Novel 2-substituted benzothiazole derivatives: synthesis, in vitro and in silico evaluations as potential anticancer agents

Abstract

Cancer remains a global health concern, demanding the development of new therapeutic medicines. This research focuses on the synthesis, in vitro evaluation, and in silico analysis of new 2-substituted benzothiazole derivatives as possible anticancer drugs. Hybrid molecules comprising benzothiazole and pyridinone rings 8a–c and 10a–c were also synthesized. Several compounds were produced and characterized, using NMR, IR and elemental analysis, with promising anticancer activity against lung H1299, liver Hepg2 and breast MCF7 cancer cell lines. Structure–activity connection investigations identified crucial structural characteristics that influence potency, with benzylidine derivatives 6a–g demonstrating higher activity. In silico ADME research revealed favorable drug-like features for chosen compounds, such as high gastrointestinal absorption and selective CYP inhibition. Toxicological projections indicated few side effects, confirming their potential as medication candidates. Inverse-docking analysis of the five potent compounds 6a–c, 6e, and 6f against 12 selected protein tyrosine kinases (PTKs) and cyclin-dependent kinases (CDKs) revealed good docking scores, strong interaction patterns, and potential inhibitory effects, particularly against ABL1, ABL2, CDK4, and CDK6 enzymes, suggesting these compounds as promising candidates for further drug development.

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1. Introduction

Cancer is one of the deadliest diseases in the world, killing nearly 10 million people in 2020. When it comes to global mortality statistics, cancer is the second most common cause of death. The most prevalent causes of cancer mortality in 2020 are lung, colon, rectum, liver, stomach, and breast.^1,2 Over the last decade, cancer-related deaths have increased by 28%, much outpacing the 9% increase in general mortality rates.³ Cancer death rates vary among regions due to a mix of hereditary and environmental factors. These factors influence the effectiveness of screening campaigns, preventive measures, and treatment options for distinct forms of cancer.⁴ The ongoing evolution of medical technologies holds up the possibility of improved screening capabilities as well as, more importantly, advancements in patient care and treatment options. For example, much emphasis has been put on anti-cancer research for developing effective agents, especially compounds that contain benzothiazole and 2-pyridinone moiety.^5–8 Several studies have been undertaken to improve the anti-cancer activity of benzothiazole by synthesizing a wide range of derivatives. Among these compounds, 2-arylbenzothiazole derivatives have shown promising antitumor activity. For instance, CJM 126, 2-(4-aminophenyl)-benzothiazole A, had excellent in vitro cytotoxicity in nanomolar concentrations and caused potent growth inhibition against human-derived breast carcinoma cell lines, including estrogen receptor-positive (ER+) MCF-7wt cells.⁹ Additionally, PMX-610, 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole B, demonstrated superior in vivo efficacy against human breast cancer cell lines MCF-7 and MDA-468 in nanomolar concentrations; however, high lipophilicity restricted its in vivo development in aqueous formulations and apparently prevented its development as a chemotherapeutic agent.¹⁰ SAR study of compound B revealed that the presence of a methoxy substituent at carbon 3 and 4 of the phenyl ring was important for its antitumor activity; replacing this group with another one resulted in the loss of activity. To overcome this problem, fluorinated analog, 4-(5-fluorobenzothiazol-2-yl)-2-methylaniline C, DF 203, has been developed and resolved the metabolic issues.¹¹ Compound C showed potent antitumor activity against wide spectrum of cancers such as ovarian, breast, collateral, and kidney, Fig. 1.¹²

Fig. 1 Examples of potent anticancer benzothiazole derivatives.

Previous research has demonstrated that benzothiazole containing compounds exhibit significant antiproliferative and apoptotic effects on various cancer types, including those of the lung, liver, and breast origin. For instance, studies have shown that substituted benzothiazoles can inhibit the growth of HepG2 liver cancer cells and MCF7 breast cancer cells by inducing apoptosis and disrupting the cell cycle.^13,14 Similarly, the anticancer activity of benzothiazole derivatives has been documented in lung cancer cell lines, such as H1299, by inhibiting cell proliferation and migration.¹⁵

Protein tyrosine kinases (PTKs) are crucial enzymes involved in cellular signaling, and their dysregulation is a significant factor in cancer development and progression.¹⁶ Benzothiazole derivatives have been identified in literature as a class of compounds that can act as potent inhibitors of PTKs.¹⁷ The benzothiazole scaffold is known to mimic the adenine portion of ATP, allowing it to compete for binding at the catalytic domain of these kinases.¹⁸ This competitive inhibition prevents the kinases from performing their normal function of phosphorylating proteins, thus disrupting critical pro-cancer signaling pathways. Specifically, several studies have shown that substituted benzothiazoles can inhibit a variety of PTKs, including ABL1 and ABL2 kinases.¹⁹ By blocking the activity of these molecular targets, benzothiazole compounds can effectively inhibit the proliferation and survival of cancer cells.

Over the last two decades, there has been a surge of interest in 2-pyridone derivatives in medicinal development efforts, with numerous FDA-approved medications working as kinase inhibitors. These include recent approvals for Tazemetostat (2020), which stands up as an effective, selective, and orally accessible small-molecule inhibitor of EZH2.²⁰ This is crucial because EZH2 inhibitors have promises in cancer treatment, especially in tackling difficulties such as drug resistance, poor distribution, and limited brain penetration reported with several current chemotherapeutic medications, Fig. 2. Additionally, Fredericamycin A is being investigated as a new lead molecule for battling human tumours,²¹ whilst Camptothecin has been proven to be effective in cancer treatment by blocking DNA topoisomerase I,²² Fig. 2.

Fig. 2 Anticancer drugs containing 2-pyrdinone.

Several 2-(benzo[d]thiazol-2-yl)acetohydrazide derivatives have been developed^23,24 and used for the synthesis of benzothiazole hybrid compounds.^25–27 Drawing on our previous successes in developing compounds containing benzothiazole and pyridinone rings, which demonstrated significant antimicrobial^28–30 and antiviral activities,^31–34 we proceeded on a new venture. Building on this foundation, we synthesized new benzothiazole hydrazide and benzylidine derivatives as well as hybrid compounds containing both benzothiazole and pyridinone and tested their potential anticancer properties. Furthermore, we conducted extensive in-silico research and docking analysis to better understand the molecular mechanisms behind their interactions and efficacy. After that, an inverse docking process was applied to the most active compounds to choose prospective molecular targets from a list of ones that are frequently dysregulated in cancer. This holistic strategy seeks to advance our understanding of these chemicals' therapeutic potential in cancer treatment, establishing the framework for future drug development initiatives.

2. Results and discussions

2.1. Chemistry

Novel N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4a–e were first synthesized and subsequently employed in the preparation of new benzothiazole derivatives N′-(2-(benzo[d]thiazol-2-yl)-3-arylacryloyl)benzohydrazide derivatives 6a–g, N-(6-amino-3-(benzo[d]thiazol-2-yl)-2-oxopyridin-1(2H)-yl)benzamide derivatives 8a–c and 2-amino-1-benzamido-5-(benzo[d]thiazol-2-yl)-6-oxo-N-aryl-1,6-dihydropyridine-3-carboxamide derivatives 10a–d, as illustrated in Scheme 1.

Scheme 1 Synthesis of 2-substituted benzothiazole derivatives.

The starting compound 2-(benzo[d]thiazol-2-yl)acetohydrazide 2 was obtained by reacting to hydrazine hydrate with ethyl 2-(benzo[d]thiazol-2-yl)acetate 1 in ethanol at room temperature for 24 hours.³² As illustrated in Scheme 1, benzothiazole hydrazide 2 was then reacted with benzoyl chloride derivatives 3a–e in the presence of pyridine at room temperature to afford the target N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide 4a–e. The reaction proceeded via nucleophilic substitution of the chloride atoms in benzoyl chloride by the hydrazide NH₂ group. The structures of the synthesized compounds were confirmed by elemental analysis and spectrum data such as IR, ¹H NMR, and ¹³C NMR. The IR spectra of compounds 4a–e revealed a broad absorption band at 3439–3175 cm⁻¹ corresponding to the NH group and two characteristic bands at 1696–1602 cm⁻¹ attributable to two C=O groups. Moreover, the ¹H NMR spectrum of compound 4a showed a singlet signal at δ 4.23 ppm, confirming the presence of a CH₂ group as well as four characteristic signals corresponding to the four protons of benzothiazole ring; two triplets at δ 7.43 and 7.58 ppm and two doublets at δ 7.98 and 8.06 ppm. In addition, the ¹H NMR showed multiplet signal at a range of δ 7.49–7.53 ppm and one doublet signal at δ 7.91 ppm corresponding to the five protons of benzene ring. The ¹³C NMR spectrum of compound 4a further supported the proposed structure, showing a CH₂ group at δ 39.4 ppm and two carbon atoms of C=O groups at δ 165.9 and 167.1 ppm. To confirm the structure of N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4 unambiguously, the X-ray crystal structure of 4a was determined, Fig. 3.²⁴

Fig. 3 The structure of compound 4a in the crystal.²⁴

Following the preparation of N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4a–e, the corresponding benzilydine derivatives were prepared via Knoevenagel condensation of benzaldehyde derivatives 5a–d with compounds 4a,d. In this process, piperidine deprotonated the active methylene group in compound 4, enabling nucleophilic attack on the carbonyl group of benzaldehyde 5, followed by dehydration to afford N′-(2-(benzo[d]thiazol-2-yl)-3-arylacryloyl)benzohydrazide derivatives 6a–g in good yields (Scheme 1). The structures of compounds 6a–g were confirmed by their spectroscopic analysis such as IR, ¹H NMR and ¹³C NMR. The IR spectrum of compound 6b exhibited a broad absorption band at 3438 cm⁻¹ due to the NH group, along with sharp absorption bands at 1692 and 1644 cm⁻¹ corresponding to the two C=O groups. The ¹H NMR spectrum of 6b displayed a singlet at δ 7.75 ppm for the CH proton and two singlets at δ 10.74 and 10.78 ppm for the two NH groups. Additionally, the ¹³C NMR spectrum of 6b revealed two characteristic resonances at δ 166.2 and 166.4 ppm, attributable to the two C=O carbons.

