Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards with potential enzymes inhibitors based on melamine framework

Figures & data

Figure 1.  Structures of triazine based antitumour agents and enzyme inhibitors.

Table 1.  Inhibitory effects of 12a–f on the growth of human tumour cell lines expressed as IC₅₀ in μg/mL.

12	cell line/IC50 [μg/mL] mean ± SD
	breast cancer T47D	prostate cancer LNCaP	colorectal cancer SW707	lung cancer A549	lymphoblastic leukemia Jurkat
a	27.86 ± 0.03	15.78 ± 6.68	32.38 ± 0.89	34.66 ± 1.55	31.29 ± 0.84
b	1.40 ± 0.33	0.99 ± 0.52	3.45 ± 0.28	2.06 ± 0.66	0.62 ± 0.15
c	96.84 ± 4.47	18.27 ± 14.32	96.84 ± 5.47	61.04 ± 20.31	25.60 ± 2.43
d	2.60 ± 0.99	1.47 ± 0.95	2.93 ± 0.81	2.67 ± 1.33	2.93 ± 0.36
e	36.22 ± 15.85%*	6.22 ± 3.34	25.35 ± 10.82%*	31.58 ± 10.43%*	4.50 ± 1.98
f	negative**	negative**	negative**	negative**	9.01 ± 0.86

*inhibition of cell proliferation in vitro expressed as percent of proliferation inhibition at concentration of 12e 10 μg/mL.

**no inhibition to the concentration of 12e 10 μg/mL.

Scheme 1.  Formation of triazines 12a-d bearing one 2-chloroethylamine fragment.

Scheme 2.  Synthesis 1,3,5-triazine derivatives bearing two 2-chloroethylamino residues 12e and three 2-chloroethylamino residues 12f.

Table 2.  Viability of MCF-7 cells treated for 24 h with different concentrations of chlorambucil (CHL) and compounds 12a–f.

Conc. [μM]	Non-viable cells (% of control ± 2)
	CHL	12a	12b	12c	12d	12e	12f
0	1a	3	2	3	4	2	2
1	11	6	9	8	23	25	23
10	31	20	24	27	46	31	52
50	48	24	39	46	57	52	62
100	96	42	51	52	63	69	65
150	100	48	61	75	74	72	73
IC50	29.14	139.78	92.02	67.47	32.13	43.95	18.70

^aMean values ± SD from three independent experiments done in duplicate are presented.

Table 3.  Alkylating activity of compounds 12a–f on comparison to chlorambucil; NBP test results.

Compound	Molar extinction coefficient (ϵ)	Absorbance (A)a 560 nm	Alkylation activityb
12a	103.6	0.1036	++
12b	119.5	0.1195	++
12c	88.3	0.0883	+
12d	119.3	0.1193	++
12e	130.2	0.1302	++
12f	148.9	0.1488	++
Chlorambucil	92.7	0.0927	+

^aAverage data from three determinations.

^bAccording to Preussmann: (−) A < 0.05, (+) A = 0.05–0.1, (++) A = 0.1-0.5, (+++) A > 0.5.

Figure 2.  Percentage of apoptotic and necrotic MCF-7 cells after treatment with 50 μM solutions of chlorambucil (CHL) and compounds 12a–12f. 100% = {apoptotic(%) + necrotic(%) + viable cells}.

Table 4.  DNA binding effect of compounds 12a–f.

Compound	Decrease of fluorescence [%]
CHL	61.73
12a	96.74
12b	98.75
12c	87.50
12d	85.00
12e	92.35
12f	55.62

Figure 3.  The relationship of apoptotic cells amount from increasing concentration of chlorambucil (CHL) and compounds 12a–f.