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Article

Novel Thiazolidine-2,4-dione-trimethoxybenzene-thiazole Hybrids as Human Topoisomerases Inhibitors

by
Maria Stefania Sinicropi
1,†,
Jessica Ceramella
1,†,
Patrice Vanelle
2,
Domenico Iacopetta
1,*,
Camillo Rosano
3,
Omar Khoumeri
2,
Shawkat Abdelmohsen
4,
Wafaa Abdelhady
4 and
Hussein El-Kashef
4,5,*
1
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Italy
2
Aix Marseille University, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin, CS30064, CEDEX 05, 13385 Marseille, France
3
U.O. Proteomica e Spettrometria di Massa, IRCCS Ospedale Policlinico San Martino, Largo R. Benzi 10, 16132 Genova, Italy
4
Department of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt
5
Faculty of Pharmacy, Sphinx University, New Assiut 71684, Egypt
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pharmaceuticals 2023, 16(7), 946; https://doi.org/10.3390/ph16070946
Submission received: 15 May 2023 / Revised: 9 June 2023 / Accepted: 26 June 2023 / Published: 29 June 2023
(This article belongs to the Topic Compounds with Medicinal Value (2nd Volume))
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Abstract

:
Cancer is a complex and heterogeneous disease and is still one of the leading causes of morbidity and mortality worldwide, mostly as the population ages. Despite the encouraging advances made over the years in chemotherapy, the development of new compounds for cancer treatments is an urgent priority. In recent years, the design and chemical synthesis of several innovative hybrid molecules, which bring different pharmacophores on the same scaffold, have attracted the interest of many researchers. Following this strategy, we designed and synthetized a series of new hybrid compounds that contain three pharmacophores, namely trimethoxybenzene, thiazolidinedione and thiazole, and tested their anticancer properties on two breast cancer (MCF-7 and MDA-MB-231) cell lines and one melanoma (A2058) cell line. The most active compounds were particularly effective against the MCF-7 cells and did not affect the viability of the normal MCF-10A cells. Docking simulations indicated the human Topoisomerases I and II (hTopos I and II) as possible targets of these compounds, the inhibitory activity of which was demonstrated by the mean of direct enzymatic assays. Particularly, compound 7e was proved to inhibit both the hTopo I and II, whereas compounds 7c,d blocked only the hTopo II. Finally, compound 7e was responsible for MCF-7 cell death by apoptosis. The reported results are promising for the further design and synthesis of other analogues potentially active as anticancer tools.

Graphical Abstract

1. Introduction

Cancer remains one of the foremost and crucial public health concerns. It is identified as the second leading cause of death after cardiovascular pathologies worldwide [1,2], where about 19.3 million new cancer events and about 10.0 million cancer deaths were recorded in the year 2020 [3]. Among women, breast cancer (BC) is the most frequently diagnosed and prevalent cancer, and has been reported as the second leading cause of death after lung cancer [4]. The World Health Organization estimated 2.3 million cases of breast cancer, with 685,000 deaths around the world, in 2020 [5]. Currently, chemotherapy is the main treatment for BC, but its toxicity to normal cells and acquired drug resistance are the major drawbacks. Thus, the development of safer and more potent anti-BC agents is highly required [6]. Thiazolidine-2,4-diones (TZDs) were proved to be privileged pharmacophore scaffolds in medicinal chemistry [7]. They possess a wide range of pharmacological properties [8,9,10,11,12,13], including anticancer activity [14]. It was described that TZDs may affect cancer onset, progression and metastasis [15,16]. These effects are due to the interference with the Raf/MEK/extracellular signal regulated kinase (ERK) [17], phosphatidylinositol 3-kinase (PI3K)/AKT [18], Wnt signal transduction pathways [19], PIM-1 and PIM-2 protein kinases overexpression [20], androgen receptor regulated genes [21,22] and peroxisome proliferator-activated receptors (PPARs) signaling cascades [23], which are often up-regulated in human cancers. Moreover, thiazole derivatives are known by their anticancer activities [24], in addition to their variety of other biological properties [25,26,27,28,29,30,31,32,33,34,35,36,37]. It is of interest to note that some thiazole-containing anticancer drugs are already available on the market, such as tiazofurin [38], dasatinib [39], and dabrafenib [40]. Considerable improvements in the biological activity were achieved by using the hybrid bioactive molecule approach, which combines two or more different pharmacophore moieties in a new molecule [41]. Thus, the combination of TZD and thiazole scaffolds in a single hybrid molecule may exhibit synergic anticancer effects [42]. We have previously synthesized a series of thiazolidine-2,4-dione derivatives as anti-breast cancer compounds [43] that were tested against MCF-7 and MDA-MB-231 cancer cell lines, as well as against normal breast cells, isolated from the same subjects. Three derivatives were found to be the most active, and they inhibited the breast cancer cells’ proliferation in a dose-dependent fashion. Encouraged by these results, and taking into consideration the hybrid bioactive molecule approach, we present herein the synthesis of new thiazolidinedione scaffolds. The latter contain the three pharmacophores, namely trimethoxybenzene, thiazolidinedione and thiazole, in a single molecule. In this way, we aimed to obtain hybrid compounds with high potential as anti-breast cancer tools. Interestingly, different compounds containing the above-mentioned pharmacophores were investigated as topoisomerase inhibitors [44,45,46]. Topoisomerases are enzymes possessing nuclease and ligase activities and are involved in many important cellular processes, such as DNA transcription and duplication, chromosome segregation and chromatin assembly [47]. There are two types of DNA topoisomerases, I and II, based on their amino acid sequences and structures, catalytic functions and reaction mechanisms. Both are implicated in tumor onset and progression, being often overexpressed in many cancer cells [48]. Thus, by the means of in silico simulations and direct enzymatic assays, we found that our compounds inhibited the human topoisomerase I (hTopo I) and II (hTopo II). Particularly, compound 7e was able to block both the hTopo I and II activities, whereas compounds 7c and 7d were found to block only hTopo II activity. Finally, MCF-7 cells exposed to compound 7e, which was the most active of the series and induced cancer cell death by activating the intrinsic apoptotic mechanism.

2. Results

2.1. Chemistry

The aim of this present work was to prepare a new series of thiazolidine-2,4-diones and evaluate their anticancer activity. Our target molecules were designed by combining three pharmacophores known for their anticancer activity, namely thiazolidine-2,4-dione, trimethoxyphenyl, and thiazole moieties, in a single molecule. Thus the thiazolidine-2,4-dione pharmacophore nucleus (1) was prepared by the reaction of chloroacetic acid with thiourea, as reported in our earlier paper [43]. The active methylene group of 1 was allowed to condense with 3,4,5-trimethoxybenzaldehyde via Knoevenagel reaction in boiling anhydrous toluene, in the presence of a catalytic amount of piperidinium acetate, using a Dean–Stark apparatus to produce the corresponding (Z)-5-(3,4,5-trimethoxybenzylidine)thiazolidine-2,4-dione (2). The Z-conformation of the latter compound has been proven by x-ray analysis [49]. Treatment of compound 2 with ethanolic KOH solution gave the corresponding potassium salt 3 (Scheme 1).
A thiazole moiety, as a third pharmacophore, was incorporated in the (3,4,5-trimethoxybenzylidene)thiazolidine-2,4-dione system. Thus, a series of 2-aminothiazoles, 4a–f, were chloroacetylated using 2-chloroacetylchloride in dry benzene to give the 2-chloroacetamides 5a–f (Scheme 2). It is to be noted that 4b–e were prepared using the method reported by Dodson and King [50]. The reaction of 2-chloroacetamides 5a–f with the potassium salt 3, in dry DMF, gave the target compounds 7a–f, however, in rather low yields (50–54%). In an endeavor to increase the reaction yield, the chlorine atom of 5a–f was displaced by the more labile iodine atom upon treatment with KI in boiling acetone to give the corresponding 2-iodoacetamides 6a–f (Scheme 2).
The reaction of the latter iodo derivatives 6a–f with the potassium salt 3 gave 7a–f in excellent yields (69–97%) (Scheme 3).
The structure of compounds 7a–f was confirmed by IR, 1H and 13C NMR spectral analyses. The IR spectra were characterized by a stretching vibration band (ν NH) at 3423–3184 cm−1 in addition to ν C=O bands at 1690–1685 and 1743–1736 cm−1. The 1H-NMR spectra of the basic skeleton showed, in addition to other protons, characteristic signals of the nine protons of the three methoxyl groups (in addition to those of the other substituents at the thiazole N atom). In DMSOd6, these nine protons appeared as two singlets at δ 3.77–3.76 and 3.87–3.86 ppm, integrated for three protons (of the 4-methoxy substituent) and six protons (of the 3,5-dimethoxy substituents), respectively. The proton of the methylidene group appeared as a singlet at δ 7.94–7.58 ppm. The two aromatic protons of the trimethoxyphenyl group appeared as a singlet at δ 7.0–6.98 ppm. The N-CH2-CO methylene protons appeared as a singlet at δ 4.69–4.65 ppm. The -NHCO- proton appeared at δ 12.76–12.58 ppm. The 13C NMR spectra were consistent with the structures of all of the compounds obtained.

2.2. Docking Studies

To visualize the poses and the possible binding modes among the new compounds and the protein targets (hTopo I and hTopo II), we decided to perform molecular docking simulations. As these molecules had never been investigated before in vitro, for our simulations, we adopted a “blind-docking approach”: no a priori information on the binding site was given to the system. This procedure had already been successfully used for other studies [51]. This approach is useful as it allows us, on the one hand, to identify the best candidates among the compounds and, on the other, to improve the atomic structure of our molecules for a better design and synthesis of improved lead compounds. We based our investigation on the compounds’ binding affinity for the two hTopos, using the Autodock software that calculates a binding affinity constant (Ki) on the basis of the binding energy, according to the Ki = exp (deltaG/(R × T) expression. The clustering of the results from the simulations was taken into account for discriminating the most effective candidates, as previously reported [52]. Finally, the binding mode was visually examined for the evaluation of protein–ligand interaction quality. We showed that our compounds can bind the hTopo I and II, creating different hydrogen and hydrophobic interactions. Concerning hTopo I, our molecules are placed in three distinct areas belonging to the region where DNA binds. Most of our compounds (specifically molecules 7a, 7b, 7d, 7e, 7f) occupy the whole protein active site region. These molecules are almost superposed, in a region (Figure 1 panel A) nested between two protein subdomains. Molecule 7a forms hydrogen bonds with residues Arg 364, Ser 423, Lys 463 and Thr 501, and 7b is bound via residues Arg 488, Lys 493, Thr 501 and Lys 532. The chlorine moiety of molecule 7d is involved in the formation of a halogen bond with Ser 423. Furthermore, this compound interacts with Arg 488, Lys 493, Thr 501, Lys 532 and Lys 590. Residues Arg 364, Ser 423 and Lys 493 form hydrogen bonds with compound 7e, while Arg 488 and Trp 416 further stabilize this compound by π–π stacking interactions. Finally, molecule 7f forms hydrogen bonds with Arg 364, Arg 488, Lys 493, Thr 501 and Arg 532. On the other hand, compound 7c is located on a distinct site in the N-terminal domain, in proximity of active site residues His 468 and Arg 590 on the opposite part of the channel hosting the DNA. Although this molecule does not occupy the whole active site area, it can still disturb the regular functions of hTopo I by interfering with the process of protein: DNA recognition and binding through the formation of hydrogen bonds with residues Lys 216, Glu 438 and Arg 590 (Figure 1 panels B–G). Two-dimensional plots for hTopo I were calculated using LIGPLOT [53] and included in the Supplementary Materials (Figures S59–S64).
The above-described molecules also bind the hTopo II near the DNA gate. Residues Gln 726, Asn 770, Asn 851, Asn 866, and Lys 893 are hydrogen-bonded to molecule 7a, while Phe 775 forms van der Waals contact. The same residues also form hydrogen bonds with compound 7b, which is stabilized by a π–π stacking with Trp 931. Molecule 7c forms hydrogen bonds with Asn 866 and Lys 893, and it is stabilized by hydrophobic interactions with residues Ile 769, Phe 755 and Leu 1178. Residue Lys 723 forms a halogen bond with the Cl moiety of molecule 7d, which also forms hydrogen bonds with Asn 866, Asn 770 and Lys 893. Furthermore, this compound is stabilized by hydrophobic interactions with residues Ile 769, Phe 755 and Leu 1178. Compounds 7e and 7f are positioned in a slightly different place. They both bind residues Ser 763, Asn 766 and Gln 726, but the longer chain of molecule 7e allows this compound to also interact with residues Lys 723 and His 759 (Figure 2 panels A–F). Two-dimensional plots for hTopo II were calculated using LIGPLOT [53] and included in the Supplementary Material (Figures S65–S70).

