•We explored the SAR by combining click chemistry and in situ screening assay.•Pro70 and Thr72 were the critical components for binding with these inhibitors.•Based on crystal structures, pyrazole was discovered as a novel C-terminus.•Pyrazole derivatives showed good activities and better selectivity for BACE1.We recently discovered and reported dual inhibitor 5 of AChE and BACE1 with N-benzylpiperidine ethyl as C-terminus. Compound 5 showed potent inhibitory activities for BACE1, and could reduce endogenous Aβ1–40 production in APP transgenic mice. In present work, we rapidly identified substituted triazole as the C-terminus of compound 5 by replacing the benzylpiperidine ethyl group with click chemistry and tested these synthesized compounds by in situ screening assay. As revealed by the crystal structures of BACE1 in complex with our triazole compound 12, we found that Pro70 and Thr72 located in the flap region were the critical components for binding with these inhibitors. With the aid of the crystal structure, a new series of five-membered heterocyclic compounds was prepared in order to explore the structure–activity relationship (SAR) of this class of molecules. From these efforts, pyrazole was discovered as a novel C-terminus of BACE1 inhibitors. After further modification of pyrazole with variable substituents, compound 37 exhibited good potency in enzyme inhibition assay (IC50 = 0.025 μM) and compound 33 showed moderate inhibition effects on Aβ production of APP transfected HEK293 cells. Moreover, these pyrazole derivatives demonstrated good selectivity versus cathepsin D. Our results indicated that the vicinity of Pro70 and Thr72 might be utilized as a subsite, and the discovered pyrazole derivatives might provide useful hints for developing novel BACE1 inhibitors as anti-AD drugs.We combined the click chemistry and in situ screening technologies to fast explore the SAR of C terminus of our previous compound. Further modification identified the pyrazole group as a good substituent for BACE1 inhibitors in the enzymatic, cellular inhibition assays.

2,6-Disubstituted pyridazinone 4 was identified by HTS as a novel acetylcholinesterase (AChE) inhibitor. Under SAR development, compound 17e stood out as displaying high AChE inhibitory activity and AChE/butyrylcholinesterase (BuChE) selectivity in vitro. Docking studies revealed that 17e might interact with the catalytic active site (CAS) and the peripheral anionic site (PAS) simultaneously. Based on this novel binding information, 6-ortho-tolylamino and N-ethyl-N-isopropylacetamide substituted piperidine were disclosed as new PAS and CAS binders.Graphical abstractA series of pyridazinones was evaluated and optimized for their AChE inhibitory activity in an enzyme assay. High AChE/BuChE selectivity of the lead compound was achieved.Highlights► Pyridazinone derivatives as novel AChE inhibitors were synthesized. ► High AChE/BuChE selectivity of the lead compound was achieved. ► Novel 6-ortho-tolylamino substitution which involved in π–π stacking and hydrophobic ‘Magic Methyl’ effect was investigated. ► Novel 4-substituted piperidine motif for CAS binding was reported. ► Docking study revealed that these pyridazinone derivatives might function as dual binding site inhibitors.

(M)-Bicelaphanol A (1) and (P)-bicelaphanol A (2), two unprecedented dimeric trinorditerpenes existing as atropisomers, together with their monomer celaphanol A (3), were isolated from the root bark of Celastrus orbiculatus. The structures and absolute configurations of 1 and 2 were determined by spectroscopic and single-crystal X-ray diffraction analyses. Compound 1 exhibited a significant in vitro neuroprotective effect against a hydrogen peroxide-induced cell viability decrease in PC12 cells at 1 μM, while compounds 2 and 3 showed such effects at 10 μM.

Oxidative stress has been implicated in pathophysiology of many neurodegenerative diseases (ND) and increased oxidative stress is closely associated with mitochondrial dysfunction. As a result, looking for potent antioxidants, especially those targeting mitochondria, has become an attractive strategy in ND therapy. In this study, we explored protective effects and potential mechanism of Ac-cel, a novel compound, against hydrogen peroxide (H2O2)-induced injury in PC12 cells. Pretreatment of PC12 cells with Ac-cel prior to 24 h of H2O2 exposure markedly attenuated cytotoxicity induced by H2O2 as evidenced by morphological changes and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ac-cel also exhibited potent antiapoptotic effect demonstrated by results of annexin V and PI staining. The above beneficial effects of Ac-cel were accompanied by improved mitochondrial function, reduced caspase-3 cleavage as well as upregulated ratio of Bcl-2/Bax protein expression. Moreover, Ac-cel pretreatment markedly reversed intracellular reactive oxygen species (ROS) accumulation following 30 min of H2O2 exposure in PC12 cells. Further, subcellular investigation indicated that Ac-cel significantly reduced production of mitochondrial ROS in isolated rat cortical mitochondria. Taken together, the present study, for the first time, reports that Ac-cel pretreatment inhibits H2O2-stimulated early accumulation of intracellular ROS possibly via reducing mitochondrial ROS production directly and leads to subsequent preservation of mitochondrial function. These results indicate that Ac-cel is a potential drug candidate for treatment of oxidative stress-associated ND.

Huperzine A, an active Lycopodium alkaloid extracted from traditional Chinese herb, is a potent, selective and reversible acetylcholinesterase (AChE) inhibitor and has been widely used in China for the treatment of Alzheimer's disease (AD). Accordingly, some new mechanisms of action for huperzine A have been discovered over the past decades. In addition to its AChE inhibitory effect, potent multifaceted neuroprotective effect through activating cholinergic system and directly acting on mitochondria have been explored. Moreover, in order to maximize the efficacy and safety of huperzine A therapy, great efforts have been made to optimize drug delivery system. In the present article, an attempt is made to discuss the current progress and future perspective for huperzine A therapy in AD.

