Discovery of novel TNNI3K inhibitor suppresses pyroptosis and apoptosis in murine myocardial infarction injury

Article abstract of DOI:10.1016/j.ejmech.2020.112314

Myocardial infarction (MI) injury is a highly lethal syndrome that has, until recently, suffered from a lack of clinically efficient targeted therapeutics. The cardiac troponin I interacting kinase (TNNI3K) exacerbates ischemia-reperfusion (IR) injury via oxidative stress, thereby promoting cardiomyocyte death. In this current study, we designed and synthesized 35 novel TNNI3K inhibitors with a pyrido[4,5]thieno[2,3-d] pyrimidine scaffold. In vitro results indicated that some of the inhibitors exhibited sub-micromolar TNNI3K inhibitory capacity and good kinase selectivity, as well as cytoprotective activity, in an oxygen-glucose deprivation (OGD) injury cardiomyocyte model. Furthermore, investigation of the mechanism of the representative derivative compound 6o suggested it suppresses pyroptosis and apoptosis in cardiomyocytes by interfering with p38MAPK activation, which was further confirmed in a murine myocardial infarction injury model. In vivo results indicate that compound 6o can markedly reduce myocardial infarction size and alleviate cardiac tissue damage in rats. In brief, our results provide the basis for further development of novel TNNI3K inhibitors for targeted MI therapy.

Full text of DOI:10.1016/j.ejmech.2020.112314

Journal Pre-proof
Discovery of novel TNNI3K inhibitor suppresses pyroptosis and apoptosis in murine
myocardial infarction injury
Haiying Pang, Ning Wang, Jinlong Chai, Xiaoyun Wang, Yuehua Zhang, Zhiang Bi,
Wenbin Wu, Gu He
PII:
S0223-5234(20)30283-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112314
EJMECH 112314
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2020
Revised Date: 6 April 2020
Accepted Date: 6 April 2020
Please cite this article as: H. Pang, N. Wang, J. Chai, X. Wang, Y. Zhang, Z. Bi, W. Wu, G. He,
Discovery of novel TNNI3K inhibitor suppresses pyroptosis and apoptosis in murine myocardial
infarction injury, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
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Discovery of novel TNNI3K inhibitor suppresses pyroptosis and
apoptosis in murine myocardial infarction injury
1
,†
1,†
1
1
1
Haiying Pang, Ning Wang, Jinlong Chai, Xiaoyun Wang, Yuehua Zhang,
1
2
1,
Zhiang Bi, Wenbin Wu, and Gu He *
1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center, Chengdu, Sichuan 610041,
PR China
2
Department of Neurology, Chongzhou People’s Hospital, Chengdu 611230, China
†
These authors contributed equally to this work.

Abstract
Myocardial infarction (MI) injury is a highly lethal syndrome that has, until recently,
suffered from a lack of clinically efficient targeted therapeutics. The cardiac troponin I
interacting kinase (TNNI3K) exacerbates ischemia-reperfusion (IR) injury via
oxidative stress, thereby promoting cardiomyocyte death. In this current study, we
designed and synthesized 35 novel TNNI3K inhibitors with a pyrido[4,5]thieno[2,3-d]
pyrimidine scaffold. In vitro results indicated that some of the inhibitors exhibited
sub-micromolar TNNI3K inhibitory capacity and good kinase selectivity, as well as
cytoprotective activity, in an oxygen-glucose deprivation (OGD) injury
cardiomyocyte model. Furthermore, investigation of the mechanism of the
representative derivative compound 6o suggested it suppresses pyroptosis and
apoptosis in cardiomyocytes by interfering with p38MAPK activation, which was
further confirmed in a murine myocardial infarction injury model. In vivo results
indicate that compound 6o can markedly reduce myocardial infarction size, decrease
circulating cardiac troponin I (cTnI) leakage, and alleviate cardiac tissue damage in
rats. In brief, our results provide the basis for further development of novel TNNI3K
inhibitors for targeted MI therapy.
Keywords: TNNI3K inhibitor, Structure-activity relationships, Pyroptosis, Myocardial
infarction

1
. Introduction
Acute myocardial infarction (AMI), a type of myocardial necrosis associated
with acute, persistent ischemia and hypoxia in coronary arteries, can cause various
symptoms including arrhythmia, adverse remodeling, and systolic dysfunction. AMI
remains a leading cause of morbidity and death worldwide.[1] Timely treatment using
percutaneous coronary intervention (PCI), the preferred treatment of AMI, can
minimize ischemic injury, limit MI size, and inhibit ventricular remodeling to protect
heart function, and improve the survival rate of MI. However, at the same time,
reperfusion injury caused by PCI promotes reactive oxygen species (ROS) generation,
2
+
intracellular Ca overload and upregulation of pro-inflammatory molecules, which
leads to death of viable cardiomyocytes and cardiac dysfunction during ischemia.[2-4]
Although a large number of studies on various drugs and methods for preventing
myocardial cell death during myocardial ischemia and reperfusion have been
published,[5-8] patients with myocardial ischemia/reperfusion injury still lack
effective therapeutic drugs, especially novel cardio-protective and myocardial
recovery agents, which makes research into this field particularly important.[9-14]
Cardiac troponin I interacting kinase (TNNI3K), a member of the
mitogen-activated protein kinase kinase kinase (MAPKKK) family and the cardiac
ankyrin repeat kinase (CARK), is selectively expressed in heart tissue and is observed
in fetal and adult hearts. TNNI3K is almost non-existent in all other tissues except for
trace expression in the brain and testes.[15-19] In humans, the longest TNNI3K
isoform encodes a 92 kD protein composed of 835 amino acids that contain three

types of recognizable domains: 10 N-terminal ankyrin repeats, a central kinase
domain and a C-terminal Ser-rich domain.[15] TNNI3K overexpression and
knockout mouse models indicate that TNNI3K can increase infarct size in ischemic
hearts, induce mitochondrial ROS generation, cause mitochondrial dysfunction and
bioenergy damage in cardiomyocytes. TNNI3K is also associated with activation of
p38 mitogen-activated protein kinase MAPK to exacerbate ischemic injury and
myocardial cell death.[16, 20] Research indicates that TNNI3K likely plays a crucial
role in significant parts of cardiac biology and cardiac diseases including
supraventricular tachycardia, viral myocarditis, cardiac conduction, cardiomyopathy,
obesity and metabolic disease, as well as pathological and physiological
hypertrophy,[21-23] suggesting that TNNI3K is a promising treatment target for
cardiac disease.
Although TNNI3K is a potential therapeutic target for heart disease, there have
only been a few reports of feasibility studies of TNNI3K inhibitors as potential agents.
All preclinical study results were reported by Brian G. Lawhorn et al. In 2015,
7
-deazapurine derivatives was first reported as dual TNNI3K/B-Raf inhibitors. As
B-raf kinase inhibition has been linked to effects in heart failure models,
-deazapurine compounds did not fully characterize the function of TNNI3K in
cardiac biology.[24] Subsequently, GSK114, which developed based on
-deazapurine, displayed excellent cellular activity (IC =25nM) and showed
7
7
5
0
appreciable selectivity over B-Raf (>40 fold).[25] GSK854 was first reported in 2018
as highly selective TNNI3K inhibitor (IC =25nM),[26] but the
,6-diaminopyrimidine structure of GSK854 has been proven to be clinically relevant
and an extensively explored structural class of kinase inhibitors, such as EGFR.[24-26]
a
5
0
4

GSK114 and GSK854 serve as useful TNNI3K inhibitors for cellular and in vivo
models and are available as probes, which can help elucidate the biological pathways
that are associated with TNNI3K in heart disease (Figure 1A). However, there is no
clinical data from GSK114 and GSK854.[24-27] We attempted to develop a novel
and sufficiently selective TNNI3K inhibitor based on the interaction between
GSK854 and the known crystal structure of TNNI3K by means of framework
transitions, ring fusion and other mechanisms (Figure 1B).
In this current study, we reported the rationale, the design, chemical synthesis,
and in vitro and in vivo evaluation of pyrido[4,5] thieno[2,3-d] pyrimidine-based
TNNI3K inhibitors as a potential lead compound against AMI. In vitro kinase
inhibition and selectivity assays, cell proliferation and programmed cell death assays
were measured, alongside the molecular mechanisms of these novel TNNI3K
inhibitors. These results suggested that the novel TNNI3K inhibitors
pyrido[4,5]thieno[2,3-d] pyrimidine derivatives efficiently suppressed ROS
accumulation, apoptotic and pyroptotic cell death in oxygen and glucose deprivation
(OGD) H9c2 cells via a p38-MAPK signaling pathway. Furthermore,
pyrido[4,5]thieno[2,3-d] pyrimidine derivatives significantly reduced cardiomyocyte
apoptosis, pyroptosis and tissue fibrosis in a rat myocardial ischemia/reperfusion
model without obvious morphological changes in the main organs, which suggests
that these TNNI3K inhibitors are worth further development.

