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Article

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

by
Ting-Ting Huang
,
Yun-Chuang Huang
,
Xiao-Yu Qing
,
Yong Xia
,
Xun Luo
,
Ting-Hong Ye
and
Luo-Ting Yu
*
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
*
Author to whom correspondence should be addressed.
Molecules 2012, 17(6), 6317-6330; https://doi.org/10.3390/molecules17066317
Submission received: 16 March 2012 / Revised: 3 May 2012 / Accepted: 17 May 2012 / Published: 25 May 2012
(This article belongs to the Section Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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].
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.
Molecules 17 06317 g001 550
Figure 1. Inhibitors of Aurora kinases.
Figure 1. Inhibitors of Aurora kinases.
Molecules 17 06317 g001
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 (IC50 = 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.
Molecules 17 06317 g002 550
Figure 2. Structure of compound 1.
Figure 2. Structure of compound 1.
Molecules 17 06317 g002

2. Results and Discussion

2.1. 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 SOCl2 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 1H-NMR, 13C-NMR and ESI-MS analysis.
Molecules 17 06317 g005 550
Scheme 1. Synthetic route to compounds 1 and 6a–w.
Scheme 1. Synthetic route to compounds 1 and 6a–w.
Molecules 17 06317 g005

2.2. 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, IC50 = 16.54 μM. As for the methoxy group, the activity of the analogue with methoxyl atmeta-position (6b, IC50 = 15.43 μM) is better than that of the para-(6c) and ortho-(6d, IC50 = 23.91 μM) substituted analogues. Compound 6e (IC50 = 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 (IC50 = 10.55 μM) and 6i improved greatly. Introduction of the electron-withdrawing groups CF3 [6k (IC50 = 17.04 μM), 6l (IC50 = 10.96 μM), 6m] and NO2 [6n (IC50 = 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 IC50 value of 2.23 μM, compound 6p, which exhibited a nearly 15-fold improvement in inhibitory activity against HepG2 cells over that of sorafenib (IC50 = 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.
Table
Table 1. Inhibition of cell proliferation by compounds 6a–w. Molecules 17 06317 i001
Table 1. Inhibition of cell proliferation by compounds 6a–w. Molecules 17 06317 i001
CompoundRIC50(µM) aStructure of the compound
HepG2
6a-phenyl16.54 Molecules 17 06317 i002
6b-phenyl-m-OCH315.43 Molecules 17 06317 i003
6c-phenyl-p-OCH346.97 Molecules 17 06317 i004
6d-phenyl-o-OCH323.91 Molecules 17 06317 i005
6e-phenyl-3,5-Di-OCH37.12 Molecules 17 06317 i006
6f-phenyl-o-Cl61.55 Molecules 17 06317 i007
6g-phenyl-m-F48.93 Molecules 17 06317 i008
6h-phenyl-2,4-Di-Cl10.55 Molecules 17 06317 i009
6i-phenyl-2,6-Di-F23.25 Molecules 17 06317 i010
6j-phenyl-2,3,4,5-Tetra-F54.24 Molecules 17 06317 i011
6k-phenyl-m-CF317.04 Molecules 17 06317 i012
6l-phenyl-p-CF310.96 Molecules 17 06317 i013
6m-phenyl-o-CF365.38 Molecules 17 06317 i014
6n-phenyl-m-NO219.12 Molecules 17 06317 i015
6o-phenyl-p- NO233.67 Molecules 17 06317 i016
6p-CH2Cl2.23 Molecules 17 06317 i017
6q-CHCl244.09 Molecules 17 06317 i018
6r-CCl394.55 Molecules 17 06317 i019
6s-CH2CH2Cl70.09 Molecules 17 06317 i020
6t-CH2CH2CH2Cl81.53 Molecules 17 06317 i021
6u-CH2CH375.54 Molecules 17 06317 i022
6v-CH2CH2CH3180.31 Molecules 17 06317 i023
6w-C(CH3)341.15 Molecules 17 06317 i024
Sorafenib 16.30
a Values are means of three independent experiments.
Molecules 17 06317 g003 550
Figure 3. Structure of Sorafenib.
Figure 3. Structure of Sorafenib.
Molecules 17 06317 g003
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 IC50 values <10 μM.
Table
Table 2. Inhibition of cell proliferation by compound 6p and Sorafenib.
Table 2. Inhibition of cell proliferation by compound 6p and Sorafenib.
CompoundIC50 (µM) a
HepG2MCF-7HCT116SW480A549SPC-A1A375U87
6p2.2335.739.148.7813.719.616.9725.53
Sorafenib16.30>10010.0940.6513.1518.6017.9662.19
a Values are means of three independent experiments.
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.
Table
Table 3. Kinase inhibitory assays of 6p.
Table 3. Kinase inhibitory assays of 6p.
Compound% inhibition at 10 μM
Aurora-AAurora-BAxlFlt3KDRPDGFRα
6p−1887−1−670