A series of 21-arylidenepregnenolone derivatives and their corresponding epoxides were synthesized. The neuroprotective effects of these steroidal compounds against amyloid-β25–35 (Aβ25–35)- and hydrogen peroxide (H2O2)-induced neurotoxicity in PC12 cells, and oxygen–glucose deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells were evaluated. The bioassay results indicated that several 3β-pregn-21-benzylidene-20-one derivatives displayed potent in vitro neuroprotective effects in different screening models, for example, compounds 2b, 3a, 3b, and 3s showing significant activities against Aβ25–35-induced neurotoxicity in PC12 cells, 2b showing significant activities against H2O2-induced neurotoxicity in PC12 cells, and 2g, 3b, and 3e showing potent protection against OGD insult. The results suggested that introduction of an arylidene group into steroidal nucleus played an important role in neuroprotective activity, while the formation of epoxy group at C-5,6 could be also important for the neuroprotective activity in some degree. The pharmacological data reported here are helpful for the design of novel steroidal neuroprotective candidates.A series of 21-arylidenepregnenolone derivatives and their corresponding epoxides were synthesized. The bioassay results indicated that compounds 2b, 3a, 3b, and 3s showing significant activities against Aβ25–35-induced neurotoxicity in PC12 cells, 2b showing significant activities against H2O2-induced neurotoxicity in PC12 cells, and 2g, 3b, and 3e showing potent protection against OGD insult.

With an ageing population, dementia has become one of the world's primary health challenges. However, existing remedies offer limited benefits with certain side effects, which has prompted researchers to seek complementary and alternative therapies. China has long been known for abundant usage of various herbs. Some of these herbal decoctions are effective in stimulating blood circulation, supplementing vital energy and resisting aging, the lack of which are believed to underlie dementia. These herbs are regarded as new and promising sources of potential anti-dementia drugs. With the rapid evolution of life science and technology, numerous active components have been identified that are highly potent and multi-targeted with low toxicity, and therefore meet the requirements for dementia therapy. This review updates the research progress of Chinese herbs in the treatment of dementia, focusing on their effective principles.

To explore novel effective drugs for the treatment of Alzheimer’s disease (AD), a series of dual inhibitors of acetylcholineterase (AChE) and β-secretase (BACE-1) were designed based on the multi-target-directed ligands strategy. Among them, inhibitor 28 exhibited good dual potency in enzyme inhibitory potency assay (BACE-1: IC50 = 0.567 μM; AChE: IC50 = 1.83 μM), and also showed excellent inhibitory effects on Aβ production of APP transfected HEK293 cells (IC50 = 98.7 nM) and mild protective effect against hydrogen peroxide (H2O2)-induced PC12 cell injury. Encouragingly, intracerebroventricular injection of 28 into amyloid precursor protein (APP) transgenic mice caused a 29% reduction of Aβ1–40 production. Therefore, 28 was demonstrated as a good lead compound for the further study and more importantly, the strategy of AChE and BACE-1 dual inhibitors might be a promising direction for developing novel drugs for AD patients.Novel dual inhibitors of acetylcholinesterase and β-secretase were design, synthesis and biological evaluation. Among them, compound 28 exhibited good dual potency in enzyme inhibitory potency assay (BACE-1: IC50 = 0.567 μM; AChE: IC50 = 1.83 μM), and also showed excellent inhibitory effects on Aβ production of APP transfected HEK293 cells (IC50 = 98.7 nM) and mild protective effect against hydrogen peroxide (H2O2)-induced PC12 cell injury. Encouragingly, intracerebroventricular injection of 28 into amyloid precursor protein (APP) transgenic mice caused a 29% reduction of Aβ1–40 production.

Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. In this study, we investigated the neuroprotective effects of (M)-bicelaphanol A, which has been the first dimeric podocarpane type trinorditerpene isolated from Celastrus orbiculatus, against hydrogen peroxide (H2O2)-induced injury in human SH-SY5Y neuroblastoma cells. Our study showed that cells pretreated with (M)-bicelaphanol A significantly attenuated H2O2-induced cell viability reduction and cell apoptosis. These neuroprotective effects of (M)-bicelaphanol A were associated with a reduction of reactive oxygen species and an increase in the level of adenosine triphosphate. In addition, (M)-bicelaphanol A pretreatment markedly increased the phosphorylation level of Akt in SH-SY5Y cells. In conclusion, our results for the first time demonstrate that the protection of (M)-bicelaphanol A on SH-SY5Y cells against H2O2-induced oxidative stress may attribute, at least partially, to its attenuation of mitochondrial dysfunction and activation of Akt signaling pathway. Above results shed more light on the molecular mechanisms involved in the neuroprotective effects of (M)-bicelaphanol A, which could be a potential drug candidate for the treatment of oxidative stress-associated neurodegenerative diseases.Download high-res image (258KB)Download full-size image

Our previous work in cells and animals showed that mitochondria are involved in the neuroprotective effect of huperzine A (HupA). In this study, the effects of HupA on isolated rat brain mitochondria were investigated. In addition to inhibiting the Aβ25-35 (40 μM)-induced decrease in mitochondrial respiration, adenosine 5′-triphosphate (ATP) synthesis, enzyme activity, and transmembrane potential, HupA (0.01 or 0.1 μM) effectively prevented Aβ-induced mitochondrial swelling, reactive oxygen species increase, and cytochrome c release. More interestingly, administration of HupA to isolated mitochondria promoted the rate of ATP production and blocked mitochondrial swelling caused by normal osmosis. These results indicate that HupA protects mitochondria against Aβ at least in part by preserving membrane integrity and improving energy metabolism. These direct effects on mitochondria further extend the noncholinergic functions of HupA.