New 2-[(4-Amino-6- N-substituted-1,3,5-triazin-2-yl)methylthio]- N-(imidazolidin-2-ylidene)-4-chloro-5-methylbenzenesulfonamide Derivatives, Design, Synthesis and Anticancer Evaluation

Abstract

In the search for new compounds with antitumor activity, new potential anticancer agents were designed as molecular hybrids containing the structures of a triazine ring and a sulfonamide fragment. Applying the synthesis in solution, a base of new sulfonamide derivatives 20-162 was obtained by the reaction of the corresponding esters 11-19 with appropriate biguanide hydrochlorides. The structures of the compounds were confirmed by spectroscopy (IR, NMR), mass spectrometry (HRMS or MALDI-TOF/TOF), elemental analysis (C,H,N) and X-ray crystallography. The cytotoxic activity of the obtained compounds toward three tumor cell lines, HCT-116, MCF-7 and HeLa, was examined. The results showed that some of the most active compounds belonged to the R¹ = 4-trifluoromethylbenzyl and R¹ = 3,5-bis(trifluoromethyl)benzyl series and exhibited IC₅₀ values ranging from 3.6 µM to 11.0 µM. The SAR relationships were described, indicating the key role of the R² = 4-phenylpiperazin-1-yl substituent for the cytotoxic activity against the HCT-116 and MCF-7 lines. The studies regarding the mechanism of action of the active compounds included the assessment of the inhibition of MDM2-p53 interactions, cell cycle analysis and apoptosis induction examination. The results indicated that the studied compounds did not inhibit MDM2-p53 interactions but induced G0/G1 and G2/M cell cycle arrest in a p53-independent manner. Furthermore, the active compounds induced apoptosis in cells harboring wild-type and mutant p53. The compound design was conducted step by step and assisted by QSAR models that correlated the activity of the compounds against the HCT-116 cell line with molecular descriptors.

Keywords: 1,3,5-triazines; QSAR; anticancer activity; apoptosis; benzenesulfonamide; cell cycle arrest; imidazole; proliferation; synthesis.

Figure 1

Structures of exemplary anticancer drugs and compounds containing a sulfonamide or 1,3,5-triazine moiety which are in clinical trials, among others, for breast or colorectal cancer.

Figure 2

The general structure of a designed molecular hybrid consisting of two pharmacophores: a 2-mercaptobenzenesulfonamide fragment (A) and a 4-amino-6-R²-1,3,5-triazin-2-yl ring (B), modified by the R¹ substituent (C) on the N-1 imidazolidine ring.

Figure 3

A pathway of the designing of 2-[(4-amino-6-R²-1,3,5-triazin-2-yl)methylthio]-N-(imidazolidin-2-ylidene)-4-chloro-5-methylbenzenesulfonamide derivatives based on QSAR models.

Scheme 1

Synthesis of ethyl 2-{2-[N-(1-R¹-imidazolidin-2-ylidene)sulfamoyl]-5-chloro-4-methylphenylthio}acetate 11–18 and ethyl 2-{2-[N-{1-[4-(trifluoromethyl)benzyl]tetrahydropyrimidin-2(1H)-ylidene}sulfamoyl]-5-chloro-4-methylphenylthio}acetate 19. Reagents and conditions: (a) appropriate diamine (0.04 mol), anhydrous MeOH, reflux 70–240 h; (b) N-(4-trifluoromethylbenzyl)propane-1,3-diamine (0.04 mol), anhydrous MeOH, reflux 123 h; (c) ethyl 2-bromoacetate (0.024 mol), TEA (0.02 mol), DCM.

Scheme 2

Synthesis of 2-[(4-amino-6-R²-1,3,5-triazin-2-yl)methylthio]-N-(1-R¹-imidazolidin-2-ylidene)-4-chloro-5-methylbenzenesulfonamides (20–157). Compounds synthesized previously [31].

Scheme 3

Synthesis of 2-[(4-amino-6-R²-1,3,5-triazin-2-yl)methylthio]-N-{1-[4-(trifluoromethyl)benzyl]tetrahydropyrimidin-2(1H)-ylidene}-4-chloro-5-methylbenzenesulfonamides (158–162).

