Synthesis, Cytotoxic Activity, mRNA Expression, Total Oxidant and Antioxidant Status, and Molecular Modeling Studies of Novel Schiff Base–Thiosemicarbazone Hybrids

Anber M Anber; Mehmet Özbay; İlhan Özer İlhan; Hüseyin Kekeçmuhammed; Rıfat Emin Bora; Emin Saripinar; Yalçın Erzurumlu; Muhammed Tilahun Muhammed; Sadeq K Alhag; Lamya Ahmed Alkeridis; Senem Akkoc

doi:10.1002/jbt.71140

. 2026 Oct 25;40(11):e71140. doi: 10.1002/jbt.71140

Synthesis, Cytotoxic Activity, mRNA Expression, Total Oxidant and Antioxidant Status, and Molecular Modeling Studies of Novel Schiff Base–Thiosemicarbazone Hybrids

Anber M Anber ^1,², Mehmet Özbay ¹, İlhan Özer İlhan ¹, Hüseyin Kekeçmuhammed ¹, Rıfat Emin Bora ¹, Emin Saripinar ^1,^✉, Yalçın Erzurumlu ³, Muhammed Tilahun Muhammed ⁴, Sadeq K Alhag ⁵, Lamya Ahmed Alkeridis ⁶, Senem Akkoc ^7,^8,^✉

PMCID: PMC13696017 PMID: 42927636

ABSTRACT

Several new Schiff‐based compounds were synthesized, and their chemical structures were confirmed usingFT‐IR, ¹H‐NMR, ¹³C‐NMR, and HRMS. The compounds were tested in the prostate cancer cell line (PC3) for 72 h. The results demonstrated that compounds NZ10, NZ9, NZ7, and NZ4 had high cytotoxic effects on the PC3 cell line with IC₅₀ values of 10.54, 14.58, 15.85, and 16.48 µM, respectively. The modulator roles of selected compounds on apoptotic signaling were tested by their effect on mRNA expression levels of BAX, BID, BCL2, and BCL‐xL using RT‐qPCR. Furthermore, the effects on oxidative stress levels were examined by measuring total oxidant and antioxidant levels in cells. The results revealed that selected compounds significantly upregulated pro‐apoptotic BAX and BID, whereas they downregulated anti‐apoptotic BCL2 and BCL‐xL levels. Besides, compounds were found to significantly increase the oxidative status in cells. The binding potential of relatively active compounds to BCL‐2 was assessed through molecular docking and molecular dynamics (MD) simulations. The docking study demonstrated that compounds had binding potential to the structure but were weaker than the standard drug, venetoclax. MD simulation analysis showed that complexes of the compounds with higher affinity formed stable complexes with the structure.

Keywords: cytotoxic activity, docking, MD simulation, mRNA expression, Schiff base, thiosemicarbazone

In this study, new Schiff base‐derived compounds were synthesized, and their structures were confirmed by FT‐IR, ¹H‐NMR, ¹³C‐NMR, and HRMS analyses. The cytotoxic effects of the compounds on a prostate cancer cell line (PC3) were investigated, and it was determined that the compounds NZ10, NZ9, NZ7, and NZ4, in particular, exhibited potent anticancer activity. Molecular analyses revealed that these compounds activate apoptotic mechanisms by upregulating pro‐apoptotic genes and downregulating anti‐apoptotic genes. Additionally, they were observed to increase oxidative stress levels in cells. Molecular docking and dynamic simulation studies also demonstrated that the active compounds form stable interactions with the BCL‐2 protein, supporting the in vitro results.

graphic file with name JBT-40-e71140-g007.webp

1. Introduction

Schiff bases constitute an important class of compounds exhibiting a broad spectrum of biological activities, including antifungal, antipyretic, antitumor, antiproliferative, and antimicrobial activities [1]. Schiff base is typically manufactured through a condensation reaction between a primary amine and an aldehyde or ketone [2]. Schiff bases, thiosemicarbazones, and their metal complexes have also shown an array of biological activities such as antitumor, antibacterial, and antifungal properties, and the antitumor activity of thiosemicarbazones has been reported to be analogous to that of cisplatin [3].

Thiosemicarbazones (TSCs) represent an important class of bioactive compounds that have been extensively investigated for the treatment of various diseases, including cancers [4]. Moreover, “hybrid” iron chelators incorporating aroyl hydrazones and TSCs have demonstrated selective antiproliferative effects against tumor cells [5].

TSCs have also been reported to exhibit antiviral and anti‐HIV activities [6, 7, 8]. The antiviral activity of amino acid‐based thiosemicarbazides has been evaluated using MTT and plaque formation reduction assays, and these compounds were reported to inhibit dengue virus infection in Vero cells; Bz‐Trp‐TSC is a well‐known thiosemicarbazide derivative investigated for the treatment of dengue fever [9].

Generally, hydrazinecarbothioamide derivatives (HCTA) are commonly synthesized from isothiocyanates and hydrazides or hydrazine. Hydrazinecarbothioamides are biologically active intermediates with various applications in the synthesis of other scaffolds [10, 11, 12, 13, 14, 15, 16].

The 4‐thiazolidinone ring represents a valuable structural motif used as a promising building block in modern medicinal chemistry for rational drug design and development [17]. 4‐Thiazolidinone derivatives have been reported to exhibit diverse biological activities, including anti‐inflammatory, analgesic, trypanocidal, anticonvulsant, antiviral, antifungal, antibacterial, and anticancer activities [18, 19, 20, 21, 22]. Therefore, due to their broad range of pharmacological activities, thiazolidinone derivatives have attracted considerable attention [23]. The presence of a 2,4‐dioxothiazolidine ring has been reported in anticonvulsant drugs used for diabetes treatment, and several heterocyclic compounds have been observed to play significant roles in the chemotherapy of malignancy [24]. Although 2,4‐dioxothiazolidines are considered a potential platform because they can stimulate PPARγ [25], they have also been reported to exert multiple PPARγ‐independent effects, and the dose required for anticancer activity is significantly lower than that required for hypoglycemic activity. In addition, 2,4‐dioxothiazolidines exhibit a broad spectrum of pharmacological activities, including anti‐inflammatory effects, and have been used in the treatment of neurological diseases such as Parkinson's and Alzheimer's disease [26].

As part of our ongoing research on the synthesis of Schiff base derivatives and the evaluation of their biological activity [27, 28, 29], in this work, we designed and synthesized several series of novel compounds whose core structure is based on Schiff base, coupled with thiosemicarbazone‐derivative units or a 4‐thiazolidinone ring. Most of the synthesized compounds were subjected to biological evaluation, including cytotoxic activity testing, gene expression analysis using RT‐qPCR, and antioxidant status assessment.

2. Materials and Methods

2.1. Chemistry

All chemicals and solvents that were used in this work were obtained from Sigma Aldrich and Merck. The purity of the compounds and reaction time were controlled on TLC with a fluorescent indicator visualizable at 254 and 365 nm. ¹H NMR (400 MHz) spectra and ¹³C NMR (100 MHz) spectra were recorded on a Bruker AM 400 MHz NMR spectrometer with DMSO‐d₆ as solvent at Erciyes University Technology Research and Application Center (TAUM). Chemical shifts were reported in δ ppm using residual solvent protons as an internal standard (δ 7.26 for CDCl₃ and δ 2.50 for DMSO‐d₆ in ¹H NMR, δ 77.16 for CDCl₃ and δ 39.52 for DMSO‐d ₆ in ¹³C NMR). Coupling constants were reported as J values in Hertz (Hz).

2.2. Synthesis of Compounds

The molecules were synthesized in four steps. In the first step, hydrazine hydrate (2 mol) and isothiocyanate derivatives (1 mol) were stirred in acetonitrile at room temperature for 5 h. The reaction was monitored with TLC. After the reaction was completed, pure water was added, and the resulting precipitate was filtered. The N‐arhydrazinecarbothioamide derivatives were used in the next step without any purification.

In the second step, isatin (1 mol) and propargyl bromide 80% (1 mol) in DMF and K₂CO₃ (1.2 mol) were stirred at room temperature for 2 days. The reaction was followed by TLC, and after the reaction was completed, extraction was performed with ethyl acetate (EtOAc/brine). The organic phase was dried with Na₂SO₄, and the solvent was removed on the rotary evaporator. The obtained 1‐(prop‐2‐(in‐1‐yl)indoline‐2,3‐dione was used in the next step without any purification.

In the third step, the HCTA (1 mol) and isatin (1 mol) synthesized in the first two steps were refluxed in methanol for 8 h, the reaction was monitored with TLC, the precipitate was filtered and washed with hot methanol, and obtained N.Z derivatives. In the same way, HCTA (1 mol) and 1‐(prop‐2‐(in‐1‐yl)indoline‐2,3‐dione (1 mol) were synthesized and obtained Y.Z derivatives.

In the fourth step, the N.Z. derivatives (1 mmol) and dimethylacetylene dicarboxylate DMAD (1–2 mmol) synthesized in the third step were refluxed in methanol for 8–12 h. The reaction was controlled by TLC, and the precipitate was filtered and washed with hot methyl alcohol to purify the N.Z.A oxathiazolidine derivatives. In the same way, Y.Z derivatives (1 mmol) and dimethylacetylene dicarboxylate DMAD (1–2 mmol) reacted in methanol for 8–12 h, and the precipitate was filtered and washed with hot methanol to obtain the Y.Z.A oxathiazolidine derivatives. The ¹H NMR, ¹³C NMR, FT‐IR, and HR‐MS spectra of the compounds are provided in the supporting information file (Figures S2–S87).

2.3. N‐(4‐Nitrophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ1)

Orange solid. Yield: 75%. M.p.: 274°C–275°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 13.00 (s, 1H, NH), 11.29 (s, 1H, NH), 11.10 (s, 1H, NH), 8.30 (d, J = 8.6 Hz, 2H, Ar‐H), 8.08 (d, J = 8.7 Hz, 2H, Ar‐H), 7.78 (d, J = 7.7 Hz, 2H, Ar‐H), 7.40 (t, J = 7.7 Hz, 1H, Ar‐H), 7.14 (d, J = 7.6 Hz, 2H, Ar‐H), 6.96 (d, J = 8.0 Hz, 1H, Ar‐H).

