Thiazolation of phenylthiosemicarbazone to access new thiazoles: anticancer activity and molecular docking

Abstract

Aim: Novel thiazole hybrids were synthesized via thiazolation of 4-phenylthiosemicarbazone (4). Materials & methods: The anticancer activity against the NCI 60 cancer cell line panel. Results: Methyl 2-(2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-4-oxo-3-phenylthiazolidin-5-ylidene)acetate (6a) showed significant anticancer activity at 10 μM with a mean growth inhibition (GI) of 51.18%. It showed the highest cytotoxic activity against the ovarian cancer OVCAR-4 with an IC₅₀ of 1.569 ± 0.06 μM. Compound 6a inhibited PI3Kα with IC₅₀ = 0.225 ± 0.01 μM. Moreover, compound 6a revealed a decrease of Akt and mTOR phosphorylation in OVCAR-4 cells. In addition, antibacterial activity showed that compounds 11 and 12 were the most active against Staphylococcus aureus. Conclusion: Compound 6a is a promising molecule that could be a lead candidate for further studies.

Plain language summary

Novel naphthalene-azine-thiazole hybrids 5-12 were synthesized via late-stage thiazolation of the corresponding 4-phenylthiosemicarbazone 4. Compound 6a showed significant anticancer activity at single-dose screening and yielded excellent inhibitory activity with a mean GI of 51.18%. Compound 6a showed the highest cytotoxic activity against OVCAR-4 with an IC₅₀ of 1.569 ± 0.06 μM. Moreover, compound 6a exhibited an IC₅₀ of 31.89 ± 1.19 μM against normal ovarian cell line (OCE1) and a selectivity index of 19.1. Compound 6a inhibited PI3Kα with IC₅₀ = 0.225 ± 0.01 μM compared with alpelisib (IC₅₀ = 0.061 ± 0.003 μM). Moreover, compound 6a revealed a powerful decrease of Akt and mTOR phosphorylation in the OVCAR-4 cell line. The cell cycle analysis showed that compound 6a caused an arrest at the G2/M phase. The compound also increased the total apoptosis by 26.8-fold and raised the level of caspase-3 by 4.34 times in OVCAR-4. In addition, antibacterial activity was estimated against Gram-positive and Gram-negative bacterial strains. Compounds 11 and 12 were the most active derivatives, with MIC value of 256 μg/ml against Staphylococcus aureus. Molecular docking was done and showed that 6a interlocked and fitted well into the ATP binding site of PI3Kα kinase (Protein Data Bank ID: 4JPS) with a fitness value (-119.153 kcal/mol) and forms the key H-bonds with Val851 and Ser854 like the marketed PI3Kα inhibitor alpelisib. Consequently, 6a is the most promising molecule that could be a lead candidate for further studies.

TWEETABLE ABSTRACT

A series of naphthalene-azine-thiazole derivatives were synthesized and evaluated for their anticancer and antibacterial activity. These findings propose that compound 6a has cytotoxic properties and is a starting structure for a new generation of PI3Kα kinase inhibitors.

GRAPHICAL ABSTRACT

Plain language summary

Summary points.

• A series of naphthalene–azine–thiazole derivatives were synthesized.

• The characterization of all the synthesized compounds was carried out using several analytical and spectroscopic techniques.

• The synthesized compounds were tested against various cancer cell lines and antibacterial.

• Benzenesulfonamide derivative 11 and the coumarin-containing derivative 12 showed the highest antibacterial activity with MIC value of 256 μg/ml against S. aureus.

• Compound 6a showed the highest activity against the ovarian cancer cell line OVCAR-4 with an IC₅₀ of 1.569 ± 0.06 μM.

• Compound 6a inhibited PI3Kα with IC₅₀ = 0.225 ± 0.01 μM compared with alpelisib (IC₅₀ = 0.061 ± 0.003 μM).

• Compound 6a inhibited the growth of OVCAR-4 cells during the G2/M phase and increased total apoptosis by 26.8-fold.

• Compound 6a interlocked and fitted well into the ATP binding site of PI3Kα kinase (Protein Data Bank ID: 4JPS) with a fitness value (-119,153 kcal/mol).

1. Background

Cancer is a complex disease with many known cancer-associated genes in humans [1–3]. Chemotherapy has become increasingly important in curing cancer, especially when used alongside local therapy. In advanced stages, when the tumor has spread from its origin, chemotherapy can help relieve cancer-related symptoms and prolong life. Despite its limitations, chemotherapy remains an important treatment in oncology and is expected to remain so for the foreseeable future [4].

In light of their importance in the chemical synthesis of promising bioactive molecules, azines have lately gained considerable motivation in the organic synthesis community [5–7]. Azines have a diversity of biological activities, among them cytotoxic effects with a low IC₅₀ value against numerous cancer cell lines. They have been exploited as key lead small-molecules in drug discovery and development due to their regulating action against target biomolecules [7–13]. In our previous article [7] we have provided an informative introduction to the diverse chemical synthesis and biological actions of symmetrical and unsymmetrical azines.

The building blocks of synthetic and naturally occurring naphthalene-based derivatives are capable of generating a diverse collection of biochemically active small-molecules [7,14]. These molecules are employed in targeted therapy (cancer pharmacotherapy) as potential tubulin polymerization inhibitors [15,16]. The naphthyl group is present in many US FDA-approved drugs, such as nafcillin (DB00607, a β-lactam antibiotic), naproxen (DB00788, a nonsteroidal anti-inflammatory drug [NSAID]), nafimidone (an imidazole anticonvulsant), bedaquiline (DB08903, an antimycobacterial tuberculosis), cinacalcet (DB01012, utilized to tackle hypercalcemia in parathyroid carcinoma), and terbinafine (an antifungal) [17].

Organic and medicinal chemists pay great attention to thiazole-heteroarenes as an important structural category for drug possibilities and other purposes. The thiazole nucleus are also present in many FDA-approved drugs, such as penicillin, ceftazidime (a third-generation cephalosporin), cefepime (a fourth-generation cephalosporin), aztreonam and sulfathiazole (as antibiotics), abafungin (as an antifungal), ritonavir (as an anti-HIV), bleomycin (a cancer medication), pramipexole (Parkinson's disease treatment), dasatinib (chronic myeloid leukemia treatment), meloxicam (a nonsteroidal anti-inflammatory medication), nizatidine (a peptic ulcer treatment), thiamine (vitamin B₁, as an essential micronutrient), etc. [18–23]. It is well documented that naphthalene-linked thiazoles via azine linker are potent bioactive small-molecules endowed with distinct biological potencies such as anti-inflammatory [24], anti-Toxoplasma gondii [25], antifungal activity with cytotoxic effect against Hep2 cells [26,27]. Molecular hybridization between the prior moieties and an azine linker to install new derivatives and explore their applications is attractive with expectations to produce perfect lead candidates for the creation of more effective and safer medications.

Moreover, thiazolidin-4-one is a useful framework for producing drugs that can inhibit the proliferation of cancer cells [28,29]. It is also present in natural products used for treating cancer [30]. Moreover, this framework shows great potential in the development of new anticancer agents, as many of its derivatives have been found to possess potent anticancer activity against different types of cancer cells [31,32].

In response to persistent resistance to the currently used antibiotics and anticancer drugs, pharmaceutical research centers and academic researchers must conduct continuous research to develop new and effective antibacterial [33–35] and anticancer [36–39] drugs. New drugs which are demonstrating simultaneous antimicrobial and anticancer activities are preferable [40–42] as cancer patients are particularly likely to benefit from this dual effect due to the fact that they have a decreased immune system, making them much more vulnerable to bacterial infections [43].

Despite the development of multiple successful medicines, cancer remains a major issue, owing mostly to the rise of multidrug resistance and patient difficulty responding to regimens of therapy. Thus, the development and discovery of new robust agents with improved qualifications to combat cancer is still a very critical research domain and has a meaningful impact on the lives of cancer patients.