To confirm the structure of the resulting benzylidene product, 2D NMR techniques for compound 6d, including ¹H–¹H COSY, ¹H–¹³C HSQC, and ¹H–¹³C HMBC, were performed, Fig. 4A–C. The ¹H–¹H COSY spectrum revealed a strong correlation between the benzothiazole proton at 7.46 ppm and proton at 8.12 ppm, as well as the proton at 7.46 ppm and proton at 8.00 ppm, indicating spin coupling within the system. Additionally, a significant correlation was observed between the benzene ring protons adjacent to the methoxy (–OMe) group at 7.95 ppm and those at 7.00 ppm, further supporting the structural connectivity.

Fig. 4 2D NMR of compound 6d, (A) COSY; (B) HMBC; (C) HSQC.

N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4a,d were reacted with ethoxymethylene compounds, 2-(ethoxymethylene)malononitrile 7a and (E)-ethyl 2-cyano-3-ethoxy-acrylate 7b to afford N-(6-amino-3-(benzo[d]thiazol-2-yl)-5-cyano-2-oxopyridin-1(2H)-yl)benzamide 8a and ethyl 2-amino-1-benzamido-5-(benzo[d]thiazol-2-yl)-6-oxo-1,6-dihydropyridine-3-carb-oxylate 8b,c, respectively, Scheme 1. The reaction was carried out in ethanol using one equivalent of potassium hydroxide. The mechanism likely involves a Michael addition of the ethoxymethylene compounds to 4a,d, followed by ethanol elimination and intramolecular cyclization through nucleophilic attack of the NH group on the cyano group, ultimately yielding the N-arylcarbamide pyridone derivatives 8a–c. The elemental analysis and spectral data confirmed the proposed structure of compounds 8a–c. For example, IR spectrum of compound 8a displayed absorption band at ν 3430 cm⁻¹ for NH₂ group, as well as a band at ν 2216 cm⁻¹ corresponding to the CN group and band at ν 1631 cm⁻¹ corresponding to C=O group. The ¹H NMR spectra of 8a–c displayed singlets at δ 8.72–8.76 ppm and δ 9.10–9.21 ppm corresponding to the pyridone CH protons of 8a and 8b,c, respectively. In addition, the spectra of 8b,c exhibited characteristic signals for the ethyl group: a triplet at δ 1.13–1.16 ppm (CH₃) and a quartet at δ 3.96–4.38 ppm (CH₂). The ¹³C NMR spectrum of 8a showed resonances at δ 118.6, 162.1, and 164.1 ppm, consistent with CN and two C=O carbons, respectively.

The target derivatives of 2-amino-1-benzamido-5-(benzo[d]thiazol-2-yl)-6-oxo-N-phenyl-1,6-dihydropyridine-3-carboxamide derivatives 14a–c were synthesized by reacting 4a,d with N-phenyl acrylamide derivatives 9a–c in basic condition, Scheme 1. The reaction proceeded via a Michael addition, followed by elimination of NH(CH₃)₂ and subsequent intramolecular cyclization through nucleophilic attack of the NH proton on the cyano group, affording the desired products 10a–c. The structures of compounds 10a–c were confirmed by IR, ¹H NMR and ¹³C NMR and elemental analysis. According to the IR spectral analysis of compounds 10a–c, the appearance of an absorption band at a range of 3489–3327 cm⁻¹ confirmed the presence of NH group. In addition, the IR spectra showed a sharp band at a range of 1660–1623 cm⁻¹ which corresponds to C=O group. The ¹H NMR of compound 10b, as an example, showed three singlet signals which assigned for the protons of CH₃ group, CH group of pyridone and NH group at δ 2.42, 9.23 and 11.68 ppm, respectively. ¹H NMR of compound 10c displayed four characteristic signals corresponding to the four protons of benzothiazole ring, two triplet signals at δ 7.26 and 7.44 ppm and two doublets at δ 7.92 and 8.02 ppm in addition to four doublet signals for two aryl groups. Additionally, the ¹³C NMR spectrum of 10b confirmed the presence of CH₃ carbon δ 21.5 ppm and two C=O carbons at δ 162.5 and 165.0 ppm.

2.2. Biological activity

2.2.1 Anticancer activity. Compounds 4a–e, 6a–g, 8a–c, 10b, and 10c were evaluated in vitro for their anticancer activity against three human cancer cell lines—lung (H1299), liver (HepG2), and breast (MCF7)—using the SRB assay, with doxorubicin as the standard drug. Cytotoxicity was evaluated at four different concentrations, and the IC₅₀ values of the tested compounds were compared to those of the reference drug, as presented in Table 1. Additionally, the surviving fraction was measured and compared with the control group. Anti-cancer activities of the synthesized compounds have shown low activity against lung H1299 cell line. While no compound exhibited higher activities than standard drugs, doxorubicin (DOX), with H1299, benzylidine derivatives 6a–c, 6e and 6f showed comparable results with standard drug against Hepg2 and MCF7 cell lines. Additionally, only benzylidine compounds 6a–g have shown high activities than starting benzoyl derivatives 4a–e as well as other compounds that have benzothiazole bonded with pyrdinone ring 8a–c, 10b and 10c against the three tested cell lines.

Table 1 Cytotoxic activity (IC₅₀, μM) of synthesized benzothiazole derivatives against three human cancer cell lines compared with doxorubicin

Compound	H1299	HEPG2	MCF7
a Doxorubicin was included as the positive control and standard reference drug.b Values reported as “>50 μM” indicate no measurable activity at the highest tested concentration.c Bold values denote compounds with IC50 ≤ 15 μM, considered comparable to the reference drug in potency.
Doxorubicin^a	7.87	8.70	7.69
4a	>50^b	>50^b	>50^b
4b	>50	>50	>50
4c	>50	>50	>50
4d	>50	>50	>50
4e	>50	>50	>50
6a	>50	12.52	15.02
6b	>50	25.35	12.67
6c	>50	12.57	14.51
6d	>50	>50	>50
6e	>50	10.88^c	24.18
6f	>50	10.00^c	16.74
6g	>50	36.07	33.82
8a	>50	>50	>50
8b	>50	>50	27.62
8c	>50	>50	>50
10b	>50	>50	>50
10c	>50	>50	>50

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Based on IC₅₀ values of the synthesized compounds against lung H1299, liver Hepg2 and breast MCF7 cancer cell lines, as shown in Table 1, the structure–activity relationships (SAR) were established. For instance, both compounds 6a and 6c with benzoyl group and either hydrogen atom or methyl group at C4 at the benzene ring, respectively, have shown almost equal activities toward liver Hepg2 (IC₅₀ = 12.52 and 12.56 μM) and breast MCF7 cell lines (IC₅₀ = 15.02 and 14.51 μM). However, introducing chlorine atom to benzene ring, compound 6b, led to decreasing the activity against the liver Hepg2 cell line and increasing activity against the breast MCF7 cell line (IC₅₀ = 25.35 and 12.67 μM, respectively). On the other hand, the presence of methoxy group at the para position of benzene ring, compound 6d, led to decreasing the activity against liver Hepg2 and breast MCF7 cell lines (IC₅₀ = 50+ μM). Alternatively, the presence of 4-methylbenzoyl group and either hydrogen or chlorine atom at C4 of the benzene ring, compounds 6e and 6f, resulted in lower activities for these compounds against the breast MCF7 cell line (IC₅₀ = 24.18 and 16.74 μM, respectively) as compared to the corresponding compounds containing non-substituted benzoyl group, 6a and 6b. Surprisingly compounds 6e and 6f, gave the highest activities against the liver Hepg2 cell line (IC₅₀ = 10.88 and 10.00 μM, respectively). Introducing the methoxy group at the para position of benzene ring, compound 6g, resulted in decreasing the activity against liver Hepg2 and breast MCF7 cell lines (IC₅₀ = 36.07 and 33.82 μM, respectively). The mentioned data indicated that compounds 6e and 6f are the most potent against liver Hepg2 as compared to doxorubicin drug (IC₅₀ = 8.70 μM).

