Synthesis and Antiproliferative Potential of Thiazole and 4-Thiazolidinone Containing Motifs as Dual Inhibitors of EGFR and BRAFV600E

Thiazole and thiazolidinone recur in a wide range of biologically active compounds that reach different targets within the context of tumors and represent a promising starting point to access potential candidates for treating metastatic cancer. Therefore, searching for new lead compounds that show the highest anticancer potency with the fewest adverse effects is a major drug-discovery challenge. Because the thiazole ring is present in dasatinib, which is currently used in anticancer therapy, it is important to highlight the ring. In this study, cycloalkylidenehydrazinecarbothioamides (cyclopentyl, cyclohexyl, cyclooctyl, dihydronapthalenylidene, flurine-9-ylidene, and indolinonyl) reacted with 2-bromoacetophenone and diethylacetylenedicarboxylate to yield thiazole and 4-thiazolidinone derivatives. The structure of the products was confirmed by using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and single-crystal X-ray analyses. The antiproliferative activity of the newly synthesized compounds was evaluated. The most effective inhibitory compounds were further tested in vitro against both epidermal growth factor receptor (EGFR) and B-Raf proto-oncogene, serine/threonine kinase (BRAFV600E) targets. Additionally, molecular docking analysis examined how these molecules bind to the active sites of EGFR and BRAFV600E.


