4-Thiazolidinone-Bearing Hybrid Molecules in Anticancer Drug Design

Oncological diseases have currently reached an epidemic scale, especially in industrialized countries. Such a situation has prompted complex studies in medicinal chemistry focused on the research and development of novel effective anticancer drugs. In this review, the data concerning new 4-thiazolidinone-bearing hybrid molecules with potential anticancer activity reported during the period from the years 2017–2022 are summarized. The main emphasis is on the application of molecular hybridization methodologies and strategies in the design of small molecules as anticancer agents. Based on the analyzed data, it was observed that the main directions in this field are the hybridization of scaffolds, the hybrid-pharmacophore approach, and the analogue-based drug design of 4-thiazolidinone cores with early approved drugs, natural compounds, and privileged heterocyclic scaffolds. The mentioned design approaches are effective tools/sources for the generation of hit/lead compounds with anticancer activity and will be relevant to future studies.


Introduction
Cancer diseases are a colossal problem for humanity, and according to data from the World Health Organization (WHO), they are the major cause of death among people under 70 years [1]. This poses a significant challenges to research focused on the development of novel and more effective pharmaceuticals without the disadvantages/adverse effects of classic/conventional anticancer chemotherapeutics, which usually include a low therapeutic index, development of resistance to treatment, and low bioavailability [2]. The modern medicinal chemistry of anticancer agents actively uses a wide set of tools for updating anticancer agent libraries and pipelines with the aim to achieve the desired biological activity and overcome the limitations of known chemotherapeutics.
Molecular-hybridization approaches are important synthetic strategies in the design of drug-like small molecules with more powerful and biologically more extensive anticancer properties than the initial compounds [3].
4-Thiazolidinone scaffolds belong to privileged structures in drug design [4]. The most successful molecules from this class of heterocycles include epalrestat [5] and glitazones [6] ( Figure 1), which has made a significant contribution to diabetes therapy. Over the last decade, 4-thiazolidinone derivatives have continued to be the focus of research by medicinal chemists and have once again returned to the world pharmaceutical market. Thus, the 5-ylidene derivative of 2-(alkyl)imino-4-thiazolidinone Ponesimod (Ponvory) (Figure 1) was approved by the FDA in 2021 as a potential drug for the treatment of multiple sclerosis and psoriasis [7,8]. 4-Thiazolidinone scaffolds are widely used for the development of anticancer agents, and a number of recent reviews have been published dedicated to this area of drug design [9,10]. The works in question include material devoted specifically to derivatives of 2-thioxothiazolidin-4-one (rhodanine) [9] and thiazolidine-2,4-dione [10], which possess antitumor properties. Therefore, this review's purpose was to highlight the anticancer properties of 2-imino-4-thiazolidinone derivatives and 2-3-disubstituted 4-thiazolidinones as well as several recent works devoted to 2-thioxothiazolidin-4-one (rhodanine) or thiazolidin-2,4-dione derivatives. For the writing of this review, the research articles, short communications, letters, and reports from the scientific databases Scopus (Elsevier), SciFinder (Chemical Abstracts), and PubMed were analyzed from the years 2017 to 2022. Information from patents was not included in the present study. Moreover, the main emphasis of the present review was on the application of molecular hybridization strategies such as the hybridization of scaffolds, the hybrid-pharmacophore approach, analogue-based drug design in synthesis, and the development of novel potent antitumor agents with 4-thiazolidinone scaffolds.

Approved Drug-4-Thiazolidinones Hybrids
A series of publications reported the successful application of molecular hybridization methods and strategies for the design of novel anticancer agents based on obtaining hybrids of the type "approved drugs-4-thiazolidinone scaffolds". The drugs from different pharmacological groups were used as parent molecules for further hybridization and development.

Anticancer Drug-4-Thiazolidinones Hybrids
Türe et al. [11] reported on the design and synthesis of a series of novel hybrid molecules containing imatinib (Gleevec) moiety. Imatinib (Figure 2) was the first-in-class protein kinase inhibitor approved by the FDA in 2001 and had a revolutionary impact on the treatment of most cases of chronic myeloid leukemia (CML) [12,13]. Obtained by Türe et al., 5-benzylidene-2-arylimino-4-thiazolidinone-bearing imatinib analogs were screened for their antimitotic activity on K562 (chronic myeloid leukemia), PC3 (prostate cancer), and SHSY-5Y (neuroblastoma) cell lines, and the three most potent cytotoxic hybrids, 1-3, were identified. Compounds 1-3 induced apoptotic cell death. Moreover, cycle arrest in the G0/G1 phase was caused by compounds 2 and 3, while compound 1 inhibited the cell cycle in the G2/M phase. Hybrid molecules 1 and 3 proved to have stronger genotoxic effects than imatinib against K562 cells [11].

