Optimization and evaluation of novel tetrahydropyrido[4,3-d]pyrimidine derivatives as ATX inhibitors for cardiac and hepatic fibrosis

Article abstract of DOI:10.1016/j.ejmech.2019.111904

Aiming to develop potent autotaxin (ATX) inhibitors for fibrosis diseases, a novel series of tetrahydropyrido[4,3-d]pyrimidine derivatives was designed and synthesized based on our previous study. The enzymatic assay combined with anti-proliferative activities against cardiac fibroblasts (CFs) and hepatic stellate cell (HSC) in vitro were applied for preliminary evaluation of anti-fibrosis potency of target compounds, resulting in two outstanding ATX inhibitors 8b and 10g with the IC₅₀ values in a nanomolar range (24.6 and 15.3 nM). Differently, 8b was the most prominent compound against CFs with inhibition ratio of 81.5%, while 10g exhibited the maximum inhibition ratio of 83.7% against t-HSC/Cl-6 cells. In the further pharmacological evaluations in vivo, collagen deposition assay demonstrated the conspicuous capacity of 8b to suppress TGF-β-mediated cardiac fibrosis. Simultaneously, H&E and Masson stains assays of mice liver validated 10g as an excellent anti-hepatofibrosis candidate, which reduced CCl₄-induced hepatic fibrosis level prominently. Besides, the molecular binding models identified the essential interactions between 8b and ATX which was coincided with the SARs.

Full text of DOI:10.1016/j.ejmech.2019.111904

Journal Pre-proof
Optimization and evaluation of novel tetrahydropyrido[4,3-d]pyrimidine derivatives as
ATX inhibitors for cardiac and hepatic fibrosis
Nan Jiang, Yuhong Zhou, Minglin Zhu, Junlong Zhang, Meng Cao, Hongrui Lei, Ming
Guo, Ping Gong, Guangyue Su, Xin Zhai
PII:
S0223-5234(19)31056-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.111904
EJMECH 111904
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2019
Revised Date: 17 November 2019
Accepted Date: 18 November 2019
Please cite this article as: N. Jiang, Y. Zhou, M. Zhu, J. Zhang, M. Cao, H. Lei, M. Guo, P. Gong, G.
Su, X. Zhai, Optimization and evaluation of novel tetrahydropyrido[4,3-d]pyrimidine derivatives as ATX
inhibitors for cardiac and hepatic fibrosis, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111904.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. 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.
©
2019 Published by Elsevier Masson SAS.

Optimization and evaluation of novel tetrahydropyrido[4,3-d]pyrimidine
derivatives as ATX inhibitors for cardiac and hepatic fibrosis
a, 1
c, 1
a
a
a
a
a
Nan Jiang , Yuhong Zhou , Minglin Zhu , Junlong Zhang , Meng Cao , Hongrui Lei , Ming Guo ,
a
b, **
a, ∗
Ping Gong , Guangyue Su , Xin Zhai
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, China.
b
School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, China.
c
The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of
Cardiovascular Research, Ministry of Education, Harbin Medical University, Harbin, China.
Abstract
Aiming to develop potent autotaxin (ATX) inhibitors for fibrosis diseases, a novel series of
tetrahydropyrido[4,3-d]pyrimidine derivatives was designed and synthesized based on our
previous study. The enzymatic assay combined with anti-proliferative activities against cardiac
fibroblasts (CFs) and hepatic stellate cell (HSC) in vitro were applied for preliminary evaluation
of anti-fibrosis potency of target compounds, resulting in two outstanding ATX inhibitors 8b and
1
0g with the IC values in a nanomolar range (24.6 and 15.3 nM). Differently, 8b was the most
50
prominent compound against CFs with inhibition ratio of 81.5%, while 10g exhibited the
maximum inhibition ratio of 83.7% against t-HSC/Cl-6 cells. In the further pharmacological
evaluations in vivo, collagen deposition assay demonstrated the conspicuous capacity of 8b to
suppress TGF-β-mediated cardiac fibrosis. Simultaneously, H&E and Masson stains assays of
mice liver validated 10g as an excellent anti-hepatofibrosis candidate, which reduced
CCl -induced hepatic fibrosis level prominently. Besides, the molecular binding models identified
4
the essential interactions between 8b and ATX which was coincided with the SARs.
Key Words: ATX; THPPs; Semicarbazones; Cardiac fibrosis; Hepaticfibrosis
1
. Introduction
Fibrosis is usually defined as an excessive production of extracellular matrix (ECM)
components by activated myofibroblasts in response to chronic injury. Fibrosis diseases have
become serious worldwide health issues with extensive morbidity and mortality, which involve
almost all chronic organ failure and multi-systemic diseases, for instance, lung, liver, heart, kidney,
cystic fibrosis and multiple sclerosis [1, 2]. The complicated pathogenesis of fibrosis diseases have
been defined gradually within the past decade years. Transforming growth factor-β (TGF-β) is one
of the most pivotal profibrotic cytokine which mediates fibrosis by promoting ECM production
and collagen deposition [3, 4]. The specific TGF-β inhibitor pirfenidone [5] (Fig. 1) was approved
∗
Corresponding author.
∗
∗
Corresponding author.
E-mail address: zhaixin_syphu@126.com (X. Zhai); suggyy@163.com (G.Y.Su).
1
These authors contributed equally to this work.

in Japan for idiopathic pulmonary fibrosis (IPF) in 2008. Besides, other heterogeneous molecular
pathways and kinases also provide potential targets for clinical therapy [6, 7], such as multi-target
RTKs inhibitor nintedanib [8]; VEGFR-2 and PDGF-β inhibitor sorafinib [9,10], Smads inhibitor
SIS3 [11], JAK1/2 inhibitor ruxolitinib [12], ATX inhibitor GLPG1690 [13] (Fig.1), and so on.
Figure 1. Representative anti-fibrosis targets and agents.
Autotaxin
(ATX),
the
only
lysophospholipase
D
of
the
ectonucleotide
pyrophosphatase/phosphodiesterase (ENPP) family, is known for the unique ability of generating
the vast majority of the extracellular lysophosphatidate (LPA) from lysophosphatidylcholine (LPC)
in blood. The ATX/LPA axis is implicated in a variety of disease processes from fibrosis to cancer,
colitis and asthma, resulting in the emergence of ATX inhibitors applied to the therapeutic for
fibrosis diseases [14-16]. GLPG1690 is the first ATX inhibitor inphase II clinic for treatment of
idiopathic pulmonary fibrosis (IPF) [17]. Also, compelling ATX inhibitor A (Fig. 1) reported by
Pfizer showed remarkable anti-fibrosis potency with IC value of 2.59 nM against ATX owing to
50
privileged tetrahydropyrido[4,3-d]pyrimidines (THPPs) scaffold and non-rigid long-chain linker
region [18, 19].
In our previous work to develop potent ATX inhibitors, compound 1 as a hit was validated to
possess considerable anti-fibrosis potency by elementary pharmacological activity screening in
vitro [20]. Although, 1 exhibited considerable potency with inhibition rate of 77% towards CFs
cell and IC value of 29.1 nM against ATX enzyme, the thorough understanding of SARs and
5
0
diversity of anti-fibrosis activity evaluation were deficient.
With aiming to obtain more potent ATX inhibitors for fibrotic diseases and to enrich the SARs,
a novel series of tetrahydropyrido[4,3-d]pyrimidine derivatives (8a-e, 9a-h and 10a-k) was
1
designed and synthesized in further optimization. Various R and Ar groups were introduced into
the terminals of phenyl fragment and semicarbazones (SCZs) linker, focusing on
halogenated-phenyl groups for the essential that halogen bonding interactions are comparable in
strength to hydrogen bonds and facilitate conformation perturbations [21, 22]. The enzymatic
assay combined with anti-proliferative activities against CFs and HSC in vitro were applied for
preliminary evaluation of inhibitory potency for cardiac and hepatic fibrosis in vitro. Moreover,
two optimal compounds 8b and 10g were further evaluated by CFs collagen assessment in vitro.