2.2.2 Pass prediction. The PASS computer program offers a means to predict the potential biological activity profile of drug-like organic compounds based on their structural formula. This estimation relies on an analysis of structure–activity relationships derived from a comprehensive training set encompassing pharmaceutical agents, chemical probes, compounds with known toxicity data, drug substances, and candidates at various stages of clinical and preclinical investigation. Biological activity is categorized as either “active” or “inactive” (Pa and Pi are ranging from zero to one for each activity category) within the PASS program. Notably, compounds 6a–g exhibited the highest anticancer activity among the synthesized compounds (as depicted in Table 2), surpassing other activities such as antitubercular, antimycobacterial, and anticonvulsant effects.

Table 2 The predicted biological activities spectrum of synthesized compounds by pass analysis Pa, Pi

Comp. No.	Antituberculosic		Antimycobacterial		Anticonvulsant		Antineoplastic
	Pa	Pi	Pa	Pi	Pa	Pi	Pa	Pi
4a	0.302	0.069	0.344	0.057	0.315	0.112	0.322	0.140
4b	0.279	0.083	0.335	0.061	0.387	0.070	0.222	0.209
4c	0.365	0.043	0.436	0.031	0.350	0.088	0.325	0.138
4d	0.305	0.067	0.348	0.056	0.287	0.129	0.307	0.148
4e	0.286	0.078	0.353	0.054	NA	NA	0.362	0.120
6a	0.360	0.045	0.410	0.037	0.213	0.182	0.714	0.024
6b	0.336	0.055	0.399	0.039	0.296	0.123	0.584	0.049
6c	0.363	0.044	0.414	0.036	NA	NA	0.673	0.031
6d	0.337	0.054	0.415	0.035	NA	NA	0.682	0.029
6e	0.363	0.044	0.414	0.036	NA	NA	0.673	0.031
6f	0.340	0.053	0.403	0.038	0.270	0.141	0.547	0.058
6g	0.341	0.052	0.419	0.035	NA	NA	0.648	0.035
8a	0.388	0.035	0.256	0.114	0.477	0.041	NA	NA
8b	0.506	0.012	0.425	0.033	0.621	0.016	NA	NA
8c	0.506	0.012	0.426	0.033	0.588	0.021	NA	NA
10a	0.476	0.015	0.393	0.041	0.582	0.022	0.276	0.167
10b	0.456	0.018	0.386	0.043	0.631	0.015	NA	NA
10c	0.480	0.014	0.397	0.040	0.554	0.026	0.269	0.172

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2.2.3 In silico ADME study.
2.2.3.1 Physicochemical, pharmacokinetic/ADME and drug likeness properties. The synthesized compounds' potential as drug candidates was explored through an in silico ADME study using the Swiss ADME online tool. Physicochemical and pharmacokinetic properties of the most potent five compounds, 6a–c, 6e, and 6f, were evaluated (Table S1, SI Document). A molecule's poor oral bioavailability in drug discovery is often associated with more than five hydrogen bond donors, ten hydrogen bond acceptors, a molecular weight exceeding 500 g mol⁻¹, and a calculated LogP above 5. Notably, the molecular weights of all tested compounds range from 399.46 g mol⁻¹ to 447.94 g mol⁻¹ which indicates good bioavailability. These compounds showed moderate numbers of hydrogen bond acceptors and donors, three and two respectively, suggesting potential hydrogen bonding interactions with biological targets. The iLogP values, ranging from 3.1 to 3.6, indicated moderate lipophilicity, essential for drug-like properties. Three compounds, 6a, 6c and 6e showed moderate solubility while two compounds that have chlorine atom, 6b and 6e, showed poor solubility. Topological polar surface area (TPSA) of all tested compounds exhibited the same value of 99.33 Ų, which is lower than 140 and indicated good oral bioavailability. Additionally, the predicted high gastrointestinal absorption (GI absorption) of the compounds suggests good potential for oral administration. BOILED-EGG diagram displayed the bioavailability property space for wlogP and TPSA, white area means that intestinal absorption, Fig. 5. All compounds are failed in the white area which suggests these molecules have lower affinity or interaction with P-gp. In the context of bioavailability, P-gp-compounds may have higher absorption rates or lower susceptibility to efflux by P-gp, leading to potentially higher bioavailability. Lack of blood–brain barrier (BBB) penetration of all five potent compounds indicates no central nervous system effects, while non-substrate predictions for P-glycoprotein suggest positive drug absorption and distribution. Moreover, potent benzothiazole derivatives inhibited CYP2C19 and CYP2C9 but not CYP1A2, CYP2D6, or CYP3A4, implying selective CYP inhibition with potential drug–drug interaction relevance. Poor skin permeability is predicted for these compounds due to the negative log K_p values. With adherence to Lipinski, Ghose, Vebe, and Egan's rules for drug-likeness, except for one Muegge violation indicating good oral bioavailability, the compounds exhibited a consistent bioavailability score of 0.55, suggesting moderate potential for oral bioavailability.

Fig. 5 BOILED-EGG diagram of tested compounds 6a–c, 6e and 6f.

2.2.4 Toxicological properties. The in silico predictions of toxicological properties of 6a–c, 6e and 6f compounds were determined using the Osiris property explorer program available online at http://www.propertyexplorer-cheminfo.org/ and summarized in Table 3. The results showed that all five compounds are predicted to be non-mutagenic and non-tumorigenic. This suggests they are unlikely to cause genetic mutations or cancer. Similarly, none of the compounds are expected to be irritants, indicating they may not cause skin or eye irritation. The compounds are also predicted to have no reproductive effects, meaning they are unlikely to interfere with reproductive health or fetal development. Druglikeness assesses how well a compound resembles known drugs in terms of its physicochemical properties. Scores above zero generally indicate drug-like characteristics. Here, all compounds show positive druglikeness scores, ranging from 2.73 to 5.88. This suggests they possess properties that could make them suitable as drugs. Drug score evaluates the overall potential of a compound to be developed as a drug, considering various factors like druglikeness and toxicity risks. The drug scores for these compounds range from 0.69 to 0.81. While these values are not exceptionally high, they still indicate some potential for drug development, especially when combined with the absence of predicted mutagenicity, tumorigenicity, and irritant effects.

Table 3 Toxicity prediction of compounds 6a–c, 6e and 6f

Compounds	6a	6b	6c	6e	6f
Mutagenic	No	No	No	No	No
Tumorigenic	No	No	No	No	No
Irritant	No	No	No	No	No
Reproductive effective	No	No	No	No	No
Druglikeness	4.09	5.88	3.75	2.73	4.39
Drug score	0.81	0.74	0.77	0.76	0.69

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2.3. Inverse-docking analysis

Protein tyrosine kinases (PTKs), which are divided into receptor and non-receptor tyrosine kinases, are essential enzymes that regulate vital cellular processes like growth, proliferation, and metabolism by transferring phosphate groups from ATP (adenosine triphosphate) to tyrosine residues on specific proteins within the cell.³⁵ Disruptions in this phosphorylation process can alter cell function and lead to various diseases, including cancer. PTK inhibitors, which block these enzymes and their uncontrolled signaling pathways, are a valuable foundation for developing new drug candidates. In recent years, the FDA has approved several kinase inhibitors for treating various cancers.³⁶ Additionally, cyclin-dependent kinases (CDKs) also play a crucial role by partnering with cyclins to phosphorylate target proteins and regulate cell cycle progression. In cancer, CDKs can become dysregulated through mutations or overexpression, resulting in unchecked cell proliferation. This loss of normal cell cycle regulation is a key factor in cancer development and progression.³⁷ Therefore, cyclin-dependent kinase inhibitors are useful therapeutic agents against many proliferative disorders, such as cancer.³⁸ Based on this understanding, the five most potent compounds, 6a–c, 6e, and 6f, identified by the SRB assay as well as doxorubicin were subjected to an inverse-docking protocol to identify their potential targets. This technique involves docking a molecule against a library of potential target proteins to identify which proteins the ligand may bind to, thereby predicting its activity. The term “inverse” refers to identifying proteins that can accommodate a specific ligand, rather than finding ligands for a specific protein.^39–41 For this inverse docking protocol, a set of 12 proteins was used, including five non-receptor tyrosine kinases (ABL1, ABL2, PTK2, CDC42, PTK6), four receptor tyrosine kinases (EGFR, c-KIT, c-Met, FGFR1), and three cyclin-dependent kinases (CDK2, CDK4, CDK6) to perform comprehensive docking analysis against the five potent compounds.

