Synthesis of Novel 2-(Pyridin-2-yl) Pyrimidine Derivatives and Study of Their Anti-Fibrosis Activity

A pyrimidine moiety exhibiting a wide range of pharmacological activities has been employed in the design of privileged structures in medicinal chemistry. To prepare libraries of novel heterocyclic compounds with potential biological activities, a series of novel 2-(pyridin-2-yl) pyrimidine derivatives were designed, synthesized and their biological activities were evaluated against immortalized rat hepatic stellate cells (HSC-T6). Fourteen compounds were found to present better anti-fibrotic activities than Pirfenidone and Bipy55′DC. Among them, compounds ethyl 6-(5-(p-tolylcarbamoyl)pyrimidin-2-yl)nicotinate (12m) and ethyl 6-(5-((3,4-difluorophenyl)carbamoyl)pyrimidin-2-yl)nicotinate (12q) show the best activities with IC50 values of 45.69 μM and 45.81 μM, respectively. Furthermore, the study of anti-fibrosis activity was evaluated by Picro-Sirius red staining, hydroxyproline assay and ELISA detection of Collagen type I alpha 1 (COL1A1) protein expression. Our study showed that compounds 12m and 12q effectively inhibited the expression of collagen, and the content of hydroxyproline in cell culture medium in vitro, indicating that compounds 12m and 12q might be developed the novel anti-fibrotic drugs.


Introduction
Construction of novel heterocyclic compound libraries with potential biological activities is an important component of medicinal chemistry and chemical biology [1]. The pyrimidine moiety has been considered as a privileged structure in medicinal chemistry [2] and the compounds containing pyrimidine as the core are reported to exhibit diverse types of biological and pharmaceutical activities. For example, pyrimidine derivatives are known as antimicrobial [3][4][5], antiviral [6,7], antitumor [8][9][10] and antifibrotic compounds [11].
The discovery of anti-fibrotic drugs has attracted great attention from organic and medicinal chemists. As shown in Figure 1a, vascular endothelial growth factor receptor 2 (VEGFR-2) and Platelet derived growth factor-β (PDGF-β) inhibitor sorafinib reduced collagen deposition by nearly 63% in a liver fibrosis model [12]; N 2 ,N 4 -bis(2-methoxyethyl)pyridine-2,4-dicarboxamide (HOE-077), which is a prodrug of pyridine-2,4-dicarboxylic acid (24PDC), can inhibit collagen synthesis in rat and dog models by inactivating hepatic stellate cells that are mainly responsible for collagen synthesis in liver fibrosis [13,14]; Ethyl 3,4-dihydroxybenzoate and S4682 were effective in inhibiting collagen synthesis in keloid fibroblasts or the CCl 4 model of chronic hepatic injury, by inhibition of collagen prolyl-4-hydroxylase (CP4H) [14,15]; 2-(benzo[d][1,3]dioxol-5-yl)thiazole (CW209292) displays anti-fibrotic activity in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis by [16]; nicotinic acid prevented fibrosis by its antioxidant properties and reducing the expression of TGF-β in thioacetamide (TAA)-induced hepatic fibrogenesis [17]. All of those small molecules contain the similarly structure fragment. CP4Hs have been considered an important target for treating fibrotic disease and a few small molecular inhibitors have been reported in the literature [18,19]. However, most of these compounds usually have poor activities in cultured cells [20]. It is our ongoing interest to discover novel compounds with potent anti-fibrotic activities [21][22][23]. In this report, the novel compounds containing pyrimidine scaffold as the core were designed by combining pyrimidine ring with the similarly structure fragment through a molecular hybridization strategy, as shown in Figure 1b. Herein, we report the preparation of pyrimidine-pyridine compounds, CP4H activity in cultured cells and their anti-fibrotic activities against immortalized rat hepatic stellate cells (HSC-T6).