NSAID-4-Thiazolidinones Hybrids
Recent decades have been marked by an increased interest in the use of potential NSAIDs for the treatment of cancer or using NSAIDs scaffolds/molecules for the design of new antimitotic agents [14].
Ramadan W.S. et al. [15] used a hybridization approach involving anti-inflammatory drug-5-aminosalicylic acid and 5-ylidene-4-thiazolidinone scaffolds with the aim of designing new promising anti-cancer agents ( Figure 3). Synthesized hybrids were tested on a panel of seven cancer cell lines, and two molecules, 4 and 5, were found to be efficient in some types of cancer compared to the effect of doxorubicin (reference drug), with the highest activity level detected on the MCF7 line, with IC 50 values of 0.31 and 0.30 µM, respectively. Furthermore, tumor specificity and minimal effects on normal fibroblasts were observed for 4 and 5. In vitro studies of the molecular mechanisms of action for 4 and 5 revealed the induction of DNA damage, cell cycle arrest in the G2/M phase, and the induction of apoptosis as indicated by annexin-V staining and the activation of caspases. Moreover, it was found that both compounds modulate the expression and activity of several factors in the DNA damage response pathway, cyclins/cyclin-dependent kinases, and CDC25 phosphatase. NSAID-diclofenac was used for the design of potential anticancer agents by Shepeta et al. [16]. A series of diclofenac-4-thiazolidinones hybrids with a hydrazine linker in the molecules were synthesized and tested on antitumor activity in a 60 line screening program (DTP NCI) [17][18][19][20]. Two novel hybrids, 6 and 7 (Figure 3), displayed slight anticancer activity, and an in vitro cytostatic effect was observed on the leukemia CCRF-CEM, with a percentage of growth (GP%) of 56.82% for molecule 6, on the non-small-cell lung cancer NCIH522, with a GP of 54.32%, and on colon cancer HCT-116, with a GP% of 57.71% of cell lines for hybrid 7.

Antibacterial Drug-4-Thiazolidinones Hybrids
A series of novel 4-thiazolidinone-sulfanilamide hybrids incorporating substituted indolin-2-one moieties were designed as possible human carbonic anhydrase (hCA) inhibitors by Eldehna et al. [22]. The new hybrids were evaluated in vitro for their inhibitory activity against hCA I, II, IV, and IX, and all molecules were active in variable degrees. Moreover, all compounds were examined for their anti-proliferative activity against breast cancer MCF-7 and colorectal cancer Caco-2 cell lines, and hybrid 9 ( Figure 4) was found to be the most potent against MCF-7, with an IC 50 of 3.96 ± 0.21 µM. In-depth studies showed that hybrid 9 provoked the intrinsic apoptotic mitochondrial pathway in MCF-7 cells, which was evidenced by the enhanced expression of the pro-apoptotic protein Bax and the reduced expression of the anti-apoptotic protein Bcl-2 as well as up-regulated active caspase-3 and caspase-9, cytochrome C, and p53 levels. Multifunctional sulfanilamide-bearing hybrids were developed and tested against HepG2 and MDA-MB-231 cancer cell lines by Sunil Kumar et al. [23]. Among the tested hybrids, compound 10 ( Figure 4) was identified as potent against the MBA-MB-231 cell line and showed the highest cytotoxic activity, with an IC 50 value of 17.45 µM, which was higher than in the case of the standard drug cisplatin.
Special attention from the point of view of analog-and fragment-based anticancer drug design is warranted in the case of the molecule ciminalum, which is a p-nitro-αchlorocinnamic aldehyde or (2Z)-2-chloro-3-(4-nitrophenyl)prop-2-enal (CAS 3626-97-9) and was used as a drug in medical practice in the former Soviet Union ( Figure 5) as an active antimicrobial agent against Gram-positive and Gram-negative microorganisms.
The diversity-oriented approach was applied for the synthesis of ciminalum-4-thiazolidinone hybrid molecules, and a series of derivatives were reported by Buzun and Finiuk [24][25][26]. For such a hybrid type, SAR analysis revealed that the presence of carboxylic acids [25], phydroxyphenyl [24], or (4-hydroxyphenyl)-pyrrolidine-2,5-dione [26] substituents at position 3 of the 4-thiazolidinone ring determines the most effective anti-tumor activity, while the absence of a substituent or presence of an additional ciminalum substituent at position 3 leads to a weakening in activity. Among the synthesized and screened hybrids, derivative 11 ( Figure 6) was cytotoxic toward MCF-7 and MDA-MB-231 breast cancer cell lines, with IC 50 values of 5.02 µM and 15.24 µM, respectively [25]. The hybrid 11 induced the extrinsic and intrinsic apoptotic pathways and caused a reduction in topoisomerase II concentration in both tested human breast cancer cell lines. The affinity of hybrid 11 to topoisomerase II was also confirmed by docking simulations. In addition, molecule 11 caused a decrease in the levels of Beklin-1, LC3A (the microtubule-associated protein 1A/1B light chain 3A), and LC3B (the microtubule-associated protein 1A/1B light chain 3B), suggesting its influence on the autophagy process.