Introduction
Cancer is a significant global health challenge, impacting the lives of millions of people worldwide [1,2].Chemotherapy remains the prevailing method for cancer treatment in current medical practice.While it offers advantages in addressing cancer, it also carries undesirable side effects that are attributable to its indiscriminate impact on healthy and cancerous cells [3,4].Combination chemotherapy is one way to simultaneously block two or more targets.However, the pharmacokinetic profiles and metabolic stabilities of two or more drugs are often different.In addition, combination chemotherapy may result in risky drug-drug interactions [5,6].Developing potent and less toxic anticancer chemotherapeutic agents can be achieved effectively by targeting multiple integrated signaling functions with a single molecule [7,8].Kinases have been shown to play an important role in regulating many fundamental cancer processes, including tumor growth, Molecules 2023, 28, 7951 2 of 18 metastasis, neovascularization, and chemotherapy resistance.As a result, kinase inhibitors have become a major focus of drug development, with several kinase inhibitors now receiving approval by the United States Food and Drug Administration (FDA) for various cancer indications [9,10].
The epidermal growth factor receptor (EGFR) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) are both well-studied kinases that play crucial roles in cancer progression [11,12].Unlike single-target inhibitors, dual inhibitors targeting both the EGFR and BRAF have the potential to provide greater efficacy and to overcome resistance.Combining EGFR and BRAF inhibition has resulted in synergistic antitumor effects in preclinical studies [13].Furthermore, dual inhibitors may help prevent the development of resistance, a common problem with single-target inhibitors [14].Therefore, developing dual EGFR/BRAF inhibitors is a promising approach in cancer therapy [15].
Thiazole and 4-thiazolidinone containing heterocyclic compounds, with a broad spectrum of pharmaceutical activities, represent a significant medicinal chemistry class.Thiazole and 4-thiazolidinone are five-membered unique heterocyclic motifs containing S and N atoms as the essential core scaffold, and they have commendable medicinal significance [16][17][18].Thiazoles and 4-thiazolidinones containing heterocyclic compounds are building blocks for the next generation of pharmaceuticals.Thiazole precursors have been frequently used, due to their capability to bind to numerous cancer-specific protein targets.Suitably, thiazole motifs have a biological suit via inhibition of different signaling pathways involved in cancer causes.The scientific community has always tried to synthesize novel thiazole-based heterocycles by replacing functional groups or skeletons around the thiazole moiety [16].
Molecules 2023, 28, x FOR PEER REVIEW 2 of 18 risky drug-drug interactions [5,6].Developing potent and less toxic anticancer chemotherapeutic agents can be achieved effectively by targeting multiple integrated signaling functions with a single molecule [7,8].Kinases have been shown to play an important role in regulating many fundamental cancer processes, including tumor growth, metastasis, neovascularization, and chemotherapy resistance.As a result, kinase inhibitors have become a major focus of drug development, with several kinase inhibitors now receiving approval by the United States Food and Drug Administration (FDA) for various cancer indications [9,10].
The epidermal growth factor receptor (EGFR) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) are both well-studied kinases that play crucial roles in cancer progression [11,12].Unlike single-target inhibitors, dual inhibitors targeting both the EGFR and BRAF have the potential to provide greater efficacy and to overcome resistance.Combining EGFR and BRAF inhibition has resulted in synergistic antitumor effects in preclinical studies [13].Furthermore, dual inhibitors may help prevent the development of resistance, a common problem with single-target inhibitors [14].Therefore, developing dual EGFR/BRAF inhibitors is a promising approach in cancer therapy [15].
Thiazole and 4-thiazolidinone containing heterocyclic compounds, with a broad spectrum of pharmaceutical activities, represent a significant medicinal chemistry class.Thiazole and 4-thiazolidinone are five-membered unique heterocyclic motifs containing S and N atoms as the essential core scaffold, and they have commendable medicinal significance [16][17][18].Thiazoles and 4-thiazolidinones containing heterocyclic compounds are building blocks for the next generation of pharmaceuticals.Thiazole precursors have been frequently used, due to their capability to bind to numerous cancer-specific protein targets.Suitably, thiazole motifs have a biological suit via inhibition of different signaling pathways involved in cancer causes.The scientific community has always tried to synthesize novel thiazole-based heterocycles by replacing functional groups or skeletons around the thiazole moiety [16].
The thiazole nucleus is a fundamental part of some clinically applied anticancer drugs, such as dasatinib (1) [17] and dabrafenib (2) [19] (Figure 1).Recently, thiazole-containing compounds have been successfully developed as possible inhibitors of several biological targets, including enzyme-linked receptor(s) located on the cell membrane (i.e., polymerase inhibitors) and the cell cycle (i.e., microtubular inhibitors) [20].Aly et al. [21] synthesized and characterized six paracyclophanyl thiazolidinonebased compounds, which were subsequently screened against 60 different cancer cell lines.Compound 3 (Figure 2) showed comparatively better anticancer activity, overall, than the rest of the series, especially against two cell lines of leukemia (RPMI-8226 and SR).