In view of the prominent superiority of 10g toward HSC, H&E and Masson stains assays of mice
liver were carried out to assess the capability of reducing CCl -induced hepatic fibrosis level in
4
vivo.
Figure 2. Design strategies for target compounds.
2
. Results and discussion
2
.1 Chemistry
The synthesis of target compounds 8a-8e, 9a-9h and 10a-10k was described in Scheme 1.
Hydrogenation of commercially available ethyl 1-benzyl-4-oxopiperidine-3-carboxylateunder
catalysis of palladium-carbon afforded intermediate 2, which was replaced by phenyl
chloroformate at 0 °C to room temperature in the presence of diisopropylethylamine (DIPEA) to
generate 3. Tetrahydropyrido[4,3-d]pyrimidine intermediate 4 was obtained via cyclization of
intermediate 3 with formamide acetate in refluxing methanol for 6 h. Subsequently, the
nucleophilic substitution of 4 with phosphorus oxychloride as chlorination agent at 100 °C for 10
minutes gave rise to chloride 5, which was further replaced by different anilines to yield 6a-6c.
Then, 7a-7c were prepared from 6a-6c by hydrazinolysis in ethanol. Eventually, the condensation
of 7a-7c with corresponding aromatic aldehyde in refluxing anhydrous ethanol for 1 h furnish
target compounds 8a-8e, 9a-9h and 10a-10k successfully without any further purification. The
1
13
structures of all target compounds were confirmed by MS and H-NMR spectra. C-NMR spectra
of partial compounds were also provided. Furthermore, the E or Z configuration of all target
compounds were demonstrated as E isomer on the strength of our preliminary research.

O
O
O
O
O
O
O
OH
N
O
•
HCl
a
b
c
O
N
O
NH•HCl
O
N
O
N
N
4
O
2
3
R1
R1
Cl
N
O
NH
O
f
NH
N
O
g
e
d
N
N
5
O
NH2
N
N
N
O
N
N
N
H
6a-6c
7a-7c
6
a, 7a, 8a-e: R1 = 3-Cl-4-F
1
9b: Ar = 3,4-diCH3Ph
10c: Ar = 2-Cl, 4-FPh
10d: Ar = Ph
R1
6b, 7b, 9a-h: R = 3-Br
9
9
c: Ar = 3,5-diCF3Ph
d: Ar = 4-SO2CH3Ph
1
NH
N
O
6c, 7c, 10a-k: R = 3-CF3
10e: Ar = 3,5-diCF3Ph
N
Ar
N
N
N
8a: Ar = 2,4,6-tirCH Ph
9e: Ar = 3-CF Ph
10f: Ar = 4-SO CH Ph
3
3
2
3
H
8
8
8
8
9
b: Ar = 4-ClPh
9f: Ar = 3-Cl, 4-FPh
10g: Ar = 4-ClPh
c: Ar = 2-Cl, 5-NO2Ph
d: Ar = 3-Br, 4-OHPh
e: Ar = 4-SO2CH3Ph
a: Ar = 4-OCF3Ph
9g: Ar= 2-Cl, 4,5-diFPh
9h: Ar = 4-ClPh
10h: Ar = 2,4-diClPh
10i: Ar = 3-pyridyl
10j: Ar = 2-furyl
8
a-7e, 9a-9h, 10a-10k
10a: Ar = 3-BrPh
10b: Ar = 4-OCF3Ph
10k: Ar = 2-pyrrolyl
Scheme 1. The synthetic rounte for target compounds. Reagents and conditions: (a) H /Pd-C, 95% ethanol 50 °C,
2
7
h; (b) ClCOOPh, DIPEA, DCM, 0 °C then r.t. 1 h; (c) formamidine acetate, CH ONa, CH OH, reflux, 6 h; (d)
3 3
POCl , 100 °C, 10min; (e) aniline, NH Cl, MeCN, reflux, 5 h; (f) NH NH H O, ethanol, reflux, 2 h; (g) aromatic
3
4
2
2
2
aldehyde, ethanol, reflux, 1 h.
2
2
.2 Biological activity and discussion
.2.1 Anti-proliferation activities in vitro and SARs study
Organ fibrosis is strictly related to the activation of myofibroblas. In order to estimate the
anti-fibrosis potency, all target compounds (8a-e, 9a-h and 10a-k) were tested anti-proliferation
activities against CFs which generated from Wistar rats and t-HSC/Cl-6 cells (American Type
Culture Collection) in MTT assay [23, 24], taking lead compounds A and 1 as positive controls.
The results were expressed as inhibition ratio at 10 µM and summarized in Table 1. All data were
generated by three independent experiments.
Table1. The structures and anti-proliferation activities of compounds 8a-e, 9a-h and 10a-k against CFs and
t-HSC/Cl-6 cells in vitro.
a
Inhibition ratio (%)
1
Compd.
R
Ar
CFs
t-HSC/Cl-6
8
8
a
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
2,4,6-triCH
4-ClPh
3
Ph
54.0
58.1
b
81.5
72.1
69.0
66.2
79.4
74.7
72.0
60.3
8
c
2-Cl-5-NO
3-Br-4-OHPh
4- CH SO Ph
2
Ph
8
d
8
e
3
2

9
9
a
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
4-OCF
3,4-diCH
3,5-diCF
4- CH SO
3-CF
3
Ph
Ph
Ph
48.8
41.7
70.1
43.7
51.8
45.1
41.7
61.1
54.6
67.1
55.2
48.3
42.5
45.9
73.8
58.5
41.2
54.1
51.3
72.6
68.4
54.8
49.2
62.1
55.0
64.8
60.3
56.7
72.5
68.1
65.0
70.6
57.4
62.9
61.5
83.7
66.3
63.2
71.5
68.8
77.2
73.8
b
3
9
c
3
9
d
3
Ph
2
9
e
3
Ph
9
f
3-Cl-4-FPh
2-Cl-4,5-diFPh
4-ClPh
9
9
g
h
1
0a
0b
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3
3-BrPh
1
3
3
3
3
3
3
3
3
3
3
3
4-OCF Ph
3
1
0c
0d
0e
0f
2-Cl-4-FPh
Ph
1
1
3,5-diCF
4- CH SO
4-ClPh
3
Ph
1
3
2
Ph
1
0g
0h
1
2,4-diClPh
3-pyridyl
2-furyl
1
0i
0j
1
1
0k
1
2-pyrrolyl
4-CH Ph
3
A
a
Data presented are the means of at least three independent experiments.
As illustrated in Table 1, all target compounds exhibited moderate to excellent activities against
two fibroblasts with inhibition ratio range from 41.7% to 83.7%. It is obvious that most
compounds presented better selectivity for t-HSC/Cl-6 cells superior to CFs, except for 8b, 8e, 9c
and 10b. Notably, compounds 8b and 10g, bearing 4-chlorophenyl at Ar moiety exerted obviously
improved activities compared to compounds 1 and A. Differently, 8b was the most potential
compound against CFs with inhibitory activity of 81.5%, while 10g exhibited the maximum
inhibition ratio of 83.7% against t-HSC/Cl-6 cells.
1
Further analysis of SARs based on inhibition ratio revealed that variations of R group led to
conspicuous divergence on anti-proliferation activities. On the whole, 3-chloro-4-fluoro
substituted derivatives exerted more active inhibition ratio against CFs than 3-trifluoromethyl
derivatives, and 3-bromo derivatives showed the worst potency, which can be illustrated by the
comparison of 8b, 9h and 10g (81.5%, 61.1%, 73.8%), as well as 8e, 9d and 10f (66.2%, 43.7%,
4
5.9%). What's more, Ar group at the terminal of SCZs moiety had a vital influence on
anti-fibrosis activities. As expected, compounds 8b and 10g bearing 4-chlorophenyl motif showed
the best potency superior to compound 1, suggesting halogen were essential for improved activity.

