Design, synthesis, and biological evaluation of a novel dual peroxisome proliferator-activated receptor alpha/delta agonist for the treatment of diabetic kidney disease through anti-inflammatory mechanisms

DOI: 10.1016/j.ejmech.2021.113388

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European Journal of Medicinal Chemistry (2021)

Update date:2022-07-30

Topics: Synthesis Biological Evaluation Design

Authors:

Liu, Kai

Zhao, Xing

Qi, Xue Qi, Xue

Hou, Dong-Liang

Li, Hao-Bin

Gu, Yu-Hao

Xu, Qing-Long

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Article abstract of DOI:10.1016/j.ejmech.2021.113388

Diabetic kidney disease (DKD) is a major feature of the final stage of nearly all cause types of diabetes mellitus (DM). To date, few safe and effective drugs are available to treat. Peroxisome proliferator-activated receptors (PPARs), comprised of three members: PPAR-α, PPAR-δ and PPAR-γ, play a protective role in the DKD through glycemic control and lipid metabolism, whereas systemic activation of PPAR-γ causes serious side-effects in clinical trials. GFT505 is a dual PPAR-α/δ agonist, and the selectivity against PPAR-γ is still to be improved. Sulfuretin has been shown to suppress the expression of PPAR-γ and improve the pathogenesis of diabetic complications. In this study, by hybridizing the carboxylic acid of GFT505 and the parent nucleus of sulfuretin, we pioneeringly designed and synthetized a series of novel dual PPAR-α/δ agonists, expecting to provide a better benefit/risk ratio for PPARs. Of all the synthesized compounds, compound 12 was identified with highly activity on PPAR-α/δ and higher selectivity against PPAR-γ than that of GFT505 (EC₅₀: hPPAR-α: 0.26 μM vs.0.76 μM; hPPAR-δ: 0.50 μM vs.0.73 μM; hPPAR-γ: 4.22 μM vs.2.79 μM). The molecular docking studies also depicted good binding affinity of compound 12 for PPAR-α and PPAR-δ compared to GFT505. Furthermore, compound 12 exhibited an evidently renoprotective effect on the DKD through inhibiting inflammatory process, which might at least partly via JNK/NF-κB pathways in vivo and in vitro. Overall, compound 12 hold therapeutic promise for DKD.

Full text of DOI:10.1016/j.ejmech.2021.113388

European Journal of Medicinal Chemistry 218 (2021) 1 13388

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design, synthesis, and biological evaluation of a novel dual

peroxisome proliferator-activated receptor alpha/delta agonist for the

treatment of diabetic kidney disease through anti-inﬂammatory

mechanisms

Kai Liu ¹, Xing Zhao ¹, Xue Qi ¹, Dong-Liang Hou, Hao-Bin Li, Yu-Hao Gu, Qing-Long Xu^*

Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia

Xiang, Nanjing, 210009, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Diabetic kidney disease (DKD) is a major feature of the ﬁnal stage of nearly all cause types of diabetes

mellitus (DM). To date, few safe and effective drugs are available to treat. Peroxisome proliferator-

Received 7 February 2021

Received in revised form

8 March 2021

Accepted 14 March 2021

Available online 20 March 2021

activated receptors (PPARs), comprised of three members: PPAR-

tive role in the DKD through glycemic control and lipid metabolism, whereas systemic activation of

PPAR- causes serious side-effects in clinical trials. GFT505 is a dual PPAR- agonist, and the selectivity

against PPAR- is still to be improved. Sulfuretin has been shown to suppress the expression of PPAR-

a, PPAR-d and PPAR-g, play a protec-

a/d

and improve the pathogenesis of diabetic complications. In this study, by hybridizing the carboxylic acid

of GFT505 and the parent nucleus of sulfuretin, we pioneeringly designed and synthetized a series of

Keywords:

PPAR

GFT505

Sulfuretin

Diabetic kidney disease

Inﬂammation

novel dual PPAR-

thesized compounds, compound 12 was identiﬁed with highly activity on PPAR-

against PPAR- than that of GFT505 (EC₅₀: hPPAR- : 0.26 M vs.0.76 M; hPPAR-

hPPAR- : 4.22 M vs.2.79 M). The molecular docking studies also depicted good binding afﬁnity of

compound 12 for PPAR- and PPAR- compared to GFT505. Furthermore, compound 12 exhibited an

evidently renoprotective effect on the DKD through inhibiting inﬂammatory process, which might at

agonists, expecting to provide a better beneﬁt/risk ratio for PPARs. Of all the syn-

and higher selectivity

: 0.50 M vs.0.73 M;

a/d

least partly via JNK/NF-

B pathways in vivo and in vitro. Overall, compound 12 hold therapeutic promise

for DKD.

1. Introduction

progression of DKD, the morbidity and mortality associated with

DKD remains high [4]. Research identifying additional therapeutic

approaches that are innocuous is urgent clinical needs.

Diabetic kidney disease (DKD), a serious complication of dia-

betes mellitus (DM), is one of the most leading causes of end-stage

renal disease worldwide [1]. According to the 9th edition of Global

Diabetes Map from the International Diabetes Federation (IDF) in

2019, approximately 463 million adults (20e79 years) were living

with diabetes, by 2045 this will rise to 700 million. Seriously, up to

40% of people with diabetes will develop into DKD. Clinically,

although several available therapeutic interventions, such as

angiotensin converting enzyme inhibitors (ACEI) [2] or angiotensin

The pathogenesis of DKD is extremely complex involving

multiply different pathways, including the glycolipid metabolism

disorder [5], advanced glycation end products (AGEs) [6], protein

kinase C [7], polyol pathway [8] and hemodynamic changes [9]. In

parallel, compelling evidence supports the fact that inﬂammation

plays a pivotal role in the development and progression of DKD

[10]. The inﬂammatory inﬁltration, accumulation of macrophages,

and increased secretion of injurious cytokines, including trans-

II type

(AT1) receptor blockers [3], can delay the onset

forming growth factor b1 (TGF-b1), which is also a key regulator of

extracellular matrix (ECM) protein synthesis in the progress of

renal ﬁbrosis, are observed in renal biopsy specimens from patients

with DKD [1 1]. Therefore, inhibition of inﬂammation and the

* Corresponding author.

E-mail address: qlxu@cpu.edu.cn (Q.-L. Xu).

resulting ECM accumulation, may be served as

a potential

These authors contributed equally to this article.

https://doi.org/10.1016/j.ejmech.2021.113388

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

therapeutic approach for preventing kidney injury in DKD.

Peroxisome proliferator-activated receptors (PPARs) are ligand-

activated transcription factors of nuclear hormone receptor su-

nucleus of sulfuretin to design and synthesize a series of novel dual

PPAR-

agonists, expecting to increase the activity of PPAR-

and simultaneously decrease the activation of PPAR-

, as well as

perfamily, which comprises of three members: PPAR-

PPAR- [12]. There is an increasing body of evidence suggesting

that PPARs play a senior role in the pathogenesis of DKD for the

function in glycemic control and lipid metabolism [13]. PPAR-

, PPAR-

and

explore the pharmacological activity for diabetes-provoked in-

duction and progression of DKD.

Taken together, in this study, we designed, synthesized a series

of novel dual PPAR-a/d agonists through hybridizing GFT505 and

agonists are used as lipid lowering agents in clinical therapy for

more than 30 years [14] and the anti-inﬂammatory effect of PPAR-

sulfuretin, and evaluated their biological activities against PPARs,

then screened promising candidates to verify potential binding

agonists has been shown in many animal studies [15]. In addition,

PPAR- agonists are widely used to control serum glucose levels of

diabetic patients [16], nevertheless, several of them were dis-

continued because of PPAR- related weight gain, hemodilution

mode for PPAR-a and PPAR-d by molecular docking, as well as

investigate the therapeutic efﬁcacy and safety on the development

of DKD in vivo and in vitro.

and ﬂuid retention in clinical trials [1 7]. It is mandatory to develop

2. Results and discussion

novel therapeutic interventions and simultaneous activation of

PPAR-a/d with higher selectivity against PPAR-g may be a potential

2.1. Chemistry

therapeutic option to prevent diabetes-associated hyperglycemia

and hyperlipidemia provoked induction and progression of DKD.

GFT505 (Elaﬁbranor, Fig. 1A), a PPARs agonist which exhibited

The target compounds 1e16 were prepared via a convergent

synthesis according to the synthetic route outlined in Schemes 1.

preferential activity on PPAR-a/d with no obvious PPAR-g associ-

The compound

B were synthesized through 3,5-dimethyl-4-

ated adverse effect, had been evaluated for the amelioration of

glycolipid disorder associated with the metabolic syndrome several

years [18] and exhibited reasonable effects on treatment of non-

alcoholic steatohepatitis (NASH, NCT02704403, phase III) in clin-

ical trials and DM in animal models [19]. But risks regarding

hydroxybenzaldehyde (A) and ethyl 2-bromoisobutyrate in aceto-

nitrile containing caesium carbonate. Then the aldehyde B was

reduced to give alcohol C in the presence of sodium borohydride.

