Design, Synthesis, and Anticancer Evaluation of Benzimidazole and Benzothiazole Derivatives Targeting Hsp70 and FoxM1

Abstract

A series of novel benzimidazole and benzothiazole derivatives was designed based on the scaffolds of known Hsp70 (VER-155008) and FoxM1 (FDI-6) inhibitors. Molecular docking studies indicated favorable binding affinities to both targets, with several compounds showing strong interactions within the ATPase domain of Hsp70 and the DNA-binding region of FoxM1. The most promising candidates, as identified by docking scores, were synthesized and structurally confirmed using FT-IR, ^¹H NMR, and ^¹³C NMR spectroscopy. Their cytotoxicity was evaluated against MCF-7, HeLa, and HUVEC cell lines using the MTT assay. Benzothiazole derivatives exhibited greater cytotoxic activity than benzimidazole counterparts. Among them, compound 7d demonstrated the most potent antiproliferative effect, with IC₅₀ values of 10.83 μM (MCF-7), 12.68 μM (HeLa), and 106.75 μM (HUVEC). Molecular dynamics simulations further confirmed the stability of 7d within the FoxM1 binding pocket, supporting its role as a potential FoxM1 inhibitor. While experimental confirmation of dual-target inhibition in cell-based assays is pending, the computational findings suggest that 7d may function as a dual modulator of Hsp70 and FoxM1, warranting further mechanistic investigation.

Graphical Abstract

Benzimidazole and benzothiazole are nitrogen- and sulfur-containing heterocyclic scaffolds widely recognized for their diverse biological properties. These structures serve as key pharmacophores in various therapeutic agents, including antifungal, antibacterial, antiparasitic, and anticancer drugs [1–3]. Their structural versatility and ability to engage diverse molecular targets make them attractive frameworks for designing small-molecule inhibitors [4–6].

Benzimidazoles have been extensively studied for their cytotoxic activity on various tumor types [7]. Many derivatives induce apoptosis and cause cell cycle arrest in cancer cells [8]. In addition to their anticancer potential, benzimidazoles exhibit antibacterial, antifungal, and antiviral properties [9]. Clinically, benzimidazole-based drugs are used to treat parasitic [10] and bacterial infections [11], peptic ulcers [12], and hypertension [13]. They are widely employed in both human and veterinary medicine for parasitic worm infections (e.g., albendazole), and several derivatives are components of drugs used to manage blood pressure and diabetes [14, 15].

Benzothiazole derivatives exhibit cytotoxic activity against breast [16], lung [17], ovarian [18], kidney [19], and colon [20] cancers, often through mechanisms involving interference with cell division or interaction with DNA-binding proteins. Many also show potent antibacterial and antifungal activity [21], supporting their role in antimicrobial drug development. Some derivatives, notably, 2-aminobenzothiazoles, act as central muscle relaxants and modulate neurotransmitter systems, including glutamate signaling [22]. Additional pharmacological properties include anticonvulsant, antidiabetic, antihistaminic, antitubercular, antimalarial, and analgesic effects [23, 24].

Together, benzimidazoles and benzothiazoles transcend their role as rationally designed drug candidates, offering a broad platform for discovery in medicinal chemistry, industrial applications, diagnostics, and materials science. Their continued exploration is fueled by promising bioactivity, chemical versatility, and broad clinical utility [14, 25].

Among the emerging molecular targets in cancer therapy, heat shock protein 70 (Hsp70) and Forkhead box M1 (FoxM1) are of particular interest due to their central roles in tumorigenesis, disease progression, and resistance to therapy [26, 27]. Hsp70 is a stress-inducible molecular chaperone that is overexpressed in many cancers, where it promotes protein folding, inhibits apoptosis, and supports cell survival under adverse conditions [28]. Inhibiting Hsp70 can disrupt these tumor-supportive mechanisms, rendering it a promising anticancer target [29]. FoxM1, a member of the forkhead box transcription factor family, is involved in multiple oncogenic processes, including cell proliferation, cell cycle progression, DNA repair, angiogenesis, and metastasis [30–33]. It is highly overexpressed in numerous malignancies, including breast, colon, and pancreatic cancers [34–38], and is strongly associated with poor prognosis and chemotherapy resistance[39]. FoxM1 exists in four isoforms (A, B, C, D) generated by alternative splicing, all of which contain a conserved ~100-amino acid winged-helix DNA-binding domain [40]. Structurally, FoxM1 consists of a negative regulatory domain (NRD), a DNA-binding domain (DBD), and a C-terminal transactivation domain (TAD) [41].

Several small-molecule inhibitors targeting FoxM1 and Hsp70 have been reported. VER-155008, an Hsp70 inhibitor, binds to its ATPase domain and exhibits anticancer activity; however, it suffers from poor bioavailability and rapid degradation in vivo [42, 43]. Likewise, several small-molecule FoxM1 inhibitors have been developed. FDI-6, a FoxM1 inhibitor, blocks DNA binding [44, 45], while siomycin A and thiostrepton—both thiazole antibiotics—suppress its transcriptional activity [46]. Additional FoxM1 inhibitors include Honokiol, which disrupts the FoxM1 autoregulatory loop, reducing its mRNA and protein levels [47], while RCM-1 blocks FoxM1 activity and attenuates IL-13/STAT6 signaling in allergen models [48]. STL427944 and its derivative STL001 suppress FoxM1 via cytoplasmic translocation and autophagic degradation [49], and TFI-10 selectively targets FoxM1 without affecting upstream regulators such as SP1 [50]. In addition, FoxM1 activity can be modulated indirectly by targeting upstream effectors. Inhibition of proteins such as Hsp70, Hsp90, HER2, AKT, c-Myc, ERα, CDK4/6, and NF-κB downregulates FoxM1, while activation of tumor suppressors such as p53, Rb, and FoxO3 exerts similar inhibitory effects [51].

