼ 235.0 Hz), 155.0, 150.2,
412.0784, found, 412.0793. Purity: 98.4%
147.7, 141.6 (d, J ¼ 5.7 Hz), 138.0, 137.1, 129.2, 127.1, 124.1, 119.2, 116.6
(d, J ¼ 27.6 Hz), 115.5 (d, J ¼ 10.5 Hz), 112.1 (d, J ¼ 9.5 Hz), 105.7,
104.4 (d, J ¼ 24.8 Hz), 64.1, 19.6. HRMS (ESI) calcd for C20H15FN4O3S,
[M þ H] þ, 411.0922, found, 411.0922. Purity: 98.0%.
6.1.22. General procedure for the synthesis of 35e36
To a solution of 26 (1 eq) in THF (5 mL) under N2 atmosphere
was added Grignard reagent (3 eq) with an ice bath. The mixture
was stirred at room temperature for 1 h H2O was added to quench
the reaction. The aqueous layer was extracted with DCM (1 mmol/
30 mL * 3). The combined organic layer was dried over Na2SO4,
filtrated and concentrated. The residue was purified by silica gel
column chromatography (DCM/MeOH ¼ 50/1) to give the desired
product 35e36.
6.1.20.2. 8-((3,4-Dimethyl-1H-pyrazol-5-yl)amino)-2,2-
dimethylthieno[2,3-g]quinolin-3(2H)-one
Compound 30 was obtained as a yellow solid (16 mg, 22%). 1H NMR
(400 MHz, DMSO‑d6) 12.35 (s, 1H), 9.47 (s, 1H), 9.32 (s, 1H), 8.64
(d, J ¼ 4.8 Hz, 1H), 8.40 (s, 1H), 6.92 (d, J ¼ 5.2 Hz, 1H), 2.21 (s, 3H),
1.84 (s, 3H), 1.56 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
194.5,
1,1-dioxide
(30).
d
d
154.6,150.3,149.9,145.7, 137.1, 136.8, 129.1, 126.9,123.6,119.0, 105.7,
104.2, 64.1, 19.6, 9.6, 7.1. HRMS (ESI) calcd for C18H18N4O3S, [M þ H]
þ, 371.1172, found, 371.1173. Purity: 95.5%.
6.1.22.1. 8-(benzo[d]thiazol-5-ylamino)-3-hydroxy-2,2,3-trimethyl-
2,3-dihydrothieno[2,3-g]quinoline 1,1-dioxide (35). Compound 35
was obtained as a yellow solid (30 mg, 52%)。1H NMR (400 MHz,
DMSO‑d6)
d
9.57 (s, 1H), 9.45 (s, 1H), 9.02 (s, 1H), 8.58 (d, J ¼ 5.6 Hz,
6.1.20.3. 8-((5-Hydroxy-2-methylphenyl)amino)-2,2-dimethylthieno
[2,3-g]quinolin-3(2H)-one 1,1-dioxide (31). Compound 31 was ob-
tained as a yellow solid (15 mg, 20%). 1H NMR (400 MHz, DMSO‑d6)
1H), 8.22 (d, J ¼ 8.4 Hz, 1H), 8.09 (s, 1H), 8.06 (s, 1H), 7.55 (d,
J ¼ 8.4 Hz, 1H), 7.06 (d, J ¼ 5.2 Hz, 1H), 6.19 (s, 1H), 1.60 (s, 3H), 1.39
(s, 3H), 1.27 (s, 3H). 13C NMR (151 MHz, DMSO‑d6)
d 157.5, 154.1,
d
9.49 (s, 1H), 9.41 (s, 1H), 9.33 (s, 1H), 8.60 (d, J ¼ 4.4 Hz, 1H), 8.40
(s, 1H), 7.19 (d, J ¼ 8.0 Hz, 1H), 6.77e6.66 (m, 2H), 6.34 (d, J ¼ 4.0 Hz,
1H), 2.05 (s, 3H), 1.57 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
194.6,
153.4, 151.2, 149.0, 144.8, 138.9, 132.1, 129.3, 125.0, 123.2, 121.5,
119.8, 117.8, 116.4, 101.9, 76.3, 67.1, 39.5, 26.6, 19.5, 15.5. HRMS (ESI)
calcd for C21H19N3O3S2, [M þ H] þ, 426.0941, found, 426.0942. Pu-
rity: 97.5%.
d
156.5, 154.6, 150.5, 150.4, 137.6, 136.7, 132.1, 129.3, 126.9, 124.5,
123.8, 119.0, 114.5, 113.6, 103.2, 64.2, 55.0, 19.6, 16.7. HRMS (ESI)
calcd for C20H18N2O4S, [M þ H] þ, 383.1060, found, 383.1060. Purity:
91.1%.
6.1.22.2. 8-(benzo[d]thiazol-5-ylamino)-3-cyclopropyl-3-hydroxy-
2,2-dimethyl-2,3-dihydrothieno [2,3-g]quinoline 1,1-dioxide (36).