Chemistry
In this work, the key intermediate 5 was obtained by a convenient four-step procedure in moderate yields and the synthetic route was depicted in Scheme 1: First, esterification of nicotinic acid was undertaken to yield 2; then, compound 2 was oxidated with 3-chloroperoxybenzoic acid (mCPBA) to afford pyridine N-Oxides 3; next, a nucleophilic substitution of the ortho-position of pyridine N-Oxides 3 with trimethylsilyl cyanide (TMSCN) was conducted to generate 4 [24]; finally, compound 4 was reacted with Na and NH4Cl in EtOH solution to get compound 5 [25]. CP4Hs have been considered an important target for treating fibrotic disease and a few small molecular inhibitors have been reported in the literature [18,19]. However, most of these compounds usually have poor activities in cultured cells [20]. It is our ongoing interest to discover novel compounds with potent anti-fibrotic activities [21][22][23]. In this report, the novel compounds containing pyrimidine scaffold as the core were designed by combining pyrimidine ring with the similarly structure fragment through a molecular hybridization strategy, as shown in Figure 1b. Herein, we report the preparation of pyrimidine-pyridine compounds, CP4H activity in cultured cells and their anti-fibrotic activities against immortalized rat hepatic stellate cells (HSC-T6 The target compounds (12a-12t and 13a-13t) were prepared according to Scheme 1, starting from the commercially available 4-pyrazolecarboxylic acid. Briefly, condensation of 4pyrazolecarboxylic acid 6 with benzyl alcohol in the presence of 1-ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBT) afforded compound 7 [26]. Intermediate 8 was prepared by using amino 4-nitrobenzoate in 1-methyl-2-pyrrolidinone (NMP). This step was followed by oxidation of 8 with NaIO4 at a low temperature [27]. Subsequently, a Diels-Alder reaction between the key intermediate 5 and Benzyl 1,2,3-triazine-5-carboxylate (9) led to the formation of the correspondent compound 10 [28], which in turn was converted to the carboxylic acid intermediate 11 by reaction with hydrogen at room temperature. Finally, EDCI and HOBT mediated an amide coupling reaction between the activated intermediate carboxylic acid 11 and various substituted amines formed the desired compounds 12a-12t in moderate to good yields and compounds 12a-12t were also converted into the desired product compounds 13a-13t by hydrolysis reactions.

MTT Assay on HSC-T6 Cell Proliferation
To evaluate the anti-proliferation activities of target compounds, HSC-T6 cells (Rat Hepatic Stellate Cell) were employed since they have been recognized as an effective and convenient method for anti-proliferation screening model in vitro [29,30]. In our experiment, HSC-T6 cells were seeded to the 96-well plate and were cultured with serum-free medium. This process resulted in HSC-T6 cells transforming into a static period and synchronizing, then the culture medium was refreshed with 2% fetal bovine serum to stimulate the activation of HSC-T6 cells. The inhibitory activities of all synthesized compounds were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with Bipy55'DC, 24PDC and clinical drug Pirfenidone (PFD) as control (Bipy55′DC and 24PDC are typical CP4H inhibitors; Pirfenidone (PFD) can treat idiopathic pulmonary fibrosis with good tolerance and little side effects [31]). It showed that the cell growth increased in speed after cells were cultured with 2% fetal bovine serum in the control group. Moreover, the floating cell is rare in the 100 μM group when co-culturing with the 12m and 12q compounds for about 48 h, indicating that the death cell is rare, but we can observe that the hepatic stellate cells become slim. The half-maximal inhibitory concentration (IC50) values of compounds are shown in Table 1. The target compounds (12a-12t and 13a-13t) were prepared according to Scheme 1, starting from the commercially available 4-pyrazolecarboxylic acid. Briefly, condensation of 4-pyrazolecarboxylic acid 6 with benzyl alcohol in the presence of 1-ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBT) afforded compound 7 [26]. Intermediate 8 was prepared by using amino 4-nitrobenzoate in 1-methyl-2-pyrrolidinone (NMP). This step was followed by oxidation of 8 with NaIO 4 at a low temperature [27]. Subsequently, a Diels-Alder reaction between the key intermediate 5 and Benzyl 1,2,3-triazine-5-carboxylate (9) led to the formation of the correspondent compound 10 [28], which in turn was converted to the carboxylic acid intermediate 11 by reaction with hydrogen at room temperature. Finally, EDCI and HOBT mediated an amide coupling reaction between the activated intermediate carboxylic acid 11 and various substituted amines formed the desired compounds 12a-12t in moderate to good yields and compounds 12a-12t were also converted into the desired product compounds 13a-13t by hydrolysis reactions.