2.3. Biology

2.3.1. Effects on Cell Viability

All of the synthesized compounds (7a–f) were studied to test their anticancer activity toward the estrogen receptor-positive (ER+) MCF-7 and the triple-negative MDA-MB-231 breast cancer cells (ER-, PR- and HER-2/Neu not overexpressed) and A2058 melanoma cells. Cells were incubated for 72 h with different concentrations of the compounds, and then the viability was checked by the MTT assay. Colchicine was adopted as the reference molecule for these assays. The results, summarized in Table 1, indicate that all of the compounds diminished the viability of both breast cancer cell lines. In particular, compound 7e proved the most active against the ER+ MCF-7 breast cancer cells, with an IC50 value of 3.1 ± 0.6 µM, followed by compound 7b, which showed good anticancer activity as well with an IC50 value of 8.5 ± 1.2 µM. However, compounds 7c, 7d and 7f were less able to reduce MCF-7 cell growth, showing IC50 values of 19.5 ± 0.6, 34.1 ± 0.7 and 17.4 ± 1.1 µM, respectively. Finally, compound 7a did not interfere with MCF-7 cell viability until the concentration of 100 µM. All of the tested compounds were less active against the triple-negative MDA-MB-231 breast cancer cells, with the exception of compound 7a. Particularly, 7b showed the best anticancer activity, with an IC50 value of 17.9 ± 1.1 µM, whereas compound 7a showed an IC50 of 27.5 ± 0.9 µM. Good anticancer activity was also found against the A2058 melanoma cells, where compound 7b was the most active, showing an IC50 value of 3.5 ± 1.1 µM. Compounds 7c, 7d and 7e exhibited good anticancer activity on the same melanoma cell line as well, with IC50 values of 4.8 ± 1.0, 6.4 ± 0.9 and 8.4 ± 0.9 µM, respectively. In contrast, compound 7f showed a higher IC50 value (24.6 ± 1.1 µM), while compound 7a did not exhibit any anticancer activity up to 100 µM. Moreover, we checked the cytotoxicity of all of the synthesized compounds against the human MCF-10A non-malignant breast epithelial cells and mouse BALB/3T3 embryonic fibroblasts, in order to evaluate their selectivity on cancer cells. Among them, only 7d and 7e exhibited a slight toxicity against the BALB/3T3 cells, with IC50 values of 36.1 ± 1.0 and 35.0 ± 0.6 µM, respectively. However, compound 7e possessed a large therapeutic window; indeed, it was about 11-fold more active against the MCF-7 breast cancer cells than against mouse BALB/3T3 embryonic fibroblasts. Moreover, all of the compounds were non-cytotoxic against the human MCF-10A mammary epithelial cells, with the exception of compound 7c (IC50 = 46.4 µM). Finally, all of the compounds exhibited less activity in comparison with the adopted reference molecules, colchicine and ellipticine; indeed, the latter had higher anticancer activity against all of the cell lines used in these assays compared to the newly synthesized compounds, but they exhibited dramatic cytotoxic effects on the normal cells (colchicine: IC50 = 5.2 (± 0.9) × 10−1 and 8.9 (± 1.2) × 10−3 µM against the BALB/3T3 and MCF-10A cells, respectively; ellipticine: IC50 = 0.97 ± 0.1 and 1.09 ± 0.1 µM against the BALB/3T3 and MCF-10A cells, respectively), contrarily to the compounds under study. It is worth noting that an improvement in anticancer activity was observed when the 4-position of the thiazole ring of compound 7a was substituted by a phenyl- or p-substituted phenyl group. Indeed, compounds 7b, 7c, 7d and 7e showed higher activity than compound 7a against MCF-7 and A2058 cells, but not against MDA-MB-231 cells. Moreover, the fusion of a benzene ring to the thiazole moiety, as in 7f, increased the anticancer activity, even if to a lesser extent, against MCF-7 and A2058 cells. Finally, we decided to investigate the mechanism of action of the compounds that showed higher anticancer activity, 7b, 7c, 7d and 7e, bearing at position 4 of their thiazole ring a non-substituted phenyl-, p-tolyl-, p-chlorophenyl-, and p-bromophenyl group, respectively.

2.3.2. Inhibition of Human Topoisomerases I and II

Thiazoles and their derivatives are extensively recognized as chemical nuclei of great interest for producing molecules with multiple biological properties [54]. In particular, several compounds bearing these heterocycles were studied for their capability to interfere with DNA topoisomerases (Topos) [54,55,56], ubiquitous enzymes essential in several key cellular processes. In recent years, these enzymes have become popular targets in cancer therapy; indeed, the topoisomerase inhibitors can induce damage to genome integrity. Consequently, the inhibition of topoisomerases leads to cell death by apoptosis and, thus, is fatal [57]. Thus, we carried out two specific tests in vitro for both of the Topos, namely the hTopo I relaxation and the hTopo II decatenation assays. For the hTopo I relaxation assay, we exposed the enzyme to the most interesting compounds, 7b–7e, at the final concentration of 10 µM for 1 h at 37 °C, using as substrate the supercoiled pHOT1 (see Section 4 Materials and Methods for more details). As evidenced in Figure 3, panel A, only compound 7e was able to inhibit hTopo I activity under the adopted experimental conditions; indeed, a single band in the lower portion of the agarose gel, representing the uncleaved plasmid pHOT1, was visible (Figure 3, lane G). Contrarily, in the CTRL lane, where the hTopo I was exposed to only vehicle (DMSO), several bands due to pHOT1 DNA cleavage were present (Figure 3, lane C). A similar behavior was obtained by incubating the enzyme with compounds 7b, 7c or 7d (Figure 3, lanes D–F), indicating that these compounds were not able to inhibit the relaxation activity of the enzyme. In Figure 3, panel B, the hTopo I inhibition results using ellipticine, as reference molecule are shown. The latter was able to block the hTopo I activity at both of the used concentrations (10 and 50 µM, lanes 3 and 4, respectively). An electrophoretic mobility shift of the pHOT1 DNA was also noticeable, due to the intercalating properties of ellipticine [58].
After that, we also tested the inhibitory activity of compounds 7b–7e toward hTopo II activity, exposing the enzyme to the mentioned compounds at a concentration of 10 µM for 1 h at 37 °C (Figure 4a). We found that none of the compounds were capable of blocking hTopo II activity, as evidenced by the two DNA bands at the bottom of the gel (decatenation products of kDNA, used as substrate, Figure 4a, lanes E–H). The same pattern was visible in the CTRL reaction, in which the enzyme was incubated with DMSO only, and for the decatenated kDNA marker (Figure 4a, lanes D and B, respectively). However, increasing the compound concentrations to 50 µM (Figure 4b), compounds 7c–7e fully blocked the enzyme activity, as confirmed by a clear band in the upper of the gel that is the catenated kDNA (Figure 4b, lanes F–H). On the contrary, the compound 7b was unable to inhibit the hTopo II activity (Figure 4b, lane E). The linear kDNA (Figure 4a,b, lanes A), decatenated kDNA (Figure 4a,b, lanes B) and the catenated circles of kDNA (Figure 4a,b, lanes C) were used as markers. A parallel experiment was performed using ellipticine as reference molecule. As visible in Figure 4c, ellipticine was able to totally block the hTopo II activity only at 50 µM and partially at 10 µM (lanes 4 and 5, respectively). As well in these experiments, ellipticine intercalates the decatenated kDNA products, producing their electrophoretic mobility shift. Finally, the obtained hTopo I and II inhibition results were resumed in Table 2.

2.3.3. Compound 7e Induced Apoptosis via Cytochrome c Release in MCF-7 Cells

Numerous data in the literature indicate that topoisomerase inhibitors are among the most efficient inducers of apoptosis [59,60,61]. Thus, we carried out the TUNEL assay to assess the capability of compound 7e, which was the most active compound and also the only one able to inhibit both of the hTopos, to trigger apoptosis. As reference molecule, we adopted ellipticine for this assay, also. As shown in Figure 5, the green nuclear fluorescence indicated that MCF-7 cells treated with compound 7e, at its IC50 value, had undergone the apoptotic process (Figure 5, 7e, panel B). Ellipticine exhibited similar behavior (Figure 5, E, panel B). In contrast, no fluorescence was visible in the DMSO-treated cells used as negative control (Figure 5, CTRL, panel B). These outcomes indicated the ability of the studied compound to induce DNA damage, as ellipticine did, triggering cancer cells death.
In order to establish whether the observed cell death could be ascribed to the induction of the apoptotic pathway, we performed immunostaining assays using an anti-cytochrome c (cyt c) antibody and the mitochondrial probe MitoTracker Deep Red FM, which would allow us to verify whether cyt c was released from mitochondria to the cell cytoplasm or not. In Figure 6, panels B,C, it is possible to visualize the intracellular localization of cyt c (green fluorescence) and mitochondria (red fluorescence). In MCF-7 cells exposed to only the vehicle (DMSO), there was a perfect merge between the two fluorescence signals (panel D), indicating that cyt c resided within the mitochondrial compartment. On the contrary, under compound 7e exposure, the mitochondria became piled up (panel C) and, most importantly, the cyt c was released into the cell cytoplasm, as indicated by the rise in a diffused green fluorescence (panel B). Indeed, as visible in the overlay channel (panel D), the two fluorescence signals (red and green) were no longer superimposable.
Once established that mitochondria were damaged and cyt c released into the cytoplasm, we evaluated the ability of compound 7e treatment (used at its IC50 value) in modulating caspase activity. Figure 7 shows that there was a noticeable increase in caspase-9 activity and no change in caspase-8 activity in MCF-7 cells treated with compound 7e compared to the vehicle-treated ones. Furthermore, MCF-7 cells treated with compound 7e exhibited higher activity of caspases 3/7 (versus vehicle-treated cells), which are substrates of the initiator caspase-9. Altogether, these data suggest that compound 7e is able to trigger the intrinsic apoptotic pathway in MCF-7 cells.

3. Discussion

Cancer therapies have been subjected to a considerable evolution in recent years, contributing to improve the quality of life and survival of patients with cancer. These advances mirror the efforts made by governments and research institutes worldwide, and the clinical use of chemotherapy has been significantly improved using personalized dosing regimens and combination therapies. Moreover, the advent of targeted therapy revolutionized cancer treatment and, for this purpose, drug design strategies still play an essential role in drug discovery and development [62]. An important strategy in medicinal chemistry is hybrid drug design, which integrates different compounds or pharmacophores together, generating new bioactive molecules [63]. Herein, we present the synthesis of new thiazolidinedione scaffolds containing three pharmacophores, namely trimethoxybenzene, thiazolidinedione and thiazole, in a single hybrid molecule. Several studies already reported the anticancer potential of the above-mentioned pharmacophores [16,64,65], but in different cases, several issues, such as low activity and eventually, selectivity or the lack of a specific target, were raised. In some cases, the hybrid derivatives may represent a valid alternative, in particular, our compounds were proved to exert good anticancer activity, particularly against the MCF-7 cells, and most interestingly, without affecting the viability of the tested normal cells. This is a very promising parameter to consider, since one of the major limitations of current anticancer chemotherapy regimens is the high incidence of side effects. Moreover, by means of in silico and in vitro assays, we individuated two intracellular targets, i.e., the human topoisomerases I and II, as candidates responsible for the observed anticancer properties. The importance of these enzymes in cancer onset and progression is currently being ascertained, and a still growing number of studies are focused on this [66]. Most importantly, targeting both of the enzymes, our compounds could also diminish the frequent onset of resistance phenomena related to the compensative pathways that cancer cells may trigger [67]. The blockade of these enzymes produced DNA damage, as demonstrated by the TUNEL assays, and induced death of the cancer cells through the intrinsic apoptotic pathway. Indeed, the treatment with 7e promoted the activation of caspases 3/7 and 9, mitochondria destabilization and cyt c migration into the cytoplasm. Summing up, we are confident that our outcomes may bring an important contribution to the field of hybrid drug design, to be further developed for cancer chemotherapy.

