3 курс / Фармакология / Синтез_и_изучение_свойств_новых_материалов_с_противоопухолевой

Fig. 3.24. Circular dichroism spectra of DNA (C = 6.1 μM) as a function of compound concentration 1.57 (C = 1–100 µM).

An increase in temperature has a strong effect on the stability of the doublestranded DNA structure, causing breakage to a single-stranded structure, the so-called

‘melting’ of the nucleic acid. The temperature at which 50 % of the duplex DNA remains in the double-stranded state is referred to as the DNA melting point (Tm). The melting temperature of DNA (Tm) directly depends on the stability of its double helix. The interaction of DNA with small molecules can stabilise the structure of the nucleic acid, causing conformational changes, which usually leads to an increase in the Tm value.

The study of DNA melting by UV spectroscopy in the presence of compound

1.57 showed that the melting temperature of DNA remains practically unchanged, although the transition width increases (Fig. 3.25), which indicates the presence of an interaction. A similar shape of the melting curve is observed in the interaction of cisplatin with DNA at similar ratios. Thus, it can be assumed that the binding in both cases is carried out with DNA bases.

Fig. 3.26. Absorption spectra of DNA solutions (C = 3.6–24.4 μM) at a constant concentration of compound 1.57 (C = 5.4 μM), as well as the dependence in coordinates [DNA] / (εa − εf) on [DNA] (R2 = 0.982) at [DNA] = 3.6, 5.4, 8.2, 11.1,

13.5, 1.89 and 24.4 µM.

The value of G was −43.03 kJ/mol.

3.5.3.Antioxidant activity

3.5.3.1.Study of antiradical activity

To quantify the reaction rate between compound 1.57 and DPPH, we used a pseudo-first-order kinetic model of the reaction described by the Eq. 3.20:

where A∞, At and A0 are the optical densities of the solution after ‘infinity’ (six days after the start of the experiment), at time t and at the initial time, respectively.

From the tangents of the slopes of the curves in the coordinates ln A - At vs t

A - A0

(Fig. 3.27), the values of the apparent rate constants at various temperatures were obtained (Table 3.9).

Fig. 3.28. Temperature dependence of the logarithm of the rate constant of the reaction of the interaction of compound 1.57 with DPPH. T is the absolute temperature, k is the pseudo first order rate constant.

3.5.3.2. Study of photodynamic properties

The study of photobleaching of Radachlorin in the presence of compound 1.57 makes it possible to evaluate the antioxidant properties of the drug upon irradiation with visible light. The practical significance of the study lies in the fact that the data obtained make it possible to evaluate the possibility of using substances for the implementation of photodynamic therapy, which is actively used in oncology [106,107] and the treatment of non-tumour diseases [108]. Carrying out photodynamic therapy requires the selection of effective photosensitisers that are selective with respect to the target and have a sufficient quantum yield of singlet oxygen upon irradiation. These requirements are met by preparations of chlorin e6, which have proven themselves in clinical practice. In this work, we used Radachlorin, a second-generation photosensitiser intended for fluorescent diagnostics and photodynamic therapy of malignant tumours. [109,110]. The method of photodynamic therapy is based on the ability of Radachlorin to selectively accumulate in a tumour upon its administration and generate singlet oxygen, which has a toxic effect on tumour cells and destroys their plasma membranes when

Fig. 3.30. Kinetic dependence of photobleaching of Radachlorin (►) in the presence of the compound 1.57 (▲ 5 µM, ▼ 10 µM, ● 25 µM, 50 µM, ▼75 µM, ■ 100 µM) compared to sodium azide (● 500 µM). A0 and At are optical densities of Radachlorin solutions at a wavelength of 664.9 nm before and after irradiation.

3.5.3.3. Photoinduced haemolysis

It is known that erythrocyte lysis is initiated by exposure to ultraviolet or visible light in the presence of photosensitisers, the most effective of which are porphyrins and their derivatives [109], in particular Radachlorin. It has been established that the photodynamic effect is primarily associated with the generation of singlet oxygen, and then other reactive oxygen species. Binding of porphyrins to cell membranes leads to a decrease in membrane photostability [110].

Fig. 3.31 shows the concentration dependence of the degree of photoinduced haemolysis in the presence of compound 1.57. As can be seen from the results obtained, compound 1.57 suppresses haemolysis induced by Radachlorin, which manifests itself in an increase in the time of haemolysis. From the obtained results, it can be concluded that compound 1.57 exhibits dose-dependent antioxidant activity.

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Fig. 3.32. The efficiency of NO-radical capture by compound 1.57 (dark grey) compared to sodium azide (light grey). C is the molar concentration of compound

1.57.

3.5.4. Genotoxicity of compound 1.57

The mean % comet tail DNA content, tail length, and comet tail momentum observed from human PBMCs incubated in the presence of H2O2 (positive control), PBS (negative control), and compound 1.57 are presented in Table 3.10. The amount of DNA damage in the presence of H2O2 is significantly higher than in control cells. As an example, Fig. 3.33 shows micrographs of DNA comets in the presence of H2O2

(C = 100 μM), PBS, and compound 1.57 in the concentration range 1–200 μM. Analysis of Table 3.10 shows that incubation of human PBMC with compound 1.57 causes dosedependent damage to DNA integrity.