2.4. N‐(4‐Methoxyphenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ3)

Yellow solid. Yield: 90%. M.p.: 255°C–256°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.77 (s, 1H, NH), 11.29 (s, 1H, NH), 10.78 (s, 1H, NH), 7.80 (d, J = 4.8 Hz, 1H, Ar‐H), 7.49 (d, J = 10.0 Hz, 2H, Ar‐H), 7.41 (d, J = 6.6 Hz, 1H, Ar‐H), 7.15 (d, J = 7.0 Hz, 1H, Ar‐H), 7.05–6.95 (m, 3H, Ar‐H), 3.82 (s, 3H, O‐CH₃).

2.5. N‐(4‐Chlorophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ4)

Yellow solid. Yield: 88%. M.p.: 257°C–258°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.84 (s, 1H, NH), 11.28 (s, 1H, NH), 10.87 (s, 1H, NH), 7.76 (d, J = 7.3 Hz, 1H, Ar‐H), 7.66 (d, J = 8.7 Hz, 2H, Ar‐H), 7.48 (d, J = 11.6 Hz, 2H, Ar‐H), 7.38 (t, J = 7.7 Hz, 1H, Ar‐H), 7.12 (t, J = 7.6 Hz, 1H, Ar‐H), 6.95 (d, J = 7.7 Hz, 1H, Ar‐H).

2.6. N‐(3‐Methoxyphenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ5)

Yellow solid. Yield: 84%. M.p.: 229°C–230°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.81 (s, 1H, NH, NH), 11.27 (s, 1H, NH), 10.78 (s, 1H, NH), 7.79 (d, J = 6.4 Hz, 1H, Ar‐H), 7.42–7.27 (m, 3H, Ar‐H), 7.24 (d, J = 8.6 Hz, 1H, Ar‐H), 7.18–7.08 (m, 1H, Ar‐H), 6.95 (d, J = 7.4 Hz, 1H, Ar‐H), 6.85 (d, J = 8.3 Hz, 1H, Ar‐H), 3.79 (s, 3H, O‐CH₃).

2.7. N‐(Naphthalen‐1‐yl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ6)

Yellow solid. Yield: 93%. M.p.: 277°C–278°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.90 (s, 1H, NH), 11.29 (s, 1H, NH), 11.15 (s, 1H, NH), 8.03 (s, 1H, Ar‐H), 7.97 (d, J = 6.7 Hz, 2H, Ar‐H), 7.89 (s, 1H, Ar‐H), 7.78 (s, 1H, Ar‐H), 7.65–7.52 (m, 4H, Ar‐H), 7.39 (d, J = 5.5 Hz, 1H, Ar‐H), 7.12 (d, J = 4.9 Hz, 1H, Ar‐H), 6.97 (d, J = 5.8 Hz, 1H, Ar‐H).

2.8. N‐(2,6‐Difluorophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ7)

Orange solid. Yield: 86%. M.p.: 228°C–229°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.86 (s, 1H, NH), 11.29 (s, 1H, NH), 10.89 (s, 1H, NH), 7.78 (d, J = 11.0 Hz, 2H, Ar‐H), 7.65 (d, J = 7.8 Hz, 1H, Ar‐H), 7.52–7.42 (m, 1H, Ar‐H), 7.37 (dt, J = 13.5, 7.7 Hz, 1H, Ar‐H), 7.16–7.08 (m, 1H, Ar‐H), 6.95 (d, J = 7.8 Hz, 1H, Ar‐H).

2.9. N‐(4‐Ethylphenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ9)

Yellow solid. Yield: 85%. M.p.: 243°C–244°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.78 (s, 1H, NH), 11.27 (s, 1H, NH), 10.78 (s, 1H, NH), 7.78 (d, J = 7.7 Hz, 1H, Ar‐H), 7.50 (d, J = 7.0 Hz, 2H, Ar‐H), 7.39 (d, J = 6.2 Hz, 1H, Ar‐H), 7.27 (d, J = 7.1 Hz, 2H, Ar‐H), 7.12 (t, J = 6.6 Hz, 1H, Ar‐H), 6.95 (d, J = 8.6 Hz, 1H, Ar‐H), 2.69–2.58 (m, 2H, CH₂), 1.21 (t, J = 6.6 Hz, 3H, CH₃).

2.10. N‐(2‐Bromophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ10)

Yellow solid. Yield: 78%. M.p.: 249°C–250°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.86 (s, 1H, NH), 11.28 (s, 1H, NH), 10.88 (s, 1H, NH), 7.82–7.74 (m, 2H, Ar‐H), 7.65 (d, J = 8.2 Hz, 1H, Ar‐H), 7.50–7.31 (m, 3H, Ar‐H), 7.12 (td, J = 7.7, 2.8 Hz, 1H, Ar‐H), 6.95 (dd, J = 7.9, 2.8 Hz, 1H, Ar‐H).

2.11. 2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)‐N‐Phenylhydrazine‐1‐Carbothioamide (YZ1)

Yellow solid. Yield: 88%. M.p.: 244°C–246°C.¹H NMR (400 MHz, DMSO‐d₆) δ 12.58 (s, 1H, NH), 10.90 (s, 1H, NH), 7.86 (d, J = 7.5 Hz, 1H, Ar‐H), 7.62 (d, J = 7.6 Hz, 2H, Ar‐H), 7.47 (dt, J = 24.9, 7.8 Hz, 3H, Ar‐H), 7.25 (dq, J = 14.6, 7.5 Hz, 3H, Ar‐H), 4.66 (s, 2H, CH₂).

2.12. N‐(4‐Methoxyphenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ2)

Yellow solid. Yield: 90%. M.p.: 216°C–218°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.56 (s, 1H, NH), 10.83 (s, 1H, NH), 8.03–7.80 (m, 1H, Ar‐H), 7.56–7.43 (m, 3H, Ar‐H), 7.30–7.20 (m, 2H, Ar‐H), 7.04–6.95 (m, 2H, Ar‐H), 4.67 (s, 2H, CH₂), 3.80 (s, 3H, CH₃), 3.51 (s, 1H, CH).

2.13. N‐(4‐Chlorophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ4)

Yellow solid. Yield: 90%. M.p.: 198°C–200°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.63 (s, 1H, NH), 10.95 (s, 1H, NH), 7.85 (d, J = 6.7 Hz, 1H, Ar‐H), 7.67 (d, J = 7.9 Hz, 2H, Ar‐H), 7.50 (d, J = 7.2 Hz, 3H, Ar‐H), 7.24 (d, J = 12.9 Hz, 2H, Ar‐H), 4.66 (s, 2H, CH₂), 3.49 (s, 1H, CH).

2.14. N‐(3‐Methoxyphenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ5)

Orange solid. Yield: 83%. M.p.: 209°C–210°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.59 (s, 1H, NH), 10.85 (s, 1H, NH), 7.87 (d, J = 7.5 Hz, 1H, Ar‐H), 7.50 (t, J = 7.8 Hz, 1H, Ar‐H), 7.28 (ddd, J = 34.0, 15.6, 7.9 Hz, 5H, Ar‐H), 6.86 (d, J = 8.1 Hz, 1H, Ar‐H), 4.66 (s, 2H, CH₂), 3.78 (s, 3H, OCH₃), 3.43 (s, 1H, CH).

2.15. N‐(Naphthalen‐1‐yl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ6)

Yellow solid. Yield: 91%. M.p.: 279°C–280°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.70 (s, 1H, NH), 11.24 (s, 1H, NH), 8.05–7.96 (m, 2H, Ar‐H), 7.87 (d, J = 8.3 Hz, 2H, Ar‐H), 7.67–7.49 (m, 5H, Ar‐H), 7.33–7.20 (m, 2H, Ar‐H), 4.70 (s, 2H, CH₂), 3.41(s, 1H, CH).

2.16. N‐(2,6‐Difluorophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ7)

Yellow solid. Yield: 77%. M.p.: 197°C–198°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.65 (s, 1H, NH), 10.95 (s, 1H, NH), 7.86 (d, J = 6.9 Hz, 1H, Ar‐H), 7.80 (s, 1H, Ar‐H), 7.66 (d, J = 7.2 Hz, 1H, Ar‐H), 7.48 (dd, J = 17.1, 8.7 Hz, 2H, Ar‐H), 7.35 (d, J = 7.5 Hz, 1H, Ar‐H), 7.26 (d, J = 5.7 Hz, 1H, Ar‐H), 4.66 (s, 2H, CH₂), 3.56 (s, 1H, CH).

2.17. N‐(4‐Fluorophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ8)

Yellow solid. Yield: 87%. M.p.: 224°C–225°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.62 (s, 1H, NH), 10.91 (s, 1H, NH), 8.06–7.99 (m, 1H, Ar‐H), 7.85 (d, J = 7.6 Hz, 1H, Ar‐H), 7.72 (d, J = 7.6 Hz, 1H, Ar‐H), 7.64 (d, J = 7.2 Hz, 1H, Ar‐H), 7.51 (t, J = 7.7 Hz, 1H, Ar‐H), 7.29–7.17 (m, 3H, Ar‐H), 4.66 (s, 2H, CH₂), 3.33 (s, 1H, CH).

2.18. N‐(4‐Ethylphenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ9)

Yellow solid. Yield: 89%. M.p.: 224°C–225°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.56 (s, 1H, NH), 10.85 (s, 1H, NH), 7.85 (d, J = 7.0 Hz, 2H, Ar‐H), 7.50 (d, J = 7.8 Hz, 3H, Ar‐H), 7.25 (dd, J = 11.4, 7.9 Hz, 4H, Ar‐H), 4.66 (s, 2H, CH₂), 3.54 (s, 1H, CH), 2.68–2.58 (m, 2H, CH₂), 1.20 (t, J = 7.5 Hz, 3H, CH₃).

2.19. N‐(2‐Bromophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ10)

Yellow solid. Yield: 79%. M.p.: 203°C–204°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 12.75 (s, 1H, NH), 10.94 (m, 1H, Ar‐H), 8.23 (s, 1H, Ar‐H), 7.85–7.76 (m, 1H, Ar‐H), 7.65 (d, J = 8.2 Hz, 1H, Ar‐H), 7.56–7.40 (m, 1H, Ar‐H), 7.38–7.30 (m, 2H, Ar‐H), 7.29–7.23 (m, 1H, Ar‐H), 7.18 (dd, J = 12.9, 7.5 Hz, 1H, Ar‐H), 5.56 (s, 2H, CH₂), 5.05 (s, 1H, CH).

