Mannich bases of 1,2,4‐triazole‐3‐thione containing adamantane moiety: Synthesis, preliminary anticancer evaluation, and molecular modeling studies

A series of 18 novel N‐Mannich bases derived from 5‐adamantyl‐1,2,4‐triazole‐3‐thione was synthesized and characterized using NMR spectroscopy and X‐ray diffraction technique. All derivatives were evaluated for their anticancer potential against four human cancer cell lines. Several tested compounds exerted good cytotoxic activities on K562 and HL‐60 cell lines, along with pronounced selectivity, showing lower cytotoxicity against normal fibroblasts MRC‐5 compared to cancer cells. The effects of compounds 5b, 5e, and 5j on the cell cycle were investigated by flow cytometric analysis. It was found that these compounds cause the accumulation of cells in the subG1 and G1 phases of the cell cycle and induce caspase‐dependent apoptosis, while the anti‐angiogenic effects of 5b, 5e, and 5j have been confirmed in EA.hy926 cells using a tube formation assay. Further, the interaction of Bax protein with compound 5b was investigated by means of molecular modeling, applying the combined molecular docking/molecular dynamics approach.

viral infections, neurodegenerative disorders, acne vulgaris and type 2 diabetes mellitus. [11] RARγ-selective adamantylbased compound induces G0/G1 cell cycle arrest and apoptosis in human breast carcinoma and human leukemia cell lines in a p53-independent fashion. [12] It is also found to be a potent growth inhibitor of cell lung cancer (NSCLC) lines with IC 50 values in the range of <0.13-0.53 μm. [13] The scientific literature regarding anticancer activity of compounds which contain both adamantoyl and 1,2,4-triazole moieties is very scarce. One of such compounds, 4-(1-ada mantyl)-5-[2-(3-hydroxynapthyl)]-2H-1,2,4-triazole-3(4 H)-thione, was evaluated for its in vitro anticancer activity against MCF-7 breast cancer cell line giving IC 50 value of 8.15 μm. [14] Most common chemotherapeutic drugs, doxorubicin, cisplatin, and 5-fluorouracil, used for treating a wide range of cancers, induce reduced cancer cell division and growth. Chemotherapy does not distinguish between a cancer and normal cells and destroys not only the fast-growing cancer cells but also healthy cells in the body. Therefore, it is of most importance to design and perform biological screening of new pharmacophores, which could potentially enhance the selectivity of cytotoxic agents against cancer cell lines and decrease their toxic effects toward normal cells. Numerous newly synthesized compounds which possess excellent cytotoxic activity display very low cancer selectivity. Having in mind these facts, in this study, we examined cytotoxicity, cancer selectivity, and plausible mechanism of action of 18 novel N-Mannich bases of 5-adamantyl-1,2,4-triazole-3-thio ne, which displayed a pronounced selectivity showing IC 50 values for MRC-5 significantly higher in comparison with cancer cell lines. In addition, molecular action mode of these compounds was investigated through Western blot analysis of key proteins involved in apoptosis, anti-angiogenic activity, and molecular docking studies.

| Physical measurements and methods
Melting points were determined on a Mel-Temp capillary melting points apparatus, model 1001, and are uncorrected. Elemental (C, H, N, S) analysis of the samples was carried out in the Center for Instrumental Analysis, Faculty of Chemistry, Belgrade. IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer with a KBr disk. 1 H and 13 C NMR spectra were recorded on a Varian Gemini 200-MHz spectrometer. Crystal structures were determined by single-crystal X-ray crystallography. Diffraction measurements were performed on an Oxford Diffraction Gemini S diffractometer. Structures were solved using the SHELXT [15] and refined with the SHELXL. [16] Details of preparation and structure determinations are given in the Supporting information.

| In vitro cytotoxicity assay
Human cervical adenocarcinoma HeLa, human chronic myelogenous leukemia K562, human acute promyelocytic leukemia HL-60, human lung carcinoma A549, and normal human lung fibroblasts MRC-5 were grown in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA). Nutrient medium was supplemented with 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin (Sigma-Aldrich). Medium for HL-60 cells was supplemented with 20% fetal bovine serum. HeLa (2,000 cells per well), K562, A549, and MRC-5 (5,000 cells per well), and HL-60 (7,000 cells per well) were seeded in 96-well plates. The tested compounds were added to the cells 20 hr later (for adherent cell lines) or 2 hr later (for cell lines that grow in a suspension). The nutrient medium was added to control cells. Final concentrations of compounds were 6.25, 12.5, 25, 50, and 100 μm. Cisplatin was used as a positive control. After 72-hr treatment, the survival of cells was measured by MTT test, as described earlier. [17,18] More details of determination of cell survival are given in the Supporting information.

