Synthesis And Biological Evaluation Of Bambuterol Analogues As Butyrylcholinesterase Inhibitors In CNS

Alzheimer's disease(AD)is a type of neurodegenerative disorder associated to a progressive impairment of cholinergic neurotransmission.It is reported that acetylcholinesterase(ACh E)and butyrylcholinesterase(BCh E),the two enzymes responsible for the hydrolysis of acetylcholine(ACh),play a significant role in the pathological process of AD.The clinical use of cholinesterase inhibitors mitigates the symptoms of the disease by increasing the level of ACh in the brain of AD patients.In healthy brain,ACh is hydrolyzed mainly by ACh E,while BCh E has only a supporting role.However,the neurodegeneration associated to AD progressively reduces the amount of ACh E,therefore in advanced AD patients BCh E acquires a more prominent role in the hydrolysis of ACh.The cholinesterase inhibitors currently used in clinic are specific ACh E inhibitors and double target inhibitors targeting both ACh E and BCh E.To date,no specific BCh E inhibitor have been approved for the treatment of AD.The potential advantages associated to the use of specific BCh E inhibitors to treat advanced AD patients are the possibility to increase ACh levels in the brain with reduced side effects caused by overinhibition of ACh E and with the concomitant possibility to slow down the progression of the disease.Bambuterol(BMB),a drug used for the treatment of asthma,has high specificity and inhibitory activity towards BCh E,but it is not suitable to be used in the treatment of AD because of its side effects on the heart and its poor blood-brain barrier permeability.The aim of this work was to discover novel specific and efficient BCh E inhibitors based on the structure of BMB with lower cardiac side effects and higher possibility to pass the blood-brain barrier than BMB.Such compounds can find application as novel drugs to treat AD.The main contents of this work can be summarized as follows:1)BMB was used as the lead compound and thirty-six BMB derivatives were prepared with the aim of reducing the side effects on the heart while maintaining the inhibitory activity for BCh E and improving the lipid solubility.The results from Chemoffice software showed that most of the coupounds prepared in this work have higher lipid solubility than BMB.2)Ellman's method was employed to measure the inhibitory activity of the prepared compounds towards cholinesterases in vitro.The IC50 values obtained with equine BCh E were significantly lower than those obtained with electric eel ACh E for all the analogues.The IC50 values of the two compounds with higher druggability(6a-2 and 6b-1)and BMB were higher than 10000 n M for human ACh E and lower than 10 n M for human BCh E.The results showed that all the analogues could inhibit BCh E and that compounds 6a-2 and 6b-1 were specific BCh E inhibitors as efficient as BMB.The second order carbamylation rate constant k I and the decarbamylation rate constant k3 of equine and human BCh E inhibited by BMB,6a-2 and 6b-1 were measured.The three drugs showed similar k I values for each enzyme which means that the three drugs have the same inhibition efficiency for BCh E.Similar k3 values were obtained for each enzyme when BMB and 6a-2 were the inhibitors,while lower values were obtained for 6b-1,which means that BCh E recovers its activity after the inhibition with the three drugs and that the recovery is slower when 6b-1 is used as the inhibitor compared to BMB and 6a-2.Taken together,the results demonstrated that compounds 6a-2 and 6b-1 are potent BCh E inhibitors as efficient as BMB and that they inhibit BCh E with the same pseudo irreversible mechanism observed for BMB.3)The drugs were administered(20 mg/kg)to anesthetized mice to evaluate the effect of BMB,6a-2 and 6b-1 on the heart.BMB increased significantly the heart rate of the mice while weaker effects were observed for the other two analogues,especially for 6a-2 which showed an effect similar to the blank group.Based on these results,we predict that analogues 6a-2 and 6b-1 have less cardiac side effects than BMB.4)The in vivo inhibition of brain cholinesterase activity by 6a-2 and 6b-1 was studied in mice with a dose of 10 mg/kg and three administration protocols: single intragastric,repeated intragastric and single intraperitoneal administration.The administration of compound 6a-2 showed BCh E inhibition and the levels of inhibition were 6.50 %,34.29 % and 34.87 % for the three protocols,respectively.The difference in BCh E activity between the group treated with 6a-2 and the blank group was significant when the repeated intragastric and the single intraperitoneal administration protocols were employed(P<0.01).There was no significant difference in ACh E activity between the group treated with 6a-2 and the blank group(P>0.05).There was no significant difference in ACh E and BCh E activity between the group treated with 6b-1 and the blank group(P>0.05).These results demonstrated that compound 6a-2 can permeate the blood-brain barrier and inhibit the activity of brain BCh E,in vivo.Thirtysix BMB analogues with inhibitory activity towards BCh E were prepared in this study and 35 of these analogues have not been previously reported.We found that the novel compound 6a-2 is a potent inhibitor of brain BCh E with activity in vivo and less cardiac side effects than the existing inhibitors.This compound represents an important stepping stone in the discovery of novel drugs for the treatment of AD.