2.20. Methyl 2‐(3‐(4‐Nitrophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.1)

Orange solid. Yield: 80%. M.p.: 335°C–337°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.78 (s, 1H, N‐H), 8.55 (d, J = 7.8 Hz, 1H, Ar‐H), 8.53 (d, J = 8.9 Hz, 1H, Ar‐H), 7.96–7.98 (t, J = 8.8 Hz, 2H, Ar‐H), 7.62–7.63 (d, J = 7.2 Hz, 1H, Ar‐H), 7.31–7.44 (d, J = 48.8 Hz, 1H, Ar‐H), 7.05–7.07 (d, J = 6.9 Hz, 1H, Ar‐H), 6.94 (s, 1H, Csp₂‐H), 6.86–6.84 (d, J = 9.5 Hz, 1H, Ar‐H), 3.85 (s, 3H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.12, 166.33, 164.82, 164.19, 151.67, 148.14, 145.33, 140.89, 139.96, 134.37, 130.19, 129.82, 128.62, 124.96, 124.40, 122.61, 116.99, 116.77, 111.29, and 53.34. FT‐IR (cm⁻¹): 3194.4 (N‐H), 3083.3 (Ar‐H), 3010.0 (C_sp2‐H), 2820.0‐2970.0 (C_sp3‐H), 2220‐2250 (C═N), 1740.0 (C═O), 1720.6 (C═O), 1613.4, 1525.0, 1465.7, 1379.9, 1327.6, 1207.4, 1100.4, 1015.1, 843.26, 743.57, and 572.55. HR‐MS m/z [M + H]⁺ Mass found 452.065 g/mol, Calculated C₂₀H₁₃N₅O₆S: 451.058 g/mol.

2.21. Methyl 2‐(3‐(4‐Cyanophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.2)

Orange solid. Yield: 66%. M.p.: 326°C–328°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.71 (s, 1H, N‐H), 8.05–8.17 (m, 2H, Ar‐H), 7.87–7.89 (t, J = 7.2 Hz, 2H, Ar‐H), 7.60–7.62 (d, J = 7.5 Hz, 1H, Ar‐H), 7.41‐7.42 (d, J = 7.2 Hz, 1H, Ar‐H), 7.08‐7.05 (m, 1H, Ar‐H), 6.92 (s, 1H, Csp₂‐H), 6.85–6.88 (s, 1H, Ar‐H), 3.85 (s, 3H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.06, 166.32, 164.83, 164.17, 151.65, 145.33, 140.85, 138.57, 134.39, 133.85, 133.32, 129.86, 128.57, 122.52, 118.78, 118.67, 117.32, 116.79, 112.67, 111.32, and 53.32. FT‐IR (cm⁻¹): 3366.1 (N‐H), 3178.6 (Ar ‐H), 3069.3 (C_sp2‐H), 2830.0‐2960.0 (C_sp3‐H), 2223.2 (C≡N), 2200‐2210 (C═N), 1722.6 (C═O), 1703.8 (C═O), 1608.2, 1525.0, 1468.0, 1436.0, 1374.8, 1332.1, 1203.9, 1101.0, 1034.5, 1018.2, 967.2, 944.0, 843.99, 744.93, 695.31, 628.40, 572.86, 550.99, and 507.33. HR‐MS m/z [M + H]⁺ Mass found 432.077 g/mol, Calculated C₂₁H₁₃N₅O₄S: 431.068 g/mol.

2.22. Methyl 2‐(3‐(4‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.3)

Orange solid. Yield: 56%. M.p.: 343°C–344°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.70 (s, 1H, N‐H), 7.58–7.59 (d, J = 7.0 Hz, 1H, Ar‐H), 7.47–7.49 (t, J = 7.9 Hz, 2H, Ar‐H), 7.41–7.38 (m, 1H, Ar‐H), 7.05–7.08 (d, J = 6.6 Hz, 3H, Ar‐H), 6.85 (s, 1H, C_sp2‐H), 6.84 (d, J = 7.7 Hz, 1H, Ar‐H), 3.83 (s, 6H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.61, 166.40, 164.88, 161.95, 160.19, 159.73, 158.76, 151.45, 148.68, 145.22, 144.55, 133.89, 129.70, 126.73, 122.48, 116.91, 116.27, 114.46, 111.18, 56.04, and 53.15. FT‐IR (cm⁻¹): 3174.9 (N‐H), 3070.0 (Ar‐H), 3058.3 (C_sp2‐H), 2860.0‐2959.6 (C_sp3‐H), 2210‐2260 (C═N), 1729.8 (C═O), 1713.0 (C═O), 1690.0, 1603.7, 1527.4, 1381.5, 1333.1, 1251.8, 1210.6, 1032.5, 758.81, 575.07, 442.91, and 421.64. HR‐MS m/z [M + H]⁺ Mass found 437.091 g/mol, Calculated C₂₁H₁₆N₄O₅S: 436.084 g/mol.

2.23. Methyl 2‐(3‐(4‐Chlorophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.4)

Orange solid. Yield: 83%. M.p.: 329°C–331°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.74 (s, 1H, N‐H), 7.72 (d, 1H, Ar‐H), 7.63 (t, 4H, Ar‐H), 7.42 (d, 1H, Ar‐H), 7.06 (d, 1H, Ar‐H), 6.88 (s, 1H, C_sp2‐H), 6.84 (d, 1H, Ar‐H), 3.85 (s, 3H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.36, 166.32, 164.84, 164.36, 151.89, 151.62, 145.34, 141.01, 134.83, 134.60, 134.32, 133.41, 130.51, 129.79, 128.70, 122.37, 120.99, 117.15, 116.92, 111.28, and 53.27. FT‐IR (cm⁻¹): 3207.5 (N‐H), 3050.0 (Ar‐H), 3035.0 (C_sp2‐H), 2880.0‐2960.6 (C_sp3‐H), 2220‐2260 (C═N), 1730.0 (C═O), 1722.6 (C═O), 1705.0, 1608.2, 1527.4, 1494.5, 1465.9, 1381.2, 1330.6, 1247.7, 1210.3, 1094.7, 1045.1, 1015.9, 928.46, 867.81, 803.13, 757.62, 645.00, 574.05, 445.27, and 418.25. HR‐MS m/z [M + H]⁺ Mass found 441.035 g/mol, Calculated C₂₀H₁₃ClN₄O₄S: 440.0346 g/mol.

2.24. Methyl 2‐(3‐(3‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.5)

Orange solid. Yield: 87%. M.p.: 318°C–321°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.76 (s, 1H, N‐H), 7.58–7.60 (d, J = 7.0 Hz, 1H, Ar‐H), 7.43–7.44 (d, J = 7.8 Hz, 1H, Ar‐H), 7.42–7.38 (m, 1H, Ar‐H), 7.24 (s, 1H, Ar‐H), 7.14‐7.12 (d, J = 7.4 Hz, 1H, Ar‐H), 7.05 (s, 2H, Ar‐H), 6.87 (s, 1H, C_sp2‐H), 6.85 (s, 1H, Ar‐H), 3.82 (s, 6H, O‐CH₃). FT‐IR (cm⁻¹): 3304.8 (N‐H), 3077.0 (Ar‐H), 3040.0 (C_sp2‐H), 2890.0‐2980.0 (C_sp3‐H), 2210‐2255 (C═N), 1734.6 (C═O), 1719.7 (C═O), 1705.0, 1675.5, 1611.5, 1545.5, 1496.0, 1372.9, 1292.6, 1262.2, 1222.5, 1044.0, 964.32, 827.58, 745.12, 679.48, 570.98, 481.01, 443.86, and 426.30. HR‐MS m/z [M + H]⁺ Mass found 437.091 g/mol, Calculated C₂₁H₁₆N₄O₅S: 436.084 g/mol.

2.25. Methyl 2‐(3‐(Naphthalen‐1‐yl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.6)

Orange solid. Yield: 85%. M.p.: 316°C–319°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.62 (s, 1H, N‐H), 8.13–8.15 (m, 1H, Ar‐H), 8.08–8.10 (m, 1H, Ar‐H), 7.76–7.80 (m, 3H, Ar‐H), 7.69–7.71 (d, J = 7.2 Hz, 1H, Ar‐H), 7.57–7.62 (m, 2H, Ar‐H), 7.37–7.39 (d, J = 6.0 Hz, 1H, Ar‐H), 7.03–7.05 (d, J = 6.4 Hz, 1H, Ar‐H), 6.93 (s, 1H, Csp₂‐H), 6.79–6.80 (d, J = 6.6 Hz, 1H, Ar‐H), 3.82 (s, 3H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 166.42, 166.25, 164.89, 164.74, 151.79, 145.15, 141.07, 134.16, 130.64, 128.86, 128.59, 128.05, 127.47, 127.33, 126.22, 122.97, 121.91, 117.41, 116.67, 111.06, and 53.33. FT‐IR (cm⁻¹): 3304.3 (N‐H), 3065.0 (Ar‐H), 3000.0 (C_sp2‐H), 2910.0‐2990.0 (C_sp3‐H), 2000–2200 (C═N), 1722.5 (C═O), 1692.1 (C═O), 1614.1, 1522.1, 1397.3, 1293.2, 1176.2, 1041.7, 955.01, 746.88, and 430.30. HR‐MS m/z [M + H]⁺ Mass found 457.096 g/mol, Calculated C₂₄H₁₆N₄O₄S: 456.089 g/mol.

2.26. Methyl 2‐(3‐(2,6‐Difluorophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.7)

Orange solid. Yield: 66%. M.p.: 335°C–337°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.73 (s, 1H, N‐H), 7.78 (d, 1H, Ar‐H), 7.93 (d, 3H, Ar‐H), 7.38‐7.42 (t, 1H, Ar‐H), 7.03‐7.06 (t, J = 6.9 Hz, 1H, Ar‐H), 6.88 (s, 1H, Csp₂‐H), 6.86 (d, J = 22.9 Hz, 1H, Ar‐H), 3.83 (s, 6H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.14, 166.31, 164.83, 164.25, 151.67, 145.36, 140.92, 135.69, 134.37, 133.70, 131.34, 129.92, 128.67, 127.42, 122.67, 122.38, 117.19, 116.89, 111.30, and 53.29. FT‐IR (cm⁻¹): 3196.4 (N‐H), 3070.0 (Ar‐H), 3020.0 (C_sp2‐H), 2880.0‐2946.4 (C_sp3‐H), 2210–2260 (C═N), 1721.7 (C═O), 1702.2 (C═O), 1611.9, 1531.0, 1470.9, 1375.4, 1334.2, 1246.5, 1208.6, 1102.2, 1013.3, 672.01, and 419.62. HR‐MS m/z [M + H]⁺ Mass found 441.041 g/mol, Calculated C₂₀H₁₂F₂N₄O₄S: 442.030 g/mol.