| Cell cycle analysis
Chronic myelogenous leukemia K562 cells were seeded into a six-well plates (2.5 × 10 5 cells/well), and after 2 hr treated with compounds, with the exception of control cells. Tested concentrations corresponded to IC 50 and 2IC 50 values. After 24-hr treatment, the cells were collected, and fixed as described elsewhere. [19] Cell cycle distribution was analyzed after incubation of cells with RNAse and propidium iodide. The statistical significance of differences in percentages of cells at specific cell cycle phases between control samples and samples exposed to tested compounds was evaluated using repeated measures ANOVA with Tukey's multiple comparison test. The p-values <0.05 were regarded as statistically significant.

| Morphological analysis of cell death mode
K562 cells were seeded into six-well plates in 2 ml of complete nutrient medium (2 × 10 5 cells/well). After 2 hr, the cells were treated with 2IC 50 concentrations of the investigated compounds. After 24-hr treatment, the cells were collected by centrifugation and stained with acridine orange/ ethidium bromide mixture (3 μg/ml AO and 10 μg/ml EB in phosphate-buffered saline), and visualized under a fluorescence microscope (Carl Zeiss PALM MicroBeam with Axio Observer.Z1 using AxioCam MRm).

| Tube formation assay
To examine the in vitro anti-angiogenic activity of selected compounds 5b, 5e, and 5j on human umbilical vein endothelial EA.hy926 cells, the tube formation assay was performed. Briefly, 24-well plates were coated with Corning ® Matrigel ® Basement Membrane Matrix, (Corning, 356234). EA.hy926 cells were seeded onto the coated wells (4 × 10 4 cells per well), with or without the investigated compounds. Concentrations of the compounds 5b, 5e, and 5j corresponded to IC 20 values (15 μm) previously determined for this cell line for 24-hr treatment. After 24 hr of incubation, the cells were photographed under the inverted microscope.

| Chemistry
The synthetic pathway for the targeted novel N-Mannich bases of 5-adamantyl-1,2,4-triazole-3-thione is presented in Scheme 1. The synthesis starts from a commercially available 1-adamantanecarboxylic acid 1 which was converted into 1-adamantanecarbonyl chloride 2 in the reaction with thionyl chloride in the presence of catalytic amounts of N,N-dimethylformamide (DMF). The corresponding acylthiosemicarbazide 3 was obtained with very high purity by stirring acid chloride 2 with thiosemicarbazide in dry tetrahydrofuran (THF) at room temperature. The intramolecular cyclization of acylthiosemicarbazide 3 was performed by addition of 2 m aqueous solution of NaOH and refluxing for 3 hr. After refluxing and cooling, the clear solution was acidified using 2 m HCl until pH 1 was reached followed with the formation of the precipitate of the 5-adamantyl-1,2,4-tri azole-3-thione 4. The final compounds 5a-r were prepared in a Mannich reaction which included stirring of the equimolar concentrations of 5-adamantyl-1,2,4-triazole-3-thio ne 4, formalin, and corresponding primary aromatic amine in THF for 2 days. Then, the solvent was evaporated, and EtOH was added to the residue. The compounds 5a-r were obtained with excellent purity excluding any need for further purification.
The structure of all synthesized compounds was determined by means of 1 H and 13 C NMR spectroscopy (see Figs S1-S38).
Theoretically, the alkylation of 5-substituted-1,2,4-triazo le-3-thione can be performed at N2 and N4 atom of triazole ring. Herein, we have obtained a suitable single crystal of 5e and by means of X-ray crystallography unambiguously demonstrated that the aminomethylation occurs exclusively at N2 atom of triazole moiety ( Figure 1, CCDC 1465502-1465503). For more details, see Supporting information (cif file and checkcif report).