The introduction of 4-methylsulfonylphenyl moiety gave rise to compounds 8e, 9d and 10f which
exhibited obviously declined activities. Remarkably, the quantity of the substituents on phenyl
group was closely related to the anti-proliferation potency. Compared with monosubstituted
derivatives (e.g. 8b, 9e, 9h, 10a and 10g), disubstitutedcompounds 8d, 9b, 9f, 10e and 10h
showed weaker activities against both fibroblasts, while embedment of trisubstituted phenyl
groups (8a and 9g) resulted in dramatically declined potency by almost 50 percent. Moreover,
replacement of sustituented phenyl moiety with heterocycle motifs (e.g. 10i-10k) weakened
anti-proliferation potency evidently.
Inspired by the preliminary pharmacological results, two optimal compounds 8b and 10g were
determined for IC50 values against CFs and t-HSC/Cl-6 cells in vitro to further ascertain the
anti-fibrosis potential. Satisfactorily, 8b (IC = 0.87 and 1.10 µM) and 10g (IC = 1.21 and 0.78
5
0
50
µM) displayed improved activities against CFs and HSC compared to compound 1 (IC = 1.51
5
0
and 1.14 µM) (Fig. 3). The results were in accord with the inhibition ratio, validating 8b as an
ideal candidate for cardic fibrosis, and 10g was worthy of further anti-hepatofibrosis evaluation.
Figure 3. Inhibitation of 8b and 10g on CFs and t-HSC/Cl-6cells.
In summary, aiming to develop more promising ATX inhibitors as anti-fibrosis agents,
compounds 8b and 10g were developed by the introduction of 4-chlorophenyl motif at the
terminal of SCZs moiety, which revealed compelling anti-proliferation potency against CFs and
t-HSC/Cl-6 cells, respectively. The in-depth analysis was performed in the following research
work.
2
.2.2 In vitro enzymatic assay
To estimate anti-fibrosis potential at the molecular level, fourteen preferred compounds 8b-8e,
c-9e, 9h, 10a-10c and 10f-10h were selected for further enzymatic assay against ATX kinase in
9
vitro based on the remarkable results in cellular assays. The results expressed as IC values were
50

summarized in Table 2. As depicted in Table 2, five compounds 8b, 8e, 9h, 10c and 10g exerted
improved inhibition against ATX compared to compound 1 with IC50 values range from 15.3 to
2
7.5 nM, seperately. Among them, the most excellent compound 10g (IC = 15.3 nM) showed
50
doubling potency against ATX compared to compound 1 (IC = 29.1 nM), and the other four
5
0
compounds exhibited a slightly difference in activity. Together with the outstanding results in
cellular assays, 8b and 10g were worthy of further investigation.
Table 2 IC50 values of compounds 8b-8e, 9c-9e, 9h, 10a-10c and 10f-10h against ATX in vitro.
a
IC50 (nM)
1
Compd.
R
Ar
ATX
24.2
43.8
31.3
21.0
29.1
34.7
30.2
27.5
35.3
41.2
26.9
35.8
15.3
33.9
29.1
10.4
8
b
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Br
4-ClPh
8
c
2-Cl-5-NO Ph
2
8
d
3-Br-4-OHPh
4- CH SO Ph
3,5-diCF Ph
4- CH SO Ph
3-CF Ph
8
9
e
c
3
2
3
9
d
3-Br
3
2
9
e
3-Br
3
9
h
3-Br
4-ClPh
3-BrPh
1
0a
0b
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3
3
3
3
3
3
3
1
4-OCF
2-Cl-4-FPh
4- CH SO Ph
4-ClPh
2,4-diClPh
4-CH Ph
Ph
3
1
0c
0f
1
3
2
1
0g
0h
1
1
3
A
a
Data presented are the means of at least two independent experiments.
2
.2.3 Collagen Assessment
TGF-β is widely recognized as a master regulator of fibrogenesis [25]. It has been demonstrated
that activated TGF-β promotes cardiac and hepatic fibrosis by inducing production of
myofibroblasts and collagen secretion [26, 27].
To further investigate the anti-fibrosis effects of 8b and 10g induced by TGF-β on the cardiac
fibroblasts, we pretreated cardiac fibroblast with 20 µmol/L TGF-β prior to the treatment with 8b
and 10g and examined the content of collagen. As shown in Fig. 4, both compounds at 10 µmol/L
successfully suppressed collagen content. Especially, compound 8b reduced collagen level by 74.4%
compared to TGF-β group (0.14 vs. 0.63), reflecting the conspicuous capacity to inhibit
TGF-β-mediated cardiac fibrosis.

Figure 4. Inhibition of collagen content by 8b and 10g in the cardiac fibroblasts. Data are expressed as the means
±
SEM; n= 5 to 6. #P < 0.05 vs. control; *P < 0.05, **P < 0.01 vs. TGF-β.
2
.2.4 Immunohistochemical analysis
To evaluate the effect of 10g on CCl -induced hepatic fibrosis and collagen deposition, liver
4
sections were stained with H&E and Masson stains following standard procedures and examined
by light microscopy. Images of liver sections using H&E staining (Fig. 5A-5D) and Masson
staining (Fig. 5E-5H) are showed in Fig. 5.
Liver sections from control group (Fig. 5A and 5E) exhibited no pathologic changes. In CCl4
group, conspicuous fat accumulation, multiple bile ductules formation, inflammatory cell
infiltration and necrosis were observed, resulting in a high fibrotic pathologic scores (Fig. 5B and
5
F). However, as seen in Fig.5C and 5G from CCl + 10g (20 mg/kg) group, the pathologic
4
changes were relieved significantly with less fiber accumulation, steatosis and inflammatory
infiltrate. Furthermore, in the 40 mg/kg treatment group, liver sections showed negligible fiber
accumulation, bile ductules and inflammatory cells, which indicated that compound 10g was a
prominent inhibitor for the therapy of CCl -induced hepatic fibrosis.
4
Figure 5. Histological analysis of liver sections. Representative images of liver sections of the different groups
stained with H&E staining (A-D) and Masson staining (E-H).
2
.3 Molecular docking
In order to expound the binding mode, the docking study of the most prominent compound 8b
with ATX was carried out using Discovery Studio Program 3.5 based on the co-crystal structure of
1
compound A in complex with ATX (PDB ID code: 4ZG6) [28].

Figure 6. The binding poses of 8b with ATX. (A) Modeled docking of leading compound A with ATX (PDB ID
ZG6). (B) Predicted binding conformation for 8b in the binding sites of ATX. (C) 2D diagram of the interaction
4
between 8b and ATX.
The binding mode and interactions of compound 8b inside the active site of ATX are showed in
Fig. 6 (B-C). On the basis of the ATX-8b binding conformations, we found a favorable hydrogen
bond interaction between C=O of SCZs fragment and TRP276. What's more, the presence of the
THPPs scaffold and the phenyl at the terminal of SCZs linker resulted in π-π interactions with
Tyr307 and Phe275. Simultaneously, the THPPs scaffold interacted with the hydrophobic pocket
made up by Tyr307, Leu214, Ala305 and Ile168. As our expected, the introducing of chlorine and
fluorine atoms at both ends of the molecule formed halogen interactions and hydrophobic
interactions with Leu214, Ile168, Phe275 and Trp255, which demonstrated the reasonability of
our optimal design.
3
. Conclusion
Aiming to develop potent ATX inhibitors for fibrosis diseases, a novel series of
tetrahydropyrido[4,3-d]pyrimidine derivatives was designed and synthesized based on our
previous study. SARs analysis based on cellular and enzymatic assay resulted in the recognition of
8
1
8
b and 10g as two outstanding ATX inhibitors with the IC value in a nanomolar range (24.6 and
50
5.3 nM). Differently, 8b was the most potential compound against CFs with inhibitory activity of
1.5%, while 10g exhibited the maximum inhibition ratio of 83.7% against t-HSC/Cl-6 cells. In
view of the different potency of 8b and 10g toward CFs and HSC, targeted anti-fibrosis
evaluations were carried out to ascertain the anti-fibrosis potential. In collagen deposition assay,
8
b revealed the conspicuous capacity to suppress TGF-β-mediated cardiac fibrosis.
Simultaneously, H&E and Masson stains assays of mice liver disclosed 10g prominently reduced
CCl -induced hepatic fibrosis level in vivo, reflecting a great potency as anti-hepatofibrosis agent.
4
The molecular binding models identified the essential interactions between 8b and ATX as
concided with the SARs analysis. Overall, the discovery of
a
novel series of
tetrahydropyrido[4,3-d]pyrimidine derivatives lead to 8b and 10g as potential candidates for
cardiac and hepatic fibrosis, separately.
4
. Experimental
4
.1. Chemistry
All materials and solvents were obtained from commercial suppliers without further purification.
All melting points were detected on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
Flawil, Switzerland) and were uncorrected. Mass spectra (MS) was recorded in electrospray
ionization (ESI) mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA) with positive