2.27. Methyl 2‐(3‐(4‐Fluorophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.8)

Yellow solid. Yield: 63%. M.p.: 325°C–327°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.75 (s, 1H, N‐H), 8.02 (s, 1H, Ar‐H), 7.85–7.86 (d, J = 7.4 Hz, 1H, Ar‐H), 7.58‐7.64 (d, J = 22.5 Hz, 2H, Ar‐H), 7.36–7.40 (t, J = 21.5 Hz, 2H, Ar‐H), 7.05–7.07 (d, J = 7.0 Hz, 1H, Ar‐H), 6.87 (s, 1H, C_sp2‐H), 6.84–6.85 (d, J = 7.5 Hz, 1H, Ar‐H), 3.83 (s, 3H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.23, 166.35, 164.85, 164.29, 152.03, 151.62, 145.37, 141.02, 138.49, 137.08, 135.33, 134.35, 131.57, 128.75, 128.02, 122.61, 117.11, 116.91, 111.28, and 53.30. FT‐IR (cm⁻¹): 3161.0 (N‐H), 3080.0 (Ar‐H), 3010.0 (C_sp2‐H), 2860.0–2980.0 (C_sp3‐H), 2150–2250 (C═N), 1726.2 (C═O), 1712.0 (C═O), 1680.0, 1610.7, 1521.1, 1469.3, 1380.9, 1335.8, 1251.4, 1210.8, 1096.3, 969.05, 673.44, and 408.50.

2.28. Methyl 2‐(3‐(4‐Ethylphenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.9)

Orange solid. Yield: 49%. M.p.: 320°C–322°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.75 (s, 1H, N‐H), 7.58–7.60 (d, J = 6.9 Hz, 1H, Ar‐H), 7.47–7.49 (d, J = 7.2 Hz, 2H, Ar‐H), 7.36–7.39 (m, 3H, Ar‐H), 7.03–7.05 (m, 1H, Ar‐H), 6.86 (s, 1H, C_sp2‐H), 6.83 (d, 1H, Ar‐H), 3.82 (s, 3H, O‐CH₃), 2.68–2.70 (q, J = 7.2 Hz 2H, CH₂), 1.25 (t, J = 7.0 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.47, 166.35, 164.90, 164.52, 151.60, 146.00, 145.21, 141.11, 134.24, 132.41, 132.12, 130.14, 129.01, 128.36, 122.23, 117.33, 117.00, 111.19, 109.21, 53.29, 28.47, and 16.37. FT‐IR (cm⁻¹): 3160.5 (N‐H), 3065.0 (Ar‐H), 3040.0 (C_sp2‐H), 2840.0‐2963.8 (C_sp3‐H), 2200–2260 (C═N), 1719.3 (C═O), 1694.1 (C═O), 1605.3, 1526.0, 1467.8, 1381.3, 1331.3, 1260.51, 1207.8, 1186.6, 1098.1, 1080.3, 1044.4, 1010.6, 966.96, 925.3, 871.80, 748.14, 720.64, 630.59, 573.99, and 510.68. HR‐MS m/z [M + H]⁺ Mass found 435.112 g/mol, Calculated C₂₂H₁₈N₄O₄S: 434.104 g/mol.

2.29. Methyl 2‐(3‐(2‐Bromophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.10)

Orange solid. Yield: 81%. M.p.: 303°C–305°C. ¹H NMR (400 MHz, DMSO‐d₆): δ 10.75 (s, 1H, N‐H), 7.78 (d, 1H, Ar‐H), 7.58 (d, 4H, Ar‐H), 7.38–7.40 (d, J = 6.9 Hz, 1H, Ar‐H), 7.05–7.06 (d, J = 6.3 Hz, 1H, Ar‐H), 6.89 (s, 1H, Csp₂‐H), 6.83‐6.85 (d, J = 6.8 Hz, 1H, Ar‐H), 3.82 (s, 3H, O‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆): 167.11, 166.31, 164.80, 164.25, 151.69, 145.41, 141.00, 135.73, 134.69, 134.34, 133.70, 131.33, 129.92, 128.70, 127.45, 122.35, 117.15, 116.93, 111.28, and 53.30. FT‐IR (cm⁻¹): 3195.8 (N‐H), 3070.0 (Ar‐H), 3030.0 (C_sp2‐H), 2920.0–2941.8 (C_sp3‐H), 2200‐2270 (C═N), 1722.1 (C═O), 1705.0 (C═O), 1611.6, 1531.2, 1471.0, 1435.2, 1375.2, 1334.0, 1246.8, 1208.5, 1099.0, 1014.7, 868.33, 722.95, 672.64, 573.53, 441.06, and 417.61.

2.30. Methyl 2‐(4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)‐3‐Phenylthiazolidin‐5‐ylidene)Acetate (Y.Z.A.1)

Orange solid. Yield: 40%. M.p.: 261°C–264°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.67–7.69 (d, J = 6.3 Hz, 1H, Ar‐H), 7.51–7.58 (dd, J = 18.0, 10.1 Hz, 6H, Ar‐H), 7.14–7.16 (d, J = 7.5 Hz, 2H, Ar‐H), 6.90 (s, 1H, C_sp2‐H), 4.49 (s, 2H, C‐H), 3.84 (s, 3H, O‐CH₃), 3.26 (s, 1H, C_SP‐H). FT‐IR (cm⁻¹): 3287.2 (C_sp‐H), 3051.0 (Ar‐H), 3030.0 (C_sp2‐H), 2956.9 (C_sp3‐H), 2000‐2250 (C═N), 1723.3 (C═O), 1688.6 (C═O), 1605.8, 1528.8, 1469.9, 1441.8, 1377.8, 1330.8, 1257.7, 1211.4, 1183.3, 1100.0, 994.62, 929.10, 877.98, 790.17, 760.79, 724.86, 684.85, 589.43, 498.64, 448.92, and 417.97. HR‐MS m/z [M + H]⁺ Mass found 445.096 g/mol, Calculated C₂₃H₁₆N₄O₄S: 444.089 g/mol.

2.31. Methyl 2‐(3‐(4‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.2)

Yellow solid. Yield: 53%. M.p.: 295°C–298°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.66–7.68 (d, J = 7.1 Hz, 1H, Ar‐H), 7.48–7.53 (m, 3H, Ar‐H), 7.14–7.16 (m, 2H, Ar‐H), 7.07–7.09 (m, 2H, Ar‐H), 6.88 (s, 1H, Csp₂‐H), 4.49 (s, 2H, C‐H), 3.84 (s, 6H, O‐CH₃), 3.26 (s, 1H, C_SP‐H). ¹³C NMR (100 MHz, DMSO‐d₆): 168.53, 166.42, 164.68, 160.23, 159.81, 156.37, 150.09, 147.32, 144.24, 141.11, 134.12, 129.68, 129.56, 128.71, 126.87, 117.17, 116.57, 114.86, 110.50, 78.20, 74.97, 56.08, 53.38, and 29.27. FT‐IR (cm⁻¹): 3291.8 (C_sp‐H), 3040.0 (Ar‐H), 3030.0 (C_sp2‐H), 2950.0 (C_sp3‐H), 2200–2270 (C═N), 1723.9 (C═O), 1690.0 (C═O), 1605.6, 1534.0, 1464.1, 1377.9, 1256.8, 1215.5, 1180.6, 1025.2, and 745.63. HR‐MS m/z [M + H]⁺ Mass found 475.107 g/mol, Calculated C₂₄H₁₈N₄O₅S: 474.099 g/mol.

2.32. Methyl 2‐(3‐Benzyl‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.3)

Orange solid. Yield: 80%. M.p.: 256°C–258°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.66–7.69 (d, J = 7.3 Hz, 1H, Ar‐H), 7.58–7.60 (d, J = 6.9 Hz, 2H, Ar‐H), 7.52–7.56 (t, J = 7.6 Hz, 1H, Ar‐H), 7.33–7.35 (t, J = 7.3 Hz, 3H, Ar‐H), 7.17–7.19 (d, J = 7.8 Hz, 2H, Ar‐H), 6.88 (s, 1H, C_sp2‐H), 5.11 (s, 2H, C‐H), 4.59 (s, 2H, C‐H), 3.82 (s, 3H, O‐CH₃), 3.18 (s, 1H, C_SP‐H). FT‐IR (cm⁻¹): 3261.8 (C_sp‐H), 3055.8 (Ar‐H), 3020.0 (C_sp2‐H), 2890.0–2960.0 (C_sp3‐H), 2100–2280 (C═N), 1718.2 (C═O), 1695.2 (C═O), 1614.2, 1532.2, 1392.1, 1337.1, 1213.7, 1173.8, 1094.7, 975.92, 790.10, 745.07, 695.99, 585,65, 418.55, and 405.95. HR‐MS m/z [M + H]⁺ Mass found 459.112 g/mol, Calculated C₂₄H₁₈N₄O₄S: 458.104 g/mol.

2.33. Methyl 2‐(3‐(4‐Chlorophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.4)

Orange solid. Yield: 93%. M.p.: 295°C–298°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.73–7.75 (d, J = 8.6 Hz, 1H, Ar‐H), 7.66 (d, 2H, Ar‐H), 7.63 (t, 2H, Ar‐H), 7.51–7.55 (t, 1H, Ar‐H), 7.14–7.18 (d, J = 8.8 Hz, 2H, Ar‐H), 6.90 (s, 1H, Csp₂‐H), 4.50 (s, 2H, C‐H), 3.84 (s, 3H, O‐CH₃), 3.24 (s, 1H, C_SP‐H). ¹³C NMR (100 MHz, DMSO‐d₆): 174.85, 166.46, 162.95, 160.47, 156.63, 151.50, 145.05, 142.58, 132.27, 130.19, 126.86,125.30, 124.49, 123.85, 121.93, 119.61, 115.08, 112.83, 111.07, 75.52, 69.78, 60.13, and 29.19. FT‐IR (cm⁻¹): 3282.3 (C_sp‐H), 3048.4 (Ar‐H), 3020.0 (C_sp2‐H), 2960.0 (C_sp3‐H), 2210‐2270 (C═N), 1726.5 (C═O), 1690.0 (C═O), 1604.8, 1531.6, 1492.4, 1373.0, 1340.0, 1253.5, 1210.2, 1090.5, 1016.3, 864.65, 795.68, 759.59, 744.11, and 468.94. HR‐MS m/z [M + H]⁺ Mass found 479.057 g/mol, Calculated C₂₃H₁₅ClN₄O₄S: 478.050 g/mol.