Molecules 2023, 28, x FOR PEER REVIEW 3 of 18 Aly et al. [21] synthesized and characterized six paracyclophanyl thiazolidinonebased compounds, which were subsequently screened against 60 different cancer cell lines.Compound 3 (Figure 2) showed comparatively better anticancer activity, overall, than the rest of the series, especially against two cell lines of leukemia (RPMI-8226 and SR).All the compounds exhibited anti-proliferative activity against RPMI-8226 and SR, with IC50 values of 1.61 µM and 1.11 µM, respectively.LV et al. [22] introduced two series of thiazolidinone derivatives and assayed for inhibitory action against EGFR and HER-2 kinases.Some of the synthesized compounds displayed potent EGFR and HER-2 inhibitory activities.Compound 4 displayed the most potent EGFR and HER-2 inhibitory activities (IC50; 0.09 µM for EGFR and IC50; 0.42 µM for HER-2) in MCF-7 cell lines, compared to erlotinib.Thiazolidin-4-one hybrids were developed by Aziz et al., and their anticancer properties were tested on breast cancer (MCF-7) and lung cancer (A549) cell lines.The most effective derivative against the lung cancer (A549) cell line was compound 5 (Figure 2), with an IC50 value of 0.72 µM and promising EGFR inhibitory activity at a concentration of 65 nM [23].Several 4-(5-methylisoxazol-3-ylamino) thiazoles were prepared and evaluated as cytotoxic agents against three human cancer cell lines (HCT-116, HePG-2, and MCF-7).Compound 6 (Figure 2), with IC50 = 20.2µg/mL against the Hep-G2 cell line, was as potent as the reference drug, doxorubicin (IC50 = 21.6 µg/mL).The in vitro kinases inhibitory assay revealed that this compound strongly inhibited 3 out of 12 kinases (EGFR, PI3K [p110b/p85a] and p38α) by 95%, 89%, and 85%, respectively.In addition, moderateto-weak inhibitory activities were observed for the rest of kinases (AKT2, CDK2/Cylin A1, PDGFRβ, VEGFR-2, BRAF[V600E], CHK1, PI3K [p110a/p85a], c-RAF, and AKT1; inhibitions of 2-64%) [24].Herein, we report the synthesis of thiazoles and 4-thiazolidinones containing scaffolds (A and B) and study their antiproliferative activity.Four human cancerous cell lines (all of ATCC cell lines) (the breast cancer (MCF-7) cell line, the epithelial cancer (A-549) Herein, we report the synthesis of thiazoles and 4-thiazolidinones containing scaffolds (A and B) and study their antiproliferative activity.Four human cancerous cell lines (all of ATCC cell lines) (the breast cancer (MCF-7) cell line, the epithelial cancer (A-549) cell line, the pancreatic cancer (Panc-1) cell line, and the colon cancer (HT-29) cell line) were subjected to an MTT assay to evaluate the newly synthesized thiazoles and 4-thiazolidinones containing motifs.All cells are obtained from The American Type Culture Collection Company, Manassas, Virginia, USA.
The most effective inhibitory compounds were tested in vitro for EGFR and BRAF V600E targets.Molecular docking analysis evaluated how these molecules attached to the active sites of EGFR and BRAF V600E .
Molecules 2023, 28, x FOR PEER REVIEW 4 of 18 cell line, the pancreatic cancer (Panc-1) cell line, and the colon cancer (HT-29) cell line) were subjected to an MTT assay to evaluate the newly synthesized thiazoles and 4-thiazolidinones containing motifs.All cells are obtained from The American Type Culture Collection Company, Manassas, Virginia, USA.The most effective inhibitory compounds were tested in vitro for EGFR and BRAF V600E targets.Molecular docking analysis evaluated how these molecules attached to the active sites of EGFR and BRAF V600E .
The disappearance of C=S, one ester group, and NH2 in the spectral data confirmed the cyclocondensation of cycloalkylidenehydrazinecarbothioamides (7a-f) with diethycetylenedicarboxylate 11. 1 H NMR of compound 12a revealed one triplet and one quartet signal associated with the ethyl group at δH 1.39 and 4.2, respectively, in addition to a singlet signal at δH 6.70 and broad one at δH 7.14, corresponding to vinyl-CH and NH, respectively.Moreover, 13 C NMR showed characteristic signals at 176.8, 164.92, 141.6, and 116.58, associated with two C=O, C=N, and vinyl-C, respectively.Furthermore, mass spectroscopy and X-ray analysis confirmed the proposed structure, Figures 5 and 6.The 4-thiazolidinone derivatives 12a-f (Scaffold B) could be formed according to the proposed mechanism (Scheme 2), which started with the initial conjugate addition of the sulfur lone pair of electrons of compounds 7a-f to the acetylenic triple bond, followed by intramolecular nucleophilic attack of the NH group on the ester carbonyl carbon in intermediate, followed by elimination of the methanol molecule to yield 12a-f.
The disappearance of C=S, one ester group, and NH 2 in the spectral data confirmed the cyclocondensation of cycloalkylidenehydrazinecarbothioamides (7a-f) with diethycetylene dicarboxylate 11. 1 H NMR of compound 12a revealed one triplet and one quartet signal associated with the ethyl group at δ H 1.39 and 4.2, respectively, in addition to a singlet signal at δ H 6.70 and broad one at δ H 7.14, corresponding to vinyl-CH and NH, respectively.Moreover, 13 C NMR showed characteristic signals at 176.8, 164.92, 141.6, and 116.58, associated with two C=O, C=N, and vinyl-C, respectively.Furthermore, mass spectroscopy and X-ray analysis confirmed the proposed structure, Figures 5 and 6