2.3. 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.
Molecules 17 06317 g004 550
Figure 4. The binding mode of compound 6p with ATP pocket of Aurora-B kinase obtained by molecular docking experiments (PDB code: 4AF3).
Figure 4. The binding mode of compound 6p with ATP pocket of Aurora-B kinase obtained by molecular docking experiments (PDB code: 4AF3).
Molecules 17 06317 g004

3. Experimental

3.1. General

The human cancer cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco’s modified Eagle medium (DMEM) and RPMI 1640 were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Melting points were determined on a SGW X-4 microscopic melting point (Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China). 1H-NMR and 13C-NMR spectra were recorded on a Bruker Varian Unity Inova-400 (400/100 MHz) spectrometer using TMS as internal reference chemical. Shifts are expressed as δ values in ppm. Mass spectra (MS) were measured on a Q-TOF Premier mass spectrometer (Micromass, Manchester, UK) utilizing electrospray ionization (ESI).

3.2. Preparation of 4-Chloropyridine-2-carbonyl Chloride Hydrochloride (4)

4-Chloropyridine-2-carbonyl chloride hydrochloride was prepared according to the reported method. Thionyl chloride (198 g, 1.68 mol) was added to a mixture of 2-picolinic acid (60 g, 0.48 mol), sodium bromide (8.08 g, 0.0785 mol) and chlorobenzene (84.8 g). Then 2.0 M methylamine solution in methanol was added to afford 4 as a white solid. Yield: 48 g, 58%; m.p. 34.0–38.0 °C; 1H-NMR (DMSO-d6): δ 3.04 (d, J = 5.2 Hz, 3H), 7.43 (dd, J = 5.2, 2.0 Hz, 1H), 7.98 (s, 1H), 8.21 (d, J = 1.6 Hz, 1H), 8.44 (d, J = 5.2 Hz, 1H); ESI-MS: m/z 193.04 (M+Na)+.

3.3. 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: 6.76 g, 87%; m.p. 112.8–115.1 °C; 1H-NMR (DMSO-d6): δ 2.81 (q, J = 10.0 Hz, 3H), 5.74 (s, 2H), 6.68 (d, J = 8.4 Hz, 2H), 7.18 (dd, J = 3.6, 23.6 Hz, 3H), 7.49 (s, 1H), 8.34 (d, J = 5.2 Hz, 1H), 8.71 (d, J = 4.4 Hz, 1H); 13C-NMR (DMSO-d6): δ 164.43, 154.72, 151.47, 150.44, 148.39, 137.46 (2C), 121.94, 117.37, 115.57 (2C), 110.60, 26.42; ESI-MS: m/z 282.29 (M+Na)+.

