Research Article Design, Synthesis, and Biological Evaluation of Novel Pyrrolo [2,3-b] Pyridine Derivatives for Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is featured with liver fat infiltration and accumulation with no connection with overdrinking. With the rising trends and no FDA-approved drugs, NAFLD attracts global attention. In this study, we successfully synthesized 18 novel SIS3 derivatives and evaluated them for pharmacological activity. F-9 and F-15 had potent weight-losing effects. Importantly, intraperitoneal administration of F-9 at 10mg · kg − 1 · day − 1 for 5 weeks had the most excellent effects to reduce the weight of the body, liver, and fat, as well as adjusting serum levels of AST, ALT, TC, TG, HDL, and LDL. H&E staining showed that F-9 could suppress fat deposition and increase the adipocyte size in the liver.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases, and the global prevalence is estimated at 24% [1]. NAFLD is a clinicopathological hepatic steatosis, featured with liver fat in ltration and accumulation with no connection with overdrinking [2]. Many diseases, including obesity, insulin resistance, in ammation, and type 2 diabetes, are closely linked to NAFLD [3][4][5]. Currently, the pathogenesis of NAFLD developing from "double hit" to "multiple hit," is still multifactorial and unclear [6,7]. Researchers mostly pick metabolism, in ammation, and brosis as targets to treat this disease [8,9]. However, there are no FDA-approved drugs for NAFLD so far. A better understanding of the NAFLD pathogenesis has led to the clinical research of various potential drugs for the treatment of NAFLD. ese investigational drugs target several aspects of metabolic disruption, oxidative stress, in ammation, and/or in ammatory signaling [10]. Seemingly, because of the complex, multidirectional pathophysiology involved in NAFLD, the perfect animal model representing the complete NAFLD spectrum in a workable time frame does not exist. e investigators should have a clear perception of what they are studying and should choose the best-suited animal model relative to their research goal, taking into account NAFLD comorbidities, grade of brosis, and the possible development of hepatocellular carcinoma (HCC) [11].
SIS3 is a selective inhibitor of Smad3 and can signicantly inhibit Smad3 phosphorylation, which was rst reported in 2006 [12]. Currently, research studies on SIS3 mainly focus on anti brosis, antiapoptosis, anti-in ammation, and inhibiting cancer progression [13][14][15]. Wang et al. reported that SIS3 can not only signi cantly reduce the bodyweight, fat mass, and fasting blood glucose in highfat diet-induced type 2 diabetes model mice but also improve insulin sensitivity and oral glucose tolerance of highfat diet-induced type 2 diabetes model mice [16]. erefore, it may be e ective to use SIS3 in the treatment of NAFLD.
In this study, we designed and synthesized 18 novel SIS3 derivatives utilizing the principle of bioelectronic isobaric (Table 1) to test their biological activity in the treatment of NAFLD.

Preparation Procedure of the Target Compounds F Series.
e novel SIS3 derivatives were prepared following the synthetic route as shown in Scheme 1. e preparation of 2phenyl-1H-pyrrolo [2,3-b] pyridine (1-1) used tetrahydrofuran, benzonitrile and LDA as starting materials. In the second step, amine hydrogen of 1-1 was replaced by methyl. Compound 1-3 was obtained by Vilsmeier-Haack reaction.

In Vivo Efficacy for NAFLD.
Male 4-week-old C57BL/ 6J mice were provided by Beijing Huafukang Bioscience Co., Inc., in China. e mice were fed a normal diet or a high-fat diet (HFD) (60% calories from fat) for 90 days. en, the HFD mice were randomly divided into 20 groups (a model group (model), a vehicle group (vehicle), and 18 treatment groups to evaluate the effects of all compounds. After 15 days of continuous administration, the mice's bodyweight, fasting blood glucose, and intraperitoneal glucose tolerance (IGTT) were measured. According to the results of pharmacological evaluation above, F-9 and F-15 were selected for further in vivo experiments.
We randomly divided 30 NAFLD model mice into 6 groups (model group, vehicle group, F-9 low-dose group: 10 mg/kg, F-9 high-dose group: 50 mg/kg, F-15 low-dose group: 10 mg/kg and F-15 high-dose group: 50 mg/kg). e mice's bodyweight, fasting blood glucose, intraperitoneal injection glucose tolerance (IGTT), and insulin tolerance (ITT) were measured after 35 days of continuous administration. e serum, liver, and epididymal fat were taken. All liver specimens were cryopreserved in liquid nitrogen to −80°C until analysis.