MTT Assay on HSC-T6 Cell Proliferation
To evaluate the anti-proliferation activities of target compounds, HSC-T6 cells (Rat Hepatic Stellate Cell) were employed since they have been recognized as an effective and convenient method for anti-proliferation screening model in vitro [29,30]. In our experiment, HSC-T6 cells were seeded to the 96-well plate and were cultured with serum-free medium. This process resulted in HSC-T6 cells transforming into a static period and synchronizing, then the culture medium was refreshed with 2% fetal bovine serum to stimulate the activation of HSC-T6 cells. The inhibitory activities of all synthesized compounds were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with Bipy55 DC, 24PDC and clinical drug Pirfenidone (PFD) as control (Bipy55 DC and 24PDC are typical CP4H inhibitors; Pirfenidone (PFD) can treat idiopathic pulmonary fibrosis with good tolerance and little side effects [31]). It showed that the cell growth increased in speed after cells were cultured with 2% fetal bovine serum in the control group. Moreover, the floating cell is rare in the 100 µM group when co-culturing with the 12m and 12q compounds for about 48 h, indicating that the death cell is rare, but we can observe that the hepatic stellate cells become slim. The half-maximal inhibitory concentration (IC 50 ) values of compounds are shown in Table 1.
a Inhibitory activity was assayed by exposure to substances for 48 h and expressed as concentration required to inhibit HSC-T6 by 50% (IC50). b Positive control.
From the screening results in Table 1, some of the target compounds displayed better antifibrosis activity than Pirfenidone (PFD), Bipy55′DC and 24PDC on HSC-T6 cells. Regarding the activity of the tested compounds, compounds (12k, 12l, 12m, 12n, 12o, 12p and 12q) with ethyl groups on the substituent group R exhibited better activity effectiveness than compounds (13k, 13l, 13m, 13n, 13o, 13p and 13q) with hydrogen atoms on the substituent group R. In addition, compounds (12k, 12l, 12m, 12n, 12o, 12q and 12r) with phenyl rings on the substituent group R1 presented more highly inhibitory activities than those (12b, 12c, 12d, 12f, 12e, 12i and 12j) with benzyl groups on the a Inhibitory activity was assayed by exposure to substances for 48 h and expressed as concentration required to inhibit HSC-T6 by 50% (IC 50 ). b Positive control.
From the screening results in Table 1, some of the target compounds displayed better anti-fibrosis activity than Pirfenidone (PFD), Bipy55 DC and 24PDC on HSC-T6 cells. Regarding the activity of the tested compounds, compounds (12k, 12l, 12m, 12n, 12o, 12p and 12q) with ethyl groups on the substituent group R exhibited better activity effectiveness than compounds (13k, 13l, 13m, 13n, 13o, 13p and 13q) with hydrogen atoms on the substituent group R. In addition, compounds (12k, 12l, 12m, 12n, 12o, 12q and 12r) with phenyl rings on the substituent group R 1 presented more highly inhibitory activities than those (12b, 12c, 12d, 12f, 12e, 12i and 12j) with benzyl groups on the substituent group R 1 . Similarly, compounds (12l, 12m) with the 4-substituent group R 1 showed better activity compared to compounds (12n, 12p) with the 3-substituent group R 1 . According to a series of chemical structural modifications, we got two highly active compounds: 12m and 12q. To summarize what has been mentioned above, the results encouraged us to further investigate the anti-fibrotic activity of the two compounds 12m and 12q.

