In Vitro, In Vivo, Ex Vivo Characterisation of Dihydroimidazotriazinones and Their Thermal Decomposition Course Studied by Coupled and Simultaneous Thermal Analysis Methods

Abstract

The biological and thermal properties of a class of synthetic dihydroimidazotriazinones were disclosed in this article for the first time. Molecules 1-6-as potential innovative antimetabolites mimicking bicyclic aza-analogues of isocytosine-were evaluated for their in vitro anticancer activity. Moreover, in vivo, in vitro, and ex vivo toxicity profiles of all the compounds were established in zebrafish, non-tumour cell, and erythrocyte models, respectively. Their antihaemolytic activity was also evaluated. Additionally, the thermal decomposition mechanism, path, and key thermal properties of heterocycles 1-6 were analysed. It was found that all the studied compounds revealed significant antiproliferative activities against tumour cells of the lung, cervix, ovary, and breast, as well as acute promyelocytic leukaemia cells, superior or comparable to that of an anticancer agent gemcitabine. Most of them were less toxic to non-tumour cells than this standard drug, and none had a haemolytic effect on red blood cells. All the tested heterocycles proved to be safer for zebrafish than a standard drug pemetrexed. Some exhibited the ability to inhibit oxidative haemolysis, suggesting their protective action on erythrocytes. The differential scanning calorimetry (DSC) analyses confirmed that all molecules melted within one narrow temperature range, proving their purity. The melting points depended solely on the type of substituent and increased as follows: 4 (R = 3-ClPh) < 2 (R = 4-CH₃Ph) = 3 (R = 4-OCH₃Ph) < 5 (R = 4-ClPh) = 1 (R = Ph) < 6 (R = 3,4-Cl₂Ph). The thermogravimetry/differential thermogravimetry (TG/DTG) studies confirmed high thermal stability of all the investigated heterocycles in inert (>230 °C) and oxidising (>260 °C) atmospheres, which depended directly on the R. The pyrolysis process included one main decomposition stage and was connected with the emission of NH₃, HCN, CH₃CN, HNCO, alkane, alkene, aromatic fragments, CO₂ (for all the compounds), and HCl (for the molecule with 3,4-Cl₂Ph), which was confirmed by FTIR and QMS analyses. In turn, the oxidative decomposition process of the tested polyazaheterocycles took place in two main stages connected with the formation of the same volatiles as those observed in an inert atmosphere and additionally with the release of N₂, NO, CO, and H₂O. These results proved that the pyrolysis and oxidative decomposition run through the radical mechanism connected with the additional reactions between radicals and oxygen in synthetic air. The favourable biological and thermal properties of this class of dihydroimidazotriazinones imply their usefulness as potential pharmaceutics.

Keywords: DSC/TG/DTG/FTIR/QMS; anticancer activity; decomposition course; dihydroimidazotriazinones; in vivo, in vitro and ex vivo toxicity profile; thermal behaviour.

Figure 1

Structures of the investigated compounds: 1. R = Ph; 2. R = 4-CH₃Ph; 3. R = 4-OCH₃Ph; 4. R = 3-ClPh; 5. R = 4-ClPh; 6. R = 3,4-Cl₂Ph.

Figure 2

Zebrafish mortality in the control and compound/standard drug-treated groups at the end of the exposure period. STD—a standard drug pemetrexed. Data represent the mean ± SD of three independent experiments under similar conditions. *—statistically different from the control group (p < 0.05, Student’s t-test).

Figure 3

Hatching rates of zebrafish embryos in the control and compound/standard drug-treated groups. STD—a standard drug pemetrexed. hpf—hours post-fertilisation.

Figure 4

Cardiac function measured by heartbeats per minute in zebrafish exposed to the tested compounds/standard drug. STD—a standard drug pemetrexed. Data represent the mean ± SD of three independent experiments under similar conditions. *—statistically different from the control group (p < 0.05, Student’s t-test).

Figure 5

The representative 96 h old larvae from the control group and groups exposed to the highest concentration of compound/standard drug that did not induce phenotypic abnormalities.

Figure 6

Phenotypic abnormalities (yolk sac swelling, pericardial oedema, and abnormal body shape) observed in 96 h old larvae treated with compounds 1–6 and the standard drug.

Figure 7

DSC curves for the tested compounds collected in an inert atmosphere.

Figure 8

TG (a) and DTG (b) curves for the tested compounds in inert conditions.

Figure 9

The gaseous FTIR spectra collected at T_max1 in inert conditions.

Figure 10

The gaseous QMS spectra collected at T_max1 in inert conditions.

Scheme 1

The course of pyrolysis of the tested compounds in inert conditions.

Figure 11

TG (a) and DTG (b) curves for the tested compounds in oxidising conditions.

Figure 12

The gaseous FTIR spectra collected at T_max1 and T_max2.

Figure 13

The exemplary QMS spectra collected at T_max1 (a) and T_max2 (b) (for compound 2) in oxidising conditions.