2.34. Methyl 2‐(3‐(3‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazineylidene)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.5)

Orange solid. Yield: 78%. M.p.: 254°C–256°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.67–7.69 (d, J = 7.4 Hz, 1H, Ar‐H), 7.53–7.56 (d, J = 7.7 Hz, 1H, Ar‐H), 7.43–7.47 (t, J = 7.9 Hz, 1H, Ar‐H), 7.22 (s, 1H, Ar‐H), 7.17 (m, 3H, Ar‐H), 7.05‐7.06 (d, J = 7.9 Hz, 1H, Ar‐H), 6.89 (s, 1H, Csp₂‐H), 4.50 (s, 2H, C‐H), 3.84 (s, 6H, O‐CH₃), 3.23 (s, 1H, C_SP‐H). ¹³C NMR (100 MHz, DMSO‐d₆): 168.11, 166.37, 164.36, 162.70, 160.25, 150.24, 144.37, 141.09, 135.54, 134.15, 130.46, 128.64, 123.21, 120.61, 117.14, 116.49, 115.68, 114.23, 110.56, 78.13, 75.09, 56.00, 53.31, and 29.17. FT‐IR (cm⁻¹): 3291.8 (C_sp‐H), 3050.0 (Ar‐H), 3010.0 (C_sp2‐H), 2920.0–2960.0 (C_sp3‐H), 2120‐2290 (C═N), 1723.9 (C═O), 1680.0 (C═O), 1605.6, 1534.0, 1464.1, 1337.9, 1256.8, 1215.5, 1180.6, 1025.2, and 745.63. HR‐MS m/z [M + H]⁺ Mass found 475.107 g/mol, Calculated C₂₄H₁₈N₄O₅S: 474.099 g/mol.

2.35. Methyl 2‐(3‐(Naphthalen‐1‐yl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.6)

Orange solid. Yield: 68%. M.p.: 216°C–219°C. ¹H NMR (400 MHz, DMSO‐d₆): 8.13–8.15 (d, J = 8.0 Hz, 1H, Ar‐H), 8.08–8.10 (d, J = 7.9 Hz, 1H, Ar‐H), 7.77–7.80 (t, 2H, Ar‐H), 7.66–7.72 (dd, J = 16.7, 8.0 Hz, 2H, Ar‐H), 7.57–7.62 (dd, J = 12.3, 7.6 Hz, 2H, Ar‐H), 7.49–7.52 (t, J = 7.7 Hz, 1H, Ar‐H), 7.09–7.17 (m, 2H, Ar‐H), 6.95 (s, 1H, C_sp2‐H), 4.38 (s, 2H, C‐H), 3.86 (s, 3H, O‐CH₃), 3.19 (s, 1H, C_SP‐H). FT‐IR (cm⁻¹): 3263.2 (C_sp‐H), 3040.0 (Ar‐H), 3020.0 (C_sp2‐H), 2970.0 (C_sp3‐H), 2000‐2300 (C═N), 1722.4 (C═O), 1690.0 (C═O), 1608.5, 1520.5, 1338.3, 1213.9, 1171.7, 989.91, 750.12, and 446.55. HR‐MS m/z [M + H]⁺ Mass found 495.112 g/mol, Calculated C₂₇H₁₈N₄O₄S: 494.104 g/mol.

2.36. Methyl 2‐(3‐(2,6‐Difluorophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.7)

Orange solid. Yield: 92%. M.p.: 261°C–264°C. ¹H NMR (400 MHz, DMSO‐d₆): 8.76 (s, 1H, Ar‐H), 7.68–7.70 (d, J = 5.5 Hz, 1H, Ar‐H), 7.60 (t, 2H, Ar‐H), 7.52–7.54 (t, J = 7.7 Hz, 1H, Ar‐H), 7.15–7.17 (d, J = 7.1 Hz, 2H, Ar‐H), 6.90 (s, 1H, C_sp2‐H), 4.50 (s, 2H, C‐H), 3.84 (s, 3H, O‐CH₃), 3.21 (s, 1H, C_SP‐H). ¹³C NMR (100 MHz, DMSO‐d₆): 166.39, 164.31, 162.74, 161.94, 160.47, 150.36, 144.39, 140.78, 139.16, 135.66, 134.31, 133.76, 131.42, 128.66, 127.42, 123.39, 117.45, 116.43, 110.64, 78.11,75.00, 53.36, and 29.29. FT‐IR (cm⁻¹): 3263.2 (C_sp‐H), 3050.0 (Ar‐H), 3030.0 (C_sp2‐H), 2960.0 (C_sp3‐H), 2000‐2260 (C═N), 1722.4 (C═O), 1700.0 (C═O), 1608.5, 1520.5, 1338.3, 1213.9, 1171.7, 989.91, 750.12, and 446.55. HR‐MS m/z [M + H]⁺ Mass found 481.057 g/mol, Calculated C₂₃H₁₄F₂N₄O₄S: 480.059 g/mol.

2.37. Methyl 2‐(3‐(4‐Fluorophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.8)

Orange solid. Yield: 90%. M.p.: 235°C–236°C. ¹H NMR (400 MHz, DMSO‐d₆): 8.00 (s, 1H, Ar‐H), 7.86–7.88 (d, J = 7.1 Hz, 1H, Ar‐H), 7.66–7.69 (t, J = 17.7 Hz, 2H, Ar‐H), 7.54–7.55 (d, J = 7.0 Hz, 1H, Ar‐H), 7.37–7.38 (d, J = 7.2 Hz, 1H, Ar‐H), 7.14–7.15 (d, J = 7.6 Hz, 2H, Ar‐H), 6.90 (s, 1H, C_sp2‐H), 4.50 (s, 2H, C‐H), 3.84 (s, 3H, O‐CH₃), 3.26 (s, 1H, C_SP‐H). FT‐IR (cm⁻¹): 3247.7 (C_sp‐H), 3050.0 (Ar‐H), 3030.0 (C_sp2‐H), 2880.0–2990.0 (C_sp3‐H), 2000–2350 (C═N), 1725.6 (C═O), 1685.0 (C═O), 1602.1, 1524.4, 1378.0, 1336.9, 1208.8, 1089.9, 989.33, and 440.0.

2.38. Methyl 2‐(3‐(4‐Ethylphenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.9)

Orange solid. Yield: 35%. M.p.: 295°C–298°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.66–7.68 (d, J = 7.4 Hz, 1H, Ar‐H), 7.47–7.49 (d, J = 7.5 Hz, 3H, Ar‐H), 7.37–7.39 (d, J = 8.3 Hz, 2H, Ar‐H), 7.13–7.16 (d, J = 8.7 Hz, 2H, Ar‐H), 6.88 (s, 1H, Csp₂‐H), 4.49 (s, 2H, C‐H), 3.82 (s, 3H, O‐CH₃), 3.26 (s, 1H, C_SP‐H), 2.70 (dd, J = 15.2, 7.6 Hz, 2H, ‐CH₂), 1.26 (t, J = 13.4 Hz, 3H, ‐CH₃). FT‐IR (cm⁻¹): 3264.0 (C_sp‐H), 3049.3 (Ar‐H), 3010.0 (C_sp2‐H), 2880.0–2950.0 (C_sp3‐H), 2100‐2280 (C═N), 1717.3 (C═O), 1690.0 (C═O), 1602.9, 1525.5, 1470.6, 1380.3, 1329.7, 1256.7, 1209.8, 1102.1, 994.37, 870.51, 693.89, 640.09, 581.88, and 417.53. HR‐MS m/z [M + H]⁺ Mass found 473.127 g/mol, Calculated C₂₅H₂₀N₄O₄S: 472.120 g/mol.

2.39. Methyl 2‐(3‐(2‐Bromophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.10)

Orange solid. Yield: 39%. M.p.: 269°C–272°C. ¹H NMR (400 MHz, DMSO‐d₆): 7.76 (s, 1H, Ar‐H), 7.69 (m, 1H, Ar‐H), 7.59 (m, 3H, Ar‐H), 7.54 (t, 1H, Ar‐H), 7.12–7.15 (d, J = 16.1 Hz, 2H, Ar‐H), 6.91 (s, 1H, C_sp2‐H), 4.50 (s, 2H, C‐H), 3.84 (s, 3H, O‐CH₃), 3.26 (s, 1H, C_SP‐H). ¹³C NMR (100 MHz, DMSO‐d₆): 166.37, 164.24, 162.23, 160.42, 158.80, 143.80, 143.85, 141.22, 135.44, 134.24, 130.98, 129.97, 128.52, 123.60, 122.42, 119.79, 117.53, 116.67, 110.52, 98.76, 78.20, 74.77, 53.34, and 29.34. FT‐IR (cm⁻¹): 3282.9 (C_sp‐H), 3060.0 (Ar‐H), 3020.0 (C_sp2‐H), 2910.0–2960.0 (C_sp3‐H), 2200–2250 (C═N), 1721.2 (C═O), 1695.0 (C═O), 1606.6, 1530.6, 1471.6, 1436.0, 1378.4, 1335.8, 1211.4, 1172.4, 1103.3, 995.90, 868.21, 745.11, 721.19, 674.95, 593.74, 443.30, and 418.54.

2.40. Cytotoxic Activity Studies

Cytotoxic activity studies of compounds were performed according to the literature [30]. The human prostate cancer cell line (PC3, ATCC CRL‐1435) was purchased from the American Type Culture Collection (ATCC, USA). Cells were cultured in RPMI supplemented with 10% fetal bovine serum and 1% glutamax. The cell seeding was done at a density of 5 × 10³ cells/well into sterile 96‐well plates. The cells were exposed to the compounds at 300, 150, 75, and 37.5 µM concentrations for 72 h. The MTT stock solution (50 µL, 5 mg/mL) was added to the plate wells and incubated for 2 h. Absorbance values were measured in the Epoch 2 ELISA plate reader device at 590 nm. IC₅₀ values were calculated by using GraphPad Prism software 5.