12m and 12q Screened for Prolyl-4-hydroxylase Content In Vitro
Initial screening for prolyl-4-hydroxylase inhibitory activity was performed by determining the hydroxyproline content in HSC-T6 cells [20] and collagen was also determined by estimating the content of hydroxyproline [32]. As shown in Figure 2, the content of hydroxyproline was significantly reduced in the presence of 12m and 12q at a concentration of 50 µM or 100 µM. The result of the hydroxyproline assay displayed that 12m and 12q are potent inhibitors of collagen prolyl-4-hydroxylase. The result also suggested that 12m and 12q do have a potential effect on suppressing the production of collagen in vitro.
hydroxyproline content in HSC-T6 cells [20] and collagen was also determined by estimating the content of hydroxyproline [32]. As shown in Figure 2, the content of hydroxyproline was significantly reduced in the presence of 12m and 12q at a concentration of 50 μM or 100 μM. The result of the hydroxyproline assay displayed that 12m and 12q are potent inhibitors of collagen prolyl-4hydroxylase. The result also suggested that 12m and 12q do have a potential effect on suppressing the production of collagen in vitro.

12m and 12q Suppressed the Total Collagen Accumulation In Vitro
Extracellular collagen deposition was established by Picro-Sirius red (PSR) staining to test inhibitory activity of 12m and 12q against total collagen accumulation [33,34]. As depicted in Figure  3, treatment with 12m and 12q reduced the cell density and inhibited the total collagen accumulation in a dose-dependent manner. In addition, it is worth noting that the total collagen accumulation was significantly decreased in the presence of 12m and 12q at a concentration of 100 μM. Extracellular collagen deposition was established by Picro-Sirius red (PSR) staining to test inhibitory activity of 12m and 12q against total collagen accumulation [33,34]. As depicted in Figure 3, treatment with 12m and 12q reduced the cell density and inhibited the total collagen accumulation in a dose-dependent manner. In addition, it is worth noting that the total collagen accumulation was significantly decreased in the presence of 12m and 12q at a concentration of 100 µM.  Collagen type I alpha 1 (COL1A1), which has been generally recognized as a fibrotic marker, is overexpressed in the common fibrotic diseases. To further study the anti-fibrotic activity of compounds 12m and 12q, their abilities of inhibiting protein expression of COL1A1 in vitro were investigated through the ELISA method. As displayed in Figure 4, it was observed that 12m could reduce the expression of COL1A1 in HSC-T6 cells, which means that the extracellular matrix molecules' deposition was also reduced. Collagen type I alpha 1 (COL1A1), which has been generally recognized as a fibrotic marker, is overexpressed in the common fibrotic diseases. To further study the anti-fibrotic activity of compounds 12m and 12q, their abilities of inhibiting protein expression of COL1A1 in vitro were investigated through the ELISA method. As displayed in Figure 4, it was observed that 12m could reduce the expression of COL1A1 in HSC-T6 cells, which means that the extracellular matrix molecules' deposition was also reduced.
Collagen type I alpha 1 (COL1A1), which has been generally recognized as a fibrotic marker, is overexpressed in the common fibrotic diseases. To further study the anti-fibrotic activity of compounds 12m and 12q, their abilities of inhibiting protein expression of COL1A1 in vitro were investigated through the ELISA method. As displayed in Figure 4, it was observed that 12m could reduce the expression of COL1A1 in HSC-T6 cells, which means that the extracellular matrix molecules' deposition was also reduced.

