Discovery of 1-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)benzyl)-5-(trifluoromethyl)pyridin-2(1H)-one, an orally active multi-target agent for the treatment of diabetic nephropathy

Article abstract of DOI:10.1016/j.bmcl.2017.07.001

Oxidative stress, inflammation and fibrosis can cause irreversible damage on cell structure and function of kidney and are key pathological factors in Diabetic Nephropathy (DN). Therefore, multi-target agents are urgently need for the clinical treatment of DN. Using Pirfenidone as a lead compound and based on the previous research, two novel series (5-trifluoromethyl)-2(1H)-pyridone analogs were designed and synthesized. SAR of (5-trifluoromethyl)-2(1H)-pyridone derivatives containing nitrogen heterocyclic ring have been established for in vitro potency. In addition, compound 8, a novel agent that act on multiple targets of anti-DN with IC₅₀ of 90 μM in NIH3T3 cell lines, t_1/2 of 4.89 ± 1.33 h in male rats and LD₅₀ > 2000 mg/kg in mice, has been advanced to preclinical studies as an oral treatment for DN.

Full text of DOI:10.1016/j.bmcl.2017.07.001

Accepted Manuscript
Discovery of 1-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)benzyl)-5-(tri-
fluoromethyl)pyridin-2(1H)-one, an orally active multi-target agent for the
treatment of diabetic nephropathy
Jun Chen, Zhangzhe Peng, Miaomiao Lu, Xuan Xiong, Zhuo Chen, Qianbin Li,
Zeneng Cheng, Dejian Jiang, Lijian Tao, Gaoyun Hu
PII:
S0960-894X(17)30694-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.001
BMCL 25114
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 April 2017
28 June 2017
1 July 2017
Please cite this article as: Chen, J., Peng, Z., Lu, M., Xiong, X., Chen, Z., Li, Q., Cheng, Z., Jiang, D., Tao, L., Hu,
G., Discovery of 1-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)benzyl)-5-(trifluoromethyl)pyridin-2(1H)-one, an
orally active multi-target agent for the treatment of diabetic nephropathy, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.001
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Discovery of 1-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)be
nzyl)-5-(trifluoromethyl)pyridin-2(1H)-one, an orally active m
ulti-target agent for the treatment of diabetic nephropathy
Jun Chena,1, Zhangzhe Pengb,1, Miaomiao Lub, Xuan Xiongb, Zhuo Chena, Qianbin Lia,
Zeneng Chenga，Dejian Jiangc，Lijian Taob, Gaoyun Hua,∗
aDepartment of Medicinal Chemistry, Xiangya School of Pharmacy, Central South University, Changsha 410013,
China
bDepartment of Nephrology, Xiangya Hospital, Central South University, Changsha 410008, China
cDrug Safety Evaluation Research Center of Hunan province, Changsha 410331,China
ABSTRACT:
Oxidative stress, inflammation and fibrosis can cause irreversible damage on cell struct
ure and function of kidney and are key pathological factors in Diabetic Nephropathy
(DN). Therefore, multi-target agents are urgently need for the clinical treatment of DN.
Using Pirfenidone as a lead compound and based on the previous research, two nove
l series (5-trifluoromethyl)-2(1H)-pyridone analogs were designed and synthesized. SAR
of (5-trifluoromethyl)-2(1H)-pyridone derivatives containing nitrogen heterocylic ring h
ave been established for in vitro potency. In addition, compound 8, a novel agent that
act on multiple targets of anti-DN with IC50 of 90uM in NIH3T3 cell lines, t1/2 of 4.
89
1.33 h in male rats and LD50 > 2000 mg/kg in mice, has been advanced to pre
clinical studies as an oral treatment for DN.
Diabetic Nephropathy (DN) is considered as one of the major complications of diabetes
mellitus, which is the leading cause of end-stage renal disease (ESRD) and has become a
serious threat to human health. There are 285 million people in 2010 and 347 million people
in 2013 with diabetes mellitus all over the world, 1and WHO projects that diabetics will be the
seventh-leading cause of death by 2030.2 Roughly one-third of the diabetic population will
develop diabetic nephropathy,3 which is a heavy social and economic burden.4
*Corresponding author.
E-mail address:hugaoyun@csu.edu.cn(GY Hu).
1Both authors contributed equally to this paper.