Natural Compound Scaffold-4-Thiazolidinones Hybrids
The structural motifs of natural products always serve as an excellent source for drug-like molecules from the perspective of the design of anticancer agents [27,28].

Monoterpene-4-Thiazolidinone Hybrids
Fawzi et al. [29] synthesized 4-acetyl-1-methylcyclohexenothiosemicarbazones and 4-acetyl-1-methylcyclohexeno-2-imino-4-thiazolidinone hybrids based on a monoterpene backbone structure, that of limona ketone, containing hydrazone linker in the molecules (Figure 7). The cytotoxicity of the obtained compounds was evaluated on HT-1080, A549, and MCF-7 cell lines. Compounds 16 and 17 ( Figure 7) proved to be the most cytotoxic on HT-1080 lines, demonstrating IC 50 s of 15.85 µM and 16.13 µM, respectively. Through molecular docking, these compounds were found to form stable ligand-caspase-3 complexes. In addition, compound 17 was revealed to be the most potent in generating apoptosis and caspases-3/7 activation, including S-phase cell cycle inhibition in HT-1080 cells, while compound 16 exhibited a lower degree of apoptosis induction compared to compound 17, in turn inducing G0/G1 phase arrest in the same cells. Oubella et al. [30] developed hybrids containing the monoterpene-carvone skeleton and 4-thiazolidinone scaffolds linked by the 1,2,3-triazole ring ( Figure 8). The novel hybrids were screened for their anticancer activity against four cell lines: breast MCF-7 and MDA-MB-231, lung A-549 and fibrosarcoma HT-1080. The highest cytotoxic activity was demonstrated by compounds 18 and 19 ( Figure 8). Expanding the study, the researchers proved that such activity is generated through the induction of apoptosis via caspase-3, as confirmed by molecular docking, and effects on the cell cycle, specifically arresting cells in S and G2/M phases in HT-1080 and A-549 cells, respectively. Applying thymol as part of the structure of novel hybrids with 4-thiazolidinone scaffolds is a promising approach. A synthesis and anticancer activity evaluation of a series of thymol-4-thiazolidinone hybrids was reported by El-Miligy et al. [31]. Among the new hybrids compounds, 20-22 ( Figure 9) exhibited the highest activity against human colorectal cancer (CRC) cell lines (Caco-2 and HCT-116) at doses less than their EC 100 on normal human cells. Furthermore, compounds 21 and 22 induced apoptosis-dependent death and caspase activation by >50% in human CRC cell lines. Furthermore, compounds 20-22 showed in vitro inhibitory activity against both PIM-1/2 kinases comparable to the reference, staurosporine.

Coumarin 4-Thiazolidinones Hybrids
The application of the coumarin motif is an attractive direction in anticancer drug design [32]. Following this strategy, Sigalapalli et al. [33] reported the synthesis of novel molecular hybrids of 4-thiazolidinone-umbelliferone (7-hydroxycoumarin) as prominent cytotoxic agents. The most active derivative, 23 ( Figure 10), showed the highest potency against A549 cells, with an IC 50 value of 0.96 ± 1.09 µM and a selectivity index of 51.7. The in-depth study of hybrid 23 showed apoptosis induction by the annexin-v/PI dual staining assay; its effect on different phases of the cell cycle; the inhibition of tubulin polymerization at an IC 50 value of 2.65 ± 0.47 µM; and its effective binding with CT-DNA. In silico experiments revealed a prominent binding affinity towards the α/β-tubulin receptor with remarkable protein-ligand interactions and binding energy in the case of 23. Thacker et al. [34] reported the synthesis of coumarin-4-thiazolidinone hybrids with pyrazole linker in the molecules as potential hCA inhibitors. The results obtained by the authors indicated the selective inhibition by synthesized hybrids of the tumor-associated isoforms hCA IX and XII but not in the case of the off-target isoforms, hCA I and II. Compound 24 ( Figure 10), with the best hCA XII inhibition constant value, 61.5 nM, was considered the lead compound for further developing selective and potent hCA IX inhibitors.
In silico studies inferred that hybrid 25 binds at the colchicine binding site of the tubulin, with prominent binding affinity.