Figure 4

Molecular structure of compound 27, showing the atom-labelling scheme. Solvent molecules omitted. Displacement ellipsoids are shown at 50% probability.

Figure 5

Crystal packing and hydrogen bonding in 27. Molecules of the main component are linked by the ring-type hydrogen bond motif $R_2^2\left(8\right)$ NH^…N (using N4 and N8 atoms) located at the inversion center (drawn as the orange ball). The two dimethylsulfoxide molecules are interacting with the sulfonamide through NH^…O, CH^…O or NH^…S hydrogen bonds.

Figure 6

Scatter plots for N-(imidazolidin-2-ylidene)benzenesulfonamides 20–44 (model 1), N-(1-benzylimidazolidin-2-ylidene)benzenesulfonamides 45–73 (model 2) and N-[1-(4-fluorobenzyl)imidazolidin-2-ylidene]benzenesulfonamides 82–101 (model 3), as well as N-[1-(3-trifluoromethylbenzyl)imidazolidin-2-ylidene]benzenesulfonamides 112–117, N-[1-(4-trifluoromethylbenzyl)imidazolidin-2-ylidene]benzenesulfonamides 118–143 and N-{1-[3,5-bis(trifluoromethyl)benzyl]imidazolidin-2-ylidene}benzenesulfonamides 144–157 (model 4) with experimental IC₅₀ and predicted IC₅₀ values of the training set (blue) and test set (red) for the HCT-116 cell line.

Figure 6

Scatter plots for N-(imidazolidin-2-ylidene)benzenesulfonamides 20–44 (model 1), N-(1-benzylimidazolidin-2-ylidene)benzenesulfonamides 45–73 (model 2) and N-[1-(4-fluorobenzyl)imidazolidin-2-ylidene]benzenesulfonamides 82–101 (model 3), as well as N-[1-(3-trifluoromethylbenzyl)imidazolidin-2-ylidene]benzenesulfonamides 112–117, N-[1-(4-trifluoromethylbenzyl)imidazolidin-2-ylidene]benzenesulfonamides 118–143 and N-{1-[3,5-bis(trifluoromethyl)benzyl]imidazolidin-2-ylidene}benzenesulfonamides 144–157 (model 4) with experimental IC₅₀ and predicted IC₅₀ values of the training set (blue) and test set (red) for the HCT-116 cell line.

Figure 7

Effects on MDM2 and p53 protein levels upon treatment. Western blot analysis showing MDM2 and p53 protein levels in MCF-7 cells, untreated and treated with the indicated compounds in concentrations 4 µM (A) and 10 µM (B) for 24 h. Reference: β-tubulin.

Figure 8

Cell cycle arrest in MCF-7 cells induced by compounds 48 and 140. Cells were treated with the indicated concentrations of the compounds, and the cell cycle distribution was analyzed with flow cytometry. (A) Histograms showing the distribution of cell populations in the sub-G1, G0/G1, S and G2/M phases of the cell cycle. (B) Graphs demonstrating the percentages of cells in the depicted phases of the cell cycle. Values represent the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test [p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)].

Figure 9

Induction of apoptosis by compounds 48 and 140 in MCF-7 cells. Cells were treated with the indicated concentrations of compounds 48 and 140 for 72 h, stained with Annexin V-PE and 7-AAD and analyzed with flow cytometry. (A) Dot plots showing early apoptotic (bottom right quadrant), late apoptotic (upper right quadrant), viable (lower left quadrant) and necrotic cell populations (upper left quadrant). (B) Graphs indicating early and late apoptotic cells. Values represent the mean ± SD of three repetitions. Data were analyzed by one-way ANOVA with Tukey’s post hoc test [p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)].

Figure 10

Induction of apoptosis by compounds 48 and 140 in T47D cells. Cells were treated with the indicated concentrations of compounds 48 and 140 for 72 h, stained with Annexin V-PE and 7-AAD, and analyzed with flow cytometry. (A) Dot plots showing early apoptotic (bottom right quadrant), late apoptotic (upper right quadrant), viable (lower left quadrant) and necrotic cell populations (upper left quadrant). (B) Graphs indicating early and late apoptotic cells. Values represent the mean ± SD of three repetitions. Data were analyzed by one-way ANOVA with Tukey’s post hoc test [p < 0.01 (**), p < 0.001 (***)].