Subsequently, In the presence of carbon tetrabromide and triphe-

nylphosphine, the compound D was obtained in 50% yield over 3

steps. On the other hand, the compound F were synthesized by

condensation of the corresponding aldehyde with 6-hydroxy-2,3-

dihydrobenzo[b]furan-3-one (E) under basic condition. Then the

compound F reacted with D in acetonitrile afforded the corre-

sponding acetate G. Finally, the acetate G was then carried out in

the presence of potassium hydroxide to afford the desired com-

pounds 1e16 (Scheme 1).

adverse effects still exist for its activity on PPAR-

g. Developing dual

PPAR agonists with minimum activity on PPAR-g

might be

provide a better beneﬁt/risk ratio in clinical practice. Moreover, it

might be an innovative point of penetration to ﬁnd a hybridizable

molecule, which can antagonize PPAR-

g or inhibit the PPAR-g

expression in metabolic diseases. Sulfuretin (Fig. 1B), one of the

natural products isolated from Rhus verniciﬂua, exerted several

pharmacological effects [20], including anti-inﬂammatory, anti-

oxidation and anti-ﬁbrosis, to improve the pathogenesis of diabetic

complications in many animal studies [21,22]. More importantly, it

is now increasingly appreciated that sulfuretin had potent anti-

2.2. PPARs agonistic activity

Compounds 1e16 were evaluated in vitro for their agonistic

adipogenic activity via suppressing the expression of PPAR-

g in

activities toward the human PPAR-a, PPAR-d and PPAR-g subtypes

metabolic diseases [23,24], which indicated that sulfuretin might

exist underlying connection with PPARs. Hence, we innovatively

tried to hybridize the carboxylic acid of GFT505 and the parent

by employing GAL4-PPAR transactivation assay in transiently

transfected COS7 cells. Commercially available PPAR agonists,

GW7647 (Fig. 1C), GW501516 (Fig. 1D) and rosiglitazone (Fig. 1E),

Fig. 1. Structure of GFT505 (Elaﬁbranor), sulfuretin, GW7647, GW501516 and rosiglitazone.

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

Scheme 1. Synthetic route for target product 1e16.

were used as reference compounds for PPAR-

, PPAR-

and PPAR-

activity, respectively. The results were summarized in Table 1.

Among all the synthesized compounds, compound 12 exhibited the

best agonistic activity towards PPAR-

and PPAR- agonistic activation 16.2-fold and 8.4-fold greater

than PPAR- activity, respectively. More importantly, compound 12

exhibited more potent than PPAR- agonistic activity with higher

selectivity against PPAR- compared to GFT505 (EC₅₀: hPPAR-

M; hPPAR- : 0.50 M vs.0.73 M; hPPAR-

a and PPAR-d. It exerted PPAR-

Table 1

In vitro activities of the human PPARs.

a/d

Compd.

hPPAR-

(EC₅₀

)

hPPAR-

(EC₅₀

)

hPPAR-

(EC₅₀

)

GFT505

0.76 0.15

3.10 0.96

2.80 0.47

0.73 0.17

1.80 0.34

1.60 0.42

2.79 0.35

1.72 0.24

0.26

4.22

M vs.0.76

>10

mM vs.2.79

mM), respectively. Above all, compound 12 was

>10

>10 mM

optimal, providing potent PPAR-

selectivity against PPAR-

activity and high PPAR-

1.73 0.48

0.79 0.01

5.21 0.12

1.98 0.16

1.72 0.45

3.93 0.62

2.25 0.61

2.31 0.18

0.52 0.02

8.94 2.65

2.45 0.03

0.50 0.10

1.63 0.29

1.17 0.16

1.26 0.25

0.98 0.24

>10

2.3. Molecular docking study

3.30 0.14

4.44 0.90

1.88 0.49

0.26 0.08

1.66 0.12

1.03 0.18

4.56 1.70

4.19 1.87

To get a more detailed view at a molecular level of compound 12

acting on PPAR- and PPAR- , molecular docking study was per-

1.00 0.02

4.22 0.18

>10 mM

6.36 1.50

1.63 0.40

formed, GFT-505 was also studied in the same protocol for com-

parison. As shown in Fig. 2, GFT-505 and compound 12 were

docked into the binding sites of PPAR-

(Fig. 2 and 2B) and PPAR-

>10

(Fig. 2C and 2D), respectively. The predicted binding modes be-

GW7647

GW501516

Rosiglitazone

2.08 0.48 nM

tween compound 12 and PPAR-a showed that hydrogen bond in-

teractions were generated between the ligand and Ser 280, Tyr314,

His 440 and Tyr464 (Fig. 2B). The predicted binding mode between

compound 12 and PPAR-d showed that hydrogen bond interactions

were generated between the ligand and Thr252, Thr253, His 287,

1.05 0.37 nM

191.50 46.67 nM

EC₅₀value represent the mean of at least two determinations, which is the

concentration giving 50% of the maximal activity determined for the tested

compound.

ND: not determined.

His 413 and Tyr437 (Fig. 2D). Compound 12 kept all the protein-

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

Fig. 2. The binding model of GFT-505 (A,C) and compound 12 (B,D) in the pocket of PPAR-a(A ~ B)and PPAR-d (C ~ D) was predicted by molecular docking. The PPAR-a or PPAR-

protein was shown in wheat carton. Green stick: small molecules, Yellow stick: key amino acid residues, Magenta dash: hydrogen bonds.

ligand interactions of GFT-505 with all the key residues of PPAR-

and PPAR-d, indicating its potential bioactivity against these two

subtypes of PPAR. Hence, we selected compound 12 as the prom-

ising candidate to investigate therapeutic efﬁcacy and safety on the

development of DKD in vivo and in vitro.

Furthermore, the evidently elevated serum cystatin C observed in

the model rats was apparently attenuated in the compound 12

treated rats (Fig. 3H). Taken together, these data revealed that DKD

occurred in rats and suggested that compound 12 could protect

renal functions in DKD rats.

2.4. Pharmacodynamic evaluation in vivo and in vitro

2.4.3. Effects on lipid metabolism in DKD rats

Excess intracellular lipid accumulation in non-adipose tissue,

such as in kidney, can induce cell injury and organ dysfunction

(termed lipotoxicity) [25]. We found that the serum levels of tri-

glyceride (TG) and total cholesterol (TC) were signiﬁcantly higher in

model rats than in control rats (Fig. 4A and 4B). However, com-

pound 12 at two doses signiﬁcantly reduced the serum TG levels

(Fig. 4A). The increase of TC was tended to be less with compound

12 treatment, but this trend was not statistically signiﬁcant

(Fig. 4B). Furthermore, the renal Oil red O staining results showed

that compound 12 treatment evidently decreased the excess

intracellular lipid accumulation in the renal of model rats (Fig. 4C).

Consistently, this ﬁnding was supported by the decreased contents

of TG and TC in serum. To verify the effect of compound 12 treat-

ment on hepatotoxicity, the serum levels of alanine aminotrans-

ferase (ALT) and aspartate aminotransferase (AST) were measured.

The results showed that compound 12 treatment did not increase

the serum levels of ALT and AST, but slightly decreased the level of

AST (Fig. 4D and 4E), indicating no obvious hepatotoxicity of

compound 12 at these two doses. Altogether, these data revealed

that compound 12 at these two doses could restore lipid metabolic

disorder in DKD rats to different degrees.

2.4.1. Effects on the general symptoms in DKD rats

Initially, we examined the effects of compound 12 on the fasting

blood glucose and body weight in model rats during the animal

procedures. Results showed that the model rats had the symptoms

of hyperglycemia and weight loss which are typical characteristics

of DM. There was no signiﬁcant difference in fasting blood glucose

between the four DKD groups (Fig. 3A). In parallel, the decrease of

body weight in model rats tended to be less with compound 12

treatment, but this trend was not statistically signiﬁcant (Fig. 3B).

However, results of renal morphology showed that the kidneys

from model rats were obvious swelling and exhibited brownish

yellow compared with the control which were vivid purple brown.

Treatment with compound 12 at two doses obviously ameliorated

this symptom (Fig. 3C). Moreover, compound 12 treatment signif-

icantly reduced the kidney/body weight ratio compared to the

model group (Fig. 3D). Collectively, these data revealed that DM

occurred in rats and suggested that compound 12 might ameliorate

nephrosis in DKD rats without depending on hypoglycemic action.

2.4.2. Effects on renal functional parameters in DKD rats

Next, we evaluated the effects of compound 12 on renal func-

tional parameters in DKD rats. We determined the 24 h albumin-

uria during the experiments and found that it was signiﬁcantly

increased in model rats as compared with that in control rats.

Interestingly, administration of compound 12 for 6 weeks signiﬁ-

cantly attenuated the DM induced increment in 24 h albuminuria

(Fig. 3E). At the end of experiments, we also determined the serum

levels of urea nitrogen, creatinine and cystatin C, three key pa-

rameters of renal function. Compound 12 at two doses signiﬁcantly

reduced the serum levels of urea nitrogen and creatinine, to

different degrees compared to the model rats (Fig. 3F and 3G).

2.4.4. Effects on oxidative stress in DKD rats

Lipid peroxidation (LPO) is always potentiated by oxidative

stress [26] and accumulating evidence also indicate that activation

of oxidative stress plays a key role in kidney further aggravating

DKD [27]. Considering compound 12 could restore lipid metabolic

disorder, we thus measured serum and kidney levels of malon-

dialdehyde (MDA) and superoxide dismutase (SOD), two major

parameters for oxidative stress. We found a marked increase in

serum and kidney levels of MDA in model rats, which was blunted

by compound 12 treatment (Fig. 4F). In parallel, the serum and

Fig. 3. Effects on the general symptoms and renal functional parameters in DKD rats. (A) Measurement of fasting blood glucose levels at weeks 0, 2, 4 and 6 during the experiments. (B) Measurement of body weight at weeks 0, 1, 2, 3, 4,

5 and 6 during the experiments. (C) Representative renal morphology of rats at the end of the experiments. (D) Measurement of right kidney/body weight ratio at the end of the experiments. (E) Measurement of 24 h albuminuria at

weeks 0, 2, 4 and 6 during the experiments. (F) Measurement of serum urea nitrogen at the end of the experiments. (G) Measurement of serum creatinine at the end of the experiments. (H) Measurement of serum cystatin C at the end

of the experiments. All data were presented as means SEM (n ¼ 8e10 per group). Statistical signiﬁcance of differences were determined by one-way ANOVA. ###p < 0.001 and ##p < 0.01 compared to Control group; ***p < 0.001,

**p < 0.01 and *p < 0.05 compared to Model group.

Fig. 4. Effects on lipid metabolism, oxidative stress and PPAR-

sections of Oil red O staining on the renal cortex of DKD rats. Scale bars, 50

of serum and kidney MDA at the end of the experiments. (G) Measurement of the levels of serum and kidney SOD at the end of the experiments. (H) RT-PCR analyses of Ppard mRNA expression in renal cortex of DKD rats.

used as the invariant control. (I) Western blot analyses of PPAR- protein expression in renal cortex of DKD rats. -actin was used as an invariant control for equal loading. Representative blots are shown with densitometry. All data were

expression in DKD rats. (A) Measurement of serum TG at the end of the experiments. (B) Measurement of serum TC at the end of the experiments. (C) Representative

m. (D) Measurement of serum ALT at the end of the experiments. (E) Measurement of serum AST at the end of the experiments. (F) Measurement of the levels

-actin was

presented as means SEM (n ¼ 8e10 per group). Statistical signiﬁcance of differences were determined by one-way ANOVA. ###p < 0.001 and ##p < 0.01 compared to Control group; ***p < 0.001, **p < 0.01 and *p < 0.05 compared

to Model group.

