Study On The Design, Synthesis And Structure - Activity Relationship Of β - Secretase Inhibitors

Alzheimer’s disease(AD) is a progressive degenerative neurodisorder that is characterized by neurofibrillary tangle(NFT) and amyloid plaque(AP). Clinical symptoms include memory loss, cognitive dysfunction. The patients may at last can’t take care of themselves and thus poses great burden on the family as well as on the society. With an ageing society in our country AD is becoming a great challenge to the medical and health care profession.The clinical drugs(Rivastigmine, galantamine, donepezil and memantine) currently used can only relieve the symptoms instead of curing. According to the β–amyloid cascade hypothesis, the peptide fragment Aβ produced from β–secretase(BACE1) and γ-secretase by sequentially cutting β-APP is neurotoxic and responsible for the occurrence of AD. Being in the up-stream position of the Aβ production, BACE1 was considered to be a novel therapeutic target for AD and the discovery of BACE1 inhibitors has became an important strategy to find novel AD drug.On the basis of comprehensively analyzing results of literature researches, a brain-penetrating weak BACE1 inhibitor 2-imino-3-methyl-5,5-diphenyl-imidazolidin-4-one, was chosen as the lead(No. I-1 in this dissertation) for structural optimization to search for novel brain penetrating BACE1 inhibitors with improved inhibition efficiency and druggability.By analyzing the interactions between the lead compound and BACE1 active binding site, three types of compounds I, II and III were first designed. The guideline of designing these compounds was that the basic scaffold of the lead compound merged with the pharmacophore of Rivastigmine, Ladostigil or Selegiline while the sizes and electrical property of other substituents were varied to determine the effect on the BACE1 inhibition efficiency. Enzymatic inhibition test results revealed that compound I-6 and III-5 showed improved inhibition efficiency than the lead and this led us to design compounds of IV and V. Altogether 18 compounds with much improved inhibitory activity(IC50<1μM) were obtained.66 target compounds, including 33 of type I, 4 of type II, 5 of type III, 12 of type IV and 12 of type V, were synthesized through 14 synthetic routes.. These target compounds were not reported in literatures. Meanwhile, 85 intermediates were obtained in the synthesis process. The structures of all target compounds and intermediates were confirmed by 1H-NMR and/or MS.The BACE1 inhibitory activity of the target compounds were tested using the time-resolved fluorescence(HTRF) method. 18 compounds showed an IC50 of below 1μM. These compounds are I-6, III-5, IV-5, IV-6, IV-7, IV-8, IV-9, IV-10, IV-11, IV-12 and V-1, V-5, V-6, V-7, V-8, V-10, V-11, V-12. The most promising compound V-10 exhibited an IC50 of 84.7 n M, 84-fold improved potency than the lead.III-5, IV-5, IV-10, V-5 and V-6 were chosen to evaluate the pharmacokinetics and ability of brain-penetration. Primary results showed that all this five compounds could cross the BBB and distributes in the brain after oral administration to mice. Compound V-6 was well absorbed and showed better metabolic properties and higher level of exposure in the brain than the rest. The mechanism of penetrating BBB of V-6 is simple and less likely to be affected by other factors. Thus, if safe and effective dosages were assigned V-6 would be a better pharmacokinetic profile than other four compounds.It should be noted that the target compounds in this dissertation contains a chiral carbon while the compounds were tested with their racemic mixtures. Enantioselective synthesis approach was studied by taking V-5 as an example and this part of work laid foundations for the preparation of the single isomers of the other target compounds.Analyzing the enzymatic inhibition test results, the main SAR of these compounds against BACE1 is concluded as follows: 1) A =NH group of the guanidine(P1′ position) is inevitable for the BACE1 inhibitory activity. 2) Linking a Rivastigmine pharmacophore to the meta-position of the P1 phenyl ring could increase the BACE1 inhibitory activity while the Ladostigil pharmacophore decreases or eliminates the inhibitory activity. 3) A rigid diphenyl fragment at the P1-P3 position was important for improving the inhibitory activity. 4) Groups with proper size and electrical property at the P3 position to interact with the S3 sub-pocket is helpful for inhibitory activity improvement. 5) Prop-2-yn-1-ol, ethynylcyclopropane and 2-fluoroethanol are suitable fragments to interact with the S3 sub-pocket. 6) Replacing the P2′ phenyl ring with a cyclopropyl group results in compounds with little bioactivity change, this implies that reducing the size of P2′ fragment(smaller than the phenyl ring) might not affect the inhibitory activity a lot, it hints that this may shall be replaced by substituents even smaller than cyclopropyl group.Compounds I-6, III-5, IV-5 and V-6 were docked into the BACE1 active binding site to further determine the interaction modes and their common interaction feathers are as follows: 1) The =NH group of the guanidine forms hydrogen bonds with the Asp228 and Asp32 and this is inevitable for the BACE1 inhibitory activity. 2) The P2′ phenyl ring(or cyclopropyl group) occupies the S2′ pocket through hydrophobic force. 3) The P1 phenyl ring interacts with the S1 pocket. 4) The P3 phenyl ring(or phenol ester group for I-6) interacts with the S3 pocket through hydrophobic force. 5) The substituent at the P3 phenyl ring interacts with the S3 sub-pocket.The compounds we have design and synthesized in this dissertation showed improved enzymatic inhibition efficiency than the lead and five of the compounds(chosen to research on animal models) were demonstrated to be brain-penetration after oral administration to mice. The aim of discovering novel BACE1 inhibitors that could cross the BBB is achieved. This work laid foundations for the design and synthesis of more promising and selective BACE1 inhibitors for the potential treatment of AD.