Figure 1. (A) Chemical structures of representative TNNI3K inhibitors; (B) The
scaffold hoping strategy of novel TNNI3K inhibitors.
2
2
. Results and Discussion
.1 Chemistry
The synthesis of target compounds is depicted on Scheme 1. A series of
differently substituted N-(5-amino-2-(dimethylamino) phenyl) amide derivatives were
generated according to Scheme 1. 2-fluoro-5-nitroaniline 1 reacted with
dimethylamine to generate compound 2, and amination of compound 2
with appropriate acid chloride generated the corresponding products 3a-h. Compound
4
was developed from
a
previously reported procedure.[28-31] First,
n-(tert-butoxycarbonyl)-4-piperidone was treated with ethyl cyanoacetate and S8
through a Gewald reaction. Then, the compound reacted with formamidine acetate to

form a pyrimidine ring. Finally, the product was chlorinated using phosphorus
oxychloride and was converted to intermediate 4.
After obtaining the key intermediates 4 and 3a-h, the nitro-compounds 3a-h were
reduced by zinc powder, NH Cl and CH COOH at 80�
to generate
amino-compounds without further purification which were then coupled with chloride
in isopropyl alcohol overnight. This was followed by Boc deprotection under acidic
4
3
4
conditions to give the desired compounds 5a-h. Finally, the compounds 5a-h were
converted to the corresponding target product 6a-p through alkylation using various
substituted halogen compounds and 7a-k through amide condensation reaction using
various substituted aromatic acids.
Scheme 1. Synthesis of novel TNNI3K inhibitors. Reagents and conditions: (a) Dimethylamine, EtOH
at RT for 15d. (b) RCOCl, DCM, Et3N at RT for 1h, (c) (1) Zn, NH4Cl, CH COOH, EtOH at 80� for
3
3
h, (2) Isopropyl alcohol, HCl at 85� overnight, (3) TFA, DCM at RT for 30min, (d) R-X, DIEA,
EtOH at 80� overnight. (e) RCOOH, HATU, DIEA, DCM at RT for 30 min.
2
.2 The SAR (structure-activity relationship) analysis and binding modes of
novel TNNI3K inhibitors

The kinase inhibitory abilities of the compounds 6a-p and 7a-k are shown in
Table 1, including the percentage of 1.0 μM inhibitor incubated with TNNI3K, EGFR,
HER2, Flt1(VEGFR1) and KDR(VEGFR2) kinase activity. Table 1 shows the
1
2
comparison between the bioactivities of different R and R fragments based on the
pyrido[4,5]thieno[2,3-d]pyrimidine scaffold. We utilized EGFRs and VEGFRs as
models to test the off-target efficiency of synthesized compounds. The results of the
kinase inhibitory assays suggest that all compounds have few off-target activities to
EGFRs and VEGFRs. Moreover, results from our study suggest that a moderate steric
2
volume at R position is favorable to TNNI3K inhibition. For example, the naphthyl
or 4-methylphenyl groups resulted in low inhibitory capacities on TNNI3K.
2
Additionally, the 2-furyl or 2-thienyl substitutions at the R position demonstrate
better inhibitory capacities than the other substitutions (also see Figure S1). A panel of
1
aromatic carboxyl substitutions on R position were prepared and their kinase
inhibitory capacities were tested. However, these aromatic carboxyl substitutions at
1
the R position did not display better TNNI3K inhibition or kinase selectivity
compared to alkylation substitutions. The alkyl chains with a positively charged
1
terminal group at R position demonstrated better TNNI3K inhibitory activities
compared to simple alkyl or aromatic substitutions.
The 11 compounds with better TNNI3K inhibition at 1.0μM were further
evaluated for their half-inhibitory concentrations on TNNI3K, as well as the
cytoprotective effects on cellular proliferation of OGD H9c2 cardiomyocyte cell lines.
The corresponding results are listed in Table 2. Compound 6o demonstrates the best
inhibitory activity on TNNI3K inhibition with an IC value of 0.41 μM. Additionally,
5
0
all 11 compounds exhibit potent cytoprotective effects on OGD H9c2 cells. The cell
viability of OGD H9c2 cells go from 40% to 70-90% after incubation with 1.0 or

1
0μM TNNI3K inhibitor. Furthermore, we evaluated the off-target kinase inhibition
of compound 6o using a panel of 104 kinases through the KINOMEscan® method. As
shown in Table S1, after incubation with 10 μM of compound 6o, only one member of
the MAP3K family, TAK1, was significantly suppressed while the other kinases
showed over 50% activity. Thus, compound 6o was selected for further studies to
understand the molecular mechanism and to evaluate its efficacy in in vivo murine
myocardial infarction models.
Table 1. The remained kinase activities (%) after 1μM compounds 6a-p and 7a-k
incubation on a panel of TKs.
No.
R1
R2
Remained kinase activities (%) @1.0μM
TNNI3K EGFR
HER2
102
Flt1
107
KDR
92
5
a
H
H
CH CH
46
78
97
2
3
5
b
4-MeC H
108
92
96
95
6
4
5
c
H
H
2-pyridy
3-pyridy
4-pyridy
42
45
39
71
35
38
49
103
108
90
98
107
109
90
96
104
103
105
96
95
90
92
88
92
95
82
5
d
5
e
H
5
f
H
1-naphthyl
2-furyl
110
100
93
5
5
g
h
H
91
H
2-thienyl
CH CH
106
109
101
95
6
a
CH3
99
2
3
3
3
6
b
CH CH3
CH CH
53
48
93
91
90
94
109
106
80
85
2
2
6
c
Bn
CH CH
2
6
d
2-pyrimidinyl
CH CH
59
71
102
108
99
92
106
97
80
83
2
3
6
e
CH3
4-MeC H
6
4
6
f
CH3
CH3
CH3
CH3
CH3
CH3
2-pyridy
3-pyridy
4-pyridy
55
47
46
77
37
40
100
105
105
98
103
93
94
89
94
84
87
89
89
6
6
g
h
103
105
101
105
108
97
6
i
j
1-naphthyl
2-furyl
96
6
93
95
6
k
2-thienyl
104
103

6
l
CH CH
37
33
38
105
90
108
107
103
90
100
98
86
88
93
2
3
6
m
2-furyl
6
n
2-thienyl
86
6
o
2-furyl
28
33
81
86
96
91
98
80
81
6
p
2-thienyl
102
7
a
CH CH
41
39
41
35
54
43
40
45
36
49
107
104
110
107
82
103
89
109
86
95
77
93
92
81
90
73
69
82
86
2
3
7
b
2-furyl
7
c
2-furyl
2-furyl
2-furyl
2-furyl
101
116
98
89
7
d
109
101
98
7
e
7
f
89
96
7
g
h
2-thienyl
2-thienyl
2-thienyl
2-thienyl
99
99
87
7
94
93
81
7
7
i
j
85
110
112
85
92
104

7
k
2-thienyl
44
80
113
98
80
Compound 6o was docked into the ATP site of TNNI3K by using CDOCKER
module of Accelrys Discovery Studio 3.5 packages. The crystalized structure of
TNNI3K kinase domain was retrieved from Protein Databank (PDB No. 4YFF), the
protein preparation and molecular docking were according to the software operating
manuals. The 3D and 2D contours of compound 6o bound to the ATP sites of
TNNI3K are shown in Figure 2A and 2B, respectively. We speculate that the N1 atom
of the pyrimidine ring forms stable hydrogen bonds with the Ile542 residue, and the
oxygen atom of the furan-carboxylic group forms a hydrogen bond with Thr539 of
TNNI3K, respectively. Moreover, the thieno[2,3-d] pyrimidine scaffold forms pi-pi
stack interactions with the Tyr541 residue. These interactions were similar to those of
the co-crystalized TNNI3K inhibitor.
Figure 2. The 3D contour (A) and 2D contour (B) of the binding mode of compound
6
o in ATP site of TNNI3K.
Table 2. The IC values (μM) of selected compounds on TNNI3K and their
5
0
cytoprotective effects on OGD H9c2 cells.