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

kidney activity of SOD was noted to be decreased in model rats

compared to control rats. However, compound 12 at the high dose

signiﬁcantly restored the decreased SOD activity in both serum and

kidney (Fig. 4G), suggesting that oxidative stress could be attenu-

ated by compound 12 treatment. Taken together, these data indi-

cated that compound 12 may improve LPO via suppressing the

oxidative stress in serum and kidney, which may contribute to the

improved renal function in DKD rats.

lesions, cells disordered arrangement, spotty necrosis and vacuolar

degeneration occurred in kidney from model rats. Nonetheless,

these pathological alterations were markedly improved by com-

pound 12 at two doses (Fig. 5F). Collectively, these data clearly

indicated that compound 12 could suppress the inﬂammatory

process in the kidney of DKD rats.

2.4.7. Effects on the ﬁbrosis response in the kidney of DKD rats

Renal ﬁbrosis is also a primary characteristic of advanced DKD

and the process is usually irreversible [36]. Notably, ECM accu-

mulation is the major cause of renal ﬁbrosis [37]. We initially used

periodic acideSchiff (PAS) staining to determine the structure of

glomerulus and Masson’s trichrome staining to assess the accu-

mulation of ECM. As shown in Fig. 6, obvious accumulation of

glycogen, mesangial expansion and collagen deposition were found

in the kidney of model rats. Intriguingly, the DKD-induced these

pathological lesions were signiﬁcantly improved by compound

12 at two doses to different degrees (Fig. 6A~6C). To further validate

the improvements of compound 12 on renal ﬁbrosis in DKD rats, we

2.4.5. Effects on the PPAR-

It is worth-mentioning that PPARs are expressed in several tis-

sues including the kidney. PPAR- is predominantly expressed in

proximal tubules and medullary thick ascending limbs, PPAR- is

selectively expressed in medullary collecting ducts, glomeruli and

pelvic urothelium, while PPAR- is equally expressed in renal cortex

and medulla [28]. Notably, numerous clinical cases and literatures

[29,30] had revealed that glomerulosclerosis in the renal cortex is

the source of the genesis and progression of DKD and the renal

cortex was widely investigated for molecular mechanisms of DKD.

d expression in the kidney of DKD rats

We therefore detected the PPAR-

renal cortex. Consistently, the mRNA level of PPAR-

tectable in the renal cortex. There was no changes in renal cortex

PPAR- mRNA expression between the control and model rats.

However, the PPAR- mRNA of compound 12 at two doses had

and PPAR-

expression in the

was unde-

detected the mRNA expression of TGF-b1, collagen4a1, laminin,

connective tissue growth factor (CTGF), osteopontin (OPN), and

plasminogen activator inhibitor-1(PAI-1), which were ﬁbrosis-

related factors during renal ﬁbrosis [38]. Consistent with the

changes of the mesangial fractional and interstitial ﬁbrosis area,

gene expression for this six parameters were markedly increased in

model rats, and dramatically suppressed by compound 12 at two

doses treatment to different degrees (Fig. 6D~6I). Noteworthy,

signiﬁcantly high expression compared to another groups, even

had signiﬁcant difference at high dose compared to the model

group (Fig. 4H). Furthermore, determination of PPAR-

d protein

expression in the renal cortex showed that the protein levels were

all signiﬁcantly depressed in the DKD rats. The decrease of PPAR-

excessive TGF-b1 is associated with ECM collagen 4 in the

protein expression in DKD rats tended to be less with compound

12 treatment, but this trend was not statistically signiﬁcant (Fig. 4I).

Currently, the literatures about PPAR- agonists on the expression

of PPAR- are few and controversial. Data from studies by Liang

et al. [28] and Lee et al. [31] were almost consistent with us. On the

contrary, Matsushita et al. [15] found that the renal PPAR- mRNA

glomerular area and both have been identiﬁed as key mediators of

glomerular and tubulointerstitial pathologies in DKD [39]. Immu-

nohistochemical analyses showed that the protein expression of

TGF-b1 and collagen 4 was evidently increased in the model rats as

compared to the control rats. However, both were expressed at

signiﬁcantly lower levels in the compound 12 groups (Fig. 6J~6L).

Above all, these results suggested that compound 12 might lead to

decrease renal ﬁbrosis in DKD rats through inhibiting relevant

ﬁbrosis-related factors pathological overexpression.

and protein expression were signiﬁcantly greater in the DM group

than in the control group. The reason for the divergence was un-

known. A possible explanation for this might be that the stability of

PPAR-d protein in DKD might be altered [32,33].

2.4.6. Effects on the inﬂammatory response in the kidney of DKD

rats

Many studies have proposed an important role of inﬂammatory

processes in the pathogenesis of DKD [34]. Tumor necrosis factor

2.4.8. Effects on the inﬂammatory and ﬁbrosis response in vitro

To further validate the anti-inﬂammatory and anti-ﬁbrosis ef-

fects of compound 12 on DKD, we examined in vitro anti-

inﬂammatory and anti-ﬁbrosis activity of compound 12 in human

glomerular mesangial cells (HGMCs), which are known to regulate

the secretion of ECM in kidney [28], and human myeloid leukemia

mononuclear cells (THP-1)-derived macrophages. We ﬁrstly eval-

uated the effects of compound 12 at different concentrations (1, 3,

(TNF- ), interleukin-1 (IL-1 ) and interleukin 6 (IL-6), as impor-

tant inﬂammatory factors, are involved in the inﬂammatory path-

ological damage process in DKD patients [35]. We thereby detected

mRNA expression of the three factors to evaluate the occurrence of

inﬂammation in vivo. As shown in Fig. 5, the mRNA expression TNF-

10, 30

mM) on cell viability. Results showed that exposure of HGMCs

in the model rats were expressively up-regulated in contrast with

the control rats. But compound 12 at two doses were signiﬁcantly

down-regulated the mRNA expression TNF- (Fig. 5A). The increase

of mRNA expression of IL-1 and IL-6 in model rats was not sta-

and THP-1-derived macrophages to 1, 3, 10 or 30 m

M of compound

12 did not affect cell viability at 48 h (Fig. 7A~7B). Then, we

examined the effects of compound 12 on the anti-inﬂammatory

and anti-ﬁbrosis activity in corresponding cells. As expected,

compound 12 signiﬁcantly inhibited high glucose (HG)-induced

tistically signiﬁcant, but this trend tended to be less with com-

pound 12 treatment (Fig. 5B and 5C). Meanwhile, we detected the

effects of compound 12 on macrophage inﬁltration in the kidney.

CD14 is a marker for all macrophages, and CD11c is speciﬁc for the

M1 subtype of macrophages [15]. We found that the mRNA levels of

CD11c and CD14, were strongly increased in the model group.

However, compound 12 treatment signiﬁcantly reduced the

expression of CD11c and slightly decreased the expression of CD14

(Fig. 5D and 5E). Further histological evidence strengthened the

protective role of compound 12 for the kidney through anti-

inﬂammatory mechanism. Data from hematoxylin and eosin (HE)

staining revealed that obvious inﬂammatory inﬁltration and

mRNA expression of TGF-

ride (LPS)-induced mRNA expression of TGF-

and IL-6, in THP-1-derived macrophages were markedly down-

regulated by compound 12 in dose-dependent manner

1 (Fig. 7C). Moreover, Lipopolysaccha-

b1, TNF- , even IL-1

(Fig. 7D~7G). However, it seemed that compound 12 could not

suppress the mRNA expression of collagen1a1, collagen4a1, laminin

and CTGF, which can be induced by TGF-b1 in HGMCs (Fig. 7H~7K).

Collectively, these data indicated that compound 12 strongly

inhibited HG and LPS induced inﬂammatory gene expression,

which might be a potential way to ameliorate renal functions in

DKD, rather than directly suppressing ﬁbrosis-related factors.

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

Fig. 5. Effects on the inﬂammatory response in the kidney of DKD rats. RT-PCR analyses of Tnfa (A), Il1b (B), Il-6 (C), Cd11c (D), Cd14 (E) mRNA expression in renal cortex of DKD rats.

-actin was used as the invariant control. (F) Representative sections of HE staining on the renal cortex of DKD rats. Scale bars, 50 m. All data were presented as means SEM

(n ¼ 8e10 per group). Statistical signiﬁcance of differences were determined by one-way ANOVA. ###p < 0.001 and ##p < 0.01 compared to Control group; ***p < 0.001, **p < 0.01

and *p < 0.05 compared to Model group.

2.4.9. Preliminary study on anti-inﬂammatory mechanism

levels of the two factors were all signiﬁcantly elevated in the model

rats, but were clearly dose dependently decreased by treatment

with compound 12 compared to the model rats (Fig. 8G~8I).

Together, these data indicated that compound 12 could suppress

Finally, we preliminarily tried to explore the anti-inﬂammatory

mechanism of compound 12 on DKD. Among the known factors

that are incriminated in the genesis and progression of DKD, the c-

Jun N-terminal protein kinase (JNK) and nuclear transcription fac-

JNK and NF-kB signaling pathways in vivo and in vitro, which may

tor kappa B (NF-kB) play crucial roles in the pathogenesis of DKD

be, at least partly, a potential molecular mechanism to ameliorate

via inﬂammatory process [40,41]. Of note, both PPARs [42] and

sulfuretin [20] had been shown to suppress inﬂammation process

through down-regulation of this two factors in many DM animal

studies. We thus checked the phosphorylation levels of JNK and NF-

inﬂammatory process for DKD.

