Discovery and optimization of tetrahydropyrido[4,3-d]pyrimidine derivatives as novel ATX and EGFR dual inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.02.023

In order to discovery autotaxin (ATX) and EGFR dual inhibitors with potential therapeutic effect on IPF-LC, a series of novel tetrahydropyrido[4,3-d]pyrimidine derivatives possessing semicarbazones moiety were designed and synthesized. The preliminary investigation at the cellular level indicated six compounds (7h, 8a, 8c, 8d, 9a and 9d) displayed preferable anti-tumor activities against A549, H1975, MKN-45 and SGC cancer cells. Further enzymatic assay against EGFR kinase identified 8a and 9a as promising hits with IC₅₀ values of 18.0 nM and 24.2 nM. Meanwhile, anti-inflammatory assessment against cardiac fibroblasts (CFs) cell and RAW264.7 macrophages led to the discovery of candidate 9a, which exhibited considerable potency both on inhibition rate of 77% towards CFs and on reducing NO production to 1.05 μM at 10 μg/mL. Simultaneously, 9a indicated preferable potency towards ATX with IC₅₀ value of 29.1 nM. Significantly, a RT-PCR study revealed the function of 9a to down-regulate the mRNA expression of TGF-β and TNF-α in a dose-dependent manner. The molecular docking analysis together with the pharmacological studies validated 9a as a potential ATX and EGFR dual inhibitor for IPF-LC treatments.

Full text of DOI:10.1016/j.bmc.2018.02.023

Accepted Manuscript
Discovery and optimization of tetrahydropyrido[4,3-d]pyrimidine derivatives
as novel ATX and EGFR dual inhibitors
Tongfei Jing, Xiuqi Miao, Feng Jiang, Ming Guo, Lingyun Xing, Junlong
Zhang, Daiying Zuo, Hongrui Lei, Xin Zhai
PII:
S0968-0896(18)30105-6
https://doi.org/10.1016/j.bmc.2018.02.023
BMC 14209
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 January 2018
8 February 2018
15 February 2018
Please cite this article as: Jing, T., Miao, X., Jiang, F., Guo, M., Xing, L., Zhang, J., Zuo, D., Lei, H., Zhai, X.,
Discovery and optimization of tetrahydropyrido[4,3-d]pyrimidine derivatives as novel ATX and EGFR dual
inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.023
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery and optimization of tetrahydropyrido[4,3-d]pyrimidine
derivatives as novel ATX and EGFR dual inhibitors
Tongfei Jing, Xiuqi Miao, Feng Jiang, Ming Guo, Lingyun Xing, Junlong Zhang, Daiying Zuo,
Hongrui Lei, Xin Zhai
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, PR China
Abstract:
In order to discovery autotoxin (ATX) and EGFR dual inhibitors with potential therapeutic
effect on IPF-LC, a series of novel tetrahydropyrido[4,3-d]pyrimidine derivatives possessing
semicarbazones moiety were designed and synthesized. The preliminary investigation at the
cellular level indicated six compounds (7h, 8a, 8c, 8d, 9a and 9d) displayed preferable anti-tumor
activities against A549, H1975, MKN-45 and SGC cancer cells. Further enzymatic assay against
EGFR kinase identified 8a and 9a as promising hits with IC50 values of 18.0 nM and 24.2 nM.
Meanwhile, anti-inflammatory assessment against cardiac fibroblasts (CFs) cell and RAW264.7
macrophages led to the discovery of candidate 9a, which exhibited considerable potency both on
inhibition rate of 77% towards CFs and on reducing NO production to 1.05 μM at 10 μg/mL.
Simultaneously, 9a indicated preferable potency towards ATX with IC50 value of 29.1 nM.
Significantly, a RT-PCR study revealed the function of 9a to down-regulate the mRNA expression
of TGF-β and TNF-α in a dose-dependent manner. The molecular docking analysis together with
the pharmacological studies validated 9a as a potential ATX and EGFR dual inhibitor for IPF-LC
treatments.
Keywords: THPPs; Semicarbazones; ATX; EGFR; inhibitor; IPF-LC
1. Introduction
Idiopathic pulmonary fibrosis with lung cancer (IPF-LC) as a frequent comorbidity was first
proposed in 1958. Recent evidence supports the view that lung cancer occurs as a late
complication of IPF rather than as an incidental or preceding finding 1-2. Pathogenesis of IPF-LC
involved genetic changes 3, overexpression of cellular signal pathway 4, oxidation and antioxidant
5
imbalance and so on. Particularly, chronic inflammation resulted in overexpression of epithelial
mesenchymal transition, which further evolved into IPF-LC 6-7. Though surgical resection was the
common strategy of IPF-LC, it was restricted due to poor prognosis and high mortality 8-9. In
terms of medication, epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib10 (Fig.
1) and erlotinib11 (Fig. 1), indicated efficiency in non-small cell lung cancer (NSCLC) and
availability in IPF12, but more than 4.5% patients developed interstitial lung disease within three
*Corresponding author. E-mail address: zhangdajun2008@126.com (D.J. Zhang); zhaixin_syphu@126.com(X. Zhai).
# Authors contributed equally to this work.

months as treatment of IPF13. Besides, pirfenidone14 (Fig. 1) and nintedanib15 (Fig. 1) with
excellent effect were approved for IPF in 2008 and 2014, respectively. Although nintedanib
manifested potency on NSCLC treatment 16, there was still lack of drugs effective on IPF-LC so
far.
Figure 1. Structures of small-molecule EGFR and anti-fibrosis agents
Autotaxin (ATX), a secreted lysophophospholipase D belonging to ENPP family of enzymes,
is a key factor which converts lysophosphatidyl choline (LPC) into the bioactive phospholipid
derivative lysophosphatidic acid (LPA) 17. The ATX-LPA signal pathway aroused high interest of
researchers due to its involvement in cancer and fibrotic diseases18. At present, the ATX inhibitor
GLPG169019 (Fig. 1) was an ideal prospect for the treatment of pulmonary fibrosis in phase II
clinical study. Meanwhile, compound A (Fig. 1), a promising ATX inhibitor reported by Pfizer,
exhibited remarkable anti-fibrotic potency against ATX (IC50 = 2.59 nM) due to non-rigid
tetrahydropyrido[4,3-d]pyrimidines (THPPs) skeleton and long-chain linker section20-21
.
Additionally, the investigation of THPPs as the bioisosterism of 4-aminoquinzaline to develop
EGFR inhibitors has been reported by our laboratory previously22. A further chemical 3D
superposition analysis manifested A was rationally overlapped with gefitinib, as revealed THPPs
skeleton could ideally imitate 4-aminoquinzaline moiety (Fig. 2) 23-24. Accordingly, it was feasible
to discovery novel THPPs-based ATX and EGFR dual inhibitor.
In view of the SARs of lead A, introduction of aliphatic ring relative to aromatic groups at
terminal R2 moiety would reduce the pharmacological activity dramatically21. Interestingly,
25
semicarbazones (SCZs) was embedded as privileged bioisosteric group of propionyl motif on
account of the presence of hydrogen bond donors and acceptors as well as its flexible skeleton.
Thus, a series of THPPs derivatives bearing the aryl SCZs moiety were designed and synthesized
as promising ATX and EGFR dual inhibitors (Fig. 2).

Figure 2. Design strategies for the novel THPP derivatives.
In the present study, twenty compounds were examined for their anti-proliferative activities in
vitro against four cancer cell lines using MTT assay, and four preferred compounds were selected
for further in vitro enzymatic evaluation. Meanwhile, all target compounds were evaluated
anti-fibrotic activity against cardiac fibroblasts (CFs) cell in MTT assay, and two optimal
compounds (7h and 9a) were further examined the IC50 values and enzyme activity towards ATX.
And twenty-two compounds were selected to evaluate the release of NO from LPS-stimulated
RAW264.7 macrophages. In addition, RT-PCR assay was applied to investigate the expression of
TGF-β and TNF-α at the transcriptional level.
2. Chemistry
Target THPP derivatives 7a-7m, 8a-8k and 9a-9f were synthesized according to the pathway
described in Scheme 1.
As
shown
in
Scheme
1,
commercially
available
material
ethyl
1-benzyl-4-oxopiperidine-3-carboxylate hydrochloride was hydrogenated under palladium-carbon
catalysis in 95% ethanol at 50°C to provided 1, which was acylated using phenyl chloroformate in
the presence of N,N-diisopropylethylamine at 0°C to room temperature to afford 2. Refluxing in
methanol, cyclization of 2 with formamide acetate proceeded to obtain the pyrimidine core 3,
which
was
treated
with
phosphorus
oxychloride
to
yield
phenyl
4-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate 4. Subsequently, condensation of
4 with a variety of substituted anilines at reflux gave rise to 5a-5c, which were reacted with
hydrazine hydrate in refluxing ethanol to furnish 6a-6c, respectively. Finally, 6a-6c were reacted
with aromatic aldehyde at reflux for 1 h to generate target compounds 7a-m, 8a-k and 9a-f.