2.41. Quantitative Real‐Time Polymerase Chain Reaction (RT‐qPCR)

RT‐qPCR studies were performed as described before [31]. Cells were treated with selected compounds at IC₅₀ concentration for 48 h. After that, total RNA was extracted from cells using a PureLink RNA Mini‐prep Kit (Invitrogen) following the manufacturer's instructions. The RNA quality and concentration were measured by a nano‐spectrophotometer (Allsheng). Complementary DNA (cDNA) was synthesized using an iScript cDNA synthesis kit (Bio‐Rad) by being included in the reaction 1 μg RNA. Real‐time PCR amplification was performed using iTaq Universal SYBR Green Supermix (Bio‐Rad) following the manufacturer's protocol on a CFX Connect Real‐Time PCR detection system (Bio‐Rad). Human‐specific primer pairs were designed to amplify BAX, BCL2, BCL‐xL, BID, and GAPDH. Primer sequences are available upon request. Quantification of relative gene expression was calculated by the Livak method [32]. GAPDH was used to normalize the target gene expression in each sample. A melting curve was used to ascertain the specificity of amplification. Each sample was analyzed using a triple technique and biological repeat in RT‐qPCR studies. Data were presented in the graph as a fold change with standard deviation.

2.42. Total Oxidant Status

Total oxidant status (TOS) was spectrophotometrically examined using the total oxidant status assay (REL Assay Diagnostics) following the manufacturer's instructions. The TOS method is based on the oxidation of ferrous ions to ferric ions in the presence of various oxidative species in the acidic medium. Ferric ion was measured by xylenol orange. The experiment was performed with three biological replicates. Results were presented in the graph as the fold change with standard deviation compared to the control group.

2.43. Total Antioxidant Status

Cells were treated with selected compounds at IC₅₀ concentration for 48 h, and then oxidative stress parameters were measured by the spectrophotometric method. Total antioxidant status (TAS) was colorimetrically examined using the TAS assay (REL Assay Diagnostics) following the manufacturer's protocol. The reaction was calibrated with Trolox, and the TAS value of the samples tested was expressed as μmol Trolox equivalent/L (μmol Trolox eq/L). The experiment was performed in three biological replicates. Results were presented in the graph as the fold change with standard deviation compared to the control group.

2.44. Molecular Docking

The crystal structure of BCL‐2 with co‐crystalline venetoclax was downloaded from the protein data bank (PDB). The human BCL‐2 structure with PDB code of 6O0 K has a resolution of 1.62 Å [33]. The protein and compounds were made ready for the docking by using Schrödinger release 2025‐3. Compound structures were optimized through the Gaussian program with the B3LYP/6‐311 G basis set. In the ligand preparation, the compounds were also optimized with possible elaboration of protonation and tautomeric states, as well as stereochemistry via the LigPrep wizard under the applications list of Maestro 14.5. The possible states were generated at a pH of 7.00 ± 2.00 by using Epik. The protein structure was prepared by using the Protein Preparation wizard. The protein was made ready by deleting water, adding missing side chain residues and hydrogens, assigning charges, optimization, and minimization through the force field of OPLS‐2005. Then, a grid box was generated through the Receptor Grid Generation wizard in the Glide module based on critical residues of interaction for the protein structure [34]. The molecular docking was undertaken with the standard protocol of Glide.

2.45. MD Simulation

The stabilities of complexes formed between the BCL‐2 structure and compounds with the highest binding affinity to it were evaluated through MD simulation analysis. Compound topologies were built through the CGenFF server [35]. Protein structure topologies were built through GROMACS using the related command line. The protein and subsequent complex structures were then made ready for the simulation analysis using the related commands. MD simulation was undertaken by using GROMACS 2024 according to the reported simulation environment [32, 36]. Then, simulation plots were sketched through QtGrace.

3. Results and Discussion

3.1. Chemistry

The chemical structure of the target precursors was confirmed using ¹H NMR, ¹³C NMR, HRMS, and FTIR, providing conclusive structural evidence for the proposed structures.

The target compounds were synthesized as shown in Scheme 1. The ¹H NMR spectra of the NZ series compounds showed characteristic signals attributed to the thiourea (NH) group protons at δ = 12.75–12.56 and 11.24–10.83 ppm, while the NH proton signal of the isadine unit appeared in the range δ = 11.29–11.27 ppm. The ¹H NMR spectra of the final NZ compounds also showed characteristic signals at δ = 13.00–12.77 and 11.15–10.78 ppm attributed to the NH protons in the thiourea group, in addition to the appearance of a signal at δ = 4.70–4.66 ppm attributed to the methylene group (CH₂), and a signal at δ = 3.56–3.33 ppm attributed to the terminal CH proton of the ethinyl group. The proton nuclear magnetic resonance (NMR) spectra of the NZA series compounds showed characteristic signals attributed to the isadiene (NH) group proton at δ = 12.75–12.62 ppm, indicating the relative acidity of this proton and its potential for hydrogen bonding. A characteristic signal attributed to the double bond (=CH) proton was also observed at δ = 6.93–6.84 ppm. For the YZA series compounds, the NMR spectra showed a signal attributed to the ethinyl group proton (–C≡CH) at δ = 3.26–3.19 ppm, a typical value for a terminal alkene proton. A signal from the double bond (=CH) proton was also observed at δ = 6.94–6.85 ppm. Some structural and physical properties of the synthesized compounds are given in Table 1.

Table 1.

Structural and physical properties of compounds and IC₅₀ results for compounds against the prostate cancer cell line.

Compd.	Ar	R	IC₅₀
Compd.	Ar	R	PC3
NZ1	4‐nitrophenyl	H	48.61
NZ3	4‐methoxyphenyl	H	179.20
NZ4	4‐chlorophenyl	H	16.48
NZ5	3‐methoxyphenyl	H	38.22
NZ6	1‐naphtyl	H	77.96
NZ7	2,6‐difluorophenyl	H	15.85
NZ9	4‐ethylphenyl	H	14.58
NZ10	2‐bromophenyl	H	10.54
YZ1	phenyl		—
YZ2	4‐methoxyphenyl		115.60
YZ4	4‐chlorophenyl		53.40
YZ5	3‐methoxyphenyl		74.97
YZ6	1‐naphtyl		—
YZ7	2,6‐difluorophenyl		23.33
YZ8	4‐fluorophenyl		38.92
YZ9	4‐ethylphenyl		68.53
YZ10	2‐bromophenyl		25.08
NZA1	4‐nitrophenyl	H	543.40
NZA2	4‐cyanophenyl	H	257.20
NZA3	4‐methoxyphenyl	H	393.70
NZA4	4‐chlorophenyl	H	748.70
NZA5	3‐methoxyphenyl	H	324.90
NZA6	1‐naphtyl	H	17.47
NZA7	2,6‐difluorophenyl	H	240.80
NZA8	4‐fluorophenyl	H	69.87
NZA9	4‐ethylphenyl	H	260.50
NZA10	2‐bromophenyl	H	95.77
YZA1	phenyl		187.20
YZA2	4‐methoxyphenyl		288.80
YZA3	benzyl		334.50
YZA4	4‐chlorophenyl		—
YZA5	3‐methoxyphenyl		260.50
YZA6	1‐naphtyl		67.94
Y.Z.A7	2,6‐difluorophenyl		85.84
Y.Z.A8	4‐fluorophenyl		153.80
Y.Z.A9	4‐ethylphenyl		83.91
Y.Z.A10	2‐bromophenyl		252.20

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3.2. Antiproliferative Activity Studies

Compounds were evaluated for their cytotoxicity in the PC3 cell line using the MTT assay method (Table 1). The IC₅₀ values in the 2‐(2‐oxoindolin‐3‐ylidene)hydrazine‐1‐carbothioamide derivative series (NZ1‐10) range from 10.54 to 179.20 µM. The highest cytotoxic activity was observed in compounds NZ10 (10.54 µM), NZ9 (14.58 µM), NZ7 (15.85 µM), and NZ4 (16.48 µM). These results indicate that halogenated phenyl substituents (2‐bromo, 4‐chloro, 2,6‐difluoro) increase activity. On the other hand, the NZ9 compound containing a 4‐ethylphenyl group was found to be more active than the NZ6 compound containing a naphthalen‐1‐yl group, the NZ5 compound containing a 3‐methoxyphenyl group, NZ3 compound containing a 4‐methoxyphenyl group. Compounds in the 4‐oxo‐2‐((2‐oxoindolin‐3‐ylidene)hydrazono)thiazolidin‐5‐ylidene) acetate derivative series (NZA1‐10) were found to be largely inactive against the prostate cancer cell line. However, compound NZA6 (17.47 µM), which contains a naphthalene‐1‐yl core, showed remarkably potent activity compared to other compounds in the NZA series.

The IC₅₀ values in the 2‐(2‐oxo‐1‐(prop‐2‐yn‐1‐yl)indolin‐3‐ylidene)hydrazine‐1‐carbothioamide derivative series (YZ1‐10) range from 23.33 to 115.60 µM. The most active compounds are YZ7 (23.33 µM), which contains a 2,6‐difluorophenyl group, and YZ10 (25.08 µM), which contains a 2‐bromophenyl group. In both cases, halogenated or ortho‐substituted phenyl structures appear to support activity. Compounds in the 4‐oxo‐2‐((2‐oxo‐1‐(prop‐2‐yn‐1‐yl)indolin‐3‐ylidene)hydrazono)thiazolidin‐5‐ylidene) acetate derivative series (YZA1‐10) generally exhibited lower antiproliferative effects compared to the YZ series. The compounds YZA6, YZA7, and YZA9 showed higher cytotoxic effects against the PC3 cell line with IC₅₀ values of 67.94, 85.84, and 83.91 μM, respectively, compared to other YZA derivatives.

3.3. Gene Expression Analysis

To analyze the effect of compounds on apoptotic cell death in PC3 cells, mRNA expression levels of pro‐apoptotic BAX and BID and anti‐apoptotic BCL2 and BCL‐xL genes were tested by RT‐qPCR. Our findings revealed that all tested compounds significantly increased the mRNA levels of pro‐apoptotic BAX and BID genes in PC3 cells (Figure 1).