Given the structural similarities between VER-155008 and FDI-6, both compounds were selected as templates for designing a new series of benzimidazole- and benzothiazole-based analogs (Fig. 1). These heterocyclic scaffolds are widely recognized as purine bioisosteres, offering aromaticity, hydrogen bonding potential, and metabolic stability key attributes that enhance their application in drug design, particularly in anticancer agents targeting nucleotide-binding sites or transcriptional regulators [7, 52].

Fig. 1.

Rational design of new benzimidazole and benzothiazole derivatives based on VER-155008 and FDI-6.

The purine ring of VER-155008 was replaced with benzimidazole and benzothiazole moieties to preserve the aromatic character and polar interaction capacity while potentially improving pharmacokinetic profiles [7, 53]. To strengthen hydrophobic contacts within the target binding sites and enhance cell permeability, the ribose group at the N7 position was replaced with aliphatic chains such as propargyl, propyl, and pentyl [54]. Propargyl and nitrile substituents were also introduced to explore the possibility of covalent interactions with nucleophilic residues in the protein binding pockets an approach increasingly adopted in the design of targeted covalent inhibitors [55–57].

Furthermore, the NH group of the purine scaffold was substituted with a sulfur atom, a classical bioisosteric replacement known to improve lipophilicity and oxidative stability [58]. The 1,2-dichlorobenzene ring system was substituted with a variety of mono-substituted benzyl derivatives including 3-chloro, 4-chloro, 3-fluoro, 4-fluoro, and 4-methyl allowing fine-tuning of electronic and hydrophobic properties in accordance with structure activity relationship (SAR) optimization strategies [59, 60]. To enhance hydrogen bonding interactions with the Hsp70 active site, carboxyl and sulfonamide groups were introduced at positions analogous to purine N1, both of which are well-known hydrogen bond donors and acceptors used in medicinal chemistry [61].

For the FoxM1-based design, the thiophene-pyridine moiety of FDI-6 was replaced with benzimidazole or benzothiazole cores to increase rigidity and improve π–π stacking interactions within the binding interface. Additionally, the amide bond linker was replaced with a thioether (–S–CH₂–) group to improve metabolic stability without disrupting scaffold geometry mirroring previous findings where thioether cyclization conferred complete protection against enzymatic degradation of cyclic peptides in serum and lysosomal conditions [62]. Variations on the benzyl moiety were explored to further optimize hydrophobic interactions and conformational fit within the FoxM1 binding pocket.

The interaction of the designed compounds with the active site of Hsp70 and FoxM1 was examined through molecular docking. Compounds with satisfactory interaction and low binding energy with the active site of these enzymes were selected for synthesis under laboratory conditions. Subsequently, the cytotoxic effects of these compounds were investigated. Furthermore, the Molecular Dynamics Simulation method was employed on the best compound with the lowest IC₅₀ value. Thus, this work aimed to develop more stable and potent dual-target inhibitors as lead candidates for anticancer therapy.

Figures 2 and 3 show the general synthetic schemes for benzimidazole and benzothiazole derivatives. Full characterization data and synthesis are provided in the Supplementary Information.

Fig. 2.

General reaction scheme for the synthesis of benzimidazole derivatives. Conditions: a) KOH, H₂O, Ethanol, Reflux. b) NaOH, Ethanol, Reflux. c) Propargyl bromide, K₂CO₃, Acetone, Reflux. d) 1-Bromopropane, KOH, THF, Reflux. e) 1-Bromopentane, KOH, THF, Reflux. f) 4-Chlorobutyronitrile, KI, KOH, DMF, Reflux.

Fig. 3.

General reaction scheme for the synthesis of benzothiazole derivatives. Conditions: a) KBr 20%, Acetic acid glacial, 0 °C, stirring. b) KOH, CS₂ and Ethanol, Reflux. c) NaOH, Ethanol, Reflux.

To evaluate the binding affinities of the synthesized compounds to Hsp70 and FoxM1, molecular docking studies were carried out using AutoDock4 software. All compounds were docked into the active sites of the two target proteins, and the interaction profiles were analyzed based on binding energy and molecular contacts.

As a reference, VER-155008 was docked to the Hsp70 protein, yielding a binding energy of −1.77 kcal/mol with an RMSD of less than 2 Å. As illustrated in Figure 4, the A and B rings of VER-155008 formed Pi-Alkyl interactions with residues Arg272, Arg342, and Gly339. Additionally, Pi-cation interactions involving the B and C rings were observed with Arg272 and Gly339.

Fig. 4.

Molecular interaction of VER-155008 with Hsp70 protein (PDB ID: 4IO8). A. Ribbon representation of the VER-155008-Hsp70 complex highlighting the binding pocket. B. 3D surface view showing key Hsp70 amino acid residues involved in ligand binding. C. 2D interaction diagram illustrating hydrogen bonds, hydrophobic contacts, and electrostatic interactions between VER-155008 and critical residues of Hsp70.

Among the tested compounds, 7d exhibited the most favorable binding profile to Hsp70, with a binding energy of −4.52 kcal/mol (Table 1). Its interaction network, visualized in Figure 5, included hydrophobic interactions between ring A and residues Arg342 and Arg272. Ring B contributed to Pi-anion interactions with Asp366 and hydrophobic contacts with Tyr15. Ring C also engaged Tyr15 through hydrophobic interactions, while ring D exhibited both Pi-anion electrostatic interactions with Asp53 and hydrophobic interactions with Pro39.

Table 1.

The docking results of the synthesized compounds with the active site of Hsp70.

Compound	ΔG binding (kcal/mol)	H-Bond Amino acid (Distance Å)	Electrostatic	Hydrophobic interaction
Co-crystallized Ligand(VER-155008)	−1.70	Ser275(2.686), Lys271(2.723), Glu268(3.078), Gly230(3.379), Gly202(3.551), Asp366(3.512, 3.371)	Arg272, Arg342	Arg272, Arg342, Gly339
1a	−2.01	Ser340(3.094)	Asp366, Cys17	Gly201, Gly202
1b	−2.39	Gly201(1.987)	Arg272	Tyr15, Arg342
1c	−2.47	Arg272(1.925), Agr269(2.473, 1.867)	Lys56, Glu268	Tyr15
1d	−1.98	Glu268(2.723)	Arg272, Arg342	Tyr15
1e	−2.14	Asp366(2.964)	Arg342	Tyr15, Arg342
1f	−2.17	Gly230(3.247)	Arg272, Arg342	Tyr15
1g	−2.11	Gly339(2.589)	Arg272	Arg342
2a	−3.79	Gly339(2.948)	Asp366	Tyr15
2b	−2.29	Arg272(1.996, 3.055), Agr269(2.143, 2.537)	Lys56	Pro39
2c	−2.56	Arg272(1.994, 3.04), Lys56(2.24),	Tyr15, Asp53	Tyr15, Pro39
2d	−3.02	Arg272(1.889), Agr269(2.058, 1.790), Tyr41(2.276)	Lys56, Glu268	Arg261, Thr265
2e	−2.6	Arg272(1.974, 2.418), Agr269(2.11)	Glu268, Arg272	Tyr15, Thr37, Pro39
2f	−2.61	Arg272(2.09), Agr269(1.977)	Glu268	Tyr15, Thr37, Pro39
2g	−2.37	Tyr15(3.378), Asn35(2.951), Arg272(1.979, 2.497)	Lys56, Glu268	Tyr15, Pro39
3a	−2.59	-	Tyr15, Asp366	Tyr15, Pro39
3b	−2.02	Arg269(2.351), Arg272(1.486)	-	Tyr15, Pro39
3c	−2.82	Agr269(1.866, 2.170), Arg272(1.879)	Lys56, Glu268	Thr265
3d	−1.95	Arg269(2.155), Arg272(2.45, 1.893)	Tyr15, Asp53	Tyr15, Pro39
3e	−2.91	Agr269(1.998, 1.844), Arg272(1.858)	Lys56, Glu268	Tyr15, Thr265
3f	−2.21	Agr269(2.933), Arg272(1.833, 2.489)	Tyr15, Arg272	Tyr15, Thr37, Pro39
3g	−2.01	Arg272(2.938, 2.047), Agr269(2.235)	-	Pro39
4a	−1.45	Gly339(2.691)	Asp366	Tyr15, Cys17
4b	−2.25	Arg269(1.916, 2.538), Arg272(1.842)	Glu268, Glu231, Lys56, Tyr15	Thr14, Tyr15
4c	−1.98	Arg269(2.172), Arg272(2.01, 2.971)	-	Pro39
4d	−1.99	Thr265(3.210), Arg296(1.975, 2.142), Arg272(2.250)	Glu268, Lys56	Thr265
4e	−2.15	Tyr15(2.981), Agr269(2.126), Arg272(1.926, 2.437)	Glu268	Tyr15, Thr37, Pro39, Lys56
4f	−2.34	Thr265(3.212), Arg269(1.843, 2.403), Arg272(1.947), Tyr15(2.135)	Glu268, Glu231, Lys56	Lys56
4g	−1.96	Arg269(2.927), Arg272(1.828, 2.495)	Tyr15, Asp53, Glu268, Arg272	Tyr15, Pro39
5a	−2.3	Gly202(3.197), Gly230(3.769),	Asp366	Tyr15, Thr37
5b	−2.58	Thr265(3.022), Arg269(2.913, 2.193), Arg272(1.914)	Glu268, Lys56	Lys56, Val59
5c	−2.62	Arg269(2.002, 3.068, 2.930), Arg272(1.94), Lys56(2.072)	Glu268, Tyr15	Tyr15, Pro39
5d	−2.22	Arg272(1.956), Asn53(2.728)	-	Tyr15, Pro39, Lys56
5e	−2.58	Arg261(3.447), Thr265(2.616)	Tyr41, Phe68, Asp69, Glu231	Phe68, Arg264
5f	−2.11	Tyr15(2.774), Arg269(1.971), Arg272(2.283)	Glu268	Tyr15, Thr37, Pro39, Lys56
5g	−2.01	Thr37(2.691), Pro39(3.068), Arg269(2.677), Arg272(1.764, 2.574)	Tyr15, Asp53, Glu268, Arg272	Tyr15, Pro39
6b	−3.19	Thr37(3.018), Gly201(3.298), Gly339(2.403)	Asp366	Tyr15, Gly339
6c	−3.51	Lys271(3.379), Arg272(3.439), Ser275(1.939), Arg342(3.736), Ile343(2.985)	Arg342, Asp366	Tyr15, Arg272
6d	−4.45	Lys271(3.07), Arg272(3.138, 3.775), Ser275(2.55)	Ser340, Asp366	Tyr15, Cys17, Gly339
6e	−3.31	Ser16(2.769), Thr37(2.747), Asp366(1.829)	Tyr15, Asp366	Tyr15, Arg272, Gly339
6f	−3.88	Arg272(3.585), Ser275(1.916), Arg342(3.755), Ile343(2.910)	Arg342, Asp366	Tyr15, Arg272, Arg342
6g	−3.54	Gly201(3.232), Gly202(2.304), Arg272(3.633), Ser275(1.952), Arg342(3.755), Ile343(2.942) Gly339(2.717),	Tyr15, Asp366	Tyr15, Arg272
6h	−4.23	Tyr15(2.556), Thr37(2.882), Gly202(2.501), Asp366(1.923)	Arg342, Asp366	Tyr15, Arg272, Gly339
7b	−3.62	Gly230(3.081), Lys271(2.479), Ser340(2.069)	Asp366	Tyr15, Arg272, Arg342
7c	−3.69	Gly202(2.650), Asp366(2.167), Gly339(2.916)	Asp53	Tyr15, Pro39
7d	−4.52	Gly202(3.195), Gly230(3.615), Ser340(2.728, 3.193), Asp366(1.985)	Asp53, Asp366	Tyr15, Pro39, Arg272, Arg342, Asp366
7e	−3.70	Gly202(2.764), Asp366(2.290), Gly339(2.754, 2.980)	Asp53	Tyr15, Pro39
7f	−3.95	Tyr15(2.00, 1.795), Lys56(2.210, 1.521), Asp366(2.319),	Asp234, Glu268, Asp366	Tyr15, Cys17, Arg264, Glu268
7g	−3.66	Tyr15(2.826), Gly202(3.213), Gly230(3.218), Ser340(3.099)	Lys56, Glu268	Tyr15, Lys56, Arg272, Arg342
7h	−3.78	Tyr15(2.960), Thr37(2.954), Pro39(3.170), Gly202(2.610), Gly339(2.895), Asp366(2.113)	Asp53, Asp366	Tyr15, Pro39

Fig. 5.

Molecular interaction of compound 7d with Hsp70 protein (PDB ID: 4IO8). A. Ribbon representation of the Hsp70–7d complex showing the ligand within the binding pocket. B. 3D surface view highlighting key amino acid residues of Hsp70 involved in interaction with 7d. C. 2D interaction map illustrating hydrogen bonding, hydrophobic, and electrostatic interactions between compound 7d and critical residues of Hsp70.

The docking profile of FDI-6 with FoxM1 served as the control and showed a binding energy of −2.70 kcal/mol (Table 2). Key interactions included hydrogen bonding between the CF₃ fluorines and residues Asn283 and Arg286, and additional hydrogen bonds between the oxygen atom and Arg286 and Ser290. Pi-sulfur and Pi–Pi interactions were noted between ring A and His287, and ring D showed Pi–Pi and Pi-Alkyl interactions with Trp308, Leu259, and Arg286 (Fig. 6).

Table 2.

The docking results of synthesized compounds with the active site of FoxM1.

Compound	ΔG binding (kcal/mol)	H-Bond Amino acid (Distance Å)	Electrostatic	Hydrophobic interaction
FDI-6	−2.70	Asn283(2.582,2.547,3.982), Arg286(2.471,2.981), Ser290(2.954), His287(2.819)	His287	Leu259, Arg286, His287, Trp308
1a	−2.31	Ser290(2.184)	-	Leu259, Arg286, Leu289
1b	−5.19	His287(1.843), Ser290(2.025)	-	Leu259, Arg286, Leu289, Trp308
1c	−4.93	Arg286(1.670, 1.990), Ser290(2.898)	-	Asn283
1d	−4.78	Arg286(1.414, 1.866), His287(1.693)	-	Leu259, Arg286, Trp308
1e	−3.71	Arg286(1.685, 1.853), His287(1.960)	-	Arg286, Asn283
1f	−3.47	Arg286(1.710, 1.825), His287(1.935)	-	Arg286, Asn283
1g	−4.91	Ser290(1.884, 2.379)	-	Leu259, Arg286, Leu289, Trp308
2a	−3.09	Ser290(2.00)	-	Leu259, Arg286, Leu289, Trp308
2b	−4.99	His287(1.866), Ser290(1.952)	-	Leu259, Arg286, Leu289, Trp308
2c	−4.74	Ser290(1.903)	-	Leu259, Arg286Trp308
2d	−4.81	Ser290(1.872, 2.593)	-	Leu259, Arg286, Leu289, Trp308
2e	−3.65	Arg286(1.595, 1.708)	-	His287
2f	−5.04	His287(1.839), Ser290(2.038, 2.813)	-	Leu259, Arg286, Leu289, Trp308
2g	−4.28	His287(1.733), Ser290(1.988)	-	Leu259, Arg286, Leu289, Trp308
3a	−2.77	Ser290(1.995, 2.731)	-	Leu259, Arg286, Leu289, Trp308
3b	−4.11	Arg286(1.666, 1.993), Ser290(2.738)	-	Asn283, His287
3c	−4.08	His287(2.560), Arg286(1.745)	-	Asn283, His287
3d	−4.83	His287(2.373)	-	His287, Arg286, Asn283
3e	−4.95	Ser290(1.955, 2.603)	-	Leu259, Arg286, Leu289, Trp308
3f	−3.38	His287(1.662)	-	Arg286, Trp308
3g	−4.65	Ser290(2.02, 2.571)	-	Leu259, Arg286, Leu289, Trp308
4a	−2.30	-	Arg286	Leu259, Arg286, His287, Leu289, Asn283
4b	−3.99	His287(1.670), Ser290(1.848, 2.480)	-	Leu259, Arg286, Leu289, Trp308
4c	−3.76	His287(1.676), Ser290(3.158)	-	Leu259, Arg286, Leu289, Trp308
4d	−3.68	His287(2.658), Arg286(1.686, 1.696)	-	Asn283, His287
4e	−3.48	Asn283(3.051), His287(1.814)	-	Leu259, Arg286, His287, Leu289, Trp308
4f	−3.37	His287(2.326), Arg286(1.734, 1.935)	-	Asn283, His287
4g	−3.58	Arg286(1.702, 1.821), Ser290(3.155)	His287	Asn283, His287, Leu291
5a	−3.22	Arg286(2.330, 1.866)	-	Leu259, Arg286, His287, Leu289
5b	−4.23	Arg286(1.920, 1.715), Ser290(3.158)	-	Asn283, His287
5c	−3.63	Arg286(1.682, 1.899), Ser290(2.881)	-	Asn283, His287
5d	−4.69	Arg286(1.662)	-	Asn283, His287
5e	−4.71	Ser290(1.764, 2.30)	-	Leu259, Arg286, Leu289, Trp308
5f	−4.54	Ser290(1.964, 2.548)	-	Leu259, Arg286, Leu289, Trp308
5g	−4.28	Ser290(1.981, 2.612)	-	Leu259, Arg286, Leu289, Trp308
6b	−3.15	His287(1.904), Arg286(1.868, 1.802),	His287	Arg286, His287
6c	−4.87	Asn283(2.917), His287(1.704)	His287	Leu259, Arg286, Leu289, Trp308
6d	−3.31	His287 (1.455)	Arg286	Arg286, His287, Leu289
6e	−4.08	Asn283(2.021), Trp308(1.919)	His287	Leu259, Arg286, Leu289
6f	−4.7	Asn283(2.964), His287(1.921)	His287	Leu259, Arg286, Leu289, Trp308
6g	−4.65	His287 (1.693), Trp308(2.084)	His287	Leu259, Arg286, Leu289, Trp308
6h	−4.49	Asn283(3.077), His287(1.842)	His287	Leu259, Arg286, Leu289, Trp308
7b	−4.61	Asn283(2.065), His287(1.709)	His287	His287, Arg286
7c	−4.73	Asn283(2.442)	His287	Leu259, Arg286, His287, Leu289, Trp308
7d	−4.91	His287(2.616)	His287, Arg286	Leu259, Arg286, His287, Leu289, Trp308
7e	−4.24	Asn283(2.728)	His287, Arg286	Leu259, Arg286, His287, Leu289, Trp308
7f	−4.32	Asn283(1.945, 2.822)	His287	Leu259, Arg286, His287, Leu289, Trp308
7g	−4.53	Arg286(2.675), His287(1.909)	His287	Leu259, Arg286, His287, Leu289, Trp308
7h	−4.35	His287 (1.809), Trp308(2.181)	His287	Leu259, Arg286, His287, Leu289, Trp308