4. Materials and Methods

4.1. Chemistry

4.1.1. General Methods

All melting points were determined on a Stuart melting point apparatus SMP3 and are uncorrected. IR spectra were recorded on a Nicolet iS10 FT-IR spectrometer using the KBr wafer technique (Thermo Fisher, Waltham, MA, USA). The 1H-NMR spectra were recorded on a Bruker Avance III spectrometer operating at 400 MHz (1H) or 100 MHz (13C) (Bruker, Billerica, MA, USA). 1H- and 13C-NMR chemical shifts were reported in parts per million (ppm) and were referenced to the residual proton peaks in a deuterated solvent, DMSO-d6 (2.50 ppm for 1H and 39.70 ppm for 13C). Multiplicities are represented by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Coupling constants (J) are reported in Hertz (Hz). The reactions were monitored by thin-layer chromatography (TLC). Elemental analyses (C, H, N) were conducted using a Perkin Elmer 240 C Microanalyzer (Perkin Elmer, Waltham, MA, USA) and found to be in good agreement with the theoretical values within ± 0.4%. 2-Aminothiazole and 2-aminobenzothiazoles were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,3-thiazolidine-2,4-dione (1), (Z)-5-(3,4,5-trimethoxy benzylidene)-1,3-thiazolidine-2,4-dione (2) and (Z)-5-(3,4,5-trimethoxy benzylidene)-1,3-thiazolidine-2,4-dione potassium salt (3) were prepared as described in our previous publication [43].

4.1.2. Preparation of 2-amino-4-aryl-1,3-thiazoles 4b–e

These compounds were prepared according to the literature [50]. To a slurry consisting of thiourea (1.522 g, 0.02 mol), acetophenone (1.1 mL, 0.01 mol), 4-methylacetophenone (1.34 g, 0.01 mol), 4-chloroacetophenone (1.25 mL, 0.01 mol), or 4-bromoacetophenone (1.9 g, 0.01 mol),iodine (2.5 g, 0.01 mol) were added all at once. The reaction mixture was heated on a boiling water bath overnight with occasional swirling. The reddish reaction product obtained was diluted with water, heated under boiling until a nearly clear solution was obtained. This was filtered, cooled and made alkaline with conc. NH4OH solution. The yellow product was filtered, washed with water, dried and crystallized from ethanol.
2-Amino-4-phenylthiazole (4b). Yellow crystals, yield 1.7 g (99%), mp 145–147 °C (lit. [50] 147 °C). IR (KBr, cm−1): 3435 and 3250 (ν NH2), 3114 (ν C-H arom.), 1637 (δNH2).
2-Amino-4-(4-methylphenyl)thiazole (4c). Yellow crystals, yield 1.6 g (87%), mp 135–137 °C (lit. [68] 135–136 °C). IR (KBr, cm−1) 3454 and 3297 (ν NH2), 3117 (ν C-H arom.), 2974 (ν C-H aliph.), 1636 δNH2).
2-Amino-4-(4-chlorolphenyl)thiazole (4d). Yellow crystals, yield 1.5 g (73%), mp 167–169 °C (lit. [68] 167–168 °C). IR (KBr, cm−1) 3438 and 3283 (ν NH2), 3111 (ν C-H arom.), 2987 (ν C-H aliph.), 1633 δNH2).
2-Amino-4-(4-bromolphenyl)thiazole (4e). Yellow crystals, yield 2.5 g (99%), mp 180–183 °C (lit. [69] 179–181 °C). IR (KBr, cm−1) 3428, 3281 (ν NH2), 3111 (ν C-H arom.), 2987 (ν C-H aliph.) and 1633 (δNH2).

4.1.3. Chloroacetylation of 2-Aminothiazoles; Preparation of 2-chloroacetamides 5a–f

To a cold solution 0–5 °C of 4a–f (0.002 mol) in dry benzene, a cold solution of chloroacetyl chloride (0.0033 mol) in dry benzene was added dropwise with stirring. The reaction mixture was then heated in a boiling water bath for 3 h. After cooling, the reaction was evaporated under reduced pressure until dryness. The solid product obtained was washed with 5% NaHCO3, then with water, filtered, dried and crystallized from ethanol.
2-Chloro-N-(thiazol-2-yl)acetamide (5a). White needles, yield 0.759 g (86%), mp 171–172 °C (lit. [70] 170–170.5 °C). IR (KBr, cm−1): 3186 (ν N-H), 3081 (ν C-H arom.), 2949 and 2882 (ν C-H aliph.), 1703 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.53 (s, 1H, NH), 7.45 (s, 1H, thiazole-H), 7.19 (s, 1H, thiazole-H), 4.20 (s, 2H, CH2). Anal. calcd. for C5H5ClN2OS (176.62): C, 34.00; H, 2.85; N, 15.86; S, 18.15. Found: C, 34.18; H, 2.79; N, 15.92; S, 18.25%.
2-Chloro-N-(4-phenylthiazol-2-yl)acetamide (5b). Colorless crystals, yield 0.327 g (65%), mp 150–151 °C (lit. [70] 164 °C). IR (KBr, cm−1): 3181 (ν N-H), 3022 (ν C-H arom.), 2983 (ν C-H aliph.), 1655 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.95 (s, 1H, NH), 7.76 (d, 2H, J = 7.2 Hz, Ph-H), 7.35 (t, 2H, J = 7.3 Hz, Ph-H), 7.27 (m, 1H, Ph-H), 7.12 (s, 1H, thiazole-H), 4.13 (s, 2H, CH2). Anal. calcd. for C11H9ClN2OS (252.72): C, 52.28; H, 3.59; N, 11.09; S, 12.69. Found: C, 52.39; H, 3.43; N, 11.39; S, 12.73%.
2-Chloro-N-(4-methylphenylthiazol-2-yl)acetamide (5c). Colorless crystals, yield 0.499 g (94%), mp 162–163 °C (lit. [69] 157–158 °C). IR (KBr, cm−1): 3157 (ν N-H), 3047 (ν C-H arom.), 2921 (ν C-H aliph.), 1700 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.07 (s, 1H, NH), 7.63 (d, 1H, J = 7.8 Hz, Ph-H), 7.15 (d, 1H, J = 7.8 Hz, Ph-H), 7.05 (s, 1H, thiazole-H), 4.08 (s, 2H, CH2), 2.30 (s, 3H, CH3). Anal. calcd. for C12H11ClN2OS (266.74): C, 54.03; H, 4.16; N, 10.50; S, 12.02. Found: C, 54.13; H, 4.01; N, 10.41; S, 12.11%.
2-Chloro-N-(4-chlorophenylthiazol-2-yl)acetamide (5d). Colorless crystals, yield 0.48 g (86%), mp 175–177 °C (lit. [71] 170–173 °C). IR (KBr, cm−1): 3373 (ν N-H), 3109 (ν C-H arom.), 2988 (ν C-H aliph.), 1692 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.82 (s, 1H, NH), 7.68 (d, 2H, J = 8.3 Hz, Ph-H), 7.30 (d, 2H, J = 8.3 Hz, Ph-H), 7.09 (s, 1H, thiazole-H), 4.17 (s, 2H, CH2). Anal. calcd. for C11H8Cl2N2OS (287.16): C, 46.01; H, 2.81; N, 9.76; S, 11.16. Found: C, 46.10; H, 2.89; N, 9.84; S, 11.21%.
2-Chloro-N-(4-bromophenylthiazol-2-yl)acetamide (5e). Colorless crystals, yield 0.408 g (62%), mp 174–175 °C (lit. [69] 169–172 °C). IR (KBr, cm−1): 3366 (ν N-H), 3109 (ν C-H arom.), 2940 (ν C-H aliph.), 1692 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.75 (s, 1H, NH), 7.62 (d, 2H, J = 8.4 Hz, Ph-H), 7.46 (d, 2H, J = 8.4Hz, Ph-H), 7.11 (s, 1H, thiazole-H), 4.17 (s, 2H, CH2). Anal. calcd. for C11H8BrClN2OS (331.61): C, 39.84; H, 2.43; N, 8.45; S, 9.67. Found: C, 39.91; H, 2.39; N, 8.44; S, 9.70%.
2-Chloro-N-(benzo[d]thiazol-2-yl)acetamide (5f). White crystals, yield 0.424 g (94%), mp 161–162 °C (lit. [72] 140–143 °C). IR (KBr, cm−1): 3507 (ν NH), 3048 (ν CH arom.), 2986 (ν CH aliph.), 1695 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.81 (s, 1H, NH), 7.76 (t, 2H, J = 7.0 Hz, Ph-H), 7.40 (t, 1H, J = 7.3 Hz, Ph-H), 7.28 (t, 1H, J = 7.2 Hz, Ph-H), 4.23 (s, 2H, CH2). Anal. calcd. for C9H7ClN2OS (226.68): C, 47.69; H, 3.11; N, 12.36; S, 14.14. Found: C, 47.78; H, 3.00; N, 12.44; S, 14.04%.

4.1.4. Preparation of 2-Iodoacetamides 6a–f

A mixture of 5a–f (5 mmole) and KI (6 mmole) in acetone (50 mL) was heated under reflux for 4 h. The solvent was then evaporated under reduced pressure. The solid product obtained was washed thoroughly with water, filtered and recrystallized from ethanol.
2-Iodo-N-(thiazol-2-yl)acetamide (6a). Colorless crystals, yield 0.925 g (69%), m.p 175.9–177.5 °C. IR (KBr, cm−1): 3166 (ν N-H), 3107 (ν C-H arom.), 2964 (ν C-H aliph.), 1647 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.42 (s, 1H, NH), 7.50 (d, J = 3.5 Hz, 1H, Ph-H), 7.26 (d, J = 3.5 Hz, 1H, Ph-H), 3.93 (s, 2H, CH2). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 165.8 (C=O), 159.7 (C), 134.9 (CH), 114.2 (CH), 3.9 (CH2). Anal. calcd. for C5H5IN2OS (268.07): C, 22.40; H, 1.88; N, 10.45; S, 11.96. Found: C, 22.19; H, 1.77; N, 10.50; S, 11.71%.
2-Iodo-N-(4-phenylthiazol-2-yl)acetamide (6b). Colorless crystals, yield 1.585 g (92%), m.p 188–190 °C. IR (KBr, cm−1): 3174 (ν N-H), 3039 (ν C-H arom.), 2988 (ν C-H aliph.), 1648 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.60 (s, 1H, NH), 7.90 (d, 2H, J = 7.2 Hz, Ph-H), 7.67 (s, 1H, thiazole-H), 7.44 (t, 2H, J = 7.6 Hz, Ph-H), 7.33 (t, 1H, J = 7.3 Hz, Ph-H), 3.97 (s, 2H, CH2). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 167.9 (C=O), 158.4 (C), 149.7 (C), 134.8 (C), 129.5 (2CH), 128.6 (CH), 126.4 (2CH), 109.2 (CH), −0.51 (CH2). Anal. calcd. for C11H9IN2OS (344.17): C, 38.39; H, 2.64; N, 8.14; S, 9.32. Found: C, 38.43; H, 2.57; N, 8.20; S, 9.25%.
2-Iodo-N-(4-methylphenylthiazol-2-yl)acetamide (6c). Colorless crystals, yield 1.535 g (89%), m.p 180–182 °C. IR (KBr, cm−1): 3182 (ν N-H), 3073 (ν C-H arom.), 2972 (ν C-H aliph.), 1646 (ν C=O), 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.58 (s, 1H, NH), 7.78 (d, J = 8.1 Hz, 2H, Ph-H), 7.56 (s, 1H, thiazole-H), 7.23 (d, 2H, J = 8.0 Hz, Ph-H), 3.97 (s, 2H, CH2), 2.31 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 167.8 (C=O). 158.2 (C), 149.8 (C), 137.8 (C), 132.1 (C), 130.1 (2CH), 126.3 (2CH), 108.2 (CH), 21.5 (CH3), −0.51 (CH2). Anal. calcd. for C12H11IN2OS (358.20): C, 40.24; H, 3.10; N, 7.82; S, 8.95. Found: C, 40.33; H, 3.22; N, 7.88; S, 9.01%.
2-Iodo-N-(4-chlorophenylthiazol-2-yl)acetamide (6d). Colorless crystals, yield 1.75 g (70%), m.p 184.7–186 °C. IR (KBr, cm−1): 3181 (ν N-H), 3071 (ν C-H arom.), 2974 (ν C-H aliph.), 1652 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.61 (s, 1H, NH), 7.91 (d, 2H, J = 8.6 Hz, Ph-H), 7.73 (s, 1H, thiazole-H), 7.49 (d, 2H, J = 8.6 Hz, Ph-H), 3.96 (s, 2H, CH2). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 168.0 (C=O). 158.7 (C), 148.6 (C), 138.8 (C), 133.1 (C), 129.5 (2CH), 128.1 (2CH), 110.6 (CH), −0.51 (CH2). Anal. calcd. for C11H8ClIN2OS (378.61): C, 34.90; H, 2.13; N, 7.40; S, 8.47. Found: C, 35.11; H, 2.09; N, 7.49; S, 8.51%.
2-Iodo-N-(4-bromophenylthiazol-2-yl)acetamide (6e). Colorless crystals, yield 1.89 g (89%), m.p 192.7–194 °C. IR (KBr, cm−1): 3179 (ν N-H), 3072 (ν C-H arom.), 2974 (ν C-H aliph.), 1652 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.61 (s, 1H, NH), 7.85 (d, 2H, J = 8.6 Hz, Ph-H), 7.74 (s, 1H, thiazole-H), 7.63 (d, 2H, J = 8.6 Hz, Ph-H), 3.93 (s, 2H, CH2). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 168.0 (C=O), 158.7 (C), 148.7 (C), 134.2 (C), 132.6 (2CH), 128.5 (2CH), 121.7 (C), 110.1 (CH), −0.51 (CH2). Anal. calcd. for C11H8BrIN2OS (423.07): C, 31.23; H, 1.91; N, 6.62; S, 7.58. Found: C, 31.32; H, 1.82; N, 6.69; S, 7.60%.
2-Iodo-N-(benzo[d]thiazol-2-yl)acetamide (6f). Yellow crystals, yield 1.295 g (82%), m.p 182–184 °C. IR (KBr, cm−1): 3171 (ν N-H), 3057 (ν C-H arom.), 2989 (ν C-H aliph.) and 1656 (ν C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.67 (s, 1H, NH), 7.98 (d, 1H, J = 7.8 Hz, Ph-H), 7.76 (d, 1H, J = 8.0 Hz, Ph-H), 7.44 (t, 1H, J = 7.2 Hz, Ph-H), 7.31 (t, 1H, J = 7.2 Hz, Ph-H), 3.99 (s, 2H, CH2). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 171.6 (C), 167.5 (C=O), 158.5 (C), 158.1 (C), 138.5 (CH), 138.3 (CH), 114.6 (CH), 114.2 (CH), −0.51 (CH2). Anal. calcd. for C9H7IN2OS (318.13): C, 33.98; H, 2.22; N, 8.81; S, 10.08. Found: C, 34.05; H, 2.29; N, 8.90; S, 10.11%.