Scheme 1. Reagents and conditions: (a) H2/Pd-C, 95% ethanol 50°C, 7 h; (b) ClCOOPh, DIPEA, DCM, 0°C then
r.t. 1 h; (c) formamidine acetate, CH3ONa, CH3OH, reflux, 6 h; (d) POCl3, 100°C, 10min; (e) aniline, NH4Cl,
MeCN, reflux, 5 h; (f) NH2NH2H2O, ethanol, reflux, 2 h; (g) aromatic aldehyde, ethanol, reflux, 1 h.
The structures of target compounds were confirmed by MS, 1H-NMR, 13C-NMR and NOESY
NMR spectra. It is worth mentioning that all target compounds exist in the E or Z isomer due to
1
the imine scaffold (-CH=N-). According to the H-NMR spectra, the imine signal was a single
peak for all compounds, indicating only one isomer was obtained. In order to further verify the
configuration of all compounds, the representative compound 7e was performed the NOESY
experiment, the NOE signal was observed between the proton of =N-NH- (δ = 10.60) and
-CH=N- (δ = 8.19) (Fig. 3). Hence, 7e was confirmed as the E isomer rather than the Z isomer
because of the larger intramolecular H1-H2 distance.
Figure 3. The 2D NOESY effects of representative compound 7e
3. Results and discussion
3.1 Anti-tumor evaluation
3.1.1 In vitro anti-proliferation activities
The cytotoxicity of target compounds 7a-7i, 8a-8g and 9a-9d were evaluated against A549
(Human lung cancer cells), H1975 (Gefitinib-resistant human NSCLC cells), MKN-45 (Human
gastric cancer cells), and SGC (Human gastric adenocarcinoma cells). Using gefitinib as positive
control, the pharmacological data presented as IC50 values were summarized in Table 1. Each

compound was tested in three independent experiments.
As illustrated in Table 1, most compounds showed acceptable to excellent cytotoxicity
against tested cancer cell lines. Six compounds (7h, 8a, 8c, 8d, 9a and 9d) were more potent than
gefitinib against A549, MKN-45 and SGC cells with IC50 values in a rational range. The
promising compound 8a displayed remarkable activities whose values were 1.71 μM, 1.61 μM
and 0.99 μM against MKN-45, A549 and SGC cell lines, which were approximately 6.1, 7.6 and
3.3 times superior to that of gefitinib, and followed by 8d and 9a. To our disappointed, all target
compounds exhibited poor inhibitory potency against H1975.
The pharmacological data in Table 1 referred to variations of R1 made a moderate impact on
the cytotoxicity. As comparing of 7e (R1 = 3-Cl-4-F, IC50 = 11.43 μM against A549), 8d (R1 =
3-Br, IC50 = 2.27 μM against A549) and 9d (R1 = 3-CF3, IC50 = 6.37 μM against A549) which
bearing the same 4-fluorophenyl group on R2 moiety, 3-bromophenyl derivative (8d) displayed the
best activity. The same trend was observed in other matched molecular pairs (7i, 8e vs.9b and 7a,
8c vs. 9c). As for the influence of R2 on the terminal of SCZs scaffold, phenyl moiety (8b)
indicated superior cytotoxicity against A549 than other aromatic groups. Meanwhile, the
electron-donating five membered groups displayed superior potency than the
electron-withdrawing pyridine group (8e, 8f vs. 8g).
In terms of phenyl group, the influence of substituents was explored further. The data
presented in Table 1 indicated that the introduction of various substituted phenyl affected
inhibitory activities notably. For tested compounds, the existence of electron-withdrawing groups
such as 3-nitro(7c), 4-cyano (7f) and 4-trifluoromethyl (7g) weakened the activity dramatically.
By contrast, the introduction of the halogen group at the C-4 position (7a and 7e) led to 2.2- and
2.8-fold improvement in anti-tumor activity than 7b without substituents (IC50 =31.53 μM against
A549). Furthermore, embedding of multi-halogen groups revealed advanced potency comparing
with mono-halogen groups (7e vs. 7h and 8a vs. 8d). Meanwhile, the introduction of the methyl
groups at the C-4 position (9a) led to remarkable improvement in anti-tumor activity. Additional
SARs and further optimization of the promising hit 9a are in progress and will be reported in due
course.
Table1 Structures and cytotoxicity of compounds 7a-7i, 8a-8g and 9a-9d against A549, H1975, MKN-45 and
SGC cancer cell lines in vitro.
aIC50 (µM) ± SD
Compd.
R1
A549
H1975
>200
MKN-45
12.05±0.75
22.86±0.85
>200
SGC
3-Cl-4-F
3-Cl-4-F
3-Cl-4-F
3-Cl-4-F
4-bromophenyl
Phenyl
14.09±0.85
31.53±2.56
43.09±2.14
15.61±0.98
13.53±0.87
7.47±0.34
34.75±1.25
11.50±0.72
7a
7b
7c
7d
28.83±1.53
86.91±1.03
6.83±0.43
3-nitrophenyl
3,4-dimethylphenyl
18.08±1.25

3-Cl-4-F
3-Cl-4-F
3-Cl-4-F
3-Cl-4-F
3-Cl-4-F
3-Br
4-fluorophenyl
4-cyanophenyl
4-trifluoromethylphenyl
2-chloro-4,5-difluorophenyl
2-thienyl
11.41±0.73
42.62±3.64
37.80±2.89
7.55±0.41
>200
64.45±4.68
67.53±4.65
36.83±1.98
46.07±2.78
33.04±2.31
15.65±0.95
37.83±2.13
51.03±3.58
37.64±1.45
121.5±3.12
>200
10.85±0.67
52.30±4.02
21.43±1.65
7.33±0.35
57.30±4.12
1.71±0.03
22.35±1.02
23.61±1.78
2.49±0.11
>200
16.91±0.95
35.77±2.78
36.43±2.11
10.41±0.76
96.27±8.44
0.99±0.02
16.42±1.56
6.93±0.87
1.79±0.37
>200
7e
7f
7g
7h
7i
2-chloro-4-fluorophenyl
Phenyl
1.61±0.21
22.73±1.32
6.56±0.58
2.27±0.24
50.25±3.14
47.06±2.56
72.13±4.66
4.96±0.34
42.36±2.98
11.47±1.45
6.37±0.57
10.45±0.75
8a
3-Br
8b
3-Br
4-bromophenyl
4-fluorophenyl
2-thienyl
8c
3-Br
8d
3-Br
8e
3-Br
2-furyl
65.68±5.86
>200
>200
8f
3-Br
3-pyridyl
>200
>200
8g
3-CF3
3-CF3
3-CF3
3-CF3
4-methylphenyl
2-thienyl
21.27±7.65
168.65±8.72
61.42±4.55
55.55±3.97
8.58±0.56
3.43±0.28
36.35±0.62
11.41±1.08
4.00±0.35
12.29±0.64
2.91±0.09
24.58±0.85
16.77±1.77
4.23±0.27
6.48±0.35
9a
9b
9c
4-bromophenyl
4-fluorophenyl
9d
b Gefitinib
a Data presented is mean ± SD value of three independent experiments.
b Used as positive control.
3.1.2. In vitro enzymatic assays
Based on the anti-proliferative activities, four compounds (7h, 7i, 8a and 9a) with remarkable
activity were subjected to enzymatic assay against EGFR, VEGFR2 and T790M/L858R/EGFR
protein kinases. The data in Table 2 showed 8a and 9a indicated promising potency with IC50
values of 18.0 and 24.2 nM against EGFR, respectively, which was comparable to the positive
control gefitinib (IC50 = 29.6 nM). Unfortunately, all compounds hardly indicated inhibitory
effects against T790M/L858R/EGFR protein kinase.
Table 2 Enzymatic assay of selected compounds 7h, 7i, 8a and 9a against EGFR, VEGFR2 and
T790M/L858R EGFR in vitro.
aIC50 (nM) ± SD
Compd.
R1
R2
EGFR
42.3±3.4
62.5±5.4
VEGFR2
169.9±15.6
237.3±21.3
T790M/L858R EGFR
3-Cl-4-F
3-Cl-4-F
2-chloro-4,5-difluorophenyl
2-thienyl
bND
bND
7h
7i

3-Br
2-chloro-4-fluorophenyl
4-methylphenyl
18.0±1.2
24.2±2.6
29.6±1.4
133.2±12.1
151.5±14.2
48.5±3.5
939.7±83.1
3217.2±201.3
2874.7±185.3
8a
9a
3-CF3
Gefitinib
a Data presented is mean ± SD value of three independent experiments
b ND: not determined
3.2 Anti-inflammatory evaluation
3.2.1 Inhibition of rat CFs proliferation and in vitro enzymatic assays
In order to evaluate the anti-fibrotic activities, the inhibition rate of compounds (7a-m, 8a-k
and 9a-f) were investigated against CFs cell which generated from Wistar rats via differential
adherence method in MTT assay26. Meanwhile, two optimal compounds (7h and 9a) were selected
to evaluated the IC50 values. The results generated by three independent experiments were
summarized in Fig. 4 and Fig. 5. Especially, enzymatic assay of 7h and 9a against ATX were
accomplished in the FS-3 assay, and the data was presented in Table 3.
As an expected trend, all target compounds revealed preferable to outstanding potency
against CFs cell. Eleven compounds (7a, 7g, 7h, 7i, 7l, 8c, 8e, 9a, 9b, 9c and 9f) exhibited
improved activity with inhibition rate range from 65% to 83%. Particularly, 7i indicated the most
promising anti-fibrotic activity with 83% inhibitory activity. Furthermore, 9a revealed promising
potency with inhibition rate of 77% as compared lead A (68% inhibition) (Fig. 4). Significantly,
two preferred compounds (7h and 9a) were further evaluated for their IC50 values to identify the
anti-fibrotic potency. To our delight, both of them disclosed remarkable inhibition against CFs cell
with IC50 values of 1.91 μM and 1.49 μM (Fig. 5). Importantly, enzymatic assay revealed 7h and
9a displayed commendable activity against ATX with IC50 values of 38.4 nM and 29.1 nM,
separately.
100
80
60
40
20
0
A
7i
7j
7l
8i
8j
7f
8f
9f
7a
7c
7e
7k
8a
8c
8e
8k
9a
9c
9e
7b
7d
7g
7h
8b
8d
8g
8h
9b
9d
7m
Figure 4. The inhibition rate of lead A and target compounds (7a-m, 8a-k and 9a-f)

样品
对照
样品
对照
100
80
60
40
20
0
100
80
60
40
20
0
9a
7h
-4
-2
0
2
4
-4
-2
0
2
4
Log concentration(μM)
Log concentration(μM)
Figure 5. Effects of 7h and 9a on TGF-induced fibroblasts.
Table 3 Enzymatic assay of selected compounds 7h and 9a against ATX in vitro.
Compd.
R1
3-Cl-4-F
3-CF3
R2
ATX aIC50 (nM) ± SD
2-chloro-4,5-difluorophenyl
4-methylphenyl
38.4±2.4
7h
9a
A
29.1±1.5
10.4±0.8
a Data presented is mean ± SD value of three independent experiments
3.2.2 Effect of target compounds on the generation of NO production
Aiming to further evaluate the anti-inflammatory activity, the effect of twenty compounds
(7e-7m, 8a-8k and 9a-9b) on the release of NO from Lipopolysaccharide (LPS)-stimulated
RAW264.7 macrophages were detected using the Griess method27-28, and each compound was
tested in three independent experiments (Fig. 6).
As illustrated in Fig. 6, most compounds displayed excellent activities in a dose-dependent
manner to reduce the NO concentration in RAW264.7 cell. Eight compounds (7g, 7i, 7l, 7m, 8e,
8h, 9a and 9b) exhibited superior inhibitory activities against RAW264.7 with the NO
concentration below 1.9 μM at 10 μg/mL. Particularly, the most promising compound 7g at 2.5, 5
and 10 μg/mL reduced the NO production significantly to 3.87, 2.10 and 0.79 μM, respectively,
and 7i and 9a displayed a descending order.