3.4. 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.
4-(4-Benzamidophenylthio)-N-methylpicolinamide (6a). Orange solid; yield: 91.87%; m.p. 139.1–140.7 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.4 Hz, 3H), 7.27 (dd, J = 2.0, 5.2 Hz, 1H), 7.56 (t, J = 7.4 Hz, 2H), 7.61–7.64 (m, 4H), 8.02 (q, J = 7.86 Hz, 4H), 8.42 (d, J = 5.2 Hz, 1H), 8.84 (d, J = 4.4 Hz, 1H), 10.63 (s, 1H); 13C-NMR (DMSO-d6): δ 166.48, 164.30, 152.61, 150.64, 148.71, 141.69, 136.56 (2C), 135.19, 132.27, 128.91 (2C), 128.25 (2C), 122.61, 122.03 (2C), 121.84, 117.91, 26.44; ESI-MS: m/z 364.44 (M+H)+.
4-(4-(3-Methoxybenzamido)phenylthio)-N-methylpicolinamide (6b). Orange solid; yield: 35.47%; m.p. 60.1–63.4 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.8 Hz, 3H), 3.84 (d, J = 8.0 Hz, 3H), 7.18 (dd, J = 2.4, 8.0 Hz, 1H), 7.25 (dd, J = 1.6, 5.2 Hz,1H), 7.45–7.63 (m, 6H), 7.99 (d, J = 8.4 Hz, 2H), 8.40 (d, J = 5.6 Hz, 1H), 8.69 (d, J = 4.4 Hz, 1H), 10.48 (s, 1H); 13C-NMR (DMSO-d6): δ 165.69, 163.78, 159.16, 152.11, 150.14, 148.18, 141.13, 136.09 (2C), 136.05, 129.57, 122.09, 121.57 (2C), 121.38, 119.94, 117.45, 117.39, 113.04, 55.31, 25.93; ESI-MS: m/z 394.45 (M+H)+.
4-(4-(4-Methoxybenzamido)phenylthio)-N-methylpicolinamide (6c). Orange solid; yield: 33.20%; m.p. 155.2–158.7 °C; 1H-NMR (DMSO-d6): δ 2.76 (t, J = 5.8 Hz, 3H), 3.85 (t, J = 9.8 Hz, 3H), 7.09 (d, J = 9.2 Hz, 2H), 7.25 (dd, J = 2.0, 5.2 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.99 (q, J = 4.0 Hz, 4H), 8.40 (d, J = 4.8 Hz, 1H), 8.75 (d, J = 4.8 Hz, 1H), 10.40 (s, 1H); 13C-NMR (DMSO-d6): δ 165.77, 164.29, 162.58, 152.69, 150.65, 148.71, 141.91, 136.55 (2C), 130.24 (2C), 127.14, 122.58, 121.96 (2C), 121.46, 117.88, 114.14 (2C), 55.93, 26.43; ESI-MS: m/z 394.43 (M+H)+.
4-(4-(2-Methoxybenzamido)phenylthio)-N-methylpicolinamide (6d). Orange solid; yield: 82.47%; m.p. 122.4–125.1 °C; 1H-NMR (DMSO-d6): δ 2.76 (d, J = 4.8 Hz, 3H), 3.91 (s, 3H), 7.08 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 2.0, 5.2 Hz, 1H), 7.51–7.54 (m, 2H), 7.60–7.64 (m, 3H), 7.95 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 5.6 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H), 10.46 (s, 1H); 13C-NMR (DMSO-d6): δ 165.55, 164.30, 156.94, 152.68, 150.65, 148.70, 141.50, 136.70 (2C), 132.62, 130.07, 125.41, 122.57, 121.61, 121.50 (2C), 120.95, 117.88, 112.44, 56.34, 26.44; ESI-MS: m/z 416.14 (M+Na)+.
4-(4-(3,5-Dimethoxybenzamido)phenylthio)-N-methylpicolinamide (6e). Orange solid; yield: 32.13%; m.p. 153.9–157.4 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.4 Hz, 3H), 3.84 (s, 6H), 6.75 (s, 1H), 7.12 (d, J = 2.0 Hz, 2H), 7.26 (dd, J = 2.0, 5.2 Hz, 1H), 7.54 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 5.2 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H), 10.49 (s, 1H); 13C-NMR (DMSO-d6): δ 165.50, 163.71, 160.36 (2C), 152.27, 150.02, 148.09, 141.08, 136.69, 136.03 (2C), 122.10, 121.64 (2C), 121.39, 117.42, 105.72 (2C), 103.47, 55.48 (2C), 25.93; ESI-MS: m/z 424.20 (M+H)+.
