Study on dual-site inhibitors of acetylcholinesterase: Highly potent derivatives of bis- and bifunctional huperzine B
Introduction
To date, the cholinergic hypothesis is still the practical approach for treating Alzheimer’s disease (AD), therefore, the clinical use of acetylcholinesterase (AChE) inhibitors is wide to alleviate symptoms of moderate AD patients.1, 2, 3 Several AChE inhibitors, such as tacrine, rivastigmine, donepezil, and galanthamine, have been approved by FDA for the treatment of AD.4 However, the clinical use of the AChE inhibitors is sometimes limited mainly due to their some adverse effects and modest benefits to AD patients. Therefore, novel more effective therapy, including AChE inhibitors, needs to be developed for AD therapy.5, 6, 7
In 1991, X-ray crystallographic structural analysis of the AChE from Torpedo californica (TcAChE) has demonstrated that active site laid near the bottom of a deep and narrow gorge, that reaches halfway into the protein, and 14 aromatic residues lined a substantial portion of the surface of the gorge.8 This cavity was named as the ‘active site gorge’ and, further, the peripheral sites existed at the gorge mouth.9
Recently, scientists have reported that AChE was also responsible for the non-cholinergic actions. It has been demonstrated that AChE might function to accelerate β-amyloid peptide (Aβ) formation and could play a role during amyloid deposition in AD brain.10 The peculiar feature of AChE was affected by peripheral site binding ligands, such as decamethonium and propidium, and was not acted by active site inhibitors, such as edrophonium. Moreover, it has been shown that molecules that were able to interact with both active and peripheral sites of AChE (i.e. a dual-site inhibitor) could prevent the aggregating activity of AChE toward Aβ besides the inhibitory activity.11 In addition, the butyrylcholinesterase (BuChE) did not affect amyloid formation. Therefore, inhibitors with the dual-site binding to AChE have recently presented a new therapeutic strategic option.12, 13
Pang et al. first reported that bis(7)-tacrine (1), the heptylene-linked tacrine dimer, possessed both optimal AChE inhibitory potency and AChE/BuChE selectivity than tacrine itself.14, 15 The derivatives of bis-HupA16 and bis-5-amino-5,6,7,8-tetrahydroquinolinone (2)17, 18 have also been reported. The bis-galanthamine linked by alkylene (3) was more potent than galanthamine in the inhibition.19 The crystal structure of TcAChE-(bis-5-amino-5,6,7,8-tetrahydroquinolinone) complexes showed that they bound to TcAChE in the bivalent binding fashion.18 It was evident that the increased affinities of inhibitors 1, 2, and 3 (Fig. 1) to AChE were presented by binding to the dual-site of the enzyme.
Natural (−)-huperzine B (HupB, 4) was a Lycopodium alkaloid isolated from Chinese medicinal club moss Huperzia serrata. HupB was less potent and selective in inhibition of AChE than (−)-huperzine A (HupA, 5).20, 21 The latter was first isolated from the same herbs and has been approved as a new drug for treating symptoms of AD in China.22 However, HupB exhibited a higher therapeutic index in comparison with HupA.23, 24 We have reported preliminary results of a series of derivatives of bis- and bifunctional HupB.25, 26
In this paper, we would present our detail research results on design and synthesis of a series of derivatives of bis- and bifunctional HupB for discovering AD drug candidates from natural HupB. Besides study on the pharmacological activities of the HupB derivatives, the docking program by computational modeling was also performed.
Section snippets
Design and synthesis of bis- and bifunctional HupB derivatives
Should HupB be a lead compound for AD drug candidate, it will be necessary first to enhance inhibitory potency of the derivatives to AChE. The design of novel inhibitors derived from HupB should bind to the dual-site of the enzyme and, further, the structures of the derivatives will be optimized for improving their pharmacological properties.
Several methods were tried during the preparation of bis-HupB derivatives. The reaction of HupB with α,ω-dihaloalkanes was carried out in the presence of
AChE inhibition results and SAR discussion
These HupB derivatives have been assayed for their AChE (rat cortex homogenate) and, partially, BuChE (rat serum) inhibition potency using the Ellman method with slight modification.29 The pharmacological inhibitory results of bis-HupB are shown in Table 1.
All of the bis-HupB derivatives have displayed more potent and selective AChE inhibition than its parental HupB. The evaluation of the pharmacological results from between 8 and 9, 10 and 11, and particularly, 12 and 13 series revealed that
Pharmacological effects of the derivative 12h
The bis-HupB derivative 12h was taken as a typical sample and has been evaluated for its pharmacological effects on AChE inhibition, cognitive enhancement, and neuroprotection in vitro and in vivo. Spectrophotometrical methods were used to determine cholinesterase (ChE) activity, cell viability, antioxidants and lipid peroxidation. Mice water maze performance was used to evaluate the effects of 12h on the acquisition and memory impairment. Apoptosis was determined by DAPI staining under a
Docking study for derivatives 12 series
To explain the interaction modes of the bis-HupB derivatives to AChE, molecular docking simulations for derivatives 12a–12h to TcAChE were performed employing the program DOCK4.030, 31 based on the X-ray crystal structure of TcAChE-HupB complex27 (PDB entry 1GPN). The binding gorge of TcAChE composed of the central catalytic pocket and peripheral sites was taken as the binding site for docking. There were two conformations of Phe330 in the binding sites and conformation A occupies 65% of the
Conclusions
On the basis of the dual-site binding strategy in rational design of AChE inhibitors, natural HupB was chosen as a lead compound to synthesize a series of derivatives of bis- and bifunctional HupB. All bis-HupB and bifunctional HupB derivatives exhibited significantly more potent inhibition of AChE as compared with its parental HupB. The bis-HupB derivatives were able to bind simultaneously to both the catalytic and peripheral dual-site of AChE in different level, supported by the docking
Chemical preparation
The 1H NMR spectra were recorded on Bruker AMX 400 MHz NMR spectrometer. The NMR data were reported in parts per million relative to TMS or referenced to the solvent in which they were run. Melting points (uncorrected) were determined on a Buchi-510 capillary apparatus. IR spectra were recorded on Nicolet Magna IR 750 spectrometers. The MS(ESI) and HRMS (ESI) spectra were obtained on an Finnigan MAT 95 mass spectrometer, EI: 70 eV. The optical rotation value [α]D was determined with Perkin-Elmer
Acknowledgments
This study was financially supported by Shanghai SK Foundation for Research and Development (Project-2004010-h) and the National Natural Science Foundation of China (No. 30472067).
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