2.5μg/mL
5μg/mL
8
6
4
2
0
#
10μg/mL
**
**
**
**
**
**
**
**
**
**
*
**
**
**
**
**
**
**
*
**
**
**
**
**
**
**
**
**
**
**
**
**
#
n
S
7i
7j
7l
7f
7e
7k
8a
7g
7h
8b
P
L
7m
Co
2.5μg/mL
5μg/mL
8
6
4
2
0
10μg/mL
#
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
*
**
**
**
**
**
**
**
*
#
*
n
S
8i
8j
8f
8c
8e
8k
9a
8d
8g
8h
9b
P
L
Co
Figure 6. Target compounds (7e-7m, 8a-8k and 9a-9b) inhibited LPS-induced NO production in RAW264.7
macrophages. RAW264.7 was treated with 5μg/mL LPS in pre-incubated mediums. Subsequently, cells were
incubated with 2.5, 5 and 10μg/mL compounds (7e-7m, 8a-8k and 9a-9b) for 1 h. After incubation for 24 h,
secreted NO in the cell-free culture media was measured by the Griess method. Each compound was tested in three
independent experiments. * p<0.01, ** p<0.05 vs. LPS group; # p<0.01 vs. control group.
As shown in Fig. 4, the anti-fibrotic activity of target compounds is parallel to the
anti-inflammatory data illustrated in Fig. 4. Initially, the SARs was conducted by the introduction
of diverse R1 groups, indicating 3-chloro-4-fluoroanilino derivatives revealed the optimum
activity comparing with 3-trifluoromethylanilino and 3-bromoanilino derivatives (7i, 8e vs. 9b and
7a, 8c vs. 9c). Meanwhile, various R2 fragments (8a-8k) on the terminal of SCZs scaffold made
evident influence on the biological properties. On one hand, 2-thienyl derivative (8e) led to a
remarkable improvement in anti-fibrotic activity as compared with five or six membered aromatic
group (8b, 8j, 8g, 8j and 8k), and the 4-methylphenyl derivative (9a) indicated ideal potency
against CFs cell. On the other hand, introduction of phenyl group (8a, 8c, 8d and 8h) resulted in a
significant promotion in anti-inflammatory activity. Further investigation displayed that
trifluoromethyl (7h) and methyl (9a) at C-4 position of phenyl ring reduced the release of NO
notably. In addition, 2-thienyl derivative (7i) displayed rational anti-inflammatory activity as well
as anti-fibrotic activity.
Overall, compound 9a bearing 3-trifluoromethylphenyl group at 4-position of THPP and
4-methylphenyl group at R2 moiety indicated superior activity both in anti-fibrotic and in
anti-proliferation assays. Herein, 9a was validated as potential dual inhibitor and will be optimized
further in due course.
3.2.3 Real-time polymerase chain reaction

Transforming growth factor-β (TGF-β), a member of multifunctional cytokine family, is a
key therapeutic target for pulmonary fibrosis (PF) 29. Meanwhile, tumor necrosis factor-α (TNF-α)
also plays an important role in the pathogenesis of PF30. To comprehend the anti-fibrotic
mechanism further, we simultaneously determined the function of 8e and 9a on the TGF-β and
TNF-α in LPS-stimulated RAW264.7 macrophages by RT-PCR. As shown in Fig. 7, two
compounds reduced the mRNA expression of TGF-β and TNF-α with dose-dependent responses.
Particularly, 9a at 2.5, 5 and 10 μg/mL inhibited the mRNA expression of TGF-β (by 46%, 65%
and 86%) and TNF-α (by 37%, 54% and 72%) significantly.
50
40
30
20
10
0
50
40
30
20
10
0
##
##
**
***
***
*
**
**
5
0
5
0
n
Co
n
Co
S
P
S
P
.0
.0
2.
5.
2.
5.
L
10
L
10
8e(μg/mL) + LPS
9a(μg/mL) + LPS
50
40
30
20
10
0
50
40
30
20
10
0
##
##
*
**
***
**
***
***
5
0
5
0
n
Co
n
Co
S
S
P
.0
.0
P
2.
5.
2.
5.
L
10
L
10
9a(μg/mL) + LPS
8e(μg/mL) + LPS
Figure 7. Effect of 8e and 9a on TGF-β and TNF-α mRNA expression in LPS-stimulated RAW264.7 cells.
RAW264.7 cells were treated with 2.5-10 μg/mL 8e and 9a for 8h. TGF-β and TNF-α were analyzed by RT-PCR.
Each compound was tested in three independent experiments. * p<0.01, ** p<0.05, ***<0.001 vs. LPS group; ##
p<0.05 vs. control group.
3.3 Molecular docking
In order to elucidate the binding mode, a detail docking analysis of 9a with ATX and EGFR
was performed using Discovery Studio Program 3.5, respectively. The co-crystal structures of
gefitinib with EGFR (PDB ID code: 4I22)31 and compound A with ATX (PDB ID code: 4ZG6)32
were selected as the docking models.
As shown in Fig. 8A-8C, 9a was docked into the ATP pocket in a similar manner to gefitinib.
In addition to the same hydrogen bond found in cocrystal model of gefitinib with Met793, 9a was
observed to interact with Cys797 at the hinge area via one hydrogen bond of 3.2 Å, validating the
rationality and importance of SCZs moiety (Fig. 8A-8B). Meanwhile, 9a bound the active site
through Lys745 to form two hydrogen bonds of 2.1 and 2.6 Å. The docking analysis of 9a
indicated that 3-trifluoromethylphenyl fragment reached into the hydrophobic pocket consist of
Val726, Lys745, Met790 and Leu788, and participated in alkyl interactions and halogen
interaction (Fig. 8C).
Fig. 8D-8F gave details of 9a occupied same active site as lead A with ATX protein. As

expected, the docking analysis indicated that 9a formed two hydrogen bonds with Ala218 and
TRP276. Concretely, the THPPs scaffold attach to Leu214, Phe274 and Tyr307 through
hydrophobic interaction. Simultaneously, 3-trifluoromethylanilino moiety formed halogen
interaction and hydrophobic interactions with the hydrophobic pocket made up by Leu214,
Phe274, Leu217, Ala218, Ala305 and Ile168. In addition, 4-methylphenyl group at the terminal of
SCZs scaffold was involved in π-π interaction and alkyl interactions with Phe275 and Trp255.
Thus, the results from the docking study were in accordance with the SARs analysis.
Figure 8. The binding poses of 9a with EGFR and ATX. (A) Modeled docking of gefitinib with EGFR (PDB ID
4I22). (B) Predicted binding conformation for 9a in the ATP site of EGFR. (C) 2D diagram of the interaction
between 9a and EGFR (D) Modeled docking of leading compound A with ATX (PDB ID 4ZG6). (E) Predicted
binding conformation for 9a in the binding site of ATX. (F) 2D diagram of the interaction between 9a and ATX.
4. Conclusion
In summary, a series of 4-anilinotetrahydropyrido[4,3-d]pyrimidine derivatives (7a-7m,
8a-8k and 9a-9f) bearing the aryl SCZs moiety were designed and synthesized as ATX and EGFR
dual inhibitors. The preliminary cytotoxic investigation showed that six compounds (7h, 8a, 8c,
8d, 9a and 9d) displayed preferable activities against a panel of cancer cell lines (A549, MKN-45
and SGC) with IC50 values in double digits micro-molar degree. Meanwhile, evaluation on the
anti-fibrotic potency of all target compounds against CFs cell resulted in the discovery of eleven
promising compounds (7g, 7h, 8c, 9a and 9f etc.) with inhibition rate over 68%, and two preferred
compounds (7h and 9a) revealed excellent activity with IC50 values of 1.91 μM and 1.49 μM,
respectively. In the further Griess assay, eight compounds (7g, 7i, 7l, 7m, 8e, 8h, 9a and 9b)
effectively alleviated the increase of LPS-induced NO release in RAW264.7 cell. Exploration of
SARs culminated in 9a, a promising EGFR (IC50 = 24.2 nM) and ATX (IC50 = 29.1 nM) dual
inhibitor, which possessed remarkable anti-tumor and anti-fibrotic potency against A549 (IC50 =
4.96 μM) and CFs (IC50 = 1.49 μM) cells superior to references gefetinib and A. Furthermore, the
RT-PCR evaluation confirmed that 9a reduced the mRNA expression of the pivotal fibrosis factors