4.1.5. General Procedure for Synthesis of the Target Compounds 7a–f

A mixture of the potassium salt 3 (0.333 g, 1 mmole) and 6a–f (1 mmole) in DMF (5 mL) was heated on a boiling water bath overnight. After cooling, the reaction mixture was diluted with ice-cold water. The solid product obtained was collected and recrystallized from a mixture of ethanol/dioxane.
(Z)-2-(5-(3,4,5-Trimethoxybenzylidene)thiazolidin-2,4-dion-3-yl)-N-(thiazol-2-yl)acetamide (7a).
Yellow crystals, yield 0.3 g (69%), mp 245–247 °C. IR (KBr, cm−1): 3189 (ν N-H), 3093 (ν C-H arom.), 2936 (ν C-H aliph.), 1742 and 1685 (ν C=O), 1604, 1577, 1504 and 1453 (C=C arom). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.58 (s, 1H, NH), 7.94 (s, 1H, -CH=), 7.50 (d, 1H, J = 3.4 Hz, thiazole-H), 7.25 (d, 1H, J = 3.4 Hz, thiazole-H), 6.98 (s, 2H, Ph-H), 4.65 (s, 2H, CH2), 3.86 (s, 6H, 2-OCH3), 3.76 (s, 3H, -OCH3). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 167.5 (C=O), 165.5 (C=O), 164.8 (C=O), 157.9 (C), 153.7 (2 C), 140.3 (CH), 138.2 (C), 134.4 (CH), 128.7 (C), 120.3 (C), 114.4 (CH), 108.3 (2 CH), 60.7 (OCH3), 56.5 (2 OCH3), 43.8 (CH2). Anal. calcd. for C18H17N3O6S2 (435.47): C, 49.65; H, 3.93; N, 9.65; S, 14.73. Found: C, 49.88; H, 4.18; N, 9.77; S, 15.03%.
(Z)-2-(5-(3,4,5-Trimethoxybenzylidene)thiazolidin-2,4-dion-3-yl)-N-(4-phenylthiazol-2-yl)acetamide (7b).
Yellow crystals, yield 0.48 g (94%), mp 222–224 °C. IR (KBr, cm−1): 3196 (ν N-H), 3077 (ν C-H arom.), 2974 (ν C-H aliph.), 1748 and 1693 (ν C=O). 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 12.75 (s, 1H, NH), 7.95 (s, 1H, thiazole-H), 7.91 (d, 2H, J = 7.5 Hz, Ph-H), 7.65 (s, 1H, -CH=), 7.44 (t, 2H, J = 7.5 Hz, Ph-H), 7.34 (t, 1H, J = 7.2 Hz, Ph-H), 6.98 (s, 2H, Ph-H), 4.69 (s, 2H, CH2), 3.86 (s, 6H, 2 -OCH3), 3.77 (s, 3H, -OCH3). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 167.5 (C=O), 165.5 (C=O), 157.8 (C), 165.1 (C=O), 153.7 (2 C), 149.5 (C), 140.3 (CH), 134.6 (C), 134.4 (C), 129.2 (C), 128.7 (2 CH), 128.3 (CH), 126.1 (2 CH), 120.3 (C), 108.9 (CH), 108.3 (2 CH), 60.7 (OCH3), 56.5 (2 OCH3), 43.8 (CH2). Anal. calcd. for C24H21N3O6S2 (511.57): C, 56.35; H, 4.14; N, 8.21; S, 12.54. Found: C, 56.56; H, 4,27; N, 8.15; S, 12,71%.
(Z)-2-(5-(3,4,5-Trimethoxybenzylidene)thiazolidin-2,4-dion-3-yl)-N-(4-(4-methylphenyl)thiazol-2-yl)acetamide (7c).
Yellow crystals, yield 0.48 g (93%), mp 213–214 °C. IR (KBr, cm−1): 3190 (ν N-H), 3070 (ν C-H arom.), 2962 (ν C-H aliph.), 1738 and 1687 (ν C=O). 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.71 (s, 1H, NH), 7.96 (s, 1H, thiazole-H), 7.80 (d, 2H, J = 7.6 Hz, Ph-H), 7.58 (s, 1H, -CH=), 7.25 (d, 2H, J = 7.7 Hz, Ph-H), 7.00 (s, 2H, Ph-H), 4.67 (s, 2H, CH2), 3.87 (s, 6H, 2 -OCH3), 3.77 (s, 3H, -OCH3), 2.34 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6): δ (ppm) 167.5 (C=O), 165.6 (C=O), 165.2 (C=O), 157.6 (C), 153.7 (2 C), 149.6 (C), 140.3 (CH), 137.7 (C), 134.4 (C), 131.9 (C), 129.7 (C), 128.7 (2 C), 126.1 (2 CH), 120.4 (C), 108.3 (CH), 108.1 (2 CH), 60.7 (OCH3), 56.5 (2 OCH3), 43.8 (CH2), 21.2 (CH3). MS (ESI) m/z 525.25. Anal. calcd. for C25H23N3O6S2 (525.59): C, 57.13; H, 4.41; N, 7.99; S, 12.20. Found: C, 57.33; H, 4.59; N, 8.11; S, 12.31%.
(Z)-2-(5-(3,4,5-Trimethoxybenzylidene)thiazolidin-2,4-dion-3-yl)-N-(4-(4-chlorophenyl)thiazol-2-yl)acetamide (7d).
Yellow crystals, yield 0.52 g (95%), mp 243–246 °C. IR (KBr, cm−1): 3188 (ν N-H), 3065 (ν C-H arom.), 2962 (ν C-H aliph.), 1736 and 1685 (ν C=O). 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.80 (s, 1H, NH), 7.95 (s, 1H, thiazole-H), 7.92 (d, 2H, J = 8.5 Hz, Ph-H), 7.74 (s, 1H, -CH=),7.50 (d, 2H, J = 8.5 Hz, Ph-H), 6.99 (s, 2H, Ph-H), 4.67 (s, 2H, CH2), 3.86 (s, 6H, 2 -OCH3), 3.75 (s, 3H, -OCH3). 13C-NMR (100 MHz, DMSO-d6): δ (ppm) 167.5 (C=O), 165.6 (C=O), 165.2 (C=O), 157.9 (C), 153.7 (2 C), 148.2 (C), 140.2 (CH), 134.4 (C), 133.4 (C), 132.8 (C), 129.2 (C), 128.7 (2 CH), 127.8 (2 CH), 120.3 (C), 109.7 (CH), 108.2 (2 CH), 60.7 (OCH3), 56.5 (2 OCH3), 43.8 (CH2). Anal. calcd. for C24H20ClN3O6S2 (546.02): C, 52.79; H, 3.69; N, 7.70; S, 11.75. Found: C, 52.94; H, 3.98; N, 7.85; S, 11.99%.
(Z)-2-(5-(3,4,5-Trimethoxybenzylidene)thiazolidin-2,4-dion-3-yl)-N-(4-(4-bromophenyl)thiazol-2-yl)acetamide (7e).
Yellow crystals, yield 0.58 g (97%), mp 250–251 °C. IR (KBr, cm−1): 3248 (ν N-H), 3097 (ν C-H arom.), 2941 (ν C-H aliph.), 1741 and 1682 (ν C=O). 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.76 (s, 1H, NH), 7.96 (s, 1H, thiazole-H), 7.85 (d, 2H, J = 8.1 Hz, Ph-H), 7.74 (s, 1H, -CH=),7.63 (d, 2H, J = 8.2 Hz, Ph-H), 6.99 (s, 2H, Ph-H), 4.68 (s, 2H, CH2), 3.86 (s, 6H, 2 -OCH3), 3.77 (s, 3H, -OCH3). 13C-NMR (100 MHz, DMSO-d6): δ (ppm) 167.5 (C=O), 165.5 (C=O), 165.2 (C=O), 158 (C), 153.7 (2 C), 148.3 (C), 140.3 (CH), 134.4 (C), 133.8 (2 CH), 132.1 (C), 128.7 (C), 128.1 (2 CH), 121.4 (C), 120.3 (C), 109.8 (CH), 108.3 (2 CH), 60.7 (OCH3), 56.6 (2 OCH3), 43.8 (CH2). Anal. calcd. for C24H20BrN3O6S2 (590.47): C, 48.82; H, 3.41; N, 7.12; S, 10.86. Found: C, 49.11; H, 3.49; N, 7.40; S, 10.92%.
(Z)-2-(5-(3,4,5-Trimethoxybenzylidene)thiazolidin-2,4-dion-3-yl)-N-(benzo[d]thiazol-2-yl)acetamide (7f).
Yellow crystals, yield 0.426 g (88%), mp 143–144 °C. IR (KBr, cm−1): 3423 (ν N-H), (ν C-H arom.), 2932 (ν C-H aliph.), 1743 and 1690 (ν C=O). 1H-NMR (400 MHz, DMSO-d6): δ (ppm) 12.87 (s, 1H, NH), 7.80 (d, 1H, J = 7.8 Hz, Ph-H), 7.96 (s, 1H, -CH=),7.78 (d, 1H, J = 8.0 Hz, Ph-H), 7.45 (t, 1H, J = 7.1 Hz, Ph-H), 7.34 (t, 1H, J = 8.0 Hz, Ph-H), 6.99 (s, 2H, Ph-H), 4.71 (s, 2H, CH2), 3.86 (s, 6H, 2 -OCH3), 3.75 (s, 3H, -OCH3), 13C-NMR (100 MHz, DMSO-d6): δ (ppm) 167.5(C), 165.9 (C=O), 165.6 (C=O), 157.8 (C=O), 153.7 (2 C), 148.9 (C), 140.2 (CH), 134.5 (C), 131.9 (C), 128.7 (C), 126.7 (CH), 124.3 (CH), 122.3 (CH), 121.2 (CH), 120.3 (C), 108.2 (2 CH), 60.4 (OCH3), 56.5 (2 OCH3), 44.0 (CH2). Anal. calcd. for C22H19N3O6S2 (485.53): C, 54.42; H, 3.94; N, 8.65; S, 13.21. Found: C, 54.79; H, 4.13; N, 8.93; S, 13.38%.