4-(4-(2-Chlorobenzamido)phenylthio)-N-methylpicolinamide (6f). Orange solid; yield: 70.04%; m.p. 191.3–193.5 °C; 1H-NMR (DMSO-d6): δ 2.76 (d, J = 5.2 Hz, 3H), 7.26 (t, J = 2.8 Hz, 1H), 7.46–7.66 (m, 7H), 7.93 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 5.6 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H), 10.86 (s, 1H); 13C-NMR (DMSO-d6): δ 165.28, 163.80, 152.04, 150.14, 148.22, 140.79, 136.63, 136.25 (2C), 131.26, 129.92, 129.66, 128.96, 127.26, 122.13, 121.66, 120.92 (2C), 117.41, 25.95; ESI-MS: m/z 398.40 (M+H)+.
4-(4-(3-Fluorobenzamido)phenylthio)-N-methylpicolinamide (6g). Orange solid; yield: 71.66%; m.p. 187.9–189.0 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.8 Hz, 3H), 7.27 (d, J = 5.2 Hz, 1H), 7.49 (t, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.60–7.65 (m, 3H), 7.83 (q, J = 7.7 Hz, 2H), 8.01 (d, J = 8.0 Hz, 2H), 8.42 (d, J = 5.2 Hz, 1H), 8.76 (d, J = 4.4 Hz, 1H), 10.62 (s, 1H); 13C-NMR (DMSO-d6): δ 165.03, 164.26, 163.60, 161.17, 152.57, 150.62, 148.66, 141.40, 137.41, 136.55 (2C), 131.03, 124.46, 122.62, 122.11 (2C), 117.94, 115.22, 114.99, 26.43; ESI-MS: m/z 380.17 (M−H)+.
4-(4-(2,4-Dichlorobenzamido)phenylthio)-N-methylpicolinamide (6h). Orange solid; yield: 59.61%; m.p. 227.3–229.7 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.8 Hz, 3H), 7.26 (dd, J = 2.0, 5.2 Hz, 1H), 7.55–7.64 (m, 5H), 7.79 (d, J = 1.6 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 5.2 Hz, 1H), 8.70 (d, J = 4.8 Hz, 1H), 10.84 (s, 1H); 13C-NMR (DMSO-d6): δ 164.35, 163.78, 151.97, 150.15, 148.21, 140.60, 136.27 (2C), 135.41, 135.07, 131.24, 130.38, 129.22, 127.47, 122.15, 121.88, 120.95 (2C), 117.41, 25.94; ESI-MS: m/z 432.23 (M+H)+.
4-(4-(2,6-Difluorobenzamido)phenylthio)-N-methylpicolinamide (6i). Orange solid; yield: 64.15%; m.p. 188.2–190.1 °C; 1H-NMR (DMSO-d6): δ 2.78 (d, J = 4.8 Hz, 3H), 7.24–7.32 (m, 3H), 7.57 (s, 1H), 7.60–7.66 (m, 3H), 7.89 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 5.2 Hz, 1H), 8.77 (d, J = 4.8 Hz, 1H), 11.15 (s, 1H); 13C-NMR (DMSO-d6): δ 163.69, 159.57, 158.53, 157.97, 152.10, 149.98, 148.17, 140.26, 136.36 (2C), 132.32, 122.23, 122.16, 120.83 (2C), 117.54, 115.11, 112.18, 112.04, 25.95; ESI-MS: m/z 422.12 (M+Na)+.