Cell Viability Assay
The human mammary gland epithelial (MCF-10A) cell line was used to test the viability of new compounds 9a-f and 12a-f.Using the MTT test, the cell viability of compounds 9a-f and 12a-f was evaluated after four days of incubation on MCF-10A cells [25,26].As shown in Table 1, none of the compounds tested were cytotoxic, and all compounds showed cell viability at 50 µM levels greater than 89%.

Cell Viability Assay
The human mammary gland epithelial (MCF-10A) cell line was used to test the viability of new compounds 9a-f and 12a-f.Using the MTT test, the cell viability of compounds 9a-f and 12a-f was evaluated after four days of incubation on MCF-10A cells [25,26].As shown in Table 1, none of the compounds tested were cytotoxic, and all compounds showed cell viability at 50 µM levels greater than 89%.

Biology 2.1.1. Cell Viability Assay
The human mammary gland epithelial (MCF-10A) cell line was used to test the viability of new compounds 9a-f and 12a-f.Using the MTT test, the cell viability of compounds 9a-f and 12a-f was evaluated after four days of incubation on MCF-10A cells [25,26].As shown in Table 1, none of the compounds tested were cytotoxic, and all compounds showed cell viability at 50 µM levels greater than 89%.

Antiproliferative Assay
The antiproliferative effect of compounds 9a-f and 12a-f was evaluated using the MTT assay against four human cancer cell lines: the colon cancer (HT-29) cell line, the pancreatic cancer (Panc-1) cell line, the lung cancer (A-549) cell line, and the breast cancer (MCF-7) cell line [27,28].Erlotinib was used as a control.Table 1 displays the median inhibitory concentration (IC 50 ) and the average inhibitory concentration (GI 50 ) against the four cancer cell lines.
In general, compounds 9a-f and 12a-f showed significant antiproliferative activity, with GI 50 values ranging from 35 nM to 84 nM, compared to that of erlotinib, which had a GI 50 value of 33 nM.Scaffold A compounds (9a-f) had GI 50 values ranging from 35 nM to 78 nM, while Scaffold B compounds (12a-f) had GI 50 values ranging from 42 nM to 84 nM, suggesting that Scaffold A compounds are more tolerated than Scaffold B compounds as antiproliferative agents.The most effective derivatives were compounds 9c, 9f, 12d, 12e, and 12f, with GI 50 values ranging from 35 nM to 47 nM, which were 1.1-to 1.4-fold less potent than erlotinib.
Compound 9c (R = cyclooctylidene, Scaffold A) was the most potent derivative of all synthesized compounds, with a GI 50 value of 35 nM, comparable to that of erlotinib (GI 50 = 33 nM).Compound 9c was even more potent than erlotinib against the breast cancer cell line (MCF-7), with an IC 50 value of 37 nM, while erlotinib had an IC 50 value of 40 nM.Replacement of the cyclooctylidene group in compound 9c with cyclopentyl, as in compound 9a (R = cyclopentyl, Scaffold A), or cyclohexyl, as in compound 9b (R = cyclohexyl, Scaffold A), resulted in a marked decrease in antiproliferative action, with GI 50 values of 78 nM and 64 nM, respectively.Similarly, replacing the cyclooctylidene group with heterocyclic moiety, as in compound 9f (R = indolin-3-one, Scaffold A), or a polycyclic group, as in compounds 9e (R = 9H-fluoren, Scaffold A) and 9d (R = 3,4-dihydronaphthalene, Scaffold A), resulted in a decrease in the antiproliferative activity, with GI 50 values 44 nM, 50 nM, and 59 nM, respectively.These findings demonstrated that the nature of the substituent at the N-2 of the hydrazinyl moiety of Scaffold A compounds plays an important role in the antiproliferative activity of 9a-f, with activity increasing in the order cyclooctylidene > indolin-3-one > 9Hfluorene > dihydronaphthalen > cyclohexyl > cyclopentyl.
Compounds 12d (R = 3,4-dihydronaphthalenyl, Scaffold B) and 12e (R = 9H-fluorenyl, Scaffold B) were ranked second and third in activity, with GI 50 values of 38 nM and 42 nM, respectively, and were 1.1-fold and 1.2-fold less potent than compound 9c.For Scaffold B compounds, the nature of the substituent at the N-2 of the hydrazinyl moiety was a bit different than that of Scaffold A compounds, with activity increased in the order of dihydronaphthalen > 9H-fluorene > indolin-3-one > cyclooctylidene > cyclohexylidene > cyclopentylidene.
Finally, the cyclopentyl derivatives 9a (R = cyclopentyl, Scaffold A) and 12a (R = cyclopentyl, Scaffold B) were the least active, with GI 50 values of 78 nM and 84 nM, respectively, demonstrating that the cyclopentyl group is not favored for antiproliferative action of these types of compounds.