TGF-β (by 46%, 65% and 86%) and TNF-α (by 37%, 54% and 72%) in a dose-dependent manner
(at 2.5, 5 and 10 μg/mL). Finally, molecular docking models of 9a with EGFR and ATX protein
revealed that 9a could form critical bonding interactions with both of proteins, which was
identical with SARs studies.
In conclusion, the SARs observations together with pharmacological assays on novel THPPs
validated 9a as a promising ATX and EGFR dual inhibitor potential for treatment of IPF-LC.
5. Experimental
5.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in ESI
1
mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). H NMR spectra was performed
using Bruker ARX-400 spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an
internal standard. Column chromatography was run on silica gel (200–300 mesh) from Qingdao
Ocean Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all materials were
obtained from commercially available sources and were used without further purification.
5.1.1 Preparation of ethyl 4-oxopiperidine-3-carboxylate hydrochloride (1)
To a solution of ethyl 1-benzyl-4-oxopiperidine-3-carboxylate (80.0g, 0.306mol) in 95%
ethanol was added 10% Pd/C (8.0 g, 7.6mmol). The reaction mixture was stirred at 40 °C under an
atmosphere of hydrogen for 7h. After completion of the reaction, the mixture was filtered through
diatomite, and the filtrate was removed under reduced pressure and dried in a vacuum tray drier at
45 °C to give compound 1 as a light red solid (54.7g, 98.2%). MS (ESI) m/z 208.3 [ M+H]+.
5.1.2 Preparation of 3-ethyl 1-phenyl 4-oxopiperidine-1,3-dicarboxylate (2)
To the mixture of ethyl 4-oxopiperidine-3-carboxylate hydrochloride (1) (53.8g, 0.206mol)
and N,N-diisopropylethylamine (DIPEA) (84.0g, 0.651mol) was added to dichloromethane (DCM)
(500mL) at -15°C, and then phenyl carbonochloridate (49.1g, 0.313mol) was added to the mixture.
Subsequently, the reaction was stirred at room temperature for 1h. After completion of reaction as
indicated by thin layer chromatography (TLC), the mixture was poured in cool water. Organic
layer was washed with dilute hydrochloric acid and brine, dried over anhydrous Na2SO4, and then
filtered and concentrated to yield the compound 2 as a white solid (74.9g 98.2%). MS (ESI) m/z
292.1 [ M+H]+
5.1.3 Preparation of phenyl 4-hydroxy-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (3)
A mixture of 3-ethyl 1-phenyl 4-oxopiperidine-1,3-dicarboxylate (2) (58.5g, 0.201mol),
formamidine acetate(25.1g, 0.241mol) and sodium methoxide(43.4g, 0.804mol) in anhydrous
methanol(1200mL) was heated at 70°C for 6h, and then concentrated immediately. Subsequently,
the resulting syrup was added purified water (1L), treated with sodium hydroxide solution to
adjust pH to 12. Organic impurities were extracted by little toluene. The aqueous layer was treated
with concentrated hydrochloric acid to adjust pH to 5, and then filtered to give compound 3 as a
white solid (32.9g,60.4%). MS (ESI) m/z 270.6 [M+H]+.
5.1.4 Preparation of phenyl 4-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)- carboxylate (4)

To a solution of 4-hydroxy-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate(10.0g,
0.037mol) (3) was added in phosphorus oxychloride(10.0mL).Subsequently, the reaction was
heated at 100°C for 10min. After completion of reaction indicated by TLC, the mixture was
cooled to room temperature and poured into ice-water. The mixture was extracted with DCM (100
mL *5), dried over anhydrous Na2SO4, filtered and concentrated to give compound 4 as a red oil
(9.0g, 99.1%). MS (ESI) m/z: 290.4 [M+H]+.
5.1.5 Preparation of phenyl 4-(substituted phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidine
-6(5H)-carboxylate (5a-c)
Concentrated phenyl 4-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)- carboxylate(4)
(9.0g,0.031 mol), substituted aniline(0.036 mol) and ammonium chloride(0.25g,6.5mmol) were
added successively to acetonitrile(90mL). The reaction mixture was heated at reflux for 5h. After
completion the reaction, the mixture was filtered to yield the compound 5a-c.
5.1.5.1
phenyl
4-((4-chloro-3-fluorophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate(5a).
Light yellow solid; yield: 75.1%. MS (ESI) m/z 399.3 [M+H]+.
5.1.5.2 phenyl 4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate
(5b). Yellow solid; yield: 71.2%. MS (ESI) m/z 425.6 [M+H]+.
5.1.5.3
phenyl
4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate(5c).
White solid; yield: 79.6%. MS (ESI) m/z 415.4 [M+H ]+.
5.1.6 Preparation of 4-(phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide
(6a-c)
To a solution of phenyl 4-(substituted phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidine-
6(5H)-carboxylate(5a-c) in anhydrous ethanol(100 mL) was added hydrazine hydrate(80%, 10mL)
drop-wise. Upon the completion of addition, the reaction mixture was heated at reflux for 2h. The
mixture was cooled and filtered to give compound 6a-c.
5.1.6.1
4-((3-chloro-4-fluorophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide
(6a). White solid; yield: 82.0%. MS (ESI) m/z 353.4 [M+H]+.
5.1.6.2 4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide(6b).
White solid; yield: 78.6%. MS (ESI) m/z 363.7 [M+H]+.
5.1.6.3
4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide
(6c). White solid; yield: 56.5%. MS (ESI) m/z 337.5 [M+H]+.
5.1.7 General procedure for the preparation of (E)-N'-substituted benzylidene-4-( substituted

phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide (7a-n,8a-l,9a-d)
To the solution
of
4-(phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide (6a-c) (0.3mmol) and
substituted benzaldehyde (6mmol) were dissolved in anhydrous ethanol(2mL). The reaction
mixture was heated at reflux. After completion of reaction, the mixture was cooled and filtrated to
give target compounds 7a-j, 8a-j and 9a-j.
5.1.7.1(E)-N'-(4-bromobenzylidene)-4-((3-chloro-4-fluorophenyl)amino)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carbohydrazide(7a)
1
Light yellow solid; yield: 52.5%; H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 8.74 (s,
1H), 8.47 (s, 1H), 8.15 (s, 1H), 7.99 (dd, J = 6.8, 2.6 Hz, 1H), 7.72 – 7.66 (m, 1H), 7.64 – 7.57 (m,
4H), 7.39 (t, J = 9.1 Hz, 1H), 4.56 (s, 2H), 3.80 (t, J = 5.7 Hz, 2H), 2.82 (t, J = 5.5 Hz, 2H). m.p.:
224.4-228.2 °C; MS (ESI) m/z: 503.3 [M+H]+. Anal. calcd. for C21H17BrClFN6O (%): C, 50.07; H,
3.40; N, 16.68. Found (%) C, 50.03; H, 3.46; N, 16.64.
5.1.7.2(E)-N'-benzylidene-4-((3-chloro-4-fluorophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin
e-6(5H)-carbohydrazide(7b)
1
Light yellow solid; yield: 58.3%; H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.88 (s,
1H), 8.47 (s, 1H), 8.26 (s, 1H), 8.05 (d, J = 4.7 Hz, 1H), 7.79 – 7.69 (m, 1H), 7.63 (d, J = 7.1 Hz,
2H), 7.46 – 7.33 (m, 4H), 4.61 (s, 2H), 3.79 (d, J = 5.0 Hz, 2H), 2.82 (s, 2H). m.p.: 162.4-164.1°C;
MS (ESI) m/z: 424.8 [M+H]+. Anal. calcd. for C21H18ClFN6O (%): C, 59.37; H, 4.27; N, 19.78.
Found (%) C, 59.35; H, 4.29; N, 19.74.
5.1.7.3(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(3-nitrobenzylidene)-7,8-dihydropyrido[4,3-d]p
yrimidine-6(5H)-carbohydrazide(7c)
1
Yellow solid; yield: 46.4%; H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.91 (s, 1H),
8.52 (s, 1H), 8.48 (s, 1H), 8.45 (s, 1H), 8.21 (d, J = 9.8 Hz, 1H), 8.11 (dd, J = 6.8, 2.4 Hz, 1H),
8.04 (d, J = 7.7 Hz, 1H), 7.85 – 7.79 (m, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.39 (t, J = 9.1 Hz, 1H),
4.68 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 2.82 (t, J = 5.4 Hz, 2H). m.p.: 174.1-175.6 °C; MS (ESI) m/z:
470.2 [M+H]+. Anal. calcd. for C21H17ClFN7O3 (%): C, 53.68; H, 3.65; N, 20.87. Found (%) C,
53.64; H, 3.68; N, 20.83.
5.1.7.4(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(3,4-dimethylbenzylidene)-7,8-dihydropyrido[4,
3-d]pyrimidine-6(5H)-carbohydrazide(7d)
White solid; yield: 43.0%;1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.74 (s, 1H), 8.47
(s, 1H), 8.11 (s, 1H), 8.00 (dd, J = 6.8, 2.4 Hz, 1H), 7.72 – 7.66 (m, 1H), 7.44 – 7.30 (m, 3H), 7.17
(d, J = 7.8 Hz, 1H), 4.56 (s, 2H), 3.79 (t, J = 5.4 Hz, 2H), 2.82 (t, J = 4.8 Hz, 2H), 2.24 (s, 6H).
m.p.: 188.8-190.5°C; MS (ESI) m/z: 453.3 [M+H]+. Anal. calcd. for C23H22ClFN6O (%): C, 60.99;
H, 4.90; N, 18.56. Found (%): C, 60.95; H, 4.94; N, 18.52.
5.1.7.5(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(4-fluorobenzylidene)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carbohydrazide(7e)
Light gray solid; yield: 37.1%; 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.75 (s, 1H),
8.47 (s, 1H), 8.19 (s, 1H), 8.04 – 7.96 (m, 1H), 7.75 – 7.62 (m, 3H), 7.40 (t, J = 9.1 Hz, 1H), 7.26
(t, J = 8.8 Hz, 2H), 4.57 (s, 2H), 3.80 (t, J = 5.4 Hz, 2H), 2.82 (t, J = 4.8 Hz, 2H). 13C NMR (101
MHz, DMSO-d6) δ 161.84, 161.17, 156.71, 155.45, 154.94, 142.38, 137.30, 131.93, 129.01,
128.93, 123.68, 122.60, 122.54, 116.97, 116.75, 116.31, 116.10, 112.10, 41.65, 41.50 31.80. m.p.:
163.2-164.6°C; MS (ESI) m/z: 443.2 [M+H]+. Anal. calcd. for C21H17ClF2N6O (%): C, 56.96; H,
3.87; N, 18.98. Found (%) C, 56.93; H, 3.91; N, 18.92.
5.1.7.6(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(4-cyanobenzylidene)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carbohydrazide(7f)
Gray solid; yield: 37.1%; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.75 (s, 1H), 8.47
(s, 1H), 8.22 (s, 1H), 8.02 – 7.96 (m, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.72