TNNI3K
No.
Cell viability (% of control)
IC (μM)
5
0
0
μM
1.0 μM
10 μM
5
e
0.96±0.09
0.73±0.10
0.78±0.08
0.56±0.04
0.48±0.06
0.64±0.07
0.33±0.03
0.24±0.03
0.57±0.05
0.82±0.08
0.98±0.07
49.11±5.94
47.98±7.01
44.98±3.73
42.40±4.32
45.53±6.64
42.08±4.58
46.22±6.47
46.02±4.55
46.58±4.42
42.74±4.70
44.62±4.37
52.97±8.10
54.15±8.27
56.85±6.59
59.62±6.13
62.41±6.69
61.08±6.48
69.07±5.20
73.82±7.80
58.51±4.17
54.33±6.23
49.14±5.74
68.13±5.56
71.01±8.83
75.88±12.45
85.56±7.90
82.18±11.17
79.86±6.63
81.78±8.17
88.95±7.84
78.51±5.49
70.94±10.07
70.68±5.27
5
g
h
5
6
6
j
l
6m
6
n
o
p
b
d
6
6
7
7
2
.3 Novel TNNI3K inhibitor 6o efficiently suppressed ROS accumulation,
apoptotic and pyroptotic cell death in oxygen and glucose deprivation (OGD)
induced H9c2 cells via the p38-MAPK signaling pathway.
To evaluate the role of TNNI3K inhibitor 6o on cell death of cardiomyocytes, we
focused on the effect of 6o on cell apoptosis in OGD-induced H9c2 rat
cardiomyocytes. Apoptosis was determined mainly by the Annexin-V/PI dual-staining
apoptosis assay, MTT method, LDH activity detection, as well as western blotting of
apoptosis and pyroptosis biomarkers.[32-34] It was observed that compound 6o
remarkably alleviated OGD-induced apoptosis in H9c2 rat cardiomyocytes in a
dosage-dependent manner, based on Annexin-V/PI dual-staining apoptosis assay and
LDH cytotoxicity assay (Figure 3A-B). Meanwhile, cytochrome C levels were

significantly decreased under the dose-dependent condition of compound 6o (Figure
C), which further indicated that compound 6o could inhibit the mitochondrial
3
apoptosis pathway.
TNNI3K is a member of the MAP3K superfamily according to structural
homology.[15] It has been reported that TNNI3K specifically activates the p38
pathway in cardiomyocytes.[16] Therefore, we examined the activation of the classic
MAPK cascade in OGD-induced H9c2 rat cardiomyocytes under a dose-dependent
condition of compound 6o. Western blot analysis shows that treatment with
compound 6o declined the phosphorylation levels of p38, while protein expression of
total TNNI3K and p38 were not affected (Figure 3E). Moreover, no significant
differences were found in the extracellular signal regulated kinase 1/2 (ERK 1/2),
c-Jun N-terminal kinase (JNK) and their activation pathway (Figure 3E). These results
indicate that compound 6o is capable of specifically reducing activation of the
p38-MAPK pathway and myocardial cell death. Pyroptoisis can be achieved by
activating the Caspase-1 pathway. NOD like receptor pyrin domain-containing protein
3
(NLRP3) binds the adaptor protein ASC and recruits Caspase-1 precursor to form
the NLRP3 inflammasome, leading to the activation of Caspase-1.[35-40]

Figure 3. (A) and (B) Apoptosis in ODG-induced H9c2 cells incubated with 0.5 or 1.0 μM compound
o for 24h, based on dual staining with annexin-V/propidium iodide (PI) followed by flow cytometry.
C) LDH activity detection in ODG-induced H9c2 cells incubated with 0.5 or 1.0 μM compound 6o for
4h. (D) Cell viability of ODG-induced H9c2 cells treated with 0.5 or 1.0 μM compound 6o was
assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. **p <
.01. (E) Levels of TNNI3K, apoptosis-, and pyroptoisis-related proteins and phosphorylation status of
6
(
2
0

ERK1/2, JNK1/2, or p38 MAPK in ODG-induced H9c2 cells incubated with 0.5 or 1.0 μM compound
o assessed using Western blotting. (F) Intracellular distribution of caspase-1 and NLRP3 were
6
detected by immunofluorescent assay.
On the one hand, activated Caspase-1 cleaves GSDMD, causing an inflammatory
reaction. Additionally, it cleaves the precursors of IL-1β and IL-18 to form active
IL-1β and IL-18, which cause inflammatory cells to aggregate and expand the
inflammatory response.[41-44] We observed that compound 6o reduced the activation
caspase-1 and GSDMD without affecting the total protein levels of caspase-1 and
GSDMD. Furthermore, we observed inflammasome-associated pyroptoisis. Protein
levels of NLRP3, ASC and IL-1β were significantly reduced after incubation with
compound 6o, while pro-IL-1β showed no significant difference. Similarly,
fluorescence microscopy showed a significant decrease in protein expression and
intracellular co-localization between caspase-1 and NLRP3, indicating a potential
mechanism of action between TNNI3K inhibitor and pyroptosis.
2
.4 In vivo myocardial ischemia model and preliminary safety evaluation
An in vivo model of myocardial infarction in C57BL/6 male mice was
established to evaluate the therapeutic efficacy of compound 6o on myocardial
infarction at a dose of 25mg/kg per mice. TTC staining was used to stain mice hearts,
in which the infarct area of LV stained yellow-white and the living myocardium
appeared red. We observed less yellow-white area of the compound 6o-treated group
compared to the NS group. In comparison to the NS group, the compound 6o-treated
group showed less collagen deposition in the LV, as indicated by the size of blue
region in Masson, indicating a striking decreasing levels of scar tissue.[45-47]
Meanwhile, results from H&E and MT staining were consistent. Compared to the NS
group, compound 6o-treated group showed fewer left ventricular inflammatory cells
and significantly inhibited ventricular dilatation.

Figure 4. (A) TTC, Masson’s trichrome and H&E staining of heart sections. (B) IF or IHC analysis of
expression TUNEL, cleaved Caspase-1, TNNI3K, phosphorylated P38 and GSDMD (N-terminal) in
the heart sections of different groups after treatment for 28 days. (C) The quantitative analysis of
positive stained cells in (B).
We further analyzed the myocardial tissue after MI using immunofluorescence
and immunohistochemistry to verify whether compound 6o-regulated myocardial cell
recovery was related to TNNI3K inhibition and apoptosis/pyroptosis alleviation.
Immunofluorescent TUNEL assay was used to detect apoptosis in LV and caspase 1
+
activation. The results showed that less TUNEL signals (green) were observed within
the LV area in compound 6o-treated group compared to the NS group, indicating that
the NS group had more left ventricular myocardial cell apoptosis, a larger infarction
area, and a decrease in the activation level of caspase. Furthermore,
immunohistochemical staining showed that the expression of TNNI3K, p-p38 and
GSDMD was decreased. Results of the in vivo experiments indicated that compound

6
o has a protective effect on cardiomyocytes and showed an inhibitory effect on
TNNI3K. These results have demonstrated that the potential mechanism of action
between TNNI3K inhibitors and pyroptosis, with results of the in vivo experiment
being consistent with those of the in vitro experiments. The ADME properties of
compound 6o were predicted by the ADMET tools from the Discovery Studio 3.5
package. As depicted in Figure S2, the predicted AlogP , absorption and BBB
9
8
penetration properties of compound 6o were located in the acceptable range. The
H&E staining of tissue sections suggest that there were no significant changes to the
morphology of main organs of mice in both the compound 6o-treated and sham
groups (Figure S3). It is also worth pointing out that our results are performed with a
limited number of experimental animals and need to be validated in carefully
designed studies.
In conclusion, we report the design, synthesis and biological evaluation of novel
TNNI3K inhibitors with a pyrido[4,5] thieno[2,3-d]pyrimidine scaffold in
OGD-induced H9c2 cardiomyocyte cell lines. The most potent inhibitor, compound
6
o, remarkably suppressed ROS accumulation, apoptotic and pyroptotic cell death in
OGD-induced H9c2 cells. The detailed binding modes of compound 6o to TNNI3K
were probed to elucidate its kinase selectivity. Furthermore, the capacity of compound
6
o to suppress apoptosis and pyroptosis were validated in a model of myocardial
infarction in C57BL/6 male mice. Collectively, these results indicate that compound
o suppressed OGD-induced H9c2 cell apoptosis and pyroptosis by interfering with
6
the p38-MAPK signaling pathway, suggesting a novel molecular mechanism of these
novel TNNI3K inhibitors that are worth further study.
3
. Experimental section

