Synthesis and Biological Evaluation of Novel 4-(4-Formamidophenylamino)- N-methylpicolinamide Derivatives as Potential Antitumor Agents

Abstract

A novel series of 4-(4-formamidophenylamino)-N-methylpicolinamide derivatives were synthesized and evaluated against different tumor cell lines. Experiments in vitro showed that these derivatives could inhibit the proliferation of two kinds of human cancer cell lines (HepG2, HCT116) at low micromolar concentrations and the most potent analog 5q possessed broad-spectrum antiproliferative activity. Experiments in vivo demonstrated that 5q could effectively prolong the longevity of colon carcinoma-burdened mice and slow down the progression of cancer cells by suppression of angiogenesis and the induction of apoptosis and necrosis.

Keywords: 4-(4-formamidophenylamino)-N-methylpicolinamide derivatives; angiogenesis inhibitors; anti-proliferation; apoptosis; pharmacology.

Scheme 1

Synthesis of compounds 5a–5v. Reagent and conditions: (a) chlorobenzene, sodium bromide, and thionyl chloride reflux for 19 h, 85 °C; (b) 25% methylamine aqueous, THF; (c) 4a: (i) N-(4-aminophenyl)acetamide, 160 °C, (ii) EtOH, concentrated HCl, reflux for 4 h; 4b: 4-aminophenol, sodium hydride, DMSO, 100 °C, stir for 3 h; (d) triphosgene, substituted aniline or cyclohexylamine, triethylamine, and CH₂Cl₂ reflux for 1 h; (e) K₂CO₃, THF, substituted benzoyl chloride, r.t.

Figure 1

Effects of 5q on tumor-bearing mice. 5q was orally administered to Balb/c mice through gastric perfusion daily at a dosage of 75 mg/kg about 10 days after inoculation of cells. The volume of tumor was calculated by the following formula: volume (mm³) = long diameter (mm) × short diameter (mm)² × 0.5236. (A) Oral treatment with 5q presents a decrease in tumor volume against untreated (p < 0.05). (B) H&E staining of tumor tissue sections of untreated group (a), 5q-treated group (b) (400×). (C) Oral treatment with 5q presents a survival advantage against untreated. Data are plotted as the percentage of survival animal.

Figure 2

5q induces the apoptosis of cancer cells and inhibits angiogenesis. (A) 5q induces the apoptosis of cancer cells. (a) Scattered positive signals in the control group. (b) Clustered positive signals in 5q-treated group. (B) Quantitative analysis of the apoptotic cells. Data are represented as means ± standard deviation of cells per hpf (* p < 0.05) (200×). (C) 5q inhibits angiogenesis. CD31 immunohistochemistry in the frozen sections from the control group (a) and 5q-treated group (b). (D) Quantitative analysis of the vessel density. Data are represented as means ± standard deviation of vessels per hpf (* p < 0.05) (200×).