– 7.65 (m, 1H), 7.40 (t, J = 9.1 Hz, 1H), 4.57 (s, 2H), 3.81 (t, J = 5.6 Hz, 2H), 2.83 (t, J = 5.4 Hz,
2H). m.p.: 231.1-233.7 °C; MS (ESI) m/z: 450.3 [M+H]+. Anal. calcd. for C22H17ClFN7O (%): C,
58.74; H, 3.81N, 21.79. Found (%) C, 58.78; H, 3.77;N, 21.83.
5.1.7.7(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(4-(trifluoromethyl)benzylidene)-7,8-dihydropyr
ido[4,3-d]pyrimidine-6(5H)-carbohydrazide(7g)
White solid; yield: 46.6%;.1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 8.91 (s, 1H), 8.46
(s, 1H), 8.03 (dd, J = 9.1, 5.1 Hz, 3H), 7.95 (s, 1H), 7.74 (d, J = 8.1 Hz, 3H), 7.38 (t, J = 9.1 Hz,
1H), 4.56 (s, 2H), 3.74 (t, J = 5.0 Hz, 2H), 2.78 (t, J = 5.1 Hz, 2H). m.p.: 209.3-211.8°C; MS (ESI)
m/z: 493.4 [M+H]+.. Anal. calcd. for C22H17ClF4N6O (%): C, 53.61; H, 3.48; N, 17.05.
Found(%):C, 53.68; H, 3.43; N, 17.02.
5.1.7.8(E)-N'-(2-chloro-4,5-difluorobenzylidene)-4-((3-chloro-4-fluorophenyl)amino)-7,8-dihydro
pyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide(7h)
1
Light yellow solid; yield: 68.3%; H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.74 (s,
1H), 8.50 – 8.45 (m, 2H), 7.99 (dd, J = 6.8, 2.6 Hz, 1H), 7.85 – 7.75 (m, 2H), 7.64-7.68 (m,1H),
7.39 (t, J = 9.1 Hz, 1H), 4.58 (s, 2H), 3.80 (t, J = 5.6 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H). m.p.:
161.9-163.1°C; MS (ESI) m/z: 495.3 [M+H]+. Anal. calcd. for C21H15Cl2F3N6O (%): C, 50.93; H,
3.05; N, 16.97. Found (%): C, 50.97; H, 3.03; N, 16.95.
5.1.7.9(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(thiophen-2-ylmethylene)-7,8-dihydropyrido[4,
3-d]pyrimidine-6(5H)-carbohydrazide (7i)
White solid; yield: 34.5%;1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.72 (s, 1H), 8.46
(s, 1H), 8.38 (s, 1H), 7.99 (dd, J = 6.8, 2.6 Hz, 1H), 7.69 (m, 1H), 7.57 (d, J = 5.0 Hz, 1H), 7.44 –
7.29 (m, 2H), 7.10 (dd, J = 4.9, 3.7 Hz, 1H), 4.54 (s, 2H), 3.78 (t, J = 5.6 Hz, 2H), 2.82 (t, J = 5.3
Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.32, 156.72, 155.47, 154.87, 141.81, 140.23,
138.89, 130.71, 129.64, 128.10, 128.05, 125.79, 124.30, 121.56, 120.85, 112.35, 41.65, 41.60,
31.84. m.p.: 173.9-175.3°C; MS (ESI) m/z: 431.3 [M+H]+. Anal. calcd. for C19H16ClFN6OS (%):
C, 52.96; H, 3.74; N, 19.50. Found (%): C, C, 52.93; H, 3.72; N, 19.56.
5.1.7.10(E)-N'-(2-chloro-4-fluorobenzylidene)-4-((3-chloro-4-fluorophenyl)amino)-7,8-dihydropy
rido[4,3-d]pyrimidine-6(5H)-carbohydrazide(7j)
Off-white solid; yield: 30.9%; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.74 (s, 1H),
8.54 (s, 1H), 8.47 (s, 1H), 7.96-8.01 (m, 2H), 7.68-7.75 (m,1H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H),
7.39 (t, J = 9.1 Hz, 1H), 7.30 (td, J = 8.5, 2.5 Hz, 1H), 4.58 (s, 2H), 3.80 (t, J = 5.7 Hz, 2H), 2.83
(t, J = 5.4 Hz, 2H). m.p.: 189.0-190.3°C; MS (ESI) m/z: 477.3 [M+H]+. Anal. calcd. for
C21H16Cl2F2N6O (%): C, 52.85; H, 3.38; N, 17.61. Found (%) C, 52.81; H, 3.36; N, 17.64.
5.1.7.11(E)-N'-(3,5-bis(trifluoromethyl)benzylidene)-4-((3-chloro-4-fluorophenyl)amino)-7,8-dihy
dropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide(7k)
White solid; yield: 55.1%;1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.75 (s, 1H), 8.47
(s, 1H), 8.33 (s, 1H), 8.29 (s, 2H), 8.09 (s, 1H), 7.99 (dd, J = 6.8, 2.5 Hz, 1H), 7.64-7.71 (m, 1H),
7.39 (t, J = 9.1 Hz, 1H), 4.58 (s, 2H), 3.82 (t, J = 5.5 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 161.13, 156.66, 155.50, 154.70, 152.29, 139.96, 138.24, 137.27, 131.36,
130.91, 126.90, 125.00, 123.72, 122.64, 122.57, 122.34, 119.19, 119.11, 116.96, 116.75, 111.97,
41.66, 41.57, 31.82. m.p.: 250.1-251.2°C; MS (ESI) m/z: 561.4 [M+H]+. Anal. calcd. for
C23H16ClF7N6O (%): C, 49.26; H, 2.88; N, 14.98. Found (%) C, 49.29; H, 2.86;; N, 14.95.
5.1.7.12(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(4-(methylthio)benzylidene)-7,8-dihydropyrido
[4,3-d]pyrimidine-6(5H)-carbohydrazide (7l)
White solid; yield: 55.1%; 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H),8.92 (s, 1H),8.51
(s, 1H),8.17 (s, 1H), 8.00 (dd, J = 6.8, 2.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.73 – 7.68 (m, 1H),
7.57 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 4.59 (s, 2H), 3.80 (t, J = 5.3 Hz, 2H), 3.36 (s,
3H), 2.83 (t, J = 4.7 Hz, 2H). m.p.: 182.6-183.9°C; MS (ESI) m/z: 471.2 [M+H]+. Anal. calcd. for
C22H20ClFN6OS (%):,C, 56.11; H, 4.28; N, 17.85. Found (%): C, 56.14; H, 4.26; N, 17.87.

5.1.7.13(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(2-(trifluoromethyl)benzylidene)-7,8-dihydrop
yrido[4,3-d]pyrimidine-6(5H)-carbohydrazide (7m)
White solid; yield: 51.0%; 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 8.75 (s, 1H), 8.55
(s, 1H), 8.47 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.99 (dd, J = 6.8, 2.5 Hz, 1H), 7.77 (d, J = 7.8 Hz,
1H), 7.68-7.72 (m, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 9.1 Hz, 1H), 4.58 (s, 2H), 3.81 (t, J =
5.5 Hz, 2H), 2.84 (t, J = 5.3 Hz, 2H). m.p.: 130.0-131.3°C; MS (ESI) m/z: 493.3 [M+H]+. Anal.
calcd. for C22H17ClF4N6O (%):C, 53.61; H, 3.48; N, 17.05. Found (%): C, 53.64; H, 3.46; N,
17.06.
5.1.7.14(E)-4-((3-bromophenyl)amino)-N'-(2-chloro-4-fluorobenzylidene)-7,8-dihydropyrido[4,3-
d]pyrimidine-6(5H)-carbohydrazide(8a)
White solid; yield: 42.0%; 1H NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H), 8.69 (s, 1H), 8.52
(s, 1H), 8.48 (s, 1H), 8.02 (t, J = 1.9 Hz, 1H), 7.96 (dd, J = 8.9, 6.4 Hz, 1H), 7.75 (dd, J = 7.7, 1.3
Hz, 1H), 7.52 (dd, J = 8.8, 2.6 Hz, 1H),7.27-7.32 (m, 2H), 7.25 – 7.22 (m, 1H), 4.58 (s, 2H), 3.80
(t, J = 5.7 Hz, 2H), 2.83 (t, J = 5.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.28, 156.70,
155.49, 154.63, 141.83, 138.41, 130.71, 129.38, 128.60, 125.81, 124.30, 121.56, 120.86, 117.51,
117.25, 115.85, 115.64, 112.27, 41.61, 41.41, 31.84. m.p.: 157.2-158.9°C; MS (ESI) m/z: 503.2
[M+H]+. Anal. calcd. for C21H17BrClFN6O (%): C, 50.07; H, 3.40; N, 16.68. Found (%): C, 50.11;
H, 3.43; N, 16.63.
5.1.7.15(E)-N'-benzylidene-4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)
-carbohydrazide (8b)
White solid; yield: 68.7%; 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.69 (s, 1H), 8.49
(s, 1H), 8.18 (s, 1H), 8.03 (t, J = 1.9 Hz, 1H), 7.76 (d, J = 8.9 Hz, 1H), 7.66 – 7.62 (m, 2H),
7.44-7.51(m, 3H), 7.30 (t, J = 8.0 Hz, 1H), 7.23 (dd, J = 7.0, 1.6 Hz, 1H), 4.58 (s, 2H), 3.81 (t, J =
5.7 Hz, 2H), 2.83 (t, J = 5.5 Hz, 2H). m.p.: 125.6-127.0°C; MS (ESI) m/z: 451.3 [M+H]+. Anal.
calcd. for C21H19BrN6O (%): C, 55.89; H, 4.24; N, 18.62. Found (%): C, 55.84; H, 4.26; N, 18.65.
5.1.7.16(E)-N'-(4-bromobenzylidene)-4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimid
ine-6(5H)-carbohydrazide(8c)
White solid; yield: 54.4%;1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.69 (s, 1H), 8.48
(s, 1H), 8.15 (s, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.63 – 7.57 (m, 4H), 7.30
(t, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 4.58 (s, 2H), 3.80 (t, J = 5.6 Hz, 2H), 2.82 (t, J = 5.4
Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.31, 156.71, 155.49, 154.77, 142.22, 141.72,
134.62, 132.18(2C), 130.72, 128.80(2C), 125.80, 124.28, 122.75, 121.52, 120.84, 112.35, 41.67,
41.52, 31.83. m.p.: 217.0-218.2°C; MS (ESI) m/z: 529.3 [M+H]+. Anal. calcd. for C21H18Br2N6O
(%): C, 47.57; H, 3.42; N, 15.85. Found (%): C, 47.52; H, 3.44; N, 15.88.
5.1.7.17(E)-4-((3-bromophenyl)amino)-N'-(4-fluorobenzylidene)-7,8-dihydropyrido[4,3-d]pyrimid
ine-6(5H)-carbohydrazide(8d)
White solid; yield: 67.1%; 1H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.69 (s, 1H), 8.48
(s, 1H), 8.17 (s, 1H), 8.02 (s, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.69 (dd, J = 8.6, 5.7 Hz, 2H), 7.32 –
7.22 (m, 4H), 4.58 (s, 2H), 3.80 (t, J = 5.6 Hz, 2H), 2.82 (t, J = 5.4 Hz, 2H). m.p.: 138.0-139.6°C;
MS (ESI) m/z: 469.2 [M+H]+. Anal. calcd. for C21H18BrFN6O (%): C, 53.74; H, 3.87; N, 17.91.
Found (%): C, 53.77; H, 3.85; N,17.96.
5.1.7.18(E)-4-((3-bromophenyl)amino)-N'-(thiophen-2-ylmethylene)-7,8-dihydropyrido[4,3-d]pyri
midine-6(5H)-carbohydrazide(8e)
Gary solid; yield: 36.0%; 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.68 (s, 1H), 8.48
(s, 1H), 8.38 (s, 1H), 8.02 (t, J = 1.8 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 5.0 Hz, 1H),
7.34 (d, J = 3.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.25 – 7.21 (m, 1H), 7.10 (dd, J = 5.0, 3.7 Hz,
1H), 4.55 (s, 2H), 3.78 (t, J = 5.7 Hz, 2H), 2.82 (t, J = 5.5 Hz, 2H). m.p.: 149.8-150.4°C; MS
(ESI) m/z: 457.3 [M+H]+. Anal. calcd. for C19H17BrN6OS (%): C, 49.90; H, 3.75; N, 18.38. Found
(%): C, 49.93; H, 3.77; N,18.41.
5.1.7.19(E)-4-((3-bromophenyl)amino)-N'-(furan-2-ylmethylene)-7,8-dihydropyrido[4,3-d]pyrimid