switching. Samples were analyzed from a 100 µM MeCN/H O (6:4) solution with a 3 µL injection
2
1
13
volume. H NMR and C NMR spectra were recorded on Bruker spectrometers (Bruker
Bioscience, Billerica, MA, USA) relative to TMS as an internal standard. Thin-layer
chromatography (TLC) analysis was carried out on silica gel plates GF254 from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Target compounds 8a-8e, 9a-9h and 10a-10k were
prepared according to the method in our previous report, as well as all intermediates 2-7 [20].
4
3
.1.1(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(2,4,6-trimethylbenzylidene)-7,8-dihydropyrido[4,
-d]pyrimidine-6(5H)-carbohydrazide (8a).
1
Light yellow solid; yield: 47.3 %; H NMR (400 MHz, DMSO-d ) δ 10.47 (s, 1H), 8.76 (s, 1H),
6
8
.48 (d, J = 11.8 Hz, 2H), 8.02 (dd, J = 6.8, 2.6 Hz, 1H), 7.72 (ddd, J = 9.1, 4.2, 2.8 Hz, 1H), 7.39
(t, J = 9.1 Hz, 1H), 6.89 (s, 2H), 4.58 (s, 2H), 3.80 (t, J = 5.7 Hz, 2H), 2.81 (t, J = 5.5 Hz, 2H),
2
.36 (s, 6H), 2.23 (s, 3H). Anal. calcd. for C H ClFN O (%): C, 61.73; H, 5.18; N, 18.00. Found
2
4
24
6
+
(%): C, 61.68; H, 5.12; N, 18.05. m.p. : 168.0-169.8 °C; MS (ESI) m/z: 466 [M+H] .
4
.1.2(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(4-chlorobenzylidene)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carbohydrazide (8b).
1
Light yellow solid; yield: 52.5 %; H NMR (400 MHz, DMSO-d ) δ 10.67 (s, 1H), 8.74 (d, J =
6
5
.1 Hz, 2H), 8.47 (s, 1H), 8.17 (s, 1H), 7.99 (dd, J = 6.8, 2.6 Hz, 1H), 7.70 (dd, J = 5.9, 3.1 Hz,
H), 7.67 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.40 (t, J = 9.1 Hz, 1H), 4.56 (s, 2H), 3.80
1
(
t, J = 5.6 Hz, 2H), 2.82 (t, J = 5.4 Hz, 2H). Anal. calcd. for C H ClFN O (%): C, 54.92; H, 3.73;
24 24 6
N, 18.30. Found (%):C, 54.96; H, 3.75; N, 18.51. m.p. : 215.9-219.6 °C; MS (ESI) m/z: 459
+
[
M+H] .
4
.1.3(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(2-chloro-5-nitrobenzylidene)-7,8-dihydropyrido
[
4,3-d]pyrimidi ne-6(5H)-carbohydrazide (8c).
1
Yellow solid; yield: 60.8 %; H NMR (400 MHz, DMSO-d ) δ 11.18 (s, 1H), 8.93 (s, 1H), 8.64
6
(
s, 2H), 8.52 (s, 1H), 8.18 (dd, J = 8.8, 2.8 Hz, 1H), 8.00 (dd, J = 6.8, 2.5 Hz, 1H), 7.70 (m, 1H),
13
7
.40 (t, J = 9.1 Hz, 1H), 4.61 (s, 2H), 3.83 (t, J = 5.1 Hz, 2H), 2.86 (t, J = 5.1 Hz, 2H). C NMR
(
101 MHz, DMSO-d6) δ 160.11, 156.92, 154.98, 154.28, 147.13, 138.76, 137.40, 137.02, 134.11,
31.95, 124.82, 124.04, 122.96, 122.89, 121.15, 117.10, 116.79, 111.97, 41.59, 41.20, 31.30. Anal.
calcd. for C H Cl FN O (%): C, 50.02; H, 3.20; N, 19.44. Found (%): C, 50.09; H, 3.12; N,
1
2
1
16
2
7
3
+
1
9.37. m.p. : 186.0-188.8 °C; MS (ESI) m/z: 504 [M+H] .
4
.1.4(E)-N'-(3-bromo-4-hydroxybenzylidene)-4-((3-chloro-4-fluorophenyl)amino)-7,8-dihydropyri
do[4,3-d]pyrimidine-6(5H)-carbohydrazide (8d).
1
Pale white solid; yield: 58.9 %; H NMR (400 MHz, DMSO-d ) δ 10.65 (s, 1H), 10.48 (s, 1H),
6
8
.73 (s, 1H), 8.46 (s, 1H), 8.07 – 7.94 (m, 2H), 7.80 – 7.64 (m, 2H), 7.50 – 7.34 (m, 2H), 6.98 (d,
J = 7.8 Hz, 1H), 4.55 (s, 2H), 3.78 (s, 2H), 2.81 (s, 2H). Anal. calcd. for C H BrClFN O (%): C,
21
17
6
2
4
8.53; H, 3.30; N, 16.17. Found (%):C, 48.52; H, 3.33; N, 16.10. m.p. : 251.0-251.7 °C; MS (ESI)
+
m/z: 519 [M+H] .
4
.1.5(E)-4-((3-chloro-4-fluorophenyl)amino)-N'-(4-(methylsulfonyl)benzylidene)-7,8-dihydropyrid
o[4,3-d]pyrimidine-6(5H)-carbohydrazide (8e).