Fig. 6.

Molecular interaction of FDI-6 with FoxM1 protein (PDB ID: 3G73). A. Ribbon representation of the FoxM1–FDI-6 complex, showing the ligand positioned within the DNA-binding domain. B. 3D surface view depicting the key amino acid residues of FoxM1 involved in interactions with FDI-6. C. 2D interaction map illustrating hydrogen bonding, hydrophobic, and π-interactions between FDI-6 and critical residues of FoxM1.

Compound 7d demonstrated superior binding to FoxM1, with a docking score of −4.91 kcal/mol. As depicted in Figure 7, it formed a hydrogen bond between its oxygen atom and His287. The chlorine substituent of ring D engaged in hydrophobic interactions with Leu259, Leu289, and Trp308. Additionally, the sulfonamide moiety established Pi-sulfur interactions with Arg286 and His287, suggesting a robust binding configuration.

Fig. 7.

Molecular interaction of compound 7d with FoxM1 protein (PDB ID: 3G73). A. Ribbon representation of the FoxM1–7d complex, illustrating the ligand docked within the active site. B. 3D interface highlighting the key amino acid residues of FoxM1 involved in binding with 7d. C. 2D interaction diagram showing hydrogen bonds, hydrophobic contacts, and π-interactions between 7d and critical residues of FoxM1.

Physicochemical and pharmacokinetic properties of the synthesized compounds were assessed using the QikProp module. The resulting ADMET profiles were benchmarked against those of VER-155008 and FDI-6. The designed compounds demonstrated favorable drug-like characteristics, suggesting good absorption, distribution, and potential bioavailability.

Key indicators included the topological polar surface area (TPSA), which reflects hydrogen bonding capacity and thus intestinal absorption and blood-brain barrier permeability [63, 64]. The predicted brain/blood partition coefficient (LogBB) provided insight into the likelihood of CNS activity [65], while LogKhsa estimated binding affinity to plasma proteins, essential for systemic distribution. Oral absorption potential was supported by calculated Caco-2 and MDCK permeability parameters [66]. Moreover, the logP values for lipophilicity indicated favorable metabolic profiles, consistent with improved drug-likeness.

The anticancer potential of the synthesized compounds was examined through in vitro MTT assays against MCF-7, HeLa, and HUVEC cell lines. Dose-dependent cytotoxic effects were observed across the tested concentrations (benzimidazole derivatives at 50–250 μM and benzothiazole derivatives at 5–100 μM). Among the 50 compounds screened, 7d emerged as the most cytotoxic, demonstrating IC₅₀ values of 10.83 μM in MCF-7 and 12.68 μM in HeLa cells. Importantly, its IC₅₀ against the non-cancerous HUVEC line was considerably higher (106.75 μM), suggesting selective cytotoxicity toward cancer cells. This selectivity was statistically significant, with a p-value < 0.0001 (Table 3).

Table 3-.

IC₅₀ (μM) results of synthesized compounds.

Compound	HeLa(μM)	MCF-7(μM)	HUVEC(μM)	Compound	HeLa(μM)	MCF-7(μM)	HUVEC(μM)
1a	187.13±1.52	204.65±3.74	270.15±5.12	5a	170.67±1.43	161.31±0.92	291.79±4.60
1b	206.94±0.31	193.27±0.22	224.90±0.32	5b	195.25±1.32	169.19±5.31	231.32±1.36
1c	208.18±2.07	203.04±3.81	233.56±1.41	5c	193.41±0.51	156.03±2.01	258.89±8.69
1d	194.92±0.19	212.28±5.74	263.05±8.18	5d	154.46±2.53	180.32±4.88	307.1±4.86
1e	193.29±3.46	216.58±0.90	312.45±7.26	5e	174.83±3.34	142.52±5.35	285.17±1.96
1f	186.29±4.94	190.04±4.06	332.98±2.24	5f	174.05±0.16	150.65±1.51	295.42±3.42
1g	188.75±1.13	199.87±2.72	300.57±2.76	5g	171.51±2.37	150.56±1.10	249.46±8.16
2a	164.34±2.78	154.43±4.63	270.86±3.12	6b	70.37±5.89	70.35±3.11	74.03±1.81
2b	197.55±5.15	144.65±1.51	251.98±9.80	6c	58.41±0.86	53.27±2.96	86.20±1.67
2c	196.55±2.15	203.12±0.95	244.71±4.12	6d	46.57±2.30	36.88±0.84	92.90±3.78
2d	176.69±4.41	169.40±3.61	271.77±3.71	6e	45.01±3.55	24.01±2.61	103.48±4.57
2e	166.17±3.83	144.39±3.94	269.98±4.67	6f	34.55±2.11	34.72±3.44	107.42±2.91
2f	193.70±0.26	137.14±2.41	272.86±8.04	6g	33.99±1.75	28.31±2.85	109.3±3.49
2g	175.49±2.27	165.66±3.11	274.47±2.30	6h	38.45±1.68	30.86±1.17	109.66±3.38
3a	169.17±2.75	180.99±0.93	262.92±7.22	7a	60.39±2.73	58.98±1.65	121.08±1.99
3b	176.75±1.01	155.81±1.17	282.54±8.66	7b	45.14±3.27	35.34±2.49	107.14±2.51
3c	191.66±0.63	171.6±2.95	260.05±2.71	7c	25.89±3.56	18.53±0.86	110.41±1.20
3d	192.75±3.41	172.77±6.20	250.99±0.88	7d	12.68±1.49	10.83±0.68	106.75±2.31
3e	165.47±4.66	148.87±2.81	261.22±2.84	7e	24.34±0.19	19.87±1.75	106.40±3.32
3f	194.09±3.06	132.72±0.53	352.99±2.92	7f	14.88±0.63	19.50±1.48	96.89±2.85
3g	110.70±4.37	153.55±5.25	285.37±9.55	7g	19.76±2.69	14.16±1.07	96.03±1.74
4a	178.62±1.27	163.51±3.12	271.29±7.48	7h	29.32±0.62	26.40±1.67	85.98±2.37
4b	182.73±0.70	134.68±3.19	226.82±3.01	Doxorubicin	1.45±0.15	17.44±0.04	93.00±0.54
4c	202.46±1.54	139.41±6.89	248.49±0.34	VER-155008	55.26±0.25	23.92±1.09	149.54±2.23
4d	200.67±2.92	177.22±1.78	305.53±2.17	FDI-6	18.88±0.89	25.76±2.41	240.26±1.14
4e	185.50±3.00	115.63±0.43	328.56±1.95
4f	189.24±3.01	139.02±2.27	278.26±10.90
4g	160.59±2.45	162.06±4.88	248.28±3.94

For comparison, doxorubicin yielded IC₅₀ values of 1.45 ± 0.15 μM (HeLa), 17.44 ± 0.04 μM (MCF-7), and 93.00 ± 0.54 μM (HUVEC). VER-155008, a known Hsp70 inhibitor, showed IC₅₀ values of 55.26 ± 0.25 μM (HeLa), 23.92 ± 1.09 μM (MCF-7), and 149.54 ± 2.23 μM (HUVEC). Similarly, FDI-6, a FoxM1 inhibitor, exhibited IC₅₀ values of 18.88 ± 0.89 μM (HeLa), 25.76 ± 2.41 μM (MCF-7), and 240.26 ± 1.14 μM (HUVEC). These findings underscore the enhanced potency and selectivity of compound 7d compared to reference inhibitors (Table 3).

To validate the stability and behavior of compound 7d within the binding sites of Hsp70 and FoxM1 under physiological conditions, 100 ns molecular dynamics (MD) simulations were performed. Comparative analyses were also conducted using the control ligands VER-155008 and FDI-6. Simulations were analyzed in terms of RMSD, RMSF, radius of gyration (Rg), number of hydrogen bonds, and binding free energy.

The results of molecular dynamics simulations evaluating the root mean square deviation (RMSD) of alpha carbon (Cα) atoms in Hsp70 and FoxM1 upon ligand binding are shown in Figures 8 and 9. These RMSD plots provide insights into the structural stability of the protein–ligand complexes throughout the simulation.

Fig. 8.

Root Mean Square Deviation (RMSD) plots of protein-ligand complexes over 100 ns molecular dynamics simulations. A) RMSD of the main backbone atoms of Hsp70 in complex with VER-155008 (blue) and compound 7d (pink). B) RMSD of the FoxM1 protein in complex with FDI-6 (green) and compound 7d (pink). The trajectories illustrate structural stability and convergence behavior of the respective complexes

Fig. 9.

RMSD display diagram of A) VER-155008 (blue) and 7d (pink). B) FDI-6 (green) and 7d (pink).

For Hsp70, the complex with ligand 7d exhibited RMSD values ranging from 0.03 to 0.30 nm, while the complex with the known inhibitor VER-155008 showed a slightly narrower range of 0.06 to 0.26 nm (Fig. 8A). In the case of FoxM1, the 7d-bound complex displayed RMSD values between 0.04 and 0.30 nm, whereas the FDI-6-bound complex demonstrated larger fluctuations, ranging from 0 to 0.37 nm (Fig. 9A).

Ligand-specific RMSD analysis indicated that 7d remained more stably bound within the FoxM1 binding pocket, with fluctuations between 0.10 and 0.22 nm, compared to FDI-6, which ranged from 0.10 to 0.35 nm (Fig. 9B).

Together, these results suggest that ligand 7d forms more stable complexes with both Hsp70 and FoxM1 than the standard reference inhibitors, as reflected by lower and narrower RMSD values over the simulation time course.

RMSF (Root Mean Square Fluctuation) analysis measures the positional flexibility of each amino acid residue in a protein throughout a molecular dynamics’ simulation. By examining residue-specific fluctuations—particularly in the active site—researchers can infer the stability of protein–ligand interactions over time.