4.2. Docking Studies

To build the three-dimensional atomic structures of the molecular targets for our compounds, we used as templates, as previously described [73], the crystal structures of the human Topoisomerase I in covalent and noncovalent complexes with DNA (PDB code 1A35) [74] and the structure of Topoisomerase IIα in complex with a short DNA fragment and etoposide (PDB Code 5gwk) [75] (hTopo I and hTopo II). The atomic coordinates of compounds 7a to 7f were built and energy-minimized using the software MarvinSketch [ChemAxon Ltd., Budapest, Hungary]. We performed all of the simulations using the Autodock v.4.2.2. [76] program suite in order to identify the possible poses and assess the binding modes and binding energies of the different compounds to these proteins. We adopted a “blind docking” simulation approach in which the docking of the small molecules to the targets was performed without a priori knowledge of the position of the binding site by the system. The simulations were conducted using the standard default values. Proteins and ligands were prepared using the ADT graphical interface [77]. For each protein, we added polar hydrogens, assigned Kollman charges and calculated solvation parameters. Each protein was considered as a rigid object, and all ligands as totally flexible. We built a searching grid and extended it all over the protein, and finally, we calculated affinity maps. The search was conducted with a Lamarckian genetic algorithm: a population of 100 individuals with a mutation rate of 0.02 was evolved for 100 generations. Amino acidic residues involved in binding are labeled and reported in Figure 1 and Figure 2. Evaluation of the results was performed by listing the different ligand poses according to their predicted binding energies. A cluster analysis was conducted on the basis of the root mean squares deviation (RMSD) values from the starting geometry. The lowest energetic conformation of the most populated cluster was considered as the best candidate. Clusters almost equipopulated and with a spread energy distribution indicated molecules considered as bad ligands [78].
The obtained docking poses were ranked in terms of increasing binding energy values and clustered using an RMSD cut-off value of 2.0 Å. From the structural analysis of the lowest energy solutions of each cluster, the protein binding site could be spotted. Figures were drawn using the Chimera software [79]. Two-dimensional plots were calculated using LIGPLOT [53]

4.3. Cell Cultures

The used cell lines (breast cancer cell lines MCF-7 and MDA-MB-231, melanoma cell line A2058 and human mammary epithelial cell line MCF-10A) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as already reported [73]. The mouse BALB/3T3 embryonic fibroblasts were also purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM high glucose supplemented with 10% bovine calf serum (BCS) and 100 U mL−1 penicillin/streptomycin.

4.4. Biology

4.4.1. MTT Assay

MTT assay (Sigma Aldrich (St. Louis, MO, USA)) was performed for evaluating the in vitro anticancer activities of the compounds, as previously indicated [80,81]. Different concentrations (0.1, 1, 10, 25, 50, and 100 μM) of the compounds were employed, with a 72 h endpoint. IC50 values were obtained from the percent (%) of control using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA).

4.4.2. hTopo I Relaxation Assay and hTopo II Decatenation Assay

hTopo I relaxation assays were performed by incubating the substrate (supercoiled pHOT1) with the recombinant hTopo I (TopoGEN, Port Orange, FL, USA) and compounds, as indicated in the supplier protocol (TopoGEN, Port Orange, FL, USA), with some revisions [80]. hTopo II decatenation assays were conducted by incubating the kinetoplast DNA substrate (kDNA) with the hTopo II (TopoGEN, Port Orange, FL) and studied compounds, as indicated in the supplier procedures (TopoGEN, Port Orange, FL, USA), with some changes as in [80].

4.4.3. TUNEL Assay

Cell apoptosis was detected by means of the TUNEL assay, following the manufacturer’s protocols (CF™488A TUNEL Assay Apoptosis Detection Kit, Biotium, Hayward, CA, USA), with few modifications, as already reported [60]. DAPI staining (0.2 μg/mL, Sigma Aldrich, Milan, Italy) was used for cell nuclei. A fluorescence microscope (Leica DM 6000) was used for the observations at 20× magnification. LAS-X software allowed the acquisition and processing of all of the images, which were representative of three separate experiments.

4.4.4. Immunofluorescence Assays

Cells grown on glass coverslips in full medium were serum-deprived for 24 h, then the compound (or only vehicle) was added (24 h, 7e at its IC50 value). Next, cells were incubated with a staining solution containing MitoTracker® Deep Red FM probe (MitoTracker® Mitochondrion-Selective Probes, Invitrogen European Headquarters, Paisley PA4 9RF, UK; fluorescence excitation = 644, fluorescence emission = 665) for 20 min, at 37 °C. After a PBS wash, cells were fixed (cold methanol, 15 min at −20 °C) and incubated with the mouse anti-cytochrome c (556 433) primary antibody, purchased from BD, diluted in the blocking solution (4 °C/overnight), as described in [60]. Alexa Fluor® 488 conjugate goat-anti-mouse (1:500 dilution), purchased from Thermo Fisher Scientific, MA, USA, was used as secondary antibody. DAPI (Sigma Aldrich, Mila, Italy), for nuclei counter-stain, was added (10 min, concentration of 0.2 μg/mL), then the mixture was washed thrice with PBS. A fluorescence microscope (Leica DM 6000) was used for visualizing images, and LAS-X software for acquisition and processing.

4.4.5. Caspase Assay

The caspase-Glo Assay (Caspase-Glo® 3/7, 8 and 9 Assay Systems, Promega Corporation, Madison, WI) was used to detect caspase-3/7, -8 and -9 activities, as reported in [82]. Briefly, cells were grown in white-walled 96-well plates, treated with the compound, and 100 µL of each caspase was added.

4.4.6. Statistical Analysis

One-way ANOVA followed by Dunnett’s test was performed by GraphPad Prism 9. Data were adopted for analysis of statistical significance (p < 0.001). Standard deviations (SD) are shown.

5. Conclusions

In this paper, we demonstrated the anticancer activity of new hybrid molecules containing three pharmacophores, namely trimethoxybenzene, thiazolidinedione and thiazole. The most active compound targets were hTopo I and II, the blockade of which induced the intrinsic apoptotic mechanism. Our data highlight the importance of the hybrid drug design and shed light on the intracellular mechanisms underlying the biological activity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph16070946/s1, Figures S1–S25, IR spectra; Figures S26–S58 1H- and 13C-NMR spectra. Figures S59–S64 2D diagrams for hTopo I; Figures S65–S70 2D diagrams for hTopo II.

Author Contributions

Conceptualization, M.S.S. and H.E.-K.; methodology, P.V.; software, C.R.; validation, J.C. and D.I.; formal analysis, O.K., S.A. and W.A.; investigation, J.C. and O.K.; resources, M.S.S., H.E.-K. and P.V.; data curation, D.I., J.C., O.K., S.A. and W.A.; writing—original draft preparation, D.I. and J.C.; writing—review and editing, M.S.S., D.I. and H.E.-K.; visualization, D.I. and C.R.; supervision, M.S.S., H.E.-K. and P.V.; project administration, M.S.S., H.E.-K. and P.V.; funding acquisition, M.S.S., H.E.-K. and P.V. All authors have read and agreed to the published version of the manuscript.