The occurrence and development of the DN is a complex process with the pathogenic
nature of kidney fibrosis. Abundant evidence suggests that genetic predisposition,5
hyperglycemia,6-8 hyperlipidemia,9 and microcirculatory disturbance are the major factors
contributing to the initiation and progression of DN.10 Although DN is associated with high
morbidity and mortality and the prevalence of this disease is continuously increasing
worldwide, there is currently no effective treatment strategy available other than symptom
control. In recent years, numerous studies have shown that the development of oxidative
stress,11 inflammatory and renal fibrosis is the key to the occurrence and development of
DN.12,13 Therefore, compounds that can bring resistance to oxidative stress, inflammation, and
fibrosis have been the focus of drug development studies on DN.
Reactive Oxygen Species (ROS) play an important role in DN which indicates that the
direct scavenge of ROS is a potential strategy for drug discovery of anti-DN agents.
Superoxide dismutase mimetic Tempol,14 a nonspecific NOX inhibitors with IC50 of 0.9 uM,
reduced the peroxide production in glomeruli. At the same time, due to increased
myeloperoxdase, this drug failed to reduce albuminuria which is a result of the damage to the
GBM. VAS-2870, a NOX specific depressor, inhibits ROS generation with an IC50 value of
10.6 mM in cell-free systems.15 However, it failed in animal experiments assessing the
pharmaceutical profile. Next, inflammation also plays a key role in the development and
progression of DN. TLK-19705,16 a novel CCR2 antagonist, has been tested in a group of
db/db mice with dosages of 30 mg/kg/day and 10 mg/kg/day for 8 weeks by Okamoto. M’
team. And the results show that the urinary ACR has improved in the animal experiment.
Finally, renal fibrosis is the final common pathway of DN. IN-1130 showed efficacy in
suppressing fibronectin in UUO rat kidneys by inhibiting Smad2 phosphorylation.17
GW788388 can diminish renal fibrosis by inhibiting the proliferation of fibrosis cell.18,19
HOE77, an inhibitor of the formation of collagen in the late stage of liver fibrosis, can cause
cataracts in phase II clinical trial, and its investigation has been terminated.20,21 The above
many compounds effect in a link or target of fibrosis, which rarely passed clinical research,
suggesting that single target inhibitor is not the ideal research direction of anti-DN drug
discovery.

Pirfenidone (PFD), a multi-target drug, has been marketed mainly for idiopathic pulmonary
fibrosis since 2008.22 And a phase clinical trial of PFD for DN has been completed with 77
patients at San Digo Medical Center of California University.23 Clinical effect of PFD is not
satisfactory because its 5-methyl can be easily metabolized.24 In addition, PFD showed a
certain acute toxicity in animal experiments.25 With PFD as the lead compound, our research
group has carried on a series of transformation. The previous research results have been
reviewed by Yi-Min Liu.26 First, we gain Fluorofenidon（e AKF-PD）by introducing fluorine in
the meta position of the phenyl group of PFD.27 AKF-PD, a “me-better” drug, show higher
inhibitory activity and more alleviated fibrosis symptoms than PFD.28 As with PDF, AKF-PD
is prone to inactivation due to the metabolism of methyl groups on pyridone ring.29 In order to
prevent the metabolism of the 5-methyl group, our research group turn methyl into aromatic
ring,30 trifluoromethyl and methyl cyclization.31,32 which greatly increase the activity of the
compound. Among them, the activity of compound ZHC-102 with an IC50 of 290uM was
obviously improved. In our early work, we found that the introduction of methylene into
pyridine ring and phenyl group can improve the activity of the target compound. For the
purpose of finding the target compounds with excellent activity and further exploring the
structure-activity relationship, our group has developed a new series of compounds by
converting the phenyl group at the 1-position of the pyridone ring to the benzyl group31.
ZHC-159, a benzyl series compound, shows excellent activity with IC50 of 80uM in NIH3T3
cell lines, but it also has high toxicity.
Table 1
The anti-DN compounds in preclinical or clinical research.
IN-1130
GW-788388
BIBF-1120
HOE-77
VAS-2870