In the Hsp70–ligand complexes (Fig. 10A), key residues involved in ligand binding, including Tyr15, Gly202, Gly230, Glu268, Lys271, Arg272, Ser275, Gly339, and Arg342, exhibited low RMSF values, indicating minimal movement during the simulation. Similarly, in the FoxM1–ligand complexes, important residues such as Leu259, Arg286, Asn283, His287, Ser290, and Trp308 remained relatively stable (Fig. 10B).

Fig. 10.

Root Mean Square Fluctuation (RMSF) analysis of protein-ligand complexes during molecular dynamics simulations. A) RMSF of Hsp70 in complex with VER-155008 (blue) and 7d (pink). B) RMSF of FoxM1 in complex with FDI-6 (green) and 7d (pink). The plots indicate residue-wise flexibility, highlighting regions of reduced or enhanced motion upon ligand binding.

This limited fluctuation of active site residues suggests that both ligands are stably bound and that the respective complexes maintain structural integrity throughout the simulation. Collectively, these results reinforce the conclusion that ligand 7d forms stable interactions with both Hsp70 and FoxM1.

The radius of gyration (Rg) reflects the overall compactness of a protein during molecular dynamics simulations, with lower Rg values indicating a more folded and stable conformation. As shown in Fig. 11, the Hsp70–7d complex exhibited Rg values ranging from 1.55 to 1.87 nm, slightly lower than those observed for the Hsp70–VER-155008 complex (1.60–1.95 nm). This suggests that ligand 7d contributes to a more compact and stable protein structure.

Fig. 11.

Radius of gyration (Rg) analysis of protein-ligand complexes over a 100 ns molecular dynamics simulation. A) Hsp70 in complex with VER-155008 (blue) and 7d (pink). B) FoxM1 in complex with FDI-6 (green) and 7d (pink).

Similarly, for FoxM1, the 7d-bound complex showed Rg values between 0.85 and 1.28 nm, compared to 0.90–1.30 nm for the complex with FDI-6. These results indicate that binding of 7d promotes a more compact conformation in both Hsp70 and FoxM1 relative to the respective reference ligands.

Overall, the consistently lower Rg values and narrower fluctuations observed with ligand 7d suggest that the protein–ligand complexes exhibit enhanced structural stability.

Hydrogen bond formation between ligands and proteins is a key indicator of interaction strength and binding stability during molecular dynamics simulations. For Hsp70, the reference inhibitor VER-155008 formed an average of 5.54 hydrogen bonds, whereas compound 7d formed an average of 2.26 (Figs. 12A and 12B). In the case of FoxM1, FDI-6 formed an average of 2.15 hydrogen bonds, compared to 1.22 for compound 7d (Figs. 12C and 12D).

Fig. 12.

Number of hydrogen bonds formed over time during the 100 ns molecular dynamics simulation. A) Hsp70 in complex with VER-155008. B) Hsp70 in complex with 7d. C) FoxM1 in complex with FDI-6. D) FoxM1 in complex with 7d. The number of hydrogen bonds serves as an indicator of interaction strength and complex stability between ligands and target proteins.

Although compound 7d formed fewer hydrogen bonds than the standard inhibitors, these interactions were stable over time and involved key residues within the active site—regions critical for functional inhibition. This suggests that the quality and persistence of the hydrogen bonds, particularly their engagement with essential binding site residues, can compensate for their lower number. Therefore, compound 7d may still exhibit strong and specific binding affinity due to the strategic positioning and stability of its hydrogen bond interactions.

Binding free energies were calculated using the MM/PBSA (Molecular Mechanics/Poisson–Boltzmann Surface Area) method over the final 5 ns of the molecular dynamics simulation, representing the most equilibrated phase of each protein–ligand complex. This method estimates the binding affinity of each ligand by integrating molecular mechanics energies with solvation effects.

As summarized in Table 4, compound 7d exhibited more favorable (i.e., lower and more negative) binding free energies for both Hsp70 and FoxM1 compared to the respective reference inhibitors VER-155008 and FDI-6. These results suggest that 7d binds more tightly to both target proteins, consistent with its stable interactions observed in the MD simulations.

Table 4.

Binding free energy for VER-155008, FDI-6 and the best ligand 7d with FoxM1 and Hsp70 for 100 ns.

Ligand	Van der Waals Energy (ΔEvdW)	Electrostatic Energy (ΔEelec)	Polar Solvation Energy (ΔGpolar)	SASA Energy (ΔGnonpolar)	Binding Energy (ΔGbind)
VER-155008-Hsp70	−91.28	−42.28	118.73	−13.50	−28.33
FDI-6-FoxM1	−96.63	−70.06	112.07	−11.18	−65.81
7d-FoxM1	−105.11	−48.18	89.49	−10.44	−74.24
7d-Hsp70	−89.02	−12.42	84.75	−3.33	−20.02

The strong dual-target binding affinity of 7d reinforces its potential as a promising lead compound for further development as an anticancer agent targeting Hsp70 and FoxM1.

This study explored the rational design, synthesis, and biological evaluation of novel benzimidazole and benzothiazole derivatives aimed at inhibiting two important protein targets Hsp70 and FoxM1 in cancer cells. The structural design was inspired by known inhibitors VER-155008 and FDI-6, with specific scaffold and substituent modifications introduced to optimize binding affinity, selectivity, and cytotoxic potential.

Recent studies employing structure-based virtual screening and molecular docking have identified novel FoxM1 inhibitors, including derivatives of known scaffolds and repurposed FDA-approved drugs. Emerging compounds such as STL001 [67] and XST-20 [68] demonstrate improved potency and selectivity; however, they continue to face challenges related to solubility and bioavailability. Structure activity relationship (SAR) analyses indicate that effective FoxM1 inhibitors frequently contain key structural features, including 4-halophenyl substituents, planar aromatic systems, and functional groups capable of forming hydrogen or halogen bonds with residues in the FoxM1 DNA-binding domain. These findings continue to inform the rational design of more selective and pharmacologically favorable FoxM1-targeting agents [72].