| In vitro cytotoxicity
The cytotoxic activity of 5-phenyl-1,2,4-triazole-3-thione was determined, proving this compound to be inactive against K562 cell line showing IC 50 value of 200.00 μm. To determine its influence on the activity, the corresponding phenyl group was substituted with adamantyl group. The cytotoxic activity of the synthesized 5-adamantyl-1,2,4-triazole-3-thione (4) was also determined against K562 cells, and the IC 50 value for this compound was 101.38 μm. This increase in the activity, caused by the incorporation of the adamantyl group, has encouraged us to perform the synthesis of the corresponding Mannich bases starting from the intermediate 4 and to investigate their cytotoxic potential. Cytotoxic activity of the newly synthesized triazoles was evaluated against four malignant cell lines (HeLa, K562, HL-60, and A549) as well as against normal, non-transformed MRC-5 cells. The obtained IC 50 values are shown in Table 1. Most of the compounds that displayed the highest cytotoxic activities against K562 cell line possess a substituent at ortho-position of the phenyl ring (5b, 5e, 5h, 5j). On the other hand, all the tested compounds showed comparable activity against HL-60 cells, where compounds containing different substituents at meta-position (5c, 5f, 5k) exerted the weakest activity. The electronic effect of the substituent does not have a noticeable influence on the activity, as both electron-donating and electron-withdrawing substituents showed similar effectiveness against HL-60 cancer cells. Some of the active and highly selective compounds against K562 cell line 5b, 5e, and 5j were chosen for further examination of the mechanisms of their anticancer activity.

| Cell cycle analysis and fluorescence microscopy
We examined the mechanisms of cytotoxic activity of the selected compounds 5b, 5e, and 5j by flow cytometric analysis, using propidium iodide to label DNA. Figure 2 shows cell cycle distribution of K562 myelogenous leukemia cells incubated in the absence or presence of investigated compounds. The obtained results demonstrate that after 24-hr treatment with IC 50 and 2IC 50 concentrations of triazoles 5e, and 5j, the percentages of K562 cells in the G1 cell cycle phase were significantly higher compared with the percentage of cells within G1 phase in the control cell sample. The G1 cell cycle phase accumulation was also observed in K562 cells exposed to compound 5b, although this increase was not statistically significant. The increase in the G1 phase cell population was accompanied by a decrease in the G2/M phase cell populations. Our results are in accordance with literature data about different triazoles which induce G1 cell cycle phase arrest. [25,26] In addition, treatment of K562 cells with 2IC 50 concentrations of 5b, 5e, and 5j led to increase of cells in the subG1 phase in comparison with control cells. After 24-h treatment with triazoles 5b, 5e, and 5j, K562 cells were stained with a mixture of acridine orange and ethidium bromide and analyzed by fluorescence microscopy. As shown in Figure 3, the morphological changes typical for apoptotic cell death were observed in K562 cells treated with 2IC 50 concentrations of compounds. In the non-treated control, the K562 cells had normal morphology. In contrast, early apoptotic cells stained green with condensed chromatin in the shrunken nuclei or even fragmented nuclei could be seen in the cell samples exposed to triazoles in addition to orange-red-stained late apoptotic cells. The obtained results indicate that 5b, 5e, and 5j are able to induce apoptotic cell death in myelogenous leukemia K562 cells.