General Information
All commercial reagents were used as received without additional purification. The melting point was uncorrected. Mass spectra were obtained using the electro spray ionization (ESI) method. The 1 H and 13 C Nuclear Magnetic Resonance (NMR) data were obtained on a 300 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) with tetramethylsilane (TMS) as the internal standard, using Chloroform-d (CDCl3), DMSO-d6 as solvents. All the NMR spectra can be found in

General Information
All commercial reagents were used as received without additional purification. The melting point was uncorrected. Mass spectra were obtained using the electro spray ionization (ESI) method. The 1 H and 13 C Nuclear Magnetic Resonance (NMR) data were obtained on a 300 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) with tetramethylsilane (TMS) as the internal standard, using Chloroform-d (CDCl 3 ), DMSO-d 6 as solvents. All the NMR spectra can be found in Supplementary  Materials (Figures S1-S101). Multiplicities are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doubled doublet; coupling constants is in Hertz (Hz); chemicalshift (δ) is in parts per million (ppm).

Synthesis of Compounds
Ethyl nicotinate (2): ethanol (50 mL) and nicotinic acid 1 (2.0 g, 16.24 mmol) was added in a 100 mL round bottomed flask. To this, a catalytic amount of concentrated H 2 SO 4 was added at room temperature and was heated under reflux with continuous stirring for 8 h at 85 • C. After 8 h, the reaction was monitored by TLC using an ethyl acetate and hexane (1:4) system. Stirring was continued until TLC indicated the completion of reaction. Then, the reaction mixture was allowed to reach room temperature. Ethanol was distilled off under reduced pressure and the residue was dissolved in water and then extracted twice with ethyl acetate. The combined organic solutions were washed with saturated NaHCO 3 solution, and finally washed with water and dried over anhydrous  (3): A solution of ethyl nicotinate 2 (2.0 g, 14.6 mmol) in DCM (150 mL) was added m-CPBA (6.5 g, 29.2 mmol) at 0 • C. After stirring at 0 • C for 1 h and then at room temperature overnight, the mixture was poured into ice water. Then, 2N NaOH was added to adjust the pH to 8-9 and the resultant mixture was extracted with DCM (3 × 200 mL). The organic layer was dried over Na 2 SO 4 , filtered and concentrated. The crude product was purified by flash column chromatography on silica gel (2% v/v MeOH in DCM) to afford the title compound  Benzyl 1,2,3-triazine-5-carboxylate (9): Compound 8 (400 mg, 1.84 mmol) was taken up in CH 2 Cl 2 (30 mL) and cooled to 0 • C. NaIO 4 (787 mg, 3.68 mmol) in H 2 O (15 mL), which was cooled to 0 • C and added to the stirring solution of 8 at 0 • C. The reaction mixture was warmed to room temperature and stirred for 2 h. After 2 h, the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (20 mL × 3). The combined organic layers were washed with saturated aqueous NaCl (30 mL), dried (Na 2 SO 4 ) and concentrated on a rotary evaporator. The crude product was purified by flash chromatography on silica gel (20% v/v ethyl acetate in Petroleum ether (60-90 • C) to afford the title compound (272 mg, 81%) as a white solid: melting point: 47-48 • C; 1 H NMR (300 MHz, CDCl 3 ) δ 9.48 (s, 2H), 7.48-7.39 (m, 5H), 5.47 (s, 2H). 13  Benzyl 2-(5-(ethoxycarbonyl)pyridin-2-yl)pyrimidine-5-carboxylate (10): benzyl 1,2,3-triazine-5-carboxylate (80 mg, 0.5 mmol) was added to a stirring solution of ethyl 6-carbamimidoylnicotinate hydrochloride (100 mg, 0.5 mmol) and K 2 CO 3 (139 mg, 1 mmol) in CH 3 CN (20 mL) followed by stirring at room temperature for 8 h. The reaction mixture was extracted three times with EtOAc (20 mL). The combined organic layers were washed with brine, dried (Na 2 SO 4 ), filtered and concentrated in vacuo. The crude product was purified by flash column chromatography on silica gel (2% v/v MeOH in DCM) to afford the title compound (127 mg, 70%) as a white solid: 1 H NMR (300 MHz, CDCl 3 165.12, 164.72,  163.28, 158.84, 156.68, 151.18, 138.11, 134.93, 128.67, 128.65, 128.39, 127.63, 123.90, 123.15, 67.54, 61.60 13 13 13  starved in a serum-free medium for about 12 h to induce HSC-T6 transformation into a static period as well as synchronization, and then compounds to be tested were added to corresponding wells with different concentrations in culture medium with 2% fetal bovine serum. After 48 h of incubation, 20 µL of 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/mL) was added to each well and the plates were incubated for an additional 4 h. Subsequently, the medium was aspirated carefully, and 150 µL of DMSO was added to dissolve the crystal. The optical density was measured at 490 nm using a RT-2100C Microplate Reader (Rayto, Shenzhen, China). The results were used to calculate IC 50 values. Each experiment was repeated three times.