Compound 36 was obtained as a yellow solid (16 mg, 11%). 1H NMR
6.1.20.4. 8-((2-Fluoro-5-hydroxyphenyl)amino)-2,2-dimethylthieno
[2,3-g]quinolin-3(2H)-one 1,1-dioxide (32). Compound 32 was ob-
tained as a white solid (25 mg, 29%). 1H NMR (400 MHz, DMSO‑d6)
(400 MHz, DMSO‑d6) d 9.56 (s, 1H), 9.44 (s, 1H), 9.04 (s, 1H), 8.58 (d,
J ¼ 5.2 Hz, 1H), 8.22 (d, J ¼ 8.4 Hz, 1H), 8.06 (s, 1H), 8.04 (s, 1H), 7.55
(d, J ¼ 8.4 Hz, 1H), 7.06 (d, J ¼ 5.2 Hz, 1H), 5.74 (s, 1H), 1.49 (s, 3H),
1.34e1.30 (m, 1H), 1.28 (s, 3H), 0.82e0.74 (m, 1H), 0.68e0.60 (m,
1H), 0.60e0.50 (m, 1H), 0.36e0.26 (m, 1H). 13C NMR (151 MHz,
d
11.13 (s, 1H), 9.98 (s, 1H), 9.67 (s, 1H), 8.79 (d, J ¼ 6.4 Hz, 1H), 8.64
(s, 1H), 7.33 (t, J ¼ 9.6 Hz, 1H), 7.00e6.85 (m, 2H), 6.78 (d, J ¼ 4.0 Hz,
1H), 1.60 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
193.8, 155.5, 154.7
d
DMSO‑d6) d 157.5, 154.1,153.3,150.8,149.1,143.9,138.5,132.4,129.4,
(d, J ¼ 1.3 Hz), 149.7 (d, J ¼ 239.5 Hz), 146.3, 141.3, 139.0, 131.9, 124.1
125.5,123.2,121.5,119.8,117.7,116.4,101.9, 76.4, 68.0,19.7,18.2,16.0,
2.1, ꢁ0.2. HRMS (ESI) calcd for C23H21N3O3S2, [M þ H] þ, 452.1097,
found, 452.1097. Purity: 96.5%.
(d, J ¼ 13.8 Hz), 122.0, 120.6, 118.8, 117.6 (d, J ¼ 20.7 Hz), 116.5 (d,
J
¼
7.0 Hz), 113.8, 102.8, 64.6, 19.5. HRMS (ESI) calcd for
C
19H15FN2O4S, [M þ H] þ, 387.0809, found, 387.0809. Purity: 97.5%.
6.1.23. 8-(benzo[d]thiazol-5-ylamino)-2,2-dimethyl-3-methylene-
6.1.20.5. 8-((2,4-Difluoro-5-hydroxyphenyl)amino)-2,2-
dimethylthieno[2,3-g]quinolin-3(2H)-one 1,1-dioxide (33).
Compound 33 was obtained as a yellow solid (10 mg, 14%). 1H NMR
(400 MHz, DMSO‑d6) 10.16 (s, 1H), 9.47 (s, 1H), 9.35 (s, 1H), 8.69 (s,
1H), 8.44 (s, 1H), 7.45 (t, J ¼ 10.4 Hz, 1H), 7.03 (t, J ¼ 8.0 Hz, 1H), 6.61
(s, 1H), 1.57 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
194.5, 154.6,
2,3-dihydrothieno[2,3-g]quinoline 1,1-dioxide (37)
To
a
solution of methyltriphenylphosphonium bromide
(260 mg, 0.73 mmol) in dry THF (5 mL) with N2 atmosphere was
added n-BuLi (0.33 mL, 0.83 mmol) dropwise at ꢁ78 ꢀC. The reac-
tion was stirred at 0 ꢀC for 1.5 h. Then cooled to ꢁ78 ꢀC, a solution of
26 (100 mg, 0.22 mmol) in THF (5 mL) was added and the mixture
was stirred at room temperature overnight. Saturated NH4Cl
aqueous solution was added to quench the solution and the
d
d
150.2,149.7,149.5 (dd, J ¼ 29.4,10.8 Hz),147.9 (dd, J ¼ 29.4,10.8 Hz),
142.1 (d, J ¼ 11.4 Hz), 137.0, 129.3, 127.0, 123.8, 122.0 (d, J ¼ 13.2 Hz),
14

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
aqueous layer was extracted with EA (20 mL * 3). The combined
organic phase was dried over Na2SO4, filtrated and concentrated.
The residue was purified by silica gel column chromatography
(DCM/MeOH ¼ 20/1) to give the desired product 37 as a yellow
(400 MHz, DMSO‑d6)
d
8.07 (s, 1H), 8.02 (d, J ¼ 8.4 Hz, 1H), 7.47 (d,
J ¼ 8.4 Hz, 1H), 5.10 (s, 1H), 3.73e3.53 (m, 4H), 3.44e3.37 (m, 2H).
þ
LCMS (ESI/APCI) m/z: 207.9 [M þ H]
.
solid (10 mg, 11%). 1H NMR (400 MHz, DMSO‑d6)
d
9.58 (s, 1H), 9.45
6.1.27. 3-(3-hydroxypropyl)-5-nitrobenzo[b]thiophene 1,1-dioxide
(42)
(s, 1H), 9.11 (s, 1H), 8.60 (d, J ¼ 5.2 Hz, 1H), 8.42 (s, 1H), 8.23 (d,
J ¼ 8.8 Hz,1H), 8.07 (s,1H), 7.56 (d, J ¼ 8.4 Hz,1H), 7.06 (d, J ¼ 5.2 Hz,
1H), 6.36 (s, 1H), 5.68 (s, 1H), 1.55 (s, 6H). 13C NMR (151 MHz,
To a solution of 41 (1.8 g, 8.7 mmol) in DCM (50 mL) with an ice
bath was added 85% m-CPBA (4.4 g, 21.8 mmol). The mixture was
stirred at room temperature overnight. The mixture was filtrated
and the filtrate was quenched with Na2SO3, washed with saturated
NaHCO3 aqueous solution, extracted with DCM (40 mL * 3). The
combined organic phase was dried over Na2SO4, filtrated and
concentrated. The residue was purified by silica gel column chro-
matography (DCM/MeOH ¼ 50/1) to give the desired product 42
DMSO‑d6) d 157.5,154.1,153.8,151.2, 149.0,144.0,138.5,133.8,132.5,
129.4, 123.4, 123.2, 121.5, 120.5, 118.1, 116.5, 112.5, 102.0, 62.8, 21.8.