2.5. Discussion

B in HG-induced HGMCs and THP-1-derived macrophages stim-

As potential targets for the therapy of DKD, PPARs are critically

involved in the regulation of metabolic syndrome which associated

with glucose and lipid metabolism. Numerous preclinical studies

ulated by LPS. Immunoblot analysis demonstrated that the DMSO

group had signiﬁcantly high phosphorylation levels of JNK and NF-

B in both HGMCs and macrophages compared to the control

detected PPAR-

brate (Fig. 9B), had positive renal effects on the animals with DKD.

The activation of PPAR- regulated responsible for LPO and oxida-

tive stress [43]. Moreover, the expression of TGF- could also be

attenuated via PPAR- activation, which led to improved renal

a agonists, such as fenoﬁbrate (Fig. 9A) and bezaﬁ-

group. The increase of phosphorylation levels of JNK and NF-

were tended to be less with compound 12 groups, but this trend

was not statistically signiﬁcant in HGMCs (Fig. 8A~8C). However,

intriguingly, when we investigated the dose-dependent effects of

compound 12 on LPS-induced macrophages, a simultaneous and

signiﬁcant decrease of phosphorylation levels of JNK and NF-kB

were observed (Fig. 8D~8F). Subsequently, to further validate the

potential anti-inﬂammatory mechanism in vitro, we detected the

phosphorylation levels of JNK and NF-

As expected, immunoblot analysis showed that phosphorylation

ﬁbrosis [44]. Meanwhile, thirteen trials of the 290 clinical studies

revealed fenoﬁbrate consistently reduced the progression of uri-

nary albumin excretion in the DM patients [45]. However, further

research on this series of agonists was prevented because of asso-

ciating with serum creatinine level increasing in clinical trials [46],

kB in the kidney of DKD rats.

indicating current therapeutic limitations of the use of PPAR-

Fig. 6. Effects on the ﬁbrosis response in the kidney of DKD rats. (A) Representative sections of PAS and Masson’s trichrome staining on the renal cortex of DKD rats. Scale bars, 50

fractional area (%). (C) Quantitative analysis of the positive interstitial ﬁbrosis area (%). RT-PCR analyses of Tgfb1(D), Col4a1 (E), Lamb 1 (F), Ctgf (G), Opn (H) and Pai-1(I) mRNA expression in renal cortex of DKD rats.

the invariant control. (J) Representative immunohistochemistry sections for TGF- 1 and collagen4 on the renal cortex of DKD rats. Scale bars, 50 m. (K) Quantitative analysis of the positive (stained) area for TGF-b

m. (B) Quantitative analysis of the positive mesangial

-actin was used as

1 area (%). (L)

Quantitative analysis of the positive (stained) area for collagen4 area (%). All data were presented as means SEM (n ¼ 8e10 per group). Statistical signiﬁcance of differences were determined by one-way ANOVA. ###p < 0.001,

##p < 0.01 compared to Control group; ***p < 0.001, **p < 0.01 and *p < 0.05 compared to Model group.

Fig. 7. Effects on cell viability, inﬂammatory and ﬁbrosis response in HGMCs and THP-1-derived macrophages. (A) Effects on cell viability in HGMCs. (B) Effects on cell viability in THP-1-derived macrophages. (C) RT-PCR analyses of

TGFB1 mRNA expression in HGMCs. RT-PCR analyses of TGFB1 (D), TNFA (E), IL1B (F) and IL6 (G) mRNA expression in THP-1-derived macrophages. RT-PCR analyses of COL1A1 (H), COL4A1 (I), LAMB1 (J) and CTGF (K) mRNA expression in

HGMC.

-actin was used as the invariant control. All data were presented as means SEM (n ¼ 5 per group). Statistical signiﬁcance of differences were determined by one-way ANOVA. ###p < 0.001 and #p < 0.05 compared to Control

group; ***p < 0.001, **p < 0.01 and *p < 0.05 compared to DMSO group.

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

Fig. 8. Preliminary study on anti-inﬂammatory mechanism. (A) Representative western blots for PeNF-

B p65, NF-

B p65/NF-

B p65, P-JNK, JNK protein expression in THP-1-derived macrophages stimulated by LPS. (E) Quantitative analyses for P-JNK/JNK in THP-1-derived macrophages

stimulated by LPS. (F) Quantitative analyses for PeNF- B p65/NF- B p65 in THP-1-derived macrophages stimulated by LPS. (G) Representative western blots for PeNF- B p65, NF-

p65, P-JNK, JNK protein expression in the renal cortex of DKD rats. (H) Quantitative analyses for P-JNK/JNK in the renal cortex of DKD rats. (I) Quantitative analyses for PeNF- B p65/

NF- B p65 in the renal cortex of DKD rats.

B p65, P-JNK, JNK protein expression in HG-induced HGMCs. (B)

Quantitative analyses for P-JNK/JNK in HG-induced HGMCs. (C) Quantitative analyses for PeNF-

PeNF- B p65, NF-

kB p65 in HG-induced HGMCs. (D) Representative western blots for

-actin was used as an invariant control for equal loading. All data were presented as means SEM (n ¼ 3 per group in cells and n ¼ 8e10

per group in rats). Statistical signiﬁcance of differences were determined by one-way ANOVA. ###p < 0.001 compared to Control group; ***p < 0.001, **p < 0.01 and *p < 0.05

compared to DMSO or Model group.

agonists. Compared with PPAR-

agonists, compound 12 at doses of

(aleglitazar, Fig. 9E) [48], dual PPAR-

1-yl] acetic acid, Fig. 9F) [49] and pan-PPAR

Fig. 9G) [50] agonists had also been developed to treat DM or DKD.

However, most of them were prevented because of PPAR- related

([5-(Benzyloxy)-1H-indol-

25 mg/kg and 50 mg/kg also effectively improved the DKD char-

acteristic symptoms. Notably, decreased the serum level of creati-

nine (Fig. 3G) was observed, which successfully circumvented the

a/d/g

(chiglitazar,

side-effect from PPAR-

Currently, studies on the effects of PPAR-

are still rare. Matsushita et al. [15] and Lee et al. [31] found GW0742

(Fig. 9C), a PPAR- agonist, could suppress the gene expression of

inﬂammatory mediators and prevent nephrin loss in the renal

cortex without altering blood glucose levels. Liang et al. [28] re-

agonists (ﬁbrates).

weight gain, ﬂuid reaction, hemodilution and edema effect [1 7,51].

Considering the current research status surrounding the PPARs

agonists’ intervention for DKD, compound 12, which was pioneer-

ingly designed and synthesized via hybridizing the carboxylic acid

of GFT505 and the parent nucleus of sulfuretin, showed distinct

advantages in not only PPARs agonistic activities but also thera-

peutic efﬁcacy for DKD. More importantly, it successfully preserved

agonists for the DKD

ported L-165041(Fig. 9D), another PPAR-

DKD in vivo associated with inhibition of AGEs and NF-

agonist, could improve

B pathways.

the advantages of PPAR-

and PPAR-d agonists while circumventing

Intriguingly, similar effects also had been demonstrated from

compound 12. It markedly suppressed the gene expression of in-

ﬂammatory cytokines and attenuated the inﬂammatory response

the side-effect from PPAR-

against DKD.

agonists, holding therapeutic potential

in kidney of DKD rats, might at least partly via JNK/NF-

in vivo and in vitro, indicating the role played by PPAR-

B pathways

3. Conclusion

agonists

was ideally manifested.

In summary, a novel dual PPAR-

through hybridizing the carboxylic acids of GFT505 and the parent

a/d agonist was discovered

For years, synthetic PPAR-g (rosiglitazone) [47], dual PPAR-a/g

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

Fig. 9. Structure of PPARs agonists.

nucleus of sulfuretin. Compound 12 exhibited more potent than

PPAR- agonistic activity with higher selectivity against PPAR-

compared to GFT505 (EC₅₀: hPPAR- : 0.26 M vs.0.76 M; hPPAR-

0.50 M vs.0.73 M; hPPAR- : 4.22 M vs.2.79 M). In addition,

molecular docking study showed that compound 12 kept all the

protein-ligand interactions of GFT-505 with all the key residues of

PPAR-a and PPAR-d, indicating its potential bioactivity against these

two subtypes of PPAR. Furthermore, compound 12 was not only

capable of attenuating the characteristic symptoms from DKD

doublets (td), quartet (q), multiplet (m), and broad (br). Low and

high resolution mass spectra (LRMS and HRMS) were given with

electrospray ionization (ESI) produced by Waters Q-TOF Micro and

Agilent G6230B, respectively. Melting points (mp) were measured

in open capillary tubes, using an RY-1G melting point apparatus

(0e300 ^ꢁC). All target compounds were conﬁrmed with over 95%

purity by UPLC. A Waters Acquity UPLC system comprising the

Quaternary Solvent manager, Sample Manager-FTN, PDA Detector

were employed. The UPLC conditions were as follows: ACQUITY

while circumventing the side-effect from PPAR-

orchestrating inﬂammation and ﬁbrosis responses, which might at

least partly via JNK/NF- B pathways in vivo and in vitro. Overall,

agonists, but also

UPLC® BEH C18 column, 2.1 ꢂ 50 mm, 1.7

mm; detection wave-

length: 254 nm. Water and methanol containing 0.05% formic acid

were used to perform elution at a ﬂow rate of 1.2 mL/min.

compound 12 successfully took the essence and discarded the dregs

from PPARs, holding therapeutic promise for DKD.

4.1.1. General synthetic procedure for intermediates D

A round-bottomed ﬂask was charged with 3,5-dimethyl-4-

4. Experimental section

hydroxybenzaldehyde

(A,

1.0

equiv.)

and

ethyl

bromoisobutyrate (3.0 equiv.), caesium carbonate (3.0 equiv.),

solved in acetonitrile. The reaction mixture was stirred at 80 ^ꢁC for

36 h. After the reaction was completed (monitored by TLC), the

mixture was diluted by water and extracted by ethyl acetate. The

combined organic layer was washed with brine, dried over anhy-

drous sodium sulfate and concentrated under the reduced pressure.