N-Methyl-4-(4-(2,3,4,5-tetrafluorobenzamido)phenylthio)picolinamide (6j). Orange solid; yield: 47.85%; m.p. 183.2–185.7 °C; 1H-NMR (DMSO-d6): δ 2.76 (d, J = 5.2 Hz, 3H), 7.27 (dd, J = 2.0, 5.2 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.83–7.87 (m, 3H), 8.41 (d, J = 5.2 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H), 10.95 (s, 1H); 13C-NMR (DMSO-d6): δ 164.02, 160.25, 158.71, 156.87, 152.15, 150.40, 148.45, 140.49, 136.53 (2C), 132.55, 122.58, 122.41, 121.43 (2C), 117.65, 114.93, 112.34, 112.13, 26.16; ESI-MS: m/z 458.04 (M+Na)+.
N-Methyl-4-(4-(3-(trifluoromethyl)benzamido)phenylthio)picolinamide (6k). Orange solid; yield: 71.09%; m.p. 168.1–169.5 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.8 Hz, 3H), 7.26 (dd, J = 2.0, 5.2 Hz, 1H), 7.56 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.81 (t, J = 7.8 Hz, 1H), 8.00 (d, J = 8.8 Hz, 3H), 8.28 (s, 1H), 8.31 (d, J = 4.4 Hz, 1H), 8.41 (d, J = 5.2 Hz, 1H), 8.70 (d, J = 4.4 Hz, 1H), 10.73 (s, 1H); 13C-NMR (DMSO-d6): δ 164.43, 163.72, 152.10, 150.06, 148.12, 140.82, 136.04 (2C), 135.53, 131.89, 129.68, 128.26, 124.80, 124.36, 124.34, 122.12, 121.82, 121.70 (2C), 117.46, 25.91; ESI-MS: m/z 432.35 (M+H)+.
N-Methyl-4-(4-(4-(trifluoromethyl)benzamido)phenylthio)picolinamide (6l). Orange solid; yield: 56.73%; m.p. 193.6–194.9 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.4 Hz, 3H), 7.27 (dd, J = 1.6, 5.2 Hz, 1H), 7.55 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H), 8.18 (d, J = 8.0 Hz, 2H), 8.42 (d, J = 5.2 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H), 10.78 (s, 1H); 13C-NMR (DMSO-d6): δ 164.79, 163.77, 152.00, 150.16, 148.19, 140.83, 138.47, 136.07 (2C), 128.68 (2C), 125.37, 125.35 (2C), 122.94, 122.12, 121.84, 121.62 (2C), 117.43, 25.91; ESI-MS: m/z 432.32 (M+H)+.
N-Methyl-4-(4-(2-(trifluoromethyl)benzamido)phenylthio)picolinamide (6m). Orange solid; yield: 71.22%; m.p. 232.3–233.5 °C; 1H-NMR (DMSO-d6): δ 2.78 (d, J = 4.4 Hz, 3H), 7.27 (d, J = 4.0 Hz, 1H), 7.56 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.73–7.85 (m, 3H), 7.89 (t, J = 6.8 Hz, 3H), 8.42 (d, J = 5.2 Hz, 1H), 8.77 (d, J = 4.4 Hz, 1H), 10.92 (s, 1H); 13C-NMR (DMSO-d6): δ 165.90, 163.80, 151.99, 150.16, 148.23, 140.76, 136.24 (2C), 135.81, 132.62, 130.21, 128.56, 126.33, 124.63, 122.82, 122.14, 121.79, 120.96 (2C), 117.43, 25.93; ESI-MS: m/z 430.00 (M−H)+.
N-Methyl-4-(4-(3-nitrobenzamido)phenylthio)picolinamide (6n). Orange solid; yield: 50.01%; m.p. 200.4–201.1 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.8 Hz, 3H), 7.26 (dd, J = 2.0, 5.2 Hz, 1H), 7.57 (d, J = 1.6 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.86 (t, J = 8.0 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 5.2 Hz, 1H), 8.46 (dd, J = 1.6, 8.4 Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.70 (d, J = 5.2 Hz, 1H), 8.82 (s, 1H), 11.02 (s, 1H); 13C-NMR (DMSO-d6): δ 164.27 (2C), 152.46, 150.65, 148.77, 148.18, 141.32, 136.49 (2C), 136.43, 134.89, 130.63, 126.79, 123.21, 122.64, 122.41, 122.36 (2C), 117.96, 26.44; ESI-MS: m/z 409.19 (M+H)+.