F3C
Cl
N
O
N
O
N H2
Tempol
TLK-19705
AKF-PD
ZHC-159
PFD
ZHC-102
The purpose of this study is to develop an anti-DN drug. Although the activity of the
compound has been improved by previous studies, the drugability of these compounds is not
sufficient because of poor water-solubility and high toxicity. Experiments revealed that the
drugability of compounds could be improved by adding the larger group to 1-N of pyridone.
Herein, our research group developed two novel series of (5-trifluoromethyl)-2(1H)-pyridone
derivatives containing nitrogen heterocyclic ring to improve its medical properties(see Fig.1).
n
n
m
Fig. 1. The map of design route of the target compounds
The mouse fibroblast cell line (NIH3T3 cells), a well-recognized anti-fibrosis drugs in vitro
screening model,33 was used to test the potency of the target compounds. Here we detected
NIH3T3 cell proliferation by MTT assay with AKF-PD as the positive control to test the
potency of the anti-fibrosis compounds and discuss the SAR on anti-fibrotic activity.
Compound 8 was finally identified in the research by conducting a series of research of SAR
and pharmacokinetic experiments.
An efficient synthesis of a wide range of (5-trifluoromethyl)-2(1H)-pyridone derivative
was reported earlier with our effort to discover NIH3T3 inhibitor. Based on this, PFD was
extended by introducing versatile nitrogen heterocyclic ring side chains. The link chain
between the aromatic amino group and the heterocycle is 2 to 3 carbon atoms in length.
Representative nitrogen heterocyclic ring are listed in Table 2. Chemical synthesis and
characterization of key compounds, which were used for SAR determination and in vivo
evaluations were described in the experimental section.

F C
3
F C
3
F C
F C
3
3
F C
3
c
d, e
a
N
O
N
O
b
N
O
N
O
N
O
H
N
H
N
R
O H
n
R
n
R
1 2-NO2
2 2-NO2
3 2-NH2
4 4-NH2
5 4-NHCH2CH2OH
6 4-NHCH2CH2CH2OH
7 2-NHCH2CH2OH
8-17
Compd. Site
n
R
Compd. Site
n
3
R
Compd. Site
n
R
Compd. Site
n
R
N
O
N H
8
9
4
3
11
12
4
4
13
14
15
4
2
2
2
16
17
2
2
2
N
N
N
N
N
O H
N
O
N
N
4
2
2
3
2
2
2
N
N
N
O
10
4
N
N
Reagents and conditions:(a) K CO ,DMSO,130
Scheme 1.
20h;(d) SOCl2,DCM,TEA,reflux,4h;(e) Acetonitrile,reflux.
4h;(b)Fe,HCl,50%EtOH,reflux,3h;(c)BuOH,K CO 95 ,
℃
2 3,
℃，
2
3
Scheme 1 illustrated the preparing process of 10 target compounds. With the intention to
prevent the metabolism of 5-site methyl group of pyridone ring, we used
5-trifluoromethyl-2(1H)pyridone as the mother moiety and starting material. The compound 1
and 2 were easily prepared through coupling of 5-trifluoromethyl-2(1H)-pyridone with
substituted benzyl halide which was carried out by stirring the mixture in DMF with K2CO3.34
The compounds 3 and 4 were subsequently synthesized via the reduction of compounds 1 and
2 in 90-95% yield, in which Fe was used as the reducing agent in the solution of
HCl/ethanol.35 Treatment of 3 and 4 with halohydrin and K2CO3 in BuOH gave the key
intermediate 5, 6 and 7.36 In the experiment, we found an interesting phenomenon: The
temperature had a great influence on the alkylation products. At temperatures below 95 , the
reaction hardly occurs; whereas at temperatures above 100 , the products were mainly
aminodialkylated. The compounds 8 to 17 were obtained by one-pot method from the
intermediate 5 to 7 through chlorination as described in literature and then refluxing and
stirring reaction with nitrogen heterocycle in acetontrile.37,38

