Angular Regioselective Synthesis of Varied Functionalized Hexahydro-1,2,4-triazolo[4,3-a]quinazolin-9-ones and Their Antiproliferative Action

New 2-thioxopyrimidin-4-ones capable of participating in regioselective reactions with functionally diverse hydrazonoyl chlorides towards angular regioisomers, rather than linear ones, were designed and synthesized to form stereoisomeric cis- and trans-hexahydro [1,2,4]triazolo[4,3-a]quinazolin-9-ones to be tested as antitumor candidates. The angular regiochemistry of the products was verified through crystallographic experiments and NMR studies. In addition, the regioselectivity of the reaction was found to be independent of the stereochemistry of the used 2-thioxopyrimidin-4-one. Only compound 4c demonstrated satisfactory growth inhibition against all the cancer cells used among all the produced drugs.

Cancer remains the second leading cause of mortality in both industrialized and developing nations, despite the great advancements in cancer therapy that have increased the cure rates for a variety of malignancies [28]. Chemotherapy is one of the main treatment options for cancer patients. The toxicity and drug resistance [29] of the existing chemotherapeutics, however, restrict their use in the treatment of cancer patients. Therefore, the development of safe chemotherapy drugs with anticancer properties is urgently needed.
Continuing our research, we designed and synthesized new 2-thioxopyrimidin-4ones achieving regioselective reactions with functionally diverse hydrazonoyl chlorides 3a-h. These new 2-thioxopyrimidin-4-ones are cis and trans stereoisomers of cyclohexenecondensed 2-thioxopyrimidin-4-ones 1 and 2. The product molecules can be reacted further to form novel angular cisand trans-1,2,4-triazoles 4a-h and 5a-h with various functionalities which have the C=C bond for further derivatization. X-ray and NMR investigations were employed to establish the stereochemistry of the compounds. Furthermore, the antiproliferative action of the prepared compounds was also examined.

Results and Discussion
The cyclohexene-based 2-thioxopyrimidin-4-one 1 and its trans stereoisomer 2 precursor molecules were synthesized according to the literature [33]. The reactions of thioxopyrimidinones 1 or 2 with functionally diverse hydrazonoyl chlorides 3a-h were performed in dioxane as a solvent using triethylamine as a basic additive under reflux conditions for 6-8 h (Scheme 1). As shown in Scheme 2, the reaction proceeds through either path A or B, depending on the tautomeric structure I or ii that directs the reaction via S-alkylation to form S-alkylated intermediates iii or v, respectively. These then undergo Smiles rearrangement [34], yielding intermediates iv or vi, followed by cyclization through the elimination of H 2 S to afford angular regioisomers 4a-h and 5a-h or linear ones 6a-h and 7a-h, respectively. Because of the conjugation of the C=N and C=O bonds, the tautomeric form i is favored in comparison to ii. Consequently, the reactions proceeded through path A, which accounts for the regioselectivity of the reactions towards the angular isomers 4a-h and 5a-h rather than the linear ones 6a-h and 7a-h. This regioselectivity was confirmed using a variety of tests, including chromatography, 1 H-NMR 13 C-NMR spectroscopy, and X-ray crystallographic analysis. TLC, following the reaction, indicated the formation of only a single product. The 1 H-NMR spectra (Supplementary Materials) showed the resonances of only a single isomer. In addition, in the case of hydrazonoyl chlorides 3a-f, the signal of the methylene (CH 2 ) moiety of the ester functional group appeared to have higher multiplicity than expected (quartet). This is due to the proximity of the ester group to the cyclohexene moiety in the angular regioisomer. The 13 C-NMR spectrum (Supplementary Materials) exhibited the resonance of the carbonyl carbon (CO) of the pyrimidinone ring at almost 176 ppm, which is in accordance with the reported values of structurally related carbonyl carbon atoms in the literature [35]. In these compounds, where the carbonyl carbon of the pyrimidinone residue in the angular structure is adjacent to the sp 2 -hybridized nitrogen (releasing less electrons), the carbonyl group is less shielded, and it resonates at 170-176 ppm. In contrast, the nitrogen atom in the linear structure is sp 3 -hybridized (releasing more electrons), and consequently the carbonyl group is more shielded and resonates at lower δ values (160-165 ppm). Finally, X-ray crystallographic analysis of 5a ( Figure 1) provided indisputable evidence of the angular stereochemistry of the product.
As concerns the antiproliferative properties of the compounds thus prepared, none of them proved to be comparable with the reference agent cisplatin ( Table 1). The most active analog was 4c, eliciting 30-50% growth inhibition at 30 µM against all the cancer cells used. Incubation with compound 5h resulted in cell growth inhibition above 30% against 3 cell lines. Though no clear tendency was observed concerning the role of stereochemistry in the antiproliferative activity of the compounds, our results indicate that the cis arrangement of the p-nitrophenyl substituent on the triazole ring may be attractive for anticancer drug candidates with a similar scaffold. Treatment with compounds 4d, 4g, and 4h resulted in less than 20% growth inhibition at the higher concentration against only a single cell line. All other molecules (4a, 4b, and 5a) elicited no relevant antiproliferative action (i.e., less than 10% growth inhibition) against the tested cancer cell lines.  As concerns the antiproliferative properties of the compounds thus prepared, none of them proved to be comparable with the reference agent cisplatin ( Table 1). The most active analog was 4c, eliciting 30-50% growth inhibition at 30 µM against all the cancer cells used. Incubation with compound 5h resulted in cell growth inhibition above 30% against 3 cell lines. Though no clear tendency was observed concerning the role of stereochemistry in the antiproliferative activity of the compounds, our results indicate that the cis arrangement of the p-nitrophenyl substituent on the triazole ring may be attractive for anticancer drug candidates with a similar scaffold. Treatment with compounds 4d, 4g, and 4h resulted in less than 20% growth inhibition at the higher concentration against only a single cell line. All other molecules (4a, 4b, and 5a) elicited  As concerns the antiproliferative properties of the compounds thus prepared, none of them proved to be comparable with the reference agent cisplatin ( Table 1). The most active analog was 4c, eliciting 30-50% growth inhibition at 30 µM against all the cancer cells used. Incubation with compound 5h resulted in cell growth inhibition above 30% against 3 cell lines. Though no clear tendency was observed concerning the role of stereochemistry in the antiproliferative activity of the compounds, our results indicate that the cis arrangement of the p-nitrophenyl substituent on the triazole ring may be attractive for anticancer drug candidates with a similar scaffold. Treatment with compounds 4d, 4g, and 4h resulted in less than 20% growth inhibition at the higher concentration against only a single cell line. All other molecules (4a, 4b, and 5a) elicited  As concerns the antiproliferative properties of the compounds thus prepared, none of them proved to be comparable with the reference agent cisplatin ( Table 1). The most active analog was 4c, eliciting 30-50% growth inhibition at 30 µM against all the cancer cells used. Incubation with compound 5h resulted in cell growth inhibition above 30% against 3 cell lines. Though no clear tendency was observed concerning the role of stereochemistry in the antiproliferative activity of the compounds, our results indicate that the cis arrangement of the p-nitrophenyl substituent on the triazole ring may be attractive for anticancer drug candidates with a similar scaffold. Treatment with compounds 4d, 4g, and 4h resulted in less than 20% growth inhibition at the higher concentration against only a single cell line. All other molecules (4a, 4b, and 5a) elicited