1
Yellow solid; yield: 57.2 %; H NMR (400 MHz, DMSO-d ) δ 10.58 (s, 1H), 8.45 (s, 1H), 8.37
6
(s, 1H), 8.15 (s, 1H), 8.02 – 7.96 (m, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.72
–
7.65 (m, 1H), 7.40 (t, J = 9.1 Hz, 1H), 4.58 (s, 2H), 3.79 (t, J = 5.7 Hz, 2H), 3.16 (s, 3H), 2.92 (t,
J = 5.7 Hz, 2H). Anal. calcd. for C H ClFN O S (%): C, 52.54; H, 4.01; N, 16.71. Found (%): C,
22
20
6
3
+
5
2.58; H, 4.12; N, 16.69. m.p. : 196.0-197.2 °C; MS (ESI) m/z: 503 [M+H] .
4
.1.6(E)-4-((3-bromophenyl)amino)-N'-(4-(trifluoromethoxy)benzylidene)-7,8-dihydropyrido[4,3-d
]
pyrimidine-6(5H)-carbohydrazide (9a).
1
Yellow solid; yield: 46.5 %; H NMR (400 MHz, DMSO-d ) δ 10.66 (s, 1H), 8.69 (s, 1H), 8.49
6
(
s, 1H), 8.20 (s, 1H), 8.02 (t, J = 1.8 Hz, 1H), 7.76 (dd, J = 8.4, 4.9 Hz, 3H), 7.41 (d, J = 8.2 Hz,
H), 7.30 (t, J = 8.0 Hz, 1H), 7.25 – 7.22 (m, 1H), 4.59 (s, 2H), 3.81 (t, J = 5.6 Hz, 2H), 2.83 (t, J
5.4 Hz, 2H). Anal. calcd. for C H BrF N O (%): C, 49.36; H, 3.39; N, 15.70. Found (%): C,
2
=
2
2
18
3
6
2
+
4
9.32; H, 3.41; N, 15.73. m.p. : 133.3-135.0 °C; MS (ESI) m/z: 535 [M+H] .
4
.1.7(E)-4-((3-bromophenyl)amino)-N'-(3,4-dimethylbenzylidene)-7,8-dihydropyrido[4,3-d]pyrimi
dine-6(5H)-carbohydrazide (9b).
1
Pale white solid; yield: 41.8 %; H NMR (400 MHz, DMSO-d ) δ 10.45 (s, 1H), 8.68 (s, 1H),
6
8
7
2
.48 (s, 1H), 8.10 (s, 1H), 8.02 (t, J = 1.9 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.41 (s, 1H), 7.34 (d, J =
.8 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 4.57 (s,
H), 3.79 (t, J = 5.7 Hz, 2H), 2.82 (t, J = 5.6 Hz, 2H). Anal. calcd. for C H BrN O (%): C, 57.63;
2
3
23
6
H, 4.84; N, 17.53. Found (%): C, 57.58; H, 4.86; N, 17.56. m.p. : 178.0-179.8 °C; MS (ESI) m/z:
+
4
79 [M+H] .
4
.1.8(E)-N'-(3,5-bis(trifluoromethyl)benzylidene)-4-((3-bromophenyl)amino)-7,8-dihydropyrido[4,
3
-d]pyrimidine-6(5H)-carbohydrazide (9c).
1
Yellow solid; yield: 47.7 %; H NMR (400 MHz, DMSO-d ) δ 111.03 (s, 1H), 8.71 (s, 1H), 8.49
6
(s, 1H), 8.33 (s, 1H), 8.30 (s, 2H), 8.09 (s, 1H), 8.02 (t, J = 1.9 Hz, 1H), 7.75 (dd, J = 8.2, 0.7 Hz,
1
2
1
H), 7.30 (t, J = 8.0 Hz, 1H), 7.24 (dd, J = 7.5, 1.1 Hz, 1H), 4.59 (s, 2H), 3.82 (t, J = 5.7 Hz, 2H),
13
.84 (t, J = 5.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 161.19, 156.72, 155.49, 154.59, 141.77,
39.95, 138.23, 131.36, 131.07, 130.71(2C), 126.94, 125.83, 125.10, 124.31(2C), 121.55, 120.86(2C),
1
12.21, 41.69, 41.51, 31.81. Anal. calcd. for C H BrF N O (%): C, 47.04; H, 2.92; N, 14.31.
23 17 6 6
+
Found (%): C, 47.01; H, 2.95; N, 14.32. m.p. : 250.0-252.8 °C; MS (ESI) m/z: 587 [M+H] .
4
.1.9(E)-4-((3-bromophenyl)amino)-N'-(4-(methylsulfonyl)benzylidene)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carbohydrazide (9d).
1
Yellow solid; yield: 61.4 %; H NMR (400 MHz, DMSO-d ) δ 10.85 (s, 1H), 8.71 (s, 1H), 8.49
6
(
s, 1H), 8.26 (s, 1H), 8.02 (t, J = 1.9 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H),
.76 (d, J = 8.8 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 4.59 (s, 2H), 3.82 (t, J
5.7 Hz, 2H), 2.84 (t, J = 5.5 Hz, 2H). Anal. calcd. for C H BrN O S (%): C, 49.91; H, 4.00; N,
7
=
2
2
21
6
3
+
1
5.87. Found (%): C, 49.92; H, 4.03; N, 15.85. m.p. : 231.0-232.7 °C; MS (ESI) m/z: 529 [M+H] .
4
.1.10(E)-4-((3-bromophenyl)amino)-N'-(3-(trifluoromethyl)benzylidene)-7,8-dihydropyrido[4,3-d
]
pyrimidine-6(5H)-carbohydrazide (9e).
1
Yellow solid; yield: 42.5 %; H NMR (400 MHz, DMSO-d ) δ 10.91 (s, 1H), 8.92 (s, 1H), 8.71 (s,
6

1
1
H), 8.48 (s, 1H), 8.02 (d, J = 9.5 Hz, 2H), 7.95 (s, 1H), 7.76 (d, J = 9.8 Hz, 2H), 7.62 (d, J = 8.0 Hz,
H), 7.29 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 4.55 (s, 2H), 3.75 (t, J = 5.6 Hz, 2H), 2.79 (t,
J = 5.4 Hz, 2H). Anal. calcd. for C H BrF N O (%): C, 50.88; H, 3.49; N, 16.18. Found (%): C,
2
2
18
3
6
+
5
0.87; H, 3.46; N, 16.19. m.p. : 128.0-129.8 °C; MS (ESI) m/z: 519 [M+H] .
4
.1.11(E)-4-((3-bromophenyl)amino)-N'-(4-chloro-3-fluorobenzylidene)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carbohydrazide (9f).
1
Yellow solid; yield: 56.2 %; H NMR (400 MHz, DMSO-d ) δ 10.95 (s, 1H), 8.70 (s, 1H), 8.48 (s,
6
2
H), 8.02 (t, J = 1.8 Hz, 1H), 7.84 – 7.79 (m, 2H), 7.77 – 7.73 (m, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.23 (d,
J = 8.5 Hz, 1H), 4.59 (s, 2H), 3.80 (t, J = 5.7 Hz, 2H), 2.83 (t, J = 5.6 Hz, 2H). Anal. calcd. for
C H BrClFN O (%): C, 50.07; H, 3.40; N, 16.68. Found (%): C, 50.08; H, 3.42; N, 16.66. m.p. :
2
1
17
6
+
1
50.1-151.9 °C; MS (ESI) m/z: 503 [M+H] .
4
.1.12(E)-4-((3-bromophenyl)amino)-N'-(2-chloro-4,5-difluorobenzylidene)-7,8-dihydropyrido[4,3
-d]pyrimidine-6(5H)-carbohydrazide (9g).
1
Yellow solid; yield: 49.4 %; H NMR (400 MHz, DMSO-d ) δ 10.95 (s, 1H), 8.70 (s, 1H), 8.48 (s,
6
2
H), 8.02 (t, J = 1.8 Hz, 1H), 7.84 – 7.79 (m, 2H), 7.77 – 7.73 (m, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.23 (d,
J = 8.5 Hz, 1H), 4.59 (s, 2H), 3.80 (t, J = 5.7 Hz, 2H), 2.83 (t, J = 5.6 Hz, 2H). Anal. calcd. for
C H BrClFN O (%): C, 50.07; H, 3.40; N, 16.68. Found (%): C, 50.08; H, 3.42; N, 16.66. m.p. :
2
1
17
6
+
1
50.1-151.9 °C; MS (ESI) m/z: 521 [M+H] .
4
.1.13(E)-4-((3-bromophenyl)amino)-N'-(4-chlorobenzylidene)-7,8-dihydropyrido[4,3-d]pyrimidin
e-6(5H)-carbohydrazide (9h).
1
Light yellow solid; yield: 65.7 %; H NMR (400 MHz, DMSO-d ) δ 10.64 (s, 1H), 8.69 (s, 1H),
6
8
7
=
.48 (s, 1H), 8.16 (s, 1H), 8.02 (t, J = 1.8 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.66 (d, J = 8.5 Hz, 2H),
.47 (d, J = 8.5 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 4.58 (s, 2H), 3.80 (t, J
13
5.7 Hz, 2H), 2.83 (t, J = 5.5 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 161.36, 156.70, 155.41,
54.81, 142.11, 141.80, 134.36, 134.09, 130.81, 129.27, 128.54, 125.76, 124.28, 121.56, 120.83,
12.29, 41.66, 41.51, 31.82. Anal. calcd. for C H BrClN O (%): C, 51.92; H, 3.74; N, 17.30.
1
1
2
1
18
6
+
Found (%): C, 51.90; H, 3.75; N, 17.32. m.p. : 215.2-216.9 °C; MS (ESI) m/z: 485 [M+H] .
.1.14(E)-N'-(3-bromobenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-d
4
]
pyrimidine-6(5H)-carbohydrazide (10a).
1
Yellow solid; yield: 47.9 %; H NMR (400 MHz, DMSO-d ) δ 10.74 (s, 1H), 8.88 (s, 1H), 8.50 (s,
6
1
H), 8.11 (dd, J = 13.9, 8.6 Hz, 3H), 7.84 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.9 Hz, 2H),
.38 (t, J = 7.9 Hz, 2H), 4.61 (s, 2H), 3.81 (t, J = 5.6 Hz, 2H), 2.84 (t, J = 5.4 Hz, 2H). Anal. calcd.
7
for C H BrF N O (%): C, 50.88; H, 3.49; N, 16.18. Found (%): C, 50.85; H, 3.46; N, 16.21.
2
2
18
3
6
+
m.p. : 137.0-139.1 °C; MS (ESI) m/z: 519 [M+H] .
.1.15(E)-N'-(4-(trifluoromethoxy)benzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydro
4
pyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide (10b).
1
Light yellow solid; yield: 51.3 %; H NMR (400 MHz, DMSO-d ) δ 10.72 (s, 1H), 8.89 (s, 1H),
6
8
.51 (s, 1H), 8.22 (s, 1H), 8.16 – 8.07 (m, 2H), 7.77 (d, J = 8.7 Hz, 2H), 7.57 (t, J = 7.9 Hz, 1H),
13
7
.41 (d, J = 7.8 Hz, 3H), 4.63 (s, 2H), 3.82 (t, J = 5.5 Hz, 2H), 2.85 (t, J = 5.2 Hz, 2H). C NMR