3
.1 Materials and Regents
The antibodies targeted towards GSDMD, NLRP3, Caspase-1, Caspase-3,
Cytochrome C, p38MAPK, JNK, ERK1/2 and TUNEL assay kit were purchased from
the Proteintech Group Inc. (Wuhan, China). Antibodies targeted against TNNI3K,
CTnI, and the phosphorylated forms of CTnI, p38MAPK, JNK and ERK1/2 were
obtained from Abcam (Cambridge, MA). The horseradish peroxidase(HRP)-labeled
secondary antibodies were obtained from Cell Signal Technology (Boston, USA).
3
.2 Chemical synthesis
All materials and solvents were purchased commercially. The purity of reagents
was greater than 95% and could be used directly without further purification.
Anhydrous reagents were processed by conventional methods or purchased directly.
Column chromatography performed on silica gel (300-400 mesh) and thin-layer
chromatography silica gel plate were both purchased from the Qingdao Marine
Chemical Co., Ltd (Qingdao, Shandong, China) in order to purify the products. TLC,
carried out on glass-backed silica plates, was visualized with UV light and I . Bruker
2
1
13
Avance III 400 MHz spectrometers were used to record all H NMR spectra. C
NMR dates were collected at 100MHz with the same instrument using TMS as an
internal standard in CDCl . The δ ppm scale relative to TMS was used to describe the
3
chemical shifts. ESI-HRMS spectrum was determined with Waters SYNAPT G2
spectrometer.
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)propionamide (5a) as a white solid (yield 54%), m.p.:
1
9
4.6-102.0 � . H NMR (400 MHz, Chloroform-d) δ 8.60 (br s, 1H), 8.49 (s, 1H), 8.36
(d, J = 2.4 Hz, 1H), 7.76 (dd, J = 8.8, 2.4 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.03 (br. s,
1
H), 4.10 (t, J = 2.0 Hz, 2H), 3.28 (t, J = 5.6 Hz, 2H), 3.06 (m, 2H), 2.65 (s, 6H), 2.47
1
3
(
q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ
72.24, 166.28 , 155.17 , 152.80 , 138.76 , 135.14 , 134.00 , 133.54 , 123.79 , 120.60 ,
16.91 , 116.42 , 112.23 , 45.15 , 45.00 (2C) , 42.90 , 31.13 , 27.40 , 9.70 . HRMS
1
1
+
+
(
ESI) m/z calculated for C H N OS [M+H] 397.1805, found 397.1807.
20 25 6
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi

din-4-yl)amino)phenyl)-4-methylbenzamide (5b) as a white solid (yield 51%), m.p.:
1
1
2
8
2
1
95.0-197.1 � . H NMR (400 MHz, Chloroform-d) δ δ 9.47 (br s, 1H), 8.52 (d, J =
.4 Hz, 1H), 8.51 (s, 1H), 7.85 – 7.78 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H), 7.27 (d, J =
.8 Hz, 1H), 7.09 (br. s, 1H), 4.10 (t, J = 2.0 Hz, 2H), 3.30 (t, J = 5.6 Hz, 2H), 3.09 (m,
1
3
H), 2.71 (s, 6H), 2.44 (s, 3H). C NMR (101 MHz, Chloroform-d) δ 166.31 ,
65.21 , 155.19 , 152.81 , 142.45 , 139.24 , 135.30 , 134.18 , 133.56 , 132.16 , 129.53
(2C) , 127.02 (2C) , 123.83 , 120.68 , 117.14 , 116.46 , 112.26 , 45.16 (3C) , 42.92 ,
+
+
2
4
7.43 , 21.52 . HRMS (ESI) m/z calculated for C H N OS [M+H] 459.1962, found
25 27 6
59.1965.
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)picolinamide (5c) as a white solid (yield 56%), m.p.:
1
2
10.5-216.9 � . H NMR (400 MHz, Chloroform-d) δ 11.03 (br s, 1H), 8.69 (dd, J =
5
.6, 1.6 Hz , 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.52 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.91
(td, J = 8.0, 1.6 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.48 (m, 1H), 7.24 (d, 1H),
7
.07 (br. s, 1H), 4.12 (t, J = 2.0 Hz, 2H), 3.31 (t, J = 5.6 Hz, 2H), 3.10 (m, 2H), 2.76
1
3
(
s, 6H). C NMR (101 MHz, Chloroform-d) δ 166.31 , 162.30 , 155.26 , 152.89 ,
1
1
50.39 , 148.41 , 140.26 , 137.49 , 134.58 , 133.49 , 133.32 , 126.29 , 123.82 , 122.36 ,
20.27 , 117.63 , 116.41 , 112.74 , 45.18 , 44.82 , 42.95 , 27.46 . HRMS (ESI) m/z
+
+
calculated for C H N OS [M+H] 446.1758, found 446.1763.
2
3
24
7
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)nicotinamide (5d) as a yellow solid (yield 52%), m.p.:
1
1
86.6-190.7 � . H NMR (400 MHz, Chloroform-d) δ 9.62 (br s, 1H), 9.15 (d, J = 2.0
Hz, 1H), 8.80 (dd, J = 4.8, 2.0 Hz, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.52 (s, 1H), 8.26 (dt,
J = 8.0, 2.0 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H),
7
2
1
1
4
4
.31 (d, J = 8.8 Hz, 1H), 7.09 (br. s, 1H), 4.13 (d, J = 2.0 Hz, 2H), 3.32 (t, J = 5.6 Hz,
13
H), 3.12 (d, J = 5.6 Hz, 2H), 2.72 (s, 6H). C NMR (101 MHz, Chloroform-d) δ
66.40 , 163.23 , 155.12 , 152.79 , 152.57 , 147.90 , 139.29 , 135.48 , 135.24 , 133.73 ,
33.63 , 130.69 , 123.79 , 123.75 , 120.98 , 117.72 , 116.47 , 112.33 , 45.25 , 45.15 ,
+
+
2.91 , 27.40 . HRMS (ESI) m/z calculated for C H N OS [M+H] 446.1758, found
2
3
24
7
46.1765.