Funding

M.S.S. was supported by PON “R&I” 2014–2020–project from Area di Specializzazione “Salute”, ARS01_00568 titled “SI.F.I.PA.CRO.DE.–Sviluppo e industrializzazione farmaci innovativi per terapia molecolare personalizzata PA. CRO.DE” for providing lab tools. C.R. was supported by the Ministero della Salute, Fondi Ricerca Corrente 2022. The support from the Agence Universitaire de la Francophonie (AUF) through PCSI, 2020–2022 is highly appreciated.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Mohan, M.; Murugan, V.; Geetha, K.; Kataria, M.K. Bis-[2-(Chloroethyl) Amino] Acetamido-4-Substituted Phenyl Thiazole Derivatives as Possible Antioxidant and Alky-lating Anticancer Agents. Int. J. Pharm. Drug Anal. 2018, 6, 352–360. [Google Scholar]
  2. Asati, V.; Thakur, S.S.; Upmanyu, N.; Bharti, S.K. Virtual Screening, Molecular Docking, and DFT Studies of Some Thiazolidine-2,4-diones as Potential PIM-1 Kinase Inhibitors. ChemistrySelect 2018, 3, 127–135. [Google Scholar] [CrossRef]
  3. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer J. Clin. 2021, 71, 209–249. [Google Scholar] [CrossRef]
  4. Edwards, B.K.; Brown, M.L.; Wingo, P.A.; Howe, H.L.; Ward, E.; Ries, L.A.; Schrag, D.; Jamison, P.M.; Jemal, A.; Wu, X.C. Annual report to the nation on the status of cancer, 1975–2002, featuring population-based trends in cancer treatment. J. Natl. Cancer Inst. 2005, 97, 1407–1427. [Google Scholar] [CrossRef] [Green Version]
  5. WHO. Breast Cancer. Available online: https://www.who.int/news-room/fact-sheets/detail/breast-cancer (accessed on 2 August 2022).
  6. Liu, J.; Ming, B.; Gong, G.-H.; Wang, D.; Bao, G.-L.; Yu, L.-J. Current research on anti-breast cancer synthetic compounds. RSC Adv. 2018, 8, 4386–4416. [Google Scholar] [CrossRef] [Green Version]
  7. Naim, M.J.; Alam, M.J.; Ahmad, S.; Nawaz, F.; Shrivastava, N.; Sahu, M.; Alam, O. Therapeutic journey of 2,4-thiazolidinediones as a versatile scaffold: An insight into structure activity relationship. Eur. J. Med. Chem. 2017, 129, 218–250. [Google Scholar] [CrossRef]
  8. Day, C. Thiazolidinediones: A new class of antidiabetic drugs. Diabet. Med. 1999, 16, 179–192. [Google Scholar] [CrossRef]
  9. Tuncbilek, M.; Altanlar, N. Synthesis of New 3-(Substituted Phenacyl)-5-[3′-(4H-4-oxo-1-benzopyran-2-yl)-benzylidene]-2, 4-thiazolidinediones and their Antimicrobial Activity. Arch. Der Pharm. Int. J. Pharm. Med. Chem. 2006, 339, 213–216. [Google Scholar] [CrossRef]
  10. Bahare, R.S.; Ganguly, S.; Choowongkomon, K.; Seetaha, S. Synthesis, HIV-1 RT inhibitory, antibacterial, antifungal and binding mode studies of some novel N-substituted 5-benzylidine-2,4-thiazolidinediones. DARU J. Pharm. Sci. 2015, 23, 6. [Google Scholar] [CrossRef] [Green Version]
  11. Reddy, K.A.; Lohray, B.; Bhushan, V.; Reddy, A.S.; Kishore, P.H.; Rao, V.V.; Saibaba, V.; Bajji, A.; Rajesh, B.; Reddy, K.V. Novel euglycemic and hypolipidemic agents: Part-2 antioxidant moiety as structural motif. Bioorganic Med. Chem. Lett. 1998, 8, 999–1002. [Google Scholar] [CrossRef]
  12. Prabhakar, C.; Madhusudhan, G.; Sahadev, K.; Reddy, C.M.; Sarma, M.; Reddy, G.O.; Chakrabarti, R.; Rao, C.S.; Kumar, T.D.; Rajagopalan, R. Synthesis and biological activity of novel thiazolidinediones. Bioorganic Med. Chem. Lett. 1998, 8, 2725–2730. [Google Scholar] [CrossRef] [PubMed]
  13. Heneka, M.T.; Landreth, G.E. PPARs in the brain. Biochim. Biophys. Acta (BBA)-Mol. Cell Biol. Lipids 2007, 1771, 1031–1045. [Google Scholar] [CrossRef]
  14. Patil, V.; Tilekar, K.; Mehendale-Munj, S.; Mohan, R.; Ramaa, C. Synthesis and primary cytotoxicity evaluation of new 5-benzylidene-2, 4-thiazolidinedione derivatives. Eur. J. Med. Chem. 2010, 45, 4539–4544. [Google Scholar] [CrossRef]
  15. Asati, V.; Mahapatra, D.K.; Bharti, S.K. Thiazolidine-2, 4-diones as multi-targeted scaffold in medicinal chemistry: Potential anticancer agents. Eur. J. Med. Chem. 2014, 87, 814–833. [Google Scholar] [CrossRef] [PubMed]
  16. Kosurkar, U.B.; Mamilla, J.; Dadmal, T.L.; Choudante, P.C.; Mali, S.N.; Misra, S.; Kumbhare, R.M. Synthesis of Novel Thiazolidine-4-One Derivatives, Their Cytotoxicity, Antifungal Properties, Molecular Docking and Molecular Dynamics. Russ. J. Bioorganic Chem. 2023, 49, 314–323. [Google Scholar] [CrossRef]
  17. Liu, K.; Rao, W.; Parikh, H.; Li, Q.; Guo, T.L.; Grant, S.; Kellogg, G.E.; Zhang, S. 3, 5-Disubstituted-thiazolidine-2, 4-dione analogs as anticancer agents: Design, synthesis and biological characterization. Eur. J. Med. Chem. 2012, 47, 125–137. [Google Scholar] [CrossRef] [PubMed]
  18. Pomel, V.; Klicic, J.; Covini, D.; Church, D.D.; Shaw, J.P.; Roulin, K.; Burgat-Charvillon, F.; Valognes, D.; Camps, M.; Chabert, C. Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase γ. J. Med. Chem. 2006, 49, 3857–3871. [Google Scholar] [CrossRef] [PubMed]
  19. Smith, A.G.; Beaumont, K.A.; Smit, D.J.; Thurber, A.E.; Cook, A.L.; Boyle, G.M.; Parsons, P.G.; Sturm, R.A.; Muscat, G.E. PPARγ agonists attenuate proliferation and modulate Wnt/β-catenin signalling in melanoma cells. Int. J. Biochem. Cell Biol. 2009, 41, 844–852. [Google Scholar] [CrossRef]
  20. Xia, Z.; Knaak, C.; Ma, J.; Beharry, Z.M.; McInnes, C.; Wang, W.; Kraft, A.S.; Smith, C.D. Synthesis and evaluation of novel inhibitors of Pim-1 and Pim-2 protein kinases. J. Med. Chem. 2009, 52, 74–86. [Google Scholar] [CrossRef] [Green Version]
  21. Trotsko, N.; Przekora, A.; Zalewska, J.; Ginalska, G.; Paneth, A.; Wujec, M. Synthesis and in vitro antiproliferative and antibacterial activity of new thiazolidine-2, 4-dione derivatives. J. Enzym. Inhib. Med. Chem. 2018, 33, 17–24. [Google Scholar] [CrossRef] [Green Version]
  22. Elancheran, R.; Saravanan, K.; Divakar, S.; Kumari, S.; Maruthanila, V.L.; Kabilan, S.; Ramanathan, M.; Devi, R.; Kotoky, J. Design, synthesis and biological evaluation of novel 1, 3-thiazolidine-2, 4-diones as anti-prostate cancer agents. Anti-Cancer Agents Med. Chem. (Former. Curr. Med. Chem. Anti-Cancer Agents) 2017, 17, 1756–1768. [Google Scholar] [CrossRef] [PubMed]
  23. Yoshizumi, T.; Ohta, T.; Ninomiya, I.; Terada, I.; Fushida, S.; Fujimura, T.; Nishimura, G.-I.; Shimizu, K.; Yi, S.; Miwa, K. Thiazolidinedione, a peroxisome proliferator-activated receptor-γ ligand, inhibits growth and metastasis of HT-29 human colon cancer cells through differentiation-promoting effects. Int. J. Oncol. 2004, 25, 631–639. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. LOZYNSKYI, A.; Zimenkovsky, B.; Lesyk, R. Synthesis and anticancer activity of new thiopyrano [2, 3-d] thiazoles based on cinnamic acid amides. Sci. Pharm. 2014, 82, 723–734. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Bharti, S.; Nath, G.; Tilak, R.; Singh, S. Synthesis, anti-bacterial and anti-fungal activities of some novel Schiff bases containing 2,4-disubstituted thiazole ring. Eur. J. Med. Chem. 2010, 45, 651–660. [Google Scholar] [CrossRef]
  26. Yang, B.V.; Weinstein, D.S.; Doweyko, L.M.; Gong, H.; Vaccaro, W.; Huynh, T.; Xiao, H.-Y.; Doweyko, A.M.; McKay, L.; Holloway, D.A. Dimethyl-diphenyl-propanamide derivatives as nonsteroidal dissociated glucocorticoid receptor agonists. J. Med. Chem. 2010, 53, 8241–8251. [Google Scholar] [CrossRef] [PubMed]
  27. Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M. Synthesis and antitubercular activity of imidazo [2, 1-b] thiazoles. Eur. J. Med. Chem. 2001, 36, 743–746. [Google Scholar] [CrossRef]
  28. Aoyama, T.; Murata, S.; Arai, I.; Araki, N.; Takido, T.; Suzuki, Y.; Kodomari, M. One pot synthesis using supported reagents system KSCN/SiO2–RNH3OAc/Al2O3: Synthesis of 2-aminothiazoles and N-allylthioureas. Tetrahedron 2006, 62, 3201–3213. [Google Scholar] [CrossRef]
  29. Bell, F.W.; Cantrell, A.S.; Hoegberg, M.; Jaskunas, S.R.; Johansson, N.G.; Jordan, C.L.; Kinnick, M.D.; Lind, P.; Morin, J.M., Jr. Phenethylthiazolethiourea (PETT) compounds, a new class of HIV-1 reverse transcriptase inhibitors. 1. Synthesis and basic structure-activity relationship studies of PETT analogs. J. Med. Chem. 1995, 38, 4929–4936. [Google Scholar] [CrossRef]
  30. Bozdağ-Dündar, O.; Ceylan-Uenluesoy, M.; Eugen, V.J.; Ertan, R. Synthesis and antidiabetic activity of novel 2,4-thiazolidinedione derivatives containing a thiazole ring. Arzneimittelforschung 2006, 56, 621–625. [Google Scholar] [CrossRef]
  31. González Cabrera, D.; Douelle, F.; Feng, T.-S.; Nchinda, A.T.; Younis, Y.; White, K.L.; Wu, Q.; Ryan, E.; Burrows, J.N.; Waterson, D. Novel orally active antimalarial thiazoles. J. Med. Chem. 2011, 54, 7713–7719. [Google Scholar] [CrossRef]
  32. Kachroo, M.; Rao, G.; Rajasekaran, S.; Pai, S.; Hemalatha, Y. Synthesis, antibacterial and antioxidant activity of N-[(4E)-arylidene-5-oxo-2-phenyl-4, 5-dihydro-1H-imidazol-1-yl]-2-(2-methyl-1, 3-thiazol-4-yl) acetamide. Der Pharma Chem. 2011, 3, 241–245. [Google Scholar]
  33. Kouatly, O.; Geronikaki, A.; Kamoutsis, C.; Hadjipavlou-Litina, D.; Eleftheriou, P. Adamantane derivatives of thiazolyl-N-substituted amide, as possible non-steroidal anti-inflammatory agents. Eur. J. Med. Chem. 2009, 44, 1198–1204. [Google Scholar] [CrossRef] [PubMed]
  34. Omarx, A.-M.M.; Eshba, N.H. Synthesis and biological evaluation of new 2, 3-dihydrothiazole derivatives for antimicrobial, antihypertensive, and anticonvulsant activities. J. Pharm. Sci. 1984, 73, 1166–1168. [Google Scholar] [CrossRef] [PubMed]
  35. Spector, F.; Liang, L.; Giordano, H.; Sivaraja, M.; Peterson, M. Inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the UL5-UL8-UL52 complex. J. Virol. 1998, 72, 6979–6987. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Ucar, H.; Van derpoorten, K.; Cacciaguerra, S.; Spampinato, S.; Stables, J.P.; Depovere, P.; Isa, M.; Masereel, B.; Delarge, J.; Poupaert, J.H. Synthesis and anticonvulsant activity of 2 (3 H)-benzoxazolone and 2 (3 H)-benzothiazolone derivatives. J. Med. Chem. 1998, 41, 1138–1145. [Google Scholar] [CrossRef]
  37. Weikert, R.J.; Bingham, S., Jr.; Emanuel, M.A.; Fraser-Smith, E.B.; Loughhead, D.G.; Nelson, P.H.; Poulton, A.L. Synthesis and anthelmintic activity of 3′-benzoylurea derivatives of 6-phenyl-2,3,5,6-tetrahydroimidazo [2, 1-b] thiazole. J. Med. Chem. 1991, 34, 1630–1633. [Google Scholar] [CrossRef]
  38. Franchetti, P.; Cappellacci, L.; Grifantini, M.; Barzi, A.; Nocentini, G.; Yang, H.; O’Connor, A.; Jayaram, H.N.; Carrell, C.; Goldstein, B.M. Furanfurin and thiophenfurin: Two novel tiazofurin analogs. Synthesis, structure, antitumor activity, and interactions with inosine monophosphate dehydrogenase. J. Med. Chem. 1995, 38, 3829–3837. [Google Scholar] [CrossRef]
  39. Li, X.; He, Y.; Ruiz, C.H.; Koenig, M.; Cameron, M.D. Characterization of dasatinib and its structural analogs as CYP3A4 mechanism-based inactivators and the proposed bioactivation pathways. Drug Metab. Dispos. 2009, 37, 1242–1250. [Google Scholar] [CrossRef] [Green Version]
  40. Hu-Lieskovan, S.; Mok, S.; Homet Moreno, B.; Tsoi, J.; Robert, L.; Goedert, L.; Pinheiro, E.M.; Koya, R.C.; Graeber, T.G.; Comin-Anduix, B. Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAF V600E melanoma. Sci. Transl. Med. 2015, 7, 279ra241. [Google Scholar] [CrossRef] [Green Version]
  41. Mishra, S.; Singh, P. Hybrid molecules: The privileged scaffolds for various pharmaceuticals. Eur. J. Med. Chem. 2016, 124, 500–536. [Google Scholar]
  42. Tilekar, K.; Shelke, O.; Upadhyay, N.; Lavecchia, A.; Ramaa, C. Current status and future prospects of molecular hybrids with thiazolidinedione (TZD) scaffold in anticancer drug discovery. J. Mol. Struct. 2022, 1250, 131767. [Google Scholar] [CrossRef]
  43. El-Kashef, H.; Badr, G.; El-Maali, N.A.; Sayed, D.; Melnyk, P.; Lebegue, N.; Abd El-Khalek, R. Synthesis of a novel series of (Z)-3, 5-disubstituted thiazolidine-2, 4-diones as promising anti-breast cancer agents. Bioorganic Chem. 2020, 96, 103569. [Google Scholar] [CrossRef] [PubMed]
  44. Li, T.; Li, W.; Yang, X.; Chen, G.; Jin, X.; Chen, W.; Ye, L. Design, Synthesis, anticancer evaluation and in silico studies of 2, 4, 6-trimethoxychalcone derivatives. Saudi Pharm. J. 2023, 31, 65–84. [Google Scholar] [CrossRef] [PubMed]
  45. Aziz, H.A.; El-Saghier, A.M.; Badr, M.; Abuo-Rahma, G.E.-D.A.; Shoman, M.E. Thiazolidine-2, 4-dione-linked ciprofloxacin derivatives with broad-spectrum antibacterial, MRSA and topoisomerase inhibitory activities. Mol. Divers. 2022, 26, 1743–1759. [Google Scholar] [CrossRef] [PubMed]
  46. Liu, J.-C.; Chen, B.; Yang, J.-L.; Weng, J.-Q.; Yu, Q.; Hu, D.-X. Design, Synthesis and Cytotoxicity of Thiazole-Based Stilbene Analogs as Novel DNA Topoisomerase IB Inhibitors. Molecules 2022, 27, 1009. [Google Scholar] [CrossRef]
  47. Mastrangelo, S.; Attina, G.; Triarico, S.; Romano, A.; Maurizi, P.; Ruggiero, A. The DNA-topoisomerase Inhibitors in Cancer Therapy. Biomed. Pharmacol. J. 2022, 15, 553–562. [Google Scholar] [CrossRef]
  48. Liang, X.; Wu, Q.; Luan, S.; Yin, Z.; He, C.; Yin, L.; Zou, Y.; Yuan, Z.; Li, L.; Song, X. A comprehensive review of topoisomerase inhibitors as anticancer agents in the past decade. Eur. J. Med. Chem. 2019, 171, 129–168. [Google Scholar] [CrossRef]
  49. Mague, J.; Mohamed, S.; Akkurt, M.; El-Kashef, H.; Lebegue, N.; Albayati, M. (5Z)-3-(2-Oxopropyl)-5-(3,4,5-trimethoxybenzylidene)-1, 3-thiazolidine-2,4-dione. IUCrData 2016, 1, x161959. [Google Scholar] [CrossRef] [Green Version]
  50. Dodson, R.; King, L.C. The reaction of ketones with halogens and thiourea1. J. Am. Chem. Soc. 1945, 67, 2242–2243. [Google Scholar] [CrossRef]
  51. Sinicropi, M.S.; Iacopetta, D.; Rosano, C.; Randino, R.; Caruso, A.; Saturnino, C.; Muià, N.; Ceramella, J.; Puoci, F.; Rodriquez, M. N-thioalkylcarbazoles derivatives as new anti-proliferative agents: Synthesis, characterisation and molecular mechanism evaluation. J. Enzym. Inhib. Med. Chem. 2018, 33, 434–444. [Google Scholar] [CrossRef] [Green Version]
  52. Viale, M.; Cordazzo, C.; De Totero, D.; Budriesi, R.; Rosano, C.; Leoni, A.; Ioan, P.; Aiello, C.; Croce, M.; Andreani, A. Inhibition of MDR1 activity and induction of apoptosis by analogues of nifedipine and diltiazem: An in vitro analysis. Investig. New Drugs 2011, 29, 98–109. [Google Scholar] [CrossRef]
  53. Wallace, A.C.; Laskowski, R.A.; Thornton, J.M. LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Protein Eng. Des. Sel. 1995, 8, 127–134. [Google Scholar] [CrossRef] [PubMed]
  54. Cascioferro, S.; Parrino, B.; Carbone, D.; Schillaci, D.; Giovannetti, E.; Cirrincione, G.; Diana, P. Thiazoles, their benzofused systems, and thiazolidinone derivatives: Versatile and promising tools to combat antibiotic resistance. J. Med. Chem. 2020, 63, 7923–7956. [Google Scholar] [CrossRef] [PubMed]
  55. Bergant Loboda, K.; Janežič, M.; Štampar, M.; Žegura, B.; Filipič, M.; Perdih, A. Substituted 4,5′-Bithiazoles as catalytic inhibitors of human DNA topoisomerase IIα. J. Chem. Inf. Model. 2020, 60, 3662–3678. [Google Scholar] [CrossRef]
  56. Zelelew, D.; Endale, M.; Melaku, Y.; Kedir, F.; Demissie, T.B.; Ombito, J.O.; Eswaramoorthy, R. Synthesis, Antibacterial, and Antioxidant Activities of Thiazolyl-Pyrazoline Schiff Base Hybrids: A Combined Experimental and Computational Study. J. Chem. 2022, 2022, 3717826. [Google Scholar] [CrossRef]
  57. Aki-Yalcin, E.; Ertan-Bolelli, T.; Taskin-Tok, T.; Ozturk, O.; Ataei, S.; Ozen, C.; Yildiz, I.; Yalcin, I. Evaluation of inhibitory effects of benzothiazole and 3-amino-benzothiazolium derivatives on DNA topoisomerase II by molecular modeling studies. SAR QSAR Environ. Res. 2014, 25, 637–649. [Google Scholar] [CrossRef]
  58. Rizza, P.; Pellegrino, M.; Caruso, A.; Iacopetta, D.; Sinicropi, M.S.; Rault, S.; Lancelot, J.C.; El-Kashef, H.; Lesnard, A.; Rochais, C.; et al. 3-(Dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (DPA-HBFQ-1) plays an inhibitory role on breast cancer cell growth and progression. Eur. J. Med. Chem. 2016, 107, 275–287. [Google Scholar] [CrossRef]
  59. Sordet, O.; Khan, Q.A.; Kohn, K.W.; Pommier, Y. Apoptosis induced by topoisomerase inhibitors. Curr. Med. Chem. Anti-Cancer Agents 2003, 3, 271–290. [Google Scholar] [CrossRef]