(
101 MHz, DMSO-d6) δ 161.41, 156.72, 155.46, 154.85, 149.11, 141.83, 140.91, 134.64, 134.23,
31.98, 129.91, 128.68, 125.46(2C), 121.73(2C), 119.35, 118.08, 115.47, 112.42, 41.69, 41.52, 31.84.
Anal. calcd. for C H F N O (%): C, 52.68; H, 3.46; N, 16.03. Found (%): C, 52.70; H, 3.47; N,
1
2
3
18
6
6
2
+
1
6.05. m.p. : 125.1-128.8 °C; MS (ESI) m/z: 524 [M+H] .
4
.1.16(E)-N'-(2-chloro-4-fluorobenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyri
do[4,3-d]pyrimidine-6(5H)-carbohydrazide (10c).
1
Yellow solid; yield: 57.7 %; H NMR (400 MHz, DMSO-d ) δ 10.86 (s, 1H), 8.87 (s, 1H), 8.51
6
(
d, J = 10.2 Hz, 2H), 8.15 – 8.05 (m, 2H), 7.96 (dd, J = 8.9, 6.4 Hz, 1H), 7.60 – 7.48 (m, 2H), 7.39
d, J = 7.6 Hz, 1H), 7.29 (td, J = 8.6, 2.5 Hz, 1H), 4.61 (s, 2H), 3.81 (t, J = 5.6 Hz, 2H), 2.84 (t, J
(
=
5.3 Hz, 2H). Anal. calcd. for C H ClF N O (%): C, 53.61; H, 3.48; N, 17.05. Found (%): C,
22 17 4 6
+
5
3.63; H, 3.51; N, 17.05. m.p. : 215.4-216.3 °C; MS (ESI) m/z: 492 [M+H] .
4
.1.17(E)-N'-benzylidene-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidi
ne-6(5H)-carbohydrazide (10d).
1
Yellow solid; yield: 52.1 %; H NMR (400 MHz, DMSO-d ) δ 10.64 (s, 1H), 8.90 (s, 1H), 8.50
6
(s, 1H), 8.22 (s, 1H), 8.15 (s, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.66 – 7.62 (m, 2H), 7.57 (t, J = 8.0
Hz, 1H), 7.44 – 7.36 (m, 4H), 4.63 (s, 2H), 3.82 (t, J = 5.7 Hz, 2H), 2.84 (t, J = 5.2 Hz, 2H). Anal.
calcd. for C H F N O (%): C, 60.00; H, 4.35; N, 19.08. Found (%): C, 60.04; H, 4.37; N, 19.07.
2
2
19
3
6
+
m.p. : 147.5-158.9 °C; MS (ESI) m/z: 440 [M+H] .
.1.18(E)-N'-(3,5-bis(trifluoromethyl)benzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihyd
4
ropyrido[4,3-d]pyrimidine-6(5H)-carbohydrazide (10e).
1
Yellow solid; yield: 48.5 %; H NMR (400 MHz, DMSO-d ) δ 11.04 (s, 1H), 8.88 (s, 1H), 8.50
6
(s, 1H), 8.33 (s, 1H), 8.30 (s, 2H), 8.12 – 8.07 (m, 3H), 7.57 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.6
Hz, 1H), 4.62 (s, 2H), 3.83 (t, J = 5.6 Hz, 2H), 2.85 (t, J = 5.7 Hz, 2H). Anal. calcd. for
C H F N O (%): C, 50.01; H, 2.97; N, 14.58. Found (%): C, 50.02; H, 2.95; N, 14.56. m.p. :
2
4
17
9
6
+
1
30.0-131.9 °C; MS (ESI) m/z: 576 [M+H] .
4
.1.19(E)-N'-(4-(methylsulfonyl)benzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyr
ido[4,3-d]pyrimidine-6(5H)-carbohydrazide (10f).
1
Lighe yellow solid; yield: 48.5 %; H NMR (400 MHz, DMSO-d ) δ 10.88 (s, 1H), 8.89 (s, 1H),
6
8
7
3
.50 (s, 1H), 8.28 (s, 1H), 8.17 – 8.05 (m, 2H), 7.96 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H),
.57 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H), 4.63 (s, 2H), 3.83 (t, J = 5.7 Hz, 2H), 3.24 (s,
H), 2.86 (t, J = 5.7 Hz,2H). Anal. calcd. for C H F N O S (%): C, 53.28; H, 4.08; N, 16.21.
2
3
21
3
6
3
+
Found (%): C, 53.28; H, 4.08; N, 16.21. m.p. : 130.0-131.3 °C; MS (ESI) m/z: 518 [M+H] .
.1.20(E)-N'-(4-chlorobenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-d
4
]
pyrimidine-6(5H)-carbohydrazide (10g).
1
Lighe yellow solid; yield: 65.3 %; H NMR (400 MHz, DMSO-d ) δ 10.66 (s, 1H), 8.87 (s, 1H),
6
8
.50 (s, 1H), 8.16 (s, 1H), 8.14 – 8.06 (m, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.58 (dd, J = 14.2, 6.4 Hz,
H), 7.48 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.8 Hz, 1H), 4.61 (s, 2H), 3.81 (t, J = 5.5 Hz, 2H), 2.84
1
1
3
(t, J = 5.3 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 161.43, 156.73, 155.46, 154.83, 142.10, 141.06,
1
34.27, 134.04, 130.49, 129.94, 129.56, 129.27 (2C), 128.54 (2C), 125.57, 119.47, 118.06, 112.44,