| Western blot analysis
Molecular mechanisms underlying cytotoxic activity of the compounds 5b, 5e, and 5j were further demonstrated by Western blot analysis (Figure 4). Activation of both principal apoptotic signal pathways [27] results in the activation of cysteine aspartic acid-containing proteases (caspases). Caspase-8 and caspase-9 are usually involved in extrinsic or death receptor-mediated pathway and the intrinsic or mitochondria-mediated pathway, respectively, while caspase-3 is downstream of both caspase-8 and caspase-9. The caspases are activated by cleavage, which further activates the downstream substrate molecules. [28] The protein levels of full-length caspase-8 and caspase-3 in cells treated with compounds 5b, 5e, and 5j were increased in comparison with untreated, control cells. As shown in Figure 4, this increase was accompanied by strong appearance of active fragments. The most intensive effect was recognized for the compound 5j, while the effects declined for compounds 5b and 5e, respectively. Although an increase in protein levels of full-length caspase-9 was also observed in treated cells, it was less prominent comparing to other procaspases examined.
We further examined whether the protein expression levels of Bax were altered following the treatment with compounds 5b, 5e, and 5j. Bax is a member of the Bcl-2 protein family.
As key regulators of apoptosis, these proteins are involved in the control of mitochondrial permeability and, in particular, the release of apoptogenic proteins from this organelle. [29] Western blot analysis showed that Bax expression was substantially upregulated in K562 cells treated with all three compounds tested. Furthermore, a smaller band of ̴ 18-kDa was detected in addition to a 21-kDa band in treated cells. Detected cleavage fragment of Bax protein, p18, has been reported to be produced by calpain and to accelerate apoptosis in numerous tumor cell lines following treatment with various chemotherapeutic or biological agents. [30] In contrast, F I G U R E 4 Induction of caspase-dependent apoptosis by compounds 5b, 5e, and 5j at IC 50 concentrations in K562 myelogenous leukemia cells after 24-hr treatment. Immunoblotting was performed using anticaspase 3, anticleaved caspase 3, anticaspase 8, anticaspase 9 and antiBax antibodies. The anti-GAPDH antibody was used as a control for protein load. Besides full-length caspases, procaspases, the fragments of activated caspase-3 (subunits p19 and p17), fragments of activated caspase-8 (the cleaved intermediates p43/p41, the active fragments/subunits p18, p16, and p10), and fragments of activated caspase-9 (subunits p37 and p35) were detected | 949 mILOŠEV Et aL.
18-kDa Bax was not detected in the control cells. We further demonstrated that caspase-dependent apoptosis is involved in compounds 5b-, 5e-, and 5jinduced cytotoxicity in K562 cells and that both intrinsic and extrinsic pathway seem to be involved in induction of apoptosis. The intrinsic pathway is characterized by mitochondrial dysfunction with release of caspase activators, followed by activation of caspase-9 and caspase-3. Mitochondrial-induced apoptosis further requires involvement of the Bcl-2 family, including Bax. Caspase-8, on the other hand, is one of the upstream mediators of cell death, and its activation is associated with an increase in TNF-like cytokines, such as TNF-α and Fas ligand (FasL; CD95). [31] Caspase-8 is inactivated in a variety of human cancers, which may promote tumor progression as well as resistance to current treatment approaches. [32,33] A significant increase in its full-length and cleaved fragments is consistent with the high cytotoxic efficacy of selected compounds. In addition, augmentation of apoptosis in treated cells can also be attributed to upregulation of calpain-produced Bax fragment. Previous studies have demonstrated that 18-kDa Bax, a characteristic feature of Bax activation, is a more potent inducer of apoptosis than 21-kDa Bax. [34] Further research confirmed that interruption of Bax cleavage reduces druginduced apoptosis, while depression of 18-kDa Bax degradation significantly augments drug-mediated apoptosis. [35]

| Anti-angiogenic activity
Angiogenesis represents the formation of new blood vessels and has a fundamental role in development, reproduction, and repair of the tissue including tumors. [36,37] Dawson et al. have showed that an adamantyl-substituted, retinoid-derived molecule effectively reduces human microvascular endothelial (HMVE) cell growth (IC 50 = 0.3 μm) and suggested its potential in vivo anti-angiogenic activity. [38] Itraconazole, a compound containing 1,2,4-triazole, has been found to possess potent and selective inhibitory activity against multiple key aspects of tumor-associated angiogenesis, both in vitro and in vivo. [39] One of the most frequently used and best characterized permanent human vascular endothelial cell line in angiogenesis research is EA.hy926. [40] To evaluate the anti-angiogenic potential of the compounds 5b, 5e, and 5j applied at subtoxic concentrations, a tube formation assay, also known as in vitro angiogenesis assay was performed. As it could be seen in the Figure 5, reduced number of elongated and connected EA.hy926 cells in addition to significantly inhibited formation of tubular and polygonal structures was observed in the cell samples treated with compounds 5b, 5e, and 5j compared with control cells. The compound 5j exerted more pronounced anti-angiogenic effect in comparison with 5b and 5e. Our results point out the significant antiangiogenic potential of investigated triazoles 5b, 5e, and 5j.