Phosphoinositide 3-kinase (PI3K) is a group of lipid kinases that can be classified into three distinct classes, namely class I, II and III. The classification is based on their sequence homology, mode of action and substrate preferences [44]. The PI3K signaling pathway regulates cellular responses and plays a critical role in maintaining the balance between cellular survival and apoptosis. Therefore, PI3K signaling activation may promote the development of malignant tumors and cause unchecked cell proliferation. The catalytic subunit of PI3K is one of the most frequently mutated or amplified oncogenes across different types of cancer, including breast, ovarian, gastrointestinal, leukemia, colon, kidney, liver, brain and other cancers [45–47].

The activation of the PI3K by receptor tyrosine kinases promotes increased cell growth, cell proliferation, cell migration and invasion, as well as angiogenesis. resulting in prolonged phosphorylation and activation of AKT. The activated AKT impacts many downstream effectors, such as mTOR and finally leads to multiple cellular processes of cell transformation, and inhibition of PI3K pathway leads to tumor regression. Importantly, the PI3K pathway is activated in approximately 70% of ovarian cancers, leading to hyperactive signaling cascades that are correlated with cellular growth, proliferation and survival [45,48].

The inhibition of the PI3K enzyme presents a promising target for the development of effective anticancer agents. Also, the development of inhibitors targeting PI3K/AKT/mTOR pathway has become a promising target for medicinal chemists around the world [49–54].

Despite the biological importance of naphthalene-azine-thiazole analogs, PI3Kα inhibitor-based hybrid derivatives of them are unknown in the literature, according to our best knowledge. Combining the building block naphthalene scaffold with a thiazole core via an azine linker could result in ideal lead candidates for the development of more efficient and safer anticancer agents. Herein, we desire to combine these two pharmacophores into a single framework in hopes of synthesizing an entirely new class of PI3Kα inhibitors with strong antitumor activities.

2. Materials & methods

2.1. Materials & instrumental techniques

See the Supplementary Data file.

2.2. Chemical synthesis

2.2.1. Synthesis of 1-(1-(naphthalen-2-yl)ethylidene)-4-phenylthiosemicarbazide (4)

The assembly of a miniscale reflux apparatus began with an oven-dry 250 ml flat-bottomed flask equipped with a magnetic stir bar. A powder funnel was used to add 2-acetylnaphthalene hydrazone (3, 5.520 g, 0.03 mol) followed by equimolar amount of phenyl isothiocyanate (PITC, 3.59 ml, 0.03 mol). Dry dioxane (30 ml) was added by the conical funnel. The flask was fitted with a water-jacketed condenser, and the reaction mixture was refluxed at 80°C through a pre-heated water bath for 4 h. While cooling, the desired 4-phenylthiosemicarbazide 4 was precipitated, filtrated off, dried and recrystallized from dioxane to afford 4 in its pure state (TLC).

Off-white amorphous solid from dioxane; 87% yield; mp 194–196°C; mid FT-IR (KBr, ν/cm^-1) 3292, 3204 (2N-H stretching), 3054 (aromatic C-H stretching), 2967 (aliphatic C-H stretching), 1599 (C=N stretching), 1190 (C=S stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 10.66 (s, 1H), 10.14 (s, 1H), 8.41 (dt, J = 7.3, 1.8 Hz, 2H), 8.10–7.88 (m, 3H), 7.64–7.51 (m, 4H), 7.40 (t, J = 7.8 Hz, 2H), 7.28–7.19 (m, 1H), 2.52 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 177.58, 149.28, 139.70, 135.75, 135.42, 134.06, 133.82, 133.24, 133.21, 129.18, 129.06, 128.58, 128.28, 128.01, 127.92, 127.54, 127.36, 127.31, 127.00, 126.84, 126.52, 125.91, 124.76, 124.14, 14.72; EI-MS for C₁₉H₁₇N₃S m/z (%): 319.39 [M]⁺ (25), 321.38 [M+2]⁺ (10). Anal. Calcd. for C₁₉H₁₇N₃S: C, 71.44; H, 5.36; N, 13.16; found C, 71.63; H, 5.50; N, 13.32.

2.2.2. General procedure for the synthesis of target naphthalene-azine-thiazole hybrids (5–12)

Setting up refluxing conditions, 4 (0.319 g, 1 mmol) was transferred into an oven-dry 100 ml flat-bottomed flask containing 5 ml of dioxane and equipped with a magnetic stir bar. Dioxane (5 ml) solution of the same equivalent (1 mmol) from halogenated compounds: ethyl chloroacetate, ethyl 2-bromopropionate, ethyl 2-bromobutyrate, arene hydrazonoyl chlorides [ethyl 2-chloro-2-(2-(p-tolyl)hydrazineylidene)acetate and 2-oxo-N-phenylpropanehydrazonoyl chloride], chloroacetone, ethyl 2-chloro-3-oxobutanoate, chloroacetonitrile, 4-(2-bromoacetyl)benzenesulfonamide and 3-(bromoacetyl)coumarin, was added individually. The reaction mixture was mediated by a catalytic amount of anhydrous CH₃COONa (1 mmol) in case of halogenated compounds [24] or dry Et₃N (5 drops) in case of hydrazonoyl chlorides [55] and refluxed at 105°C through a pre-heated oil bath for 3–12 h (TLC) [24,55]. For unsaturated di-carbonyl reagents different solvents were used; MeOH with DMAD (stirring at room temperature for 2 h), EtOH with DEAD (reflux at 80°C for 3 h), toluene/DMF (4:1) with maleic anhydride (reflux at 115°C for 4 h). During reflux (or stirring at room temperature [rt]), the desired thiazoles were formed and the work-up procedures were implemented for each case.

2.2.2.1. 2-((1-(Naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenylthiazolidin-4-one (5a)

Colorless amorphous solid from methanol-dioxane; 95% yield; mp 230–232°C; mid FT-IR (KBr, ν/cm^-1) 3055 (aromatic C-H stretching), 2974 (aliphatic C-H stretching), 1726 (C=O stretching), 1606 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.29 (d, J = 1.8 Hz, 1H), 8.14 (dd, J = 8.7, 1.8 Hz, 1H), 8.04–7.97 (m, 1H), 7.94 (dd, J = 9.0, 3.8 Hz, 2H), 7.62–7.52 (m, 4H), 7.52–7.41 (m, 3H), 4.13 (s, 2H), 2.31 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 172.39, 164.17, 162.04, 157.95, 135.65, 135.40, 134.13, 133.17, 129.42, 129.14, 129.01, 128.53, 128.23, 128.00, 127.58, 127.49, 127.32, 127.02, 124.14, 123.79, 32.83, 14.93; EI-MS for C₂₁H₁₇N₃OS m/z (%): 359.29 [M]⁺ (14). Anal. Calcd. for C₂₁H₁₇N₃OS: C, 70.17; H, 4.77; N, 11.69; found C, 70.33; H, 4.65; N, 11.84.

2.2.2.2. 5-Methyl-2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenylthiazolidin-4-one (5b)

Light brown amorphous solid from methanol-dioxane; 98% yield; mp 170–172°C; mid FT-IR (KBr, ν/cm^-1) 3057 (aromatic C-H stretching), 2975 (aliphatic C-H stretching), 1714 (C=O stretching), 1603 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.29 (d, J = 1.7 Hz, 1H), 8.14 (dd, J = 8.8, 1.8 Hz, 1H), 8.02–7.99 (m, 1H), 7.96–7.92 (m, 2H), 7.56 (ddd, J = 7.0, 5.1, 3.1 Hz, 4H), 7.48 (td, J = 6.2, 1.4 Hz, 3H), 4.44 (q, J = 7.2 Hz, 1H), 2.31 (s, 3H), 1.66 (d, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 175.44, 162.09, 135.67, 135.36, 134.13, 133.16, 129.41, 129.14, 129.06, 128.54, 128.21, 127.99, 127.80, 127.60, 127.48, 127.03, 123.77, 41.81, 19.37, 14.91; EI-MS for C₂₂H₁₉N₃OS m/z (%): 373.23 [M]⁺ (51), 374.71 [M+1]⁺ (12). Anal. Calcd. for C₂₂H₁₉N₃OS: C, 70.75; H, 5.13; N, 11.25; found C, 70.63; H, 5.21; N, 11.43.