4
1.66, 41.49, 31.85. Anal. calcd. for C H ClF N O (%): C, 55.64; H, 3.82; N, 17.70. Found (%):
22 18 3 6
+
C, 55.65; H, 3.84; N, 17.73. m.p. : 131.0-131.7 °C; MS (ESI) m/z: 474 [M+H] .
4
.1.21(E)-N'-(2,4-dichlorobenzylidene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4
,3-d]pyrimidine-6(5H)-carbohydrazide (10h).
1
Yellow solid; yield: 55.4 %; H NMR (400 MHz, DMSO-d ) δ 8.87 (s, 1H), 8.53 (d, J = 12.5
6
Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 1.6 Hz, 1H), 7.50 (d, J =
.6 Hz, 1H), 7.40 (dd, J = 8.4, 1.6 Hz, 1H), 4.64 (s, 2H), 3.85 (t, J = 5.0 Hz, 2H), 2.81 (t, J = 5.0
Hz, 2H). Anal. calcd. for C H Cl F N O (%): C, 52.30; H, 3.34; N, 14.52. Found (%): C, 52.34;
8
2
1
16
2
3
5
+
H, 3.32; N, 14.48. m.p. : 225.3-227.0 °C; MS (ESI) m/z: 509 [M+H] .
.1.22(E)-N'-(furan-2-ylmethylene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3-d
4
]
pyrimidine-6(5H)-carbohydrazide (10i).
1
Yellow solid; yield: 43.7 %; H NMR (400 MHz, DMSO-d ) δ10.60 (s, 1H), 8.79 (s, 1H), 8.62
6
(
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),
.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). Anal. calcd. for C H F N O (%): C, 55.81; H, 3.98; N,
7
=
2
0
17
3
6
2
+
1
9.53. Found (%): C, 55.85; H, 3.96; N, 19.55. m.p. : 129.2-129.6 °C; MS (ESI) m/z: 430 [M+H] .
4
.1.23(E)-N'-((1H-pyrrol-2-yl)methylene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido
[
4,3-d]pyrimidine-6(5H)-carbohydrazide (10j).
1
Yellow solid; yield: 43.7 %; H NMR (400 MHz, DMSO-d ) δ 11.30 (s, 1H), 10.18 (s, 1H), 8.83
6
(
s, 1H), 8.49 (s, 1H), 8.14 – 8.07 (m, 2H), 8.03 (s, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.39 (d, J = 7.6
Hz, 1H), 6.84 (s, 1H), 6.36 (s, 1H), 6.09 (d, J = 2.7 Hz, 1H), 4.58 (s, 2H), 3.78 (t, J = 5.6 Hz, 2H),
.83 (t, J = 5.3 Hz, 2H). Anal. calcd. for C H F N O (%): C, 55.94; H, 4.23; N, 22.83. Found
2
2
0
18
3
7
+
(%): C, 55.94; H, 4.23; N, 22.83. m.p. : 208.9-210.0 °C; MS (ESI) m/z: 429 [M+H] .
4
.1.24(E)-N'-(pyridin-3-ylmethylene)-4-((3-(trifluoromethyl)phenyl)amino)-7,8-dihydropyrido[4,3
-d]pyrimidine-6(5H)-carbohydrazide (10k).
1
Yellow solid; yield: 39.6 %; H NMR (400 MHz, DMSO-d ) δ10.76 (s, 1H), 8.87 (s, 1H), 8.79
6
(
d, J = 1.8 Hz, 1H), 8.56 (dd, J = 4.7, 1.5 Hz, 1H), 8.50 (s, 1H), 8.22 (s, 1H), 8.12 (s, 1H), 8.06
ddd, J = 9.3, 8.0, 5.1 Hz, 2H), 7.57 (t, J = 8.0 Hz, 1H), 7.44 (dd, J = 7.9, 4.8 Hz, 1H), 7.39 (d, J =
(
7
.7 Hz, 1H), 4.61 (s, 2H), 3.82 (t, J = 5.7 Hz, 2H), 2.85 (t, J = 5.5 Hz, 2H). Anal. calcd. for
C H F N O (%): C, 57.14; H, 4.11; N, 22.21. Found (%): C, 57.12; H, 4.13; N, 22.22. m.p. :
2
1
18
3
7
+
1
4
4
54.5-156.0 °C; MS (ESI) m/z: 441 [M+H] .
.2. Pharmacology
.2.1. Assay for anti-proliferative activities against CFs
Cardiac fibroblasts were isolated from wistar/SD rats (1-3 days old). Briefly, excised hearts
3
were washed in ice-cold DMEM media, and then heart tissues were minced into 1~3 mm small
pieces by use of scissors in 2.5 g/L trypsin. Minced tissue was then placed into a sterile 10-ml tube
and incubated with agitation at 37°C for 50 min. The supernatant was collected, and the remaining
tissue was immersed in digestion solution. The supernatants were pooled and filtered through a
2
00-mm nylon mesh filter, then centrifuged at 1,500 r/min for 5 min. The supernatant was

removed, and cells were placed into DMEM containing 10% heat-inactivated fetal bovine serum
FBS), and incubated for 1.5-2 h at 37°C with 5% CO . Cells were examined for positivity for
(
2
vimentin. Fibroblasts between passages 3 to 4 were used for experiments.
Cell proliferation was measured by MTT assay. Briefly, CFs were seeded in 96-well culture
4
plates a density of 1×10 cells per well with DMEM containing 10% FBS and 5.6 mM glucose.
After starvation for 6 h, CFs were incubated in DMEM containing different test concentrations for
2
4 h. Then, 20 µL of MTT solution (5 mg/ml in phosphate-buffered saline, PBS) and 180 µL of
DMEM (5 mg/mL, 0.5% MTT) were added to each well. After incubation at 37 °C in the dark for
h, the culture medium was removed and 150 µL of DMSO was then added. Oscillate on a shaker
4
until the formazan crystals were dissolved. The optical density (OD) for each well was determined
at 490 nm by microplate reader (imark, BIO-RAD, USA) to calculate inhibition ratio and 50%
inhibition concentration (IC ). Blank control wells were designed without cells, but with the
5
0
addition of culture medium in parallel to the experimental wells.
4
.2.2. Assay for anti-proliferative activities against HSC
The t-HSC/Cl-6 cell line was purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). t-HSC/Cl-6 cells were incubated in DMEM, supplemented with 10% FBS,
00 µg/ml streptomycin and 1% 100 µg/ml penicillin with 5% CO at 37°C. Cell viability was
1
2
tested by MTT assay. Cells were seeded in in 96-well plates at identical conditions and each well
containing 100 µl medium. All tested compounds were dissolved and further diluted in DMSO,
respectively. After overnight incubation, cells were treated with carrier solvent alone or different
test concentrations in a final volume of 100 µl with two replicates each. The concentration of
DMSO did not exceed 0.1%. After 48 h, 10 µl of MTT (5 mg/ml) was added to each well and the
plate was incubated in the dark at 37 °C for 4 h. The culture medium was removed and the
formazan crystals were dissolved in DMSO (100 µl/well). The solution was stirred with a vortex
mixer for 10 min, and the absorbance was measured by microplate reader (imark, BIO-RAD, USA)
at 490 nm to calculate inhibition ratio.
4
.2.3. Enzymatic assays of ATX
Starting from 25 µM highest concentration, 10 µL of a dilution series of tested 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
MgCl , 1.5 mM CaCl , and 0.1% fatty acid free BSA in a total volume of 25 µL. The enzyme
2
2
mixture was added to tested 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
apparatus after 45 min incubation at room temperature (excitation 485 nm, emission 520 nM).
4
.2.4. CFs collagen assessment
Mice neonatal cardiac fibroblasts were isolated and cultured. Briefly, hearts from 2-to-3-day old
mice were finely minced and digested using type II collagenase (120 units/ml) for 10 minutes
every time, supernatant fluid were collected. Fibroblasts were isolated by removal of myocytes
through selective adhesion of non-myocytes at a 1.5 h preplating interval. CFs were maintained in

DMEM supplemented with penicillin and streptomycin (1%) and fetal bovine serum(10%).
Studies were conducted on CFs (passage 2-3) that were grown to subconfluence in
serum-containing media. After starvation in serum-free medium for 24 h, CFs were administered
recombinant human TGF-β (20 µmol/L) for 24 h. Tested compound 10 µmol/L was added to the
culture medium. After 12 h of pretreatment, content of collagen was determined.
To assess collagen secretion, 50 µL aliquots of culture media were collected from fibroblasts
plated at equal cell density, mixed with Laemmli sample buffer, heated, and loaded on precast 7.5 %
SDS-PAGE gels. Following migration, proteins were transferred electrophoretically onto
nitrocellulose membranes and blocked by incubating with 3% BSA in PBS overnight at 4 °C with
gentle mixing. Membranes were probed with the primary antibodies for 1hour at room temprature
and washed and incubated with horseradish peroxidase-conjugated secondary antibodies for an
extra 1 hour at room temperature. Signals were detected with enhanced chemiluminescence
reagent and band intensities were quantified with Bio-Rad Quantity One software.
4
.2.5 Immunohistochemical staining
Male Konmin mice (8-week old, 22-25 g) were kept in an air-conditioned animal quarter at a
temperature of 20 ± 2 °C and a relative humidity of 40 ± 10% with 12 h light/dark cycles for 1
week. Mice were randomly divided into 4 groups with 5 mice including blank, model and drug
(10g, 20 mg/kg or 40 mg/kg) groups. All the mice were allowed to drink and eat freely during the
experiment. All mice were intraperitoneally (i.p.) injected CCl (2% CCl /mineral oil; 10 mL/kg)
4
4
twice per week for 6 weeks except blank group. From the 7th week, 10g at 20 mg/kg and 40
mg/kg, respectively, was suspended in 0.5% sodium carboxymethyl cellulose and orally given to
the drug groups once daily for two weeks, coupled with molding. Blank and model groups were
orally given 0.5% sodium carboxymethyl cellulose (0.1 mL/10g) and positive control group was
orally given silymarin (0.3 mg/kg).
Slices of mice livers were cut off and fixed in formalin for 48 h. Liver sections were stained
with H&E and Masson stains following standard procedures and examined under a light
microscope for histopathological evaluation.
Acknowledgments
This work was supported by National Natural Science Foundationof China (No. 81872751 and
No.81872863), Natural Science Foundation of Liaoning Province (No. 2019-MS-301), Disruptive
Technology Innovation Guidance Fund of Shenyang Pharmaceutical University (No.
DFJJ2018201) and Development Project of Ministry of Education Innovation Team (No.
IRT1073).
References
[
[
[
1]. K. Sun, J. Tordjman, K. Cle´ment, P.E. Scherer, Fibrosis and adipose tissue dysfunction, Cell Metab. 18
(2013) 470–477.
2]. N. Pottier, C. Cauffiez, M. Perrais, P. Barbry, B. Mari, FibromiRs: translating molecular discoveries into new
anti-fibrotic drugs, Trends Pharmacolo. Sci. 35 (2014) 119-126.
3]. A.J. Edgley, H. Krum, D.J. Kelly, Targeting fibrosis for the treatment of heart failure: a role for transforming
growth factor-β, Cardiovasc. Ther. 30 (2012) 30-40.