| The mechanism of 5b proapoptotic activity
The treatment of chronic myelogenous leukemia K562 cells with compound 5b elevated levels of pro-apoptotic protein Bax, caspase-8, and main effector caspase-3, demonstrating that this triazole triggered both intrinsic and extrinsic apoptotic pathways. [41] In modern apoptosis-based anticancer therapy, one of the recognized approaches is to identify small molecules that F I G U R E 5 Effects on in vitro angiogenesis of EA.hy926 cells. Photomicrographs of control EA.hy926 cells and cells exposed to subtoxic IC 20 concentrations (15 μm) of compounds 5b, 5e, and 5j after 20-hr treatment 950 | mILOŠEV Et aL.
activate pro-apoptotic Bcl-2 proteins [42] like Bax. Consistently, 5b, as confirmed inducer of apoptosis, might exert its anticancer action as small-molecule Bax agonist. [43] Thus, 5b pro-apoptotic mode of action is exerted most likely via the displacement model pathway [44] which involves: (i) the Bax activation, as an executioner protein, with agonist, (ii) the interaction of Bax-5b pro-apoptotic complex with antiapoptotic enzymes Bcl-XL and/or Bcl-2, (iii) the extrusion of Bax-5b from anti-apoptotic enzymes with BH3 sensitizer proteins like Bad, Bik, Bmf, Bnip3, Noxa, or Hrk, (iv) the Bax-5b penetration into the outer mitochondrial membrane (MOM) and dimerisation, (v) membrane disruption to release intermembrane space (IMS) proteins such as cytochrome c, OMI/HTRA2, SMAC/DIABLO, and endonuclease G, [45] and (vi) the activation of caspase-3 as an effector of apoptosis. [46] The displacement model steps (i), (ii) (see Supporting information), and (iv) (see Supporting information) were considered in this report by means of molecular modeling to reveal the pharmacology of 5b, applying the combined molecular docking/molecular dynamics approach.
Bax agonists target the C-terminal α9 helix. [42,43] To the best of our knowledge, there is no available crystal data regarding the binding modes of any Bax agonists, while there is limited structure-based derived information about smallmolecule Bax agonists positioning. [42,43] Therefore, the blind docking procedure had been applied to outline the 5b bioactive conformation ( Figure 6) using the scoring function available in autODOck4.2 [47] program. During the Bax activation (step 1), 5b was stabilized within the α9 helix [42] mainly due to the established advantageous hydrophobic interactions with the helix residues. Hence, the adamantyl moiety was involved in the steric clash with Val180 side chain. The o-tolyl scaffold was encircled by Ala183 and Ile187 where the hydrophobic interference is constituted via the aromatic ring inasmuch as the o-methyl group was pointed away from the C-terminal domain. The orientation of adamantyl and o-tolyl groups provided some more interesting observations. Both of the functionalities were positioned in front of the Ser184, a key amino acid appointed as a target for post-translational phosphorylation during the prevention of apoptotic process [42] by anti-apoptotic proteins. If this post-translational modification is avoided, Ser184 provides the C-terminal domain stabilization by means of C-terminus helix merging with the outer mitochondrial membrane. Consequently, the pro-apoptotic protein Bax prevails in effort to induce apoptosis. [48] Small-molecule Bax agonists induce conformational changes in Bax by blocking Ser184 phosphorylation, facilitating Bax insertion into mitochondrial membranes and forming Bax oligomers. [43] The role of the 5b as the smallmolecule Bax agonist in the induction of apoptosis will be discussed lately in text, but at this point, it is crucial to accentuate that adamantyl and o-tolyl groups both served as a sort of spatial gate in front of the Ser184 and therefore prevented the amino acid phosphorylation before the actual interaction of Bax with the anti-apoptotic proteins.
The second level of Bax-5b complex stabilization was provided by 1,2,4-triazole-3(4H)-thione ring where sulfanylidene group as hydrogen-bond (HB) acceptor was narrow to Ser101 forming a strong hydrogen bond (d HB = 2.501 Å), while the HB donor nitrogen at position 4 of heterocyclic ring was in the close proximity to Asp102 (d HB = 1.767 Å). At last, the imino nitrogen at position 1 was electrostatically attracted by the α4 helix residue Asp98 and during the rigid docking simulation, this interaction has not disturbed the hydrogen bond between the Asp98 and Ser184. [21] Accordingly, 5b positioning within the Bax C-terminal domain was characterized by the binding affinity of K d = −9.34 kcal/mol and inhibition constant of Ki = 0.76 μm.