The docking protocol was validated by redocking the co-crystallized ligand into the catalytic domain of the selected target proteins. Most of the redocked poses of the co-crystallized ligands maintained their binding conformations relative to the original poses, with an RMSD value of less than 1 Å. The 3D structures of the target proteins used for molecular docking were sourced from the Protein Data Bank (PDB). The binding energy and interactions for potent compounds are presented in Table 4, Fig. 6A–E, 7A–D and 8A–C while the interaction of doxorubicin with target protein are presented in “Supplementary File”, Fig. 49. Based on the docking results for the selected non-receptor tyrosine kinase enzymes, the potent compounds showed good interactions with both ABL1 and ABL2, with comparable docking scores to the 1N1 ligand for ABL1, Fig. 6. In contrast, doxorubicin showed a lower binding score than both binding ligand and potent compounds, Table 4. For other selected enzymes such as PTK2 (PDB: 6I8Z), CDC42 (PDB: 3EQR), and PTK6 (PDB: 6CZ3), the docking scores were weaker compared to the co-crystallized ligands H82, T74, and FLJ, respectively, though PTK6 still demonstrated good binding interactions, Table 4. The data indicated that for ABL1 (PDB: 2GQG), all compounds primarily formed hydrogen bonds with Met318, with additional arene interactions involving Asp381 for compounds 6e and 6f. This suggests strong interactions like the co-crystallized ligand 1N1. The docking scores ranged from −8.7770 to −8.6343 kcal mol⁻¹, compared to −9.2914 kcal mol⁻¹ for 1N1. For ABL2 (PDB: 3HMI), the docked molecules maintained one hydrogen bond with Asp371 and arene interactions with Gly296 for all docked molecules, and extra one hydrogen bond with Met364 for compounds 6a–c. The docking scores ranged from −6.0620 to −6.1636 kcal mol⁻¹, compared to −7.5292 kcal mol⁻¹ for the co-crystallized ligand DKI. Despite the docking scores being lower than the co-crystallized ligand, the interaction patterns were still consistent and similar to those of the co-crystallized ligand DKI.

Table 4 Inverse docking studies of the potent compounds 6a–c, 6e, and 6f

Target enzyme (PDB)		6a	6b	6c	6e	6f	DOX	Co-crystalline ligand
Non-receptor tyrosine kinases
								1N1
ABL1 (2GQG)	Docking score (kcal mol^−1)	−8.7770	−8.8507	−8.8166	−8.6343	−8.7165	−6.0284	−9.2914
	Types of interactions	H-bond	H-bond	H-bond	H-bond	H-bond	2 H-bond	3 H-bond
					Arene	Arene		Arene
	Residues	Met318	Met318	Met318	Met318	Met318	Thr319, Tyr320	2 Met318, Thr315
					Asp381	Asp381		Leu248
								DKI
ABL2 (3HMI)	Docking score (kcal mol^−1)	−6.0677	−6.0620	−6.0978	−6.1215	−6.1636	−4.8389	−7.5292
	Types of interactions	2 H-bond	2 H-bond	2 H-bond	H-bond	H-bond	—	3 H-bond
		Arene	Arene	Arene	Arene	Arene		Arene
	Residues	Met354, Asp371	Met354, Asp371	Met354, Asp371	Asp371	Asp371	—	Glu362, Gly296, Met364
		Gly296	Gly296	Gly296	Gly296	Gly296		Thy299
								H82
PTK2 (6I8Z)	Docking score (kcal mol^−1)	−7.9650	−6.3871	−7.1521	−7.0425	−7.1957	−5.7019	−11.1462
	Types of interactions	Arene	2 H-bond	3 H-bond	Arene	3 H-bond	3 H-bond	5 H-bond
				Arene		Arene
	Residues	Arg426	2 Glu506	2 Glu506, Arg246	Arg426	2 Glu506, Arg246	Glu506, Gly505, Arg426	2 Cys502, Lie428, Glu500, Asp564
				Asn551		Asn551
								T74
CDC42 (3EQR)	Docking score (kcal mol^−1)	−8.1533	−8.1731	−8.2297	−7.9906	−8.3175	−5.2157	−11.5928
	Types of interactions	Arene	Arene	Arene	—	Arene	Arene	4 H-bond
	Residues	Thr205	Thr205	Thr205	—	Thr205	Leu122	2 Ala208, Glu205, Thr205
								FLJ
PTK6 (6CZ3)	Docking score (kcal mol^−1)	−6.5834	−6.6710	−6.2771	−6.6924	−6.7636	−5.5634	−9.12444
	Types of interactions	H-bond	H-bond	Arene	H-bond	H-bond	2 H-bond	4 H-bond
		Arene	Arene		Arene	Arene	Arene	Arene
	Residues	Met267	Met267	Leu197	Met267	Met267	Ala268, Leu197	Asp330, Lys219, Met267, Arg195
		Leu197	Leu197		Leu197	Leu197	Leu197	Leu197
Receptor tyrosine kinases
								YW5
EGFR (8GB4)	Docking score (kcal mol^−1)	−8.0287	−7.2188	−6.7622	−8.1569	−7.1292	−6.3271	−11.3781
	Types of interactions	3 H-bond	4 H-bond	Arene	3 H-bond	H-bond	Arene	3 H-bond
		Arene			Arene	3 Arene		Arene
	Residues	2 Met766, Asp855	Asp855, Met766, Phe856	Leu747	2 Met766, Asp855	Glu749	Lie759	2 Asp855, Met766
		Lie759	Lys745		Lie759	Phe723, Leu747, NME860		Phe856
								F82
c-KIT (6GQJ)	Docking score (kcal mol^−1)	−6.0324	−5.2508	−6.0765	−5.9900	−5.1218	−4.5458	−12.7068
	Types of interactions	H-bond	2 Arene	Arene	Arene	3 H-bond	H-bond	4 H-bond
								2 Arene
	Residues	Cys637	Gly676, Leu595	Leu595	Leu595	Gly676, Cys673, Tyr672	Gly676	Glu671, Cys673, Asp810, Glu640
								Leu595, Phe811
								L5G
c-Met (3CD8)	Docking score (kcal mol^−1)	−7.7871	−8.0868	−7.9845	−7.8021	−8.1638	−7.4166	−9.5806
	Types of interactions	3 H-bond	3 H-bond	3 H-bond	H-bond	2 H-bond	2 H-bond	3 H-bond
		3 Arene	3 Arene	2 Arene	2 Arene	3 Arene	2 Arene	Arene
	Residues	Tyr1230, Arg1208, Arg1086	Tyr1230, Arg1208, Arg1086	Tyr1230, Arg1208, Arg1086	Arg1086	Arg1208, Arg1086	Arg1208, Asn1167	Arg1208, Asp1222, Met1160
		2 Tyr1230, Met1211	2 Tyr1230, Lys1232	Tyr1230, Lys1232	Tyr1230, Met1211	2 Tyr1230, Lys1232	2 Tyr1230	Tyr1230
								0LI
FGFR1 (4V01)	Docking score (kcal mol^−1)	−7.3972	−7.4252	−7.4194	−7.7772	−7.8165	−6.9857	−9.0744
	Types of interactions	H-bond	2 H-bond	2 H-bond	2 H-bond	2 H-bond	2 H-bond	3 H-bond
								2 Arene
	Residues	Glu531	Glu531, Asp641	Glu531, Asp641	Glu531, Asp641	Glu531, Asp641	Cys619, Met535	Glu531, Asp641, His621
								2 Leu484
Cyclin-dependent kinases (CDKs)
								RRC
CDK2 (2 A4L)	Docking score (kcal mol^−1)	−6.4452	−6.9243	−7.1135	−6.8315	−6.9462	−7.1800	−8.0278
	Types of interactions	2 H-bond	2 H-bond	3 H-bond	H-bond	2 H-bond	H-bond	2 H-bond
		2 Arene		Arene	Arene		2 Arene
	Residues	Lys89, Leu83	Lys89, His84	Lys89, Leu83, His84	Lys89	Lys89, His84	Leu83	Glu81, Leu83
		Lys89, Lie10		Lie10	Lys89		Phe80, Gly11
								1PU
CDK4 (1GIH)	Docking score (kcal mol^−1)	−8.0098	−7.5669	−7.6984	−8.8194	−8.0811	−8.1383	−7.5254
	Types of interactions	H-bond	2 Arene	4 Arene	H-bond	4 Arene	2 H-bond	3 H-bond
		Arene			Arene		Arene
	Residues	Leu83	Gln131, Val18	2 Gln131, Glu12, Ala144	Leu83	2 Gln131, Glu12, Ala144	Lie10, Glu12	2 Leu83, Lys33
		Ala144			Ala144		Ala144	Val18
								NJ6
CDK6 (8I0M)	Docking score (kcal mol^−1)	−7.5315	−7.8884	−7.9575	−7.2575	−7.4880	−7.2081	−7.6397
	Types of interactions	Arene	2 Arene	2 Arene	H-bond	Arene	3 H-bond	3 H-bond
					Arene
	Residues	Phe164	Phe164, Glu21	Phe164, Glu21	Asp163	Gln149	2 Val101, Asp104	Asp163, Glu99, Val101
					Glu21

---

Fig. 6 3D interaction representation of 6a–c, 6e and 6f compounds within the binding pocket of selected non-receptor tyrosine kinases enzymes. (A) ABL1, (B) ABL2, (C) PTK2, (D) CDC42, (E) PTK6. Compounds are color-coded as follows: 6a (green), 6b (red), 6c (blue), 6e (yellow), and 6f (orange).