2.2.2.3. 5-Ethyl-2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenylthiazolidin-4-one (5c)

Brown amorphous solid from methanol-dioxane; 96% yield; mp 176–178°C; mid FT-IR (KBr, ν/cm^-1) 3054 (aromatic C-H stretching), 2967 (aliphatic C-H stretching), 1722 (C=O stretching), 1606 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.29 (d, J = 1.8 Hz, 1H), 8.15 (dd, J = 8.7, 1.8 Hz, 1H), 8.03–8.00 (m, 1H), 7.96–7.93 (m, 2H), 7.58–7.54 (m, 4H), 7.50–7.48 (m, 1H), 7.46–7.43 (m, 2H), 4.47 (dd, J = 7.6, 4.4 Hz, 1H), 2.31 (s, 3H), 2.11 (dtd, J = 14.7, 7.3, 4.4 Hz, 1H), 1.99 (dp, J = 14.5, 7.3 Hz, 1H), 1.07 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 174.51, 162.27, 157.95, 135.77, 135.59, 135.37, 134.16, 133.18, 129.45, 129.16, 129.08, 128.51, 128.21, 128.00, 127.53, 127.31, 126.99, 124.14, 123.83, 48.70, 26.27, 14.94, 10.73; EI-MS for C₂₃H₂₁N₃OS m/z (%): 387.69 [M]⁺ (38). Anal. Calcd. for C₂₃H₂₁N₃OS: C, 71.29; H, 5.46; N, 10.84; found C, 71.51; H, 5.62; N, 11.09.

2.2.2.4. Methyl 2-(2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-4-oxo-3-phenylthiazolidin-5-ylidene)acetate (6a)

Deep yellow amorphous solid from methanol-dioxane; 99% yield; mp 204–206°C; mid FT-IR (KBr, ν/cm^-1) 3062 (aromatic C-H stretching), 2957 (aliphatic C-H stretching), 1712 (C=O stretching), 1606 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.36 (d, J = 1.8 Hz, 1H), 8.16 (dd, J = 8.8, 1.8 Hz, 1H), 8.10–8.02 (m, 1H), 8.00–7.94 (m, 3H), 7.60–7.57 (m, 6H), 6.84 (s, 1H), 3.84 (s, 3H), 2.38 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 166.49, 164.53, 159.65, 142.33, 135.74, 134.83, 134.36, 133.12, 129.48, 129.17, 128.43, 128.27, 128.16, 128.04, 127.30, 127.14, 127.00, 124.12, 123.61, 115.48, 53.05, 15.20; EI-MS for C₂₄H₁₉N₃O₃S m/z (%): 429.91 [M]⁺ (34), 432.34 [M+2]⁺ (18). Anal. Calcd. for C₂₄H₁₉N₃O₃S: C, 67.12; H, 4.46; N, 9.78; found C, 67.29; H, 4.58; N, 9.91.

2.2.2.5. Ethyl 2-(2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-4-oxo-3-phenylthiazolidin-5-ylidene)acetate (6b)

Deep yellow amorphous solid from acetic acid; 99% yield; mp 239–241°C; mid FT-IR (KBr, ν/cm^-1) 3062 (aromatic C-H stretching), 2971 (aliphatic C-H stretching), 1712, 1692 (2C=O stretching), 1606 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.37–8.32 (m, 1H), 8.15 (dd, J = 8.7, 1.8 Hz, 1H), 8.09–7.92 (m, 4H), 7.65–7.54 (m, 6H), 6.79 (s, 1H), 4.29 (q, J = 7.1 Hz, 2H), 2.37 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 165.93, 164.50, 159.74, 142.11, 134.83, 134.36, 134.06, 133.13, 129.43, 129.27, 128.38, 128.13, 128.02, 127.85, 127.51, 127.30, 127.09, 126.96, 123.62, 115.77, 61.91, 15.12, 14.47; EI-MS for C₂₅H₂₁N₃O₃S m/z (%): 443.82 [M]⁺ (54). Anal. Calcd. for C₂₅H₂₁N₃O₃S: C, 67.70; H, 4.77; N, 9.47; found C, 67.82; H, 4.85; N, 9.62.

2.2.2.6. 2-((1-(Naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-5-(2-(p-tolyl)hydrazineylidene) thiazolidin-4-one (7)

Orange amorphous solid from methanol-dioxane; 88% yield; mp 208–210°C; mid FT-IR (KBr, ν/cm^-1) 3055 (aromatic C-H stretching), 2926 (aliphatic C-H stretching), 1603 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 10.64 (s, 1H), 8.41 (d, J = 1.8 Hz, 1H), 8.34 (d, J = 1.9 Hz, 1H), 8.22 (ddd, J = 12.7, 8.7, 1.8 Hz, 2H), 8.08–8.03 (m, 1H), 7.97 (dd, J = 9.4, 5.9 Hz, 3H), 7.61–7.55 (m, 5H), 7.50 (ddt, J = 10.3, 5.0, 2.3 Hz, 1H), 7.26–7.20 (m, 1H), 7.14 (d, J = 8.4 Hz, 1H), 2.38 (s, 3H), 2.26 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 163.04, 161.87, 156.89, 141.44, 135.01, 134.62, 133.88, 132.75, 130.90, 129.79, 129.04, 128.80, 128.77, 128.20, 127.78, 127.64, 127.55, 127.39, 127.14, 126.89, 126.74, 126.60, 123.72, 123.33, 122.80, 114.06, 20.39, 14.99; EI-MS for C₂₈H₂₃N₅OS m/z (%): 477.16 [M]⁺ (7.86). Anal. Calcd. for C₂₈H₂₃N₅OS: C, 65.12; H, 4.05; N, 14.06; found C, 65.07; H, 4.23; N, 14.28.

2.2.2.7. 5-Methyl-5-(naphthalen-2-yl)-N-phenyl-1,3,4-thiadiazol-2(5H)-imine (8)

Light brown amorphous solid; 87% yield; mp 214–216°C; mid FT-IR (KBr, ν/cm^-1) 3051 (aromatic C-H stretching), 2917 (aliphatic C-H stretching), 1600 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.43 (s, 1H), 8.24 (d, J = 9.0 Hz, 1H), 8.07 (s, 1H), 7.98 (s, 3H), 7.62–7.59 (m, 6H), 2.47 (s, 3H); EI-MS for C₂₃H₁₇N₃O₃S m/z (%): 317.48 [M+ (14.33). Anal. Calcd. for C₂₃H₁₇N₃O₃S: C, 71.90; H, 4.76; N, 13.24; found C, 71.68; H, 4.81; N, 13.43.

2.2.2.8. 4-Methyl-2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-2,3-dihydrothiazole (9a)

Brown amorphous solid from methanol-dioxane; 92% yield; mp 204–206°C; mid FT-IR (KBr, ν/cm^-1) 3054 (aromatic C-H stretching), 2911 (aliphatic C-H stretching), 1598 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.22–8.11 (m, 2H), 7.97–7.93 (m, 1H), 7.88 (dd, J = 9.1, 4.6 Hz, 2H), 7.63–7.42 (m, 7H), 6.24 (s, 1H), 2.24 (s, 3H), 1.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 170.13, 157.95, 154.77, 137.59, 136.45, 135.69, 133.58, 133.42, 129.77, 128.98, 128.86, 127.93, 127.89, 126.86, 126.77, 125.95, 123.85, 98.27, 15.09, 14.39; EI-MS for C₂₂H₁₉N₃S m/z (%): 359.97 [M+2]⁺ (27). Anal. Calcd. for C₂₂H₁₉N₃S: C, 73.92; H, 5.36; N, 11.75; found C, 73.79; H, 5.47; N, 11.99.

