A series of novel 1,3,5-substituted 2-pyrazoline derivatives (IIIa-j) comprising benzo[7]annulene moiety and aromatic substitutions were synthesized via the reaction of (E)-3-(9-chloro-2,3-dimethyl-6,7-dihydro-5H-benzo[7]annulen-8-yl)-1-(substituted phenyl)prop-2- n-1-one with phenylhydrazine. Using spectroscopic methods, the structures of the synthesized derivatives were confirmed. The newly synthesized pyrazoline derivatives were put to the test in vitro on two cancer cell lines (HT-29 and MCF-7). After 72 h of incubation, the results showed that compounds IIId and IIIb altered the morphology of treated cell lines more than untreated or vehicle-treated cells, possibly as a result of the presence of halogen groups (chloro and fluoro) at the para position of the phenyl ring. The nature of the interaction between the synthesized pyrazoline derivatives and the quinoxaline inhibitor bound to the JAK2 enzyme crystal structure were investigated using the molecular docking studies. These substances displayed the least amount of binding energy to the enzyme, according to the in silico investigations. As a result, the synthesized pyrazoline derivatives might be used as lead molecules for the development of new anticancer drugs.