N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)isonicotinamide (5e) as a white solid (yield 55%), m.p.:
2
20.3-223.0 � . 1H NMR (400 MHz, Chloroform-d) δ 9.63 (br s, 1H), 8.88 – 8.83 (m,
H), 8.55 (d, J = 2.4 Hz, 1H), 8.52 (s, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.76 – 7.73
2
(m, 2H), 7.31 (d, J = 8.8 Hz, 1H), 7.09 (br s, 1H), 4.12 (t, J = 2.0 Hz, 2H), 3.31 (t, J =
1
3
5
.6 Hz, 2H), 3.10 (m, 2H), 2.73 (s, 6H). C NMR (101 MHz, Chloroform-d) δ
66.41 , 163.04 , 155.09 , 152.75 , 150.88 (2C) , 141.97 , 139.32 , 135.51 , 133.77 ,
33.45 , 123.73 , 120.97 , 120.75 (2C) , 117.94 , 166.48 , 112.38 , 45.26 (2C) , 45.17 ,
1
1
+
+
4
2.92 , 27.43 . HRMS (ESI) m/z calculated for C H N OS [M+H] 446.1758,
23 24 7
found 446.1761.
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)-1-naphthamide (5f) as a grey solid (yield 63%), m.p.:
1
1
29.5-135.6 � . H NMR (400 MHz, Chloroform-d) δ 9.20 (br s, 1H), 8.67 (d, J = 2.4
Hz, 1H), 8.53 (s, 1H), 8.44 (dd, J = 7.2, 2.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.91
dd, J = 7.2, 2.4Hz, 1H), 7.82 (dd, J = 8.8, 2.4 Hz, 1H), 7.75 (dd, J = 6.8, 1.2 Hz, 1H),
(
7
.60 – 7.50 (m, 3H), 7.28 (d, J = 8.8 Hz, 1H), 7.14 (br s, 1H), 4.08 (t, J = 2.0 Hz, 2H),
1
3
3
.29 (t, J = 5.6 Hz, 2H), 3.09 (m, 2H), 2.63 (s, 6H). C NMR (101 MHz,
Chloroform-d) δ 167.60 , 166.38 , 155.22 , 152.80 , 139.26 , 135.38 , 134.63 , 134.35 ,
1
33.83 , 133.58 , 131.09 , 130.20 , 128.46 , 127.30 , 126.59 , 125.35 , 125.10 , 124.82 ,
23.86 , 120.92 , 117.61 , 116.52 , 112.47 , 45.16 (3C) , 42.93 , 27.40 . HRMS (ESI)
1
+
+
m/z calculated for C H N OS [M+H] 495.1962, found 495.1963.
2
8
27
6
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)furan-2-carboxamide (5g) as a grey solid (yield 57%), m.p.:
1
2
8
7
3
07.7-213.1 � . H NMR (400 MHz, Chloroform-d) δ 9.54 (br s, 1H), 8.50 (s, 1H),
.48 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.57 (dd, J = 1.6, 0.8 Hz, 1H),
.25 (d, J = 8.8 Hz, 1H), 7.23 (dd, J = 3.6, 0.8 Hz, 1H), 7.06 (br s, 1H), 6.57 (dd, J =
.6, 1.6 Hz, 1H), 4.10 (t, J = 2.0 Hz, 2H), 3.29 (t, J = 5.6 Hz, 2H), 3.08 (m, 2H), 2.72
1
3
(
s, 6H). C NMR (101 MHz, Chloroform-d) δ 166.30 , 156.20 , 155.14 , 152.80 ,
1
48.23 , 144.47 , 139.32 , 135.10 , 133.50 , 133.48 , 123.79 , 120.65 , 117.32 , 116.43 ,

1
15.11 , 112.51 , 112.33 , 45.13 , 45.03 (2C) , 42.89 , 27.36 . HRMS (ESI) m/z
+
+
calculated for C H N O S [M+H] 435.1598, found 435.1605
2
2
23
6
2
N-(2-(dimethylamino)-5-((5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimi
din-4-yl)amino)phenyl)thiophene-2-carboxamide (5h) as a grey solid (yield 65%),
1
m.p.: 201.6-207.1 � . H NMR (400 MHz, Chloroform-d) δ 9.35 (br s, 1H), 8.51 (s,
1H), 8.43 (d, J = 2.4 Hz, 1H), 7.82 (dd, J = 8.8, 2.4 Hz, 1H), 7.63 (dd, J = 3.6, 1.2 Hz,
1H), 7.56 (dd, J = 5.2, 1.2 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.16 (dd, J = 5.2, 3.6 Hz,
1H), 7.08 (s, 1H), 4.11 (t, J = 2.0 Hz, 2H), 3.30 (t, J = 5.6 Hz, 2H), 3.08 (m, 2H), 2.72
1
3
(
s, 6H). C NMR (101 MHz, Chloroform-d) δ 166.33, 159.78 , 155.12 , 152.80 ,
1
1
39.80 , 138.98 , 135.37 , 133.79 , 133.49 , 130.84 , 128.30 , 127.94 , 123.79 , 120.72 ,
17.17 , 116.44 , 112.10 , 45.16 , 45.12 , 42.88 , 27.35 . HRMS (ESI) m/z calculated
+
2
+
for C H N OS [M+H] 451.1369, found 451.1371.
2
2
23
6
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)propionamide (6a) as a white solid (yield 70%), m.p.:
1
1
8
24.6-127.6 � . H NMR (400 MHz, Chloroform-d) δ 8.60 (br s, 1H), 8.50 (s, 1H),
.37 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 8.8, 2.4 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.02
(br s, 1H), 3.71 (t, J = 2.0 Hz, 2H), 3.16 (m, 2H), 2.89 (t, J = 5.6 Hz, 2H), 2.65 (s, 6H),
13
2
.54 (s, 3H), 2.47 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). C NMR (101 MHz,
Chloroform-d) δ 172.21, 166.57 , 155.16 , 152.90 , 138.73 , 135.17 , 134.03 , 131.71 ,
1
2
4
23.16 , 120.60 , 116.89 , 116.02 , 112.20 , 54.02 , 51.64 , 45.27 , 45.01 (2C) , 31.12 ,
+
+
6.72 , 9.69 . HRMS (ESI) m/z calculated for C H N OS [M+H] 411.1962, found
2
1
27
6
11.1961.
N-(2-(dimethylamino)-5-((7-ethyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-
d]pyrimidin-4-yl)amino)phenyl)propionamide (6b) as a white solid (yield 69%), m.p.:
1
25.1-128.6 � . 1H NMR (400 MHz, Chloroform-d) δ 8.60 (br s, 1H), 8.50 (s, 1H),
.37 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.02
8
(br s, 1H), 3.77 (t, J = 2.0 Hz, 2H), 3.16 (m, 2H), 2.94 (t, J = 5.6 Hz, 2H), 2.69 (q, J =
7
.2 Hz, 2H), 2.65 (s, 6H), 2.47 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H), 1.22 (t, J
1
3
=
7.2 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 172.20 , 166.59 , 155.12 ,
1
52.90 , 138.68 , 135.23 , 134.05 , 132.00 , 123.57 , 120.62 , 116.77 , 116.04 , 112.09 ,

5
1.83 , 51.27 , 49.43 , 45.02 (2C) , 31.13 , 26.79 , 12.54 , 9.68 . HRMS (ESI) m/z
+
+
calculated for C H N OS [M+H] 425.2118, found 425.2115.
2
2
29
6
N-(5-((7-benzyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)a
mino)-2-(dimethylamino)phenyl)propionamide (6c) as a white solid (yield 64%), m.p.:
1
1
58.4-163.3 � . H NMR (400 MHz, Chloroform-d) δ 8.59 (br. s, 1H), 8.49 (s, 1H),
8
.36 (d, J = 2.4 Hz, 1H), 7.77 (dd, J = 8.6, 2.4 Hz, 1H), 7.43 – 7.28 (m, 5H), 7.21 (d, J
=
8.6 Hz, 1H), 7.02 (br. s, 1H), 3.77 (s, 2H), 3.75 (t, J = 2.0 Hz, 2H), 3.16 (m, 2H),
2
.96 (t, J = 5.6 Hz, 2H), 2.65 (s, 6H), 2.46 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H).
C NMR (101 MHz, Chloroform-d) δ 172.18 , 166.58 , 155.15 , 152.90 , 138.67 ,
37.70 , 135.22 , 134.05 , 131.99 , 129.02 (2C) , 128.51 (2C) , 127.47 , 123.57 ,
20.61 , 116.75 , 116.10 , 112.11 , 61.68 , 52.05 , 49.47 , 45.02 (2C) , 31.13 , 26.70 ,
1
3
1
1
9
4
+
+
.69 . HRMS (ESI) m/z calculated for C H N OS [M+H] 487.2275, found
2
7
31
6
87.2283.
N-(2-(dimethylamino)-5-((7-(pyrimidin-2-yl)-5,6,7,8-tetrahydropyrido[4',3':4,5]t
hieno[2,3-d]pyrimidin-4-yl)amino)phenyl)propionamide (6d) as a white solid (yield
1
5
4%), mp: 158.4-163.3 � . H NMR (400 MHz, Chloroform-d) δ 8.58 (br s, 1H), 8.50
(
s, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.37 (d, J = 4.8 Hz, 2H), 7.69 (dd, J = 8.8, 2.4 Hz,
H), 7.21 (d, J = 8.8 Hz, 1H), 7.00 (br s, 1H), 6.57 (t, J = 4.8 Hz, 1H), 5.10 (t, J = 2.0
Hz, 2H), 4.28 (t, J = 5.6 Hz, 2H), 3.22 (m , 2H), 2.65 (s, 6H), 2.47 (q, J = 7.6 Hz, 2H),
1
1
3
1
1
1
9
4
.27 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 172.16 , 166.75 ,
61.46 , 157.92 (2C) , 155.17 , 153.01 , 138.81 , 135.06 , 134.06 , 131.94 , 124.30 ,
20.58 , 116.89 , 116.12 , 112.35 , 110.76 , 45.00 (2C) , 44.04 , 40.97 , 31.14 , 26.03 ,
+
+
.70 . HRMS (ESI) m/z calculated for C H N OS [M+H] 475.2023, found
2
4
27
8
75.2028.
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)-4-methylbenzamide (6e) as a orange solid (yield
1
7
8
7
3%), m.p.: 105.6-109.7 � . H NMR (400 MHz, Chloroform-d) δ 9.48 (br s, 1H),
.52 (d, J = 2.4 Hz, 1H), 8.51 (s, 1H), 7.86 - 7.77 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H),
.27 (d, J = 8.8 Hz, 1H), 7.08 (br s, 1H), 3.71 (t, J = 2.0 Hz, 2H), 3.20 (m, 2H), 2.90 (t,
13
J = 5.6 Hz, 2H), 2.71 (s, 6H), 2.54 (s, 3H), 2.44 (s, 3H). C NMR (101 MHz,