The corresponding compound B was used in the next step without

further puriﬁcation.

In a dry round-bottomed ﬂask, compound B (1.0 equiv.) in

ethanol was cooled at 0 ^ꢁC. sodium borohydride (1.0 equiv.) was

added in portions. The mixture was reacted for 4 h at room tem-

perature. After the reaction was completed (monitored by TLC),

4.1. General chemistry

All reagents solvents used in this study were commercially

purchased products with purity ꢀ95%. Analytical thin-layer chro-

matography (TLC) was performed on silica gel 60 F254 plates

(Qingdao Ocean Chemical Company, China). Column chromatog-

raphy was carried out on silica gel (200e300 mesh, Qingdao Ocean

Chemical Company, China). ¹H and ¹³C NMR spectra were per-

formed on an ACF*300Q Bruker spectrometer with Me4Si as the

internal reference. Proton coupling patterns were described as

singlet (s), doublet (d), doublet of doublets (dd), triplet (t), triplet of

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

water was added slowly at 0 ^ꢁC. The mixture was stirred for 15 min,

then the mixture was extracted by ethyl acetate. The combined

organic layer was washed with brine, dried over anhydrous sodium

sulfate and concentrated under the reduced pressure. The corre-

sponding compound C was used in the next step without further

puriﬁcation.

In a dry round-bottomed ﬂask, compound C (1.0 equiv.) and

carbon tetrabromide (1.5 equiv.) solved in dichloromethane was

cooled at 0 ^ꢁC. Triphenylphosphine (1.3 equiv.) solved in dichloro-

methane was added dropwise. The mixture was reacted for 4 h at

room temperature. The reaction was removed solid by ﬁltration,

then the ﬁltrate was evaporated under reduced pressure and pu-

riﬁed by ﬂash chromatography on silica gel (petroleum ether/ethyl

acetate ¼ 10:1) to afford the corresponding product D.

methylpropanoic acid (3). 38% Yield, white solid. ¹H NMR

(500 MHz, DMSO‑d₆)

d 12.90 (br s, 1H), 8.15e7.98 (m, 2H), 7.69 (d,

J ¼ 8.5 Hz, 1H), 7.35 (t, J ¼ 8.5 Hz, 2H), 7.21 (s, 1H), 7.13 (s, 2H), 6.91

(d, J ¼ 8.4 Hz, 1H), 6.87 (s, 1H), 5.12 (s, 2H), 2.20 (s, 6H), 1.38 (s, 6H).

¹³C NMR (126 MHz, DMSO‑d₆)

d 181.57, 175.12, 167.21 (d,

J ¼ 179.9 Hz), 163.61, 161.63, 152.61, 146.93, 133.43 (d, J ¼ 8.5 Hz),

132.62, 130.87, 128.66 (d, J ¼ 2.5 Hz), 128.50, 125.52, 116.09 (d,

J ¼ 21.6 Hz), 113.92, 113.19, 109.85, 97.79, 80.17, 70.20, 24.97, 17.58.

HRMS (ESI) exact mass calcd for C₂₈H₂₆FO₆requires m/z 477.1713,

found 477.1710.

4.1.2.4. (Z)-2-(4-(((2-(4-cholobenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy)meth yl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (4). 38% Yield, white solid. ¹H NMR

(500 MHz, DMSO‑d₆)

12.84 (br s, 1H), 7.98 (d, J ¼ 8.1 Hz, 1H), 7.71

4.1.2. General synthetic procedure for targets 1-16

(d, J ¼ 8.5 Hz, 1H), 7.57 (d, J ¼ 8.1 Hz, 1H), 7.23 (s, 1H), 7.13 (s, 2H),

In a dry round-bottomed ﬂask, 6-hydroxy-2,3-dihydrobenzo[b]

furan-3-one (E, 1.0 equiv.) and substituted benzaldehyde (1.5

equiv.), potassium hydroxide (1.0 equiv.) solved in ethanol, then the

mixture was reacted overnight at room temperature. After the re-

action was completed (monitored by TLC), con. hydrochloric acid

was added slowly at 0 ^ꢁC. The solid was precipitated from the

mixture, the corresponding product F was obtained by ﬁltration.

In a dry round-bottomed ﬂask, compound F (1.0 equiv.), com-

pound D (1.5 equiv.) and potassium carbonate (2.0 equiv.) was

solved in acetonitrile, then the mixture was reacted overnight. The

mixture was evaporated under reduced pressure and puriﬁed by

ﬂash chromatography on silica gel (petroleum ether/ethyl

acetate ¼ 10:1) to afford the corresponding product G.

6.93 (d, J ¼ 8.1 Hz, 1H), 6.87 (s, 1H), 5.14 (s, 2H), 2.21 (s, 6H), 1.38 (s,

6H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 181.52, 167.95, 166.58, 152.63,

147.45, 134.35, 132.64, 132.62, 130.94, 130.83, 129.03, 128.47, 125.57,

113.81, 113.28, 109.53, 97.82, 80.20, 70.23, 24.99, 17.58. HRMS (ESI)

exact mass calcd for C₂₈H₂₆ClO₆requires m/z 493.1411, found

493.1418.

4.1.2.5. (Z)-2-(4-(((2-(4-bromobenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy)meth yl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (5). 35% Yield, yellow solid. ¹H NMR

(500 MHz, DMSO‑d₆)

12.89 (br s, 1H), 7.88 (d, J ¼ 8.2 Hz, 2H), 7.68

(d, J ¼ 8.4 Hz, 2H), 7.19 (s, 1H), 7.12 (s, 1H), 6.90 (d, J ¼ 8.3 Hz, 1H),

6.82 (s, 1H), 5.12 (s, 2H), 2.19 (s, 6H), 1.37 (s, 6H). ¹³C NMR (126 MHz,

In a dry round-bottomed ﬂask, compound G (1.0 equiv.), po-

tassium hydroxide (3.0 equiv.) was solved in tetrahydrofuran/

methanol/water, then the mixture was reacted at room tempera-

ture. After the reaction was completed (monitored by TLC), 1.0 M

hydrochloric acid was added to adjust the mixture to PH ¼ 5e6.

Then the mixture was extracted by dichloromethane. The com-

bined organic layer was washed with brine, dried over anhydrous

sodium sulfate and concentrated under the reduced pressure, then

puriﬁed by ﬂash chromatography on silica gel (dichloromethane/

methanol ¼ 200:1) to afford the product (1e16).

DMSO‑d₆) d 182.01, 175.60, 168.42, 167.06, 153.10, 148.00, 133.31,

133.11, 132.44, 131.74, 131.34, 128.97, 126.05, 123.75, 114.29, 113.77,

110.10, 98.28, 80.66, 70.71, 25.46, 18.07. HRMS (ESI) exact mass

calcd for C₂₈H₂₆BrO₆requires m/z 537.0913, found 537.0914.

4.1.2.6. (Z)-2-(2,6-dimethyl-4-(((2-(4-methylbenzylidene)-3-oxo-

2, 3-dihy drobenzofuran-6-yl) oxy)methyl)phen oxy)-2-

methylpropanoic acid (6). 33% Yield, white solid. ¹H NMR (500 MHz,

DMSO‑d₆)

12.81 (br s, 1H), 7.88 (d, J ¼ 8.0 Hz, 2H), 7.70 (d,

J ¼ 8.6 Hz, 1H), 7.33 (d, J ¼ 7.9 Hz, 2H), 7.26 (s, 1H), 7.13 (s, 2H), 6.92

(d, J ¼ 6.6 Hz, 1H), 6.83 (s, 1H), 5.14 (s, 2H), 2.37 (s, 3H), 2.20 (s, 6H),

4.1.2.1. (Z)-2-(2,6-dimethyl-4-(((2-(4-(methylthio)benzylidene)-3-

oxo-2,3-dihydrobenzo furan-6-yl)oxy)methyl)phenoxy)-2-

methylpropanoic acid (1). 32% Yield, yellow solid. ¹H NMR

1.38 (s, 6H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 181.57, 175.10, 167.83,

166.42, 152.59, 146.82, 139.94, 132.61, 131.17, 130.91, 129.64, 129.19,

128.49, 125.44, 114.02, 113.15, 111.18, 97.79, 80.16, 70.19, 24.97, 21.12,

17.58. HRMS (ESI) exact mass calcd for C₂₉H₂₉O₆requires m/z

473.1964, found 473.1960.

(500 MHz, DMSO‑d₆)

7.90 (d, J ¼ 8.3 Hz, 2H), 7.69 (d, J ¼ 8.6 Hz,

1H), 7.37 (d, J ¼ 8.3 Hz, 2H), 7.22 (s, 1H), 7.12 (s, 2H), 6.92 (d,

J ¼ 8.4 Hz,1H), 6.83 (s,1H), 5.13 (s, 2H), 2.54 (s, 3H), 2.20 (s, 6H),1.36

(s, 6H). ¹³C NMR (126 MHz, DMSO‑d₆)

181.41, 167.67, 166.39,

4.1.2.7. (Z)-2-(4-(((2-(4-methoxybenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy)methyl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (7). 37% Yield, white solid. ¹H NMR (500 MHz,

152.84, 146.84, 141.38, 132.67, 131.49, 130.70, 128.45, 128.20, 125.61,

125.43, 114.05, 113.12, 110.88, 97.78, 80.51, 70.23, 25.19, 17.64, 14.09.

HRMS (ESI) exact mass calcd for C₂₉H₂₉O₆S requires m/z 505.1685,

found 505.1681.

DMSO‑d₆)

12.89 (br s, 1H), 7.96 (d, J ¼ 8.7 Hz, 2H), 7.70 (d,

J ¼ 8.5 Hz, 1H), 7.23 (s, 1H), 7.14 (s, 1H), 7.09 (d, J ¼ 8.7 Hz, 2H),

6.98e6.91 (m, 1H), 6.86 (s, 1H), 5.14 (s, 2H), 3.85 (s, 3H), 2.21 (s, 6H),

4.1.2.2. (Z)-2-(4-(((2-benzylidene-3-oxo-2,3-dihydrobenzofuran-6-

yl)oxy)methyl)-2,6-dimethylphenoxy)-2-methylpropanoic acid (2).