Hybrids of 4-Thiazolidinones with Steroidal Skeleton
Steroids are important and physiologically significant natural compounds, opening up the possibility of structural tuning with the aim of achieving and developing valuable pharmacological properties including potent anticancer activity [36].
All the obtained molecules exhibited selective concentration-dependent cytotoxicity on all tested lines. The strongest response to the tested compound was observed on the K562 and HeLa cell lines, where the IC 50 of the majority oscillated around 10 µM, with the IC 50 of the cisplatin reference compound for these lines being 5.7 µM and 5.2 µM, respectively. Compared to the positive control, these compounds exhibited lower toxicity to normal cells. Hybrids 26 and 27 were found to be the most active ( Figure 11) and inhibited the cell cycle in HeLa cells in subG1, S, and G2/M phases and induced apoptosis by an intrinsic and extrinsic pathway.

Privileged Heterocyclic Scaffolds-4-Thiazolidinones Hybrid Molecules
Hybrids with 4-thiazolidinone scaffolds as potent anticancer agents are the most intensively studied type of hybrids in recent years and represents the largest group of molecules among recently published data.

Furan-4-Thiazolidinone Hybrids
Tahmasvand et al. [38] reported on the design and synthesis of eight novel 4-thiazolidinone hybrids which were evaluated for their in vitro anticancer activity against MDA-MB-231, HT-29, and HepG2 cell lines. The furan-bearing hybrid 28 ( Figure 12) demonstrated very strong activity against the three cell lines after 72 h with IC 50 values of 1.9 µM, 6.5 µM, and 5.4 µM, respectively, which was comparable to the control sample with the reference compound doxorubicin.

Pyrazole-4-Thiazolidinone Hybrids
A series of 4-thiazolidinone-pyrazole hybrids connected via a ylidene linker were synthesized by Bhat et al. [40]. An  Furthermore, molecule 34 exhibited superior protection of normal HDF cells than 33. Additionally, the anticancer potency of the compounds was justified by using in silico studies. SAR analysis concluded by the authors suggests the importance of the presence of dichlorosubstituted arylamino moiety in position two of the 4-thiazolidinone core in enhancing potency.
The most potent cytotoxic activity among all the analyzed hybrids was shown by compound 36 (Figure 15), with IC 50 values of 18.85 µM, 23.43 µM, 23.08 µM, 23.08 µM, and 18.50 µM for the A549, MCF-7, HepG-2, Caco-2, and PC-3 cell lines, respectively, whereas the values for the reference compound 5-fluorouracil oscillated around 90 µM. As with cytotoxic activity, compound 36 showed the strongest antioxidant properties; however, these were slightly weaker than in the case of the reference compound, ascorbic acid.

Pyrazole-Piperazine-4-Thiazolidinone Hybrids
In study [43], new pyrazole-based 4-thiazolidinones hybrids linked with piperazine moiety were reported as promising selective VEGFR2 tyrosine kinase inhibitors. Among all the tested compounds, the derivatives 37-39 ( Figure 15) were found to be the most active and selective against the HepG-2 cancer cell line, with IC 50 values 0.06 ± 0.003 µM, 0.03 ± 0.006 µM, and 0.06 ± 0.004 µM, respectively, and with the significant selectivity indices: 8.09, 11.40, and 4.37, respectively. Also, the synthesized hybrids showed potent VEGFR2 tyrosine kinase inhibitory activities, with lower IC 50 values than that of the reference drug, staurosporine, with this potency being confirmed by additional docking studies which demonstrated that 37-39 could act as inhibitors of VEGFR2 tyrosine kinase via the stabilization of the enzyme inactive DFG-out conformation.

