3-Acyloxy-1,4-benzodiazepine-2-ones have found application as hypnosedative anxiolytics. Created in A.V. Bogatsky Physico-Chemical Institute of the NAS of Ukraine and used in medical practice hypnosedative preparation with anxiolytic effect cinazepam (levana® IC) is a representative of this series of compounds. In order to investigate interaction with the central benzodiazepine CNS receptors, affinity for these receptors and GABA shift of previously synthesized 3-acyloxy-1,4- benzodiazepine-2-ones (1-6), as well as crystal structure of 1, 3, 5 and 6 was studied by us. N H N O Br O (CH2-CH2) O R R n 1 2 R1 R2 n I, % Ki, nM GS 1 H C2H5 1 99 6,06±0,6 1,6 2 Cl C2H5 1 99 2,6±0,3 1,3 3 H C6H11 cycl 0 74 >1000 - 4 Cl C6H11 cycl 0 96 72±7,6 1,15 5 H C6H11 cycl 1 38 >10000 - 6 Cl C6H11 cycl 1 79 96.4±9,3 1,14 It has been established that introduction of cyclohexyl residue into the acyl fragment leads to a decrease in affinity for the CNS CBR. At the same time, introduction of chlorine atom in the 5- phenyl substituent increases the affinity of the compounds 2, 4 and 6. It should also be noted that the GABA shifts for these compounds are in the range from 1.14 to 1.30, indicating that they are partial agonists. Crystal structures of 1, 3, 5 and 6 revealed that acyloxy substituent at C3 atom reside in equatorial position of boat-like 1,4-benzodiazepine cycle. The peculiarities of molecular conformation and intermolecular interactions will be discussed.