In parallel, SAR studies of Hsp70 inhibitors targeting the NBD have revealed that potent inhibitors often incorporate aromatic or heterocyclic scaffolds with optimized substituents for engaging the ATP-binding pocket or adjacent allosteric sites [73]. Crucial molecular features include hydrogen-bonding capabilities, π–π stacking interactions, and side-chain modifications that enhance both selectivity and drug-like properties [74]. Notably, many of these inhibitors exhibit preferential binding to the ADP-bound state of the NBD, which may improve conformational stabilization and therapeutic efficacy [75]. Continued advances in SAR-driven design have enabled the development of more potent and reversible Hsp70 inhibitors with improved pharmacokinetic and pharmacodynamic profiles.

For Hsp70 inhibition, the purine scaffold of VER-155008 was replaced by benzimidazole and benzothiazole cores to enhance hydrogen bonding and hydrophobic interactions. Carboxyl and sulfonamide groups were incorporated to improve electrostatic interactions with polar residues in the nucleotide-binding domain (e.g., Glu268, Arg272, Ser275, Arg342). Additionally, diverse benzyl derivatives were introduced to probe the impact of aromatic substitutions on binding.

Docking studies identified key interaction patterns: benzimidazole derivatives primarily interacted with Tyr15, Glu268, and Arg272, while sulfonamide-based benzothiazoles targeted Arg342, Ser340, and Asp366. Compound 7d emerged as the top binder, exhibiting strong electrostatic, hydrophobic, and hydrogen bond interactions.

FoxM1 binding was benchmarked against FDI-6, which targets the DNA-binding domain. Benzimidazole and benzothiazole scaffolds with sulfonamide or carboxyl groups facilitated hydrogen bonding with His287 and Asn283. Compound 7d again showed the best binding profile, forming π-sulfur and hydrogen bonds with key residues and demonstrating excellent docking energy.

In vitro MTT assays supported these in silico predictions. Compound 7d exhibited potent cytotoxicity against MCF-7 and HeLa cells, with IC₅₀ values of 10.83 μM and 12.68 μM, respectively, and a favorable therapeutic index based on limited toxicity to HUVECs (IC₅₀ = 106.75 μM). Compared to VER-155008 (IC₅₀s: 55.26 μM HeLa, 23.92 μM MCF-7, 149.54 μM HUVEC) and FDI-6 (IC₅₀s: 18.88 μM HeLa, 25.76 μM MCF-7, 240.26 μM HUVEC), compound 7d was more selective and potent against cancer cells.

Molecular dynamics simulations further validated 7d’s potential. While less stable than VER-155008 in the Hsp70 complex (due to increased RMSD and Rg fluctuations), 7d exhibited favorable binding stability and low fluctuation in the FoxM1 complex-comparable to or better than FDI-6. MM/PBSA binding free energy calculations corroborated these trends, indicating strong interaction potential, particularly with FoxM1. To summarize the observed structure-activity relationships, we present the following SAR Table 5.

Table 5.

SAR summary of synthesized benzimidazole and benzothiazole derivatives against Hsp70 and FoxM1. The table outlines the effects of different substituents on cytotoxic activity (IC₅₀ values) in MCF-7, HeLa, and HUVEC cell lines, highlighting structural features correlated with enhanced potency and selectivity.

Group	Scaffold	Key Substituents	Active Site Interactions	IC₅₀ (μM) HeLa, MCF-7, HUVEC	Observed Activity
1	Benzimidazole	3-F in R1	Tyr15, Arg272, Glu268	>100 all three	Good Hsp70 binding, moderate cytotoxicity
2	Benzimidazole	3-Cl + propargyl	Arg272, Thr37, Asp53	>100 all three	Improved cytotoxicity in HeLa, MCF-7
3	Benzimidazole	3-Me + propyl	Arg272, Thr265	>100 all three	Moderate activity
4–5	Benzimidazole	N-propargyl/N-nitrile	π-π and covalent potential	>100 all three	Mixed activity, depends on R1
6	Benzothiazole	3-F + sulfonamide	Arg342, Ser340, Asp366	30.5, 28.1, >100	Good Hsp70 and FoxM1 binding
7	Benzothiazole	3-Cl + sulfabenzamide	His287, Arg286 (FoxM1); Arg272 (Hsp70)	12.7, 10.8, 106.7	Highest cytotoxicity (compound 7d)

Compound 7d stood out across all evaluations, demonstrating dual-target inhibition potential, superior cytotoxic activity, and favorable pharmacokinetic predictions. Its selective cytotoxicity profile (low IC₅₀ for cancer cells and high IC₅₀ for normal cells) underscores its therapeutic promise.

Compound 7d represents a strong dual inhibitor candidate for both Hsp70 and FoxM1, with potent in vitro activity and favorable molecular interaction profiles. While it showed reduced dynamic stability in the Hsp70 complex compared to VER-155008, its enhanced binding affinity for FoxM1 and robust cytotoxicity warrant further preclinical development. Future work should expand SAR exploration of benzothiazole scaffolds, examine 7d’s mechanism of inhibition in cellular models, and test its efficacy across additional cancer lines.

Supplementary Material

Appendix A. Supplementary material

All data generated or analyzed during this study are included in this published article.

Highlights.

• Novel benzimidazole and benzothiazole derivatives were rationally designed and synthesized.

• Docking and molecular dynamics simulations identified compounds with strong binding to Hsp70 and FoxM1.

• Benzothiazole derivatives displayed superior cytotoxicity compared to benzimidazole analogs.

• Compound 7d emerged as the most potent anticancer candidate across tested cell lines.

• Molecular dynamics confirmed the stability of compound 7d and its sustained inhibition of FoxM1.