Isoxazole-4-Thiazolidinone Hybrids
The synthesis of the hybrid 2-(4-fluorophenyl)-3-(5-methylisoxazol-3-yl)thiazolidin-4-one, 42 (Figure 17), as a potential antimitotic agent was reported by Ramesh et al. [45]. The results of an in vitro cytotoxicity assessment employing the MTT assay as per the ATCC protocol, using three human cancer cell lines, HeLa (cervical cancer), MCF-7 (breast cancer), A549 (human lung cancer), and HEK-293 (normal human embryonic kidney cells), indicated that hybrid 42 exhibited excellent anticancer activity against MCF7 and displayed less cytotoxicity towards normal HEK293 cell lines. Hybrid molecule 42 inhibited the cell proliferation of HeLa by 58%, 87%, and 94% at 6.25 µM, 25 µM, and 50 µM concentrations, respectively. In terms of inhibition, 59% and 65% inhibition were observed at a 50 µM concentration on breast cancer (MCF7) and lung cancer (A549) cell lines, respectively, by compound 42. At lower concentrations, compound 42 has less effect on normal cell lines (HEK293). Additionally, a molecular docking study explored the binding mode and possible interactions between the synthesized compound and the ATP binding site of the EGFR kinase domain.

Thiazole-4-Thiazolidinone Hybrids
A series of 5-enamine-4-thiazolidinones with phenylalanine moiety in the molecules was designed and reported by Holota et al. [46]. Among the synthesized derivatives, the hybrid 43 (Figure 18), with a thiazole-bearing scaffold in the molecule, was found to be highly active in the 60 lines screening. Compound 43 demonstrated inhibition activity against (GI 50   Preliminary SAR provided by the authors revealed that the presence of thiazole substituent is crucial for anticancer activity whereas a change in the hydrogen atom, aniline derivatives, or 1,2,4-triazole nucleus leads to the disappearance of the effect.

Triazole-4-Thiazolidinone Hybrids
Two series of merged 1,2,4-triazole-4-thiazolidinone hybrids with ylidene and enamine linkers in the molecules ( Figure 19) were designed and synthesized by Holota et al. [47]. The obtained hybrids were studied for their antitumor activity in an NCI 60 lines screening [17][18][19][20] and a number of the compounds presented excellent anticancer properties at 10 µM. Derivatives 44 and 45 were found to be the most active against cancer cell lines, with total mean GI 50  An SAR analysis performed by authors revealed that both the presence and position of a chlorine atom in the benzylidene area of molecules play a crucial role in the realization of the anticancer effect [47].

Pyridine-4-Thiazolidinone Hybrids
A series of 17 novel pyridine-thiazolidinone hybrids were synthesized, characterized, and evaluated as potential hCA inhibitors by Ansari et al. [48]. Of all the synthesized hybrids, derivatives 46 and 47 ( Figure 20  In [49], the synthesis and study of molecules 48 and 49 (Figure 20), as modified analogues of hybrids 46 and 47, was reported. The authors applied the bioisosteric replacement of the pyridine core by the substituted benzene ring for the design of novel target molecules with desirable anticancer properties. Despite the promising evaluated in silico features, the newly synthesized analogues 48 and 49 were found to be less active compared to the hybrids 46 and 47 against the HepG-2 cell line.
The molecular hybridization approach was applied for the synthesis of pyridine-4thiazolidinone hybrids with aim of the synthesis of potential antiglioblastoma agents by Campos et al. [50]. Among sixteen synthesized compounds, derivatives 50-52 ( Figure 20) displayed potent antitumor activities against the tested glioblastoma cell lines and exhibited IC 50 values 2.17, 6.24, and 2.93 µM, respectively, in the C6 cell line, well below the standard drug temozolomide. The mechanism of action studies demonstrated that derivatives 50 and 52 induced apoptosis, significantly increasing the percentage of cells in the Sub-G1 phase in the absence of necrosis. Consistent with these results, the caspase-3/7 assay revealed that derivative 50 presents pro-apoptotic activity due to the significant stimulation of caspases-3/7. Moreover, hybrids 50-52 increased antioxidant defense, decreased reactive oxygen species (ROS) production, and modulated redox status.

