Synthesis and Biological Evaluation of Novel N-Methyl-picolinamide-4-thiol Derivatives as Potential Antitumor Agents

A novel series of N-methylpicolinamide-4-thiol derivatives were synthesized and evaluated on human cancer cell lines. Among them, compound 6p displayed potent and broad-spectrum anti-proliferative activities in vitro on some human cancer cell lines, even better than sorafenib. The advanced kinase inhibitory assays showed that compound 6p could selectively inhibit Aurora-B kinase. The biological results were rationalized by the molecular docking study, which indicated the stable interactions of 6p with the Aurora-B kinase.


Introduction
Aurora proteins-A, -B, and -C, a small family of serine/threonine kinases [1], play distinct roles in the regulation of mitosis [2]. Aurora-A and -B are known to be frequently overexpressed in a wide range of different human tumors, including breast, colon, lung, ovarian, and pancreatic cancers [3][4][5][6], suggesting their potential role in tumorigenesis [7].

OPEN ACCESS
In recent years, the Aurora proteins have been actively pursued as anticancer targets for the discovery of new cancer chemotherapeutics. As a result, several small-molecule inhibitors of the Aurora kinases have been identified, some of which have reached clinical evaluation, including MK-0457(VX-680) [8,9], MLN8054 [10], PHA-739358 [11] and AZD-1152 [12] (Figure 1). However, the ideal inhibitor profile for therapeutic use is still unclear, and these inhibitors with complex structures are difficult to synthesize. Our group has been interested in the design, screening, synthesis and biological evaluation of novel tumor growth inhibitors. In a previous cell-based screening of our privileged small molecule library, we found that a drug-like compound, N-methylpicolinamide-4-thiol (Figure 2), exhibited moderate in vitro cytotoxicity against human hepatocellular carcinoma cell line HepG2 (IC 50 = 62.96 µM). In order to find more potent antiproliferative compounds, we designed and synthesized a series of novel N-methylpicolinamide-4-thiol derivatives based on compound 1, employing the structure-activity relationship (SAR) study.

Chemistry
The synthetic route for the target compounds 1 and 6a-w is shown in Scheme 1. Compound 3 was synthesized according to the reported method with a small change [13]. Treatment of 2-picolinic acid (2) with SOCl 2 in the presence of NaBr and chlorobenzene afforded acid chloride 3 as the corresponding HCl salt. This HCl salt was then treated with methylamine solution (2.0 mol/L) in methanol to yield 4. To obtain 5 [14], compound 4 and potassium carbonate were treated with a solution of 4-aminothiophenol, which had been stirred at room temperature for 3.5 h in the presence of potassium tert-butoxide in dry N,N-dimethylformamide. The contents were then heated to 85 °C under argon for 15 h. Acylation of the amino group of 5 with different substituted benzoyl chlorides or alkyl acyl chlorides yielded the target compounds 1 and 6a-w [15]. The structures of compounds 1 and 6a-w were fully characterized by 1 H-NMR, 13 C-NMR and ESI-MS analysis. Scheme 1. Synthetic route to compounds 1 and 6a-w. Reagents

Biological Evaluation
As shown in Table 1, twenty-three N-methylpicolinamide-4-thiol derivatives were synthesized to survey the SAR by evaluating the cell growth inhibitory activity in human liver hepatocellular carcinoma (HepG2) cells. Sorafenib (Figure 3), which was found to significantly prolong the survival of advanced hepatocellular carcinoma (HCC) patients, was selected as the positive control.
Just as seen in the phenyl series 6a-o, unsubstituted phenyl analog 6a showed good inhibitory activity, IC 50 = 16.54 μM. As for the methoxy group, the activity of the analogue with methoxyl at meta-position (6b, IC 50 = 15.43 μM) is better than that of the para-(6c) and ortho-(6d, IC 50 = 23.91 μM) substituted analogues. Compound 6e (IC 50 = 7.12 μM), with two methoxy groups substituted at the meta-position, was one of the most potent inhibitors in this series. In terms of halogen atoms (compounds 6f-j), the location had little effect on the activities. However, when the number of the halogen atoms was two, the activity of 6h (IC 50 = 10.55 μM) and 6i improved greatly. Introduction of the electron-withdrawing groups CF 3 [6k (IC 50 = 17.04 μM), 6l (IC 50 = 10.96 μM), 6m] and NO 2 [6n (IC 50 = 19.12 μM), 6o] on the phenyl ring at the meta-and para-position was tolerated.
Replacement of the benzyl group with an aliphatic group was also tolerated. With the IC 50 value of 2.23 μM, compound 6p, which exhibited a nearly 15-fold improvement in inhibitory activity against HepG2 cells over that of sorafenib (IC 50 = 16.30 μM), was the most potent analogue in this series.
The number of chloride atom and the length of the carbon chain had a great effect on the activities. The activity was lost when the carbon chain was extended to chloropropyl or butyl chloride structures (compounds 6s,t). The introduction of two or three chloride atoms (compounds 6q,r) also had a detrimental effect on the potency. In addition, unsubstituted aliphatic derivatives 6u-w were also tested, but their activities were not notable.