N-Methyl-4-(4-(4-nitrobenzamido)phenylthio)picolinamide (6o). Orange solid; yield: 78.93%; m.p. 215.9–216.6 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.4 Hz, 3H), 7.27 (d, J = 5.2 Hz, 1H), 7.55 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.23 (d, J = 8.8 Hz, 2H), 8.41 (t, J = 7.2 Hz, 3H), 8.76 (d, J = 4.4 Hz, 1H), 10.93 (s, 1H); 13C-NMR (DMSO-d6): δ 164.28, 163.83, 151.93, 150.07, 149.15, 148.23, 140.70, 140.13, 135.96 (2C), 129.40 (2C), 123.43 (2C), 122.12, 122.02, 121.77 (2C), 117.45, 25.91; ESI-MS: m/z 409.25 (M+H)+.
4-(4-Acetamidophenylthio)-N-methylpicolinamide (1). Orange solid; yield: 74.82%; m.p. 181.7–184.1 °C; 1H-NMR (DMSO-d6): δ 2.10 (s, 3H), 2.76 (d, J = 4.8 Hz, 3H), 7.23 (dd, J = 2.0, 5.2 Hz, 1H), 7.51 (d, J = 1.6 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 8.39 (d, J = 5.2 Hz, 1H), 8.74 (d, J = 4.8 Hz, 1H), 10.27 (s, 1H); 13C-NMR (DMSO-d6): δ 168.77, 163.77, 152.24, 150.10, 148.14, 141.25, 136.21 (2C), 121.98, 120.44, 120.24 (2C), 117.31, 25.92, 24.09; ESI-MS: m/z 324.11 (M+Na)+.
4-(4-(2-Chloroacetamido)phenylthio)-N-methylpicolinamide (6p). Orange solid; yield: 43.93%; m.p. 203.1–205.7 °C; 1H-NMR (DMSO-d6): δ 2.77 (t, J = 6.2 Hz, 3H), 4.35 (s, 2H), 7.22 (dd, J = 2.0, 4.8 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 8.40 (d, J = 5.2 Hz, 1H), 8.75 (d, J = 4.4 Hz, 1H), 10.90 (s, 1H); 13C-NMR (DMSO-d6): δ 165.67, 164.22, 152.63, 150.51, 148.67, 141.10, 136.69 (2C), 122.54, 121.97, 121.15 (2C), 117.97, 44.01, 26.45; ESI-MS: m/z 336.34 (M+H)+.
4-(4-(2,2-Dichloroacetamido)phenylthio)-N-methylpicolinamide (6q). Orange solid; yield: 70.01%; m.p. 195.5–196.9 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.4 Hz, 3H), 6.66 (s, 1H), 7.25 (d, J = 4.4 Hz, 1H), 7.54 (s, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 8.41 (d, J = 5.2 Hz, 1H), 8.75 (d, J = 5.2 Hz, 1H), 11.01 (s, 1H); 13C-NMR (DMSO-d6): δ 163.73, 162.07, 151.68, 150.15, 148.26, 139.48, 136.29 (2C), 122.89, 122.21, 121.16 (2C), 117.55, 67.22, 25.94; ESI-MS: m/z 368.02 (M−H)+.
N-Methyl-4-(4-(2,2,2-trichloroacetamido)phenylthio)picolinamide (6r). Orange solid; yield: 28.15%; m.p. 197.0–197.8 °C; 1H-NMR (DMSO-d6): δ 2.77 (d, J = 4.8 Hz, 3H), 7.27 (dd, J = 1.6, 5.2 Hz, 1H), 7.55 (d, J = 1.2 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 8.42 (d, J = 5.2 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H), 11.15 (s, 1H); 13C-NMR (DMSO-d6): δ 164.28, 160.34, 152.00, 150.63, 148.80, 139.57, 136.46 (2C), 124.38, 123.08 (2C), 122.84, 118.14, 93.31, 26.44; ESI-MS: m/z 401.97 (M−H)+.