[
4]. A.H. Györf, A.E. Mate, J.H. W. Distler, Targeting TGF-β signaling for the treatment of fibrosis, Matrix Biol.
8–69 ( 2018) 8-27.
6
[
5]. T.E.J. King, W.Z. Bradford, S. Castro-Bernardini, E.A. Fagan, I. Glaspol, M.K. Glassberg, E. Gorina, P.M.
Hopkins, D. Kardatzke, L. Lancaster, D.J. Lederer, S.D. Nathan, C.A. Pereira, S.A. Sahn, R. Sussman, J.J.
Swigris, A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis, N. Engl. J. Med. 370
(
2014) 2083-2092.
6]. J. Rosenbloom, F.A. Mendoza, S.A. Jimenez, Strategies for anti-fibrotic therapies, Biochim. Biophys. Acta,
832 (2013) 1088–1103.
[
[
[
[
[
[
[
1
7]. L. Juillerat-Jeanneret, J. Aubert, J. Mikulic, D. Golshayan, Fibrogenic disorders in human diseases: from
inflammation to organ dysfunction, J. Med. Chem. 61 (2018) 9811–9840.
8]. G.J. Roth, R. Binder, F. Colbatzky, C. Dallinger, R. Schlenker-Herceg, F. Hilberg, S.L. Wollin, R. Kaiser,
Nintedanib: from discovery to the clinic, J. Med. Chem. 58 (2015) 1053-1063.
9]. E. Borkham-Kamphorst, R. Weiskirchen, The PDGF system and its antagonists in liver fibrosis, Cytokine
Growth Factor Rev. 28 (2016) 53−61.
10]. R. Ma, J. Chen, Y. Liang, S. Lin, L. Zhu, X. Liang, X. Cai, Sorafenib: a potential therapeutic drug for hepatic
fibrosis and its outcomes, Biomed. Pharmacother, 88 (2017) 459−468.
11]. H. Huang, Y. Kang, X. Huang, Effect of malotilate on expression of Smads protein in rats with
dimenthylnitrosamine-induced hepatic fibrosis, J. Prac. Hepatol, 18 (2015) 525−529.
12]. B.S. Wilkins, D. Radia, C. Woodley, S.E. Farhi, C. Keohane, C.N. Harrison, Resolution of bone marrow
fibrosis in a patient receiving JAK1/JAK2 inhibitor treatment with ruxolitinib, Haematologica, 98 (2013)
1
872–1876.
[13]. M. Ongenaert, S. Dupont, R. Blanqué, E. van der Aar, B. Heckmann, Strong reversal of the lung fibrosis
disease signature by autotaxin inhibitor GLPG1690 in a mouse model for IPF, European Respiratory J. 48
(2016) 4540-4552.
[
14]. H. Ikeda, Y. Yatomi, Autotaxin in liver fibrosis, Clinica Chimica Acta, 413 (2012) 1817–1821.
15]. J. Hausmann, A. Perrakis, W.H. Moolenaar, Structure–function relationships of autotaxin,a secreted
lysophospholipase D, Adv. Biol. Regul. 53 (2013) 112–117.
[
[
16]. M.G. Benesch, Y.M. Ko, T.P. McMullen, D.N. Brindley, Autotaxin in the crosshairs: Taking aim at cancer
and other inflammatory conditions, FEBS Lett. 588 (2014) 2712–2727.
[
17]. T.M. Maher, E.M. van der Aar, O.V. de Steen, L. Allamassey, J. Desrivot, S. Dupont, L. Fagard, P. Ford, A.
Fieuw, W. Wuyts, Safety, tolerability, pharmacokinetics, and pharmacodynamics of GLPG1690, a novel
autotaxin inhibitor, to treat idiophic pulmonary fibrosis (FLORA): a phase 2a randomised placebo-controlled
trial, Lancet Respir. Med. 6 (2018) 672-635.
[
[
[
18]. E. Barbayianni, V. Magrioti, P. Moutevelis-minakakis, G. Kokotos, Autotaxin inhibitors: a patent review.
Expert Opin. Ther. Pat. 23 (2013) 1123-1132.
19]. S. Long, A. Thorarensen, M.E. Schnute, Pyrimidine and pyridine derivatives useful in therapy, 2013, WO
2
013054185A1.
20]. T.F. Jing, X.Q. Miao, F. Jiang, M. Guo, L.Y. Xing, J.L. Zhang, D.Y. Zuo, H.R. Lei, X. Zhai, Discovery and
Optimization of tetrahydropyrido[4,3-d]pyrimidine derivatives as novel ATX and EGFR dual inhibitors,
Bioorg. Med. Chem. 26 (2018) 1784-1796.
[
21]. Y. Lu, T. Shi, Y. Wang, H. Yang, X. Yan, X. Luo, H. Jiang, W. Zhu, Halogen bondings-a novel interaction for
rational drug design? J. Med. Chem. 52 (2009) 2854–2862.
[
22]. Y. Liu, Z. Xu, Z. Yang, K. Chen, W. Zhu, A knowledge-based halogen bonding scoring function for
predicting protein-ligand interactions, J. Mol. Model. 19 (2013) 5015–5030.

[
23]. J.H. Liu, X.Z. Zhuo, W.M. Liu, Z.F. Wan, X. Liang, S.S. Gao, Z.Y. Yuan, Y. Wu, Resveratrol inhibits
highglucose induced collagen upregulation in cardiac fibroblasts through regulating TGF-β1–Smad3
signaling pathway, Chem. Biol. Interact. 227 (2015) 45-52.
[
24]. J. Xu, X.D. Wang, G.Y. Su, J.Y. Yu, Y.Y. Sun, J.Q. Cao, X.S. Zhan, Y.Q. Zhao, The antioxidant and
anti-hepatic fibrosis activities of acorns (Quercus liaotungensis) and their natural galloyl triterpenes, J. Funct
Foods, 46 (2018) 567-578.
[
25]. J. Pannu, M. Trojanowska, Recent advances in fibroblast signaling and biology in scleroderma, Curr. Opin.
Rheumatol. 16 (2004) 739-745.
[
26]. J.H. Yang, S.C. Kim, K.M. Kim, C.H. Jang, S.S. Cho, S.J. Kim, S.K. Ku, I.J. Cho, S.H. Ki, Isorhamnetin
attenuates liver fibrosis by inhibiting TGF-beta/Smad signaling and relieving oxidative stress, Eur. J.
Pharmacol. 783 (2016) 92-102.
[
27]. Y. Yue, K. Meng, Y. Pu, X. Zhang, Transforming growth factor beta (TGF-β) mediates cardiac fibrosis and
induces diabetic cardiomyopathy, Diabetes Res. Clin. Pract. 133 (2017) 124–130.
[
28]. A.J. Stein, G. Bain, P. Prodanovich, A.M. Santini, J. Darlington, N.M. Stelzer, R.S. Sidhu, J. Schaub, L.
Goulet, D. Lonergan, I. Calderon, J.F. Evans, J.H. Hutchinson, Structural basis for inhibition of human
autotaxin by four potent compounds with distinct modes of binding, Mol. Pharmacol. 88 (2015) 982-992.

Highlights
‹
‹
‹
‹
‹
A novel series of tetrahydropyrido[4,3-d]pyrimidine derivatives was synthesized
8b and 10g displayed outstanding inhibition against ATX (IC = 24.6 and 15.3 nM)
5
0
8b and 10g exhibited prominent inhibition ratio against CFs and t-HSC/Cl-6 cells
8b suppressed collagen deposition in TGF-β-mediated cardiac fibrosis
10g reduced CCl -induced hepatic fibrosis in vivo
4

Declaration of interests
•
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: