Synthesis and Structure Determination of Substituted Thiazole Derivatives as EGFR/BRAFV600E Dual Inhibitors Endowed with Antiproliferative Activity

2,3,4-trisubstituted thiazoles 3a–i, having a methyl group in position four, were synthesized by the reaction of 1,4-disubstituted thiosemicarbazides with chloroacetone in ethyl acetate/Et3N at room temperature or in ethanol under reflux. The structures of new compounds were determined using NMR spectroscopy, mass spectrometry, and elemental analyses. Moreover, the structure of compound 3a was unambiguously confirmed with X-ray analysis. The cell viability assay of 3a–i at 50 µM was greater than 87%, and none of the tested substances were cytotoxic. Compounds 3a–i demonstrated good antiproliferative activity, with GI50 values ranging from 37 to 86 nM against the four tested human cancer cell lines, compared to the reference erlotinib, which had a GI50 value of 33 nM. The most potent derivatives were found to be compounds 3a, 3c, 3d, and 3f, with GI50 values ranging from 37 nM to 54 nM. The EGFR-TK and BRAFV600E inhibitory assays’ results matched the antiproliferative assay’s results, with the most potent derivatives, as antiproliferative agents, also being the most potent EGFR and BRAFV600E inhibitors. The docking computations were employed to investigate the docking modes and scores of compounds 3a, 3c, 3d, and 3f toward BRAFV600E and EGFR. Docking computations demonstrated the good affinity of compound 3f against BRAFV600E and EGFR, with values of −8.7 and −8.5 kcal/mol, respectively.