HRMS (ESI) calcd for C21H17N3O2S2, [M þ H] þ, 408.0835. found
408.0835. Purity: 96.0%.
6.1.24. General procedure for the synthesis of 38e39
To a solution of 15b (1 eq) in ethylene glycol or propane-1,2-diol
(1 mmol/10 mL) was added Cs2CO3 (2.5 eq). The reaction was
stirred at 60 ꢀC for 5 h. The mixture was concentrated and purified
by silica gel column chromatography (DCM/MeOH ¼ 50/1) to give
the desired product 38e39.
(800 mg, 34%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
d 8.43 (d,
J ¼ 7.6 Hz, 1H), 8.31 (s, 1H), 7.90 (d, J ¼ 8.0 Hz, 1H), 6.66 (s, 1H),
3.90e3.76 (m, 2H), 2.84 (t, J ¼ 6.8 Hz, 2H), 2.04e1.91 (m, 2H). LCMS
-
(ESI/APCI) m/z: 267.8 [M - H] .
6.1.28. 5-Nitro-40,50-dihydro-2H,30H-spiro[benzo[b]thiophene-3,20-
furan] 1,1-dioxide (43)
6.1.24.1. 8-(benzo[d]thiazol-5-ylamino)-2H-spiro[thieno[2,3-g]quin-
oline-3,2’- [1,3]dioxolane] 1,1-dioxide (38). Compound 38 was ob-
tained as a hydrochloride salt (55 mg, 13%)。1H NMR (400 MHz,
To a solution of 42 (800 mg, 3.0 mmol) in MeOH (40 mL) was
added Cs2CO3 (1.47 g, 4.5 mmol). The mixture was turned into dark
yellow quickly and stirred at room temperature for 2 h. The solvent
was removed in vacuum and the residue was purified by silica gel
column chromatography (PE/EA ¼ 5/1) to give the desired product
43 (200 mg, 25%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
DMSO‑d6) d 15.02 (s, 1H), 11.48 (s, 1H), 9.55 (s, 1H), 9.46 (s, 1H), 8.63
(d, J ¼ 6.8 Hz, 1H), 8.40 (d, J ¼ 9.2 Hz, 1H), 8.38 (s, 1H), 8.25 (s, 1H),
7.63 (d, J ¼ 8.4 Hz, 1H), 6.99 (d, J ¼ 6.4 Hz, 1H), 4.38e4.30 (m, 2H),
4.28e4.20 (m, 2H), 4.14 (s, 2H). 13C NMR (151 MHz, DMOS-d6)
d
158.6, 155.9, 154.0, 145.1, 142.1, 141.3, 138.1, 135.3, 133.1, 124.1,
d
8.45e8.32 (m, 2H), 7.90 (d, J ¼ 8.4 Hz, 1H), 4.36e4.12 (m, 2H), 3.61
122.9, 119.5, 119.3, 118.9, 117.1, 106.3, 101.3, 66.3, 59.8, 39.9, 39.8,
39.7, 39.5, 39.4, 39.2, 39.1. HRMS (ESI) calcd for C20H15N3O4S2, [M þ
H] þ, 426.0577. found 426.0577. Purity: 98.8%.
(q, J ¼ 13.0 Hz, 2H), 2.63e2.25 (m, 2H), 2.25e2.07 (m, 2H).
6.1.29. 5-Amino-40,50-dihydro-2H,30H-spiro[benzo[b]thiophene-
3,20-furan] 1,1-dioxide (44)
6.1.24.2. 8-(benzo[d]thiazol-5-ylamino)-40-methyl-2H-spiro[thieno
[2,3-g]quinoline-3,2’-
Compound 39 was obtained as a yellow solid (15 mg, 7%). 1H NMR
(400 MHz, DMSO‑d6) 9.64 (s, 1H), 9.45 (s, 1H), 9.08 (s, 1H), 8.62 (d,
J ¼ 4.8 Hz,1H), 8.23 (d, J ¼ 8.8 Hz,1H), 8.13 (d, J ¼ 8.4 Hz,1H), 8.08 (s,
1H), 7.56 (d, J ¼ 8.4 Hz, 1H), 7.09 (s, 1H), 4.75e4.26 (m, 2H),
4.20e3.91 (m, 2H), 3.91e3.65 (m, 1H), 1.53e1.29 (m, 3H). HRMS
(ESI) calcd for C21H17N3O4S2, [M þ H] þ, 440.0733. found 440.0733.
Purity: 99.7%.