ine-6(5H)-carbohydrazide(8f)
1
Brown solid; yield: 46.9%; H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.79 (s, 1H),
8.62 (d, J = 0.8 Hz, 1H), 8.49 (s, 1H), 8.33 (s, 1H), 8.13 (s, 1H), 8.09 (d, J = 8.3 Hz, 1H), 7.82 (s,
1H), 7.77 (d, J = 1.2 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 4.54 (s, 2H), 3.74
(t, J = 5.7 Hz, 2H), 2.80 (t, J = 5.3 Hz, 2H). m.p.: 157.5-160.3 °C; MS (ESI) m/z: 441.4 [M+H]+.
Anal. calcd. for C19H17BrN6O2 (%): C, 51.71; H, 3.88; N, 19.04. Found (%): C, 51.74; H,3.83;
N,19.06.
5.1.7.20(E)-4-((3-bromophenyl)amino)-N'-(pyridin-3-ylmethylene)-7,8-dihydropyrido[4,3-d]pyrim
idine-6(5H)-carbohydrazide(8g)
1
White solid; yield: 67.1%; H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 8.79 (d, J = 1.7
Hz, 1H), 8.70 (s, 1H), 8.56 (dd, J = 4.7, 1.6 Hz, 1H), 8.49 (s, 1H), 8.22 (s, 1H), 8.07 – 8.01 (m,
2H), 7.75 (d, J = 8.8 Hz, 1H), 7.44 (dd, J = 7.9, 4.8 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.23 (d, J =
8.5 Hz, 1H), 4.59 (s, 2H), 3.81 (t, J = 5.7 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H). m.p.: 124.3-125.8°C;
MS (ESI) m/z: 452.3 [M+H]+. Anal. calcd. for C20H18BrN7O (%): C, 53.11; H, 4.01; N, 21.68.
Found (%): C, 53.16; H,3.98; N, 21.71.
5.1.7.21(E)-N'-(3-bromobenzylidene)-4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimid
ine-6(5H)-carbohydrazide(8h)
Light yellow solid; yield: 43.0%; 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.70 (s,
1H), 8.48 (s, 1H), 8.15 (s, 1H), 8.03 (s, 1H), 7.83 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 7.8
Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.23 (d, J =
8.0 Hz, 1H), 4.58 (s, 2H), 3.80 (t, J = 5.6 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 161.28, 156.71, 155.49, 154.74, 141.81, 141.63, 137.85, 132.18, 131.38, 130.79,
128.95, 126.08, 125.80, 124.30, 122.59, 121.56, 120.79, 112.29, 41.68, 41.47, 31.78. m.p.:
140.3-141.7°C; MS (ESI) m/z: 529.1 [M+H]+. Anal. calcd. for C21H18Br2N6O (%): C, 47.57; H,
3.42; N, 15.85;. Found (%):C, 47.54; H, 3.46; N, 15.84;.
5.1.7.22(E)-4-((3-bromophenyl)amino)-N'-(naphthalen-1-ylmethylene)-7,8-dihydropyrido[4,3-d]p
yrimidine-6(5H)-carbohydrazide (8i)
White solid; yield: 71.3%; 1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 8.86 (s, 1H), 8.77
– 8.68 (m, 2H), 8.50 (s, 1H), 8.03 (t, J = 1.9 Hz, 1H), 7.98 (t, J = 9.2 Hz, 2H), 7.86 (d, J = 6.7 Hz,
1H), 7.76 (dd, J = 5.0, 4.0 Hz, 1H), 7.65 – 7.55 (m, 3H), 7.30 (t, J = 8.0 Hz, 1H), 7.26 – 7.22 (m,
1H), 4.63 (s, 2H), 3.86 (t, J = 5.7 Hz, 2H), 2.87 (t, J = 5.6 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 161.35, 156.74, 155.51, 154.93, 143.14, 141.81, 134.01, 130.74, 130.62, 130.56,
130.10, 129.20, 127.48, 126.93, 126.61, 126.05, 125.82, 124.53, 124.31, 121.57, 120.87, 112.38,
41.69, 41.46, 31.83. m.p.: 167.5-169.2°C; MS (ESI) m/z: 501.3 [M+H]+. Anal. calcd. for
C25H21BrN6O (%): C, 59.89; H, 4.22; N, 16.76. Found (%): C, 59.86; H, 4.24; N, 16.74.
5.1.7.23(E)-4-((3-bromophenyl)amino)-N'-(pyridin-4-ylmethylene)-7,8-dihydropyrido[4,3-d]pyrim
idine-6(5H)-carbohydrazide (8j)
White solid; yield: 46.2%; 1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 8.71 (s, 1H), 8.61
– 8.59 (m, 2H), 8.49 (s, 1H), 8.16 (s, 1H), 8.02 (t, J = 1.8 Hz, 1H), 7.78 – 7.73 (m, 1H), 7.58 (d, J
= 6.0 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 4.60 (s, 2H), 3.82 (t, J = 5.7 Hz,
2H), 2.84 (t, J = 5.5 Hz, 2H). m.p.: 157.5-159.0°C MS (ESI) m/z: 452.5 [M+H]+. Anal. calcd. for
C20H18BrN7O (%):C, 53.11; H, 4.01; N, 21.68. Found (%): C, 53.14; H, 4.03; N, 21.62.
5.1.7.24(E)-N'-((1H-pyrrol-2-yl)methylene)-4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,3-d]p
yrimidine-6(5H)-carbohydrazide (8k)
Reddish brown solid; yield: 36.4%; 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 10.19
(s, 1H), 8.68 (s, 1H), 8.50 (s, 1H), 8.05 (d, J = 3.1 Hz, 2H), 7.78 (d, J = 8.9 Hz, 1H), 7.32 (t, J =
8.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 1.4 Hz, 1H), 6.38 (s, 1H), 6.12 (t, J = 4.1 Hz,
1H), 4.58 (s, 2H), 3.79 (t, J = 5.7 Hz, 2H), 2.84 (t, J = 5.5 Hz, 2H). m.p.: 150.8-151.7°C; MS
(ESI) m/z: 440.3 [M+H]+. Anal. calcd. for C19H18BrN7O (%):C, 51.83; H, 4.12; N, 22.27. Found
(%): C, 51.85; H, 4.14; N, 22.23.