EGFR Inhibitory Assay
Using erlotinib as the reference drug, the most effective derivatives, 9c, 9f, 12d, 12e, and 12f, were further explored for their inhibitory action against EGFR as a possible mechanistic target for their antiproliferative action [29].The IC 50 values for each compound and for erlotinib are shown in Table 2. Compared to erlotinib (IC 50 = 80 nM), the evaluated compounds displayed good anti-EGFR efficacy, with IC 50 values ranging from 86 nM to 100 nM.All of the studied compounds were less effective than erlotinib against EGFR.The results of this assay are comparable with the results of the antiproliferative assay, in which the most effective antiproliferative agent, compound 9c (R = cyclooctylidene, Scaffold A), was also the most potent EGFR inhibitor, with an IC 50 value of 86 nM, compared to erlotinib's IC 50 value of 80 nM, being 1.1-fold less potent than erlotinib.Compounds 12d (R = 3,4-dihydronaphthalen, Scaffold B) and 12e (R = 9H-fluoren, Scaffold B) ranked second and third in EGFR inhibitory effect, with IC 50 values of 89 nM and 91 nM, respectively.Finally, compounds 9f (R = indolin-3-one, Scaffold A) and 12f (R = indolin-3-one, Scaffold B) had moderate anti-EGFR activity, with IC 50 values of 97 nM and 100 nM, respectively.These findings indicated that compounds 9c and 12d are potential antiproliferative agents with EGFR inhibitory activity.
Once again, compounds 9c and 12d, the most active antiproliferative derivatives, were the most potent BRAF V600E inhibitors, with IC 50 values of 94 nM and 98 nM, respectively, compared to erlotinib (IC 50 = 60 nM), being roughly 1.6-fold less potent than erlotinib.The other three derivatives, 9f, 12e, and 12f, displayed weak to moderate efficacy against BRAF V600E , with IC 50 values of 117 nM, 105 nM, and 125 nM, respectively.These findings indicate that the examined compounds require structural modifications in order to yield more effective variants.Moreover, these in vitro experiments revealed that compounds 9c and 12e could be effective antiproliferative agents with dual targeting action against EGFR and BRAF V600E .