2.2.2.9. Ethyl 4-methyl-2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-2,3-dihydrothiazole-5-carboxylate (9b)

Pale yellow amorphous solid from methanol-dioxane; 90% yield; mp 209–211°C; mid FT-IR (KBr, ν/cm^-1) 3057 (aromatic C-H stretching), 2977 (aliphatic C-H stretching), 1726 (C=O stretching), 1605 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.42 (d, J = 1.8 Hz, 1H), 8.28 (d, J = 1.8 Hz, 1H), 8.08–7.96 (m, 3H), 7.94 (d, J = 8.7 Hz, 2H), 7.56–7.52 (m, 3H), 7.46–7.43 (m, 2H), 4.27 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 2.23 (d, J = 4.0 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 172.05, 163.78, 161.69, 135.24, 135.00, 133.74, 132.78, 129.71, 129.06, 128.76, 128.66, 128.14, 127.85, 127.62, 127.23, 127.10, 126.66, 123.39, 60.77, 32.43, 14.54, 13.95; EI-MS for C₂₅H₂₃N₃O₂S m/z (%): 428.72 [M]⁺ (45). Anal. Calcd. for C₂₅H₂₃N₃O₂S: C, 69.91; H, 5.40; N, 9.78; found C, 70.12; H, 5.63; N, 9.94.

2.2.2.10. 2-((1-(Naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-2,3-dihydrothiazol-4-amine (9c)

Brown amorphous solid from methanol-dioxane; 90% yield; mp 278–280°C; mid FT-IR (KBr, ν/cm^-1) 3250, 3171 (NH2 stretching), 3052 (aromatic C-H stretching), 2916 (aliphatic C-H stretching), 1605 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.44–8.29 (m, 2H), 8.32–8.16 (m, 2H), 8.16–7.86 (m, 5H), 7.70–7.55 (m, 2H), 7.55 (q, J = 2.4, 1.6 Hz, 2H), 7.49–7.32 (m, 2H), 2.32–2.28 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 171.90, 163.68, 161.55, 135.16, 134.90, 134.36, 133.64, 132.92, 132.71, 128.93, 128.66, 128.52, 128.04, 127.76, 127.51, 127.08, 126.51, 125.25, 123.30, 97.81, 14.44; EI-MS for C₂₁H₁₈N₄S m/z (%): 358.82 [M]⁺ (27), 360.04 [M+2]⁺ (15). Anal. Calcd. for C₂₁H₁₈N₄S: C, 70.36; H, 5.06; N, 15.63; found C, 70.54; H, 5.20; N, 15.76.

2.2.2.11. 4-Methyl-2-((1-(naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-5-(phenyldiazenyl)-2,3-dihydrothiazole (10)

Reddish brown amorphous solid from methanol-dioxane; 88% yield; mp 178–180°C; mid FT-IR (KBr, ν/cm^-1) 3054 (aromatic C-H stretching), 2921 (aliphatic C-H stretching), 1597 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.43 (s, 1H), 8.26 (s, 1H), 8.14 (s, 2H), 8.07 (d, J = 5.6 Hz, 1H), 7.99 (h, J = 10.2, 7.5 Hz, 4H), 7.66 (d, J = 5.9 Hz, 1H), 7.64–7.48 (m, 6H), 7.46–7.35 (m, 1H), 2.51 (s, 3H), 2.47 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 164.84, 145.55, 142.76, 132.82, 129.63, 129.44, 129.42, 129.40, 128.65, 128.46, 127.59, 126.60, 123.39, 121.78, 99.87, 14.75, 14.38; EI-MS for C₂₈H₂₃N₅S m/z (%): 461.26 [M]⁺ (16). Anal. Calcd. for C₂₈H₂₃N₅S: C, 72.86; H, 5.02; N, 15.17; found C, 73.01; H, 5.19; N, 15.40.

2.2.2.12. 4-(2-((1-(Naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-2,3-dihydrothiazol-4-yl)benzene-sulfonamide (11)

Brown amorphous solid from methanol-dioxane; 92% yield; mp 210–212°C; mid FT-IR (KBr, ν/cm^-1) 3372, 3259 (NH2 stretching), 3052 (aromatic C-H stretching), 2919 (aliphatic C-H stretching), 1600 (C=N stretching), 1365, 1128 (SO2 stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.42 (d, J = 1.8 Hz, 1H), 8.29–8.13 (m, 2H), 8.09–8.02 (m, 1H), 7.97 (dd, J = 9.3, 5.8 Hz, 3H), 7.94–7.89 (m, 1H), 7.74 (qd, J = 7.4, 6.2, 4.1 Hz, 1H), 7.69–7.64 (m, 1H), 7.60–7.50 (m, 3H), 7.40–7.32 (m, 2H), 7.27–7.17 (m, 1H), 7.09 (t, J = 9.4 Hz, 1H), 7.01–6.91 (m, 1H), 6.88–6.83 (m, 1H), 2.46 (s, 3H), ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 157.51, 135.33, 133.64, 132.82, 128.77, 128.47, 127.87, 127.60, 127.14, 126.89, 126.60, 123.72, 118.26, 14.75, EI-MS for C₂₇H₂₂N₄O₂S₂ m/z (%): 498.24 [M]⁺ (23), 499.89 [M+2]⁺ (13). Anal. Calcd. for C₂₇H₂₂N₄O₂S₂: C, 65.04; H, 4.45; N, 11.24; found C, 65.21; H, 4.64; N, 11.45.

2.2.2.13. 3-(2-((1-(Naphthalen-2-yl)ethylidene)hydrazineylidene)-3-phenyl-2,3-dihydrothiazol-4-yl)-2H-chromen-2-one (12)

Brown amorphous solid from methanol-dioxane; 94% yield; mp 180–182°C; mid FT-IR (KBr, ν/cm^-1) 3057 (aromatic C-H stretching), 2969 (aliphatic C-H stretching), 1725 (C=O stretching), 1604 (C=N stretching); ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 8.41 (dd, J = 8.8, 2.2 Hz, 1H), 8.30–8.22 (m, 1H), 8.21–8.17 (m, 1H), 8.09–8.04 (m, 1H), 8.03–7.88 (m, 4H), 7.57 (dtd, J = 21.1, 6.5, 2.7 Hz, 4H), 7.45–7.30 (m, 3H), 7.25 (dt, J = 9.8, 7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.90 (s, 1H), 2.34 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 157.53, 153.30, 135.33, 133.65, 132.82, 129.02, 128.77, 128.68, 128.52, 128.21, 128.12, 128.04, 127.88, 127.61, 127.14, 126.89, 126.60, 126.46, 126.10, 125.01, 123.72, 123.42, 116.21, 103.79, 92.28, 14.75, EI-MS for C₃₀H₂₁N₃O₂S m/z (%): 487.92 [M]⁺ (34), 490.59 [M+2]⁺ (12). Anal. Calcd. for C₃₀H₂₁N₃O₂S: C, 73.90; H, 4.34; N, 8.62; found C, 73.81; H, 4.50; N, 8.89.

2.3. Biological evaluation

2.3.1. Biological assays

The biological assays were performed using previously reported procedures and are provided in the Supplementary Data; antibacterial susceptibility testing [56,57], minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurement [57], antiproliferative activity screening by National Cancer Institute (NCI) [58–61], MTT assay procedure [62], in vitro enzymatic PI3Kα assay [63], phosphorylation assay [63], cell cycle analysis [64], apoptosis assay [65] and caspase-3 enzyme assay [66].