Introduction
Kinases control many essential cancer processes, including tumor growth, metastasis, neovascularization, and treatment resistance. Hence, the development of kinase inhibitors has become a top priority, with several of them receiving FDA approval for a variety of cancer purposes [1][2][3][4][5].
One approach for simultaneously inhibiting two or more targets is combination chemotherapy. However, two or more drugs' pharmacokinetic profiles and metabolic stabilities frequently differ. Furthermore, drug-drug interactions may occur during combination chemotherapy [6][7][8]. An alternative method for addressing these issues is to stabilities frequently differ. Furthermore, drug-drug interactions may occur during combination chemotherapy [6][7][8]. An alternative method for addressing these issues is to use a single drug to suppress two or more targets [9][10][11][12]. This approach may even make patients' treatment easier. The FDA has approved many dual-target or multi-target cancer treatments. Dasatinib is a multi-targeted kinase inhibitor that can potentially be a highly effective anticancer medication [13][14][15][16][17].
The acquired BRAF V600E mutation was suggested as a resistance mechanism after therapy with an EGFR inhibitor [18,19]. The development of resistance in colorectal cancer was also linked to the feedback stimulation of EGFR signaling [20][21][22]. Additionally, BRAF inhibition can cause EGFR to become active, promoting tumor growth [23,24]. A BRAF/EGFR combination was used to adopt these issues. In a number of studies, the BRAF/EGFR combination was found to have a significant therapeutic effect in patients with metastatic colorectal cancer that had BRAF V600E mutations [18,25,26]. As a result, sequentially inhibiting the two kinases may provide a solution to the EGFR activation problem.
We recently reported on the design and synthesis of two series of thiazole-based compounds as potent antiproliferative agents targeting EGFR and BRAF V600E [36]. Compound V (Figure 1) was shown to be the most potent derivative of all synthesized compounds, with a GI50 value of 0.90 µM against the four evaluated cancer cell lines when compared to the reference doxorubicin (GI50 = 1.10 µM). Compound V inhibited EGFR and BRAF V600E Abdel-Maksoud et al. [35] investigated several thiazole-based compounds as potential BRAF V600E inhibitors. Compound IV (Figure 1) had the most potent antiproliferative activity, with a competitive BRAF V600E inhibitory action (IC 50 = 0.05 µM). Furthermore, compound IV significantly affected dose-dependent apoptosis.
We recently reported on the design and synthesis of two series of thiazole-based compounds as potent antiproliferative agents targeting EGFR and BRAF V600E [36]. Compound V (Figure 1) was shown to be the most potent derivative of all synthesized compounds, with a GI 50 value of 0.90 µM against the four evaluated cancer cell lines when compared to the reference doxorubicin (GI 50 = 1.10 µM). Compound V inhibited EGFR and BRAF V600E with IC 50 values of 74 ± 7 and 107 ± 10 nM, respectively, and was more effective than erlotinib against EGFR (IC 50 = 80 nM). Moreover, the sulfonamide moiety is commonly employed in medicinal chemistry as efficient bioisosteres of the carboxylic group [37,38]. The sulfonamide motif could build a network of hydrogen bonds similar to the carboxylic group. As the carboxylic group's bioisosteres, it could avoid some of the carboxylic group's limitations, such as metabolic instability, toxicity, and limited passive diffusion across biological membranes [37]. As a result, the sulfonamide moiety gained popularity in medicinal chemistry, and a wide range of sulfonamide derivatives with diverse biological properties, such as anticancer activity [39][40][41], were produced.
In light of the aforementioned information, and as part of our enduring effort to develop potent antiproliferative agents that are dual inhibitors of EGFR and BRAF V600E [42][43][44][45], we describe the synthesis of a new set of thiazole-based compounds 3a-i ( Figure 2) in this article as antiproliferative agents that target EGFR and/or mutant BRAF. Scaffold A and B molecules had a methyl group in position 4, a hydrazo group in position 2, a physiologically active tosyl group for the scaffold B compounds, and a 2,4-dinitrophenyl group for the scaffold A compounds ( Figure 2). The cell viability of the novel derivatives was tested against a normal human mammary gland epithelial (MCF-10A) cell line. The antiproliferative action of 3a-i was tested on a panel of four human cancer cell lines. The ability to inhibit EGFR and mutant BRAF was further assessed for the most active antiproliferative derivatives. Finally, the most potent compounds' binding modes and docking scores toward BRAF V600E and EGFR targets were investigated. with IC50 values of 74 ± 7 and 107 ± 10 nM, respectively, and was more effective than erlotinib against EGFR (IC50 = 80 nM). Moreover, the sulfonamide moiety is commonly employed in medicinal chemistry as efficient bioisosteres of the carboxylic group [37,38]. The sulfonamide motif could build a network of hydrogen bonds similar to the carboxylic group. As the carboxylic group's bioisosteres, it could avoid some of the carboxylic group's limitations, such as metabolic instability, toxicity, and limited passive diffusion across biological membranes [37]. As a result, the sulfonamide moiety gained popularity in medicinal chemistry, and a wide range of sulfonamide derivatives with diverse biological properties, such as anticancer activity [39][40][41], were produced.
In light of the aforementioned information, and as part of our enduring effort to develop potent antiproliferative agents that are dual inhibitors of EGFR and BRAF V600E [42][43][44][45], we describe the synthesis of a new set of thiazole-based compounds 3a-i ( Figure 2) in this article as antiproliferative agents that target EGFR and/or mutant BRAF. Scaffold A and B molecules had a methyl group in position 4, a hydrazo group in position 2, a physiologically active tosyl group for the scaffold B compounds, and a 2,4-dinitrophenyl group for the scaffold A compounds (Figure 2). The cell viability of the novel derivatives was tested against a normal human mammary gland epithelial (MCF-10A) cell line. The antiproliferative action of 3a-i was tested on a panel of four human cancer cell lines. The ability to inhibit EGFR and mutant BRAF was further assessed for the most active antiproliferative derivatives. Finally, the most potent compounds' binding modes and docking scores toward BRAF V600E and EGFR targets were investigated.
Furthermore, the structures for the obtained products were confirmed via X-ray crystallography. Moreover, the X-ray measurements of compound 3b showed that the molecule (except the C-atom of the ethyl substituent, C21) is virtual planar. The aromatic ring is coplanar with the thiazole ring, and the ethyl group has the hours conformation structure. The angle between the thiazole and the aromatic ring is 6.37 (7) • , between the thiazole and the hydrazinylidene moiety is 2.98 (14) • , and between the aromatic ring and the hydrazinylidene moiety is 6.56 (9) • (angle between the L.S. planes of the moieties). In addition, the geometric structure around the exocyclic C=N has cissoid geometry concerning the thiazole S-atom and the hydrazo-group (Figure 4). The geometrical parameters (selected bond distance, bond angles, and dihedral angles; see Table 2) are in good correlation with the theoretical values.
doublet at δH = 6.40 (d, 2H) and 7.31 ppm (d, 2H), which were assigned respectively. Moreover, by comparing the data for the two compounds, ure 3, it is clear that the reaction behaves the same with the difference and that the difference is a slight difference in the chemical shift's results only in the nature of the substituted groups.
Furthermore, the structures for the obtained products were confirme tallography. Moreover, the X-ray measurements of compound 3b show cule (except the C-atom of the ethyl substituent, C21) is virtual planar. T is coplanar with the thiazole ring, and the ethyl group has the hours con ture. The angle between the thiazole and the aromatic ring is 6.37 (7)°, bet and the hydrazinylidene moiety is 2.98(14)°, and between the aromatic drazinylidene moiety is 6.56(9)° (angle between the L.S. planes of the m tion, the geometric structure around the exocyclic C=N has cissoid geom the thiazole S-atom and the hydrazo-group (Figure 4). The geometrica lected bond distance, bond angles, and dihedral angles; see Table 2) are in with the theoretical values.   (17) C4-C5-S1 N6-C2-S1 128.53 (15) C4-C5-H5 N3-C2-S1 110.03 (13) S1-C5-H5   N3-C2-N6-N7 −179.55 (16) N6-N7-C8-C9 1.6 (3) S1-C2-N6-N7 0.1 (3) N6-N7-C8-C13 −178.62 (16) C2-N6-N7-C8 −174.03 (17) Based on the above results and the X-ray confirmation of our obtained products, the proposed mechanism is as follows. Based on the above results and the X-ray confirmation of our obtained products, the proposed mechanism is as follows. First, the nucleophilic attack of sulfur on the primary carbon atom results in the formation of the intermediate, B (S-alkylation), via the transition state, A. Another nucleophilic attack on the nitrogen atom on the carbonyl carbon gives the intermediate, C, followed by water molecule (dehydration) loss to give the target product. The reaction mechanism proceeds via the SN 2 reaction type (Scheme 2). Scheme 2. The hypothesized mechanism for the synthesis of thiazole compounds 3a-i.

Cell Viability Assay
The human mammary gland epithelial (MCF-10A) cell line was used to test the viability of the novel compounds [50,51]. After four days of incubation on MCF-10A cells, the vitality of compounds 3a-i was determined using the MTT method. According to Table 3, the cell viability at 50 µM was greater than 87% for all tested agents, and none of the tested substances were cytotoxic. The human mammary gland epithelial (MCF-10A) cell line was used to test the viability of the novel compounds [50,51]. After four days of incubation on MCF-10A cells, the vitality of compounds 3a-i was determined using the MTT method. According to Table 3, the cell viability at 50 µM was greater than 87% for all tested agents, and none of the tested substances were cytotoxic.

Antiproliferative Assay
The MTT assay was used to investigate the antiproliferative activity of 3a-i against four human cancer cell lines: the colon cancer (HT-29) cell line, pancreatic cancer (Panc-1) cell line, lung cancer (A-549) cell line, and breast cancer (MCF-7) cell line, using erlotinib as the reference [52,53]. Table 3 shows the median inhibitory concentration (IC 50 ).
In general, the examined compounds 3a-i displayed good antiproliferative activity, with average IC 50 (GI 50 ) values ranging from 37 to 86 nM against the four tested human cancer cell lines, compared to the reference erlotinib (GI 50 = 33 nM).
The most potent derivatives were compounds 3a, 3c, 3d, and 3f, with GI 50 values ranging from 37 nM to 54 nM. Compound 3f (Ar = 2,4-di-NO 2 -C 6 H 3 , R = 4-CH 3 -C 6 H 5 ) was the most potent derivative of all synthesized compounds, with a GI 50 value of 37 nM against the four tested human cancer cell lines, comparable to the reference erlotinib (GI 50 = 33 nM). By replacing the p-tolyl group in compound 3f with a cyclohexyl moiety, compound 3d (Ar = 2,4-di-NO 2 -C 6 H 3 , R = C 6 H 11 ) was found to be the second-most potent compound, with a GI 50 value of 46 nM, being 1.3-fold less potent than compound 3f, demonstrating the importance of the p-tolyl moiety in antiproliferative activity.
The benzyl derivative, 3a (Ar = 2,4-di-NO 2 -C 6 H 3 , R = CH 2 -C 6 H 5 ), was less potent than 3f and 3d, with a GI 50 value of 48 nM against the tested four cancer cell lines, while the allyl derivatives, 3c (Ar = 2,4-di-NO 2 -C 6 H 3 , R = CH 2 CH = CH 2 ), showed moderate antiproliferative activity, with a GI 50 value more than 50 nM. These findings show that allyl and benzyl groups are not preferred for the antiproliferative activity of scaffold A compounds 3a-f.
With GI 50 values of 60 nM, 65 nM, and 86 nM, scaffold B compounds 3g, 3h, and 3i demonstrated moderate-to-weak antiproliferative activity. Compound 3i (Ar = p-CH 3 -C 6 H 4 -SO 2 , R = CH 2 CH 3 ) was the least potent derivative of any of the synthesized compounds, with a GI 50 value of 86 nM, which is less potent than its congeners, 3b (scaffold A), which has the same structure, but the aryl moiety was p-CH 3 -C 6 H 4 -SO 2 , while in 3b it was 2,4-di-NO 2 -C 6 H 3 . These findings demonstrated that 2,4-di-NO 2 -C 6 H 3 significantly affects the antiproliferative action of the newly synthesized compounds.

Assay for EGFR Inhibition
The most promising antiproliferative compounds, 3a, 3c, 3d, and 3f, were further evaluated for their suppressive impact on EGFR as a probable target for their mechanism of action [50,54,55]. Table 4 compares the IC 50 values to erlotinib, which worked as a control. The EGFR-TK inhibitory assay results matched the antiproliferative assay results, with the most potent derivatives, as antiproliferative agents, also being the most potent EGFR inhibitors. Compounds 3a, 3c, 3d, and 3f inhibited EGFR, with IC 50 values ranging from 89 to 98 nM, but the tested compounds were less potent than erlotinib (IC 50 = 80 nM). The most potent antiproliferative agent, compound 3f (Ar = 2,4-di-NO 2 -C 6 H 3 , R = 4-CH 3 -C 6 H 5 ), was also the most potent EGFR inhibitor, with an IC 50 value of 89 ± 7, being 1.1-fold less potent than standard erlotinib.

BRAF V600E Inhibitory Assay
Derivatives 3a, 3c, 3d, and 3f were further investigated as possible BRAF V600E inhibitors [56]. Table 4 displays the IC 50 values compared to erlotinib, which was used as a control. According to Table 4, the evaluated derivatives had a promising BRAF V600E suppressive action, with IC 50 values ranging from 93 to 126 nM, making them approximately 1.5-fold less effective than erlotinib (IC 50 = 60 nM). Compound 3f, the most potent derivative in the antiproliferative and EGFR suppressive assays, was also the most effective derivative as anti-BRAF V600E (IC 50 = 93 ± 8 nM). These findings show that compound 3a has potent antiproliferative activity as a dual EGFR/BRAF V600E inhibitor, implying that further structural modifications may be required to obtain a more potent lead compound for future development.

In Silico Study
AutoDock4.2.6 software was used to investigate the binding scores and poses of compounds 3a, 3c, 3d, and 3f against BRAF V600E and EGFR. The estimated docking features and scores are listed in Table 5. As tabulated in Table 5, all inspected compounds revealed good docking scores against BRAF V600E and EGFR targets, ranging from −7.8 to −8.7 kcal/mol and from −7.9 to −8.5 kcal/mol, respectively. The good docking scores of the inspected compounds toward BRAF V600E and EGFR may be imputed to their capability of forming H-bonds and vdW, pi-based, and hydrophobic interactions with the proximal residues within the active sites of the investigated targets (Table 5). Compound 3f demonstrated superior docking scores of −8.7 and −8.5 kcal/mol against BRAF V600E and EGFR, respectively. Inspecting the docking mode of compound 3f with the EGFR active site unveiled that this compound formed one H-bond with CYS773 (2.18 Å). Moreover, compound 3f exhibited two carbon-hydrogen bonds with LEU768 and pi-anion interaction with ASP831. On the other hand, compound 3f, complexed with BRAF V600E , demonstrated three H-bonds with LYS483 (2.15 Å), THR529 (2.38 Å), and ASP594 (2.12 Å). Additionally, compound 3f established pi-cation interaction with LYS483 and pi-pi stacking interaction with PHE583 and TRP531 residues ( Figure 5).   Compound 3d showed the second-lowest docking score, with values of −8.5 and −8.1 kcal/mol against BRAF V600E and EGFR, respectively. Compound 3d displayed one hydrogen bond with the CYS773 (2.17 Å) within the active site of EGFR. However, compound 3d demonstrated four H-bonds with the LYS483 (1.93Å), GLY596 (2.32 Å), THR529 (2.66 Å), and ASP594 (2.14 Å) of BRAF V600E . Compound 3a exposed the third-lowest docking score, with values of −8.4 and −8.0 kcal/mol against BRAF V600E and EGFR, respectively (Table 5). Compound 3a made one H-bond with the CYS773 (2.21 Å) of EGFR, while compound 3a exhibited three H-bonds with the LYS483 (2.15 Å), THR529 (2.38 Å), and ASP594 (2.12 Å) within the BRAF V600E binding pocket.