5.1.7.25(E)-N'-(4-methylbenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3
-d]pyrimidine-6(5H)-carbohydrazide(9a)
White solid; yield: 36.0%; 1H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 8.07 (s, 1H), 8.05
(s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.51 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.8
Hz, 1H), 7.16 (d, J = 8.0 Hz, 2H), 4.64 (s, 2H), 3.86 (t, J = 5.8 Hz, 2H), 2.89 (t, J = 5.7 Hz, 2H),
2.32 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.35, 156.75, 155.39, 155.02, 143.58, 140.96,
139.33, 132.60, 129.91, 129.78 (2C), 129.44, 126.90 (2C), 126.13, 125.58, 119.47, 118.12, 112.48,
41.71, 41.49, 31.80, 21.40. m.p.: 192.6-194.8°C; MS (ESI) m/z: 455.3 [M+H]+. Anal. calcd. for
C23H21F3N6O (%): C, 60.79; H, 4.66; N, 18.49. Found (%): C, 60.74; H,4.69; N, 18.52.
5.1.7.26(E)-N'-(thiophen-2-ylmethylene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[
4,3-d]pyrimidine-6(5H)-carbohydrazide(9b)
White solid; yield: 68.3%; 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.86 (s, 1H), 8.49
(s, 1H), 8.38 (s, 1H), 8.14 – 8.06 (m, 2H), 7.57 (dd, J = 9.9, 5.9 Hz, 2H), 7.37 (dd, J = 16.4, 5.5 Hz,
2H), 7.14 – 7.06 (m, 1H), 4.58 (s, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.84 (t, J = 5.4 Hz, 2H). m.p.:
156.0-157.2°C; MS (ESI) m/z: 447.4 [M+H]+. Anal. calcd. for C20H17F3N6OS (%): C, 53.81; H,
3.84; N, 18.82. Found (%): C, C, 53.84; H, 3.82; N, 18.86.
5.1.7.27(E)-N'-(4-bromobenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3
-d]pyrimidine-6(5H)-carbohydrazide(9c)
1
Yellow solid; yield: 65.8%; H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.89 (s, 1H),
8.50 (s, 1H), 8.12 (dd, J = 16.5, 11.2 Hz, 3H), 7.64 – 7.54 (m, 5H), 7.39 (d, J = 7.6 Hz, 1H), 4.61
(s, 2H), 3.81 (t, J = 5.3 Hz, 2H), 2.84 (d, J = 4.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ
161.43, 156.73, 155.47, 154.81, 142.18, 141.34, 134.63, 132.18 (2C), 132.14, 131.97, 129.93,
128.79 (2C), 125.58, 123.12, 119.72, 118.43, 112.43, 41.68, 41.60, 31.96. m.p.: 159.0-161.2°C;
MS (ESI) m/z: 519.2 [M+H]+. Anal. calcd. for C22H18BrF3N6O (%): C, 50.88; H, 3.49; N, 16.18.
Found (%): C, 50.84; H,3.44; N, 16.21.
5.1.7.28(E)-N'-(4-fluorobenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-
d]pyrimidine-6(5H)-carbohydrazide(9d)
White solid; yield: 67.1%;1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.87 (s, 1H), 8.50
(s, 1H), 8.17 (s, 1H), 8.10 (d, J = 12.6 Hz, 2H), 7.73 – 7.66 (m, 2H), 7.57 (t, J = 7.9 Hz, 1H), 7.39
(d, J = 7.7 Hz, 1H), 7.25 (t, J = 8.8 Hz, 2H), 4.60 (s, 2H), 3.81 (t, J = 5.5 Hz, 2H), 2.84 (t, J = 5.3
Hz, 2H). m.p.: 163.7-164.9°C; MS (ESI) m/z: 459.3 [M+H]+. Anal. calcd. for C22H18F4N6O (%):
C, 57.64; H, 3.96; N, 18.33. Found (%): C, 57.66; H,3.94; N, 18.37.
5.1.7.29(E)-4-((3-(trifluoromethyl)phenyl)amino)-N'-vinyl-7,8-dihydropyrido[4,3-d]pyrimidine-6(
5H)-carbohydrazide (9e)
White solid; yield: 85.5%;1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.76 (d, J = 3.4 Hz,
2H), 8.49 (s, 1H), 8.12 – 8.05 (m, 2H), 7.56 (t, J = 7.9 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 6.29 (dd,
J = 17.1, 10.1 Hz, 1H), 6.18 (dd, J = 17.1, 2.2 Hz, 1H), 5.70 (dd, J = 10.1, 2.2 Hz, 1H), 4.53 (s,
2H), 3.74 (t, J = 5.6 Hz, 2H), 2.80 (t, J = 5.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.94,
161.37, 157.34, 156.70, 155.46, 140.93, 130.05, 129.95, 126.98, 125.50, 119.45, 118.02, 112.47,
79.94, 41.30, 40.98, 31.42. m.p.: 176.3-177.8°C; MS (ESI) m/z: 379.3 [M+H]+. Anal. calcd. for
C17H17F3N6O (%): C, 53.97; H, 4.53; N, 22.21. Found (%): C, 53.94; H, 4.56; N, 22.24.
5.1.7.30(E)-N'-(4-(trifluoromethyl)benzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydro
pyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide (9f)
White solid; yield: 55.1%; 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.06 (s, 1H), 8.54
(s, 1H), 8.33 (s, 1H), 8.17 (d, J = 11.5 Hz, 3H), 7.88 (s, 3H), 7.80 (d, J = 7.8 Hz, 2H), 7.60 (t, J =
7.8 Hz, 1H), 7.42 (d, J = 6.9 Hz, 1H), 4.71 (s, 2H), 3.86 (s, 2H), 2.88 (s, 2H). m.p.: 121.0-122.6°C;
MS (ESI) m/z: 509.3 [M+H]+. Anal. calcd. for C23H18F6N6O (%): C, 54.33; H, 3.57; N, 16.53.
Found (%): C, 54.37; H, 3.54; N, 16.54.
5.2. Pharmacology

5.2.1. MTT assay in vitro
The cytotoxic activities of compounds 7a-7i, 8a-8g and 9a-9d were evaluated against A549,
H1975, MKN-45 and SGC by the standard MTT assay in vitro, with gefitinib as the positive
controls. The cancer cell lines were cultured in minimum essential medium (MEM) supplement
with 10% fetal bovine serum (FBS).
Approximate 4 × 103 cells, suspended in MEM medium, were plated into each well of a 96-well
o
plate and incubated in 5% CO2 at 37 C for 24 h. The tested compounds at the indicated final
concentrations were added to the culture medium and incubated for 72 h. Fresh MTT was added to
each well at the terminal concentration of 5 μg/mL, and incubated with cells at 37 oC for 4 h. The
formazan crystals in each well were dissolved in 100 μL DMSO, and the absorbency at 492 nm
(for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with
an ELISA reader. All of the compounds were tested three times in each of the cell lines. The
results, expressed as IC50 (inhibitory concentration 50%), were the averages of three
determinations and calculated relative to the vehicle (DMSO) control by the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software.
5.2.2. In vitro enzymatic assays
The in vitro enzymatic assays versus EGFR, VEGFR2 and T790M/L858R/EGFR were
evaluated by homogeneous time-resolved fluorescence (HTRF) assay.
In enzymatic assay, the solution of peptide substrates, ATP, appropriate kinase, and diluted
compound was mixed with the kinase reaction buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35,
10 mM MgCl2, 2 mM DTT), with blank DMSO solution as the negative control. The kinase
reaction was initiated by the addition of tyrosine kinase proteins diluted in 39 μL of kinase
reaction buffer solution and incubated at 28 °C for 60 min. And then add 25 μL of stop buffer (100
mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) to stop reaction.
The plate was read by Caliper at 320 nm and 615 nm. IC50 values were calculated from the
inhibition curves.
5.2.3. Inhibition rate assay
Cardiac fibroblasts were isolated from Wistar rats (1-2 days old). Briefly, excised hearts were
washed in ice-cold 1×phosphate buffer saline (1×PBS) solution, and then ventricular tissues were
minced into small pieces by use of scissors in a mixture of 0.2% collagenase type 2 and 0.25%
trypsin. Minced tissue was then placed into a sterile 50-ml tube and incubated with agitation at
37uC for 50 min. The supernatant was collected, and the remaining tissue was immersed in
digestion solution. The supernatants were pooled and filtered through a 400-mm nylon mesh filter,
then centrifuged at 1,500 r/min for 5 min at 4uC. The supernatant was removed, and cells were
placed into fibroblast culture media (DMEM, 10% normal bovine serum and 100 U/ml
penicillin/streptomycin) and incubated for 2 h at 37°C with 5% CO2 and 95% air. Cells were
examined for positivity for vimentin. Fibroblasts between passages 6 to 12 were used for
experiments.
Cell proliferation was measured by MTT assay. Briefly, CFs were seeded in 96-well culture
plates a density of 1×104 cells per well with DMEM containing 10% FBS and 5.6 mM glucose.
After starvation in serum-free medium for 24 h, CFs were incubated in DMEM containing 5.6
mM glucose (normal glucose), 25 mM glucose (HG), 25 mM glucose plus 20 lM Res, 25 mM
glucose plus 20 μM target compounds, with 25 mM glucose plus 0.1% DMSO as the control.
Following incubation for 12, 20 μL of MTT solution (5 mg/ml in phosphate-buffered saline, PBS)

was added to each well. After incubation at 37 °C for 4 h, the culture medium was removed and
150 μL of DMSO was then added. The optical density (OD) for each well was determined at 490
nm using a spectrophotometer (Bio-Rad, CA, USA). Blank control wells were designed without
cells, but with the addition of culture medium in parallel to the experimental wells.
5.2.4. enzymatic assays of ATX
Starting from 25 μM highest concentration, 10 μL of a dilution series of compound, 1/5
dilution, was added to the wells. Glycosylated human ATX protein was used at a final
concentration of 0.4 or 0.54 μg/mL. The enzyme was diluted in 50 mM Tris-HCl (2-amino-2-
(hydroxymethyl)-1,3-propanediol hydrochloride) pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1.5 mM CaCl2, and 0.1% fatty acid free BSA in a total volume of 25 μL. The enzyme
mixture was added to compounds, and the resulting mixture was incubated for 45 min at room
temperature under shaking. The reaction was started by the addition of 20 μL of 0.75 μM FS-3
diluted in the same buffer as described above. Fluorescence was read on an Envision
apparatusafter 45 min incubation at room temperature (excitation 485 nm, emission 520 nM).
5.2.4. Nitric oxide determination
RAW264.7 cells (5 × 105 cells/mL) in logarithmic phase were pretreated with different
concentrations of target compound for 2h, and then incubated with 5 μg/mL LPS for 18h. And all
control groups were not treated with anything. The nitrite accumulation in the supernatant was
determined by Griess reagent. Briefly, equal volume of cell-free culture media (50 μL) was
reacted with Griess reagent I (50 μL) and Griess reagent II (50 μL), and measuring the absorbance
at 540nm.
5.2.5. Real-time polymerase chain reaction
RAW264.7 cells (1 × 106 cells/mL) were pretreated with different concentrations of 8e and
9a for 2h, followed by treatment with LPS (1 μg/mL) and incubation for an additional 8 h. Total
RNA from RAW264.7 cells were extracted using Total RNA Extraction Reagent (Vazyme, China)
according to the manufacturer's instructions. The concentration and purity of RNA were measured
by spectrophotometric analysis with a Gene Quant Pro spectrophotometer (Amer-sham
Biosciences,USA). Total RNA (2 μg) was reverse-transcribed to cDNA by using the HiScript™ Q
RT Super Mix (Vazyme, China) in a total volume of 20 μL. RT-PCR was performed on a
iCycleriQ™5 Multicolor Reai-Time PCR Detection System (Bio-Rad, USA) with SYBRs Green
Master Mix (Vazyme, China). The sequences of primers used for RT-PCR analyses are shown
below. Amplification conditions were as follows:95 °C initial denaturation for 5 min followed by
40 cycles of 95 °C for 15s and 60 °C for 30s. Relative expression levels of the target genes were
calculated based on 2-ΔΔCt according to the manufacture's specifications by using the
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a reference housekeeping gene.
Gene
Primer sequence
5’-CTGCOGATCTCTGTGTCAUT-3’
5’-CTCAGAGTGUTOCTATGGTG-3’
5’-GTAGCCCACGTCGTAGCAA-3’
5’-GTGAGGAGCACGTAGTCGG-3’
TGF-β Forward:
TGF-β Reverse:
TNF-α Forward:
TNF-α Reverse:
Acknowledgments
This work was supported by Development Project of Ministry of Education Innovation Team
(IRT1073).