Investigation of the mRNA expression level of pro‐apoptotic genes in PC3 cells. Cells were treated with compounds at IC₅₀ concentration for 48 h and then mRNA levels of apoptosis‐related genes, (a) *BAX* and (b) *BID* were analyzed by RT‐qPCR. *GAPDH* was used as a reference gene. Data were presented as a fold change with standard deviation in the graph. Control was set to 1. The statistical significance of differences between groups was analyzed by a two‐tailed equal variance Student's t‐test with a minimum 95% confidence interval. (*p < 0.05, **p < 0.001) (n = 3).

Anti‐apoptotic BCL2 and BCL‐xL levels were also significantly decreased with all tested compounds (Figure 2). These results suggested that the tested compounds importantly triggered the apoptotic signaling in PC3 cells.

Investigation of the mRNA expression level of anti‐apoptotic genes in PC3 cells. Cells were treated with compounds at IC₅₀ concentration for 48 h and then mRNA levels of apoptosis‐related genes, (a) *BCL2* and (b) *BCL‐xL* were analyzed by RT‐qPCR. *GAPDH* was used as a reference gene. Data were presented as a fold change with standard deviation in the graph. Control was set to 1. The statistical significance of differences between groups was analyzed by a two‐tailed equal variance Student's t‐test with a minimum 95% confidence interval. (*p < 0.05, **p < 0.001) (n = 3).

The BCL2/BAX mRNA expression ratio represents a cell death or survival signaling, which determines the life or death of cells in response to an apoptotic stimulus. Thus, this ratio is generally used for understanding the balance of apoptotic induction in cells [37]. In this context, we also evaluated the ratio of our BCL2 and BAX mRNA expression results. Our data represent that all tested compounds significantly decreased the BCL2/BAX ratio (Figure 3). Overall, these data suggested that all tested compounds effectively induced apoptotic signaling in PC3 cells through the reciprocal regulation of apoptosis‐related genes. Specifically, the observed upregulation of pro‐apoptotic genes together with the downregulation of anti‐apoptotic genes indicates a shift in the cellular balance toward apoptosis, highlighting the ability of these compounds to activate the apoptotic machinery. Collectively, these results support the potential of the tested compounds to promote apoptotic cell death in PC3 cells through the coordinated modulation of key apoptotic regulators.

*BCL2*/*BAX* mRNA expression ratio in PC3 cells. Data were presented as a fold change with standard deviation in the graph. Control was set to 1. The statistical significance of differences between groups was analyzed by a two‐tailed equal variance Student's t‐test with a minimum 95% confidence interval. (*p < 0.05, **p < 0.001) (n = 3).

3.4. Oxidative Stress Analysis

Many natural products or synthesized compounds are known to exhibit anticancer effects by affecting oxidative stress status in cells [38]. Therefore, we aimed to test the effects of the compounds on oxidant and antioxidant content in PC3 cells. To evaluate the effect of compounds on the oxidant and antioxidant status in PC3 cells, we analyzed the total oxidant and antioxidant content by using spectrophotometric methods. Also, oxidative stress status was calculated to understand the oxidative environment of cells. Our findings revealed that all tested compounds importantly increased the total oxidant content in PC3 cells (Figure 4). In contrast to the TOS results, the antioxidant content significantly decreased depending on the compounds in PC3 cells (Figure 4). Moreover, OSI calculation revealed that compounds broadly increased the oxidative status at the cellular level (Figure 4). Collectively, these results suggested that all tested compounds importantly induced oxidative stress in PC3 cells by conversely regulating oxidant and antioxidant systems.

Evaluation of the oxidative stress‐related parameters in PC3 cells. Cells were treated with compounds at IC₅₀ concentration for 48 h and then oxidative stress‐related parameters, such as (a) total oxidant status (TOS) and (b) total antioxidant status (TAS), and were investigated by spectrophotometrically using commercial kits. (c) OSI values were calculated by dividing TOS by TAS. Data were presented as a fold change with standard deviation in the graph. Control was set to 1. The statistical significance of differences between groups was analyzed by a two‐tailed equal variance Student's t‐test with a minimum 95% confidence interval. (*p < 0.05, **p < 0.001) (n = 3).

3.5. Molecular Docking

The in vitro lab study showed that some of the compounds were active on the cancer cell line. The experimental study on the effect of compounds on apoptotic‐related targets revealed the inhibition potential of some derivatives on anti‐apoptotic genes, including BCL‐2. Compounds with higher cytotoxic efficacy on the cancer cell line and inhibition potential on BCL‐2 were selected. The probable mode of interaction to inhibit BCL‐2 was explored through molecular docking and MD simulation studies. The docking procedure to be employed was validated by re‐docking the co‐crystalline venetoclax on the BCL‐2 structure (6O0K). The docking procedure via Glide has given interaction of venetoclax to the structure through the Asp103 and Gly145 amino acid residues. An H‐bond interaction was observed with Gly145, and a salt bridge was formed with the Asp103 residue (Figure 5). The interaction residues were assessed considering reported docking studies using the same BCL‐2 structure (6O0K). A computational study conducted with Glide reported interactions of ZINC59647158 hit with the same structure, including H‐bonding with the Gly145 residue [39]. Another study supported with docking of midostaurin (MDS) and its derivative using AutoDock 4.2 reported the interaction of the compounds to the same structure. The compounds have formed diverse interactions with the structure, including pi and sigma interactions with the Asp103 residue [40]. Hence, the interactions of venetoclax observed in this study were affirmed by previous studies. So, the docking procedure is anticipated to give reliable interactions for active compounds too. In addition, an MD simulation study was performed to evaluate the formation of stable complexes in the docking process.

Binding profiles of compounds **NZ7**, **NZA6**, and venetoclax in their interaction with the BCL‐2 structure (6O0K).

Docking studies of relatively active compounds were undertaken after the procedure was validated. The compounds have given energy values with various ranges in the docking study. In the study of cytotoxic activity, NZ‐coded compounds have had relatively higher potency in general. The docking score values of the compounds haven't given a sharp difference among the active compounds. Together with this, some of the active compounds have resulted in lower docking scores and other related energy values (Table 2). In this regard, compounds NZ7 and NZA6 have resulted in relatively lower docking scores (Table 2). Compound NZA6 interacted just with a pi‐pi stacking with Phe104 residue, unlike NZ7, which formed H‐bonding and pi‐pi stacking with the Asp103 and Tyr108 residues, respectively (Figure 5). On the other hand, NZA6 has resulted in the lowest Glide energy value among the active derivatives (Table 2). Hence, compounds NZ7 and NZA6 are anticipated to show higher binding affinity to the BCL‐2 structure based on energy values in the docking study. With this being noted, all active compounds have given higher energy values than the co‐crystalline venetoclax, implying lower binding affinity for them (Table 2). The stabilities of the complexes formed with compounds with higher binding affinity were evaluated through MD simulation and then compared to the stability of the complex formed with venetoclax.

Table 2.

Docking score and other related energy values of compounds with relatively high cytotoxic efficacy and venetoclax.

Compounds	Docking score	Glide energy	Glide gscore	Glide emodel
NZ1	−4.85	−36.83	−4.85	−45.55
NZ4	−4.66	−35.13	−4.66	−44.64
NZ5	−4.60	−35.48	−4.60	−44.27
NZ7	−5.24	−34.71	−5.24	−44.08
NZ9	−4.61	−34.89	−4.61	−42.31
NZ10	−4.72	−34.69	−4.72	−42.21
NZA6	−5.40	−37.65	−5.40	−49.78
YZ7	−4.20	−34.76	−4.20	−41.21
YZ8	−4.32	−34.25	−4.32	−40.96
YZ9	−4.81	−36.45	−4.81	−41.80
YZ10	−4.80	−35.72	−4.80	−42.39
Venetoclax	−6.00	−61.23	−6.11	−70.02

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The docking study of NZ‐coded active compounds emphasized the importance of the hydrogen tethered to the nitrogen of the oxoindoline ring in the formation of H‐bonding with the BCL‐2 structure. All NZ‐coded active compounds formed H‐bonds with this hydrogen (Figure 6). In addition, the phenyl tethered to the nitrogen of the carbothioamide or its substituents took part in the interaction of the compounds with the BCL‐2 structure. In this regard, pi‐pi stacking interaction was observed between this phenyl ring of compounds NZ5, NZ7, and NZ9 with the BCL‐2 structure (Figures 5 and 6). Similarly, salt bridge was formed with nitro substituents of the phenyl ring in compound NZ1, and halogen bond was formed with chloro substituents of the phenyl ring in compound NZ4 (Figure 6). The H‐bonds between NZ coded active compounds and the BCL‐2 structure were achieved through with the involvement of Asp103 residue. The interactions of compounds with the same structure via Asp103 were observed in reported studies, and the interaction of venetoclax. On the other hand, pi‐pi stacking was formed with involvement of Tyr108, and other interactions were observed with Arg146 (Figure 6). H‐bonding interactions of ZINC14166367 and ZINC38847833 hits with Tyr108 and Arg146 residues of the BCL‐2 structure were observed in previous studies [39]. Hence, interaction residues of NZ‐coded active compounds with the BCL‐2 structure were in line with reported studies.

Binding profiles of NZ coded derivatives (**NZ1**, **NZ4**, **NZ5**, **NZ9**, **NZ10**) in their interaction with the BCL‐2 structure (6O0K).

Docking study of YZ coded active compounds emphasized importance of hydrogen tethered to nitrogen of carbothioamide group in their interaction with BCL‐2 structure. The H‐bonds of compounds YZ7, YZ8, and YZ10 with the structure were formed with the involvement of this hydrogen (Figure 7). Compound YZ7 formed the H‐bond with the Asp103 residue, as observed in earlier studies and this study. Compounds YZ8 and YZ10 formed H‐bonding with the involvement of Tyr202 residue of the structure. A crystallographic study reported pi‐pi stacking interaction of venetoclax with the same structure via Tyr202 [33]. So, the interaction residues of YZ‐coded compounds were in harmony with reported experimental and computational studies. In general, active YZ‐coded compounds formed lower number of interactions in relative to NZ‐coded ones (Figures 5, 6, and 7). In addition, YZ7 and YZ8 have given higher docking scores implying lower binding affinity for them. The docking study implied that NZ‐coded active compounds would have slightly higher binding potential to the BCL‐2 structure relative to YZ‐coded compounds.

Binding profiles of YZ coded derivatives (**YZ7**, **YZ8**, **YZ9**, **YZ10**) in their interaction with the BCL‐2 structure (6O0K).