Docking Study
The most effective molecules, 9c, 9f, 12d, 12e, and 12f, were selected for further investigation regarding their potential to interact with the active sites of EGFR and BRAF, using erlotinib as a reference compound [31,32].
The study aimed to evaluate the efficacy of Scaffold A (compounds 9c,f) and Scaffold B (compounds 12d,e,f) as inhibitors of EGFR and BRAF.The results revealed a positive interaction pattern for these molecules within the active sites of EGFR and BRAF, as outlined in Tables 3 and 4. Notably, among the five test compounds, compound 3c exhibited the highest docking scores, −6.97 and −7.90 (S; kcal/mol), compared to those of the reference compound erlotinib, which had scores of −7.38 and −8.04, respectively.The top docking poses of the five test compounds, when compared with that of erlotinib, indicated that these compounds exhibited stability within the active site cavity, with several H-bonds and pi-H hydrophobic interactions with the several residues of amino acids around the active site, as illustrated in Figure 7. Compound 9c within the active sites of EGFR has two hydrogen bonds with Asp 831 and pi-H hydrophobic interaction with Cys 773, while erlotinib forms two hydrogen bonds with Met 769 and water molecule and pi-H hydrophobic interaction with Lys 721.
On the other hand, compound 9c within active sites of BRAF has two hydrogen bonds with Gln 530 and Cys 532 (Figure 8).The order of the docking scores fits with the results of the biochemical tests.Compound 9c (with R = cyclooctylidene, Scaffold A) emerged with the highest docking score, about 1.1-fold less than that of erlotinib.
Compounds 12d (with R = 3,4-dihydronaphthalenylidene, Scaffold B) and 12e (with R = 9H-fluoren, Scaffold B) had the second and third highest docking scores, respectively.Therefore, it was obvious that the stated docking results agreed with the biological findings.On the other hand, compound 9c within active sites of BRAF has two hydrogen bonds with Gln 530 and Cys 532 (Figure 8).The order of the docking scores fits with the results of the biochemical tests.Compound 9c (with R = cyclooctylidene, Scaffold A) emerged with the highest docking score, about 1.1-fold less than that of erlotinib.Melting point apparatus (i.e., the Gallenkamp melting point apparatus) was used.Infrared spectra (IR) were performed with Bruker Alpha instruments with a wavelength ranging from 4000 to 600 cm −1 as KBr disks.NMR spectra were recorded on a Bruker AM 400 MHz spectrometry using CDCl 3 as a solvent, with TMS as the internal standard (δ = 0).The data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, and q = quartet). 13C NMR, TMS (S=O) was used as the internal solvent, and spectra were observed with complete proton decoupling.Mass spectrometers were recorded on a Finnegan MAT instrument (70 ev, EI-mode).Elemental analyses for C, H, N, and S were obtained using Elmyer 306, and preparative layer chromatography (plc) was carried out using glass plates covered with a 1.0 mm thick silica gel (Merk Pf 254 ).
Synthesis of thiazole derivatives (9a-f), and 1,3,4-thiadiazole derivatives (10).A solution of 7a-f (1.0 mmol) and 2-bromoacetophenone (8) (1.0 mmol) in absolute ethanol was used as the solvent.The reaction mixture was refluxed for three hours or until the starting solution was fully consumed (the reaction was monitored by TLC analyses).The reaction mixture was filtered, and the precipitate was washed several times with ethanol.The filtrate was evaporated and subjected to chromatographic plates, using toluene-ethylacetate (10:5) as the eluent.The fastest migration zone contained thiazole derivatives (9a-f), and the slowest zone contained 1,3,4-thiadiazole derivatives (10).The isolated products obtained were recrystallized from suitable solvents.To assess the viability of new compounds 9a-f and 12a-f, the human mammary gland epithelial (MCF-10A) cell line was employed.The cell viability of compounds 9a-f and 12a-f was assessed using the MTT test [25,26].For more details, see the Supplementary Materials.

Assay for Antiproliferative Activity
The MTT assay was used to assess the antiproliferative activity of compounds 9a-f and 12a-f against four human cancer cell lines, using erlotinib as a control [27,28].See the Supplementary Materials for more details.

Assay for EGFR Inhibitory Effect
Using erlotinib as a reference medication, the most effective derivatives, 9c, 9f, 12d, 12e and 12f, were investigated further for their inhibitory action against EGFR as a potential mechanistic target for their antiproliferative action [29].Refer to the Supplementary Materials for more details.

Assay for BRAF V600E Inhibitory Effect
The most potent antiproliferative derivatives, 9c, 9f, 12d, 12e and 12f, were tested as BRAF V600E inhibitors, using erlotinib as the reference drug [29].Refer to the Supplementary Materials for more details.

Protocol of Docking Studies
The automated docking simulation study was performed using Molecular Operating Environment (MOE ® ) version 2014.09.The X-ray crystallographic structure of the target EGFR and BRAF was obtained from the protein data bank (PDB: 1M17, 5JRQ, respectively).The target compounds were constructed in a three-dimensional model using the builder

Figure 2 .
Figure 2. Structures of some thiazoles and 4-thiazolidinones containing scaffolds of potent anticancer activity.

Figure 2 .
Figure 2. Structures of some thiazoles and 4-thiazolidinones containing scaffolds of potent anticancer activity.
a H-acceptor; b pi-H; c H-donor.