Synthesis of novel piperazine-based bis(thiazole)(1,3,4-thiadiazole) hybrids as anti-cancer agents through caspase-dependent apoptosis

Abstract

A series of novel piperazine-based bis(thiazoles) 13a-d were synthesized in moderate to good yields via reaction of the bis(thiosemicarbazones) 7a, b with an assortment of C-acetyl-N-aryl-hydrazonoyl chlorides 8a-f. Similar treatment of the bis(thiosemicarbazone) 7a, b with C-aryl-N-phenylhydrazonoyl chlorides 10a, b afforded the expected bis(thiadiazole) based piperazine products 13b-d in reasonable yields. Cyclization of 7a, b with two equivalents of α-haloketones 14a-d led to the production of the corresponding bis(4-arylthiazol)piperazine derivatives 15a-h in good yields. The structures of the synthesized compounds were confirmed from elemental and spectral data (FTIR, MALDI-TOF, ¹H, and ¹³C NMR). The cytotoxicity of the new compounds was screened against hepatoblastoma (HepG2), human colorectal carcinoma (HCT 116), breast cancer (MCF-7), and Human Dermal Fibroblasts (HDF). Interestingly, all compounds showed promising cytotoxicity against most of the cell lines. Interestingly, compounds 7b, 9a, and 9i exhibited IC₅₀ values of 3.5, 12.1, and 1.2 nM, respectively, causing inhibition of 89.7%, 83.7%, and 97.5%, compared to Erlotinib (IC₅₀ = 1.3 nM, 97.8% inhibition). Compound 9i dramatically induced apoptotic cell death by 4.16-fold and necrosis cell death by 4.79-fold. Compound 9i upregulated the apoptosis-related genes and downregulated the Bcl-2 as an anti-apoptotic gene. Accordingly, the most promising EGFR-targeted chemotherapeutic agent to treat colon cancer was found to be compound 9i.

Fig. 1. Examples of some piperazine-based marketed drugs.

Fig. 2. Marketed anti-cancer drugs employing thiazole and 1,3,4-thiadiazole motifs.

Fig. 3. Marketed bis-carboxamide anti-cancer medication.

Fig. 4. Rationale design for molecular hybridization of our targets toward new anti-cancer agents.

Scheme 1. Synthetic route to the bis-thiosemicarbazones 7a, b.

Fig. 5. The tautomeric form of compounds 7a, b with restricted rotation of thioamide group.

Scheme 2. Synthetic route to piperazine-based bis(thiazole) derivatives 9a–j.

Scheme 3. Synthetic route to piperazine-based bis(1,3,4-thiadiazole) hybrids 13a–d.

Scheme 4. Synthetic route to the piperazine-based bis(thiazol-2-ylidene) derivatives 15a–h.

Fig. 6. Cytotoxicity of selected compounds on HCT116, HEPG2, and MCF7 cell lines after 48 h treatment by sulphorhodamine assay. Graphs and data analysis were performed using GraphPad InStat, version 8.

Fig. 7. (A) Cryptographs of Annexin-V/propidium iodide staining of untreated and 9i-treated HCT-116 cells with (IC₅₀ = 8.5 μM, 48 h). (B) Percentage of cell population at each cell cycle “G1, and G2” using DNA content-flow cytometry aided cell cycle analysis. Cell arrest happened at the P2 phase (arrow indicated).

Fig. 8. Quantitative RT-PCR results analysis of the apoptosis-related genes; P53, bax, caspases 3, 8, 9, and Bcl-2, respectively in HCT-116 cells treated with compound 9i with (IC₅₀ = 8.5 μM, 48 h). The data illustrated is the average of 3 independent experimental runs (mean ± SD). Fold change of untreated control = 1.