Chloroform-d) δ 166.58 , 165.20 , 155.18 , 152.91 , 142.43 , 139.23 , 135.31 , 134.19 ,
1
1
32.18 , 131.70 , 129.52 (2C) , 127.03 (2C) , 123.20 , 120.68 , 117.14 , 116.06 ,
12.27 , 54.01 , 51.65 , 45.26 , 45.17 (2C) , 26.73 , 21.52 . HRMS (ESI) m/z
+
+
calculated for C H N OS [M+H] 473.2118, found 473.2120.
2
6
29
6
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)picolinamide (6f) as a light-yellow solid (yield 79%),
1
m.p.: 105.6-110.2 � . H NMR (400 MHz, Chloroform-d) δ 11.03 (br s, 1H), 8.69 (m,
1
8
7
6
1
1
H), 8.59 (d, J = 2.4 Hz, 1H), 8.52 (s, 1H), 8.30 (dt, J = 8.0, 1.2 Hz, 1H), 7.91 (td, J =
.0, 1.6 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.48 (m, 1H), 7.25 (d, J = 8.8 Hz, 1H),
.07 (br s, 1H), 3.72 (t, J = 2.0 Hz, 2H), 3.20 (m, 2H), 2.90 (t, J = 5.6 Hz, 2H), 2.76 (s,
1
3
H), 2.54 (s, 3H). C NMR (101 MHz, Chloroform-d) δ 166.55 , 162.29 , 155.25 ,
52.96 , 150.38 , 148.41 , 140.26 , 137.48 , 134.58 , 133.30 , 131.59 , 126.29 , 123.22 ,
22.35 , 120.25 , 117.66 , 116.01 , 112.78 , 54.00 , 51.65 , 45.25 , 44.82 (2C) , 26.71 .
+
+
HRMS (ESI) m/z calculated for C H N OS [M+H] 460.1914, found 460.1923.
2
4
26
7
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)nicotinamide (6g) as a white solid (yield 78%), m.p.:
1
2
09.8-211.9 � . H NMR (400 MHz, Chloroform-d) δ 9.62 (br s, 1H), 9.15 (d, J = 2.0
Hz, 1H), 8.80 (dd, J = 4.8, 2.0 Hz, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.53 (s, 1H), 8.26 (dt,
J = 8.0, 2.0 Hz, 1H), 7.80 (dd, J = 8.8, 2.4 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H),
7
.30 (d, J = 8.8 Hz, 1H), 7.07 (br s, 1H), 3.73 (t, J = 2.0 Hz, 2H), 3.21 (m, 2H), 2.91 (t,
1
3
J = 5.6 Hz, 2H), 2.72 (s, 6H), 2.55 (s, 3H). C NMR (101 MHz, Chloroform-d) δ
1
1
4
4
66.67, 163.21 , 155.11 , 152.87 , 152.57 , 147.91 , 139.26 , 135.50 , 135.23 , 133.74 ,
31.84 , 130.70 , 123.78 , 123.13 , 120.97 , 117.70 , 116.07 , 112.31 , 54.01 , 51.65 ,
+
+
5.28 (2C) , 45.25 , 26.74 . HRMS (ESI) m/z calculated for C H N OS [M+H]
2
4
26
7
60.1914, found 460.1913.
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)isonicotinamide (6h) as a white solid (yield 73%),
1
mp: 221.4-224.3 � . H NMR (400 MHz, Chloroform-d) δ 9.63 (br s, 1H), 8.88 – 8.81
(m, 2H), 8.55 (d, J = 2.4 Hz, 1H), 8.53 (s, 1H), 7.80 (dd, J = 8.8, 2.4 Hz, 1H), 7.77 –
7
.71 (m, 2H), 7.31 (d, J = 8.8 Hz, 1H), 7.07 (br s, 1H), 3.72 (t, J = 2.0 Hz, 2H), 3.20

1
3
(
m, 2H), 2.91 (t, J = 5.6 Hz, 2H), 2.73 (s, 6H), 2.55 (s, 3H). C NMR (101 MHz,
Chloroform-d) δ 116.70 , 163.04 , 155.09 , 152.85 , 150.89 (2C) , 141.99 , 139.31 ,
1
5
35.53 , 133.47 , 131.94 , 123.11 , 120.98 , 120.76 (2C) , 117.96 , 116.09 , 112.39 ,
4.03 , 51.66 , 45.30 (2C) , 45.27 , 26.76 . HRMS (ESI) m/z calculated for
+
+
C H N OS [M + H] 460.1914, found 460.1917.
24
26
7
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)-1-naphthamide (6i) as a light-yellow solid (yield
1
6
0%), m.p.: 118.7-125.0 � . H NMR (400 MHz, Chloroform-d) δ 9.20 (br s, 1H),
8
.66 (d, J = 2.4 Hz, 1H), 8.53 (s, 1H), 8.45 (dd, J = 7.2, 2.4 Hz, 1H), 8.00 (d, J = 8.0
Hz, 1H), 7.92(dd, J = 7.2, 2.4 Hz, 1H), 7.82 (dd, J = 8.8, 2.4 Hz, 1H), 7.77 (dd, J =
.8, 1.2 Hz, 1H), 7.62 – 7.51 (m, 3H), 7.29 (d, J = 8.8 Hz, 1H), 7.12 (br s, 1H), 3.73 (t,
J = 2.0 Hz, 2H), 3.22 (t, J = 5.6 Hz, 2H), 2.91 (t, J = 5.6 Hz, 2H), 2.64 (s, 6H), 2.55 (s,
6
1
3
3
1
1
H). C NMR (101 MHz, Chloroform-d) δ 167.60 , 166.65, 155.24 , 152.92 , 139.30 ,
35.34 , 134.66 , 134.36 , 133.85 , 131.76 , 131.10 , 130.23 , 128.45 , 127.32 , 126.60 ,
25.37 , 125.12 , 124.85 , 123.22 , 120.93 , 117.68 , 116.10 , 112.53 , 54.03 , 51.67 ,
+
4
5.28 , 45.16 (2C) , 26.75 . HRMS (ESI) m/z calculated for C H N NaOS [M + Na]
2
9
28
6
+
531.1938, found 531.1942.
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide (6j) as a white solid (yield
1
7
8
0
6
8%), m.p.: 159.1-160.3 � . H NMR (400 MHz, Chloroform-d) δ 9.55 (br s, 1H),
.51 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 8.8, 2.4 Hz, 1H), 7.57 (dd, J = 1.6,
.8 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.24 (dd, J = 3.6, 0.8 Hz, 1H), 7.05 (br s, 1H),
.57 (dd, J = 3.6, 1.6 Hz, 1H), 3.71 (t, J = 2.0 Hz, 2H), 3.18 (m, 2H), 2.89 (t, J = 5.6
1
3
Hz, 2H), 2.72 (s, 6H), 2.54 (s, 3H). C NMR (101 MHz, Chloroform-d) δ 166.59 ,
1
1
2
4
56.18 , 155.13 , 152.90 , 148.27 , 144.43 , 139.30 , 135.13 , 133.52 , 131.75 , 123.16 ,
20.65 , 117.30 , 116.04 , 115.08 , 112.49 , 112.32 , 54.02 , 51.65 , 45.28 , 45.04 (2C) ,
+
+
6.73 . HRMS (ESI) m/z calculated for C H N O S [M + H] 449.1754, found
2
3
25
6
2
49.1757.
N-(2-(dimethylamino)-5-((7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3
d]pyrimidin-4-yl)amino)phenyl)thiophene-2-carboxamide (6k) as a white solid (yield
-