Pyridine-Piperazine-4-Thiazolidinone Hybrids
The attempt to design potential antimitotic agents following the hybridization of pyridine, piperazine, and 4-thiazolidinone scaffolds was reported by Demirci et al. [51]. The synthesized hybrids were evaluated on the PC-3, DU145, and LNCaP prostate cancer cell lines. All the studied molecules possessed satisfactory calculated pharmacokinetic properties. Compound 53 (Figure 21) showed the most potent activity level inside the tested series, with an IC 50 of 36.75 µM on the PC-3 cancer line, while the other cancer cells tested showed no response to the compounds used (IC 50 > 500 µM).

Pyridine-Thiazole-4-Thiazolidinone Hybrids
Hussein et al. [52] applied a hybrid-pharmacophore approach for the design of novel biologically active 4-thiazolidinones. Among the synthesized molecules, compound 54 ( Figure 21) exhibited the most potent cytotoxic activity and was found to be more effective on the WI-38 normal human lung fibroblast cell line, with an IC 50 of 92 µg/mL, but not as effective on the A549 and MDA-MB-31 cancer cells, with IC 50 values of 357 µg/mL and 505 µg/mL, respectively.

Indole-4-Thiazolidinone Hybrids
Indole scaffolds are often used for the design and development of 4-thiazolidinone hybrids as anticancer agents. Sigalapalli et al. [53] reported on the synthesis of a small library of hybrid molecules containing 4-thiazolidinone and indole scaffolds linked by an aliphatic linker with aim of the designing tubulin polymerization inhibitors. The novel compounds were tested on five human cell lines of the lung (A549, NCI-H460), breast (MDA-MB-231), and colon (HCT-29, HCT-15), using podophyllotoxin as a reference compound. The synthesized compound 55 (Figure 22) showed the strongest activity against both colon cancer cell lines, with an IC 50 value of 0.92µM for HCT-15. Meanwhile, against the normal human lung epithelial cell line (L132), the IC 50 value for the compound was 10.84 µM, indicating that compound 55 is 10-fold more selective against the colon cell line. SARs analysis performed by the authors revealed that the antiproliferative activity of phenethylthiazolidinone-indole hybrids is higher than that of benzyl-thiazolidinone-indole ones. Furthermore, it was observed that hybrids without a substituent at the nitrogen atom into indole core are more active than N-alkyl/aryl-substituted ones. In addition, the presence of the methoxy group at the phenyl ring of the side chain of hybrid compounds leads to higher activity than halogen substituents. Extended analyses on compound 55 revealed that the compound inhibited the cell cycle of HCT-15 cells in sub G1 and G2/M phases, additionally inhibited tubulin more strongly compared to the reference compound, induced intracellular ROS, and decreased the mitochondrial potential, causing cell death by apoptosis [53]. A series of novel indole-azolidinone hybrids with 5-fluoro-3-formyl-1H-indole-2carboxylic acid methyl ester scaffolds were synthesized via the Knoevenagel reaction [55]. Among the synthesized compounds, hybrid 57 ( Figure 22) was found to be the most active and exhibited toxicity in the 60 lines screening, with total GI 50 of 0.45/0.65 µM and with IC 50 values toward cancer cells MCF-7-0.70 µM, HCT116-0.80 µM, A549-9.70 µM, and HepG2-12.00 µM. Meanwhile, the non-malignant cells (human keratinocytes of HaCaT line and murine embryonic fibroblasts of Balb/c 3T3 line) possessed moderate sensitivity to hybrid 57. Furthermore, compound 57 induced apoptosis in studied tumor cells via caspase-3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. Additionally, hybrid 57 impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much fewer DNA damage events were induced by 57 in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells.

Isatine-4-Thiazolidinone Hybrids
Isatin ( Figure 23) is an endogenously occurring molecule in human tissues, based on the structure of which tyrosine kinase inhibitor Sunitinib (Sutent ® ) [56] was established. Sunitinib was approved by the FDA in 2006 for the treatment of the imatinib-resistant cancers advanced metastatic renal cell carcinoma and gastrointestinal stromal tumors.
El-Naggar et al. [57] reported a series of isatine-4-thiazolidinone hybrids, among which molecule 58 (Figure 23  Szychowski et al. [59] reported studies on the anticancer potential of an isatine-4thiazolidinone hybrid modified with a 3,5-diaryl-2-pyrazoline scaffold 60 ( Figure 23). The impact of hybrid 60 on cytotoxicity, the apoptotic process, and metabolism in the human squamous carcinoma (SCC-15) cell line was evaluated. The results obtained by the authors showed that the studied molecule 60 exhibits both cytotoxic and proapoptotic properties at a 10-100 µM concentration range. The activation of caspase-3 was caused by 60 and was accompanied by an LDH release and resazurin reduction. Additionally, increased DCF fluorescence in a wide range of concentrations after 6, 24, and 48 h of exposure was observed after treatment with 60, which suggests the stimulation of ROS production. The properties of 60 established by the authors make it a promising tool for further research as an anti-cancer agent.