1.39 (s, 6H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 181.40, 175.07, 167.59,

166.23,160.61,152.56,146.06,133.06,132.60,130.95,128.50,125.35,

124.49, 114.62, 114.22, 113.01, 111.39, 97.76, 80.14, 70.15, 55.36,

24.95, 17.57. HRMS (ESI) exact mass calcd for C₂₉H₂₉O₇requires m/z

489.1913, found 489.1911.

36% Yield, white solid. ¹H NMR (500 MHz, DMSO‑d₆)

d 12.91 (s, 1H),

7.98 (d, J ¼ 7.4 Hz, 2H), 7.71 (d, J ¼ 8.5 Hz, 1H), 7.55e7.45 (m, 3H),

7.25 (s, 1H), 7.14 (s, 2H), 6.93 (d, J ¼ 8.4 Hz, 1H), 6.86 (s, 1H), 5.14 (s,

2H), 2.21 (s, 6H), 1.39 (s, 6H). ¹³C NMR (126 MHz, DMSO‑d₆)

181.64,175.08, 167.98,166.52,152.59,147.26,132.61,131.98,131.12,

4.1.2.8. (Z)-2-(4-(((2-(4-ethoxybenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy) methyl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (8). 32% Yield, yellow solid. ¹H NMR

130.89, 129.79, 128.96, 128.50, 125.52, 113.91, 113.18, 110.92, 97.83,

80.15, 70.20, 24.95, 17.57. HRMS (ESI) exact mass calcd for C₂₈H₂₇O₆

requires m/z 459.1808, found 459.1804.

(500 MHz, DMSO‑d₆)

12.90 (br s, 1H), 7.95 (d, J ¼ 8.6 Hz, 2H), 7.70

(d, J ¼ 8.6 Hz, 1H), 7.24 (s, 1H), 7.14 (s, 2H), 7.07 (d, J ¼ 8.6 Hz, 2H),

6.93 (d, J ¼ 8.5 Hz, 1H), 6.85 (s, 1H), 5.14 (s, 2H), 4.12 (d, J ¼ 7.0 Hz,

2H), 2.21 (s, 6H), 1.38 (s, 6H), 1.36 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR

4.1.2.3. (Z)-2-(4-(((2-(4-ﬂuorobenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy) methyl)-2,6-dimethylphenoxy)-2-

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

(126 MHz, DMSO‑d₆)

181.40, 175.10, 167.59, 166.24, 159.93, 152.60,

J ¼ 7.5 Hz), 123.10 (q, J ¼ 274.6 Hz), 113.48, 113.46, 103.84, 98.00,

146.02, 133.10, 132.61, 130.93, 128.50, 125.35, 124.33, 115.01, 114.23,

113.03, 111.46, 97.75, 80.18, 70.16, 63.35, 24.98, 17.58, 14.50. HRMS

(ESI) exact mass calcd for C₃₀H₃₁O₇requires m/z 503.2070, found

503.2066.

80.17, 70.30, 24.96, 17.56. HRMS (ESI) exact mass calcd for

₂₉H₂₆F₃O₆requires m/z 527.1681, found 527.1677.

4.1.2.14. (Z)-2-(2,6-dimethyl-4-(((3-oxo-2-(4-(triﬂuoromethoxy)ben-

zylidene)-2,3-dihydrobenzofuran-6-yl)oxy)methyl)phenoxy)-2-

methylpropanoic acid (14). 38% Yield, yellow solid. ¹H NMR

4.1.2.9. (Z)-2-(4-(((2-(4-isopropylbenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy) methyl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (9). 40% Yield, yellow solid. ¹H NMR

(500 MHz, DMSO‑d₆)

12.90 (br s, 1H), 8.08 (d, J ¼ 8.0 Hz, 2H), 7.71

(d, J ¼ 8.2 Hz, 1H), 7.50 (d, J ¼ 7.6 Hz, 2H), 7.21 (s, 1H), 7.13 (s, 2H),

(500 MHz, DMSO‑d₆)

12.91 (br s, 1H), 7.91 (d, J ¼ 7.8 Hz, 2H), 7.72

7.02e6.82 (m, 3H), 5.14 (s, 2H), 2.21 (s, 6H), 1.38 (s, 6H). ¹³C NMR

(d, J ¼ 8.4 Hz, 1H), 7.40 (d, J ¼ 7.8 Hz, 1H), 7.25 (s, 1H), 7.15 (s, 2H),

6.94 (d, J ¼ 8.4 Hz, 1H), 6.85 (s, 1H), 5.16 (s, 2H), 3.03e2.90 (m, 1H),

2.21 (s, 6H), 1.39 (s, 6H), 1.25 (d, J ¼ 6.7 Hz, 6H). ¹³C NMR (126 MHz,

(126 MHz, DMSO‑d₆) d 181.58, 175.10, 168.04, 166.62, 152.61, 148.75,

147.54, 132.94, 132.63, 131.32, 130.85, 128.49, 125.63, 121.31, 120.00

(q, J ¼ 257.0 Hz), 113.80, 113.28, 109.19, 97.83, 80.17, 70.23, 24.96,

17.57. HRMS (ESI) exact mass calcd for C₂₉H₂₆F₃O₇requires m/z

543.1631, found 543.1626.

DMSO‑d₆)

d 181.58, 175.09, 167.85, 166.44, 152.60, 150.67, 146.89,

132.61, 131.30, 130.90, 129.58, 128.50, 127.00, 125.48, 114.03, 113.13,

111.18, 97.81, 80.17, 70.19, 33.41, 24.97, 23.55, 17.58. HRMS (ESI)

exact mass calcd for C₃₁H₃₃O₆requires m/z 501.2277, found

501.2267.

4.1.2.15. (Z)-2-(2,6-dimethyl-4-(((3-oxo-2-(4-((triﬂuoromethyl)thio)

benzylidene)-2,3-dihydrobenzofuran-6-yl)oxy)methyl)phenoxy)-2-

methylpropanoic acid (15). 28% Yield, white solid. ¹H NMR

4.1.2.10. (Z)-2-(4-(((2-(4-cyanobenzylidene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy) methyl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (10). 35% Yield, white solid. ¹H NMR

(500 MHz, DMSO‑d₆)

12.88 (br s, 1H), 8.09 (d, J ¼ 7.4 Hz, 2H), 7.84

(d, J ¼ 7.4 Hz, 2H), 7.73 (d, J ¼ 8.2 Hz, 1H), 7.24 (s, 1H), 7.14 (s, 2H),

7.00e6.79 (m, 2H), 5.15 (s, 2H), 2.21 (s, 6H), 1.39 (s, 6H). ¹³C NMR

(500 MHz, DMSO‑d₆)

12.89 (br s, 1H), 8.11 (s, 2H), 7.96 (s, 2H), 7.71

(126 MHz, DMSO‑d₆) d 181.57, 175.07, 168.12, 166.74, 152.60, 148.29,

(d, J ¼ 8.0 Hz, 1H), 7.25 (d, J ¼ 5.4 Hz, 1H), 7.14 (d, J ¼ 6.3 Hz, 2H),

136.31, 134.98, 132.63, 132.01, 130.84, 129.40 (q, J ¼ 308.7 Hz),

128.49, 125.72, 124.02, 113.68, 113.38, 108.95, 97.91, 80.16, 70.25,

24.95, 17.57. HRMS (ESI) exact mass calcd for C₂₉H₂₆F₃SO₆requires

m/z 559.1402, found 559.1392.

7.01e6.85 (m, 2H), 5.15 (s, 2H), 2.21 (s, 6H), 1.39 (s, 6H). ¹³C NMR

(126 MHz, DMSO‑d₆) d 181.54, 175.08, 168.16, 166.80, 152.61, 148.60,

136.71, 132.63, 131.35, 130.80, 128.50, 125.75, 118.61, 113.54, 113.45,

111.36, 108.55, 97.95, 80.16, 70.28, 24.96, 17.57. HRMS (ESI) exact

mass calcd for

506.1569.

₂₉H₂₅NO₆Na requires m/z 506.1580, found

4.1.2.16. (Z)-2-(2,6-dimethyl-4-(((2-(naphthalen-2-ylmethylene)-3-

oxo-2,3-dihydrobenzofuran-6-yl)oxy)methyl)phenoxy)-2-

methylpropanoic acid (16). 37% Yield, white solid. ¹H NMR

4.1.2.11. (Z)-2-(4-(((2-([1,1⁰-biphenyl]-4-ylmethylene)-3-oxo-2,3-

dihydrobenzofuran-6-yl)oxy)methyl)-2,6-dimethylphenoxy)-2-

methylpropanoic acid (11). 31% Yield, white solid. ¹H NMR

(500 MHz, DMSO‑d₆) d 12.95 (br s, 1H), 8.52 (s, 1H), 8.19 (d,

J ¼ 9.1 Hz, 1H), 8.11e7.92 (m, 3H), 7.77 (d, J ¼ 8.4 Hz, 1H), 7.65e7.55

(m, 2H), 7.35 (s, 1H), 7.17 (s, 2H), 7.05 (s, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H),

5.19 (s, 2H), 2.24 (s, 6H), 1.40 (s, 6H). ¹³C NMR (126 MHz, DMSO‑d₆)

(500 MHz, DMSO‑d₆)

12.88 (br s, 1H), 8.04 (d, J ¼ 8.0 Hz, 2H), 7.80

(d, J ¼ 8.0 Hz, 2H), 7.74 (d, J ¼ 7.5 Hz, 2H), 7.69 (d, J ¼ 8.5 Hz, 1H),

7.49 (t, J ¼ 7.4 Hz, 2H), 7.41 (d, J ¼ 7.2 Hz, 1H), 7.23 (s, 1H), 7.13 (s,

2H), 6.98e6.83 (m, 2H), 5.13 (s, 2H), 2.20 (s, 6H), 1.38 (s, 6H). ¹³C

d 181.60, 175.12, 168.00, 166.54, 152.64, 147.49, 133.11, 132.88,

132.63, 131.81, 130.87, 129.68, 128.49, 127.67, 127.58, 127.28, 126.84,

125.57, 113.97, 113.27, 111.07, 97.93, 80.22, 70.24, 25.01, 17.59. HRMS

(ESI) exact mass calcd for C₃₂H₂₉O₆requires m/z 509.1964, found

509.1952.