1
7
8
1
3
2
1
1
2
4
5%), m.p.: 101.3-105.0 � . H NMR (400 MHz, Chloroform-d) δ 9.37 (br s, 1H),
.52 (s, 1H), 8.43 (d, J = 2.4 Hz, 1H), 7.85 (dd, J = 8.8, 2.4 Hz, 1H), 7.64 (dd, J = 3.6,
.2 Hz, 1H), 7.57 (dd, J = 5.2, 1.2 Hz, 1H), 7.28 (d, J = 8.8 Hz , 1H), 7.16 (dd, J = 5.2,
.6 Hz, 1H), 7.06 (s, 1H), 3.72 (t, J = 2.0 Hz, 2H), 3.18 (m, 2H), 2.90 (t, J = 5.6 Hz,
1
3
H), 2.72 (s, 6H), 2.54 (s, 3H). C NMR (101 MHz, Chloroform-d) δ 166.61 ,
59.77 , 155.11 , 152.89 , 139.84 , 138.96 , 135.39 , 133.82 , 131.79 , 130.82 , 128.29 ,
27.92 , 123.15 , 120.73 , 117.15 , 116.06 , 112.10 , 54.02 , 51.65 , 45.27 , 45.17 (2C) ,
+
2
+
6.73 . HRMS (ESI) m/z calculated for C H N OS [M + H] 465.1526, found
2
3
25
6
65.1528.
N-(2-(dimethylamino)-5-((7-(3-morpholinopropyl)-5,6,7,8-tetrahydropyrido[4',3'
:4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)propionamide (6l) as pink solid (yield
1
4
5%), m.p.: 118.1-122.5. H NMR (400 MHz, Chloroform-d) δ 8.60 (br s, 1H), 8.50
(
s, 1H), 8.36 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.22 (d, J = 8.8 Hz,
H), 7.02 (br s, 1H), 3.76 (t, J = 2.0 Hz, 2H) , 3.73 (t, J = 4.8 Hz, 4H), 3.15 (t, J = 5.6
Hz, 2H), 2.94 (t, J = 5.6 Hz, 2H), 2.65 (m, 8H), 2.53 – 2.39 (m, 8H), 1.79 (q, J = 7.6
1
13
Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 172.24 ,
66.55 , 155.14 , 152.91 , 138.72 , 135.18 , 134.03 , 131.88 , 123.57 , 120.62 , 116.82 ,
16.04 , 112.12 , 67.00 (2C) , 56.86 , 55.24 , 53.80 (2C) , 52.25 , 49.73 , 45.02 (2C) ,
1
1
3
5
+
+
1.12 , 26.64 , 24.52 , 9.68 . HRMS (ESI) m/z calculated for C H N O S [M + H]
2
7
38
7
2
24.2802, found 524.2806.
N-(2-(dimethylamino)-5-((7-(3-(4-methylpiperazin-1-yl)propyl)-5,6,7,8-tetrahydr
opyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide
1
(
6m) as a red solid (yield 35%), m.p.: 90.5-97.1 � . H NMR (400 MHz, Chloroform-d)
δ 9.55 (br s, 1H), 8.51 (s, 1H), 8.47 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 8.8, 2.4 Hz, 1H),
7
.57 (dd, J = 1.6, 0.8 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.24 (dd, J = 3.6, 0.8 Hz, 1H),
.05 (br s, 1H), 6.57 (dd, J = 3.6, 1.6 Hz, 1H), 3.77 (t, J = 2.0 Hz, 2H), 3.16 (t, J = 5.6
7
Hz, 2H), 2.95 (t, J = 5.6 Hz, 2H), 2.72 (s, 6H), 2.65 (t, J = 7.6 Hz, 2H), 2.62 – 2.33 (m,
13
1
1
1
0H), 2.29 (s, 3H), 1.81 (p, J = 7.6 Hz, 2H). C NMR (101 MHz, Chloroform-d) δ
66.58 , 159.78 , 155.09 , 152.87 , 139.82 , 138.94 , 135.39 , 133.81 , 132.01 , 130.82 ,
28.29 , 127.92 , 123.56 , 120.73 , 117.11 , 116.08 , 112.07 , 56.42 , 55.36 , 55.12

(2C) , 53.25 (2C) , 52.21 , 49.75 , 46.03 , 45.17 (2C) , 26.64 , 24.84 . HRMS (ESI)
+
+
m/z calculated for C H N O S [M + H] 575.2911, found 575.2910.
3
0
39
8
2
N-(2-(dimethylamino)-5-((7-(3-(4-methylpiperazin-1-yl)propyl)-5,6,7,8-tetrahydr
opyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)thiophene-2-carboxamid
1
e (6n) as a white solid (yield 37%), m.p.: 48.9-55.7 � . H NMR (400 MHz,
Chloroform-d) δ 9.38 (br s, 1H), 8.52 (s, 1H), 8.44 (d, J = 2.4 Hz, 1H), 7.86 (dd, J =
8
.8, 2.4 Hz, 1H), 7.65 (dd, J = 3.6, 1.2 Hz, 1H), 7.58 (dd, J = 5.2, 1.2 Hz, 1H), 7.28
(d , J = 8.8 Hz, 1H), 7.17 (dd, J = 5.2, 3.6 Hz, 1H), 7.08 (s, 1H), 3.78 (t, J = 2.0 Hz,
2
2
H), 3.17 (t, J = 5.6 Hz, 2H), 2.96 (t, J = 5.6 Hz, 2H), 2.74 (s, 6H), 2.66 (t, J = 7.6 Hz,
1
3
H), 2.63 – 2.34 (m, 10H), 2.31 (s, 3H), 1.82 (p, J = 7.6 Hz, 2H). C NMR (101 MHz,
Chloroform-d) δ 166.53 , 159.70 , 155.03 , 152.81 , 139.81 , 138.87 , 135.38 , 133.76 ,
1
5
31.97 , 130.80 , 128.23 , 127.90 , 123.56 , 120.67 , 117.02 , 116.04 , 112.00 , 56.39 ,
5.34 , 55.10 (2C) , 53.22 (2C) , 52.19 , 49.72 , 46.02 , 45.14 (2C) , 26.63 , 24.83 .
+
2
+
HRMS (ESI) m/z calculated for C H N OS [M + H] 591.2683, found 591.2681.
3
0
39
8
N-(2-(dimethylamino)-5-((7-(3-(dimethylamino)propyl)-5,6,7,8-tetrahydropyrido
[
4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide (6o) as a
1
white solid (yield 40%), m.p.: 64.9-70.5 � . H NMR (400 MHz, Chloroform-d) δ
9
7
7
2
2
1
1
4
.55 (br s, 1H), 8.51 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 8.8, 2.4 Hz, 1H),
.57 (dd, J = 1.6, 0.8 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.24 (dd, J = 3.6, 0.8 Hz, 1H),
.06 (br s, 1H), 6.57 (dd, J = 3.6, 1.6 Hz, 1H), 3.77 (t, J = 2.0 Hz, 2H), 3.17 (m, 2H),
.95 (t, J = 5.6 Hz, 2H), 2.72 (s, 6H), 2.65 (t, J = 7.6 Hz , 2H), 2.41 (t, J = 7.6 Hz ,
13
H), 2.28 (s, 6H), 1.80 (p, J = 7.6 Hz, 2H). C NMR (101 MHz, Chloroform-d) δ
66.59 , 156.18 , 155.11 , 152.90 , 148.27 , 144.43 , 139.27 , 135.15 , 133.53 , 131.95 ,
23.58 , 120.66 , 117.23 , 116.05 , 115.07 , 112.49 , 112.26 , 57.60 , 55.26 , 52.28 ,
9.74 , 45.36 (2C) , 45.04 (2C) , 26.70 , 25.39 . HRMS (ESI) m/z calculated for
+
+
C H N O S [M + H] 520.2489, found 520.2486.
27
34
7
2
N-(2-(dimethylamino)-5-((7-(3-(dimethylamino)propyl)-5,6,7,8-tetrahydropyrido
[
4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)thiophene-2-carboxamide (6p)
1
as a white solid (yield 40%), m.p.: 39.8-43.5 � . H NMR (400 MHz, Chloroform-d) δ
9
.36 (br s, 1H), 8.51 (s, 1H), 8.44 (d, J = 2.4 Hz, 1H), 7.84 (dd, J = 8.8, 2.4 Hz, 1H),