Quinoline-4-Thiazolidinone Hybrids
Batran et al. [60] reported on the synthesis of quinoline-4-thiazolidinone hybrids as potential antimitotic agents. Molecule 61 ( Figure 24) was active against the MCF-7 breast cancer cell line, with an IC 50 value of 98.79 µM. Nafie et al. [61] studied another series of quinolone-4-thiazolidinone hybrids. Five of the ten compounds showed strong cytotoxic activity against the HCT-116 cell line, comparable to 5-FU, while compound 62 (Figure 24) was the most potent cytotoxic, excluding effects on normal intestinal FHC cells. The compound's inhibitory activity against EGFR was also confirmed, where it acted at a similar level to the reference compound erlotinib. In addition, hybrid 62 caused the induction of apoptosis and cell cycle arrest in the G2 and S phases. Moreover, it was confirmed to increase the expression levels of p53, PUMA, Bax, caspase-3, -8, and -9 protein genes, and the downregulation of Bcl-2. In in vivo studies, the LD 50 value was established, which was 6 mg/kg BW. It was observed that the use of this hybrid causes a reduction in tumor size comparable to 5-FU (the inhibition ratio was 52.92% for 62 and 57.16% for 5-FU), with the preservation of biochemical and histochemical structures being close to normal.
A new series of quinolone-4-thiazolidinone hybrid molecules were reported by Kumar et al. [62]. All the compounds were screened in vitro for their anticancer activities against MDA-MB-231 and MCF-7 cell lines using an MTT assay. Derivative 63 (Figure 24) was found to be the most potent against the MDA-MB-231 cell line, with an IC 50  Qi et al. [63][64][65][66][67] reported on the application of the hybrid-pharmacophore approach using a 4-phenoxy-6,7-dimethoxyquinoline moiety as a key part of foretinib and cabozatinib molecules in the design of novel multi-tyrosine kinase inhibitors based on 4-thiazolidinone core ( Figure 25). Structural modification of phenyl rings A and B as well as quinoline ring C was applied ( Figure 25) in the molecular design. As a result, hit-compounds with inhibitory activity toward mesenchymale epithelial transition factor (c-Met) (64,65,68) and toward HT-29 (human colon cancer cell line) (66)(67)(68) were identified.

Quinazoline-4-Thiazolidinone Hybrids
A series of hybrids with a quinazoline scaffold was synthesized by Samridhi Thakral et al. [68] ( Figure 26). The synthesized compounds were screened for their in vitro cytotoxic and growthinhibitory activities against MCF-7 and Hep-G2 cell lines using the MTT assay method in comparison with the activity of the known anticancer drug doxorubicin. The hybrid with a chlorine atom at the m-position in the phenyl ring attached to the 4-thiazolidinone nucleus at position two (69) was found to be the most active against the hepatic cell line, with an IC 50 of 1.79 µg/mL. The presence of the methoxy-group at the p-position in the mentioned phenyl ring (70) leads to activity against breast cancer cell line MCF-7, with an IC 50 of 1.94 µg/mL. The IC 50 value for the standard drug in this study was found to be 0.09 µg/ml.

Benzimidazole-4-Thiazolidinone Hybrids
Sharma et al. [69] reported design and synthesis as potential apoptotic agents of a series of new benzimidazole-4-thiazolidinedione hybrid molecules. All the new twenty synthesized hybrids were evaluated for their in vitro cytotoxic potential against selected human cancer cell lines:  In-depth studies of the impact of 71 and 72 on A549 lung cancer cells revealed a remarkable inhibition of cell migration through the disruption of the F-actin assembly.
Moreover, treatment with 71 and 72 led to the collapse of the mitochondrial membrane potential (DJm) and increased the levels of ROS in A549 cells. Reported in [69] were the results of different studies suggesting that such hybrids have the potential to be developed as cytotoxic agents and their structural modifications could lead to a new generation of promising anticancer agents.