  60. Ceramella, J.; Mariconda, A.; Sirignano, M.; Iacopetta, D.; Rosano, C.; Catalano, A.; Saturnino, C.; Sinicropi, M.S.; Longo, P. Novel Au carbene complexes as promising multi-target agents in breast cancer treatment. Pharmaceuticals 2022, 15, 507. [Google Scholar] [CrossRef]
  61. Mariconda, A.; Iacopetta, D.; Sirignano, M.; Ceramella, J.; Costabile, C.; Pellegrino, M.; Rosano, C.; Catalano, A.; Saturnino, C.; El-Kashef, H. N-Heterocyclic Carbene (NHC) Silver Complexes as Versatile Chemotherapeutic Agents Targeting Human Topoisomerases and Actin. ChemMedChem 2022, 17, e202200345. [Google Scholar] [CrossRef]
  62. Bousbaa, H. Novel Anticancer Strategies. Pharmaceutics 2021, 13, 275. [Google Scholar] [CrossRef]
  63. Alkhzem, A.H.; Woodman, T.J.; Blagbrough, I.S. Design and synthesis of hybrid compounds as novel drugs and medicines. RSC Adv. 2022, 12, 19470–19484. [Google Scholar] [CrossRef] [PubMed]
  64. Romagnoli, R.; Baraldi, P.G.; Salvador, M.K.; Prencipe, F.; Lopez-Cara, C.; Schiaffino Ortega, S.; Brancale, A.; Hamel, E.; Castagliuolo, I.; Mitola, S.; et al. Design, Synthesis, in Vitro, and in Vivo Anticancer and Antiangiogenic Activity of Novel 3-Arylaminobenzofuran Derivatives Targeting the Colchicine Site on Tubulin. J. Med. Chem. 2015, 58, 3209–3222. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Jain, S.; Pattnaik, S.; Pathak, K.; Kumar, S.; Pathak, D.; Jain, S.; Vaidya, A. Anticancer Potential of Thiazole Derivatives: A Retrospective Review. Mini-Rev. Med. Chem. 2018, 18, 640–655. [Google Scholar] [CrossRef]
  66. Buzun, K.; Bielawska, A.; Bielawski, K.; Gornowicz, A. DNA topoisomerases as molecular targets for anticancer drugs. J. Enzym. Inhib. Med. Chem. 2020, 35, 1781–1799. [Google Scholar] [CrossRef]
  67. Skok, Ž.; Zidar, N.; Kikelj, D.; Ilaš, J. Dual Inhibitors of Human DNA Topoisomerase II and Other Cancer-Related Targets. J. Med. Chem. 2019, 63, 884–904. [Google Scholar] [CrossRef] [Green Version]
  68. Banothu, J.; Vaarla, K.; Bavantula, R.; Crooks, P.A. Sodium fluoride as an efficient catalyst for the synthesis of 2, 4-disubstituted-1, 3-thiazoles and selenazoles at ambient temperature. Chin. Chem. Lett. 2014, 25, 172–175. [Google Scholar] [CrossRef]
  69. Wolf, L.; Quoos, N.; Mayer, J.C.; de Souza, D.; Sauer, A.C.; Meichtry, L.; Bortolotto, V.; Prigol, M.; Rodrigues, O.E.; Dornelles, L. Synthesis and free radical scavenging activity of 2-alkyl/arylchalcogenyl-N-(4-aryl-1,3-thiazol-2-yl) acetamide compounds. Tetrahedron Lett. 2016, 57, 1031–1034. [Google Scholar] [CrossRef]
  70. Geronikaki, A.; Theophilidis, G. Synthesis of 2-(aminoacetylamino) thiazole derivatives and comparison of their local anaesthetic activity by the method of action potential. Eur. J. Med. Chem. 1992, 27, 709–716. [Google Scholar] [CrossRef]
  71. Liu, H.-L.; Li, Z.; Anthonsen, T. Synthesis and fungicidal activity of 2-imino-3-(4-arylthiazol-2-yl)-thiazolidin-4-ones and their 5-arylidene derivatives. Molecules 2000, 5, 1055–1061. [Google Scholar] [CrossRef] [Green Version]
  72. Srivastava, S.; Sen, J. Synthesis and biological evaluation of 2- aminobenzothiazole derivatives. Indian J. Chem. 2008, 47B, 1583–1586. [Google Scholar]
  73. Iacopetta, D.; Mariconda, A.; Saturnino, C.; Caruso, A.; Palma, G.; Ceramella, J.; Muià, N.; Perri, M.; Sinicropi, M.S.; Caroleo, M.C. Novel gold and silver carbene complexes exert antitumor effects triggering the reactive oxygen species dependent intrinsic apoptotic pathway. ChemMedChem 2017, 12, 2054–2065. [Google Scholar] [CrossRef] [PubMed]
  74. Redinbo, M.R.; Stewart, L.; Kuhn, P.; Champoux, J.J.; Hol, W.G. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 1998, 279, 1504–1513. [Google Scholar] [CrossRef] [PubMed]
  75. Wang, Y.-R.; Chen, S.-F.; Wu, C.-C.; Liao, Y.-W.; Lin, T.-S.; Liu, K.-T.; Chen, Y.-S.; Li, T.-K.; Chien, T.-C.; Chan, N.-L. Producing irreversible topoisomerase II-mediated DNA breaks by site-specific Pt (II)-methionine coordination chemistry. Nucleic Acids Res. 2017, 45, 10861–10871. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  76. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785–2791. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  77. Sanner, M.F.; Duncan, B.S.; Carrillo, C.J.; Olson, A.J. Integrating computation and visualization for biomolecular analysis: An example using python and AVS. In Biocomputing’99; World Scientific: Singapore, 1999; pp. 401–412. [Google Scholar]
  78. Rosano, C.; Lappano, R.; F Santolla, M.; Ponassi, M.; Donadini, A.; Maggiolini, M. Recent advances in the rationale design of GPER ligands. Curr. Med. Chem. 2012, 19, 6199–6206. [Google Scholar] [CrossRef] [PubMed]
  79. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612. [Google Scholar] [CrossRef] [Green Version]
  80. Iacopetta, D.; Rosano, C.; Puoci, F.; Parisi, O.I.; Saturnino, C.; Caruso, A.; Longo, P.; Ceramella, J.; Malzert-Fréon, A.; Dallemagne, P. Multifaceted properties of 1, 4-dimethylcarbazoles: Focus on trimethoxybenzamide and trimethoxyphenylurea derivatives as novel human topoisomerase II inhibitors. Eur. J. Pharm. Sci. 2017, 96, 263–272. [Google Scholar] [CrossRef]
  81. Sirignano, E.; Saturnino, C.; Botta, A.; Sinicropi, M.S.; Caruso, A.; Pisano, A.; Lappano, R.; Maggiolini, M.; Longo, P. Synthesis, characterization and cytotoxic activity on breast cancer cells of new half-titanocene derivatives. Bioorganic Med. Chem. Lett. 2013, 23, 3458–3462. [Google Scholar] [CrossRef]
  82. Iacopetta, D.; Rosano, C.; Sirignano, M.; Mariconda, A.; Ceramella, J.; Ponassi, M.; Saturnino, C.; Sinicropi, M.S.; Longo, P. Is the Way to Fight Cancer Paved with Gold? Metal-Based Carbene Complexes with Multiple and Fascinating Biological Features. Pharmaceuticals 2020, 13, 91. [Google Scholar] [CrossRef]
Pharmaceuticals 16 00946 sch001 550
Scheme 1. Synthetic route for preparation of the potassium salt 3. Reagents and conditions: (a) i. water, 0–5 °C, 20 min, ii. HCl, reflux, 12 h; (b) anhydrous toluene, catalytic amount of piperidinium acetate, reflux, 15 h; (c) KOH/ethanol, 80 °C, 20 min.
Scheme 1. Synthetic route for preparation of the potassium salt 3. Reagents and conditions: (a) i. water, 0–5 °C, 20 min, ii. HCl, reflux, 12 h; (b) anhydrous toluene, catalytic amount of piperidinium acetate, reflux, 15 h; (c) KOH/ethanol, 80 °C, 20 min.
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Scheme 2. Synthesis of 2-chloro- and 2-iodoacetamides 5a-f and 6a-f respectively. Reagents and conditions: (i) 1. ClCH2COCl, anhydrous benzene, boiling water bath, overnight, 2. NaHCO3 solution, (ii) KI, acetone, reflux, 4 h.
Scheme 2. Synthesis of 2-chloro- and 2-iodoacetamides 5a-f and 6a-f respectively. Reagents and conditions: (i) 1. ClCH2COCl, anhydrous benzene, boiling water bath, overnight, 2. NaHCO3 solution, (ii) KI, acetone, reflux, 4 h.
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Scheme 3. Synthesis of the target hybrid molecules. Reagents and conditions:(i) 6a, DMF, boiling water bath, overnight; (ii) 6b, DMF, boiling water bath, overnight; (iii) 6c, DMF, boiling water bath, overnight; (iv) 6d, DMF, boiling water bath, overnight; (v) 6e, DMF, boiling water bath, overnight; 6f, DMF, boiling water bath, overnight.
Scheme 3. Synthesis of the target hybrid molecules. Reagents and conditions:(i) 6a, DMF, boiling water bath, overnight; (ii) 6b, DMF, boiling water bath, overnight; (iii) 6c, DMF, boiling water bath, overnight; (iv) 6d, DMF, boiling water bath, overnight; (v) 6e, DMF, boiling water bath, overnight; 6f, DMF, boiling water bath, overnight.
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Figure 1. Panel (A) The three-dimensional structure of a monomeric hTopo I is drawn as a tanned ribbon. The active site and the binding site for molecule 7c are highlighted and indicated by arrows. Close-up visualizations of the active site show the binding modes of molecule 7a (Panel (B)), 7b (Panel (C)), 7c (Panel (D)), 7d (Panel (E)), 7e (Panel (F)) and 7f (Panel (G)). The principal amino acids interacting with the different compounds are evidenced as sticks and properly labeled.
Figure 1. Panel (A) The three-dimensional structure of a monomeric hTopo I is drawn as a tanned ribbon. The active site and the binding site for molecule 7c are highlighted and indicated by arrows. Close-up visualizations of the active site show the binding modes of molecule 7a (Panel (B)), 7b (Panel (C)), 7c (Panel (D)), 7d (Panel (E)), 7e (Panel (F)) and 7f (Panel (G)). The principal amino acids interacting with the different compounds are evidenced as sticks and properly labeled.
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Figure 2. Panel (A) A ribbon representation of hTopo II binding cleft in complex with compound 7a. Panel (B) reports the binding mode of compound 7b to hTopo II. Panel (C) illustrates the pose of compound 7c. Panel (D) shows how compound 7d adopts a position that is head-to-tail rotated with respect to molecule 7c. Panel (E) illustrates the interactions of compound 7e with hTopo II. Panel (F) reports the pose of compound 7f. In all the figures, we adopted the same orientation of the protein.
Figure 2. Panel (A) A ribbon representation of hTopo II binding cleft in complex with compound 7a. Panel (B) reports the binding mode of compound 7b to hTopo II. Panel (C) illustrates the pose of compound 7c. Panel (D) shows how compound 7d adopts a position that is head-to-tail rotated with respect to molecule 7c. Panel (E) illustrates the interactions of compound 7e with hTopo II. Panel (F) reports the pose of compound 7f. In all the figures, we adopted the same orientation of the protein.
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Figure 3. hTopo I relaxation assay. Plasmid pHOT1, used as substrate, was incubated with hTopo I with or without the studied compounds 7b–7e (10 µM concentration), panel (A), and with or without ellipticine (10 and 50 µM concentration), panel (B). Lane A, relaxed DNA marker; lane B, pHOT1 supercoiled DNA, lane C, CTRL (DMSO), lane D–G, compounds 7b–7e 10 µM, respectively. Lane 1, pHOT1 supercoiled DNA; lane 2, CTRL (DMSO), lanes 3–4, ellipticine 10 and 50 µM, respectively.
Figure 3. hTopo I relaxation assay. Plasmid pHOT1, used as substrate, was incubated with hTopo I with or without the studied compounds 7b–7e (10 µM concentration), panel (A), and with or without ellipticine (10 and 50 µM concentration), panel (B). Lane A, relaxed DNA marker; lane B, pHOT1 supercoiled DNA, lane C, CTRL (DMSO), lane D–G, compounds 7b–7e 10 µM, respectively. Lane 1, pHOT1 supercoiled DNA; lane 2, CTRL (DMSO), lanes 3–4, ellipticine 10 and 50 µM, respectively.
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Figure 4. hTopo II decatenation assay. The catenated circles of kDNA, used as substrate, incubated with hTopo II with or without the studied compounds 7b–7e used at the concentrations of 10 µM (a) and 50 µM (b). Lane A, linear kDNA marker; lane B, decatenated kDNA marker; lane C, catenated circles of kDNA; lane D, CTRL (DMSO), lanes E–H, compounds 7b–7e, respectively. The same experiment was performed using ellipticine (c). Lane 1, decatenated kDNA marker; lane 2, linear kDNA marker; lane 3, CTRL (DMSO), lanes 4–5, ellipticine 10 and 50 µM, respectively.
Figure 4. hTopo II decatenation assay. The catenated circles of kDNA, used as substrate, incubated with hTopo II with or without the studied compounds 7b–7e used at the concentrations of 10 µM (a) and 50 µM (b). Lane A, linear kDNA marker; lane B, decatenated kDNA marker; lane C, catenated circles of kDNA; lane D, CTRL (DMSO), lanes E–H, compounds 7b–7e, respectively. The same experiment was performed using ellipticine (c). Lane 1, decatenated kDNA marker; lane 2, linear kDNA marker; lane 3, CTRL (DMSO), lanes 4–5, ellipticine 10 and 50 µM, respectively.
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Figure 5. TUNEL assay. MCF-7 breast cancer cells were exposed to compounds 7e and ellipticine (E) at their IC50 values, or to the vehicle (DMSO) for 24 h. Then, they were further processed, as reported in Section 4, and imaged under a fluorescence microscope (20× magnification). Panels (A), DAPI λex,em= 350 nm/460 nm; panels (B), CFTM488A, λex,em = 490 nm/515 nm; panels (C) show the merge channel.
Figure 5. TUNEL assay. MCF-7 breast cancer cells were exposed to compounds 7e and ellipticine (E) at their IC50 values, or to the vehicle (DMSO) for 24 h. Then, they were further processed, as reported in Section 4, and imaged under a fluorescence microscope (20× magnification). Panels (A), DAPI λex,em= 350 nm/460 nm; panels (B), CFTM488A, λex,em = 490 nm/515 nm; panels (C) show the merge channel.
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Figure 6. Mitochondria and cytochrome c immunostaining in MDA-MB-231 cells. In DMSO-treated cells, cytochrome c (panel (C), CTRL) resides within mitochondria (panel (B), CTRL), as visible in the overlay channel (panel (D), CTRL). MCF-7 cells treated with 7e (concentration equal to its IC50 value, for 24 h) show cytochrome c release in the cytosol (panel (C), 7e) and changes in mitochondrial structure (panel (B), 7e). (A) Nuclear stain with DAPI (excitation/emission wavelength 350 nm/460 nm). (B) MitoTracker Deep Red FM probe (excitation/emission wavelength 644 nm/665 nm). (C) Alexa Fluor CF 488 (excitation/emission wavelength 490 nm/515 nm). (D) Merge channels. Images were taken at 40× magnification, and representative fields are shown.
Figure 6. Mitochondria and cytochrome c immunostaining in MDA-MB-231 cells. In DMSO-treated cells, cytochrome c (panel (C), CTRL) resides within mitochondria (panel (B), CTRL), as visible in the overlay channel (panel (D), CTRL). MCF-7 cells treated with 7e (concentration equal to its IC50 value, for 24 h) show cytochrome c release in the cytosol (panel (C), 7e) and changes in mitochondrial structure (panel (B), 7e). (A) Nuclear stain with DAPI (excitation/emission wavelength 350 nm/460 nm). (B) MitoTracker Deep Red FM probe (excitation/emission wavelength 644 nm/665 nm). (C) Alexa Fluor CF 488 (excitation/emission wavelength 490 nm/515 nm). (D) Merge channels. Images were taken at 40× magnification, and representative fields are shown.
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Figure 7. Caspase activity in MCF-7 cells. The treatment of MCF-7 cells with the compound 7e, at a concentration equal to its IC50 value for 24 h, induced caspase 3/7 and 9 activation (compared to DMSO-treated cells, CTRL). Columns mean, bars SD; * p < 0.001; n.s., not significant (treated vs. CTRL).
Figure 7. Caspase activity in MCF-7 cells. The treatment of MCF-7 cells with the compound 7e, at a concentration equal to its IC50 value for 24 h, induced caspase 3/7 and 9 activation (compared to DMSO-treated cells, CTRL). Columns mean, bars SD; * p < 0.001; n.s., not significant (treated vs. CTRL).
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Table
Table 1. IC50 values, expressed in µM, of the studied compounds 7a–f and colchicine and ellipticine against different cell lines.
Table 1. IC50 values, expressed in µM, of the studied compounds 7a–f and colchicine and ellipticine against different cell lines.
IC50 (µM)
Compounds MDA-MB-231 MCF-7 A2058 3T3 BALB MCF-10A
7a 27.5 ± 0.9 >100 >100 89.2 ± 1.1 >100
7b 17.9 ± 1.1 8.5 ± 1.2 3.5 ± 1.1 >100 >100
7c 32.8 ± 0.6 19.5 ± 0.6 4.8 ± 1.0 >100 46.4 ± 1.1
7d 33.4 ± 1.0 34.1 ± 0.7 6.4 ± 0.9 36.1 ± 1.0 >100
7e 41.2 ± 0.9 3.1 ± 0.6 8.4 ± 0.9 35.0 ± 0.6 >100
7f 50.5 ± 0.8 17.4 ± 1.1 24.6 ± 1.1 >100 >100
colchicine 8.1 (± 1.0) × 10−2 1.6 (± 0.8) × 10−2 3.4 (± 0.7) × 10−2 5.2 (± 0.9) × 10−1 8.9 (± 1.2) × 10−3
ellipticine 1.73 ± 0.4 1.15 ± 0.5 1.33 ± 0.9 0.97 ± 0.1 1.09 ± 0.1
Table
Table 2. Overview of the hTopo assays: (+) inhibition; (−) no inhibition; (+/−) partial inhibition.
Table 2. Overview of the hTopo assays: (+) inhibition; (−) no inhibition; (+/−) partial inhibition.
Topoisomerase Inhibition
Compounds [µM] hTopo I hTopo II
7b 10 − −
50 −
7c 10 − −
50 +
7d 10 − −
50 +
7e 10 − −
50 +
7f 10 + −
50 +
ellipticine 10 + +/−
50 + +
The color indicates the degree of inhibition (null = orange, intermediate = yellow, total = green).
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MDPI and ACS Style