3. Results & discussion

3.1. Design strategy, physicochemical descriptors & drug-likeness

Aiming to open the anticancer profile and enhance the activity of naphthalene–azine–thiazole hybrids, as well as being encouraged by the promising antifungal and anti-Toxoplasma gondii potencies of A [25–27], as well as the anti-inflammatory and analgesic activities of B [24], new thiazoles-linked naphthalene via azine linker (5–12, as numbered in the organic synthesis part) were designed employing a variation of substituents and ring isostere strategies (Figure 1). Afterwards, the target compounds 5–12 were sketched and in silico evaluated to predict their physicochemical features and druglike nature through the free ADMETlab 2.0 online platform [7,67]. Supplementary Table S1 shows that the tested compounds have been displayed in the optimal range for each physicochemical property according to the Drug-Like Soft rule [67], where the size parameter is indicated by the molecular weight (MW), which ranged from 358 to 498 g/mol (optimal: 100–600), the number of the rotatable bonds (nRot) that defined the flexibility is 3–6 (optimal: 0–11), and the topological polar surface area (TPSA) that affected the polarity is between 29.65 and 89.81 Å² (optimal: 0–140). Lipophilicity is a critical physicochemical characteristic assessed by the partition coefficients logP and logD (logP at physiological pH 7.4) between water and n-octanol, which serves as an important indicator of permeability across the cell wall [68]. All the compounds have logP and logD values within the optimum ranges according to the Lipinski and Golden Triangle filters. As a result, they may have strong permeability and absorption capabilities through the cell membrane of infected cells and pass both the drug-likeness rules of Lipinski and the Golden Triangle. Hence, they could be potential drug lead candidates and encourage us to synthesize them in the organic lab.

Figure 1.

Design strategy of target compounds.

3.2. Organic synthesis

The synthetic pathways required to furnish the target naphthalene-azine-thiazole hybrids (5–12) are illustrated in Figures 2 & 3. The molecular structure of these compounds was proved by FT-IR, NMR and EI-MS instrumental analysis (See Experimental Section and Supplementary Data).

Figure 2.

Chemical synthesis of compounds 4–8.

Figure 3.

Chemical synthesis of compounds 9–12.

To put this effort into action, we start with the chemical synthesis of our substrate [1-(1-(naphthalen-2-yl)ethylidene)-4-phenylthiosemicarbazide, 4] that was previously reported by a one-step straightforward protonic acid-catalyzed condensation reaction [69–71] between commercially available 2-acetylnaphthalene (1) and antibacterial 4-phenylthiosemicarbazide (4-PTSC, 2) [72]. In our new approach, 1 was transformed into the corresponding hydrazone 3 in the first step by treatment with hydrazine hydrate [7]. In the second step, 3 was reacted with Edman's reagent (phenyl isothiocyanate, PITC) that is available in our feedstock to furnish 4 in high yield (Figure 2). The physical properties and the obtained experimental data of 4 are matched very well with those reported in the literature [69–71].

Afterward, we focused on the thiazolation of phenylthiosemicarbazone 4 by reaction first with commercially available α-halo ethyl esters (ethyl chloroacetate, ethyl 2-bromopropionate and ethyl 2-bromobutyrate) in refluxing dioxane containing a catalytic quantity of sodium acetate [24] to install the corresponding 4-thiazolone derivatives 5a–c in excellent yields (Figure 2). The generation of 5a–c was conducted via heterocyclization process [73,74]. The aliphatic and aromatic regions of 5a–c compounds are clearly distinguished in the 1D NMR spectra. Where, the key ¹H NMR signals of 5a–c proves the absence of thiosemicarbazone-2NH at δ_H = 10.66 and 10.14 ppm and appearance of new CH₂ at δ_H = 4.13 ppm for 5a, CH₃ at δ_H = 1.66 ppm for 5b, and CH₂CH₃ at δ_H = 2.11, 1.99 ppm (geminal CH₂, J = 14.7 Hz) with δ_H = 1.07 (CH₃) ppm for 5c. The C=S ¹³C resonance at δ_C = 177.58 ppm of 4 was replaced with C=O counterpart at δ_C = 172.39–175.44 ppm of 5a–c compounds. Nicely, in the IR spectra of 5a–c compounds, the C=O stretching bands are strongly vibrated at 1726, 1714 and 1722 cm^-1, respectively. Moreover, the EI-MS data of 5a–c supported the skeletal formulas by displaying the molecular ion peaks [M]⁺ at m/z = 359 (17%), 374 (8%) and 387 (37%), respectively.

Next, thia-Michael 1,4-conjugate addition has been shown to be an extremely useful technique in the chemical synthesis of organosulfur compounds [75]. As a synthetic utility of thia-Michael, the reaction between our Michael donor 4 and electrophilic di-ester called dimethyl acetylenedicarboxylate (DMAD) or diethyl acetylenedicarboxylate (DEAD) as a Michael acceptor, followed by cyclization via nucleophilic acyl substitution reaction between NH and ester groups, afforded the 4-thiazolone derivatives 6a,b in quantitative yields. Glancing at the ¹H NMR spectra of 6a,b at δ_H = 6.84 and 6.79 ppm, we find singlet signals that must be assigned to the vinyl protons in these molecules respectively. Moving to the aliphatic region, the methoxy group signal of 6a resonated with high intensity at δ_H = 3.84 ppm. The ethoxy group of 6b appears as correlated two signals at δ_H = 4.29 (q, CH₂) and 1.30 (t, CH₃) ppm with the same J value (7.1 Hz). In the ¹³C NMR spectra of 6a,b, the ¹³C chemical shifts of carbonyl amide and ester are closely resonated at δ_C ranging from 166.49 to 164.50 ppm, while the methoxy ester of 6a is revealed at δ_C = 53.05 ppm. The ¹³C resonance of the ethoxy group of 6b is manifested at δ_C = 61.91 (CH₂) and 14.47 (CH₃) ppm. The C=O stretching bands in the IR spectra are strongly absorbed around 1712 (COOR) and 1692 (thiazolidinone) cm^-1. In addition, the EI-MS spectra validated the chemical structure of 6a,b by exhibiting the molecular ion [M]⁺ peaks at m/z = 429 (34%) and 443 (54%), respectively.

Following that, triethylamine (Et₃N)-catalyzed the functionalization of phenylthiosemicarbazone 4 by treatment with easily accessible ethyl 2-chloro-2-(2-(p-tolyl)hydrazineylidene)acetate [55,76] in refluxing dioxane to obtain the corresponding hydrazineylidene thiazolidin-4-one 7 in a high yield (88%) (Figure 2). The preparation of 7 was conducted via the proposed mechanistic routes that were reported in the literature [55,77]. The 1D NMR of 7 shows the hydrazineylidene NH at δ_H = 10.64 ppm, while in the up-field region, the methyl group of the p-tolyl fragment is resonated at δ_H = 2.26 ppm, and its corresponding ¹³C resonance appears at δ_C = 20.39 ppm. The IR solid spectrum of 7 as a KBr disc shows the distortion of NH and C=O bands that could be due to hydrogen bond formation.

Also, the reaction between 4 and maleic anhydride proceeds according to the Michael reaction to afford the corresponding Michael adduct intermediate which transformed to 1,3,4 thiadiazole derivative 8 in a good yield (87%) (Figure 2). The reaction proceeds via [4+2] cycloaddition between our Michael donor 4 and dienophile maleic anhydride as a Michael acceptor (α,β-unsaturated carbonyl compound) which transformed to 8, through the elimination of maleic anhydride and, cyclization through the 1,3-proton shift, followed by dehydrogenation (Supplementary Figure S1) [78].