For the four selected receptor tyrosine kinase enzymes, the results indicated that all potent compounds as well as doxorubicin had lower docking scores compared to the co-crystallized ligands, Table 4 and Fig. 7A–D. Meanwhile, the potent compounds and doxorubicin showed comparable docking scores. For instance, in EGFR (PDB: 8GB4), the docking score of doxorubicin is −6.3271 kcal mol⁻¹ and of the potent compounds ranged from −6.7622 kcal mol⁻¹ to −8.1569 kcal mol⁻¹, whereas the co-crystallized ligand scored −11.3781 kcal mol⁻¹. Similarly, for c-KIT (PDB: 6GQJ), the scores of docked compounds 6a–c, 6e and 6f ranged from −5.1218 kcal mol⁻¹ to −6.0765 kcal mol⁻¹, with the co-crystallized ligand scoring −12.7068 kcal mol⁻¹, while maintaining consistent interaction patterns with key residues. In c-Met (PDB: 3CD8), the docked compounds showed several interactions with residues within the binding pocket. Compounds 6a–c have three H-bond interactions with residues, Tyr1230, Arg1208, Arg1086, as well as arene interaction with Tyr1230 and Lys1232 residues. Additionally, for FGFR1 (PDB: 4V01), the docked compounds showed consistent interaction patterns involving Glu531 and Asp641 with lower docking scores compared to the co-crystallized ligand 0LI.

Fig. 7 3D interaction representation of 6a–c, 6e and 6f compounds within the binding pocket of selected receptor tyrosine kinases enzymes. (A) EGFR, (B) c-Kit, (C) c-Met, (D) FGFR1. Compounds are color-coded as follows: 6a (green), 6b (red), 6c (blue), 6e (yellow), and 6f (orange).

For cyclin-dependent kinases, Table 4 and Fig. 8A–C, the docking scores of doxorubicin and 6a–c, 6e and 6f compounds for CDK4 (PDB: 1GIH) ranged from −7.5669 kcal mol⁻¹ to −8.8194 kcal mol⁻¹, slightly higher than the co-crystallized ligand 1PU, which scored −7.5254 kcal mol⁻¹. This result indicated strong binding potential like the co-crystallized ligand. For CDK6 (PDB: 8I0M), docking scores ranged from −7.2575 kcal mol⁻¹ to −7.9575 kcal mol⁻¹, comparable to the co-crystallized ligand, which scored −7.6397 kcal mol⁻¹. Key interactions involved Phe164 and Glu21, suggesting these compounds could effectively inhibit CDK6. However, for CDK2 (PDB: 2A4L), the docked compounds showed lower docking scores, and different residue interactions compared to the co-crystallized ligand RRC.

Fig. 8 3D interaction representation of 6a–c, 6e and 6f compounds within the binding pocket of selected cyclin-dependent kinases enzymes. (A) CDK2, (B) CDK4, (C) CDK6. Compounds are color-coded as follows: 6a (green), 6b (red), 6c (blue), 6e (yellow), and 6f (orange).

Based on docking results, the five potent compounds have strong binding affinities to CDK4 and CDK6, suggesting that they can effectively inhibit these kinases. Since CDKs play a vital role in controlling the cell cycle, their inhibition would disrupt the cell cycle and potentially lead to the cessation of cell proliferation. This mechanism is consistent with the known effects of doxorubicin, which interferes with CDK activity and disrupts cell cycle regulation.⁴² This highlights the potential of these compounds to be used as CDK inhibitors, contributing to their anti-cancer effects by halting cell cycle progression.

A future study will be conducted to investigate the in vitro and in vivo anti-proliferative effects of these compounds against CDK enzymes, comparing their efficacy to doxorubicin and assessing their potential for disrupting cell cycle progression.

3. Conclusions

In this study, a novel series of functionalized benzothiazole derivatives, including benzylidene and N-carboxamide 2-pyridone derivatives, was synthesized from N-aryl carboxohydrazides bearing a benzothiazole moiety. The anticancer activities of the newly synthesized compounds against three cell lines lung H1299, liver HEPG2 and breast cancers MCF7 revealed that five compounds 6a–c, 6e, and 6f exhibited notable cytotoxicity against HepG2 and MCF7 cell lines, in some cases approaching the potency of the reference drug doxorubicin. According to the in-silico study, potent compounds demonstrated promising properties for drug development, including high GI absorption and selective CYP inhibition and exhibited promising characteristics for further investigation as potential drug candidates. The inverse-docking study demonstrated that the potent compounds exhibited consistent and significant interactions with key residues in both PTKs and CDKs, although with lower docking scores than the co-crystallized ligands. This indicates their potential as effective inhibitors, particularly for enzymes like ABL1, ABL2, CDK4, and CDK6.

To further elucidate the anticancer mode of action, future work will involve in vitro and in vivo mechanistic studies focusing on kinase inhibition, cell cycle arrest, and apoptotic pathways, thereby validating the predicted PTK/CDK interactions. Moreover, structural optimization will be explored through systematic variation of substituents on the benzylidene ring and by designing hybrid analogues incorporating alternative heterocycles to improve potency and selectivity. Together, these findings highlight the potential of benzothiazole derivatives as promising anticancer candidates and establish a foundation for targeted structural refinement and mechanistic validation in future studies.

4. Experimental section

4.1. Chemistry

All melting points were measured using a SMP3 melting point apparatus. IR spectra were recorded on an FTIR plus 460 or pyeunicam SP-1000 spectrophotometer using KBr pellets. The ¹H and ¹³C NMR spectra were done in the Center of Drug Discovery Research and Development at Ain Shams University and recorded on a Bruker Avance (III)-400 Spectrometer (400 and 100 MHz, respectively) in DMSO-d₆ as a solvent using Si(CH₃)₄ as an internal standard and chemical shifts are reported as δ ppm units. Progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F254 (Merck), and UV lamp.

Spectra of compounds 4b, 4c, 6f, 6g, 8c and 10c were set on the APT system (125 MHz) using a Bruker Advance (III)-500 MHz Spectrometer at Helwan University's Central Laboratory, Hub of Creativity and Scientific Research which produces even signals along with DMSO-d₆ signals.

4.2. General procedures for preparation compounds (4a–e)

A mixture of 2-(benzo[d]thiazol-2-yl)acetohydrazide 2 (0.08 mol) and pyridine (10 ml) were stirred for 15 min. Benzoyl chloride derivatives 3a–e (0.16 mol) were added gradually to the reaction mixture in ice bath and stirred for 15 min. The reaction mixture was left at room temperature for 3 h. After the completion of the reaction, the solution was poured onto ice water and neutralized with HCl. The solid formed was filtered off and dried to produce a solid product. The solid product formed was washed using suitable solvent.

The preparation of compound N′-(2-(Benzo[d]thiazol-2-yl)acetyl)benzohydrazide (4a) has previously been described.²⁴

4.2.1 N′-(2-(Benzo[d]thiazol-2-yl)acetyl)-4-chlorobenzohydrazide (4b). White solid, yield 80%, m.p: 225–226 °C; IR (KBr, cm⁻¹): ν 3264 (NH), 3033 (CH-Ar), 1683, 1655 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 4.23 (s, 2H, CH₂), 7.44 (t, J = 7.8 Hz, 1H, benzothiazole-H), 7.51 (t, J = 6.6 Hz, 1H, benzothiazole-H), 7.58 (d, J = 10 Hz, 2H, Ar–H), 7.90 (d, J = 7.2 Hz, 2H, Ar–H), 7.98 (d, J = 8.4 Hz, 1H, benzothiazole-H), 8.09 (d, J = 10.8 Hz, 1H, benzothiazole-H), 10.60 (s, 2H, NH); ¹³C-APT (125 MHz, DMSO-d₆): δ 39.3 (CH₂), 122.5, 122.7, 125.5, 126.5, 129.1, 129.9, 131.5, 135.8, 137.2, 152.6, 164.9 (11C, Ar–C), 165.9, 167.0 (2CO); anal. calcd for C₁₆H₁₂ClN₃O₂S (345.80): C% 55.57; H% 3.50; N% 12.15; found: C% 55.60; H% 3.48; N% 12.13.

4.2.2 N′-(2-(Benzo[d]thiazol-2-yl)acetyl)-4-bromobenzohydrazide (4c). White solid, yield 80%, m.p: 238–239 °C; IR (KBr, cm⁻¹): ν 3265 (NH), 3033 (CH-Ar), 1683, 1656 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 4.21 (s, 2H, CH₂), 7.44 (t, J = 7.2 Hz, 1H, benzothiazole-H), 7.51 (t, J = 7.4 Hz, 1H, benzothiazole-H), 7.73 (d, J = 8.4 Hz, 2H, Ar–H), 7.83 (d, J = 8.4 Hz, 2H, Ar–H), 7.97 (d, J = 8 Hz, 1H, benzothiazole-H), 8.09 (d, J = 7.2 Hz, 1H, benzothiazole-H), 10.49 (s, 1H, NH), 10.63 (s, 1H, NH); ¹³C-APT (125 MHz, DMSO-d₆): δ 39.5 (CH₂), 122.5, 122.7, 125.5, 126.2, 126.5, 130.0, 131.8, 132.0, 135.8, 152.6, 164.9 (11C, Ar–C), 165.0, 167.0 (2CO); anal. calcd for C₁₆H₁₂BrN₃O₂S (390.25): C% 49.24; H% 3.10; N% 10.77; found: C% 49.20; H% 3.13; N% 10.79.

4.2.3 N′-(2-(Benzo[d]thiazol-2-yl)acetyl)-4-methylbenzohydrazide (4d). White solid, yield 83%, m.p: 193–195 °C; IR (KBr, cm⁻¹): ν 3190 (NH), 3027 (CH-Ar), 1669, 1602 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.36 (s, 3H, CH₃), 4.21 (s, 2H, CH₂), 7.30 (d, J = 8.0 Hz, 2H, Ar–H), 7.43 (t, J = 6.0 Hz, 1H, benzothiazole-H), 7.51 (t, J = 8.0 Hz, 1H, benzothiazole-H), 7.80 (d, J = 8.0 Hz, 2H, Ar–H), 7.98 (d, J = 8.0 Hz, 1H, benzothiazole-H), 8.08 (d, J = 8.0 Hz, 1H, benzothiazole-H), 10.45 (s, 1H, NH), 10.46 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 21.4 (CH₃), 39.4 (CH₂), 122.4, 122.7, 125.5, 126.5, 127.9, 129.5, 129.8, 135.7, 142.5, 152.6, 165.0 (11C, Ar–C), 165.9, 167.1 (2CO); anal. calcd for C₁₇H₁₅N₃O₂S (325.38): C% 62.75; H% 4.65; N% 12.91; found: C% 62.78; H% 4.67; N% 12.89.

4.2.4 N′-(2-(Benzo[d]thiazol-2-yl)acetyl)-3-methoxybenzohydrazide (4e). White solid, yield 70%, m.p: 171–172 °C; IR (KBr, cm⁻¹): ν 3272 (NH), 3027 (CH-Ar), 1693, 1661 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 3.81 (s, 3H, OCH₃), 4.20 (s, 2H, CH₂), 7.40 (d, J = 8.8 Hz, 1H, Ar–H), 7.39–7.53 (m, 5H, 3Ar–H & 2-benzothiazole-H), 7.98 (d, J = 8.8 Hz, 1H, benzothiazole-H), 8.09 (d, J = 10.8 Hz, 1H, benzothiazole-H), 10.46 (s, 1H, NH), 10.51 (s, 1H, NH); anal. calcd for C₁₇H₁₅N₃O₃S (341.38): C% 59.81; H% 4.43; N% 12.31; found: C% 59.84; H% 4.40; N% 12.34.

4.3. General procedures for preparation compounds (6a–g)

A mixture of N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4a,d (0.01 mol) and benzaldehyde derivatives 5a–d (0.01 mol) were stirred at room temperature in ethanol containing a catalytic amount of piperidine (3 drops) for 5 h. After the completion of the reaction, the solution was poured onto ice water. The solid formed was filtered, dried, and washed using suitable solvent.

4.3.1 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-phenylacryloyl)benzohydrazide (6a). Yellowish white solid, yield 75%, m.p: 216–217 °C; IR (KBr, cm⁻¹): ν 3267 (NH), 2993 (CH-Ar), 1641 (CO); ¹H NMR (400 MHz, DMSO-d₆): δ 7.45–7.58 (m, 7H, 6 Ar–H & 1-benzothiazole-H), 7.62 (t, J = 6.6 Hz, 1H, benzothiazole-H), 7.75 (s, 1H, CH), 7.97–8.04 (m, 5H, 4Ar–H & 1-benzothiazole-H), 8.15 (d, J = 8.4 Hz, 1H, benzothiazole-H), 10.73 (s, 1H, NH), 10.89 (s, 1H, NH); anal. calcd for C₂₃H₁₇N₃O₂S (399.46): C% 69.15; H% 4.29; N% 10.52; found: C% 69.19; H% 4.28; N% 10.50.

4.3.2 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-(4-chlorophenyl)acryloyl)benzohydrazide (6b). Yellowish white solid, yield 75%, m.p: 232–233 °C; IR (KBr, cm⁻¹): ν 3268 (NH), 2923 (CH-Ar), 1692, 1644 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 7.47–7.62 (m, 7H, 6 Ar–H & 1-benzothiazole-H), 7.75 (s, 1H, CH), 7.98–8.04 (m, 5H, 4Ar–H & 1-benzothiazole-H), 8.15 (d, J = 8.4 Hz, 1H, benzothiazole-H), 10.70 (s, 2H, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 122.6, 123.2, 126.4, 127.2, 128.1, 128.9, 129.1, 130.9, 132.4, 132.5, 132.8, 132.8, 134.5, 134.8, 134.9, 153.4, 165.5 (Ar–C), 166.2, 166.4 (2CO); anal. calcd for C₂₃H₁₆ClN₃O₂S (433.91): C% 63.66; H% 3.72; N% 9.68; found: C% 63.69; H% 3.70; N% 9.72.

4.3.3 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-(p-tolyl)acryloyl)benzohydrazide (6c). Yellowish white solid, yield 73%, m.p: 232–233 °C; IR (KBr, cm⁻¹): ν 3269 (NH), 2921 (CH-Ar), 1686, 1643 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.37 (s, 3H, CH₃), 7.27 (d, J = 6.4 Hz, 2H, Ar–H), 7.47 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.53–7.57 (m, 3H, Ar–H), 7.62 (t, J = 7.0 Hz, 1H, benzothiazole-H), 7.70 (s, 1H, CH), 7.88 (d, J = 7.6 Hz, 2H, Ar–H), 7.99–8.02 (m, 3H, 2Ar–H & benzothiazole-H), 8.14 (d, J = 8.0 Hz, 1H, benzothiazole-H), 10.72 (s, 1H, NH), 10.82 (s, 1H, NH); anal. calcd for C₂₄H₁₉N₃O₂S (413.50): C% 69.71; H% 4.63; N% 10.16; found: C% 69.74; H% 4.60; N% 10.15.

4.3.4 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)acryloyl)benzohydrazide (6d). Yellow solid, yield 73%, m.p: 215–217 °C; IR (KBr, cm⁻¹): ν 3270 (NH), 2924 (CH-Ar), 1685, 1645 (CO); ¹H NMR (400 MHz, DMSO-d₆): δ 3.82 (s, 3H, OCH₃), 7.00 (d, J = 7.6 Hz, 2H, Ar–H), 7.46 (t, J = 8.0 Hz, 1H, benzothiazole-H), 7.52–7.57 (m, 3H, Ar–H), 7.62 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.67 (s, 1H, CH), 7.95–8.01 (m, 5H, 4Ar–H & 1-benzothiazole-H), 8.12 (d, J = 8 Hz, 1H, benzothiazole-H), 10.70 (s, 1H, NH), 10.81 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆):δ 55.8 (OCH₃), 114.6, 122.5, 122.9, 125.8, 126.4, 127.0, 127.8, 128.2, 128.9, 132.3, 132.9, 133.0, 134.7, 135.8, 153.6, 161.1, 165.9 (17C, Ar–C), 166.3, 166.9 (2CO); anal. calcd for C₂₄H₁₉N₃O₃S (429.11): C% 67.12; H% 4.46; N% 9.78; found: C% 67.16; H% 4.49; N% 9.74.

4.3.5 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-phenylacryloyl)-4-methylbenzohydrazide (6e). White solid, yield 65%, m.p: 200–203 °C; IR (KBr, cm⁻¹): ν 3265 (NH), 2971 (CH-Ar), 1684, 1641 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.40 (s, 3H, CH₃), 7.35 (d, J = 8.0 Hz, 2H, Ar–H), 7.44–7.50 (m, 4H, Ar–H & benzothiazole-H), 7.56 (t, J = 7.2 Hz, 1H, benzothiazole-H), 7.74 (s, 1H, CH), 7.90 (d, J = 7.6 Hz, 2H, Ar–H), 7.97–7.99 (m, 2H, Ar–H), 8.02 (d, J = 8.0 Hz, 1H, benzothiazole-H), 8.15 (d, J = 8.4, 1H, benzothiazole-H), 10.71 (s, 2H, NH); anal. calcd for C₂₄H₁₉N₃O₂S (413.49): C% 69.71; H% 4.63; N% 10.16; found: C% 69.75; H% 4.65; N% 10.13.