7
4
1
5
7
4
3
2
5
3
2
6
6
1
8
8
1-1
1-2
Fig. 2. The reaction route map of first step.
Fig. 3. HMBC of compound 8.
On the basis of the reaction mechanism analysis, there could be two productions of
N-alkylation (1-1) and O-alkylation (1-2) in the first step (see Fig. 2). C13-NMR and H1-NMR
of the two compounds are similar in theory, so the two compounds cannot be judged just by
C13-NMR and H1-NMR. In order to further elucidate the structure, HMBC of compound 8
was determined. As all know, there is a correlation between 8-H and 6-C of 1-1 but 1-2. The
HMBC result shown: 8-H (5.1597 ppm) and 6-C (139.39 ppm) had a long rang correlation
(see Fig. 3). It indicated that compound 8 belonged to productions of N-alkylation.
Chemical structures of all others were confirmed by IR, MS, H1-NMR and C13-NMR.
F3C
F3C
F3C
F3C
N
O
N
O
N
O
F3C
N
O
c
a
b
Cl
d, e
Cl
Cl
Cl
N
H
O
NO2
HN
NH2
HN
R
n
O H
18
19
20
21-28
Compd.
21
n
R
Compd.
23
n
R
Compd.
25
n
2
R
Compd.
27
n
R
N
N
3
3
3
2
2
N
N
N
N
OH
O
N H
O
N
22
24
26
2
28
2
N
N
N
N
Reagents and conditions:(a) K CO ,DMSO,130
℃，
4h;(b)Fe,HCl,50%EtOH,reflux,3h;(c)BuOH,K CO 95
2
,
℃
Scheme 2.
2
3
3,
20h; (d) SOCl2,DCM, TEA,reflux,4h;(e) Acetonitrile reflux, 4h.
According to Scheme 2, 8 designed phenyl substituted compounds were synthesize
d. There is a small amount of 1-(3-chloropropyl)piperidine, 4-(3-chloropropyl)morpho
line and 1-(3-chloropropyl)-4-methylpiperazine in the laboratory. So the target compo
unds 21, 22 and 23 were directly obtained by treatment of 19 with the above react

ants and K2CO3 in BuOH. The synthesis methods of the other compounds was the
same with Scheme 1.
The mouse fibroblast cell line(NIH3T3 cells), which has been used as routine screening
model of anti-fibrosis drugs in vitro,39 was used to test the potency of the desired compounds.
Compounds 8 to 17 and 21 to 28 were examined for their anti-proliferative activity by MTT
assay using the NIH3T3 cell line with AKF-PD as the positive control. The results of these
assays are presented in Table 2. The inhibition ratio was processed by Ascent Software™ and
IC50 values were obtained from the dose-response curves. Compound 8 is currently one of the
most active compounds that inhibit NIH3T3 cell lines with IC50 value of 90uM.
Firstly, it could be seen that the introduction of the heterocyclic side chain further enhanced
the activity of the target compound. As these data illustrate in Table 2, all the desired
compounds showed significantly improved inhibition activities relative to the positive control
AKF-PD.
The effect of heterocycles side chains on activity was different in benzyl and phenyl series
target compounds. As shown in the table 2, the activity of compound 25, 21 of phenyl series
were better than compound 26, 22 and 23, 27, respectively. It can be concluded that
piperidine ring was favor to morphine ring and 4-methyl piperazine ring in phenyl series;
while compounds 8, 9 of benzyl series was superior to compound 10, 11 and 13, 14. So the
activity of the 4-methylpiperazine derivative in the benzyl series is superior to the morphine
and piperidine derivatives.
From Table 2. It could also be concluded that the length of the link chain between
heterocyclic and amino group has an effect on the activity of the benzyl series compounds.
However, this effect was not observed in the phenyl series. In the article the number of
carbon in link chain was investigated ranging from 2 to 3，the anti-fibrosis activity of
compound 13 with IC50 of 580uM was better than compound 14 with IC50 of 2550uM, while
the anti-fibrosis activity of compounds 25 and 26 were roughly equal to compounds 21 and 22,
respectively. The results showed that the activity of the 3 carbon atom link chains in the
benzyl series is better than that of the 2 carbon atom link chains; the link chain length was 2
carbons or 3 carbons in the phenyl series made no significantly difference to inhibit NIH3T3
cell.

From the previous study we found that:31 5-trifluoromethy l-substituted derivatives are
superior to 5-methyl or 5-chloro-substituted derivatives. The ideas have been extended and
confirmed in this study. In the earlier studies the activity of the target compounds in the
benzyl series was better than that in the phenyl series; however, this phenomenon was not
reflected in this study. Our analysis showed that the activity of the target compound was
greatly improved after introducing the heterocyclic side chain, which masked the difference
of the activity of the two series of compounds.
The compound to be highlighted was compound 8 with an IC50 value of 90uM. The
improved inhibition may be attributed to the facts that compound 8 can form more hydrogen
bond with the related receptors, higher solubility and excellent membrane permeability.
Further studies need to be conducted on these derivatives to evaluate their potent.
Table 2
The structures of 5- trifluoromethyl -2(1H)-pyridone target derivatives and their inhibitory
activities against NIH3T3 cells.
Substituent
R1
Benzyl series
compound
IC50
Phenzyl series
compound
IC50
R2
(uM)
(uM)
site
4
N
8
90
100
220
260
330
580
2550
100
21
22
23
24
25
26
27
28
80
150
170
60
9
4
4
4
4
4
4
2
10
11
12
13
14
15
70
110
200
1300

