Design, Synthesis And Bioactivity Of Meptazinol Derivatives As AChE Inhibitors And Analgesic Prodrugs

Alzheimer's disease (AD), which is characterized by progressive loss of memory and impairment in cognition, is becoming a serious threat to life expectancy for elderly people. Current clinical therapy for AD patients is mainly palliative treatment targeting acetylcholinesterase (AChE). Recently AChE, particularly the peripheral anionic sites (PAS), was indicated to be involved in Aβpeptide aggregation-promoting action. AChE inhibitors, which simultaneously block both the catalytic and PAS, might not only alleviate the cognitive deficit of AD patients by elevating ACh levels, but also act as disease-modifying agents delaying amyloid plaque formation.Meptazinol (MEP) is a racemic marketed opioid analgesic with low addiction liability, the (-)-enantiomer of which has demonstrated moderate inhibition to AChE. In this thesis, starting with investigations on the action mechanism of (-)-MEP on TcAChE, we designed and synthesized series of MEP and (-)-MEP derivatives or carbamates and bivalent (-)-nor-MEP analogues by means of molecular docking and pharmacophore-based drug design. Aiming to improve the oral bioavailability of MEP, a coumarin-based esterase-sensitive prodrug of MEP was synthesized. Moreover, an RP-HPLC method for the determination of (2-amine-6-methyl)-benzoic acid meptazinol ester prodrug was established.1. Investigation of the binding mode of (-)-MEP on TcAChE by molecular dockingA reliable and practical molecular docking method for AChE ligand investigation was established by comparing two widely used docking programs FlexX and GOLD. Compared with FlexX, GOLD reproduces the experimental binding poses of known AChE ligands more accurately (RMSD < 1.5 A). GOLD fitness scores of these ligands also correlated with their activities.(-) and (+) isomers of MEP were docked separately into three gate conformations of TcAChE by using GOLD protocol. Comparing the fitness scores and interactions of both isomers, we revealed the binding modes of (-)- and (+)-MEPs on TcAChE. (-)-MEP bound with the enzyme catalytic site in an open-gated conformation through strong hydrophobic interactions and a hydrogen bond. The absence of the key hydrogen bond might be the reason for the low affinity of (+)-MEP to AChE. To our knowledge, this is the first exploration of the mechanism of action of MEP on AChE. Present study provides an alternate tool for the structural optimization of (-)-MEP in search of new AChE inhibitors. 2. Structural modification and optimization of MEP targeting the azepane ringThe leading compound MEP was structurally modified on two points (1-N-position and 4-position) in the azepane ring. Carbalkoxy was substituted to 1-N-position of (-)-nor-MEP. Hydroxyl, amino, and guanidine groups were introduced into the 4-position of MEP. Beckmann rearrangement reactions were carried out to obtain structurally diverse derivatives with eight-member ring. The aim was to improve AChE inhibitory activities by exploring the effectiveness of additional hydrogen bond donors and acceptors. We synthesized totally 33 compounds, including 10 target compounds, 18 intermediates, and 5 unexpected products. Among them 25 are new compounds. We found lots of interesting byproducts produced by abnormal rearrangement and theoretically explained the reaction mechanism.The results showed that N-demethylation does not harm the AChE inhibition. Activities were improved when basic groups were substitueted in 4-position. Both (-)-N-carbalkoxy-nor-MEPs and Beckmann rearrangement products lose their potencies. It demonstrated that 1-N-positive ionizable amino group is the key component for the AChE-ligand cation-Ï€interactions. A bulky eight-member ring might unfavorably conflict with the enzyme. 