Sinicropi, M.S.; Ceramella, J.; Vanelle, P.; Iacopetta, D.; Rosano, C.; Khoumeri, O.; Abdelmohsen, S.; Abdelhady, W.; El-Kashef, H. Novel Thiazolidine-2,4-dione-trimethoxybenzene-thiazole Hybrids as Human Topoisomerases Inhibitors. Pharmaceuticals 2023, 16, 946. https://doi.org/10.3390/ph16070946

AMA Style

Sinicropi MS, Ceramella J, Vanelle P, Iacopetta D, Rosano C, Khoumeri O, Abdelmohsen S, Abdelhady W, El-Kashef H. Novel Thiazolidine-2,4-dione-trimethoxybenzene-thiazole Hybrids as Human Topoisomerases Inhibitors. Pharmaceuticals. 2023; 16(7):946. https://doi.org/10.3390/ph16070946

Chicago/Turabian Style

Sinicropi, Maria Stefania, Jessica Ceramella, Patrice Vanelle, Domenico Iacopetta, Camillo Rosano, Omar Khoumeri, Shawkat Abdelmohsen, Wafaa Abdelhady, and Hussein El-Kashef. 2023. "Novel Thiazolidine-2,4-dione-trimethoxybenzene-thiazole Hybrids as Human Topoisomerases Inhibitors" Pharmaceuticals 16, no. 7: 946. https://doi.org/10.3390/ph16070946

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