To a solution of 43 (200 mg, 0.74 mmol) in EtOH (10 mL) and
H2O (5 mL) was added iron powder (166 mg, 2.96 mmol) and NH4Cl
(157 mg, 2.96 mmol). The mixture was stirred at 80 ꢀC for 1 h. The
solid was filtered off through diatomaceous earth and the cake was
washed with DCM (100 mL). The resulting filtrate was extracted
with DCM (20 mL * 3), dried over Na2SO4 and concentrated. The
residue was purified by silica gel column chromatography (PE/
EA ¼ 1/1) to give the desired product 44 (120 mg, 68%) as a yellow
[1,3]
dioxolane]
1,1-dioxide
(39).
d
solid. 1H NMR (400 MHz, CDCl3)
d
7.47 (d, J ¼ 8.0 Hz, 1H), 6.71 (d,
J ¼ 8.0 Hz, 1H), 6.66 (s, 1H), 4.18 (s, 2H), 4.15e4.04 (m, 2H), 3.46 (dd,
6.1.25. Methyl (E)-3-(5-nitrobenzo[b]thiophen-3-yl)acrylate (40)
To a solution of 10 (5.0 g, 19.5 mmol) in DMF (50 mL) was added
methyl acrylate (3.4 g, 39 mmol)，NaHCO3 (3.3 g, 39 mmol), Xphos
(954 mg, 2.0 mmol), Pd(OAc)2 (220 mg, 1.0 mmol). The reaction was
stirred at 120 ꢀC under N2 atmosphere for 4 h. The solvent was
removed in vacuum and the residue was purified by silica gel col-
umn chromatography (DCM) to give the desired product 40 (3.6 g,
J ¼ 41.6, 12.8 Hz, 2H), 2.56e2.16 (m, 3H), 2.16e2.01 (m, 2H). LCMS
þ
(ESI/APCI) m/z: 239.8 [M þ H]
.
6.1.30. 5-(((1,1-Dioxido-40,50-dihydro-2H,30H-spiro[benzo[b]
thiophene-3,20-furan]-5-yl)amino) methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (45)
To a solution of 44 (120 mg, 0.5 mmol) in EtOH (5 mL) was added
5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione
(140 mg, 0.75 mmol). The mixture was stirred at room temperature
for 20 min. The resulting solid was collected via filtration, washed
with EtOH (5 mL) and dried in vacuum to give the desired product
70%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
d 8.89 (s, 1H), 8.28
(d, J ¼ 8.0 Hz, 1H), 8.07e7.87 (m, 3H), 6.58 (d, J ¼ 16.0 Hz, 1H), 3.87
(s, 3H).
6.1.26. 3-(5-aminobenzo[b]thiophen-3-yl)propan-1-ol (41)
45 (140 mg 72%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
d 11.38
To a solution of 40 (3.6 g, 13.7 mmol) in dry THF (100 mL) with
an ice bath was added NaBH4 (4.2 g, 109.6 mmol) slowly. The re-
action was stirred at room temperature overnight. AlCl3 (3.6 g,
27.4 mmol) was added with an ice bath and the reaction was stirred
at reflux temperature for 2 days. The reaction was quenched by
saturated NH4Cl aqueous solution under ice bath. The solid was
filtrated off, washed with EA (50 mL) and the resulting filtrate was
extracted with EA (50 mL * 3). The combined organic phase was
dried over Na2SO4, filtrated and concentrated. The residue was
purified by silica gel column chromatography (DCM/MeOH ¼ 50/1)
to give the desired product 41 (1.8 g. 64%) as a yellow solid. 1H NMR
(d, J ¼ 13.6 Hz, 1H), 8.70 (d, J ¼ 13.6 Hz, 1H), 7.78 (d, J ¼ 8.0 Hz, 1H),
7.40 (s, 1H), 7.37 (d, J ¼ 8.8 Hz, 1H), 4.30e4.08 (m, 2H), 3.54 (dd,
J ¼ 24.4, 12.8 Hz, 2H), 2.62e2.24 (m, 2H), 2.24e2.02 (m, 2H), 1.77 (s,
6H).
6.1.31. 80-hydroxy-4,5-dihydro-20H,3H-spiro[furan-2,30-thieno[2,3-
g]quinoline] 10,10-dioxide (46)
The diphenyl ether (10 mL) was added to a round-bottomed
flask and the solvent was heated to 240 ꢀC for 5 min. Intermedi-
ate 45 (140 mg, 0.36 mmol) was added slowly to the solution. The
mixture was stirred for 5 min. After cooling to room temperature,
15

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
the resulting solid was collected via filtration, washed with ether
(5 mL) and dried in vacuum to give the desired product 46 (52 mg,
10 min. Precipitate was filtered off and washed with MeOH. The
resulting filtrate was evaporated. The residue was purified by silica
gel column chromatography (PE/EA ¼ 2/1) to give the desired
product 51 (1.8 g, 25%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
50%) as a yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 12.08 (s, 1H),
8.29 (s, 1H), 8.03 (t, J ¼ 6.0 Hz, 1H), 7.73 (s, 1H), 6.14 (d, J ¼ 7.2 Hz,
1H), 4.13e4.00 (m, 2H), 3.97 (d, J ¼ 13.6 Hz, 1H), 3.61 (d, J ¼ 13.2 Hz,
1H), 2.46e2.37 (m, 1H), 2.23e2.11 (m, 3H). LCMS (ESI/APCI) m/z:
d
7.47e7.38 (m, 1H), 7.38e7.29 (m, 3H), 7.29e7.20 (m, 3H),
7.17e7.08 (m, 1H), 7.07e6.96 (m, 1H), 4.53 (s, 2H), 4.16 (d,
J ¼ 11.2 Hz, 2H), 4.07 (d, J ¼ 10.4 Hz, 2H), 3.94 (s, 2H). LCMS (ESI/
291.8 [M þ H] þ
.
APCI) m/z: 290.9 [M þ H] þ
.