Imidazopyridine-4-Thiazolidinone Hybrids
Iqbal et al. [70] reported the synthesis of two series of imidazopyridine-4-thiazolidinone hybrid molecules and an evaluation of their anticancer activity on a panel of three human cancer cell lines: MCF-7 (human breast cancer), A549 (human lung cancer), and DU145 (human prostate cancer).
Among the synthesized hybrids, derivatives 73-75 ( Figure 28) were found to be the most active, with IC 50 values at the micromolar level. The preliminary SAR provided by the authors revealed that anticancer activity was connected to the R1 nature ( Figure 28). The docking results suggested that the synthesized hybrids 73-75 could be potential EGFR kinase inhibitors.

Empirical SAR, Mechanisms of Action, and Molecular Targets for 4-Thiazolidinone-Bearing Hybrid Molecules with Anticancer Activity
Summarizing the structure-activity relationship analysis, the following trends can be observed. As reported in this review, 4-thiazolidinone-bearing hybrid molecules with anticancer activity mostly belong to the linked type of hybrids (Figure 29), and only molecules, 44 and 45, could be characterized as merged/fused hybrids. Hybrids mostly contain two or three potential pharmacophores (approved drugs and natural or privileged heterocyclic scaffolds) in their molecules. Most parts of the hybrids contain linked pharmacophores in positions C2 or C5, whereas substitution at the position N3 was used less often for design. Applying imino/amino-and hydrazone-linkers between pharmacophores and a 4thiazolidinone core at position two was often used in the design of novel hybrid molecules with potential anticancer activity ( Figure 29). Good results were obtained using a pyrazoline linker at position two in the 4-thiazolidinone ring. It is worth noting that using of pyrazolines is a promising direction in the construction of hybrid molecules with anticancer properties. Pyrazolines can be considered cyclic bioisosteres of the hydrazone-linker which possess their own pharmacological profile and could be additionally substituted with potential anticancer pharmacophores. This makes pyrazoline scaffolds a desirable aspect of the hybrid molecules. In some cases, pharmacologically attractive substituents were linked to the C2 position of the 4-thiazolidinone core without a linker through the carbon-carbon bond. At the N3 position, as methylene-and amino groups were used as linkers, urea moiety or the linker was absent. Often position N3 was unsubstituted in the structures of reported molecules or was substituted with alkyl/aryl-groups or amino acids. The introduction of the enamine linker at C5 of the 4-thiazolidinone ring was used in molecules 43-45; however, in the case of hybrids 44 and 45, such a structure modification led to the disappearance of activity. The design using a ylidene linker at C5 was much more popular and resulted in the majority of reported hit/lead compounds.
The analysis of the mechanisms of anticancer effects reported in this review for hybrid molecules showed that they possess a complex mechanism of action and additional indepth studies are needed in this area ( Figure 30). A large number of hybrid molecules were characterized as potent apoptosis inducers via multiple mechanisms. Caspase-dependent (intrinsic) and extrinsic pathways, with the upregulation of anti-apoptotic signals and downregulation of pro-apoptotic signals, were the most often indicated. An important reported mechanism for antimitotic activity was an impact on the cell cycle with arrest following in different phases. Moreover, enzymes such as PIM-and tyrosine kinases as well as carbonic anhydrases were used as molecular targets for the design of novel 4-thiazolidinones with anticancer activity. Some reported hybrids were found to be promising tubuline inhibitors and inducers of ROS generation.

Conclusions
The molecular hybridization strategy is a popular trend and an attractive research direction, providing endless source of significant opportunities in the design of new 4-thiazolidinone-bearing hybrid molecules with potential anticancer activity. A combination of potential pharmacophores in one molecule is one of the possible approaches used to achieve multi-target and polypharmacological effects for small molecules and is especially essential in the case of antimitotic agents. The hybridization of scaffolds, the hybrid-pharmacophore approach, and analogue-based drug design are key tools that are widely used by synthetic and medicinal chemists in this field. The hybridization of the 4-thiazolidinone core with early approved drugs, natural compounds, and privileged heterocyclic scaffolds is the most frequently used method in the development of novel molecules with antimitotic effects. The application of a molecular hybridization methodology allowed for identification of the new "hit" and "lead" hybrid compounds which target valid and important targets in carcinogenesis. The mentioned strategies and methodologies will remain relevant to studies over the next decade, and new wave/direction in the design of potential antimitotic agents using the hybridization of 4-thiazolidione scaffolds with biomolecules such as monoclonal antibodies, etc., can be expected. The data presented in this review contribute to the SAR profile of 4-thiazolidinone-based hybrid molecules for further exploration in the design, improvement, and optimization of new molecules with antitumor activity.