16
17
2
2
100
340
AKF-PD
/
2750
For a follow-up study of one of the preferred compounds, the LD50 of compounds 8, 23,
and 24 in mice were assayed. From the experimental results, we could see that compound 8
was the least toxicity with a LD50 more than 2000 mg/kg. Compound 8 was selected as the
preferred compound for further study because of higher activity and lower toxicity.
To determine the therapeutic effect in vivo, we performed an in vivo study of compound 8
against diabetic nephropathy in the db/db mouse model. db/db mice (C57BLKs/J Leprdb/db
mice) is a mutant strain from C57BLKs/J mice with leptin receptor gene mutation that causes
type 2 diabetes. The disease progression in this model is very similar with the human type 2
DN, which makes it widely used as a research animal model for DN.40-42 Angiotensin II
receptor blockers (ARBs) show good kidney protection effects in a variety of experimental
animal models and humans for the treatment of kidney disease. Losartan is the first
anti-hypertensive drugs of AIIA class and has been used in the treatment of diabetic
nephropathy.43 So Losartan was selected as the positive control drug in the experiment.
Losartan, PFD and compound 8 were selected for in vivo assessment of the anti-DN effect in
db/db mice of 8 week-old. The effect of compound 8 on anti-glomerulosclerosis was superior
to that of the Losartan in db/db mice ranging from 8 to 24 week-old. The blood glucose of
model group was significantly higher than that of diabetes model (DBM) group (P<0.05). The
difference was statistically significant, and the diabetic model was justified to be successful
by comparing with model group. Then, 24-hour urine was collected from the model mice and
urine albumin measured by ELISA. The model group 24-hour urinary albumin was
significantly higher than that of the normal group, and the difference was statistically
significant, indicating the success of diabetic nephropathy model. Compounds were
dissolved in 0.5% CMC-Na in water by gavage administration to db/db mice once a day
continuing to 24 weeks of age (except for DBM pathological control group). The dose of
Losartan, PFD, and compound 8 were 20 mg/kg.d, 250 mg/kg and 40 mg/kg.d, respectively.
The therapeutic effect of anti-DN was assessed by determination of serum creatinine, blood
urea nitrogen, creatinine clearance ratio, urinary albumin, albumin-to–creatinine ratio,

glomerular sclerosis index, kidney weight/body weight and pathological section dying with
PAS and score in treated groups compared to the control group.44
Table 3
The measurement of BUN, Scr, Ccr, Urinary Albumin, ACR in each experimental groups.
BUN
Scr
Ccr
Urinary
ACR
Groups
(mmol/L)
(umol/L)
(ul/min)
albumin(ug)
(ug/mg)
DBM
Model
10.31 0.31
28.25 8.25
216.96 16.96
18.746
0.19 .194
★
★
☆☆
☆☆
☆
☆☆☆
11.22 1.22
38.86 8.86
120.77 20.77
155.14 55.14
136.36 36.36
244.98 44.98/
112.32 12.32
105.85 05.85
2.72 0.729
△△
1.17 0.173
▲
▲
▲
▲
△
△
Losartan
PFD
11.70 1.70
41.13 1.13t
▲
▲
▲
13.08 3.08
41.67 1.67
1.16 0.168
▲◆
▲◆
△◆
△△△
41.96 1.962
△△△\△△△
Compd. 8
11.58 1.58
29.83 9.83
222.92 22.92
0.37 0.376
Note：Compared to DBM group ★P>0.05,☆P<0.05, ☆☆P<0.01 ☆☆☆P<0.001; Compared to modle group,▲P>0.05,△P<0.05;
△△P<0.01; △△△P<0.001; Compared to Losartan group with 8 weeks age ◆P>0.05 △P<0.05 △△P<0.01,△△△P<0.001.
The four compounds in Table 3 were assessed: Losartan, PFD and compound 8. Given
equal doses after treatment, the model group serum creatinine, blood urea nitrogen, 24 hours
urinary albumin, urine albumin creatinine ratio (ACR), creatinine clearance rate (Ccr)
significantly decreased (P < 0.05) compared with DBM group. Serum creatinine and blood
urea nitrogen have no significant difference between each treatment group and model group
with same ages (P value of 0.05), but the amount of 24 hours urinary albumin, ACR, (except
compound 8 8 weeks) reduced to some extent, and the decrease in creatinine clearance was
significantly delayed.
In comparison with the DBM group, the result of urinary albumin in 24 hours and ACR of
Losartan group fell by 54.15%(P=0.021), 56.87%(p=0.005), respectively, and Ccr decreased
slowly by 28.45% (p=0.397). compound 8 and PFD 24-hour urinary albumin has declined by
82.87% (p=0.002) and 56.79% (p=0.023); ACR decreased by 86.22% and 57.23% （p=0.000
and 0.070）; while the decline of Ccr slowed by 84.57% and 12.9% (p=0.050 and 0.686).
The urinary albumin in 24 hours, ACR of compound 8 group dropped significantly
compared with Losarton group at
8 weeks and the difference had statistical
significance(p<0.005). The rest of result had changed but with no statistical difference.
Renal tissue PAS staining slice was examined under ordinary optical microscope 400 times
the microscopic. The results show that DBM mice kidney pathological control group and