The general stability of Bax-5b was confirmed by molecular dynamics study (Fig. S41, Supporting information). Thus, the complex was stabile after 0.82 ns by means of RMSD (Fig. S41A) and RMSF values (ligand interactions in range F I G U R E 6 The best docked pose of 5b (agonist colored in blue) as predicted by autODOck4.2. The Bax α1 helix is colored in red, α2 helix in forest green, α3 helix in cornflower blue, α4 helix in magenta, α5 helix in gold, α6 helix in steel blue, α7 helix in olive grab, α8 helix in siena, and α9 helix in orange. from 0.597 to 1.638 Å; Fig. S41B). The energy decomposition procedure for Bax-5b complex outlined that calculated binding affinity of 5b (Table 2) correctly predicted the possibility of agonist to interact with the Bax C-terminus. The low value of binding energy of 5b (ΔG bind = −51.78 kcal/ mol) suggests that the agonist is easily bound to the α9 helix. As expected, hydrophobic interactions were dominant for 5b binding (ΔE vdw = −54.13 kcal/mol) while the significant contribution was perceived for electrostatic interactions too (ΔE ele = −33.52 kcal/mol). The relatively low value for the solvation energy (ΔE solv = 2.33 kcal/mol) showed that 5b was stable but no so tightly bound to the C-terminus. In that manner, 5b suffered conformational perturbation immediately after the start of simulation (0.01 ns) in manner that hydrogen bonds between the sulfanylidene group and Ser101 as well as between nitrogen at position 4 and Asp102 were broken and 1,2,4-triazole-3(4H)-thione ring was rotated toward Asp98. This advantageous alignment of 1,2,4-triazole-3(4H)-thione was supported by the establishment of new hydrogen bond between the nitrogen at position 4 and Asp98-Met99 peptide bond carbonyl oxygen, which arouse after 0.149 ns and remained stabile until the end. The disagreement between the pose obtained by molecular dynamics (5b-MD) and the best docked one (5b-BD) however cannot be related to false docking outcome, but can be attributed to applied rigid docking protocol where no flexibility for amino acids was allowed. The RMSD between the 5b-BD and 5b-MD amounted 1.748 Å, suggesting that both 5b-BD and 5b-MD are actually borderline solutions of 5b physiologically conformation and the active structure itself is in between the 5b-MD and 5b-BD. As a result of docking/dynamics-coupled simulation, Asp98 was estranged from Ser184 and kept away from latter residue due to hydrophobic repulsion between the 5b sulfanylidene group and Asp98 side-chain methylene group initially observed after 0.28 ns of simulation and occurred during the whole period of stabilization. This predicted conformational change for protein is vital for the Bax proapoptotic efforts inasmuch as it resulted is the degeneration of Asp98-Ser184 hydrogen bond [21] occurring in agonist-free Bax structure. Particularly, if this hydrogen bond remains stabile, Bax may actually inhibit itself even before the interaction with anti-apoptotic proteins as it becomes incapable to invade the mitochondrial membrane via the C-terminus. [21,49] On the other hand, the elimination of Ser184-Asp98 hydrogen bond promotes the dissociation of the Bax C-terminal helix from the hydrophobic groove through major conformational change, making it more accessible to mitochondrial membrane. [21,49] In that manner, 5b initially exerts pro-apoptotic potential by forcing the Asp98 away from Ser184 and facilitates Bax-mediated apoptotic event.
Utilizing the molecular modeling approach, it was decidedly proven that Bax-5b complex as apoptosis initiator. The remaining steps of the displacement model pathway still remain poorly understood by means of luck of experimental support, but for the purpose of 5b pro-apoptotic potential understanding, the contribution in the form of molecular modeling (Supporting information) was given for steps (ii) and (iv), outlining the interaction of Bax-5b with antiapoptotic proteins Bcl-XL and Bcl-2 and Bax-5b facilitation of MOM disturbance-based apoptosis, respectively.
An additional discussion relating to molecular docking and molecular dynamics studies of Bax-5b-Bcl-XL /Bax-5b-Bcl2 complexes formation is presented in Supporting information.

| CONCLUSION
A novel class of 1,2,4-triazole N-Mannich bases containing adamantane moiety has been synthesized. The confirmation of the structure of these compounds and determination of the exact nitrogen atom at which Mannich reaction occurs were obtained by X-ray diffraction technique. The investigated compounds showed moderate-to-good cytotoxic activities against four malignant cell lines, with K562 and HL-60 cells being significantly more sensitive. The low toxicity of tested derivatives toward normal MRC-5 cell line indicated their prominent selectivity in the anticancer action. The selected active compounds 5b, 5e, and 5j induced accumulation of K562 cells in subG1 and G1 phase. Western blot analysis demonstrated that selected compounds exert their cytotoxic activity through the caspase-dependent apoptosis and T A B L E 2 Binding free energies and individual energy terms of ATP-rTopIIα complex with various inhibitors upregulation of Bax expression levels. Also, it was found, using a tube formation assay, that tested derivatives exhibit anti-angiogenic effects. Finally, the molecular modeling study revealed, applying the combined molecular docking/ molecular dynamics approach, that compound 5b has a role in protection of Bax Ser184 from phosphorylation to facilitate the pro-apoptotic event.