4.3.6 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-(4-chlorophenyl)acryloyl)-4-methylbenzohydrazide (6f). White solid, yield 75%, m.p: 223–224 °C; IR (KBr, cm⁻¹): ν 3269 (NH), 2923 (CH-Ar), 1686,1645 (CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.40 (s, 3H, CH₃), 7.35 (d, J = 8.0 Hz, 2H, Ar–H), 7.47–7.50 (m, 3H, 2Ar–H & 1-benzothiazole-H), 7.56 (t, J = 7.8 Hz, 1H, benzothiazole-H), 7.75 (s, 1H, CH), 7.90 (d, J = 8.4 Hz, 2H, Ar–H), 8.00–8.04 (m, 3H, 2Ar–H & 1-benzothiazole-H), 8.15 (d, J = 8.4 Hz, 1H, benzothiazole-H), 10.73 (s, 2H, NH); ¹³C-APT (125 MHz, DMSO-d₆): δ 21.5 (CH₃), 122.6, 123.2, 126.2, 127.2, 128.2, 129.1, 129.5, 130.1, 131.0, 132.5, 132.8, 134.8, 135.0, 142.4, 153.5, 165.5 (17C, Ar–C), 166.2, 166.3 (2CO); anal. calcd for C₂₄H₁₈ClN₃O₂S (447.94): C% 64.35; H% 4.05; N% 9.38; found: C% 64.34; H% 4.06; N% 9.36.

4.3.7 (E)-N′-(2-(Benzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)acryloyl)-4-methylbenzohydrazide (6g). Off white solid, yield 75%, m.p: 241–243 °C; IR (KBr, cm⁻¹): ν 3278 (NH), 2953 (CH-Ar), 1681, 1636 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.39 (s, 3H, CH₃), 3.86 (s, 3H, OCH₃), 6.99 (d, J = 9.2 Hz, 2H, Ar–H), 7.35 (d, J = 13.6 Hz, 2H, Ar–H), 7.45 (t, J = 11.6 Hz, 1H, benzothiazole-H), 7.54 (t, J = 9.8 Hz, 1H, benzothiazole-H), 7.69 (s, 1H, CH), 7.93 (d, J = 11.6 Hz, 2H, Ar–H), 7.97–8.02 (m, 3H, 2Ar–H & benzothiazole-H), 8.12 (d, J = 9.0 Hz, 1H, benzothiazole-H), 10.64 (s, 1H, NH), 10.77 (s, 1H, NH); ¹³C-APT (125 MHz, DMSO-d₆): δ 21.5 (CH₃), 55.8 (OCH₃), 114.6, 122.5, 122.9, 125.8, 126.5, 127.0, 127.9, 128.2, 129.5, 130.2, 132.9, 134.8, 135.7, 142.3, 153.6, 161.1, 165.9 (17C, Ar–C), 166.2, 166.9 (2CO); anal. calcd for C₂₅H₂₁N₃O₃S (443.52): C% 67.70; H% 4.77; N% 9.47; found: C% 67.73; H% 4.75; N% 9.44.

4.4. General procedures for preparation of compounds (8a–c)

A mixture of N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4a,d (0.01 mole) and 2-(ethoxymethylene)malononitrile 7a or (E)-ethyl 2-cyano-3-ethoxyacrylate 7b (0.017 mole) were refluxed in ethanol (30 ml) containing sodium ethoxide (0.01 mole) for 5 hours. The formed precipitate was filtered then washed using suitable solvent after drying.

4.4.1 N-(6-Amino-3-(benzo[d]thiazol-2-yl)-5-cyano-2-oxopyridin-1(2H)-yl)benzamide (8a). Orange solid, yield 70%, m.p: over 350 °C; IR (KBr, cm⁻¹): ν 3430 (NH, NH₂), 2924 (CH-Ar), 2216 (CN), 1631 (CO); ¹H NMR (400 MHz, DMSO-d₆): δ 7.30 (t, J = 9.8 Hz, 1H, benzothiazole-H), 7.45 (t, J = 8.4 Hz, 1H, benzothiazole-H), 7.50–7.56 (m, 3H, 3Ar–H), 7.89 (d, J = 8.4 Hz, 1H, benzothiazole-H), 8.04 (d, J = 7.6 Hz, 1H, benzothiazole-H), 8.23 (d, J = 7.2 Hz, 2H, Ar–H), 8.73 (s, 1H, pyridone-H); ¹³C NMR (100 MHz, DMSO-d₆): δ 118.6 (CN), 76.7, 103.9, 121.2, 122.0, 123.6, 126.1, 127.2, 129.2, 130.2, 131.4, 135.0, 136.8, 152.4, 153.7, 156.4 (15C, Ar–C), 162.1, 164.1 (2CO); anal. calcd for C₂₀H₁₃N₅O₂S (387.41): C% 62.00; H% 3.38; N% 18.08; found: C% 62.02; H% 3.40; N% 18.06.

4.4.2 Ethyl 2-amino-1-benzamido-5-(benzo[d]thiazol-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxylate (8b). Orange solid, yield 65%, m.p: over 350 °C; IR (KBr, cm⁻¹): ν 3433 (NH, NH₂), 2925 (CH-Ar), 1685, 1614 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 1.40 (t, J = 7.1 Hz, 3H, CH₃), 4.36 (q, J = 7.1 Hz, 2H, CH₂), 7.29 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.42–7.56 (m, 4H, 3Ar–H & 1benzothiazole-H), 7.91 (d, J = 8.1 Hz, 1H, benzothiazole-H), 8.02 (d, J = 7.8 Hz, 1H, benzothiazole-H), 8.26 (d, J = 7.4 Hz, 2H, Ar–H), 9.15 (s, 1H, pyridone-H); anal. calcd for C₂₂H₁₈N₄O₄S (434.47): C% 60.82; H% 4.18; N% 12.90; found: C% 60.83; H% 4.20; N% 12.91.

4.4.3 Ethyl 2-amino-5-(benzo[d]thiazol-2-yl)-1-(4-methylbenzamido)-6-oxo-1,6-dihydropyridine-3-carboxylate (8c). Orange solid, yield 65%, m.p: over 350 °C; IR (KBr, cm⁻¹): ν 3433 (NH, NH₂), 2920 (CH-Ar), 1685, 1614 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 1.39 (t, J = 7.4 Hz, 3H, CH₃), 2.44 (s, 3H, CH₃), 4.38 (q, J = 7.0 Hz, 2H, CH₂), 7.34 (t, J = 8.8 Hz, 1H, benzothiazole-H), 7.42 (d, J = 12.0 Hz, 2H, Ar–H), 7.48 (t, J = 8.8 Hz, 1H, benzothiazole-H), 7.95–8.00 (m, 3H, 2Ar–H & 1-benzothiazole-H), 8.04 (d, J = 8.0 Hz, 1H, benzothiazole-H), 8.86 (s, 2H, NH₂), 9.12 (s, 1H, pyridone-H), 11.05 (s, 1H, NH); ¹³C-APT (125 MHz, DMSO-d₆): δ 14.9, 21.6 (2CH₃), 61.1 (CH₂), 89.8, 106.6, 121.8, 122.2, 124.4, 126.5, 128.8, 129.1, 129.4, 135.1, 140.3, 143.3, 152.3, 157.2, 158.4 (15C, Ar–C), 162.5, 166.3, 166.9 (3CO); anal. calcd for C₂₃H₂₀N₄O₄S (448.49): C% 61.59; H% 4.49; N% 12.49; found: C% 61.61; H% 4.47; N% 12.47.

4.5. General procedures for preparation of compounds (10a–c)

A mixture of N′-(2-(benzo[d]thiazol-2-yl)acetyl)benzohydrazide derivatives 4a,d (0.01 mole) and (Z)-2-cyano-3-(dimethylamino)-N-arylacrylamide derivatives 9a–c (0.01 mol) were refluxed in dioxane containing equimolar of KOH (0.01 mol) for 7 hours. The precipitate formed was filtered then after drying it was washed using a suitable solvent.

4.5.1 2-Amino-1-benzamido-5-(benzo[d]thiazol-2-yl)-6-oxo-N-phenyl-1,6-dihydropyridine-3-carboxamide (10a). Offwhite solid, yield 65%, m.p: over 350 °C; IR (KBr, cm⁻¹): ν 3430, 3328 (NH, NH₂), 2938 (CH-Ar), 1631 (CO); ¹H NMR (400 MHz, DMSO-d₆): δ 7.12 (t, J = 7.0 Hz, 1H, benzothiazole-H), 7.29 (t, J = 7.0 Hz, 1H, benzothiazole-H), 7.44–7.61 (m, 6H, Ar–H), 7.86–7.93 (m, 3H, 2Ar–H & 1-benzothiazole-H), 8.03 (d, J = 7.6 Hz, 1H, benzothiazole-H), 8.32 (d, J = 8.0 Hz, 2H, Ar–H), 9.25 (s, 1H, pyridone-H), 11.60 (s, 1H, NH); anal. calcd for C₂₆H₁₉N₅O₃S (481.53): C% 64.85; H% 3.98; N% 14.54; found: C% 64.87; H% 3.97; N% 14.53.