DBM group had no obvious pathological changes; model group showed glomerular
hypertrophy, thickening of basement membrane, broadening mesangial area, including
increased mesangial cell and mesangial matrix. In contrast, the lesions in the treatment groups
were significantly reduced. (see Fig. 4).
DBM
Model
Losartan 20mg/kg.d
PDF 250mg/kg.d
Compd.8 40mg/kg.d
Fig. 5. GSI chart of mice in each
Fig. 4. PAS staining of mice kidney in each groups
experimental group
The glomerular sclerosis lesions were clearly visible and GSI increased significantly in
renal tissue pathological examination in model group(see Fig. 5). The kidney tissues of all
DBM pathological control group and DBM group had no serious glomerular sclerosis.
Compared with the DBM group, the GSI of the model group increased significantly. And
compared with the model group, GSI of each treatment group decreased obviously, the
difference was statistically significant. GSI of Losartan, PFD and compound 8 group declined
and fell by 47.25%(p<0.01), 55.04%(p<0.001) and 67.31%(p<0.001), respectively. GSI of
compound 8 group dropped significantly compared with Losartan and PFD group at 8 weeks
and the difference had statistical significance(p<0.005). The results shown that 40 mg/kg.d
compound 8 had better anti-glomerular sclerosis effect than 20 mg/ kg.d Losartan and 250 mg
/ kg.d PFD at 8 weeks of age. It can be concluded from overall analysis that Losartan, PFD,
AKF-PD and compound 8 had obviously curative effect on glomerular sclerosis. Moreover,
compound 8 shows efficacy with a dosage of 40 mg/kg. D was superior to that of other
groups.
In early renal fibrosis and anti-inflammatory studies, we found that compound 8
may interact with multiple targets. The reasearch about the target and mechanism of

compound 8 has been published by other researchers in our study group.45-47 Although
we observed the effects of compound 8, its mechanism of action remain unclear. Fibrosis is
the central pathology of DN that manifests from the initiation to the end-stage. Since we
observed that compound 8 treatment exerts curative effect on DN in mouse model and
anti-proliferation effect on NIH3T3 cells, we hypothesize that the mechanism underlying such
effects involves the regulation of fibrosis. To test this hypothesis, we firstly assessed the
effect of compound 8 treatment on the fibrotic conversion of NRK-52E cell, a rat renal
tubular epithelial cell line. Previous study has demonstrated that TGF-β1 is increased in DN
and induces the expression of kidney fibrotic markers. Therefore, we used TGF-β1 to treat the
cell in the presence or absence of 24.5 mol/L compound 8 or SB431542, which is a TGF-β1
inhibitor. After 24 hour treatment, we firstly examined the cell morphology change. We
observed that TGF-β1 treatment induced the NRK-52E cells to be elongated and changed the
spearhead shape which is a fibrotic feature. Interestingly, this cell morphology change can be
blunted by treatment of both compound 8 and TGF-β1 inhibitor SB431542, suggesting
similar effect of compound 8 in blocking TGF-β1 mediated fibrotic signaling.
Next, it is well-known that once undergoing fibrotic transformation, kidney cells over
express α-SMA and decrease the expression of E-cadherin. And these changes are mediated
by TGF-β1. To further validate if compound 8 exerts anti-fibrotic effect through TGF-β1, we
determined the expression of α-SMA and E-cadherin after 48 hour treatment by TGF-β1.
Intriguingly, we found that the expression of α-SMA is induced by TGF-β1 but this induction
can be blocked by compound 8 treatment. On the contrary, the inhibition of E-cadherin
expression by TGF-β1 can be reversed by compound 8 back to that of normal levels. These
data clearly suggest that compound 8 has an anti-fibrosis effect via inhibiting the effect of
TGF-β1 in inducing renal tubular epithelial cell fibrotic conversion.(see Fig. 6.)
α-SMA
E-cadherin
α-SMA
*
2.5
2.0
1.5
1.0
0.5
0.0
E-cadherin
#
#
#
#
*
*
β-actin
*

