Synthesis and Anti-Leukemia Activity of N-[1-(1-Adamantyl)ethyl]-3-hydroxylup-20(29)-en-28-amide

Abstract

Objective: The aim of this work was to synthesize a new amide derivative of betulinic acid—N-[1-(1-adamantyl)ethyl]-3-hydroxylup-20(29)-en-28-amide, containing in the structure the pharmacophoric 1-(1-adamantyl)ethylamine group (active substance of the antiviral drug Rimantadine) and to evaluate its antiviral action in vitro and in vivo. Methods: modification of betulinic acid was carried out by the interaction of betulinic acid with 1-(adamantan-1-yl)ethan-1-amine hydrochloride through the stage of in situ (without isolation) production of the corresponding betulinic acid chloride with thionyl chloride in a solution of dimethylformamide in the presence of potash. At the initial stage of the study, the antiviral activity of the drug was determined in vitro in 96-well immunological plates with a freshly obtained culture of peripheral blood lymphocytes from a cow with leukemia, onto which different concentrations of the studied substance were layered in one row of wells, and rimantadine hydrochloride in another row with a dilution factor of 1 : 2 (from 1000 to 0.48 μg/mL) and subjected to 24 h incubation in a thermostat, followed by direct immunofluorescence reaction (DIF). Then an in vivo experiment was conducted on agouti guinea pigs (n = 25). Animals of the experimental groups (n = 5) were infected intraperitoneally with a cell suspension of lymphocytes isolated from a cow sick with leukemia, and then the substance was administered subcutaneously: group 1—once 2 h before infection at a dose of 40 μg/mg; group 2—twice at the same dose (2 h before and on the 7th day after infection); group 3—three times at the same dose (2 h before and on the 7th and 14th days after inoculation of the viral suspension); Group 4 did not receive the drug (positive control). Animals of the control group (n = 5) were administered physiological saline. Results and Discussion: The structure of the synthesized N-[1-(1-adamantyl)ethyl]-3-hydroxylup-20(29)-en-28-amide was proven using ¹H, ¹³C NMR spectroscopy and high-resolution mass spectrometry. It was found that the minimum inhibitory dose of the substance was 31.2 μg/mL, as evidenced by the absence of specific luminescence of the antigen-antibody complex on 3–4 pluses in the in vitro experiment taking into account the DIF. At the same time, rimantadine did not have an inhibitory effect on the bovine leukemia virus. Based on the results of in vivo diagnostic studies in direct and indirect immunofluorescence (IIF) and PCR, it was shown that the drug demonstrated the highest anti-leukemic activity in the 2nd and 3rd groups of guinea pigs, in which after 2-3-fold administration of the test substance, positive samples were noted in only 20% of animals by the 150th day from the start of the experiment. Conclusions: Based on the diagnostic studies, it can be concluded that the compound N-[1-(1-adamantyl)ethyl]-3-hydroxylup-20(29)-en-28-amide had a pronounced inhibitory effect on the bovine leukemia virus both in a freshly obtained culture of peripheral blood lymphocytes from a cow with leukemia and in the body of guinea pigs experimentally infected with BLV. The positive results obtained suggest its possible use in the field of veterinary medicine, virology and pharmacology for the prevention of the development and treatment of the clinical and hematological form of lymphoid leukemia in animals spontaneously infected with the bovine leukemia virus.