After our success in the synthesis of new 4-thiazolones, we turned our attention to preparing some substituted thiazoles by the same analogy. Thus, when 4 was treated with α-halo divergent reagents (chloroacetone, ethyl 2-chloro-3-oxobutanoate and chloroacetonitrile) in dioxane containing sodium acetate [24], the corresponding thiazoles (9a–c) were obtained in excellent yields (Figure 3). The 1D NMR spectra of 9a demonstrates the chemical shift of the thiazolyl methyl group at δ_H = 1.88 and δ_C = 15.09 ppm, while the thiazolyl–CH is resonated at δ_H = 6.24 and δ_C = 98.27 ppm. In the aliphatic region, the ¹H NMR spectrum of 9b exhibits the ethoxy protons at δ_H = 4.27 (q, J = 7.1 Hz, CH₂) and 1.29 (t, J = 7.1 Hz, CH₃). The IR spectrum of 9b shows the carbonyl ester absorption band at 1726 cm^-1. The EI-MS analysis of 9c supported its skeletal structure by displaying the molecular ion peaks at m/z = 358 [M]⁺ (27%) and 360 [M+2]⁺ (15%) that owing to the presence of primary and tertiary amine groups [24].

Also, hydrazineylidene-2,3-dihydrothiazole 10 was installed in the same manner as 7. Whereas treatment of 4 with an equimolar amount of 2-oxo-N-phenylpropanehydrazonoyl chloride in the dioxane–TEA system [55] furnished 10 in a good yield (88%) (Figure 3). The EI-MS spectrum validated the chemical structure of derivative 10 by exhibiting its molecular ion peak at m/z = 461 [M]⁺ (20%). Thiazole-containing sulfonamides are potent bioactive small-molecules endowed with distinct inhibitory activities against several target enzymes such as carbonic anhydrase (as antiglaucoma, anticancer, etc.) [19], SMYD3 (as anticancer) [79], aldose reductase (as an antidiabetic cataract) [22] and acetylcholinesterase with butyrylcholinesterase (as anti-Alzheimer's) [18]. In addition, thiazole-based sulfonamides serve as promising antimicrobial agents with low MICs and superior activity as antibiofilms against multidrug-resistant organisms (MDROs) [80]. Thus, 4 was treated with 4-(2-bromoacetyl)benzenesulfonamide in refluxing dioxane containing basic sodium acetate [24] to yield 11 in excellent yield (92%) (Figure 3). The reaction was conducted via a three-step cascade S_N2-nucleophilic addition-condensation process [55]. The 1D NMR spectra of 11 manifest the chemical shift of the thiazolyl–CH at δ_H = 6.86 and δ_C = 118.26 ppm. The IR measurement of 11 shows the stretching vibration bands of the SO₂ group at 1365 and 1128 cm^-1. The EI-MS stick diagram of 11, the line created by the heaviest ion flowing via the instrument at m/z = 498 [M]⁺ (20%) correlates with the parent ion (C₂₇H₂₂N₄O₂S₂).

3-(Bromoacetyl)coumarins are versatile precursors to access a variety of bioactive and polyfunctionalized heterocyclic organic molecules [81]. Therefore, we devoted our attention to 3-(bromoacetyl)coumarin as a useful synthon to construct thiazole derivative 12 in the same manner as 11. In the presence of a dioxane-CH₃COONa medium [24], a new thiazolyl-coumarin derivative 12 was obtained in excellent yield (94%). Owing to the coumarin moiety, the aromatic region became crowded in the NMR data. The stretch absorption band of coumarinyl C=O is vibrated at 1725 cm^-1 in the IR spectrum. Furthermore, the EI-MS analysis of 12 supported the chemical architecture by displaying a molecular ion peak at m/z = 487 [M]⁺ (38%).

3.3. Biological evaluation

3.3.1. Evaluation of antibacterial activity

3.3.1.1. Antibacterial susceptibility testing

The synthesized compounds were evaluated for their antibacterial activities against four pathogenic strains using the agar well diffusion method. The strains included Staphylococcus aureus ATCC 33592 and Staphylococcus epidermidis ATCC 35984, which are representatives of Gram-positive bacteria, and Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 4352, which are representatives of Gram-negative bacteria [40,56,57] (Supplementary Table S2). The standard antibiotic utilized in the study was Ofloxacin (OFX) from Oxoid. In addition, DMSO was employed as a solvent control. To ensure statistical significance, we conducted the experiment in triplicate. The recorded values for the inhibition zones were expressed as the mean ± standard error (SE). Compounds 11 and 12 showed antibacterial activity against S. aureus, with inhibition zones measuring 1.4 ± 0.3 and 1.2 ± 0.3 cm, respectively. These results were compared with a standard OFX, which showed an inhibition zone of 4.2 ± 0.3 cm. Compounds 4 and 11 demonstrated antibacterial activity against S. epidermidis, with inhibition zones measuring 0.9 ± 0.2 and 1.0 ± 0.3 cm, respectively compared with OFX which displayed an inhibition zone of 4.1 ± 0.2 cm. Compounds 4 and 12 had lower antibacterial activity against E. coli, with inhibition zones of 0.9 ± 0.3 and 0.9 ± 0.2 cm, respectively, compared with OFX with inhibition zone = 4.3 ± 0.3 cm. Compound 4 was the only compound to show antibacterial activity against K. pneumoniae with an inhibition zone of 1.0 ± 0.1 compared with 3.9 ± 0.2 cm for OFX. The control solvent, DMSO, did not show any antibacterial activity against the used strains.

3.3.1.2. Minimum inhibitory concentration & minimum bactericidal concentration

To evaluate the effectiveness of an antibacterial agent against a particular strain of bacteria, in vitro MIC (μg/ml) and MBC (μg/ml) assays are typically utilized. While MBC is the lowest dosage needed to kill 99.9% of a particular strain of bacteria, MIC is the lowest concentration of an antibacterial drug that will stop observable bacterial growth during an overnight incubation measurement [41,57]. MIC and MBC were established for derivatives 4, 11 and 12 that inhibited the growth of any of the tested bacterial strains (Supplementary Table S3). Compound 11 with benzenesulfonamide moiety and the coumarin derivative 12 scored the same MIC value of 256 μg/ml and MBC value (>256) μg/ml against S. aureus. Also, 4-phenylthiosemicarbazone derivative 4 scored MIC and MBC values >256 μg/ml against S. epidermidis, E. coli and K. pneumoniae. Similarly, compounds 11 and 12 scored MIC and MBC values >256 μg/ml against S. epidermidis, and E. coli, respectively.

3.3.2. Evaluation of the anticancer activity

3.3.2.1. Antiproliferative activity on 60 cancer cell lines

The cytotoxic activity of the synthesized compounds, including 4, 5a–c, 6a,b, 7, 8, 9a–c and 10–12, was estimated at a single dosage of 10 μM. The evaluation was conducted using 60 different human tumor cell lines under the Developmental Therapeutic Program (DTP) of the National Cancer Institute (NCI) Therapeutic Program [58–61]. The NCI-60 panel includes 60 cancer cells demonstrating various histology. The antiproliferative activity is presented as a growth inhibition percentage (GI%) achieved by the examined compounds. The results are shown in Supplementary Tables S4 & S5.

Substituted phenylthiosemicarbazide 4, the starting material for all the new derivatives showed low cytotoxic activity with a mean GI% of 2.75. The heterocyclization of 4 to yield the thiazolidin-4-one derivatives 5a-c largely improved the cytotoxic activity only for 5a which is unsubstituted thiazolone at C5. Compound 5a showed a mean GI of 16.67% and exhibited moderate antiproliferative activity against leukemia HL-60(TB); CNS (SNB-19, SNB-75 and U251); ovarian cancer OVCAR-8; renal (ACHN, and CAKI-1) cancer cells with GI% ranging between 32.24 and 53.01%. Compound 5c with 5-ethyl-thiazolidin-4-one showed low anticancer activity with GI = 3.71% and showed moderate inhibition with seven tumor cell lines with a GI% range of 31.82–58.24. While compound 5b with methyl at C5 failed to have any antiproliferative activity with mean GI% <0.