4.5.2 2-Amino-5-(benzo[d]thiazol-2-yl)-N-(4-chlorophenyl)-1-(4-methylbenzamido)-6-oxo-1,6-dihydropyridine-3-carboxamide (10b). Offwhite solid, yield 73%, m.p: over 350 °C; IR (KBr, cm⁻¹): ν 3489, 3224 (NH, NH₂), 2916 (CH-Ar), 1660, 1614 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.42 (s, 3H, CH₃), 7.30 (t, J = 10.4 Hz, 1H, benzothiazole-H), 7.40–7.49 (m, 5H, 4Ar–H & 1-benzothiazole-H), 7.89–7.94 (m, 3H, 2Ar–H & 1-benzothiazole-H), 8.03 (d, J = 9.6 Hz, 1H, benzothiazole-H), 8.20 (d, J = 14.0 Hz, 2H, Ar–H), 9.23 (s, 1H, pyridone-H), 11.68 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 21.5 (CH₃), 98.9, 103.9, 121.1, 121.9, 123.3, 125.9, 126.8, 127.2, 128.4, 129.4, 129.9, 134.4, 135.2, 138.9, 139.9, 152.7, 156.6 (17C, Ar–C), 160.7, 162.5, 165.0 (3CO); anal. calcd for C₂₇H₂₀ClN₅O₃S (530.00): C% 61.19; H% 3.80; N% 13.21; found: C% 61.22; H% 3.84; N% 13.20.

4.5.3 2-Amino-5-(benzo[d]thiazol-2-yl)-1-(4-methylbenzamido)-6-oxo-N-(p-tolyl)-1,6-dihydropyridine-3-carboxamide (14c). Beige solid, yield 62%, m.p: over 350 °C; IR (KBr, cm⁻¹): ν 3500, 3330 (NH, NH₂), 2915 (CH-Ar), 1624, 1610 (2CO); ¹H NMR (400 MHz, DMSO-d₆): δ 2.32 (s, 3H, CH₃), 2.42 (s, 3H, CH₃), 7.23 (d, J = 8.4 Hz, 2H, Ar–H), 7.29 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.40–7.46 (m, 3H, 2Ar–H & benzothiazole-H), 7.74 (d, J = 9.2 Hz, 2H, Ar–H), 7.92 (d, J = 7.6 Hz, 1H, benzothiazole-H), 8.02 (d, J = 6.0 Hz, 1H, benzothiazole-H), 8.19 (d, J = 8.0 Hz, 2H, Ar–H), 9.23 (s, 1H, pyridone-H), 11.54 (s, 1H, NH); ¹³C-APT (125 MHz, DMSO-d₆): δ 20.9, 21.5 (2CH₃), 99.4, 103.7, 119.5, 121.1, 121.9, 123.3, 125.9, 127.2, 128.5, 129.9, 130.0, 132.3, 134.3, 135.3, 137.5, 139.9, 152.7, 152.8, 156.6 (19C, Ar–C), 160.6, 162.2, 165.2 (3CO); anal. calcd for C₂₈H₂₃N₅O₃S (509.15): C% 66.00; H% 4.55; N% 13.74; found: C% 66.02; H% 4.53; N% 13.72.

4.6. Anticancer activity

Human tumor carcinoma cell lines (H1299- HEPG2- MCF7) were used in this study were obtained from the American Type Culture Collection (ATCC, Minisota, U.S.A.). The tumor cell lines were maintained at the National Cancer Institute, Cairo, Egypt, by serial sub-culturing. Samples were prepared by dissolving 1 : 1 Stock solution and stored at −20 °C in DMSO at 100 mM. Different concentrations of the drug were used 0.00, 6.25, 12.5, 25, 50 μg ml⁻¹.

The cytotoxicity was carried out using SRB (used as a protein dye) assay.⁴³ Cells were seeded in 96-well microtiter plates at initial concentration of 3 × 10³ cell per well in a 150 μl fresh medium and left for 24 h for attachment. Different concentrations 0, 6.25, 12.5, 25, 50 μg ml⁻¹ of drug were added in triplicate for each drug concentration. The plates were incubated for 48 h at 37 °C, 5% CO₂. By the end of incubation, cells were fixed with 50 μl cold trichloroacetic acid 10% final concentration for 1 h at 4 °C. The plates were washed with distilled water using (automatic washer Tecan, Germany) and stained with 50 μl 0.4% SRB dissolved in 1% acetic acid for 30 minutes at room temperature. The plates were washed four times with 1% acetic acid and air-dried, followed by addition of 200 ml 10 mM Tris base solution (pH 10.5) to each well and shake the plate on a gyratory shaker for 5 min to solubilize the protein-bound dye. Optical density (O.D.) of each well was measured spectrophoto metrically at 570 nm with an ELISA microplate reader (Sunrise Tecan reader, Germany). The mean background absorbance was automatically subtracted and mean values of each drug concentration were calculated. The experiment was repeated 3 times. The percentage of cell survival was calculated after subtraction of background blank O.D. as follows:

Surviving fraction = O.D. (treated cells)/O.D. (untreated cells).

The IC₅₀ values (the concentrations of drug required to produce 50% inhibition of cell growth) were also calculated using GraphPad Prism 8.

4.7. In silico ADME study

Drug-likeness is a qualitative notion in drug design that predicts a drug-like feature. Therapeutic-like qualities such as solubility, permeability, transporter effects, and metabolic stability are essential for therapeutic candidates' success. They have an influence on oral bioavailability, toxicity, metabolism, clearance, and in vitro pharmacology. The drug-likeness of the synthesized compounds was evaluated using five independent filters, including the Lipinski,⁴⁴ Ghose,⁴⁵ Muegge,⁴⁶ Veber,⁴⁷ and Egan⁴⁸ guidelines, as well as bioavailability and drug-likeness scores using the Swiss ADME program.

4.8. Pass analysis

The PASS system provides users with a list of expected activity forms in addition to estimates of the likelihood that each type of activity is either active (Pa) or inactive (Pi), measured on a range of 0 to 1. These probabilities are used as markers of the compound's possible membership in imprecise categories of chemical compounds that are either active or inactive. Based on these probabilities, various criteria can be developed to evaluate prediction results pertinent to real-world problems. The PASS program's efficacy has been demonstrated in a variety of domains, including the identification of compounds with multitarget action against hypertension and inflammation, the discovery of novel biological activities for medically used drugs, and the evaluation of pharmaceutical side effects and toxicity mechanisms. This computer-based software, available through an online platform called PASS (Prediction of Probable Activity Spectra of Substances) analysis (https://www.pharmaexpert.ru/passonline), allows for the examination of a compound's biological and pharmacological potential, as well as the elucidation of its interactions with other biological entities. The PASS application accepts data in the Mol file format for individual structures or the SDF file format for collections of structures.

4.9. Molecular docking study

All the invers docking analysis was conducted using the Molecular Operating Environment (MOE 2014). The ligand molecules were pulled by the building molecules, and their energy was reduced. All minimizations were performed until the MMFF94X force field achieved an RMSD gradient of 0.01 kcal mol⁻¹, at which point the partial charges were calculated automatically. Docking simulations were carried out utilizing the Protein Data Bank's crystal structure of twelve enzymes, ABL1, ABL2, PTK2, CDC42, PTK6, EGFR, c-KIT, c-Met, FGFR1, CDK2, CDK4, and CDK6, in association with co-crystallin ligand 1N1, DKI, H82, T74, FLJ, YW5, F82, L5G, 0LI, RRC, 1PU, and NJ6 (PDB ID: 2GQG, 3HMI, 6I8Z, 3EQR, 6CZ3, 8GB4, 6GQJ, 3CD8, 4V01, 2 A4L, 1GIH, and 8I0M). The MOE protonate 3D application was used to add the missing hydrogens and assign the right ionization states. The MOE-Alpha site finder was used to create an active site. Re-docking the co-crystallized ligands into their respective protein binding sites was performed. The reproduced poses were in close agreement with the experimental conformations, with root mean square deviation (RMSD) values of less than 2.0 Å, confirming the validity of the docking procedure. Ligands were then docked within the active sites using the MOE-Dock. The GBVI/WSA DG free-energy estimates were used to rank the optimized poses and docking poses were examined visually. The interactions with binding pocket residues were finally investigated.

Author contributions

Conceptualization: GHE, RAA; methodology: GHE, RAA, MMS, MAE; writing-original draft preparation: GHE, RAA; writing-review and editing: GHE, RAA and MAE.

Conflicts of interest

The authors have no conflicts of interest to declare.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5ra03277f.

Acknowledgements

The authors express their gratitude to the Central Laboratory of Helwan University (CLHU) at the headquarters of the Centers of Excellence and Scientific Innovation at Helwan University for conducting ¹³C-APT analysis.

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