References
[1] Kakerina MA, Yara T, Eliza T, Carlo V. Idiopathic pulmonary fibrosis and lung cancer: a clinical and
pathogenesis update. Curr. Opin. Pulm. Med. 2015; 21: 626- 633.
[2] Tomassetti S, Gurioli C, Ryu JH, Decker PA, Ravaglia C, Tantalocco P, Buccioli M, Piciucchi S, Sverzellati N,
Dubini A, Gavelli G, Chilosi M, Poletti V. The impact of lung cancer on survival of idiopathic pulmonary
fibrosis. Chest. 2015; 147:157-164.
[3] Hwang JA, Kim D, Bae SH, Chun SM, Song JS, Kim MY, Song JW, Kim WS, Lee JC, Park S, Kim HR, Choi
CM, Jang SJ. MA05.05 Genomic Profiles of Lung Cancer Associated with Idiopathic Pulmonary Fibrosis. J.
Thorac. Oncol. 2017; 12: S366-S366.
[4] Bowley E, O'Gorman DB, Gan BS. Beta-catenin signaling in fibroproliferative disease. J. Surg. Res. 2007;
138:141-150.
[5] Day BJ. Antioxidants as potential therapeutics for lung fibrosis. Antioxid. Redox Sign. 2008; 10: 355-370.
[6] López-Novoa JM, Nieto MA. Inflammation and EMT: an alliance towards organ fibrosis and cancer
progression. Embo. Mol. Med. 2009; 1:303-314.
[7] Heinrich EL, Walser TC, Krysan K, Liclican EL, Grant JL, Rodriguez NL, Steven M. The Inflammatory Tumor
Microenvironment, Epithelial Mesenchymal Transition and Lung Carcinogenesis. Cancer Microenvironment.
2012; 5:5-18.
[8] Suzuki H, Sekine Y, Yoshida S, Suzuki M, Shibuya K, Yonemori Y, Hiroshima K, Nakatani Y, Mizuno S,
Takiguchi Y, Yoshino I. Risk of acute exacerbation of interstitial pneumonia after pulmonary resection for lung
cancer in patients with idiopathic pulmonary fibrosis based on preoperative high-resolution computed
tomography. Surg. Today. 2011; 41:914-921.
[9] Watanabe A, Miyajima M, Mishina T, Nakazawa J, Harada R, Kawaharada N, Higami T. Surgical treatment for
primary lung cancer combined with idiopathic pulmonary fibrosis. Gen. Thorac. Cardiovasc. Surg. 2013;
6:254-261.
[10] Wada K, Lee JY, Hung HY, Shi Q, Lin L, Zhao Y, Goto M, Yang PC, Kuo SC, Chen HW, Lee KH. Novel
curcumin analogs to overcome EGFR-TKI lung adenocarcinoma drug resistance and reduce
EGFR-TKI-induced GI adverse effects. Bioorg. Med. Chem. 2015; 23:1507-1514.
[11] F. Cappuzzo, T. Ciuleanu, L. Stelmakh, S. Cicenas, A. Szczésna, E. Juhász, E. Esteban, O. Molinier, W.
Brugger, I. Melezínek, G. Klingelschmitt, B. Klughammer, G. Giaccone. Erlotinib as maintenance treatment in
advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study. Lancet
Oncol. 2010; 11:521-529.
[12] WF Li, L Li, WF Yuan, WJ Huang. Comparison of interventional effects of two epidermal growth factor
receptor tyrosine kinase inhibitors on pulmonary fibrosis in mice. Chin. J. Respir. Critic. Care Med. 2010;
09:426-429.
[13] Voltolinia L, Bongiolattia S, Luzzia L, Bargaglib E, Fossib A, Ghiribellia C, Rottolib P, Gottia G. Impact of
interstitial lung disease on short-term and long-term survival of patients undergoing surgery for
non-small-cell lung cancer: analysis of risk factors. Eur. J. cardio-thorac. 2013; 43:17-23.
[14] King TEJ, Bradford WZ, Castro-Bernardini S, Fagan EA, Glaspol I, Glassberg MK, Gorina E, Hopkins PM,
Kardatzke D, Lancaster L, Lederer DJ, Nathan SD, Pereira CA, Sahn SA, Sussman R, Swigris JJ. A Phase 3
Trial of Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis. New Engl. J. Med. 2014; 370:2083-2092.
[15] Roth GJ, Binder R, Colbatzky F, Dallinger C, Schlenker-Herceg R, Hilberg F, Wollin SL, Kaiser R.
Nintedanib: From Discovery to the Clinic. J. Med. Chem. 2015; 58:1053-1063.
[16] Grimminger F, Gunther A, Vancheri C. The role of tyrosine kinases in the pathogenesis of idiopathic
pulmonary fibrosis. Eur. Respi.r J. 2015; 45:1426–1433.

[17] Kang TS, Wang W, Zhong HJ, Liang JX, Ko CN, Lu JJ, Chen XP, Ma DL, Leung CH. A rhodium(III)-based
inhibitor of autotaxin with antiproliferative activity. Biochim. Biophys. Acta. 2017; 1861:256-263.
[18] Ragle LE, Palanisamy DJ, Joe MJ, Stein RS, Norman DD, Tigyi G, Baker DL, Parrill AL. D Discovery and
synthetic optimization of a novel scaffold for hydrophobic tunnel-targeted autotaxin inhibition. Bioorg. Med.
Chem. 2016; 24:4660-4674.
[19] Ongenaert M, Dupont S, Blanqué R, Aar EVD. Strong reversal of the lung fibrosis disease signature by
autotaxin inhibitor GLPG1690 in a mouse model for IPF. European Respiratory J, 2016; 48: 4540-4552.
[20] Barbayianni E, Magrioti V, Moutevelisminakakis P, Kokotos G. Autotaxin inhibitors: a patent review. Expert.
Opin. Ther. Pat. 2013; 23:1123-1132.
[21] Long S A, Atli T, Schnute M E. Pyrimidine and pyridine derivatives useful in therapy. 2013; WO 2013054185
A1.
[22] Zhang Y, Zhang K, Zhao M, Zhang L, Qin M, Guo S, Zhao Y, Gong P. Discovery of a novel class
anti-proliferative agents and potential inhibitors of EGFR tyrosine kinases based on
4-anilinotetrahydropyrido[4,3-d]pyrimidine scaffold: Design, synthesis and biological evaluations. Bioorg.
Med. Chem. 2015; 23:4591-4607.
[23] Totrov M. Atomic Property Fields: Generalized 3D Pharmacophoric Potential for Automated Ligand
Superposition, Pharmacophore Elucidation and 3D QSAR. Chem. Biol.Drug Design. 2008; 71:15-27.
[24] Cheeseright T, Mackey M, Rose S, Vinter A. Molecular field extrema as descriptors of biological activity:
definition and validation. J. Chem. Inf. Model.. 2006; 46:665-676.
[25] Rati K. P. Tripathia , Vishnu M. Sasia , Sukesh K. Guptab , Design, synthesis, and pharmacological evaluation
of 2 amino-5 nitrothiazole derived semicarbazones as dual inhibitors of monoamine oxidase and
cholinesterase: effect of the size of aryl binding site. J. Enzyme Inhib. Med. Ch. 2018, 33: 37–57.
[26] JH Liu, XZ Zhuo, WM Liu, ZF Wan, X Liang, SS Gao, ZY Yuan, Y Wu. Resveratrol inhibits high
glucose induced collagen upregulation in cardiac fibroblasts through regulating TGF-β1–Smad3 signali
ng pathway. Chem-Biol. Int. 2015; 227:45-52.
[27] Li B, Cai S, Yang YA, Chen SC, Chen R, Shi JB, Liu XH, Tang WJ. Novel unsaturated glycyrrhetic acids
derivatives: Design, synthesis and anti-inflammatory activity. Eur. J. Med. Chem. 2017; 139: 337-348.
[28] Chen X, Miao J, Wang H, Zhao F, Hu J, Gao P, Wang Y, Zhang L, Yan M. The anti-inflammatory activities of
Ainsliaea fragrans Champ. extract and its components in lipopolysaccharide-stimulated RAW264.7
macrophages through inhibition of NF-κB pathway. J. Ethnopharmaco. 2015; 170:72-80.
[29] Froese AR, Shimbori C, Bellaye PS, Inman M, Obex S, Fatima S, Jenkins G, Gauldie J, Ask K, Kolb M.
Stretch-induced Activation of Transforming Growth Factor-β1 in Pulmonary Fibrosis. Am. J. Resp. Crit. Care.
2016; 194:84-96.
[30] Tsavlis D, Tektonidou A, Papadopoulou A, Tzoumaka A. T Erythropoietin (EPO) Inhibits the Expression of
Tumor Necrosis Factor-Alpha (TNF-α) in Bleomycin (BLM)-Induced Pulmonary Fibrosis in Rats. Chest.
2014; 146:245A-245A.
[31] Gajiwala KS, Feng J, Ferre R, Ryan K, Brodsky O, Weinrich S, Kath JC, Stewart A. Insights into the Aberrant
Activity of Mutant EGFR Kinase Domain and Drug Recognition. Structure. 2013, 21:209.
[32] Stein AJ, Bain G, Prodanovich P, Santini AM, Darlington J, Stelzer NM, Sidhu RS, Schaub J, Goulet L,
Lonergan D, Calderon I, Evans JF, Hutchinson JH. Structural Basis for Inhibition of Human Autotaxin by
Four Potent Compounds with Distinct Modes of Binding. Mol. Pharmacol. 2015; 88:982-992.



A series of novel THPPs derivatives (7a-7m, 8a-8k & 9a-9f) were designed and synthesized.
9a showed better cytotoxicity and anti-fibrosis activity against tested cells than positive
control.



Enzymatic assays ascertained 9a with promising EGFR (24.2 nM) and ATX (29.1 nM))
inhibitory potency.
RT-PCR study revealed 9a could reduce the mRNA expression of TGF-β and TNF-α
significantly.
Docking mode indicated 9a could form critical bonding interactions with both of EGFR and
ATX.

Graphical Abstract