6.1.32. 8’-(benzo[d]thiazol-5-ylamino)-4,5-dihydro-20H,3H-spiro
[furan-2,30-thieno[2,3-g]quinoline] 10,10-dioxide (47)
6.1.36. 3-((benzyloxy)methyl)-3-(2-fluorophenyl)oxetane (52)
To a solution of 51 (1.8 g, 6.2 mmol) in dry THF (20 mL) with an
ice bath under N2 atmosphere was added n-BuLi (2.5 mL, 6.2 mmol)
dropwise. The reaction was stirred at 0 ꢀC for 10 min. A solution of
p-TsCl (1.2 g, 6.2 mmol) in the THF (5 mL) was added and the
mixture was stirred for 10 min at 0 ꢀC. A solution of n-BuLi in
hexanes (2.5 mL, 6.2 mmol) was added dropwise and the reaction
was stirred at 55 ꢀC overnight. The reaction mixture was cooled to
Intermediate 46 (20 mg, 0.07 mmol) was added to POCl3 (2 mL)
and then the mixture was stirred at reflux for 2 h to afford a light
brown solution. After cooling to room temperature, the excess
POCl3 was removed in vacuum. The residue was dissolved in EtOH
(2 mL) and benzo[d]thiazol-5-amine (13 mg, 0.08) was added
subsequently. The mixture was stirred at reflux for 2 h. After
cooling to room temperature. the solid was precipitated out of the
solution. The solid was collected via filtration, washed with EtOH
and dried in vacuum to give the desired product 47 (15 mg, 51%) as
0
ꢀC and saturated NH4Cl aqueous solution was added to quench
the reaction. The mixture was extracted with EA (30 mL * 3). The
combined organic phase was dried over Na2SO4, filtered and
concentrated. The residue was purified by silica gel column chro-
matography (PE/EA ¼ 20/1) to give the desired product 52 (1.1 g,
a hydrochloride salt. 1H NMR (400 MHz, DMSO‑d6)
d 14.67 (s, 1H),
11.41 (s, 1H), 9.55 (s,1H), 9.36 (s,1H), 8.61 (d, J ¼ 6.4 Hz,1H), 8.40 (d,
J ¼ 8.4 Hz, 1H), 8.25 (s, 2H), 7.62 (d, J ¼ 8.0 Hz, 1H), 6.97 (d,
J ¼ 6.8 Hz, 1H), 4.21e4.03 (m, 3H), 3.79 (d, J ¼ 13.5 Hz, 1H),
2.49e2.43 (m, 1H), 2.29e2.17 (m, 3H). 13C NMR (151 MHz,
61%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
d 7.32e7.21 (m,
4H), 7.21e7.15 (m, 2H), 7.15e7.08 (m, 1H), 7.08e6.96 (m, 2H), 5.00
(d, J ¼ 5.6 Hz, 2H), 4.79 (d, J ¼ 5.2 Hz, 2H), 4.52 (s, 2H), 3.88 (s, 2H).
DMSO‑d6) d 161.6, 158.5, 155.9, 153.9, 147.3, 144.7, 141.2, 137.4, 135.2,
132.9, 124.1, 122.9, 119.6, 118.6, 118.1, 116.4, 100.9, 83.4, 69.5, 61.4,
39.9, 39.8, 39.7, 39.5, 39.5, 39.4, 39.2, 39.1, 25.9. HRMS (ESI) calcd for
LCMS (ESI/APCI) m/z: 272.9 [M þ H] þ
.
C
21H17N3O3S2, [M þ H] þ, 424.0784, found, 424.0784. Purity: 96.8%.
6.1.37. (3-(2-fluorophenyl)oxetan-3-yl)methanol (53)
To a solution of 52 (1.1 g, 4.0 mmol) in MeOH (20 mL) was added
10% Pd/C (21 mg, 0.2 mmol). The reaction was stirred at room
temperature under H2 atmosphere for 12 h. The Pd/C was filtrated
off and the cake was washed with MeOH. The resulting filtrate was
concentrated to give the desired product 53 as a colorless oil
6.1.33. Dimethyl 2-(2-fluorophenyl)malonate (49)
To a solution of 48 (10.3 g, 61.3 mmol) and dimethyl carbonate
(16.6 g,183.9 mmol) in dry THF (200 ml) with an ice bath was added
60% NaH (9.8 g, 245.2 mmol). The mixture was stirred at 70 ꢀC
overnight. The reaction was quenched by saturated NH4Cl aqueous
solution with an ice bath. The aqueous layer was extracted with
DCM (100 mL * 3). The combined organic phase was dried over
Na2SO4, filtered and concentrated. The residue was purified by
silica gel column chromatography (PE/EA ¼ 20/1) to give the
desired product 49 (11.2 g, 81%) as a yellow oil. 1H NMR (400 MHz,
(650 mg, 89%). 1H NMR (400 MHz, CDCl3)
d 7.32e7.27 (m, 1H),
7.20e7.12 (m, 1H), 7.10e6.96 (m, 2H), 5.01 (d, J ¼ 6.0 Hz, 2H), 4.76
(d, J ¼ 6.0 Hz, 2H), 4.10 (s, 2H).
6.1.38. 3-(chloromethyl)-3-(2-fluorophenyl)oxetane (54)
To a solution of 53 (420 mg, 2.3 mmol) in CCl4 (20 mL) was
added PPh3 (1.2 g, 4.6 mmol). The reaction was stirred at 90 ꢀC for
24 h. The solvent was removed in vacuum and the residue was
purified by silica gel column chromatography (PE/EA ¼ 40/1) to
give the desired product 54 (440 mg, 96%) as a colorless oil. 1H NMR
CDCl3)
d 7.50e7.39 (m, 1H), 7.37e7.27 (m, 1H), 7.20e7.12 (m, 1H),
7.12e7.01 (m, 1H), 5.01 (s, 1H), 3.77 (s, 6H).
