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of various processes of degradation of substances (hydrolysis, oxidation, tautomerisation, enzymatic transformations, etc.) [11]. These processes can lead to the formation of products with less pharmacological activity up to its loss.
The stability of aqueous solutions of compound 1.57 was studied by 1H NMR spectroscopy. Fig. 3.17 shows the 1H NMR spectra of an aqueous solution of compound
1.57, recorded at certain intervals in automatic mode. In a neutral medium, there was no decrease in the intensity or shift of the signals corresponding to the protons of the starting compound 1.57 during the day; therefore, we can conclude that the structure of
1.57 is stable in an aqueous medium for 24 h [98].
Fig. 3.17. 1H NMR spectra of compound 1.57 in D2O after 12, 16, 20, and 24 h. An analysis of the literature data shows that compounds containing a 1,3-dioxane
fragment in their structure can undergo hydrolysis in the presence of acid catalysts. At the same time, electron-donating substituents in position 2 accelerate the rate of hydrolysis. Thus, compound 1.57 can undergo hydrolysis according to Scheme 3.7 to 2-((4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl)amino)-2-(hydroxymethyl)propan-1,3-diol (3.7) [98].
202
Scheme 3.7
To confirm the hydrolysis proceeding according to the mechanism presented in Scheme 3.7, 20 mg of compound 1.57 were dissolved in 40 ml of aqueous solutions of hydrochloric and acetic acids (pH 1 and 3). The resulting solutions were stirred at room temperature for 45 min. After completion of the reaction, the solvent was removed at room temperature and normal pressure. The product was air dried.
Using NMR spectroscopy, it was found that at pH values of 1 and 3, the signals of the protons of the CH3-groups disappear (δ = 1.41–1.42 ppm), and there is also a shift in the chemical shifts of the CH2 protons of the aziridine rings to a strong field at pH 1 and 3 (from δ = 2.33 to 2.16 and 2.07 ppm, respectively) (Fig. 3.18). The formation of product (2.8) was also confirmed by mass spectrometry [98].
Fig. 3.18. 1H NMR spectrum of compound 1.57 at pH 1.
Similarly, 20 mg of compound 1.57 was dissolved in 40 ml of an aqueous solution of 0.1 M NaOH (pH 10). The resulting solution was stirred at room temperature
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for 1 h. After the reaction, the solvent was removed at room temperature and normal pressure. The product was air dried. However, by NMR spectroscopy, it was found that there is no noticeable shift of the signals. Thus, we can conclude that compound 1.57 is stable in an alkaline medium (Fig. 3.19) [98].
Fig. 3.19. 1H NMR spectrum of compound 1.57 at pH 10.
3.5.Biocompatibility of compound 1.57
3.5.1.Haemocompatibility
3.5.1.1.Haemolysis
To assess the haemocompatibility of compound 1.57, its effect on spontaneous haemolysis was studied. In the case of substances compatible with blood, the erythrocyte membrane remains intact, and the contents of the cell are not released. The effect of compound 1.57 on haemolysis was determined by spectrophotometric measurement of released haemoglobin.
On Fig. 3.20 shows that compound 1.57, when incubated for 1 and 3 hours with erythrocytes, does not cause hemolysis in the concentration range from 10 to 200 μM.
Therefore, compound 1.57 is hemolytically inactive.
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Fig. 3.20. Effect of compound 1.57 on haemolysis (light grey after 1 h, dark grey after 3 h).
3.5.1.2. Platelet aggregation
As can be seen from the data presented in Table 3.6, in the test of ADP-induced platelet aggregation, compound 1.57 in the studied concentration range significantly increases platelet aggregation compared to the control; effect is not dose-dependent.
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Table 3.7. Influence of compound 1.57 on parameters of plasma-coagulation haemostasis.
Test |
Standard |
Control |
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C / µM |
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10 |
25 |
50 |
75 |
100 |
200 |
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TT / s |
15–19 |
17.1 ± 1.2 |
15.0 ± 0.9 |
15.2 ± 1.0 |
15.4 ± 0.8 |
15.6 ± 1.4 |
15.6 ± 0.7 |
15.8 ± 1.2 |
15.1 ± 2.1 |
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APTT / s |
28–40 |
36.5 ± 2.0 |
41.9 ± 1.7* |
42.3 ± 2.2* |
44.5 ± 1.3* |
46.7 ± 1.8* |
43.3 ± 1.6* |
45.2 ± 2.1* |
42.7 ± 1.4* |
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PT / s |
13–18 |
13.6 ± 1.5 |
18.6 ± 2.1 |
18.5 ± 1.8 |
18.1 ± 2.0 |
18.0 ± 1.9 |
18.2 ± 2.2 |
18.5 ± 2.4 |
18.2 ± 2.1 |
*p < 0.05 in relation to control.
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206
Table 3.6. Effect of compound 1.57 on ADP-induced platelet aggregation in plateletrich plasma.
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60.2 ± 2.4 69.9 ± 2.2* |
67.8 ± 3.1* |
68.3 ± 2.8* |
69.6 ± 3.3* |
71.4 ± 2.9* |
68.7 ± 2.7* |
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*p < 0.05 in relation to control.
3.5.1.3. Plasma coagulation haemostasis
Compound 1.57 in the concentration range of 5–200 μM exhibits anticoagulant properties in the APTT test, which are statistically significant compared to the control (Table 3.7). At the same time, compound 1.57 had no effect on TT and PT in the studied concentration range.