4-(4-(3-Chloropropanamido)phenylthio)-N-methylpicolinamide (6s). Orange solid; yield: 58.10%; m.p. 145.0–147.2 °C; 1H-NMR (DMSO-d6): δ 2.77 (t, J = 6.4 Hz, 3H), 2.88 (t, J = 6.2 Hz, 2H), 3.90 (t, J = 6.4 Hz, 2H), 7.21 (dd, J = 2.0, 5.2 Hz, 1H), 7.53 (d, J = 1.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 8.39 (d, J = 5.6 Hz, 1H), 8.68 (d, J = 4.4 Hz, 1H), 10.35 (s, 1H); 13C-NMR (DMSO-d6): δ 168.96, 164.26, 152.64, 150.61, 148.67, 141.37, 136.77 (2C), 122.52, 121.44, 120.90 (2C), 117.86, 60.21, 26.42, 14.52; ESI-MS: m/z 372.19 (M+Na)+.
4-(4-(4-Chlorobutanamido)phenylthio)-N-methylpicolinamide (6t). Orange solid; yield: 76.45%; m.p. 106.4–108.9 °C; 1H-NMR (DMSO-d6): δ 2.03–2.10 (m, 2H), 2.54 (q, J = 7.6 Hz, 2H), 2.76 (d, J = 5.2 Hz, 3H), 3.72 (t, J = 6.4 Hz, 2H), 7.23 (d, J = 4.0 Hz, 1H), 7.51 (s, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 8.40 (d, J = 5.2 Hz, 1H), 8.75 (d, J = 4.8 Hz, 1H), 10.33 (s, 1H); 13C-NMR (DMSO-d6): δ 170.71, 163.53, 152.83, 149.67, 147.84, 141.22, 136.62, 136.20 (2C), 122.01, 120.37 (2C), 117.43, 44.94, 33.47, 27.76, 25.95; ESI-MS: m/z 398.05 (M+Cl)−.
N-Methyl-4-(4-propionamidophenylthio)picolinamide (6u). Orange solid; yield: 53.49%; m.p. 184.6–187.0 °C; 1H-NMR (DMSO-d6): δ 1.11 (t, J = 7.6 Hz, 3H), 2.38 (q, J = 7.6 Hz, 2H), 2.76 (d, J = 4.8 Hz, 3H), 7.23 (dd, J = 1.6, 5.2 Hz, 1H), 7.50 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 8.39 (d, J = 5.2 Hz, 1H), 8.75 (d, J = 4.4 Hz, 1H), 10.20 (s, 1H); 13C-NMR (DMSO-d6): δ 172.73, 164.05, 152.57, 150.35, 148.38, 141.61, 136.50 (2C), 122.22, 120.53 (2C), 117.55, 29.90, 26.21, 26.18, 9.77; ESI-MS: m/z 314.10 (M−H)+.
4-(4-Butyramidophenylthio)-N-methylpicolinamide (6v). Orange solid; yield: 84.96%; m.p. 144.8–147.0 °C; 1H-NMR (DMSO-d6): δ 0.94 (t, J = 7.4 Hz, 3H), 1.59–1.67 (m, 2H), 2.34 (t, J = 7.4 Hz, 2H), 2.77 (d, J = 4.8 Hz, 3H), 7.23 (d, J = 4.8 Hz, 1H), 7.51 (s, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 8.39 (d, J = 5.2 Hz, 1H), 8.74 (d, J = 4.8 Hz, 1H), 10.21 (s, 1H); 13C-NMR (DMSO-d6): δ 171.61, 163.77, 152.26, 150.10, 148.13, 141.27, 136.20(2C), 121.97, 120.37(2C), 120.30, 117.31, 38.40, 25.91, 18.45, 13.57; ESI-MS: m/z 328.12 (M−H)+.
N-Methyl-4-(4-pivalamidophenylthio)picolinamide (6w). Orange solid; yield: 51.57%; m.p. 108.5–110.5 °C; 1H-NMR (DMSO-d6): δ 1.26 (s, 9H), 2.76 (d, J = 4.8 Hz, 3H), 7.24 (d, J = 5.2 Hz, 1H), 7.51 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 8.40 (d, J = 5.2 Hz, 1H), 8.75 (d, J = 4.8 Hz, 1H), 9.50 (s, 1H); 13C-NMR (DMSO-d6): δ 177.35, 164.28, 152.76, 150.62, 148.64, 141.92, 136.42 (2C), 122.53, 121.82 (2C), 121.12, 117.83, 27.55 (4C), 26.42; ESI-MS: m/z 366.23 (M+Na)+.