NMR (126 MHz, DMSO‑d₆)

d 181.53, 175.10, 167.88, 166.47, 152.59,

147.32, 141.17, 139.12, 132.61, 131.73, 131.11, 130.90, 129.00, 128.50,

128.01, 127.08, 126.73, 125.49, 113.98, 113.17, 110.60, 97.81, 80.15,

70.21, 24.96, 17.58. HRMS (ESI) exact mass calcd for C₃₄H₃₁O₆re-

quires m/z 535.2121, found 535.2108.

4.2. Molecular docking

All molecular docking procedures were completed in

€

4.1.2.12. (Z)-2-(2,6-dimethyl-4-(((3-oxo-2-(2-(triﬂuoromethyl)ben-

zylidene)-2,3-dihydrobenzofuran-6-yl)oxy)methyl)phenoxy)-2-

methylpropanoic acid (12). 35% Yield, yellow solid. ¹H NMR

Schroodinger 2009. The crystal structures of PPAR-

(PDB ID: 4CI4)

and PPAR-

d (PDB ID: 3SP9) obtained from RCSB (http://www.rcsb.

org/) were applied for molecular docking. The proteins in the

crystal structures were prepared using the Protein Preparation

Wizard workﬂow. The binding sites were deﬁned with the crystal

ligands in each crystal structure. Small molecules were prepared

with LigPrep and then docked into the binding sites using the Glide

module with extra precision (XP) mode.

(500 MHz, DMSO‑d₆)

12.90 (br s, 1H), 8.10 (d, J ¼ 8.0 Hz, 2H), 7.80

(d, J ¼ 8.2 Hz, 2H), 7.68 (d, J ¼ 8.5 Hz, 1H), 7.18 (s, 1H), 7.12 (s, 2H),

6.97e6.83 (m, 2H), 5.12 (s, 2H), 2.20 (s, 6H), 1.38 (s, 6H). ¹³C NMR

(126 MHz, DMSO‑d₆) d 181.54, 175.09, 168.13, 166.72, 152.61, 148.30,

136.04, 132.63, 131.38, 130.84, 129.10 (q, J ¼ 31.5 Hz), 128.45, 125.67,

125.60 (q, J ¼ 2.5 Hz), 123.0 (q, J ¼ 273.4 Hz), 113.62, 113.35, 108.73,

97.82, 80.15, 70.24, 64.89, 24.94, 17.56. HRMS (ESI) exact mass calcd

for C₂₉H₂₆F₃O₆requires m/z 527.1681, found 527.1673.

4.3. In vivo evaluation

4.3.1. Materials and instruments

4.1.2.13. (Z)-2-(2,6-dimethyl-4-(((3-oxo-2-(4-(triﬂuoromethyl)ben-

zylidene)-2,3-dihydro benzofuran-6-yl)oxy)methyl)phenoxy)-2-

methylpropanoic acid (13). 28% Yield, white solid. ¹H NMR

STZ was obtained from Sigma (USA). Ramipril was obtained

from aladdin (Shanghai, China). Accu-Chek Active System was ob-

tained from Roche (Germany). Rat metabolic cage was obtained

from Zhenghua biologic apparatus facility (Huaibei, China). Test kits

of albuminuria, urea nitrogen, creatinine, ALT, AST, MDA and SOD

were obtained from Jiancheng Bioengineering Institute (Nanjing

China). Test kits of cystatin C, TC, TG were obtained from Elabs-

cience (Wuhan, China). Ts2R inverted ﬂuorescence microscope was

obtained from Nikon (Japan). Multimode plate reader was obtained

(500 MHz, DMSO‑d₆)

12.92 (br s, 1H), 8.36 (d, J ¼ 7.8 Hz, 1H),

7.94e7.74 (m, 2H), 7.73 (d, J ¼ 8.5 Hz,1H), 7.65 (t, J ¼ 7.5 Hz,1H), 7.22

(s, 1H), 7.13 (s, 2H), 6.94 (d, J ¼ 8.2 Hz, 1H), 6.85 (s, 1H), 5.13 (s, 2H),

2.20 (s, 6H), 1.38 (s, 6H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 181.42,

175.09, 168.39, 166.90, 152.63, 148.50, 132.88, 132.63, 132.03,

130.78, 129.64, 129.48, 128.49, 127.70 (q, J ¼ 28.8 Hz), 126.42 (q,

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

from PerkinElmer (USA). Trizol, PimeScript™ RT Master Mix (Per-

fect Real Time) and SYBR® Premix Ex Taq were obtained from

Vazyme (Nanjing, China). BCA protein quantitative kit was obtained

separated, the left kidney was stored in liquid nitrogen immedi-

ately and then stored at ꢃ80 ^ꢁC, the right kidney was photographed

and weighed to calculate the ratio of kidney/body weight, then

ﬁxed in 4% paraformaldehyde for subsequent examinations.

from Beyotime (Shanghai, China). Rat

obtained from Sangon (Shanghai, China). 4% paraformaldehyde,

Anti-TGF- 1 were obtained from Servicebio (Wuhan. China). Anti-

collagen4 and anti-PPAR- were obtained from Proteintech

(Wuhan. China). Anti-Phospho-JNK1/2/3, anti-JNK1/2/3, anti-

PhosphoeNFe p65, antieNFe p65 were obtained from

ABclonal (Wuhan, China). Anti- -actin was obtained from arigo

b-actin gene primer pair was

4.3.5. Estimation of urine and serum biochemical parameters

Urine was obtained from each rat and centrifuged under

3000 rpm for 10 min. Levels of 24 h albuminuria in the supernatant

were determined according to the protocol of urinary protein test

kit. The blood was centrifuged at 3000 g for 15 min after 3 h at room

temperature, serum was prepared and stored at ꢃ80 ^ꢁC until use.

Serum levels of urea nitrogen, creatinine, cystatin C, TC, TG, ALT,

AST, levels of serum and kidney MDA and SOD were determined

using corresponding assay kits according to their protocols.

(Shanghai, China). HRP-labeled goat anti-mouse IgG secondary

antibody was obtained from Fcmacs (Nanjing, China). HRP-labeled

goat anti-rabbit IgG secondary antibody was obtained from CoWin

(Beijing, China). Western blot detected kit (Tanon™ High-sig ECL

Western Blotting Luminol/Enhancer Solution and Tanon™ High-sig

ECL Western Blotting Peroxide Buffer) was obtained from Tanon

Science & Technology Co., Ltd (Shanghai, China).

4.3.6. Renal histology analysis

The right kidney tissue samples were ﬁxed in 4% para-

formaldehyde, subjected to standard histological processing and

4.3.2. Animals

embedded in parafﬁn. The sample was cut to 5 mm thick, stained

8-week-old male Sprague-Dawley rats were supplied by the

Charles River Laboratory Animal Technology Co., Ltd. (Pinghu,

China) (Certiﬁcate No. SCXK (Zhe) 2019e001). Experimental pro-

cedures involving the use of animals complied with the Guidelines

for Animal Experimentation of the China Pharmaceutical University

(Nanjing, China). The protocol was approved by the Animal Ethics

Committee of that institution. All animals were maintained under

standard environmental conditions and provided with feed and

water ad libitum.

with HE, PAS and Masson’s trichrome following standard protocols.

Lipid content in kidney was determined by Oil Red O staining with

kidney frozen sections as described [54]. Brieﬂy, frozen sections of

kidney were rinsed with Oil Red O solution for 10 min in the dark.

And then sections rinsed with 60% isopropanol for differentiation.

Subsequently, sections were stained with hematoxylin solution,

and then were sealed to slices with glycerin gelatin. Photographs

were taken in a blinded fashion at random ﬁelds. Representative

views of kidney sections were shown. Quantiﬁcation of staining

was performed using software Image J and was expressed as a

percentage of the positive region [55]. All images were analyzed by

two investigators blinded to the identity of the samples.

4.3.3. Establishment of rat models of DKD

After one week of acclimatization, rats were randomly assigned

into control (n ¼ 8) and model (n ¼ 40) group. After fasting for 12 h,

the rats of diabetes were induced by injecting a single dose of STZ

(55 mg/kg, intraperitoneally) dissolved in ice-cold sodium citrate

buffer (0.01 M, pH 4.4). The rats of control were received only so-

dium citrate buffer. After 3 days of STZ injection, peripheral blood

was harvested from vena caudalis and measured by an Accu-Chek

Active System, the fasting blood glucose between 16.7 mM and

33 mM were considered diabetes. Sequentially, each rat fasting

blood glucose were measured, 24 h urine were collected using

metabolic cage and the 24 h albuminuria were detected every

week. The signs of fasting blood glucose between 16.7 mM and

33 mM, 24 h urine volume >150% raw urine, and 24 h albuminuria

>30 mg can be seen as the successful establishment of the DKD rat

model [52].

4.3.7. Immunohistochemistry analysis

Sections were dewaxed and rehydrated, followed by antigen

retrieval, blocking with 3% H₂O₂and BSA. After that, sections were

incubated overnight with anti-TGF-b1 (1:200) or anti-collagen4

(1:500) in a humidiﬁed chamber at 4 ^ꢁC. The sections were

washed with PBS and exposed to secondary antibody (HRP-labeled)

from the corresponding species of primary antibody and incubated

at room temperature for 50 min. Meanwhile, negative control was

set up. Diaminobenzidine (DAB) was used as a chromogen. The

positive (stained) area for each marker was calculated using colour

thresholding and by measuring area fractions with software Image J

[55]. All images were analyzed by two investigators blinded to the

identity of the samples.