3.5.2.Study of binding to DNA and HSA
3.5.2.1.Study of binding to DNA and HSA by calorimetric method
Human serum albumin (HSA) is the major plasma protein. Binding to HSA controls the free active concentration of the drug and can significantly affect the overall pharmacodynamic and pharmacokinetic profile. [99,100]. Studies of drug binding to proteins are important both from a theoretical and practical point of view, as they allow a better understanding of the processes underlying the distribution and excretion of biologically active substances.
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HSA has three main ligand binding sites: (1) site I, located in subdomain IIA (warfarin binding site); (2) site II located in subdomain IIIA (ibuprofen binding site);
(3) site II located in subdomain IB (digitonin binding site).
The dependence of the thermal effect of the reaction of the interaction of compound 1.57 with HSA at 298.15 K on the volume of the added titrant (compound
1.57) is shown in Fig. 3.21. It can be seen that in this case there is an endothermic thermal effect, which indicates the absence of interaction between compound 1.57 and HSA. This thermal effect characterises the heat of mixing of the titrant and the titrated substance. Thus, when compound 1.57 is introduced into the bloodstream, HSA will not interact with it and, therefore, will perform a transport function. This fact may be the reason for the high systemic toxicity of compound 1.57.
Another common method for studying the mechanism of antitumour activity is the in vitro study of the interaction of biologically active substances with polynucleotides.
Fig. 3.22 shows the dependence of the thermal effect of the reaction of the interaction of compound 1.57 with DNA at 298.15 K depending on the volume of the titrant (compound 1.57). Based on the obtained experimental data, the parameters of the interaction of compound 1.57 with DNA were calculated using the thermodynamic model of independent binding (Independent model) [100].
The obtained value of the interaction stoichiometry shows that at the equivalence point, 1 mol of compound 1.57 accounts for 10 mol of DNA (Table 3.8). This can be explained by the association of DNA molecules in solution. Most likely, 1 molecule of compound 1.57 interacts with a conditional associate containing 10 DNA molecules.
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0.125 |
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Fig. 3.21. Dependence of the thermal |
Fig. 3.22. Dependence of the thermal |
effect of the reaction of the interaction of |
effect of the reaction of the interaction of |
compound 1.57 with HSA at 298.15 K. |
compound 1.57 with DNA at 298.15 K. |
H is the enthalpy of the reaction, C1.57 / |
H is the enthalpy of the reaction, C1.57 / |
CHSA is a concentration ratio of |
CDNA is a concentration ratio of |
compound 1.57 and HSA. |
compound 1.57 and DNA. |
The calculated values of binding constant of compound 1.57 to DNA (Kbin = 6.65 107 M–1) indicates the formation of a covalent adduct. From the presented values of thermodynamic parameters (Table 3.8) the interaction of compound 1.57 with DNA is a highly exothermic process. In turn, the negative value of S indicates that the addition of compound 1.57 leads to ordering in solution.
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Table 3.8. Thermodynamic characteristics of the binding of compound 1.57 to DNA at 298.15 K. Kd is the dissociation constant, Kbin is the association constant, n is the stoichiometric coefficient of the binding of compound 1.57 to DNA, H, S, G are the change in enthalpy, entropy, and Gibbs energy in the interaction reaction of the
compound 1.57 with DNA, T is absolute temperature.
Thermodynamic parameter |
Value |
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Kd / M |
1.51·10−8 |
N |
0.1 |
H / kJ/mol |
−788.5 |
S / J/mol∙K |
−2495.0 |
G / kJ/mol |
−44.65 |
−T S / kJ/mol |
743.8 |
Kbin / M−1 |
6.65·107 |
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3.5.2.2. Study of DNA binding by spectrophotometric method
The interaction of compound 1.57 with DNA was studied by UV and CD spectroscopy. In the presence of interaction with DNA in aqueous solutions of 0.9 % NaCl at pH 7.4, characteristic changes in the electronic absorption spectra are usually observed. The nature of the above changes significantly depends on the type of interaction. Fig. 3.23 shows the absorption spectra of DNA in physiological saline (0.9 % NaCl) at a constant DNA concentration (6.1 μM) and various concentrations of compound 1.57.
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0.6 |
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0.4 |
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220 |
230 |
240 |
250 |
260 |
270 |
280 |
290 |
300 |
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Fig. 3.23. Absorption spectra of DNA solutions (6.1 µM) containing various concentrations of compound 1.57 (1–100 µM) in physiological saline (0.9 % NaCl).
A noticeable hypochromic shift of the DNA absorption band is observed with increasing concentration of compound 1.57. Such changes may indicate that compound
1.57 interacts with DNA, causing a change in its spectral characteristics.
Circular dichroism spectroscopy is one of the most sensitive methods for studying conformational changes caused by the interaction of small molecules with DNA [101] and is often used to establish the types of interactions of biologically active molecules with DNA [102].
Fig. 3.24 shows the circular dichroism spectra of DNA solutions, which include positive bands at 275 nm, due to nucleotide stacking interactions, and negative bands at 246 nm, associated with the chirality of the right-handed B-form of the DNA double helix [103]. In this case, an increase in the concentration of compound 1.57 leads to a decrease in the absorption intensity of the positive band and an increase in the absorption intensity of the negative band. The observed changes upon addition of compound 1.57 were concentration dependent. Thus, it can be assumed that the observed effect is the result of a combination of several types of interactions of compound 1.57 with DNA.
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