Intraperitoneal Glucose Tolerance Test (IGTT).
e mice were weighed after 35 days of treatment after fasting for 15 h, the mice were intraperitoneally injected with 20% glucose solution (0.01 mL/g). Also, then, the blood glucose of each mouse was measured and recorded at 0, 30, 60, and 120 min, respectively.

Insulin Tolerance Test (ITT).
e mice were fasted for 4 h after IGTT, and then the insulin (0.05 U/mL) was injected intraperitoneally (injection volume was calculated according to the weight of the mice, 0.01 mL/g), and blood glucose was measured and recorded at 0, 30, 60, and 120 min, respectively.

Hematoxylin-Eosin (H&E) Staining.
e liver samples were fixed in formalin and embedded in wax. 3 μm sections were obtained from each paraffin block using a microtome and stained with H&E. e pathological state of liver tissue was observed under a light microscope.

Statistical Analysis.
e data were expressed as mean ± SD of three independent experiments. e twotailed Student's t-test was used for statistical analysis and statistically significant P values were labeled as follows: * P < 0.05; * * P < 0.01; * * * P < 0.001.

General Experimental.
Nonaqueous reactions were performed in oven-dried round bottom flasks under an inert nitrogen atmosphere. Commercial reagents were purchased from Sigma-Aldrich, Alfa Aesar, or TCI Chemicals. Reaction progress was monitored by thin-layer chromatography (TLC) using UV light (254 nm) as a visualizing agent. Flash chromatography was performed on silica gel (200-300 mesh) with EtOAc/PE or DCM/MeOH as eluent. 1 H NMR spectra were recorded on a Bruker AMX400 (400 MHz) NMR spectrometer. Dimethyl sulfoxide (DMSO) was purchased from Tianjin Kemio Chemical Reagent Co. Ltd., in China. PEG400 were obtained from Sigma-Aldrich, (Merck KGaA, Darmstadt, Germany). Glucose was purchased from Jinshan Chemical Reagent Co. (Chengdu, China). Novolin R (Biosynthetic Human insulin) was prepared by Novo Nordisk A/S, Copenhagen. e remaining chemicals and reagents were supplied from commercial sources.
After stirring for 0.5 h, benzonitrile (5 g) was added dropwise into the above solution. e reaction mixture was stirred at 0°C for 1.5 h and was then allowed to reach 40°C while stirring for another 4 h. e reaction was quenched with water and extracted with EtOAc. e combined organic layers were dried over Na 2 SO 4 and concentrated to give a crude product. e crude was purified by column Table 1: SAR for novel SIS3 derivatives.

Preparation of Compound 1-2.
To the solution of compound 1-1 (3.5 g) in DMF (100 mL) was added NaH (1 g) under nitrogen. After stirring at 0°C for 10 min, CH 3 I was slowly added dropwise and stirred at room temperature overnight. e reaction was quenched with water and extracted with EtOAc. e combined organic layers were dried over Na 2 SO 4 . e crude product was puri ed by column chromatography to give a pale-yellow product (2.8 g, 75%).

Preparation of Compound 1-3.
To the solution of compound 1-2 (2 g) in DMF (100 mL), POCl 3 (2.4 mL) was slowly added dropwise under nitrogen and stirred at 0°C for 3 h. After the reaction was completed, the pH was adjusted to higher than 14 with a 1N NaOH solution. e reaction was washed with water and extracted with EtOAc. e combined organic layers were dried over Na 2 SO 4 . e solvent was removed via rotary evaporation, and the crude product was puri ed by column chromatography to obtain the compound 1-3 (2.8 g, 88.3%). 1

