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3.5.5. Cytotoxicity of compound 1.57

After 48 h of incubation of human embryonic kidney line (HEK293) cells with compound 1.57 [112], a decrease in cell survival by almost 50 % was observed in the studied concentration range (Fig. 3.34a). At the same time, a decrease in the survival of tumour cell lines was observed: human liver adenocarcinoma (SK-HEP-1; Fig. 3.34b) and human glioblastoma (T98G; Fig. 3.34c) in the presence of compound 1.57.

Fig. 3.34. Effect of compound 1.57 on the survival of HEK293 (a), SK-HEP-1 (b), and T98G (c) cell lines. C is the molar concentration of the compound 1.57.

Table 3.11 shows a comparison of IC50 values for compound 1.57 and known cytostatics: doxorubicin and cisplatin on various cell lines. As can be seen from Table 3.11, the most pronounced cytotoxic effect of compound 1.57 is observed in relation to the A549

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3.6.Study of the physicochemical properties of aqueous solutions of compound 3.6

3.6.1.Stability of compound 3.6 in aqueous solutions

At the next stage of the work, we studied the stability of the tetrazole-containing derivative of 1,3,5-triazine, compound 3.6, in a solution of DCl in D2O (C = 10–3 M, pH 10). Significant changes were observed in the 1H NMR spectra in an acidic medium. Thus, a decrease in the intensity of the signals corresponding to the protons of CH3-groups (1.40– 1.42 ppm) was recorded, up to their complete disappearance in the spectrum. These changes in the spectral characteristics indicate hydrolysis of the dioxane ring with the formation of 2-((4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl)amino)-3-hydroxy-2- (hydroxymethyl)propyl-2-(5-phenyl-2H-tetrazol-2-yl)acetate (compound 3.7) (Scheme 3.8). A shift of the methylene group proton signal from 5.48 ppm to 5.53 ppm was also observed [111].

Scheme 3.8

3.7.Compound 3.6 biocompatibility study

3.7.1.Haemocompatibility study

3.7.1.1.Haemolysis

To assess the haemocompatibility of compound 3.6, its effect on spontaneous haemolysis was studied. The effect of compound 3.6 on haemolysis was determined by assessing the released haemoglobin. Fig. 3.35 shows the effect of compound 3.6

(C = 10–100 µM) on erythrocyte haemolysis after 1 and 3 h of incubation. It was shown that compound 3.6 upon incubation for 1 and 3 h caused insignificant haemolysis in the studied concentration range, while the intensity of haemolysis depended on the dose and

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Table 3.12. Effect of compound 3.6 on ADP-induced platelet aggregation in platelet-rich plasma.

Amplitude / %

C / µM

3.7.1.3. Plasma coagulation haemostasis

The data presented in Table 3.13 demonstrate the presence of anticoagulant properties in compound 3.6, which is characterised by a statistically significant increase in APTT and PT in the studied concentration range.

Table 3.13. Influence of compound 3.6 on parameters of plasma-coagulation haemostasis.

C is the molar concentration of compound 3.6.

*p < 0.05 in relation to control.

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3.7.2.Study of the binding of compound 3.6 to DNA and HSA

3.7.2.1.Study of thermodynamic parameters of DNA and HSA binding by

calorimetric method

Fig. 3.36 shows the dependence of the thermal effect of the reaction of the interaction of the tetrazole-containing derivative of 1,3,5-triazine (compound 3.6) with DNA at 298.15 K, depending on the volume of the added titrant (compound 3.6). Based on the thermodynamic model of independent binding, the parameters of the interaction of compound 3.6 with DNA were calculated. The calculated binding constant of compound 3.6 with DNA, equal to 4.08 108 M–1, indicates the formation of a stable complex. The obtained values of thermodynamic parameters: H = −17.18 kJ∙mol−1; S = 108.6

J∙(mol∙K)−1; G = −49.55 kJ∙mol−1) show that the interaction of compound 3.6 with DNA is an exothermic process. In this case, the negative value of G indicates the spontaneity of the process of formation of the complex of compound 3.6 with DNA [111].

Fig. 3.36. Dependence of the thermal effect of the reaction of the interaction of compound 3.6 with DNA at 298.15 K. H is the thermal effect of the reaction, C3.6 /

CDNA is the concentration ratio of compound 3.6 and DNA.

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3.7.2.2. Study of thermodynamic parameters of DNA binding by spectrophotometric

method

It was previously shown that one of the possible mechanisms of the antitumour effect of the 1,3,5-triazine derivative, compound 1.57, is due to binding to the DNA molecule, leading to its damage [98].

Fig. 3.37 shows the absorption spectra of DNA in physiological saline (0.9 % NaCl) at a constant DNA concentration (C = 6.1 μM) and various concentrations of compound

3.6. It was found that with an increase in the concentration of compound 3.6, a hypochromic effect is observed in the UV spectra, indicating that compound 3.6 interacts with DNA. UV spectra of solutions with different concentrations of DNA (2.3, 4.9, 8.3, 12.0, 13.0 and 13.9 μM) were also obtained in the presence of compound 3.6 (C = 2.34

μM; Fig 3.38).

Fig. 3.37. Absorption spectra of DNA solutions (C = 6.1 μM) depending on the concentration of compound 3.6 (C = 3.89–12.3 μM).

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Fig. 3.39. Melting curves of DNA (C = 6.1 μM) in the absence and presence of compound 3.6 at a ratio of [compound 3.6] / [DNA] = 1.5.

The CD spectrum of a DNA solution consists of a positive band at 275 nm, due to the stacking interaction of nucleotides within one strand of DNA, and a negative band at 246 nm, associated with the chirality of the right-handed helix of the B-form DNA [101]. The CD spectroscopy method can be used to determine the types of interactions of various ligands with DNA. The CD spectra of the solutions were obtained at a constant DNA concentration (C = 6.1 μM) and at various concentrations of compound 3.6 (3.9, 6.1, 8.1, and 12.3 μM). As can be seen from Fig. 3.40, an increase in the concentration of compound

3.6 in solution leads to a decrease in the absorption intensity of the positive band and a slight decrease in the absorption intensity of the negative band. The observed changes in the CD spectra may indicate that the tetrazole-containing derivative of 1,3,5-triazine (3.6) is capable of disrupting stacking interactions between nucleotides in the DNA duplex [103].

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