3.5. Cell Culture

Cell lines including HepG2, A375 and U87 were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 mg/L). Cell lines including HCT116, MCF-7, SPC-A1, A549 and SW480, were maintained in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 mg/L). Cells were grown in a 5% CO2 incubator at 37 °C.

3.6. Cell Proliferation Assay (MTT Assay)

Cells (3 × 103/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 IC50 of each cell line.

3.7. 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.

4. 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 IC50 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.

Acknowledgments

This work was supported by National Science and Technology Major Project of China (2009ZX09103-132). The authors are grateful to the State Key Laboratory of Biotherapy and Cancer Center for supporting this research project.

References and Notes

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MDPI and ACS Style

Huang, T.-T.; Huang, Y.-C.; Qing, X.-Y.; Xia, Y.; Luo, X.; Ye, T.-H.; Yu, L.-T. Synthesis and Biological Evaluation of Novel N-Methyl-picolinamide-4-thiol Derivatives as Potential Antitumor Agents. Molecules 2012, 17, 6317-6330. https://doi.org/10.3390/molecules17066317

AMA Style

Huang T-T, Huang Y-C, Qing X-Y, Xia Y, Luo X, Ye T-H, Yu L-T. Synthesis and Biological Evaluation of Novel N-Methyl-picolinamide-4-thiol Derivatives as Potential Antitumor Agents. Molecules. 2012; 17(6):6317-6330. https://doi.org/10.3390/molecules17066317

Chicago/Turabian Style

Huang, Ting-Ting, Yun-Chuang Huang, Xiao-Yu Qing, Yong Xia, Xun Luo, Ting-Hong Ye, and Luo-Ting Yu. 2012. "Synthesis and Biological Evaluation of Novel N-Methyl-picolinamide-4-thiol Derivatives as Potential Antitumor Agents" Molecules 17, no. 6: 6317-6330. https://doi.org/10.3390/molecules17066317

APA Style

Huang, T. -T., Huang, Y. -C., Qing, X. -Y., Xia, Y., Luo, X., Ye, T. -H., & Yu, L. -T. (2012). Synthesis and Biological Evaluation of Novel N-Methyl-picolinamide-4-thiol Derivatives as Potential Antitumor Agents. Molecules, 17(6), 6317-6330. https://doi.org/10.3390/molecules17066317

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