7
.64 (d, J = 3.6 Hz, 1H), 7.56 (d, J = 5.2 Hz, 1H), 7.27 (d , J = 8.8 Hz , 1H), 7.16 (dd,
J = 5.2, 3.6 Hz, 1H), 7.07 (br s, 1H), 3.77 (t, J = 2.0 Hz, 2H), 3.16 (t, J = 5.6 Hz, 2H),
2
2
1
1
4
.95 (t, J = 5.6 Hz, 2H), 2.72 (s, 6H), 2.65 (t, J = 7.6 Hz, 2H), 2.39 (t, J = 7.6 Hz, 2H),
1
3
.27 (s, 6H), 1.79 (p, J = 7.6 Hz, 2H). C NMR (101 MHz, Chloroform-d) δ 166.60 ,
59.76 , 155.08 , 152.87 , 139.84 , 138.92 , 135.42 , 133.82 , 132.03 , 130.81 , 128.28 ,
27.92 , 123.57 , 120.73 , 117.07 , 116.08 , 112.03 , 57.67 , 55.35 , 52.28 , 49.75 ,
5.49 (2C) , 45.17 (2C) , 26.70 , 25.55 . HRMS (ESI) m/z calculated for
+
+
C H N OS [M + H] 536.2261, found 536.2252.
27
34
7
2
N-(2-(dimethylamino)-5-((7-(4-(trifluoromethyl)benzoyl)-5,6,7,8-tetrahydropyrid
o[4',3':4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)propionamide (7a) as a light
1
yellow (yield 85%), m.p.: 208.1-210.6. H NMR (400 MHz, Chloroform-d) δ 8.59 (br
s, 1H), 8.55 – 8.22 (m, 2H), 7.87 – 7.52 (m, 5H), 7.22 (d, J = 8.8 Hz, 1H), 6.98, 6.90
(2br s, rotamers, 1H), 5.02, 4.69 (2s, rotamers, 2H), 4.17, 3.80 (2s, rotamers, 2H),
3
.28, 3.17 (2s, rotamers, 2H), 2.65 (s, 6H), 2.47 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6
1
3
Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 172.29 , 169.37 , 166.98 , 155.21 ,
153.35 , 139.01 , 138.69 , 134.84 , 134.05 , 132.30 (q, J = 30 Hz, 1C) , 127.46 –
127.34 (m, 2C) , 126.35 (q, J = 273 Hz, 1C ), 125.96 (2C) , 122.28 , 120.64 , 117.15 ,
116.93 , 115.73 , 112.04 , 44.98 (2C) , 44.43, 42.33 , 31.12 , 27.33 , 9.67 . HRMS
+
+
(
ESI) m/z calculated for C H F N O S [M + H] 569.1941, found 569.1937.
28 28 3 6 2
N-(2-(dimethylamino)-5-((7-picolinoyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[
2
,3-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide (7b) as a light yellow (yield
1
8
6%), m.p.: 124.5-132.2 � . H NMR (400 MHz, Chloroform-d) δ 9.57, 9.54(2 br s,
rotamers, 1H), 8.69, 8.64 (2d, J =4.8 Hz, rotamers, 1H), 8.61 – 8.38 (m, 2H), 7.89 –
7
.60 (m, 3H), 7.57 (s, 1H), 7.46 – 7.38 (m, 1H), 7.28, 7.27(2s, rotamers, 1H), 7.26 –
7
.22(m, 1H), 7.02, 7.01 (2s, rotamers, 1H), 6.58 (dd, J = 3.5, 1.8 Hz, 1H), 5.06, 5.03
(2s, rotamers, 2H), 4.22, 4.05 (2t, J = 5.6 Hz, rotamers, 2H), 3.32 (t, J = 5.6 Hz, 2H),
1
3
2
1
1
.74 (s, 6H). C NMR (101 MHz, Chloroform-d) δ 167.71 , 166.96 , 156.24 , 155.15 ,
53.37 , 153.30 , 153.13 , 148.46 , 148.26 , 148.19 , 144.53 , 137.30 , 133.47 , 130.36 ,
25.10 , 124.80 , 124.40 , 123.93 , 120.68 , 117.49 , 115.95 , 115.12 , 112.50 , 45.03

+
(
2C) , 44.38 , 42.70 , 27.50 . HRMS (ESI) m/z calculated for C H N NaO S [M +
28 25 7 3
+
Na] 562.1632, found 562.1630.
N-(2-(dimethylamino)-5-((7-nicotinoyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[
2
7
8
2
1
,3-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide (7c) as a light yellow (yield
1
7%), m.p.: 127.5-130.1 � . H NMR (400 MHz, Chloroform-d) δ 9.51 (br s, 1H),
.77 (br s, 1H), 8.74 (dd, J = 4.8, 1.6 Hz 2H), 8.64 – 8.35 (m, 2H), 7.84 (dt, J = 8.0,
.0 Hz, 1H), 7.74, 7.63 (2s, rotamers, 1H), 7.58 – 7.54 (m, 1H), 7.43 (t, J = 6.0 Hz,
H), 7.25 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 3.6 Hz, 1H), 7.12 – 6.89 (m, 1H), 6.57 (dd,
J = 3.6, 1.6 Hz, 1H), 5.02, 4.78 (2s, rotamers, 2H), 4.16, 3.86 (2s, rotamers, 2H), 3.24
1
3
(
s, 2H), 2.72 (s, 6H). C NMR (101 MHz, Chloroform-d) δ 168.26 , 166.99 , 156.14 ,
1
1
2
5
55.25 , 153.36 , 151.39 , 148.19 , 148.01 , 144.48 , 139.58 , 134.79 , 133.51 , 131.08 ,
30.09 , 123.64 , 120.64 , 117.49 , 115.77 , 115.12 , 112.51 , 44.99 (3C) , 42.45 ,
+
+
9.70 . HRMS (ESI) m/z calculated for C H N NaO S [M + Na] 562.1632, found
2
8
25
7
3
62.1630.
N-(2-(dimethylamino)-5-((7-isonicotinoyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thie
no[2,3-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide (7d) as a light yellow
1
(
yield 90%), m.p.: 134.1-137.5 � . H NMR (400 MHz, Chloroform-d) δ 9.53 (br s,
1
1
H), 8.86 – 8.71 (m, 2H), 8.61 – 8.36 (m, 2H), 7.72, 7.62 (2d, J = 8.4 Hz, rotamers,
H), 7.59 – 7.54 (m,1H), 7.26 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 3.2 Hz, 1H), 7.01, 6.90
(2s, rotamers, 1H), 6.58 (dd, J = 3.6, 1.6 Hz, 1H), 5.03, 4.67 (2s, rotamers, 2H), 4.19,
1
3
3
.79 (2s, rotamers, 2H), 3.31, 3.18 (2s, rotamers, 2H), 2.72 (s, 6H). C NMR (101
MHz, Chloroform-d) δ 168.20 , 167.05 , 156.19 , 155.15 , 153.32 , 150.61 (2C) ,
1
48.15 , 144.52 , 142.82 , 139.56 , 134.76 , 133.51 , 130.00 , 123.16 , 121.02 , 120.66 ,
17.49 , 115.72 , 115.16 (2C) , 112.53 , 112.34 , 44.99 (2C) , 44.27 , 42.16 , 27.26 .
1
+
+
HRMS (ESI) m/z calculated for C H N NaO S [M + Na] 562.1632, found
2
8
25
7
3
5
62.1632.
N-(2-(dimethylamino)-5-((7-(pyrimidine-2-carbonyl)-5,6,7,8-tetrahydropyrido[4'
,
3':4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)furan-2-carboxamide (7e) as a light
1
yellow (yield 93%), m.p.: 151.7-157.1 � . H NMR (400 MHz, Chloroform-d) δ 9.56,
9
.54 (2br s, 1H), 8.92, 8.87 (2d, J = 4.8 Hz, rotamers, 2H), 8.63 – 8.33 (m, 2H), 7.94 –