Normal
TGF-β1
Compound 8
SB431542
Fig. 6. Effect of on TGF-β1 induced expression of a-SMA and E-cadherin in NRK-52E cell
Note: *p＜0.05 VS normal; #p＜0.05 VS TGF-β1
Besides tubular epithelial cells, mesangial cells can also undergo fibrotic transformation
with the challenge of TGF-β1 and high glucose. To determine if compound 8, PFD and
AKF-PD can prevent fibrotic change of mesangial cells, we treated the mouse MES-13 cell
line with TGF-β1 and different concentrations of glucose, respectively. CTGF is an important
downstream component of TGF-β1 induced pro-fibrosis effect by increasing the expression of
Collagen I and FN in fibroblasts. Because CTGF level is generally much lower with very
singular biological effects, it serves as a more specific target for the treatment of kidney
fibrosis than TGF-β1. First, we found that after 48 hour treatment with TGF-β1, both CTGF
and FN increased significantly in MES-13 cells by 3.99 and 2.76 folds respectively.
Interestingly, all the three compounds can dramatically decrease the levels of CTGF and FN
induced by TGF-β1 with a more potent effect than TGF-β1 inhibitor SB431542, with
compound 8 being the most potent one. These results strongly suggest that compound 8 is a
very promising anti-fibrosis drug candidate.
Next, to assess if compound 8 can prevent the pro-fibrosis effect of high glucose, we
treated the MES-13 cells with different concentration of glucose for 48 hours. High glucose is
a major contributor to the pathology of DN. Mesangial cells are particularly susceptible to
high glucose challenge and can easily undergo fibrotic transformation. To determine if
compound 8, PFD and AKF-PD can block such pro-fibrosis effect induced by high glucose,
we treated the cells with all the three compounds at different concentrations with Losartan as
the positive control. After carefully assessing the expression levels of CTGF and FN, we
found that in comparison to the no-treatment group, FN levels decreased by 2.42 folds, 0.80
fold, 1.78 folds and 1.23 folds respectively with the treatment of compound 8, PFD, AKF-PD
and Losartan; while CTGF levels decreased 1.85 folds, 1.39 folds, 1.38 folds and 1.66 folds,
respectively. These data suggest that at a concentration of 24.5 mol/L(10-5), compound 8 can
bring the elevated FN and CTGF levels induced by high glucose back to normal and thus
prevent the progression of pro-fibrosis change of mesangial cells and kidney as a whole,
which will in turn prevent DN.

