Benzo[e][1,2,4]triazino[2,3-c][1,2,3]triazin-2-ones electro-deficient heterocyclic compounds with promising anticancer activity

Chemistry

Substituted 3-(2-amino-3-R²-5-R³_-phenyl)-6-R1-1,2,4-triazin-5(2H)-ones 2a–j were used as starting materials for synthesis of target 3-R¹-8-R²-10-R³-2H-benzo[e][1,2,4]triazino[2,3-c][1,2,3]triazin-2-ones (3a–j, Scheme 1). The starting anilines were obtained according to a known synthetic protocol [19] via transformation of 3-R-8-R¹-10-R²-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones 1a–j [20].

Scheme 1

Synthetic route for 3-R¹-8-R²-10-R³-2H-benzo[e][1,2,4]triazino[2,3-c][1,2,3]triazin-2-ones 3a–j.

Purity of the synthesized compounds was analyzed by LC-MS (APCI) method and their structure was established by using physicochemical methods including ¹H NMR, ¹³C NMR, IR and mass spectrometry. In particular, the protons in positions 8 and 10 of 3a–j are significantly deshielded in comparison to the corresponding protons of [1,2,4]triazino[2,3-c]quinazoline system [20]. Signals caused by the presence of a substituent in position 3 are also registered in proper fields. The carbons are also more deshielded in comparison to those of previously described [1,2,4]triazino[2,3-c]quinazolines [20]. The mass spectrum of compound 3b is characterized by a molecular ion peak of low intensity (2%). One of the mass fragmentation routes includes elimination of molecular nitrogen (m/z 247) from the molecular ion.

The structure of compound 3h was additionally determined by X-ray diffraction study (Figure 1). The tricyclic fragment is planar within 0.01 Å. The H4…N1 (2.63 Å) and H16c…N2 (2.54 Å) attractive interactions can be seen because the van der Waals radii sum is 2.67 Å) [21]. The phenyl substituent is twisted relatively to the triazine ring with the C1-C9-C10-C15 torsion angle of -32.6(2)°. This is probably due by the repulsion between the phenyl group and the adjacent atoms; the shortened intramolecular contacts are 2.49 Å for H11…N5 and 2.87 Å for H15…C1 2.86 Å. It can also be suggested that the repulsion between the phenyl substituent and the triazine moiety promotes the elongation of the C1-C9 bond to 1.497(1) Å as compared with its mean value of 1.46 Å [22].

Figure 1

Structure of compound 3h according to X-ray structural study.

Anticancer activity

Compounds 3b–d and 3f–h were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov) for the in vitro cell line screening to investigate their anticancer activity. Anticancer assays were performed according to the US NCI protocol, which was described elsewhere [23–25].

The experimental data show that some of the studied compounds reveal high antitumor activity. Compound 3d is the most active with a broad cytostatic activity spectrum. The range of cancer cells growth for 3d is 13–101% with a mean growth of 53.7%. Compounds 3b (mean growth 92%, range of growth, 56–115%), 3c (mean growth 91%, range of growth, 29–113%) and 3g (mean growth 86%, range of growth, 18–108%) show moderate anticancer activity. Compounds 3f and 3h do not show significant antitumor activity. It should be noted that antitumor activity of synthesized benzo[e][1,2,4]triazino[2,3-c][1,2,3]triazin-2-ones is selective. Breast cancer cell line MDA-MB-468 is the most sensitive. Compounds 3b, 3c and 3g inhibit growth of MDA-MB-468 cells line by 43%, 70% and 81%, respectively. The most active compound 3d shows cytotoxic activity and reduced initial quantity of MDA-MB-468 cells at 13%. Compound 3d was evaluated for dose-depended antitumor activity according to phase 2 of NCI protocol. Dose-dependency was studied at five concentrations in 10-fold dilution (100–0.01 μm) at 60 lines of nine cancer cell types at 10-fold dilution (100–0.01 μm, Table 1). As can be seen, the most sensitive line is breast cancer line MDA-MB-468. The GI₅₀ value for this line is 0.41 μm.

Conclusion

The treatment of 3-(2-amino-4-R²-phenyl)-6-R¹-1,2,4-triazin-5(2H)-ones with sodium nitrite in acetic acid yielded 3-R¹-8-R²-10-R³-2H-benzo[e][1,2,4]triazino[2,3-c][1,2,3]triazin-2-ones derivatives. Some of the synthesized compounds exhibit anticancer activity against human tumor cells, especially breast cancer cell lines. The most active compound 3d is highly active against breast cancer MDA-MB-468 cell line (GI₅₀=0.41 μm).

General method for the preparation of 3-R¹-8-R²-10-R³-2H-benzo[e][1,2,4]triazino[2,3-c][1,2,3]triazin-2-ones 3a–j

Compound 2a–j (5 mmol) was suspended in acetic acid (20 mL) and treated with sodium nitrite (0.52 g, 7.5 mmol). The mixture was stirred at room temperature for 2 h, then cooled, and the resultant precipitate was filtered off, washed by water, dried and crystallized from acetic acid.

Cytotoxic activity against malignant human tumor cells

Primary anticancer assay was performed at 60 human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [23–25]. Three dose response parameters were calculated for each compound. Growth inhibition of 50% (GI₅₀) – drug concentration resulting in a 50% lower net protein increase in the treated as compared to the net protein increase seen in the control cells, the drug concentration resulting in total growth inhibition (TGI), and LC₅₀ concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning. The lowest values were obtained with the most sensitive cell lines.

X-ray crystallographic study

The colorless crystals 3h (C₁₆H₁₁N₅O) are monoclinic. At 193 K, a=7.9168(4), b=11.0274(4), c=15.7059(6) Å, β=102.618(4)°, V=1338.0(1) ų, M_r=289.30, Z=4, space group P2₁/n, d_calc=1.436 g/cm³, μ(MoK_α)=0.096 mm^-1, F(000)=600. Intensities of 12 976 reflections (3889 independent, R_int=0.022) were measured on the Xcalibur-3 diffractometer (graphite monochromated MoK_α radiation, CCD detector, ω-scaning, 2Θ_max=60°).

The structure was solved by direct method using SHELXTL package [26]. Positions of the hydrogen atoms were located from electron density difference maps and refined by a riding model with U_iso=nU_eq (n=1.5 for methyl group and n=1.2 for other hydrogen atoms) of the carrier atom. Full-matrix least-squares refinement against F² in anisotropic approximation for non-hydrogen atoms using 3805 reflections was converged to wR₂=0.125 (R₁=0.043 for 2714 reflections with F>4σ(F), S=1.025). The final atomic coordinates, and crystallographic data for molecule 3h were deposited to with the Cambridge Crystallographic Data Centre, 12 Union Road, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk). They are available on request quoting the deposition number CCDC1408960).