Cyclization of compound 4 into the substituted methyl 4-oxo-3-phenylthiazolidin-5-ylidene acetate 6a yielded excellent inhibitory activity with a mean GI of 51.18%. It showed excellent inhibitory activities against Non-Small Cell Lung Cancer (A549/ATCC (74.85%), HOP-92 (81.56%), NCI-H226 (80.65%) and NCI-H460 (76.48%)); CNS cancer (SNB-19 [77.33%] and U251 [82.04%]); ovarian cancer (OVCAR-4 [98.81%] and OVCAR-8 [85.35%]); renal (CAKI-1 [90.69%], SN12C [86.09%] and UO-31 [90.84%]) and breast cancer HS 578T (82.63%). It also showed moderate antiproliferative activity against 32 cancer cell lines from all panels with a GI% range of 32.12–68.26. It also showed a lethal effect against CNS cancer SNB-75 and renal cancer ACHN with GI% above 100. On the other hand, compound 6b bearing ethyl ester showed low inhibitory activity with a mean GI of 1.33%.

The substituted thiazoles 9a–c, 11 and 12 showed an improvement in anticancer activity according to the position of the substituents. Compound 9a exhibited a mean growth inhibition of 17.14% and showed antiproliferative activity against 16 cancer cell lines with a GI range of 32.16–54.91.

The presence of 4-benzenesulfonamide moiety in derivative 11 and 4-coumarin moiety in compound 12 improved the anticancer activity. Compound 12 exhibited a mean GI of 16.25% and showed moderate antiproliferative activity against 9 cancer cell lines with a GI% range of 31.65–57.06, while compound 11 displayed a mean GI of 7.68% and By examining the structural variations and antiproliferative effects of the new derivatives, it was found that the presence of a thiazol-4-one ring with a methyl acrylate at C5 yielded the most active compound 6a with a mean GI% of 51.18. and a detailed structure activity relationship is presented in Figure 4.

Figure 4.

The structure–activity relationship for the synthesized compounds.

3.3.2.2. Measurement of IC₅₀ of compound 6a against different cell lines

Based on the inhibition percentage obtained from the NCI single-dose screening, the antiproliferative activity of compound 6a was determined against the ovarian cell line OVCAR4, the renal cell line CAKI-1, and the breast cell line HS 578T. We used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) colorimetric test method, following Mosmann's instructions [62]. Staurosporine was used as a reference drug in these experiments. The data were presented as IC₅₀, which is the measure of the concentration needed to inhibit 50% of cell growth after 72 h of incubation. The IC₅₀ values were determined using the concentration–inhibition response curve and are listed in Table 1 Evaluation of the cytotoxic activity uncovered that 6a showed the highest activity against the OVCAR4 cell line with an IC₅₀ of 1.569 ± 0.06 μM versus staurosporine with IC₅₀ 8.76 ± 0.33. Also, compound 6a showed lower activity with IC₅₀ values of 2.71 ± 0.16 and 8.72 ± 0.46 μM against renal cell line CAKI-1 and breast cell line HS 578T, respectively compared with staurosporine with IC₅₀ 7.37 ± 0.23 and 14.25 ± 0.77 μM, respectively. Compound 6a exhibited the highest activity against ovarian cancer among the tested cell lines. To evaluate the potential toxicity of 6a on normal human epithelial cell line OCE1, the selectivity index (SI) was determined, and the findings are presented in Table 1. The OCE1 human epithelial cell line was used in the investigation on selectivity in the estimation of SI, and staurosporine was used as a positive control for the calculation of SI. The ratio of cytotoxicity (IC₅₀) on the normal ovarian cell line (OCE1) to the ovarian cancer cell line (OVCAR-4) was calculated. Compound 6a was compared with staurosporine, which had an IC₅₀ of 11.77 ± 0.65 μM against OCE1 cells and an SI of 1.34. Compound 6a had an IC₅₀ of 31.89 ± 1.19 μM against OCE1 normal cells but showed great selectivity to cancer cells (SI = 19.1).

Table 1. In vitro assessment of antiproliferative effects on cancer and normal ovarian cell lines, IC₅₀ inhibition against PI3Kα and calculation of selectivity index.

Compound	OVCAR-4	CAKI-1	HS 578T	OCE1	PI3Kα	SI
6a	1.67 ± 0.06	2.71 ± 0.16	8.72 ± 0.46	31.89 ± 1.19	0.225 ± 0.01	19.1
Staurosporine	8.76 ± 0.33	7.37 ± 0.23	14.25 ± 0.77	11.77 ± 0.65	–	1.34
Alpelisib	–	–	–	–	0.061 ± 0.003	–

SI: Selectivity index.

3.3.2.3. In vitro enzymatic PI3Kα inhibitory assay

This study aimed to elucidate the cytotoxicity mechanism of the most active compound 6a. Based on their structural similarity to alpelisib, a selective PI3Kα inhibitor, compound 6a was tested to evaluate its ability to inhibit PI3Kα and alpelisib is used as a reference control. The results showed that compound 6a had an IC₅₀ value of 0.225 ± 0.01 μM, while alpelisib had an IC₅₀ value of 0.061 ± 0.003 μM (as shown in Table 1). The findings suggest that compound 6a is a potential antiproliferative agent and PI3Kα inhibitor.

3.3.2.4. Assessment of the effect of compound 6a on Akt/mTOR pathway

The PI3K/Akt/mTOR pathway plays a crucial role in promoting cancer progression by facilitating cell division, migration, angiogenesis and metastasis. To understand the molecular mechanism behind the induction of apoptosis in cancer cells by compound 6a and identify the targets of the significant signaling pathway, the PI3K/Akt/mTOR pathway was studied. In the OVCAR-4 cell line of ovarian cancer, compound 6a was evaluated at its IC₅₀ concentration (1.67 μM) to directly inhibit Akt/mTOR phosphorylation. The results revealed that compound 6a strongly inhibits AKT phosphorylation by 76.2% compared with the control OVCAR-4 cells (Figure 5), meanwhile, staurosporine inhibited AKT phosphorylation by 71.9%. Moreover, compound 6a inhibits mTOR phosphorylation by 81.2%, while staurosporine inhibits mTOR by 72.5%. These findings demonstrate that compound 6a specifically targets Akt and mTOR protein phosphorylation.

Figure 5.

A Graph revealing the impact of compound 6a on phosphorylation of Akt/mTOR pathway in OVCAR-4 cells in comparison to staurosporine and control cells.

3.3.2.5. Cell cycle analysis

Anticancer drugs work by blocking cellular proliferation at identifiable checkpoints, thereby causing cytotoxicity. These checkpoints are different phases of the cell cycle that, when suppressed, lead to the termination of cell proliferation. Flow cytometry is used in cell cycle examinations to recognize cells in various cell cycle phases [64]. To investigate the effects of compound 6a on OVCAR-4 cell cycle development and apoptosis initiation, it was applied to OVCAR-4 cells at its IC₅₀ value (1.67 μM) for 24 h, and the effects on the cell cycle profile and apoptosis induction were assessed. The results showed that exposure to compound 6a disrupted the normal cell cycle distribution of OVCAR-4 cells. According to Figure 6, after exposure to compound 6a, OVCAR-4 cells decreased from 62.03 to 54.79% (11.67% reduction) in the G0/G1, and from 21.73 to 16.31% (24.9% reduction) at the S phase. Additionally, the percentage of cells in the G2/M phase increased by 1.77-fold compared with the control (from 16.24 to 28.9%).