The interaction of between inflammatory response and fibrosis runs through DN.48-50The
inflammation has a pivotal role in kidney interstitial fibrosis, and the essence of DN is renal
fibrosis. Compound 8 could effectively restrain inflammatory and the proliferation of renal
fibroblasts. It was reported that compound 8 could obviously inhibit TNF-α or LPS-induced
production of proinflamatory cytokines from our team’s previous study. This effect can be
attributed to the inhibition of phosphorylation of STAT3, ERK, and NF-kb.51
PFD liability decreased with 5-methyl of pyridone, in particular the phase metabolic
liability as demonstrated by the short half-lives in liver microsomal assays. The elimination
half-life of the tablet of PFD is 1.90 0.13 h from six rats by single-dose.52The main purpose
of this study was to improve the metabolic stability, extend the biological half-life, and thus
improve the effect of these compounds. To determine whether the pharmacokinetics of target
compound 8 was improved, we performed a preliminary study on the pharmacokinetic
properties of compound 8. In a preliminary study, LC-MS assay was established to detect the
plasma concentration of compound 8 and its metabolites with PFD as the internal standard by
WinNonlin® software. The pharmacokinetics profile of compound 8 was evaluated in male
rats using LC/MS analysis with a standard curve formulation: y=0.0113x-0.0043, R2=0.999.
As shown in Table 4, we obtained the pharmacokinetic parameters of compound 8.
Compared to PFD, the elimination half-life of compound 8 was prolonged to 2.58-fold, and
Cmax and AUC0-t of compound 8 increased by 1.29 times and 2.66 times, respectively. So the
pharmacokinetics properties of compound 8 has made significant improvements.
Table 4
Main pharmacokinetic parameters of compound 8 and pirfenidone.
Compd.
PFD
Dose(mg/kg)
10.0
t1/2(h)
Cmax(ng/mL)
1020.3 220.1
2032 620.4
AUC0–t(h.ng/mL)
2722.0 731.9
1.90 0.13
4.89 1.33
Compd. 8
15.5
11214.77 5375.2
By optimization of the lead compound PFD, 18 novel pyridone derivatives were designed.
In the present study, we have developed methods for efficient preparation of
5-trifluoromethyl-2(1H)-(1-nitrogen heterocycle alkyl amino benzyl or phenyl) pyridone
derivatives and evaluated their inhibitory activity against NIH3T3 cell proliferation. As
expected, all the target derivatives showed sharply improvement in their inhibitory activity

against NIH3T3 cell lines proliferation. Based on the results of biological evaluation,
preliminary SAR of pyridone derivatives could be presented for NIH3T3 cell.
As mentioned above, cellular activity of compound 8 has obviously improved effect against
NIH3T3 cell line proliferation. In the follow-on test, we conducted a series of studies on the
activity of compound 8, toxicity, in vivo anti-DN efficacy, PK and mechanism. As a
multi-target agent, the parameters of compound 8 were tuned and balanced by modifying
substituents at positions 1 and 5 of the pyridone ring. The pharmacokinetic and toxicological
properties of compound 8 were superior to those of PFD, and it was better than Losartan in
diabetic nephropathy. In general, compound 8 was a potent NIH3T3 inhibitor with excellent
druggability properties, and had high efficacy in in vivo DN models, moderate dose
proportional and wide therapeutic window and pharmaceutical properties.
Compound 8 was advanced to preclinical studies as an oral treatment for diabetic
nephropathy.
ABBREVIATIONS USED
ACR, albumin-to-creatinine ratio; AUC0-t, area under the curve; BuOH, n-butanol; BUN,
blood urea nitrogen; Cmax, peak concentration; C13-NMR, C13-nuclear magnetic resonance;
CCr, creatinine clerance rate; CCR2, CCchemkin receptor2; CDCl3, Deuterotrichloromethane;
DBM, diabetes model, DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DN,
diabetic nephropathy; ECM, extracellular matrixc; ELISA, enzyme-linked immunosorbent
assay; ERSD, end stage renal disease; GC-MS, Gas Chromatograph-Mass
Spectrometer-computer; GLP, Good laboratory practice of drug; GSI, glomerular sclerosis
index; HMBC, heteronuclear multiple bond correlation; HPLC, high performance liquid
chromatography; IR, infrared radiation; LC-MS, liquid chromatography-mass spectrum; LPS,
lipopolysaccharide; MTT, methylthiazolyl diphenyl tetrazolium bromide; NIH3T3, mouse
embryonic fibroblast cell line; NOX, nicotinamide adenine dinucleotide phosphate oxydaes;
PD, pharmacodynamics; PDGF, platelet-derived growth factor; PFD, pirfenidone; PK,
pharmacokinetics ; ROS, reactive oxygen species; SAR, structure activity relationships; SCr,
serum creatinin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factors-α;
UUO, unilateral ureteral occlusion.

Acknowledgments
This study was supported by grants from the National Science and Technology Major
Project of China(no.2012ZX09103101-025) and the National Natural Science
Foundation(no.20972194). We thanked Zhi-Yu Li Ph.D in the Department of Medicinal
Chemistry of China Pharmaceutical University for chemical support and acknowledge
Laboratory of NMR of Modern Analysis and Testing Center of Central South University
for the determination of the chemical structure of the target compounds.
Supplementary data
Supplementary data associated with this article can be found in the online version, at DOI:
xxxxx.
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