Phenolic compounds are a new class of Carbonic Anhydrase inhibitors (CAIs). Despite numerous advancements in treatment 

approaches, cancer continues to be a growing health problem worldwide. In our study, we tested the eff ects of 4-hydroxy3-methoxyacetophenone

(1) , doxycycline hydrochloride (2) , 5,7-dichloro-8-hydroxyquinoline (3) , methyl 3,4,5-trihydroxybenzoate

(4) , 2-hydroxy-4-methylacetophenone (5) , 6-hydroxy-4-methylcoumarin (6) , and 2,5-dihydroxyacetophenone (7) 

on Achetylcholynesterase (AChE), Butrycholynesterase (BChE), and Human Carbonic anhydrase I (hCA I) enzymes. The 

U2OS human osteosarcoma cell line was used to determine the anticancer potential of these phenolic compounds. The 

eff ects of the compounds on proliferation and colony formation were analyzed using the Neutral Red Uptake (NRU) assay 

and the clonogenic assay. The K i values of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde, and 3,4-dihydroxy5-methoxybenzoic

acid were 203.80, 1170.00, and 910.00 mM, respectively, for hCA I, and 75.25, 354.00, and 1510.00 mM, 

respectively, for Human Carbonic anhydrase II (hCA II). Additionally, IC 50 values from in vivo studies were found to range 

from 173.25 to 1360.00 mM for CA I and CA II, respectively, using CO 2 -hydratase activity methods. The NRU assay results 

revealed that the compounds had a dose-dependent cytotoxic eff ect on U2OS cells. The IC 50 values of the compounds in 

U2OS osteosarcoma cells were determined to be > 100, 93.7, 81.4, 26.9, > 100, 53.1, and > 100 μM, respectively. Notably, 

methyl 3,4,5-trihydroxybenzoate (4) , the compound with the lowest IC 50 value, signifi cantly suppressed colony formation at 

5 and 10 μM concentrations. These results demonstrated that the phenolic compounds used in in vivo studies could inhibit 

approximately 30% of the CO2-hydratase activity of the total CA enzyme of rat erythrocytes. Furthermore, the anticancer 

potential of the tested compounds suggests that these molecules could pave the way for the development of new approaches in 

cancer treatment. The activities of the seven molecules studied were compared against AChE (PDB ID: 4M0E), BChE (PDB 

ID: 5NN0), hCA I (PDB ID: 2CAB), and E3 ubiquitin-protein ligase (PDB ID: 4HG7) proteins. The binding free energy of 

the molecule with the highest docking score is computed using MM/GBSA techniques. Finally, molecular dynamics simulations

were performed between 6-hydroxy-4-methylcoumarin and the 4M0E protein over a 0–200 ns interval.