6.1.34. Dimethyl 2-((benzyloxy)methyl)-2-(2-fluorophenyl)
malonate (50)
(400 MHz, CDCl3)
d
7.34e7.27 (m, 1H), 7.16 (t, J ¼ 7.6 Hz, 1H),
To a solution of 49 (11.2 g, 49.6 mmol) and Cs2CO3 (32.3 g,
99.2 mmol) in DMF (130 mL) with an ice bath was added ((chlor-
omethoxy)methyl)benzene (11.7 g, 74.4 mmol) dropwise. The
mixture was stirred at room temperature for 1 h. The solvent was
concentrated and water (400 mL) was added. The aqueous layer
was extracted with EA (200 mL * 3). The combined organic phase
was dried over Na2SO4, filtered and concentrated. The residue was
purified by silica gel column chromatography (PE/EA ¼ 20/1) to
give the desired product 50 (12.6 g, 73%) as a colorless oil. 1H NMR
7.10e6.99 (m, 2H), 5.02 (d, J ¼ 6.4 Hz, 2H), 4.74 (d, J ¼ 6.4 Hz, 2H),
4.11 (s, 2H).
6.1.39. 3-(chloromethyl)-3-(2-fluoro-5-nitrophenyl)oxetane (55)
To a solution of 54 (440 mg, 2.2 mmol) in conc.H2SO4 (5 mL)
with an ice bath was added fuming nitric acid (1 mL) dropwise. The
reaction was stirred at room temperature for 5 min. The mixture
was poured into cooled water (50 mL). The aqueous was extracted
with EA (10 mL * 3). The combined organic phase was dried over
Na2SO4, filtered and concentrated to give the desired product 55
(520 mg, 93%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
d 7.51e7.41 (m, 1H), 7.35e7.27 (m, 3H), 7.25e7.18
(m, 3H), 7.16e7.09 (m, 1H), 7.07e6.98 (m, 1H), 4.58 (s, þ2H), 4.19 (s,
2H), 3.78 (s, 6H). LCMS (ESI/APCI) m/z: 346.8 [M þ H]
.
d 8.30e8.19 (m, 1H), 8.03e7.95 (m, 1H), 7.24e7.18 (m, 1H), 5.03 (d,
J ¼ 5.2 Hz, 2H), 4.75 (d, J ¼ 5.6 Hz, 2H), 4.16 (s, 2H).
6.1.35. 2-((benzyloxy)methyl)-2-(2-fluorophenyl)propane-1,3-diol
(51)
6.1.40. 5-Nitro-2H-spiro[benzo[b]thiophene-3,30-oxetane] (56)
To a solution of 55 (520 mg, 2.0 mmol) in DMSO (30 mL) was
added Na2S.9H2O (768 mg,3.2 mmol). The reaction was stirred at
room temperature for 3 h. Water (80 mL) was added to the reaction
and the aqueous was extracted with EA (20 mL * 2). The combined
organic phase was dried over Na2SO4, filtered and concentrated.
The residue was purified by silica gel column chromatography (PE/
To a solution of 50 (8.6 g, 24.9 mmol) in dry THF (100 mL) with
an ice bath was added LiAlH4 (3.8 g, 99.6 mmol) slowly. The reac-
tion was stirred at room temperature for 2 h. The reaction mixture
was cooled to 0 ꢀC and H2O (3.8 mL) was added dropwise and
stirred for 5 min then 15% NaOH sol. (3.8 mL) was added and stirred
for 10 min after that H2O (11.4 mL) was added and stirred for
16

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
EA ¼ 10/1) to give the desired product 56 (240 mg, 51%) as a yellow
subsequently. Pd2(dba)3 (18 mg, 0.02 mmol), Xantphos (12 mg,
0.02 mmol), Cs2CO3 (359 mg, 1.1 mmol) was added. The reaction
was stirred at 100 ꢀC for 1 h under N2 atmosphere. The solvent was
removed in vacuum and the resulting residue was purified by silica
gel column chromatography (DCM/MeOH ¼ 20/1) to give the
desired product 61 (15 mg, 33%) as a yellow solid. 1H NMR
sold. 1H NMR (400 MHz, CDCl3)
d
8.43 (s,1H), 8.11 (d, J ¼ 8.8 Hz,1H),
7.31 (d, J ¼ 8.8 Hz, 1H), 4.85 (d, J ¼ 6.4 Hz, 2H), 4.82 (d, J ¼ 6.4 Hz,
2H), 3.78 (s, 2H).
6.1.41. 5-Nitro-2H-spiro[benzo[b]thiophene-3,30-oxetane] 1,1-
dioxide (57)
(400 MHz, DMSO‑d6) d 9.59 (s, 1H), 9.45 (s, 1H), 8.99 (s, 1H), 8.63 (d,
To a solution of 56 (240 mg, 1.08 mmol) in DCM (15 mL) with an
ice bath was added 85% m-CPBA (543 mg, 2.69 mmol). The mixture
was stirred at room temperature overnight. The solid was filtered
off and the filtrate was quenched with Na2SO3, extracted with DCM
(20 mL * 3). The combined organic phase was washed with satu-
rated NaHCO3 aqueous solution, dried over Na2SO4, filtered and
concentrated. The residue was purified by silica gel column chro-
matography (DCM/MeOH ¼ 50/1) to give the desired product 57
J ¼ 5.6 Hz,1H), 8.47 (s,1H), 8.23 (d, J ¼ 8.8 Hz,1H), 8.07 (d, J ¼ 1.7 Hz,
1H), 7.56 (dd, J ¼ 8.8, 1.6 Hz, 1H), 7.09 (d, J ¼ 5.6 Hz, 1H), 4.97 (d,
J ¼ 6.4 Hz, 2H), 4.94 (d, J ¼ 6.4 Hz, 2H), 4.23 (s, 2H). 13C NMR
(151 MHz, DMSO‑d6)
d 157.5, 154.1, 153.6, 151.5, 149.2, 141.2, 138.5,
135.2, 129.4, 124.9, 123.2, 121.5, 119.6, 116.7, 116.4, 102.1, 80.7, 59.3,
43.9, 39.9, 39.8, 39.7, 39.5, 39.5, 39.4, 39.2, 39.1. HRMS (ESI) calcd for
C
20H15N3O3S2, [M þ H] þ, 410.0628, found, 410.0628. Purity: 98.3%.