Hydroxyproline Assay
Cells were plated on 96-wells with a density of 7000 cells/well. After attachment (24 h), each compound, at different concentrations, was added to cells. After 48 h of incubation, cell culture supernatants were taken out for the next step. The hydroxyproline contents in cell culture supernatants were measured with a conventional hydroxyproline assay kit (A030-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Subsequently, the hydroxyproline analysis was performed using Chloramine-T spectrophotometric absorbance.

Picro-Sirius Red (PSR) Staining
The HSC-T6 cells cultured in 6-well plates were carefully washed twice with phosphate buffered saline (PBS), xylene, ethanol and incubated in the PSR staining solution (SIRIUS RED.0.1% in saturated picric acid 26357-02, Electron Microscopy Organizer Sciences, Lot no.:190628-01) at room temperature for 1 h. The staining solution was then removed and the cells were washed three times with ethanol. Then, the HSC-T6 cells were incubated in the hematoxylin staining solution at room temperature for 10 min. The hematoxylin staining solution was removed and the cells were washed three times with ethanol and kept in xylene for 5 min. The stained cells were dried and a picture was taken with a microscope.

ELISA Detection for COL1A1
HSC-T6 cells were plated on 96-wells with a density of 7000 cells/well. After attachment (24 h), each compound, at different concentrations, was added to the remaining cell lines mentioned above. After 48 h of incubation, cell culture supernates were centrifuged for 20 min at 1000× g. A quantity of 50 µL of standard or cell culture supernates was added to the appropriate wells and 100 µL of Enzymeconjugate was added to standard wells and sample wells, except for the blank well. After 1 h of incubation at 37 • C, the Microtiter Plate was washed 4 times. Then, Substrate A 50 µL and Substrate B 50 µL were added to each well (Rat ColIELISA KIT, RUIXIN Biology, Guangzhou, China, lot:07/2020). After 15 min of incubation at 37 • C, 50 µL Stop Solution was added to each well and the optical density was measured at 490 nm using RT-2100C Microplate Reader.

Conclusions
In conclusion, we have successfully prepared a representative library of 2-(pyridin-2-yl)pyrimidine derivatives by starting from the readily available nicotinic acid and 1H-pyrazole-4-carboxylic acid. In addition, those compounds were evaluated for their anti-fibrotic activities in vitro. Among them, fourteen compounds exhibited inhibitory activities stronger than Pirfenidone, 24PDC and Bipy55 DC, with 12m and 12q displaying activities with IC 50 values of 45.69 µM and 45.81 µM against HSC-T6, respectively. Furthermore, the results of hydroxyproline assay displayed that 12m and 12q might be inhibitors of collagen prolyl-4-hydroxylase. In addition, hydroxyproline assay and Picro-Sirius red (PSR) staining of compounds 12m and 12q exhibited potent anti-fibrosis abilities in alleviating the total collagen accumulation in HSC-T6 cells in a dose-dependent manner. Further, ELISA results for compounds 12m and 12q showed better activity in alleviating COL1A1 in HSC-T6 cells. The in-depth mechanisms of action of the compounds 12m and 12q remain to be further investigated.