4.3.4. Experimental protocol

4.3.8. Reverse transcription-polymerase chain reaction (RT-PCR)

The RT-PCR analysis were performed as described previously

[56]. Brieﬂy, total RNA was extracted from 10 mg of the renal cortex

using 500 mL Trizol according to the manufacturer’s instructions.

One microgram of total RNA was reverse transcribed into cDNA

using HiScript II Q RT SuperMix kit according to the manufacturer’s

protocol. The Power SYBR Green Master Mix was used for real-time

PCR analysis. Differences in gene expression between groups were

calculated using cycle threshold (Ct) values. Relative expression of

genes was calculated by the 2^-△△Ctmethod. The necessary primers

were purchased from Sangon and all primer pairs could be seen in

the supplementary data (Table S1). Gene expression values were

To intuitively compare the therapeutic efﬁcacy with currently

clinical drug (ACEI) for DKD, based on the fasting blood glucose and

24 h albuminuria, DKD rats were divided into four groups: model

group, ramipril group (2 mg/kg, positive control), low dose com-

pound 12 group (25 mg/kg) and high dose compound 12 group

(50 mg/kg), 10 rats in each group. The dosages of compound 12

were consulted and optimized as described previously [19,23].

Ramipril, an ACEI, which can inhibit aldosterone secretion to exert

favorable protective effects on renal functions in clinical practice

and often used as positive control in the therapeutic efﬁcacy for

DKD [53]. All agents were prepared with carboxymethyl cellulose

sodium (CMC-Na) as suspension (0.5%) and orally given for 6

weeks, in which compound 12 groups (both low group and high

group) were gavaged twice a day and another groups were gavaged

once a day. During the experiments, body weight was measured

every week, fasting blood glucose and 24 h albuminuria were

measured every two weeks. At the end of experiments, all rats were

scariﬁed and blood was obtained via orbital cavity, kidneys were

normalized to the expression levels of b-actin.

4.3.9. Western blotting analysis

The Western blot analysis were performed as described previ-

ously [57]. Brieﬂy, a piece of the renal cortex were lysed using a lysis

buffer [120 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%

SDS, 50 mM Tris-HCl (pH 7.6)], and proteins were obtained after

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

centrifugation. The concentrations of proteins were determined

using a BCA protein assay kit. The equal amounts of proteins were

then electrophoresed on 10% SDS polyacrylamide gels. After

transferring to PVDF membranes, the membranes were blocked

with a 5% BSA solution for 1 h and incubated overnight with pri-

mary antibodies. Then membranes were incubated continually

with secondary antibodies, and the bands were detected using a

Western blot detection kit.

supplemented with 10% FBS and 1% penicillin-streptomycin solu-

tion. For the experiments, HGMCs were plated in 6-well dishes at a

density of 2 ꢂ 10⁵cells/well overnight, and then were incubated

with serum-free medium for 24 h to arrest and synchronize cell

growth. THP-1 cells were plated in 12-well dishes at a density of

5 ꢂ 10⁵cells/well, and then were treated with 100 ng/mL PMA to

differentiate. After 48 h, differentiated THP-1 cells were washed

with PBS twice and rested for another 24 h in the culture medium

to obtain the resting state of macrophage. For the RT-PCR analysis,

4.4. In vitro evaluation

HGMCs were exposed in 5.5 mM

trol), 30 mM -glucose (high glucose, HG) or 5.5 mM

24.5 mM mannitol (as an osmotic control for 30 mM

with or without the additional 24 h application of the compound

12 at different concentrations (1, 3 or 10 M). Meanwhile, HGMCs

were exposed in 10 ng/mL TGF- 1 with or without the additional

6 h application of the compound 12 at different concentrations (1, 3

or 10 M). The differentiated THP-1 cells were treated with 100 ng/

mL LPS or LPS plus the compound 12 at different concentrations (1,

3 or 10 M) for 6 h. For the Western blot analysis, cells were pre-

incubated with compound 12 at different concentrations (1, 3 or

10 M) for 2 h followed by post incubation with HG or LPS for 0.5 h.

-glucose (low glucose, as con-

-glucose plus

-glucose)

4.4.1. Evaluation for PPARs agonist activities

4.4.1.1. Materials. COS7 cell was obtained from Cellcook (Guangz-

hou, China). Dulbecco’s modiﬁed Eagle medium (DMEM), fetal

bovine serum (FBS), and penicillin-streptomycin solution were

obtained from Biological Industries (Israel). The pGL4.35 with

9 ꢂ Gal4 UAS Luc reporter plasmid was purchased from Promega

(USA). Opti-MEM was obtained from Gibco (USA). Transfection

reagent was obtained from ABclonal (Wuhan, China). Fireﬂy lucif-

erase reporter gene assay kit was obtained from Beyotime

(Shanghai, China).

4.4.1.2. Plasmids. The cDNA sequences of human PPARs ligand-

binding domain were subcloned to pBIND vector to construct

chimeric human PPARs-Gal4 receptor expression plasmids. Site-

speciﬁc mutants of PPARs-Gal4 were generated by PCR-based,

site-directed mutagenesis kit according to the manufacturer’s in-

structions [58].

4.4.2.3. Cell viability test. Cell viability was determined using the

CCK-8 assay. HGMCs (1 ꢂ 10⁴cells/well) were incubated in a 96-

well plate in DMEM (containing 5.5 mM of

D-glucose) supple-

mented with 10% FBS and 1% penicillin-streptomycin solution. After

culturing overnight, HGMCs were starved in serum-free DMEM for

24 h. THP-1 cells (4 ꢂ 10⁵cells/well) were seeded in 96-well plate in

RPMI-1640 containing 10% FBS and 100 ng/mL PMA to differentiate.

After 48 h, differentiated THP-1 cells were washed with PBS twice

and rested for another 24 h in the culture medium to obtain the

resting state of macrophage. After that, two types of cells were

exposed to compound 12 at different concentrations (0, 1, 3, 10,

4.4.1.3. Cell culture and transactivation assay. COS7 cells were

cultured in DMEM supplemented with 10% FBS and 1% penicillin-

streptomycin solution. The main function of PPARs relies on its

transcriptional activity, which is regulated by the ligand binding to

PPARs ligand-binding domain (LBD). Detailed descriptions on

30 mM) for 48 h, CCK-8 solution (10 mL) was then added to each well,

followed by incubation for 1 h. Absorbance at 450 nm was

measured.

transfection and cell-based evaluation for PPAR-

PPAR- were given in our previously reported literature [58].

Brieﬂy, COS7 cells were transfected with expression plasmids

encoding the fusion protein GAL4-PPAR- -LBD, GAL4-PPAR- -LBD

or GAL4-PPAR- -LBD, mixed with transfection reagent in Opti-

a, PPAR-d and

4.5. Statistical analysis

MEM according to the manufacturer’s instruction. 4 h after trans-

fection, cells were distributed in 96-well plates to recover for 24 h.

After an overnight incubation, the three luciferase activities were

measured using ﬁreﬂy luciferase reporter gene assay kit on a

multimode plate reader, respectively. Dose-response curves were

constructed with GraphPad Prism version 8.0 to determine the EC₅₀

value.

All data are presented as the means SEM, and analyses were

performed with Graphpad Prism 8 software. Comparisons between

experimental groups were conducted using One-way ANOVA,

Values of p < 0.05 were considered signiﬁcant.

Declaration of competing interest

4.4.2. Evaluation for pharmacological activity in HGMCs and THP-

1-derived macrophages

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.

4.4.2.1. Materials. HGMCs were obtained and maintained accord-

ing to distributor guidelines from ScienCell Research Laboratories

(USA). THP-1 cells were obtained from Stem Cell Bank, Chinese

Academy of Sciences. Roswell Park Memorial Institute-1640 (RPMI-

1640) medium was obtained from Biological Industries (Israel).

Acknowledgements

Recombinant human TGF-b1 was obtained from Peprotech (USA).

Phorbol 12-myristate 13-acetate (PMA) was obtained from Med-

ChemExpress (USA). Dimethyl sulfoxide (DMSO) and LPS were

obtained from Sigma (USA). Cell Counting Kit-8 (CCK-8) was ob-

Financial support from National Natural Science Foundation of

China (grants 21971256, 21977118 and 81773584) is gratefully

acknowledged. This project was also supported by the Jiangsu

Provincial Key Research and Development Project (BE2018710), the

tained from Beyotime (Shanghai, China). Human

b-actin gene

primer pair was obtained from Sangon (Shanghai, China).

“Double

First-Class”

University

project

(CPU2018GY01,

CPU2018GY35, CPU2018GY45) and the Program for Jiangsu Prov-

ince Innovative Research Team. The authors appreciated Jie Zhao,

who works in the pharmaceutical animal experimental center of

China Pharmaceutical University, for her kind help in the animal

feeding.

4.4.2.2. Cell culture and intervention. HGMCs were cultured in

DMEM (containing 5.5 mM of D-glucose) supplemented with 10%

FBS and 1% penicillin-streptomycin solution and were used in

passage of 6e10. THP-1 cells were cultured in RPMI-1640 medium

K. Liu, X. Zhao, X. Qi et al.

European Journal of Medicinal Chemistry 218 (2021) 113388

streptozotocin-induced diabetes, Exp. Mol. Med. 42 (2010) 628e638, https://

doi.org/10.3858/emm.2010.42.9.062.

Appendix A. Supplementary data

[21] S. Kim, N. Song, S. Chang, G. Bahn, Y. Choi, D. Rhee, U. Yun, J. Choi, J. Lee, J. Yoo,

D. Shin, K. Park, H. Kang, S. Lee, J. Ku, Y. Cho, K. Park, Sulfuretin prevents

obesity and metabolic diseases in diet induced obese mice, Biomol. Ther.

(Seoul). 27 (2019) 107e116, https://doi.org/10.4062/biomolther.2018.090.

[22] Y. Lu, Y. Xiao, Y. Li, J. Li, F. Nan, J. Li, Sulfuretin protects hepatic cells through

regulation of ROS levels and autophagic ﬂux, Acta Pharmacol. Sin. 40 (2019)

908e918, https://doi.org/10.1038/s41401-018-0193-5.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13388.

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