General Methods
NMR characterization of the product compounds was carried out in CDCl 3 at room temperature (500.20 MHz for 1 H-NMR, 125.62 MHz for 13 C-NMR) using a Bruker AV NEO Ascend 500 spectrometer with a Double-Resonance Broad-Band Probe (Bruker Biospin, Karlsruhe, Germany). As an internal standard, tetramethylsilane (TMS) was used. Thinlayer chromatography (TLC) was conducted to monitor the reaction progress (aluminum sheets, silica gel coating (POLYGRAM ® SIL G/UV254, Merck, Darmstadt, Germany) with evaluations upon UV illumination. Melting points were measured using Hinotek-X4 micro melting point equipment (Hinotek, Ningbo, China). The HRMS flow injection study was carried out using a Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer linked to a Waters Acquity I-Class UPLCTM (Thermo Fisher Scientific, Waltham, MA, USA) (Waters, Manchester, UK).
The synthesis of cisand trans-thioxopyrimidinones (1 and 2) was carried out by transforming the corresponding amino esters, as described in the literature [36,37]. The synthesis of hydrazonoyl chlorides 2a-h was performed in accordance with the previously described methods [38,39].

Determination of Antiproliferative Properties of the Prepared Compounds
The antiproliferative actions of the selected compounds (4a-e, 4g,h, and 5a-h) were investigated through the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using a set of adherent cell lines isolated from human (SiHa), ovarian (A2780), and breast (MCF-7 and MDA-MB-231) cancers [43]. All cell lines were obtained from the European Collection of Cell Cultures (ECCAC, Salisbury, UK), except for SiHa, which was purchased from the American Tissue Culture Collection (Manassas, VA, USA). The cells were grown in minimal essential medium supplemented with fetal bovine serum (10%), non-essential amino acids (1%), and a penicillin-streptomycin-amphotericin B mixture (1%). All cell culture components were purchased from Lonza Group Ltd. (Basel, Switzerland). Malignant cells were plated into 96-well plates at the density of 5000/well, and the next day, the test substance was added in 10 µM or 30 µM final concentrations. After 72 h of incubation, MTT solution (5 mg/mL, 20 µL) was added to each well and incubated for 4 h. Finally, the medium was discarded, and the formazan was solubilized in 100 µL DMSO during 60 min of shaking at 37 • C. The absorbance was determined at 545 nm using a microplate reader (SpectoStarNano, BMG Labtech, Ortenberg, Germany). Two independent experiments were carried out with five wells for each condition, and cisplatin (Ebewe GmbH, Unterach, Austria) was included as a reference agent.

Conclusions
New angular 1,2,4-triazolo[4,3-a]quinazolin-9-ones with various functionalities were prepared in a simple and regioselective manner by reacting variously functionalized hydrazonoyl chlorides and cyclohexene-based 2-thioxopyrimidin-4-ones 1 and 2 that favored the formation of angular products rather than linear ones. Both cis and trans isomers of 2-thioxopyrimidin-4-ones led to the formation of angular products. Among all the prepared compounds, only the cis isomer of triazole 4c with a p-nitrophenyl substituent of the ring revealed significant growth inhibition against all the used cancer cells.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/molecules28093718/s1, NMR spectra of all synthesized compounds and crystallographic details of 5a and Table S1: Crystal Data.