6i
-phenyl-2,6-Di-F 23.25 -phenyl-p-NO 2 33.67   To further study the cytotoxic profile, the most potent compound 6p was selected for further evaluation of its inhibitory activity against a panel of human cancer cell lines. Interestingly, compound 6p showed broad-spectrum antiproliferative activities in vitro (Table 2). It had significant cytotoxicity against colon cancer cell lines HCT-116 and SW480, lung cancer cell line SPC-A1 and melanotic cancer cell line A375 with IC 50 values <10 μM. Compound 6p was evaluated on six kinases at a concentration of 10 μM (Table 3). It was found that 6p could selectively inhibit Aurora-B kinase to a significant level (87% inhibition at 10 μM). This result provided a possible reason for its broad-spectrum antiproliferative activities.

Molecular Docking Study
In order to further investigate the interactions between compound 6p and the Aurora-B kinase, a docking study was performed using the Genetic Optimization for Ligand Docking (GOLD) 4.0 program. The crystal structure of Aurora-B (PDB Code: 4AF3) was used as the reference receptor.
According to the docking result, we can discern that two hydrogen bonds were formed between compound 6p and the Aurora-B kinase. As shown in Figure 4, the chlorine of the inhibitor formed one hydrogen bond with the residue Phe219 (2.452 Å, 32.69°), and another hydrogen bond was found between the residue Lys106 and the carbonyl group near the chlorine of the inhibitor (2.178 Å, 37.57°). In addition, there was a π-π conjugation interaction between the benzene ring group of compound 6p and the Phe88 residue in the binding mode. The stable interactions between the inhibitor and Aurora-B kinase rationalize the obtained biological results.

Preparation of 4-(4-Aminophenylthio)-N-methylcarboxamide (5)
Potassium tert-butoxide (4.04 g, 36.00 mmol) was added to a stirred solution of 4-aminothiophenol (3.76 g, 30.00 mmol) in dry N,N-dimethylformamide (58.68 mL), and the reddish-brown mixture was stirred at room temperature for 3.5 h. To the mixture was added 4 (5.89 g, 34.50 mmol) and potassium carbonate (25.44 g, 180.00 mmol), and then stirred at 85 °C under nitrogen for 15 h. The mixture was cooled to room temperature and poured into the mixture of ethyl acetate (200 mL) and brine (200 mL). The aqueous layer was extracted with ethyl acetate (150 mL). The combined organic layers were washed with brine (3 × 400 mL), dried over sodium sulfate, and concentrated to afford 5 as an orange solid. Yield

General Procedure for Preparing Compounds 1 and 6a-w
Compound 5 (0.52 g, 2.00 mmol) and anhydrous potassium carbonate (0.69 g, 5.00 mmol) were suspended in THF (7.00 mL), and then different substituted benzoyl chlorides or alkyl acyl chlorides (2.10 mmol) was added dropwise at 0-5 °C. The mixture was stirred at room temperature for 2 h and poured into the mixture of ethyl acetate (50 mL) and brine (50 mL). The aqueous layer was back extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine (3 × 50 mL), dried over sodium sulfate, and concentrated to afford compounds 6a-w.

Cell Proliferation Assay (MTT Assay)
Cells (3 × 10 3 /well) were seeded in 96-well plates and cultured for 24 h, followed by treatment with the compounds for 48 h. Ten microliters of 10 mg/mL MTT was added per well and incubated for another 2.5 h at 37 °C. Then the supernatant fluid was removed and 150 μL/well DMSO was added for 15-20 min. The absorbance (OD) of each well was measured at 570 nm using an ELISA reader (Thermo). The effect of compounds on tumor cells viability was expressed by IC 50 of each cell line.

Kinase Inhibitory Assay
In vitro kinase inhibitory assays were performed against recombinant human Aurora-B kinase at the Km of ATP (15 μM) and at a fixed concentration of 10 μM of test compound. Each assay was repeated twice. All the inhibitory assays against Aurora-B were carried out through kinase profiling services provided by Millipore (America), in which radiometric protein kinase assays were used.

Conclusions
In conclusion, a series of novel N-methylpicolinamide-4-thiol derivatives has been synthesized and evaluated on human cancer cell lines. Among them, compound 6p was found to be the most potent, displaying broad-spectrum in vitro antiproliferative activities. The results of the MTT assay showed that compound 6p had significant cytotoxicity against liver cancer cell line HepG2, colon cancer cell lines HCT-116 and SW480, lung cancer cell line SPC-A1 and melanotic cancer cell line A375 with IC 50 values <10 μM. All these antiproliferative activities were better than those of the reference compound sorafenib. The advanced kinase inhibitory assays, which were performed on six kinases at a concentration of 10 μM, indicated that 6p could selectively inhibit Aurora-B kinase. A molecular docking study showed the stable interactions of 6p with the Aurora-B kinase, which rationalized the obtained biological results. Our ongoing work aimed at researching the advanced mechanism of action and explore the efficacy of compound 6p in a range of in vivo models, will be the subjects of future reports.