Figure 6.

Effect of compound 6a on cell cycle stages and apoptosis in OVCAR-4 cells. (A) Graphical chart of the influence of 6a on the cell cycle of OVCAR4 compared with the control cells, (B) Control OVCAR-4 cells, (C) Influence of 6a (1.67 μM) on DNA-ploidy flow cytometric analysis of ovarian OVCAR-4 cells after 24 h. (D) Chart for apoptotic and necrotic cells in treated ovarian OVCAR-4 cells and untreated control cells, (E) Control OVCAR-4 cells, (F) OVCAR-4 cells treated with compound 6a.

3.3.2.6. Apoptosis evaluation

An effective assay for evaluating necrosis and apoptosis is the Annexin V-based flow cytometry. The potential for compound 6a to induce apoptosis was determined by cytofluorimetric evaluation using propidium iodide (PI), which dyes DNA and only enters dead cells. After interacting with the fluorochrome-labeled Annexin-V, Annexin-V binds to phosphatidylserine (PS) expressed only on the surface of the apoptotic cells and fluoresces green. Meanwhile, PI only enters dead cells and stains DNA [65,82]. Distinguishing between living cells, early apoptotic cells, late apoptotic cells and necrotic cells can be achieved through dual labeling for Annexin-V and PI. To estimate its effects on the OVCAR-4 cell cycle, Compound 6a was selected and studied. After treatment of OVCAR-4 cells with compound 6a at its IC₅₀ (1.67 μM) for 24 h, a decrease in the proportion of surviving cells was observed, as depicted in Figure 6. There was a marked increase in the late apoptosis stage from 0.2 to 14.43% compared with control cells (68.7-fold greater than control). and an increase in the early apoptosis stage from 0.29 to 26.41% (101.4-fold greater than control). Furthermore, this led to an increase in the total apoptotic percentage from 1.76 to 47.23% (26.8-fold greater than control).

3.3.2.7. Evaluation of caspase-3 activation

Using the ovarian OVCAR-4 cell line, the level of active caspase-3 was determined. When the ovarian cells were treated with compound 6a at its IC₅₀ value of 1.67 μM, it resulted in an increase of 4.34-fold in active caspase-3 levels in comparison to the untreated control cells. Likewise, staurosporine also increased the caspase-3 level by 5.91-fold compared with the control cells, as shown in Supplementary Figure S2.

3.4. Molecular docking

A flexible molecular docking simulation was then run to acquire insight into the interactions and binding mode of the bioactive N-phenyl-4-thiazolone derivative 6a and alpelisib (NVP-BYL719, a reference drug) into the ATP binding domain of PI3Kα kinase (Protein Data Bank [PDB] ID: 4JPS) as an important targeted therapy. The iGEMDOCK program version 2.1 [7,83,84] was manipulated to perform molecular docking, and the results are shown in Supplementary Table S6. The 3D structure of PI3Kα complexed with alpelisib (ligand ID: 1LT) was downloaded from RCSB Protein Data Bank (www.rcsb.org) and the reference ligand 1LT was extracted and re-docked to the corresponding active site, to validate the ability of docking protocol to reproduce the binding mode of the inhibitor observed in the crystal structure (iGEMDOCK validation) (Figure 7). The docked ligand (1LT) displays the same binding mode as the crystal one and the Root Mean Square Distance (RMSD) was within the reliable range (0.543 Å), validating the strength of our protocol.

Figure 7.

The interactions and binding mode of compound 6a and alpelisib into the ATP binding domain of PI3Kα kinase. (A) iGEMDOCK validation. Crystal NVP-BYL719 (green sticks) and the re-docked one (gray sticks) are superimposed and show similar binding orientation in the ATP pocket of PI3Kα (PDB ID: 4JPS) with RMSD equals to 0.543 Å. Fitting into the ATP pocket (B & E), 2D interactions (C & F) and 3D interactions (D & G) of 6a and alpelisib within the binding site of PI3Kα (PDB ID: 4JPS). Compound 6a is displayed as grey balls and sticks, while alpelisib is shown in green. H-bonds are indicated by dashed lines in green. All images were produced with Discovery Studio Visualizer Client 2020 and are simple for clarity of presentation.

PDB: Protein Data Bank.

As presented in Supplementary Table S6 & Figure 7, compound 6a that bears an N-phenyl-4-thiazolone moiety is aligned in the ATP pocket of PI3Kα with a fitness value of -119.153 kcal/mol slightly lower than alpelisib (-124.453 kcal/mol) and through different intermolecular forces, including conventional H-bonds, Van der Waals (London forces) and hydrophobic interactions. Fragments including N-phenyl-4-thiazolone and the azine linker in 6a play a vital role in the interactions with PI3Kα kinase [85]. The sulfur atom of the thiazolone ring and the nitrogen atom of the azine linker form three H-bonds with Val851 and Ser854. These interactions are similar to those of the marketed reference drug alpelisib [85], confirming the potency of our bioactive compound 6a. Also, the N-phenyl-4-thiazolone unit produced important hydrophobic interactions with Met922 (π–S) and Val850 (π–alkyl), like those formed by the 4-methylthiazole moiety of alpelisib. Thus, we can consider the 4-thiazolone-azine fragment as a bioisostere or alternative for the 2-amino-4-methylthiazole unit of PI3Kα inhibitors, validating 6a as a starting point (lead structure) for a new generation of PI3Kα kinase inhibitors.

Owing to the different terminal direction shift inside the ATP binding pocket, the potency of our bioactive small compound 6a was enhanced by Van der Waals interactions with a set of amino acids, while the activity of alpelisib was enhanced by several water H-bonds through the substituted pyridine moiety. Exhaustively, in silico molecular docking findings are well-matched with in vitro enzymatic assay (6a: 0.225 ± 0.01 μM, alpelisib: 0.061 ± 0.003 μM against PI3Kα) and will guide us to optimize 6a lead structure by nucleophilic substitution reaction of the ester group to incorporate nitrogenous aromatic units, aiming to obtain a new generation of PI3Kα inhibitors with the optimum interaction that results in a completely induced fit.

4. Conclusion

In summary, new naphthalene-azine-thiazole hybrids (5–12) were successfully installed through thiazolation of 4-phenylthiosemicarbazide 4 by reaction with a set of halogenated and unsaturated carbonyl compounds and the synthesized derivatives were evaluated as PI3Kα inhibitors. According to the results, compound 6a exhibited excellent anticancer activity against most of the tumor cell lines that were tested. It showed the highest activity against the OVCAR4 cell line with an IC₅₀ of 1.569 ± 0.06 μM and a promising PI3Kα inhibition with IC₅₀ of 0.225 ± 0.01 μM in comparison to alpelisib. Also, compound 6a showed strong selectivity to ovarian cancer cells with SI = 19.1. Moreover, compound 6a inhibited the phosphorylation of Akt by 76.2% and mTOR by 81.2%. It was demonstrated that compound 6a inhibited the growth of OVCAR-4 cells during the G2/M phase and increased total apoptosis by 26.8-fold. Also, compound 6a increased the level of caspase-3 by 4.34-fold which showed that compound 6a may cause apoptosis through a caspase-dependent mechanism. On the other hand, benzenesulfonamide derivative 11 and the coumarin-containing derivative 12 showed the highest antibacterial activity with MIC value of 256 μg/ml against S. aureus. Molecular docking simulations also confirmed the antiproliferative effect of compound 6a, and it interlocked and fitted well into the ATP binding site of PI3Kα kinase (PDB ID: 4JPS) with a fitness value (-119,153 kcal/mol). These findings propose that compound 6a has cytotoxic properties and validates it as a starting structure for a new generation of PI3Kα kinase inhibitors.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2342668

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The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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