(220 mg, 80%) as a white solid. 1H NMR (400 MHz, CDCl3)
d
8.85 (s,
6.2. In vitro biological assays
1H), 8.44 (d, J ¼ 7.6 Hz, 1H), 7.92 (d, J ¼ 8.4 Hz, 1H), 5.04 (d,
J ¼ 6.8 Hz, 2H), 4.95 (d, J ¼ 6.8 Hz, 2H), 3.91 (s, 2H).
6.2.1. Cell culture
Human colon cancer HT-29 (from ATCC) and mouse embryonic
fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s
medium (Hyclone) supplemented with 10% fetal bovine serum
6.1.42. 5-Amino-2H-spiro[benzo[b]thiophene-3,30-oxetane] 1,1-
dioxide (58)
To a solution of 57 (220 mg, 0.86 mmol) in EtOH (8 mL) and H2O
(3 mL) was added iron powder (193 mg, 3.44 mmol) and NH4Cl
(182 mg, 3.44 mmol). The mixture was stirred at 80 ꢀC for 2 h. The
solid was filtered through diatomaceous earth and the cake was
washed with DCM. The resulting filtrate was extracted with DCM
(20 mL * 2). The combined organic phase was dried over Na2SO4,
filtrated and concentrated to give the desired product 58 (200 mg,
(Invitrogen), 2 mM L-glutamine (Invitrogen) and 100-units/ml
penicillin/streptomycin (Hyclone) in a humidified incubator at
37 ꢀC and 5% CO2.
6.2.2. Cell viability assay
HT-29 cells or MEFs seeded in 96-well plates were treated with
or without the indicated compound for 1h prior to the treatment of
93%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
d
7.47 (d,
DMSO or TNFa, Smac mimetic and z-VAD (Bachem). After 24h, the
J ¼ 8.4 Hz,1H), 7.10 (s,1H), 6.74 (d, J ¼ 8.0 Hz,1H), 4.95 (d, J ¼ 6.4 Hz,
cell viability was determined by assessment of ATP levels using the
CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).
Luminescence was measured with SpectraMax i3x (Molecular De-
vices). Smac mimetic was kindly provided by Dr. Xiaodong Wang
(National Institute of Biological Sciences, Beijing).
2H), 4.87 (d, J ¼ 6.0 Hz, 2H), 4.30 (s, 2H), 3.75 (s, 2H). LCMS (ESI/
APCI) m/z: 225.8 [M þ H] þ
.
6.1.43. 5-(((1,1-Dioxido-2H-spiro[benzo[b]thiophene-3,30-oxetan]-
5-yl)amino)methylene)-2,2-dimethylꢁ1,3-dioxane-4,6-dione (59)
To a solution of 58 (200 mg, 0.8 mmol) in EtOH (4 mL) was
6.2.3. Kinase binding (Kd) assay [45]
added
5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-
The binding affinity of the test compounds for kinases was
detected by a KINOMEscan assay. Kinase-tagged T7 phage strains
were prepared in an E. coli host derived from the BL21 strain. E. coli
were grown to log-phase and infected with T7 phage and incubated
with shaking at 32 ꢀC until lysis. The lysates were centrifuged and
filtered to remove cell debris. The remaining kinases were pro-
duced in HEK-293 cells and subsequently tagged with DNA for qPCR
detection. Streptavidin-coated magnetic beads were treated with
biotinylated small molecule ligands for 30 min at room tempera-
ture to generate affinity resins for kinase assays. The liganded beads
were blocked with excess biotin and washed with blocking buffer
(SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove
unbound ligand and to reduce non-specific binding. Binding re-
actions were assembled by combining kinases, liganded affinity
beads, and test compounds in 1x binding buffer (20% SeaBlock,
0.17x PBS, 0.05% Tween 20, 6 mM DTT). All reactions were per-
formed in polypropylene 384-well plate. Each was a final volume of
0.02 ml. The assay plates were incubated at room temperature with
shaking for 1 h and the affinity beads were washed with wash
buffer (1x PBS, 0.05% Tween 20). The beads were then re-
dione (223 mg, 1.2 mmol). The mixture was stirred at room tem-
perature for 20 min. The resulting solid was collected via filtration,
washed with EtOH (5 mL) and dried in vacuum to give the desired
product 59 (240 mg, 79%) as a yellow solid. 1H NMR (400 MHz,
CDCl3)
d
11.47 (d, J ¼ 12.8 Hz,1H), 8.76 (d, J ¼ 13.6 Hz,1H), 7.87e7.74
(m, 2H), 7.43 (d, J ¼ 6.8 Hz, 1H), 5.03 (d, J ¼ 6.0 Hz, 2H), 4.89 (d,
J ¼ 6.0 Hz, 2H), 3.84 (s, 2H), 1.79 (s, 6H).
