A multicomponent synthesis was empolyed for the synthesis of ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1. An interesting cyclization was obtained when the amino-ester 1 reacted with ethyl isothiocyanate to give the benzo[4,5]thieno[2,3-d][1,3]thiazin-4-one 3. Acylation of the amino-ester 1 with chloroacetyl chloride in DCM and Et₃N afforded the acylated ester 4. The amino-ester 1 was cyclized to benzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one 8, which was reacted with some alkylating agents leading to alkylation at nitrogen 9–13. Hydrazide 14 was utilized as a synthon for the synthesis of the derivatives 15–19. Chloro-thieno[2,3-d]pyrimidine 20 was synthesized and reacted with the hydrazine hydrate to afford the hydrazino derivative 21, which was used as a scaffold for getting the derivatives 22–28. Nucleophilic substitution reactions were used for getting the compounds 29–35 from chloro-thieno[2,3-d]pyrimidine 20. In the way of anticancer therapeutics development, the requisite compounds were assessed for their cytotoxicity in vitro against MCF-7 and HepG-2 cancer cell lines. Twelve compounds showed an interesting antiproliferative potential with IC₅₀ from 23.2 to 95.9 µM. The flow cytometric analysis results showed that hit 4 induces the apoptosis in MCF-7 cells with a significant 26.86% reduction in cell viability. The in vivo study revealed a significant decrease in the solid tumor mass (26.6%) upon treatment with compound 4. Moreover, in silico study as an agonist for inhibitors of JAK2 and prediction study determined their binding energies and predicted their physicochemical properties and drug-likeness scores.

Herein, the mono and dialkylation of pyridazino[4,5-b]indole were achieved with a set of alkylating agents, including amyl bromide, allyl bromide, benzyl bromide and ethyl chloroacetate in the presence of K₂CO₃/acetone or KOH/DMSO. The hydrazinolysis of mono and di-esters 10 and 11 gave the target hydrazides 12 and 13, which displayed promising, potent, and significant cytotoxic activity against the MCF-7 cell line with IC₅₀ values of 4.25 and 5.35 μm compared to that of the standard drug 5-FU (IC₅₀ 6.98 μm), respectively. RT-PCR analysis of the most active compound 12 was performed to determine its mode of action through the up-regulation of pro-apoptotic genes and inhibition of anti-apoptotic and PI3K/AKT/mTOR genes. The findings were consistent with the proposed mechanism illustrated in the in silico study. Further, the in vivo analysis exhibited its potent anti-cancer activity through the prolongation of survival parameters, and inhibition of ascetic fluid parameters in EAC-bearing mice.