Chemistry
General information: refer to Supplementary Information. The starting materials, 1a-i, were synthesized in accordance with the documented methods [46][47][48] 3.1.1. General Procedure of Synthesis of Trisubstituted Thiazoles 3a-i Method A In a conical flask containing 10 mL ethyl acetate and two drops of Et 3 N as a catalyst, 0.092 gm of chloroacetone were dissolved (2). To this mixture, 1 mmol of thiosemicarbazides 1a-i in 10 mL ethyl acetate was added drop by drop while stirring. After addition was complete, the reaction mixture was stirred for 24 h. The reaction mixture was monitored with TLC. After the reaction was completed, the formed precipitate was filtered off and recrystallized from ethanol to afford products 3a-i as fine crystals.

Method B
In a 50 mL round-bottom flask containing 20 mL absolute ethanol, a molar ratio (1:1) mixture of chloroacetone and substituted thiosemicarbazides 1a-i was added. The flask was fitted with a condenser and was refluxed for 6-10 h. The reaction was monitored with TLC to assure the reaction completion. Then, the reaction mixture was cooled to room temperature, and the formed precipitate was filtered off and recrystallized from ethanol to afford products 3a-i.

Crystal X-ray Structure Determination of 3b
Compound 3b was obtained as single crystals by recrystallization from methanol. Bruker D8 Venture diffractometer with Photon II detector at 298(2) K using Cu-Kα radiation (λ = 1.54178 Å) was used to study the single-crystal X-ray diffraction. Moreover, we used dual space methods (SHELXT for 5a) [57,58] for the structure solution, and refinement was carried out using SHELXL-2014 (full-matrix least-squares on F 2 ) [59]. Hydrogen atoms were localized by difference electron density determination and refined using a riding model. Semi-empirical absorption corrections and a general RIGU restraint were applied. CCDC 2265616 (3b) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif (accessed on 24 June 2023).

Cell Viability Assay
The human mammary gland epithelial (MCF-10A) cell line was used to test the viability of compounds 3a-i [50,60]. See Supplementary Information.

Antiproliferative Assay
The MTT assay was used to investigate 3a-i's antiproliferative activity versus four human cancer cell lines: colon cancer (HT-29) cell line, pancreatic cancer (Panc-1) cell line, lung cancer (A-549) cell line, and breast cancer (MCF-7) cell line, using erlotinib as the reference [52,53]. See Supplementary Information.

EGFR Inhibitory Assay
Compounds 3a, 3c, 3d, and 3f were further evaluated for their suppressive effect versus EGFR as a probable molecular target for their mechanism of action [50,54]. See Supplementary Information.

In Silico Study
The crystal structures of BRAF V600E and EGFR, with PDB codes 3OG7 [62] and 1M17 [63], respectively, were prepared for all docking computations. All heteroatoms, water molecules, ligands, and ions were removed to prepare the PDB files. Modeler software was applied to construct all missing amino acids [64,65]. The protonation state of titratable residues of the investigated targets was estimated using PropKa software at pH 7.0 [66]. The 3D structure of the investigated compounds was energetically minimized using the MMFF94S force field within SZYBKI software [67,68].
For docking computations, AutoDock4.2.6 software was utilized [69]. All docking parameters were set to default values, except GA run and energy evaluation, which were 250 and 25,000,000, respectively. The active site of the investigated targets was inspected by a grid box with a size of 50 Å × 50 Å × 50 Å. The grid maps were generated using the AutoGrid program with a spacing of 0.375 Å. Gasteiger-Marsili method was employed to assign the atomic charges of the chemical compounds [70]. Discovery Studio module of Biovia software 17.1.0.115 was utilized to visualize all drug-protein interactions [71].

Conclusions
Using simple interactions between thiosemicarbazides and chloroacetone, a novel set of heterocycles with thiazole rings was developed. All obtained derivatives were validated using various spectral data such as IR, NMR, mass spectrometry, elemental analysis, and X-ray crystallography. The newly synthesized compounds, 3a-i, were evaluated against a panel of four human cancer cell lines, with compounds 3a, 3c, 3d, and 3f being the most potent variants. The in vitro assay results demonstrated that compound 3f possesses potent antiproliferative activity as a dual EGFR/BRAF V600E inhibitor, signaling that further structural modifications may be needed to establish a more potent lead molecule for future development. Finally, the docking analysis results showed that all inspected compounds revealed good docking scores toward BRAF V600E and EGFR.