6.1.44. 80-hydroxy-20H-spiro[oxetane-3,30-thieno[2,3-g]quinoline]
10,10-dioxide (60)
The diphenyl ether (6 mL) was added to a round-bottomed flask
and the solvent was heated to 240 ꢀC for 5 min. Intermediate 59
(240 mg, 0.63 mmol) was added slowly to the solution. The mixture
was stirred for 5 min. After cooling to room temperature, the
resulting suspension was then filtered, washed with ether (2 mL)
and dried in vacuum to give the desired product 60 (110 mg, 63%) as
a yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 12.34 (s, 1H), 8.27 (s,
1H), 8.17 (s, 1H), 8.05 (d, J ¼ 7.6 Hz, 1H), 6.15 (d, J ¼ 7.2 Hz, 1H), 4.96
(d, J ¼ 6.0 Hz, 2H), 4.77 (d, J ¼ 6.4 Hz, 2H), 4.13 (s, 2H). LCMS (ESI/
þ
APCI) m/z: 277.8 [M þ H]
.
suspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 mM
non-biotinylated affinity ligand) and incubated at room tempera-
ture with shaking for 30 min. The kinase concentration in the el-
uates was measured by qPCR.
6.1.45. 8’-(benzo[d]thiazol-5-ylamino)-20H-spiro[oxetane-3,30-
thieno[2,3-g]quinoline] 10,10-dioxide (61)
To a solution of 60 (30 mg, 0.11 mmol) and pyridine (0.1 mL,
1.0 mmol) in dry DCM (4 mL) was added trifluoromethanesulfonic
anhydride (0.1 mL, 0.6 mmol) dropwise at 0 ꢀC. The reaction was
stirred at room temperature for 30 min. The solvent was removed
in vacuum and the residue was dissolved in dioxane (4 mL)
6.2.4. Kinase functional activity assay
6.2.4.1. ADP-Glo Luminescent Assay. Inhibition of ALK4, PI3Ka,
RIPK1 or RIPK3 was determined using an ADP-Glo Luminescent
Assay. Kinase was incubated with the indicated compound or
17

S. Wu, C. Xu, K. Xia et al.
European Journal of Medicinal Chemistry 217 (2021) 113327
DMSO for around 15 min in the assay buffer (20 mM MgCl2, 25 mM
Declaration of competing interest
HEPES pH 7.2, 12.5 mM b-glycerol phosphate, 5 mM EGTA, 12.5 mM
MnCl2, 2 mM EDTA and 2 mM DTT for RIPK1 and RIPK3; 40 mM Tris,
pH 7.5 20 mM MgCl2,1 mM DTT, 0.10%BSA for ALK4; 50 mM HEPES,
pH 7.5, 3 mM MgCl2, 1 mM EGTA,0.03%CHAPS, 100 mM NaCl,2 mM
Sudan He and Xiaohu Zhang of the manuscript entitled “Ring
Closure Strategy Leads to Potent RIPK3 Inhibitors” declare the
following conflict of interest: S. He and X. Zhang are co-founders,
consultants and shareholders of Accro Bioscience Inc., which sup-
ports research in their labs.
DTT for PI3K
ALK4; 25 M for PI3K
at room temperature (120 min for ALK4, RIPK1, RIPK3 and 60 min
for PI3K ). The luminescence was calculated to determine the ki-
a
). Substrate and ATP (50
mM for RIPK1, RIPK3; Km for
m
a
) were added and the mixture was incubated
a
Acknowledgements
nase activity using the ADP-Glo Kinase Assay kit following the
manufacture’s instructions (Promega).
This work was supported by National Natural Science Founda-
tion of China (Grant No. 81973161, 81773561, 21502133, 31671436,
31830051), the Priority Academic Program Development of the
Jiangsu Higher Education Institutes (PAPD), the Jiangsu Key Labo-
ratory of Neuropsychiatric Diseases (BM2013003), the CAMS
Innovation Fund for Medical Sciences (CIFMS; 2019-I2M-1e004,
2019-I2M-1e003, and 2016-I2M-1e005) and Non-profit Central
Research Institute Fund of Chinese Academy of Medical Sciences
(2019PT310028, 2017NL31004, 2017NL31002). We are grateful to
Prof. Youyong Li in the Institute of Functional Nano & Soft Materials
(FUNSOM) at Soochow University for providing Schrodinger soft-
ware for molecular docking.
6.2.4.2. Mobility shift assay. Inhibition of EGFR, PDGFRa or CDK4/
CycD3 was determined using a mobility shift assay. Kinase was
incubated with the indicated compound for 10 min at room tem-
perature. FAM-labeled peptide and ATP (Km) were added and the
mixture was incubated at 28 ꢀC for 60 min on the 384-well assay
plate. Data was collected on Caliper.
6.2.4.3. Lance Ultra Assay. Inhibition of m-TOR protein kinase was
determined using a Lance Ultra Assay. Kinase was incubated with
the indicated compound in the assay buffer (50 mM HEPES, pH 7.5,
10 mM MgCl2, 3 mM MnCl2, 1 mM EGTA, 0.01% Tween-20). ATP
(Km) and substrate were added and the mixture was incubated at
28 ꢀC for 60 min on the 384-well assay plate. Kinase quench buffer
and antibody were prepared as detection solution and the solution
was added to the reaction. After 60 min at room temperature,
values of Lance signal ratio were collected from Envision program
(665nm/615 nm) and converted to percent inhibition values.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113327.
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