Preparation of Compound 1-4.
To the solution of compound 1-3 (2.8 g) in pyridine (30 mL) were added hexahydropyridine (7 mL) and malonic acid (1.5 g). en, the mixture was stirred under re ux for 4 h. After the reaction was completed, the pH was adjusted to 6 with 1N HCl solution. e reaction was washed with water and extracted with EtOAc. e combined organic layers were dried over Na 2 SO 4 and removed via rotary evaporation, and the crude product was puri ed by column chromatography to obtain

General Procedure for Compounds F Series.
To the solution of compound 1-4 (1eq) in DMF were added EDCI (3eq), HOBT (1eq), and triethylamine (3eq). e mixture was stirred for 0.5 h at room temperature, and then the corresponding amine (0.6eq) was added. e reaction was washed with water and extracted with EtOAc. e combined organic layers were dried over Na 2 SO 4 and removed via rotary evaporation. e crude product was puri ed by column chromatography to obtain corresponding products.      F-15 exhibited higher potency of regulating bodyweight and blood glucose than others (Figures 1(a) and 1(b)) ( * p < 0.05, * * p < 0.01). e results of the intraperitoneal glucose tolerance test (IGTT) indicated that F-9 and F-15 could significantly antagonize the increase and fluctuation of blood glucose in mice caused by exogenous glucose (Figures 1(c)-1(f )). erefore, the two compounds were selected for further in vivo pharmacological evaluation.
Because of the close relationship between blood glucose and fat metabolism, we further evaluated the serum level of blood glucose in HFD-induced NAFLD rats. As shown in Figure 3, the serum level of blood glucose increased during the 5 weeks high-fat feeding. After the treatment of F-9 and F-15, the concentration of fasting blood glucose was reduced to the normal level. Among them, F-9 (50) had the lowest blood glucose value, which means that it has the best hypoglycemic effect (Figure 3(a)). To further investigate the integrity of β cell function in pancreatic islets and the ability of the body to regulate blood glucose in high-fat diet-induced obese mice. After 35 days of treatment, IGTT and ITT were tested in mice. Compared with the model group, the treatment group significantly reduced blood glucose. Especially F-9 (50) is even lower than the normal group of mice (Figure 3(b)). In the ITT test, the mice of the treatment groups increased their sensitivity to exogenous insulin. At 120 min, the blood glucose in the F-9 (50) group was the lowest (Figure 3(c)).
We also measured another six serum levels of triglycerides, AST, ALT, total cholesterol, HDL, and LDL, which played a vital role in the fat liver. e levels of AST and ALT in the model group and vehicle group were higher than the normal group, indicating the liver function of model rats was impaired. After 35 days of treatment, compared to the data of the model group, F-9 and F-15 could significantly reduce the serum concentrations of AST, ALT, LDC, HDL, TC, and TG ( Figure 4). Importantly, F-9 showed a more potent ability to regulate HFD-induced abnormal serum biomarkers to normal levels than F-15.
According to the result of H&E staining of the liver (Figure 5), the liver of the normal group had no obvious histological changes. However, numerous lipid droplets in liver tissues from HFD-induced NAFLD rats severely ruined liver histology, presented as ballooning degeneration and further led to the formation of steatosis. Fortunately, the liver tissue structure and liver cell size and shape of rats in all treatment groups basically returned to normal, and fatty liver symptoms were alleviated. e most remarkable reduction was observed in the F-9 treatment group.

Conclusions
In this study, 18 novel small-molecule SIS3 derivatives have been designed, synthesized, and evaluated for pharmacological activity. F-9 and F-15 were found to possess a potent weight-lowering effect on NAFLD rats. Importantly, intraperitoneal administration of F-9 and F-15 improved effectively the progression of NAFLD and reduced the weight of the body and fat as well as modulating serum parameters of LDL, HDL, ALT, AST, TC, and TG. e results of H&E staining also revealed that F-9 and F-15 reduced fat deposition in liver tissues and suppressed the increase of adipocyte size in adipose tissues from NAFLD rats. Especially, F-9 alleviated the clinical symptoms of obesity and regulated serum biomarkers to appropriate ranges. ese surprisingly pharmacological activities demonstrate that F-9 as a candidate constitutes an attractive approach to the treatment of obesity-related NAFLD and is valued for further development.

Data Availability
All the data included in this study are available upon request by contact with the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.