3.6. MD Simulation

In the docking study, compounds NZ7 and NZA6 have exhibited higher binding affinity towards the BCL‐2 structure. Relative stabilities of the complexes formed with these compounds were measured through MD simulation analysis. Then, the stabilities of the two complexes were compared to the complex of the co‐crystalline venetoclax. For this purpose, root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond number plots of the complexes were sketched and compared thereafter. Backbone RMSD plots of the complexes of BCL‐2 structure with NZ7, NZA6, and venetoclax were drawn from simulation trajectories. The general change in overall stability of the complexes was measured by using RMSD changes in the backbone structure. The protein‐compound complexes gained stability after rising RMSD values at the beginning of the simulation. The complex with complexed NZ7 has gained stability after 30 ns time. This complex has given higher RMSD values even after this time. The complexes of compound NZA6 and venetoclax have had lower RMSD values implying higher stability (Figure 8A). The three complexes have given RMSD values below 0.3 nm all over the simulation. Hence, the resulting complexes are anticipated to be stable with slight differences among them. A compound RMSD plot was sketched to figure out the status of the compounds to stay inside the binding region persistently during simulation [41]. The compounds have had different compound RMSD values during the simulation. Compound NZ7 has had higher RMSD values, which might force it to move in and out of the binding region. Compound NZA6 and venetoclax have given lower RMSD values during the simulation. The two compounds are anticipated to remain in the binding region based on the RMSD values. Venetoclax has given lower RMSD values than compound NZA6 implying higher probability to remain bound inside the binding region of the BCL‐2 structure (Figure 8B).

Backbone RMSD (A), compound RMSD (B), Rg (C), and hydrogen bond number (D) plots of compounds **NZ7**, **NZA6**, and venetoclax complexed with BCL‐2.

Rg plot is used to measure compactness of apo protein structures or their complexes during simulation. The Rg values of the three complexes remained within a given range during simulation with mean value of nearly 1.47 nm. Complex structures of NZ7 and NZA6 have resulted in similar Rg values. The Rg values of the complex containing venetoclax were slightly higher implying lower compactness for it (Figure 8C). H‐bond number is important in determining strength of binding between a compound and target structure [42]. In the simulation study, compound NZ7 formed single H‐bond predominantly. Moreover, it also formed up to three H‐bonds as observed in the last 60 ns. Compound NZ6 formed a single H‐bond but with less frequency. Venetoclax formed mainly two H‐bonds during simulation. Together with this, it formed a higher number of H‐bonds in some time intervals, as observed in the 6‐35 ns interval and in the last 15 ns. In general, the compounds have had a higher number of H‐bonds in the simulation. Venetoclax has had the highest number of H‐bonds in the simulation (Figure 8D, Figure S1). In short, the MD simulation study demonstrated that the compounds could form stable complexes with the BCL‐2 structure. In addition, venetoclax formed a complex with higher stability.

4. Conclusions

Collectively, the present findings suggested that selected compounds have a substantially potent anticancer activity on metastatic prostate adenocarcinoma PC3 cells by modulating apoptotic signaling and elevating oxidative stress status. Also, binding potentials of relatively active compounds to the BCL‐2 structure to form stable complexes were evaluated through molecular modeling. Molecular docking study disclosed that the compounds have binding potential to the structure. However, the binding potentials of the compounds have been found to be lower than that of the co‐crystalline venetoclax. The stabilities of the complexes formed with the compounds were anticipated to be stable according to MD simulation analysis. Similarly, venetoclax has relatively higher stability than the investigated compounds. Although the comprehensive mechanisms of action of these molecules could not be fully defined in the present study, their potent anticancer effects hold significant potential for further research into their use in therapeutic regimens.

Author Contributions

Anber M Anber: formal analysis, investigation, resources, methodology, software, validation. Mehmet Özbay: methodology, resources. İlhan Özer İlhan: validation, visualization, supervision, funding acquisition. Hüseyin Kekeçmuhammed: investigation, formal analysis, resources. Rıfat Emin Bora: formal analysis, investigation. Emin Sarıpınar: investigation, formal analysis, resources, validation, visualization, supervision, writing – review and editing, data curation. Yalçın Erzurumlu: Formal analysis, investigation, methodology, resources, validation, visualization, writing – review and editing. Muhammed Tilahun Muhammed: methodology, validation, visualization, software, resources. Sadeq K. Alhag: funding acquisition, investigation. Lamya Ahmed Alkeridis: investigation, funding acquisition. Senem Akkoc: project administration, formal analysis, investigation, funding acquisition, writing – review and editing, writing – original draft, resources, validation.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File

JBT-40-e71140-s001.docx^{(166.7MB, docx)}

Acknowledgments

The authors would like to thank Erciyes University Scientific Research Projects Unit for financial support with project number FYL‐2024‐13664. SA would like to thank Süleyman Demirel University for financial support with project number TSG‐2024‐9516. This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R82), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Large Groups Project under grant number (R.G.P.2/7/47). YE would like to thank Suleyman Demirel University internal funds (TSG‐2021‐8302, TAB‐2020‐8253) for financial support.

Contributor Information

Emin Saripinar, Email: emin@erciyes.edu.tr.

Senem Akkoc, Email: senemakkoc@sdu.edu.tr.

Data Availability Statement

The data that support the findings of this study are available from the authors upon reasonable request.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File

JBT-40-e71140-s001.docx^{(166.7MB, docx)}

Data Availability Statement

The data that support the findings of this study are available from the authors upon reasonable request.

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[jbt71140-bib-0021] 21. Patel N. B. and Patel M. D., “Synthesis and Evaluation of Antibacterial and Antifungal Activities of 4‐Thiazolidinones and 2‐Azetidinones Derivatives From Chalcone,” Medicinal Chemistry Research 26 (2017): 1772–1783. [Google Scholar]

[jbt71140-bib-0022] 22. Iqbal M. A., Husain A., Alam O., et al., “Design, Synthesis, and Biological Evaluation of Imidazopyridine‐Linked Thiazolidinone as Potential Anticancer Agents,” Archiv der Pharmazie 353 (2020): 2000071. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Synthesis, Cytotoxic Activity, mRNA Expression, Total Oxidant and Antioxidant Status, and Molecular Modeling Studies of Novel Schiff Base–Thiosemicarbazone Hybrids

Anber M Anber

Mehmet Özbay

İlhan Özer İlhan

Hüseyin Kekeçmuhammed

Rıfat Emin Bora

Emin Saripinar

Yalçın Erzurumlu

Muhammed Tilahun Muhammed

Sadeq K Alhag

Lamya Ahmed Alkeridis

Senem Akkoc

ABSTRACT

1. Introduction

2. Materials and Methods

2.1. Chemistry

2.2. Synthesis of Compounds

2.3. N‐(4‐Nitrophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ1)

2.4. N‐(4‐Methoxyphenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ3)

2.5. N‐(4‐Chlorophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ4)

2.6. N‐(3‐Methoxyphenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ5)

2.7. N‐(Naphthalen‐1‐yl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ6)

2.8. N‐(2,6‐Difluorophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ7)

2.9. N‐(4‐Ethylphenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ9)

2.10. N‐(2‐Bromophenyl)‐2‐(2‐Oxoindolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (NZ10)

2.11. 2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)‐N‐Phenylhydrazine‐1‐Carbothioamide (YZ1)

2.12. N‐(4‐Methoxyphenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ2)

2.13. N‐(4‐Chlorophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ4)

2.14. N‐(3‐Methoxyphenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ5)

2.15. N‐(Naphthalen‐1‐yl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ6)

2.16. N‐(2,6‐Difluorophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ7)

2.17. N‐(4‐Fluorophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ8)

2.18. N‐(4‐Ethylphenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ9)

2.19. N‐(2‐Bromophenyl)‐2‐(2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazine‐1‐Carbothioamide (YZ10)

2.20. Methyl 2‐(3‐(4‐Nitrophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.1)

2.21. Methyl 2‐(3‐(4‐Cyanophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.2)

2.22. Methyl 2‐(3‐(4‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.3)

2.23. Methyl 2‐(3‐(4‐Chlorophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.4)

2.24. Methyl 2‐(3‐(3‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.5)

2.25. Methyl 2‐(3‐(Naphthalen‐1‐yl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.6)

2.26. Methyl 2‐(3‐(2,6‐Difluorophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.7)

2.27. Methyl 2‐(3‐(4‐Fluorophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.8)

2.28. Methyl 2‐(3‐(4‐Ethylphenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.9)

2.29. Methyl 2‐(3‐(2‐Bromophenyl)‐4‐Oxo‐2‐((2‐Oxoindolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (N.Z.A.10)

2.30. Methyl 2‐(4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)‐3‐Phenylthiazolidin‐5‐ylidene)Acetate (Y.Z.A.1)

2.31. Methyl 2‐(3‐(4‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.2)

2.32. Methyl 2‐(3‐Benzyl‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.3)

2.33. Methyl 2‐(3‐(4‐Chlorophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.4)

2.34. Methyl 2‐(3‐(3‐Methoxyphenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazineylidene)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.5)

2.35. Methyl 2‐(3‐(Naphthalen‐1‐yl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.6)

2.36. Methyl 2‐(3‐(2,6‐Difluorophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.7)

2.37. Methyl 2‐(3‐(4‐Fluorophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.8)

2.38. Methyl 2‐(3‐(4‐Ethylphenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.9)

2.39. Methyl 2‐(3‐(2‐Bromophenyl)‐4‐Oxo‐2‐((2‐Oxo‐1‐(Prop‐2‐yn‐1‐yl)Indolin‐3‐ylidene)Hydrazono)Thiazolidin‐5‐ylidene)Acetate (Y.Z.A.10)

2.40. Cytotoxic Activity Studies

2.41. Quantitative Real‐Time Polymerase Chain Reaction (RT‐qPCR)

2.42. Total Oxidant Status

2.43. Total Antioxidant Status

2.44. Molecular Docking

2.45. MD Simulation

3. Results and Discussion

3.1. Chemistry

Scheme 1.

Table 1.

3.2. Antiproliferative Activity Studies

3.3. Gene Expression Analysis

Figure 1.

Figure 2.

Figure 3.

3.4. Oxidative Stress Analysis

Figure 4.

3.5. Molecular Docking

Figure 5.

Table 2.

Figure 6.

Figure 7.

3.6. MD Simulation

Figure 8.

4. Conclusions