4-basic group substitueted derivatives might also be involved in a similar cation-Ï€interaction with the enzyme. Among them 4-Amino MEP 23 and 4-guanidine MEP methylether 25 are more potent than the leading compound MEP 1.3. Design, synthesis, bioactivities and molecular modeling of bis-(-)-nor-MEP analoguesBased on the (-)-MEP-AChE interaction mode, a series of 11 bis-(-)-nor-MEPs, connected by alkylene linker of different length (n=2-12) through the secondary amino groups, were designed and synthesized as novel inhibitors of AChE and BChE. The AChE inhibitory potency within the series was closely related to the length of alkylene chain. On the contrary, BChE inhibitory potency was less impacted by chain length. The distinction in inhibition-linker length relationship was explained, based on structural analysis of both enzymes, by the absence of peripheral site and the enlargement of the active site in BChE: some small aliphatic residues replace bulky aromatic ones.The most potent 40h (IC₅₀ = 3.9 nM) was, respectively, 10,000 and 1400 times more potent in inhibiting AChE than the monmor drug (-)-MEP and a marketed drug rivastigmine. It also showed 1500- and 150-fold increases in inhibiting BChE (IC₅₀=10 nM) compared with (-)-MEP and rivastigmine. Molecular modeling of 40h was performed to elucidate their binding poses and interactions with the active sites of both enzymes. 40h was found to simultaneously bind both the catalytic and peripheral sites of AChE throughÏ€-Ï€and cation-Ï€interactions with Trp86 and Trp286. In the case of BChE, 40h folded in the large gorge along the aliphatic residue wall.Compared with the reference compound propidium iodide, 40h markedly prevented the AChE-induced Aβaggregation by a factor of 2. In animal model, it was 400 times more potent than rivastigmine in reversing the cognitive impairment caused by scopolamine. It was demonstrated that 40h is a promising drug candidate with disease-modifying action.4. Pharmacophore-based design of MEP carbamatesA pharmacophore model was built using Catalyst/HypoGen module, based upon carbamates AChE inhibitor, including physostigmine and rivastigmine analogues. The best pharmacophore hypothesis consisted of four features: one hydrogen bond acceptor, one ring aromatic feature, one hydrophobic center and one positive ionizable center. The pharmacophore model was validated by highly predictive ability.Novel meptazinol carbamates were designed and estimated according to the established pharmacophore model. Two compounds with high predicted activities were synthesized. They showed higher AChE and BChE inhibitory potency than the drug rivastigmine in vitro. The most potent (-)-meptazinol dimethylcabamate 83 proved 787- and 500-fold increases in inhibiting AChE and BChE. (-)-Meptazinol phenylcabamate 84 was slightly less active and also less toxic than 83. Both (-)-meptazinol dimethylcabamate 83 and phenylcabamate 84 are novel potent AChE inhibitors. Further pharmacological study in animal models is currently underway to select ideal candidates.5. Studies on prodrugs of MEPSimilar to some drugs with phenol groups, MEP was easily metabolized, which caused serious first-pass effect. The clinical uses of MEP were still restricted by its low oral bioavailability (8.69%). A coumarin-based esterase-sensitive prodrug system was applied for masking the phenol group of MEP. A (Z)-3-[2-(Propionyloxy) phenyl]-2-propenoic meptazinol ester 94 was designed and synthesized as a potential prodrug against the enzyme metabolism. Biological evaluation results indicated that there was a four-fold increase in oral bioavailability of this prodrug compared to the parent drug MEP. The first-pass effect was dramatically decreased. It can be further developed into Class l-(3) new drug.An RP-HPLC method with ultraviolet detection for the determination of the prodrug (2-Amine-6-methyl)-benzoic acid meptazinol ester 97 hydrochloride was established. The method was proved to be sensitive and accurate in content determination and quality control.In this thesis, we totally synthesized 57 compounds, including 24 target compounds, 26 intermediates, and 7 unexpected products. Among them 39 are new compounds. Using molecular docking method, we established a set of evaluation system able to reliably and accurately investigate the binding mode of small inhibitors with AChE. A pharmacophore model with high predictive power was built based on carbamate AChE inhibitors, which can be used for 3D-database searching and lead identification. Here we discovered two kinds of AChE inhibitors as promising drug candidates for AD treatment. One of them is bis-(-)-nor-MEP 40h and another two are (-)-MEP dimethylcarbamate 83 and phenylcarbamate 84. Otherwise, a MEP prodrug 94 was disclosed to effectively decrease the first-pass effect and improve the oral bioavailability by a factor of 4.