In this paper, we have assessed the design, synthesis, characterization, anticancer properties, toxicity, and in silico study of 8-piperazinyl caffeinyl-triazolylmethyl derivatives as new caffeine hybrid conjugates. These compounds consist of four moieties comprising 8-caffeinyl, piperazinyl, 1,2,3-triazolyl, and alkyl substituents. The synthesis of these compounds was started by bromination of caffeine to attain 8-BC, SNAr reaction with piperazine to acquire 8-PC, N-propargylation of 8-PC and finally click Huisgen cycloaddition with diverse alkyl azides. These compounds were in vitro tested against two significant cancer cell lines comprising breast cancer MCF-7 (ATCC HTB-22) and melanoma cancer A-375 (ATCC CRL-1619) cell lines and activities compared with methotrexate (MTX) as a reference drug. Anticancer assessments indicated 12j (IC50 = 323 ± 2.6) and 12k (IC50 = 175 ± 3.2) were the most potent compounds against A-375 and MCF-7 cell growth, respectively and their activities were even stronger than MTX (IC50 = 418 ± 2 for A375 and IC50 = 343 ± 3.6 for MCF-7). Toxicities were determined by screening compounds against normal cell line HEK-293 (ATCC CRL-11268) and indicated that except 12i (IC50 = 371 ± 2.3), 12j (IC50 = 418 ± 2.4), and MTX (IC50 = 199 ± 2.4), all compounds are non-toxic. Docking was conducted for 12j and 12k and determined the strong binding affinities to B-RAF kinase and hDHFR enzymes, respectively. In silico pharmacokinetic and physiochemical profiles of tested compounds were investigated which indicated that most compounds obeyed Lipinski's rule of five (RO5). The DFT study on M06-2X/6-311G (d,p) was used to indicate HOMO, LUMO, MEP, and other parameters for a better understanding of 12j and 12k reactivity. Owing to anticancer properties, toxicity, and in silico data, 12j and 12k can be proposed for further research in the future.
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